**Investigation on the Mechanism of Qi-Invigoration from a Perspective of Effects of Sijunzi Decoction on Mitochondrial Energy Metabolism**

Xing-Tai Li

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54760

## **1. Introduction**

Traditional Chinese medicine (TCM) is an ancient Chinese medical system that takes a deep understanding of the laws and patterns of nature and applies them to the human body. TCM, which is also considered as an alternative medicine, is gradually being accepted and is practiced even in the Western world, is the quintessence of the Chinese cultural heritage, has made an everlasting contribution to the survival, propagation and prosperity of all ethnic groups in China, thereby enhancing the fertility and prosperity of the nation. TCM has been practiced by the Chinese for five thousands of years and is rooted in meticulous observation of how nature, the cosmos, and the human body are interacting. Major theories include: Qi, Yin and Yang, the Five Phases (Wu Xing), the human body Meridian system (Jingluo) and viscera and bowels (Zang Fu organs) theory.

Western medicine places strong emphasis on the physical structures of the body, which are made up of different organic and inorganic substances, proteins, cells and tissues. These substances form the physiological basis of humans. Western medicine treats disease at microscopic point of view. TCM, on the other hand, views life differently. Instead of empha‐ sizing discrete body components, the body is seen as a whole entity with connecting parts that work together to sustain life. TCM studies the world from the macroscopic point of view, and its target is to maintain the original harmony of human being [1]. Qi, Blood and Body Fluids are the most important fundamental substances necessary for life. Western Medicine is different from TCM because the TCM has a concept of Qi as a form of energy. It is suggested that Qi was "born" at the same instant as the rest of the universe, and that we are all born from the Qi of the universe. The ancient concepts of Qi are the foundation of TCM and accordingly,

© 2012 Li; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

disease or sickness is caused by a disruptive flow of energy or the imbalance of the Yin and Yang energies around our human bodies. Hence, TCM provides a holistic treatment [2]. Qi is said to be the unseen vital force that nourishes one's body and sustains one's life. An individual would become ill or dies if one's Qi in the body is imbalanced or exhausted [1].

universe at the very root of reality. It's impossible to really understand TCM and its in‐ credible healing power throughout the millennia without realizing the importance of Qi. The unseen power and intelligence that animates and orchestrates all physical and meta‐ physical processes is Qi. It is Qi that delivers the necessary information and messages be‐ tween all body structures and systems, and it is Qi that enables us to connect with the natural world and the Universal. Qi is the life force that permeates everything in the uni‐ verse. Without it there can be no growth and change. Your physical body cannot exist

Investigation on the Mechanism of Qi-Invigoration from a Perspective of Effects of Sijunzi Decoction on...

http://dx.doi.org/10.5772/54760

249

The theory of Qi in ancient philosophy was introduced into the medical field, the basic theory of Qi in TCM was formed, i.e., the concept of Qi in TCM was established during the mutual penetration between the materialist philosophy and medicine in ancient China. Qi has been used as a healing technique in China for 4000 years. The origin of the character of Qi was traced back to 3500 years ago. Confucius (who lived approximately 2500 years ago), taught moral and ethical behavior. In his Analects, the character of Qi appeared in four locations. It expressed

Taoism, which was founded by Lao-Tze (who was believed to have lived around the time of Confucius or 100 later), have had more influence on Qi and Qigong. In the book "Zhuangzi", which compiled the thoughts of Lao-Tze in the third century BC, the character of Qi appeared 39 times. What it explained was: "Qi exists throughout the universe, when Qi assembles, it appears as a human life; when Qi disassembles, the human will die. Therefore, do not worry about life and death. Live naturally and freely as you are". When one studies the principle of the Life Force, the Qi and the Tao (Yin and Yang); one would understand how this Life Force manifests in nature. Through self-cultivation, one basically enriches one's Qi for optimum health and longevity. This happens when one subscribes to this Life Force from nature that flows freely into one's mind and body. However, this requires one to live freely from desires, worries and emotions. To live freely, one has to detach from the worldly possessions. For the instance, money is to be spent, there is to-ing and fro-ing, thus a going and a coming of it; and there is a non-attachment to the money or the material things for better flow. Furthermore, one is required to discipline oneself by having a proper diet, sleep and exercise so that one would not disturb and interrupt with the movement of Life Force which may cause the Qi to dissipate in one's body. This dissipation of Qi would result one to fall into sickness, disease, physical and mental sufferings. It is the Taoist's belief that the practice of Qigong, Tai Chi movements and meditation helps one to harmonize one's Life force with one's environment and nature [2].

"Two classic medical texts, the Nei Jing (compiled from 100 B.C. to 100 A.D.) and the Nan Jing (written circa 100 to 200 A.D.) were important early documents that presented the core concepts of TCM, and they have informed generations of scholars and practitioners ever since. These core concepts suggest that disease is the result of imbalances in the flow of the body's Qi, and that the human body is a microcosm of the basic natural forces at work in the universe. Generally speaking, Qi is an essential substance that is full of vigor and flows fast. The Yellow Emperor's Inner Canon (Nei Jing) teaches us: "It is from calm, indifference, emptiness, and nondesiring that true Qi arises. If the spirit is harboured inside, whence can illness arise? When the will is at rest and wishes little, when the heart is at peace and fears nothing, when the body

without Qi. When you die your Qi leaves–it's transformed.

the concept related to breathe, food and vitality.

The total loss of Qi is what Chinese medicine refers to as death. And here are Chinese people from all walks of life as they seek relief, through a rebalancing of their Qi, their vi‐ tal energy, for ailments from colds to cancer. The ultimate goal of Tai Chi is to control and direct the Qi within the body. But does Qi really exist? It has no place in Western medical practice, but is essential to the practice of traditional medicine in China. Have ev‐ er you had a dramatic spiritual or emotional experience and felt energy literally rushing through your body? I believe this is Qi energy at work, moving through the body. The two types of medical practice existed side by side in China, and had little intercourse with one another. And from the Chinese perspective, Qi is the origin of true strength and pow‐ er as well as genuine health—body, mind, and spirit.

All kinds of diseases and ailments are born from Qi in TCM, Qi deficiency is the common cause of a variety of diseases and can lead to energy metabolism dysfunction, and Qi-invigoration is the basic principle for treatment of Qi deficiency. Due to the popularity and therapeutic values of "Qi-invigorating" herbal formulae, the investigation of biological activities and the underlying mechanisms in relation to "Qi-invigoration" is of great pharmacological interest. However, the mechanisms for Qi-invigoration in TCM remain elusive. In this regard, our previous studies show that all the four widely used Qi-invigorating herbal medcines (includ‐ ing ginseng, astragalus root, pilose asiabell root, white atractylodes rhizome) can increase bioenergy level of liver cells *in vivo*. We propose a hypothesis that Qi is closely related to bioenergy according to the ancient concept of Qi and modern bioenergetics [3]. The Qiinvigorating representative prescription Sijunzi Decoction (SD) is widely used for treatment of Qi deficiency. However, the pharmacological basis of "Qi-invigorating" action has yet to be established. This chapter aims to provide a comprehensive overview of Qi and Qi-invigoration in TCM, analyze mitochondrial protection and energy metabolic improvement of SD, find its underlying mechanism, thus further reveal the nature of Qi-invigoration in TCM from mitochondrial energy metabolism perspective, help to interpret the concept of Qi scientifically, and provide a new way of thinking and scientific evidence in guiding Qi-invigorating prescriptions for the treatment of energy metabolism- and mitochondria-related diseases. The mechanism of SD on energy metabolism improvement will be explored from mitochondrial oxidative phosphorylation and intracellular adenylates levels.

## **2. The ancient Chinese philosophy background in formation of Qi concept in TCM**

Perhaps the greatest genius of the ancient Chinese sages, and the insight that has given TCM its uninterrupted longevity and effectiveness as a complete medical system, was their discovery of Qi that gives life to everything in the universe and is everything in the universe at the very root of reality. It's impossible to really understand TCM and its in‐ credible healing power throughout the millennia without realizing the importance of Qi. The unseen power and intelligence that animates and orchestrates all physical and meta‐ physical processes is Qi. It is Qi that delivers the necessary information and messages be‐ tween all body structures and systems, and it is Qi that enables us to connect with the natural world and the Universal. Qi is the life force that permeates everything in the uni‐ verse. Without it there can be no growth and change. Your physical body cannot exist without Qi. When you die your Qi leaves–it's transformed.

disease or sickness is caused by a disruptive flow of energy or the imbalance of the Yin and Yang energies around our human bodies. Hence, TCM provides a holistic treatment [2]. Qi is said to be the unseen vital force that nourishes one's body and sustains one's life. An individual

The total loss of Qi is what Chinese medicine refers to as death. And here are Chinese people from all walks of life as they seek relief, through a rebalancing of their Qi, their vi‐ tal energy, for ailments from colds to cancer. The ultimate goal of Tai Chi is to control and direct the Qi within the body. But does Qi really exist? It has no place in Western medical practice, but is essential to the practice of traditional medicine in China. Have ev‐ er you had a dramatic spiritual or emotional experience and felt energy literally rushing through your body? I believe this is Qi energy at work, moving through the body. The two types of medical practice existed side by side in China, and had little intercourse with one another. And from the Chinese perspective, Qi is the origin of true strength and pow‐

All kinds of diseases and ailments are born from Qi in TCM, Qi deficiency is the common cause of a variety of diseases and can lead to energy metabolism dysfunction, and Qi-invigoration is the basic principle for treatment of Qi deficiency. Due to the popularity and therapeutic values of "Qi-invigorating" herbal formulae, the investigation of biological activities and the underlying mechanisms in relation to "Qi-invigoration" is of great pharmacological interest. However, the mechanisms for Qi-invigoration in TCM remain elusive. In this regard, our previous studies show that all the four widely used Qi-invigorating herbal medcines (includ‐ ing ginseng, astragalus root, pilose asiabell root, white atractylodes rhizome) can increase bioenergy level of liver cells *in vivo*. We propose a hypothesis that Qi is closely related to bioenergy according to the ancient concept of Qi and modern bioenergetics [3]. The Qiinvigorating representative prescription Sijunzi Decoction (SD) is widely used for treatment of Qi deficiency. However, the pharmacological basis of "Qi-invigorating" action has yet to be established. This chapter aims to provide a comprehensive overview of Qi and Qi-invigoration in TCM, analyze mitochondrial protection and energy metabolic improvement of SD, find its underlying mechanism, thus further reveal the nature of Qi-invigoration in TCM from mitochondrial energy metabolism perspective, help to interpret the concept of Qi scientifically, and provide a new way of thinking and scientific evidence in guiding Qi-invigorating prescriptions for the treatment of energy metabolism- and mitochondria-related diseases. The mechanism of SD on energy metabolism improvement will be explored from mitochondrial

**2. The ancient Chinese philosophy background in formation of Qi concept**

Perhaps the greatest genius of the ancient Chinese sages, and the insight that has given TCM its uninterrupted longevity and effectiveness as a complete medical system, was their discovery of Qi that gives life to everything in the universe and is everything in the

would become ill or dies if one's Qi in the body is imbalanced or exhausted [1].

er as well as genuine health—body, mind, and spirit.

oxidative phosphorylation and intracellular adenylates levels.

**in TCM**

248 Alternative Medicine

The theory of Qi in ancient philosophy was introduced into the medical field, the basic theory of Qi in TCM was formed, i.e., the concept of Qi in TCM was established during the mutual penetration between the materialist philosophy and medicine in ancient China. Qi has been used as a healing technique in China for 4000 years. The origin of the character of Qi was traced back to 3500 years ago. Confucius (who lived approximately 2500 years ago), taught moral and ethical behavior. In his Analects, the character of Qi appeared in four locations. It expressed the concept related to breathe, food and vitality.

Taoism, which was founded by Lao-Tze (who was believed to have lived around the time of Confucius or 100 later), have had more influence on Qi and Qigong. In the book "Zhuangzi", which compiled the thoughts of Lao-Tze in the third century BC, the character of Qi appeared 39 times. What it explained was: "Qi exists throughout the universe, when Qi assembles, it appears as a human life; when Qi disassembles, the human will die. Therefore, do not worry about life and death. Live naturally and freely as you are". When one studies the principle of the Life Force, the Qi and the Tao (Yin and Yang); one would understand how this Life Force manifests in nature. Through self-cultivation, one basically enriches one's Qi for optimum health and longevity. This happens when one subscribes to this Life Force from nature that flows freely into one's mind and body. However, this requires one to live freely from desires, worries and emotions. To live freely, one has to detach from the worldly possessions. For the instance, money is to be spent, there is to-ing and fro-ing, thus a going and a coming of it; and there is a non-attachment to the money or the material things for better flow. Furthermore, one is required to discipline oneself by having a proper diet, sleep and exercise so that one would not disturb and interrupt with the movement of Life Force which may cause the Qi to dissipate in one's body. This dissipation of Qi would result one to fall into sickness, disease, physical and mental sufferings. It is the Taoist's belief that the practice of Qigong, Tai Chi movements and meditation helps one to harmonize one's Life force with one's environment and nature [2].

"Two classic medical texts, the Nei Jing (compiled from 100 B.C. to 100 A.D.) and the Nan Jing (written circa 100 to 200 A.D.) were important early documents that presented the core concepts of TCM, and they have informed generations of scholars and practitioners ever since. These core concepts suggest that disease is the result of imbalances in the flow of the body's Qi, and that the human body is a microcosm of the basic natural forces at work in the universe. Generally speaking, Qi is an essential substance that is full of vigor and flows fast. The Yellow Emperor's Inner Canon (Nei Jing) teaches us: "It is from calm, indifference, emptiness, and nondesiring that true Qi arises. If the spirit is harboured inside, whence can illness arise? When the will is at rest and wishes little, when the heart is at peace and fears nothing, when the body labours but does not tire, then Qi flows smoothly from these states, each part follows its desires, and the whole gets everything it seeks'. The Chinese philosopher, Mencius (372–289 BC) described Qi in terms of moral energy, related to human excellence. This reinforces the argument that Qi is contextual, fluid in nature and not a fixed entity.

completely describes the nature of Qi. Most often, Qi is best defined according to its functions and properties. In the human body, Qi flows through meridians, or energy pathways. Twelve major meridians run through the body, and it is over this network that Qi travels through the

Investigation on the Mechanism of Qi-Invigoration from a Perspective of Effects of Sijunzi Decoction on...

Man depends on nature for his production and growth and must observe the common laws of the world. As everything in the world comes from the interaction of Heaven Qi and Earth Qi, man must breathe to absorb Heaven Qi and eat to absorb Earth Qi. The food Essence

air to produce the nutrients necessary for man's life activities. Qi of the human body comes from the combination of three kinds of Qi, Primordial Qi inherited from parents, the fresh air inhaled by the Lung and the refined food Essence transformed by the Spleen. Both the inherited and the acquired vital energies are further processed and transformed by the organs. The kidney first sends the innate vital substance upwards where it combines with food essence derived from the spleen. It further mixes with the fresh air from the lungs where it finally forms

By understanding how Qi is formed, TCM has identified two important factors necessary for maintaining health. By eating a healthy diet and breathing fresh air, the body extracts their most valuable essences and uses them to help form the vital energy. Following these simple principles are the first steps towards creating a healthy balance in the body. By keeping your daily source of energy—Acquired Qi—strong and balanced, energy is saved because a healthy Spleen and Stomach can extract more Qi from food and drink. Choosing food wisely and eating

Generally speaking, Qi of the human body has five functions: promoting, warming, defending, controlling and transforming. Qi provides the active, vital energy necessary for the growth and development of the human body and to perform the physiological functions of the organs, meridians and tissues. In addition, Qi promotes the formation and circulation of blood and

1 Both TCM and western medicine have the name of"spleen" organ, but connections and differences between them were perceived. TCM practitioners pay more attention to the function than the organ entity in the viscera concept. The Spleen is one of the viscera (zàng) organs stipulated by TCM, it is a functionally defined entity and not equivalent to the anatomical organ of the same name in Western medicine. The Spleen transforms and transports food Essence from the food after it has been preprocessed by the Stomach and the Small Intestine, and then distributes it to the whole body, especially upwards to the Lung and Heart, where food Essence is transformed into Qi and Blood. In this spirit, the Spleen is also called "root of the postnatal". Thus, TCM also describes the Spleen as the source of "production and mutual transformation" of Qi and Blood. The function "the Spleen governs transportation and absorption" and that of the pancreas have many things in common, therefore, the Spleen in TCM should include spleen and pancreas in Western medicine. The Spleen also assists the body's water metabolism, exercises control over the blood inside the vessels and governs muscles and limbs. Whereas spleen is the largest lymphoid organ in the human body in Western medicine, and its main function is to participate in the function of the immune response of the lymphoid tissue and it is closely related to cellular and humoral immunity.The author considers that the core connotation of the Spleen in TCM is energy metabolism, i.e.,the process of cellular energy metabolism is the function of the Spleen, where organelles that in charge

of the energy metabolism in the cell (i.e., mitochondria) may attribute to the Spleen in TCM.

must be sent up to the Lung to combine with fresh

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body and that the body's various organs send messages to one another.

**3.2. The sources of Qi**

into Qi of the body.

**3.3. The functions of Qi**

transformed and transported by the Spleen1

at regular intervals helps accomplish this.

## **3. The concept of Qi**

Qi is the hub from basic theory to clinical practice and health longevity in TCM. The true foundation of TCM is Qi. Qi, is an important category in the ancient Chinese philosophy, is a simple understanding of natural phenomena. In TCM, Qi is constantly in motion, is the subtle substance with a strong vitality which constitute the human body and maintain the activities of human life, is one of the most basic material, it is also known as the "es‐ sence Qi". When the concept Qi in TCM was used to discuss the human body, it often has the meaning of both life material and physiological functions. Therefore, Qi in TCM is one of the most important basic concepts. Qi is the basis for unifying theories of TCM, and Qi theory is the core of the basic theories of TCM. According to TCM, " Qi is fundamental to human, both life and death of human all depend on Qi, if Qi gets together, it will result to the birth; if Qi is harmonious, then the human body is healthy; if Qi decline, the body is weak; if Qi is disordered, the human will be sick; if Qi is depleted, the human will die; therefore, unharmonious Qi is fundamental to the disease." The concept of Qi is complex and messy, connotation of Qi is colorful, extension of Qi is all-pervasive and unlimited, Qi becomes the enigma of Chinese medicine. Because modern medicine has not the concept of "Qi", Qi is the biggest difference between Chinese and Western medicine, which caused communication barriers between the two systems of medicine.

#### **3.1. The meanings of Qi**

What is Qi? The concept of Qi is based on the ancient Chinese initial understanding of natural phenomena. That is, Qi is the most basic substance of which the world is comprised. Everything in the universe results from the movements and changes of Qi. This concept was introduced into TCM and became one of its characteristics. The meaning of Qi in TCM has two aspects. One refers to the vital substances comprising the human body and maintaining its life activities, such as the Qi of water and food (food essence), the Qi of breathing (breathing nutrients) and so on. The other refers to the physiological functions of viscera and bowels, Meridian system, such as the Qi of the heart, the lung, the spleen and the stomach and so on.

The ancient Chinese people believed Qi was the most fundamental entity making up the world. The Chinese character for "Qi" is the same word used for air or gas, and it is thought to have the same properties as these substances. While Qi is often described in the West as energy, or vital energy, the term Qi carries a deeper meaning. Qi has two aspects: one is energy, power, or force; the other is conscious intelligence or information. Qi can be interpreted as the "life energy" or "life force," which flows within us. Sometimes, it is known as the "vital energy" of the body. In fact, it may be difficult to find one equivalent English word or phrase that completely describes the nature of Qi. Most often, Qi is best defined according to its functions and properties. In the human body, Qi flows through meridians, or energy pathways. Twelve major meridians run through the body, and it is over this network that Qi travels through the body and that the body's various organs send messages to one another.

#### **3.2. The sources of Qi**

labours but does not tire, then Qi flows smoothly from these states, each part follows its desires, and the whole gets everything it seeks'. The Chinese philosopher, Mencius (372–289 BC) described Qi in terms of moral energy, related to human excellence. This reinforces the

Qi is the hub from basic theory to clinical practice and health longevity in TCM. The true foundation of TCM is Qi. Qi, is an important category in the ancient Chinese philosophy, is a simple understanding of natural phenomena. In TCM, Qi is constantly in motion, is the subtle substance with a strong vitality which constitute the human body and maintain the activities of human life, is one of the most basic material, it is also known as the "es‐ sence Qi". When the concept Qi in TCM was used to discuss the human body, it often has the meaning of both life material and physiological functions. Therefore, Qi in TCM is one of the most important basic concepts. Qi is the basis for unifying theories of TCM, and Qi theory is the core of the basic theories of TCM. According to TCM, " Qi is fundamental to human, both life and death of human all depend on Qi, if Qi gets together, it will result to the birth; if Qi is harmonious, then the human body is healthy; if Qi decline, the body is weak; if Qi is disordered, the human will be sick; if Qi is depleted, the human will die; therefore, unharmonious Qi is fundamental to the disease." The concept of Qi is complex and messy, connotation of Qi is colorful, extension of Qi is all-pervasive and unlimited, Qi becomes the enigma of Chinese medicine. Because modern medicine has not the concept of "Qi", Qi is the biggest difference between Chinese and Western medicine, which caused

What is Qi? The concept of Qi is based on the ancient Chinese initial understanding of natural phenomena. That is, Qi is the most basic substance of which the world is comprised. Everything in the universe results from the movements and changes of Qi. This concept was introduced into TCM and became one of its characteristics. The meaning of Qi in TCM has two aspects. One refers to the vital substances comprising the human body and maintaining its life activities, such as the Qi of water and food (food essence), the Qi of breathing (breathing nutrients) and so on. The other refers to the physiological functions of viscera and bowels, Meridian system, such as the Qi of the heart, the lung, the spleen and the stomach and so on.

The ancient Chinese people believed Qi was the most fundamental entity making up the world. The Chinese character for "Qi" is the same word used for air or gas, and it is thought to have the same properties as these substances. While Qi is often described in the West as energy, or vital energy, the term Qi carries a deeper meaning. Qi has two aspects: one is energy, power, or force; the other is conscious intelligence or information. Qi can be interpreted as the "life energy" or "life force," which flows within us. Sometimes, it is known as the "vital energy" of the body. In fact, it may be difficult to find one equivalent English word or phrase that

argument that Qi is contextual, fluid in nature and not a fixed entity.

communication barriers between the two systems of medicine.

**3. The concept of Qi**

250 Alternative Medicine

**3.1. The meanings of Qi**

Man depends on nature for his production and growth and must observe the common laws of the world. As everything in the world comes from the interaction of Heaven Qi and Earth Qi, man must breathe to absorb Heaven Qi and eat to absorb Earth Qi. The food Essence transformed and transported by the Spleen1 must be sent up to the Lung to combine with fresh air to produce the nutrients necessary for man's life activities. Qi of the human body comes from the combination of three kinds of Qi, Primordial Qi inherited from parents, the fresh air inhaled by the Lung and the refined food Essence transformed by the Spleen. Both the inherited and the acquired vital energies are further processed and transformed by the organs. The kidney first sends the innate vital substance upwards where it combines with food essence derived from the spleen. It further mixes with the fresh air from the lungs where it finally forms into Qi of the body.

By understanding how Qi is formed, TCM has identified two important factors necessary for maintaining health. By eating a healthy diet and breathing fresh air, the body extracts their most valuable essences and uses them to help form the vital energy. Following these simple principles are the first steps towards creating a healthy balance in the body. By keeping your daily source of energy—Acquired Qi—strong and balanced, energy is saved because a healthy Spleen and Stomach can extract more Qi from food and drink. Choosing food wisely and eating at regular intervals helps accomplish this.

#### **3.3. The functions of Qi**

Generally speaking, Qi of the human body has five functions: promoting, warming, defending, controlling and transforming. Qi provides the active, vital energy necessary for the growth and development of the human body and to perform the physiological functions of the organs, meridians and tissues. In addition, Qi promotes the formation and circulation of blood and

<sup>1</sup> Both TCM and western medicine have the name of"spleen" organ, but connections and differences between them were perceived. TCM practitioners pay more attention to the function than the organ entity in the viscera concept. The Spleen is one of the viscera (zàng) organs stipulated by TCM, it is a functionally defined entity and not equivalent to the anatomical organ of the same name in Western medicine. The Spleen transforms and transports food Essence from the food after it has been preprocessed by the Stomach and the Small Intestine, and then distributes it to the whole body, especially upwards to the Lung and Heart, where food Essence is transformed into Qi and Blood. In this spirit, the Spleen is also called "root of the postnatal". Thus, TCM also describes the Spleen as the source of "production and mutual transformation" of Qi and Blood. The function "the Spleen governs transportation and absorption" and that of the pancreas have many things in common, therefore, the Spleen in TCM should include spleen and pancreas in Western medicine. The Spleen also assists the body's water metabolism, exercises control over the blood inside the vessels and governs muscles and limbs. Whereas spleen is the largest lymphoid organ in the human body in Western medicine, and its main function is to participate in the function of the immune response of the lymphoid tissue and it is closely related to cellular and humoral immunity.The author considers that the core connotation of the Spleen in TCM is energy metabolism, i.e.,the process of cellular energy metabolism is the function of the Spleen, where organelles that in charge of the energy metabolism in the cell (i.e., mitochondria) may attribute to the Spleen in TCM.

supports the metabolism of body fluid. If there is a deficiency of Qi, its promoting functions are weakened. As a result, growth and development can be affected or delayed, the organs and meridians cannot function properly and blood formation is hampered, leading to a series of health problems. Qi also contains heat energy for the body. Being a heat source, Qi warms the body and keeps it at a constant temperature so normal physiological functions can take place. Deficiency of Qi can lead to a lowered body temperature, intolerance of cold and cold hands and feet. In TCM, "Evils" are environmental factors that lead to illness. They are classified as wind, summer heat, dampness, dryness, cold and fire. One of the main causes of disease is the invasion of "Evils". By resisting the entry of "illness evils" into the body, Qi defends against their attack and maintains healthy physiological functions.

of the human body and in the maintenance of its life activities. Therefore, any substantial matter can be regarded as a special process of the movement of Qi, and life, in essence, is the course of Qi's ascending, descending, exiting and entering movements in given conditions.

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In TCM theory, blood and Qi are inseparable. Blood is the "mother" of Qi; it carries Qi and also provides nutrients for its movement. In turn, Qi is the "commander" of the blood. This means that Qi is the force that makes blood flow throughout the body and provides the intelligence that guides it to the places where it needs to be. Losing too much blood causes an overall Qi deficiency. When there is a Qi deficiency, the body cannot function properly and therefore presents with a fever. In the treatment of such Blood Deficiency, supplementing Qi plays an even more important role than nourishing Blood. Bleeding, for another example, may be the result of Qi deficiency because Qi controls Blood flow, so such bleeding should be treated by strengthening Qi. TCM understands that everything is composed of two complementary energies; one energy is Yin and the other is Yang. They are never separate; one cannot exist without the other. Yin and Yang come from Qi. Qi is required to harmonize Yin and Yang.

**4. Mitochondrial energy metabolism – Its related diseases and ageing**

and death of eukaryotic cells, and are the main switch of cell apoptosis.

After the symbiotic engulfment of aerobic α-proteobacteria by pre-eukaryotic cells more than 1.5 billion years ago, mitochondria evolved as specialized organelles with a plethora of cellular functions. Over recent years, mitochondria have taken center stage as remarkably autonomous and dynamic cellular organelles that are intimately involved in orchestrating a diverse range of cellular activities. Mitochondria regulate the life and death of cells by manipulating several factors, including bioenergetics, mitochondrial permeability transition, and mitochondrial redox-status, they are usually regarded as specialized organelles for cellular respiration and oxidative phosphorylation (OXPHOS). Mitochondria are the driving force behind life, over 80% of the energy which is required by an adult is produced by OXPHOS under normal physiological condition. Energy metabolism would be regulated by the relative amount of adenosine triphosphate (ATP) available, as described by adenylate energy charge (AEC). ATP has been called the energy "currency" of the cell. The electron transport chain (ETC) in the mitochondrial inner membrane is actively involved in ATP synthesis in combination with respiration. The impaired ETC works less efficiently in ATP synthesis and generates more reactive oxygen species (ROS), which will cause further oxidative damage to various biomo‐ lecules. In the aging process, oxidative damage ultimately leads to a progressive decline in bioenergetic function and enhanced mitochondrial oxidative stress. Lower ATP levels can decrease the efficiency of energy-dependent processes and ATP-mediated signal transduc‐ tions. Inadequate ATP availability would initiate and accentuate the adverse consequences of energy-dependent pathways. The energy depletion and enhanced oxidative stress can lead to the aging process. As the "hubs" for cellular metabolism, mitochondria are crucial for both life

**3.5. The relations between Qi and blood, Yin and Yang**

Qi consolidates and retains the body's substances and organs by holding everything in its proper place. Qi keeps the blood flowing within the vessels and prevents it leaking out, controls and adjusts the secretion and excretion of sweat, urine and saliva, and keeps body fluids from escaping the body, consolidates and stores sperm to prevent premature ejaculation, and consolidates the organs and stops them from descending into a position where they cannot function properly. If Qi is deficient, the consolidating function is weakened, leading to various kinds of health problems such as haemorrhage, frequent urination, premature ejaculation and stomach or kidney prolapses. The promoting and consolidating functions work in a comple‐ mentary manner. For example, Qi promotes blood circulation and the distribution of body fluids, but it also controls and adjusts the secretion of fluid substances. The balance between these two functions is essential for maintaining a healthy blood circulation and water metab‐ olism. Qi also possesses vaporization or "transformation" functions, which are important for the metabolism of fundamental substances. As suggested by these words, Qi may "vaporize" substances in the body and transform them into essence or vital energy. For example, certain actions of Qi allow food to be changed into food essence, which is in turn transformed into different types of Qi and blood. Indigestible food and waste are also transformed by Qi into urine and stools for excretion.

#### **3.4. The movement of Qi**

Qi flows throughout the whole body because of its strength and vigor. The movement of Qi is called Mechanism of Qi, which can be generalized as four aspects: ascending, descending, entering and exiting movements, which are based on directions. Qi was originally a philo‐ sophic concept. Through out the ages, the Chinese have developed working constructs which serve to explain the observable phenomenon of the natural world. The idea of Qi is one of the most basic building blocks upon which the Chinese, of both ancient and modern times, conceive the universe. The concept of Qi is a fundamental stratagem in the practice of any Chinese art and is at the root of Chinese medical theory. According to Chinese thought, Qi is an invisible energy-like phenomenon which is present in every animate or inanimate object in the universe. It is a difficult concept to explain but Qi can almost be thought of as an adhesive which holds the cosmos together; the inertia through which all is create and destroyed. The ancient Chinese philosophy holds that Qi is this most basic substance constituting the world. Accordingly, TCM also believes that Qi is the most fundamental substance in the construction of the human body and in the maintenance of its life activities. Therefore, any substantial matter can be regarded as a special process of the movement of Qi, and life, in essence, is the course of Qi's ascending, descending, exiting and entering movements in given conditions.

#### **3.5. The relations between Qi and blood, Yin and Yang**

supports the metabolism of body fluid. If there is a deficiency of Qi, its promoting functions are weakened. As a result, growth and development can be affected or delayed, the organs and meridians cannot function properly and blood formation is hampered, leading to a series of health problems. Qi also contains heat energy for the body. Being a heat source, Qi warms the body and keeps it at a constant temperature so normal physiological functions can take place. Deficiency of Qi can lead to a lowered body temperature, intolerance of cold and cold hands and feet. In TCM, "Evils" are environmental factors that lead to illness. They are classified as wind, summer heat, dampness, dryness, cold and fire. One of the main causes of disease is the invasion of "Evils". By resisting the entry of "illness evils" into the body, Qi defends against

Qi consolidates and retains the body's substances and organs by holding everything in its proper place. Qi keeps the blood flowing within the vessels and prevents it leaking out, controls and adjusts the secretion and excretion of sweat, urine and saliva, and keeps body fluids from escaping the body, consolidates and stores sperm to prevent premature ejaculation, and consolidates the organs and stops them from descending into a position where they cannot function properly. If Qi is deficient, the consolidating function is weakened, leading to various kinds of health problems such as haemorrhage, frequent urination, premature ejaculation and stomach or kidney prolapses. The promoting and consolidating functions work in a comple‐ mentary manner. For example, Qi promotes blood circulation and the distribution of body fluids, but it also controls and adjusts the secretion of fluid substances. The balance between these two functions is essential for maintaining a healthy blood circulation and water metab‐ olism. Qi also possesses vaporization or "transformation" functions, which are important for the metabolism of fundamental substances. As suggested by these words, Qi may "vaporize" substances in the body and transform them into essence or vital energy. For example, certain actions of Qi allow food to be changed into food essence, which is in turn transformed into different types of Qi and blood. Indigestible food and waste are also transformed by Qi into

Qi flows throughout the whole body because of its strength and vigor. The movement of Qi is called Mechanism of Qi, which can be generalized as four aspects: ascending, descending, entering and exiting movements, which are based on directions. Qi was originally a philo‐ sophic concept. Through out the ages, the Chinese have developed working constructs which serve to explain the observable phenomenon of the natural world. The idea of Qi is one of the most basic building blocks upon which the Chinese, of both ancient and modern times, conceive the universe. The concept of Qi is a fundamental stratagem in the practice of any Chinese art and is at the root of Chinese medical theory. According to Chinese thought, Qi is an invisible energy-like phenomenon which is present in every animate or inanimate object in the universe. It is a difficult concept to explain but Qi can almost be thought of as an adhesive which holds the cosmos together; the inertia through which all is create and destroyed. The ancient Chinese philosophy holds that Qi is this most basic substance constituting the world. Accordingly, TCM also believes that Qi is the most fundamental substance in the construction

their attack and maintains healthy physiological functions.

urine and stools for excretion.

**3.4. The movement of Qi**

252 Alternative Medicine

In TCM theory, blood and Qi are inseparable. Blood is the "mother" of Qi; it carries Qi and also provides nutrients for its movement. In turn, Qi is the "commander" of the blood. This means that Qi is the force that makes blood flow throughout the body and provides the intelligence that guides it to the places where it needs to be. Losing too much blood causes an overall Qi deficiency. When there is a Qi deficiency, the body cannot function properly and therefore presents with a fever. In the treatment of such Blood Deficiency, supplementing Qi plays an even more important role than nourishing Blood. Bleeding, for another example, may be the result of Qi deficiency because Qi controls Blood flow, so such bleeding should be treated by strengthening Qi. TCM understands that everything is composed of two complementary energies; one energy is Yin and the other is Yang. They are never separate; one cannot exist without the other. Yin and Yang come from Qi. Qi is required to harmonize Yin and Yang.

## **4. Mitochondrial energy metabolism – Its related diseases and ageing**

After the symbiotic engulfment of aerobic α-proteobacteria by pre-eukaryotic cells more than 1.5 billion years ago, mitochondria evolved as specialized organelles with a plethora of cellular functions. Over recent years, mitochondria have taken center stage as remarkably autonomous and dynamic cellular organelles that are intimately involved in orchestrating a diverse range of cellular activities. Mitochondria regulate the life and death of cells by manipulating several factors, including bioenergetics, mitochondrial permeability transition, and mitochondrial redox-status, they are usually regarded as specialized organelles for cellular respiration and oxidative phosphorylation (OXPHOS). Mitochondria are the driving force behind life, over 80% of the energy which is required by an adult is produced by OXPHOS under normal physiological condition. Energy metabolism would be regulated by the relative amount of adenosine triphosphate (ATP) available, as described by adenylate energy charge (AEC). ATP has been called the energy "currency" of the cell. The electron transport chain (ETC) in the mitochondrial inner membrane is actively involved in ATP synthesis in combination with respiration. The impaired ETC works less efficiently in ATP synthesis and generates more reactive oxygen species (ROS), which will cause further oxidative damage to various biomo‐ lecules. In the aging process, oxidative damage ultimately leads to a progressive decline in bioenergetic function and enhanced mitochondrial oxidative stress. Lower ATP levels can decrease the efficiency of energy-dependent processes and ATP-mediated signal transduc‐ tions. Inadequate ATP availability would initiate and accentuate the adverse consequences of energy-dependent pathways. The energy depletion and enhanced oxidative stress can lead to the aging process. As the "hubs" for cellular metabolism, mitochondria are crucial for both life and death of eukaryotic cells, and are the main switch of cell apoptosis.

Dysfunction of mitochondria has severe cellular consequences and is linked to ageing and neurodegeneration in human. Since discovery of the first case of mitochondrial disease in 1959, with the depth of mitochondrial research and the rapid development of mitochondrial medicine, the number of mitochondria-related diseases is rapidly amplified, mitochondrial dysfunction would undermine the function of cells, tissues and organs, thereby causing cancer, myasthenia gravis, obesity, stroke, cardiovascular disease (ischemic reperfusion injury, hypertension, coronary heart disease, heart failure, diabetes and atherosclerosis, etc.), agerelated diseases, neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, depression etc.), and aging etc. These diseases is today's major diseases that threaten human health, mitochondria has become a new target for the treatment of diseases, because mito‐ chondrial oxidative damage is the main reason for cell damage and death, the general treatment program to treat a variety of mitochondrial diseases is the reduction in mitochon‐ drial oxidative damage. Therefore, mitochondrial protection is an important mechanism for the treatment of mitochondrial-related diseases.

rion is a critical factor for the normal ATP production in the cell. The end product of gly‐ colysis, pyruvate, is transported into the mitochondria by a specific carrier protein. The pyruvate is transformed, in the matrix of the mitochondria, into acetyl coenzyme A that activates the tricarboxylic acid (TCA) (Krebs) cycle. In the absence of oxygen, the end product of pyruvate is lactate that may leave the cell and pass into the microcirculatory blood stream via the monocarboxylase transporter located in the plasma membrane [6].

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**Figure 1.** Overview of the cellular energy metabolic pathways. Mitochondria can metabolize fuels, such as fatty acids, amino acids and pyruvate, derived from glucose. When glucose enters the cell via glucose transporters, it is metabolized by glycolysis to pyruvate. Pyruvate prevalently enters mitochondria through its specific carrier (PC), with only a small amount being metabolized to lactate due to the excess of NADH. In mitochondria, pyruvate de‐ hydrogenase complex (PDH) converts pyruvate into acetyl-CoA, which feeds into the Krebs cycle, of which the net reactive result is to generate NADH and FADH2. The respiratory chain consists of four enzyme complexes (com‐ plexes I–IV) (yellow), and two mobile carriers (coenzyme Q and cytochrome c) along which the electrons liberated by the oxidation of NADH and FADH2 are passed, and ultimately transferred to oxygen. This respiratory process which electrons pass through generates membrane potential (Δψm)– the main driving force for ATP synthesis used by the ATP synthase to phosphorylate ADP and produce ATP, that in turn is carried to the cytosol by adenine nu‐

cleotide translocase (ANT) in exchange for ADP.

Bioenergetics research in life sciences have played an important role, Mitchell's chemiosmotic theory earned the 1978 Nobel Prize in Chemistry, as the coupling between electron transport in the respiratory chain and adenosine diphosphate (ADP) phosphorylation which is caused by electrochemical gradient of protons between inner and outer mitochondrial membrane was expounded; Nobel Prize in Chemistry in 1997 was awarded academician PD Boyer in the U.S. Academy of Sciences for elucidating generation mechanism of ATP—the most important energy molecules. The work was closely related to the energy production and consumption which is required for life activities. According to the modern life science, energy metabolism is the center for life activity, if the energy metabolism is normal, the body can carry out normal vital activities, if no bio-energy is supplied for the body, the life activities cease immediately. Therefore, Qi and bioenergy have identical functions.

#### **4.1. Energy metabolism in mammalian cells**

Mitochondria have been described by cytologists since the mid 19th century. According to Scheffler [4], the term mitochondrion was coined by Benda in 1898. However, only in the mid 20th century the role of the mitochondria in oxidative energy metabolism was estab‐ lished in detail [5]. All cells in the body depend on a continuous supply of ATP in order to perform their different physiological and biochemical activities. Mitochondria have a central role in the energy metabolism. Part of the free energy derived from the oxidation of food inside mitochondria is transformed to ATP, energy currency of the cell. This proc‐ ess depends on oxygen. When oxygen is limited, glycolytic products are metabolized di‐ rectly in the cytosol by the less efficient anaerobic respiration that is independent of mitochondria. The following describes the basic processes occurring in a typical normal cell, using glucose as a major source of energy (Figure 1). The breakdown of glucose into water and CO2 includes two steps, namely, glycolysis (the anaerobic phase) taking place in the cytoplasm, and OXPHOS (the aerobic phase) occurring in the mitochondria. Of the total yield of 38 ATP per mole of glucose, two are produced in the glycolysis process and 36 during the OXPHOS. It is important to note that oxygen availability in the mitochond‐ rion is a critical factor for the normal ATP production in the cell. The end product of gly‐ colysis, pyruvate, is transported into the mitochondria by a specific carrier protein. The pyruvate is transformed, in the matrix of the mitochondria, into acetyl coenzyme A that activates the tricarboxylic acid (TCA) (Krebs) cycle. In the absence of oxygen, the end product of pyruvate is lactate that may leave the cell and pass into the microcirculatory blood stream via the monocarboxylase transporter located in the plasma membrane [6].

Dysfunction of mitochondria has severe cellular consequences and is linked to ageing and neurodegeneration in human. Since discovery of the first case of mitochondrial disease in 1959, with the depth of mitochondrial research and the rapid development of mitochondrial medicine, the number of mitochondria-related diseases is rapidly amplified, mitochondrial dysfunction would undermine the function of cells, tissues and organs, thereby causing cancer, myasthenia gravis, obesity, stroke, cardiovascular disease (ischemic reperfusion injury, hypertension, coronary heart disease, heart failure, diabetes and atherosclerosis, etc.), agerelated diseases, neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, depression etc.), and aging etc. These diseases is today's major diseases that threaten human health, mitochondria has become a new target for the treatment of diseases, because mito‐ chondrial oxidative damage is the main reason for cell damage and death, the general treatment program to treat a variety of mitochondrial diseases is the reduction in mitochon‐ drial oxidative damage. Therefore, mitochondrial protection is an important mechanism for

Bioenergetics research in life sciences have played an important role, Mitchell's chemiosmotic theory earned the 1978 Nobel Prize in Chemistry, as the coupling between electron transport in the respiratory chain and adenosine diphosphate (ADP) phosphorylation which is caused by electrochemical gradient of protons between inner and outer mitochondrial membrane was expounded; Nobel Prize in Chemistry in 1997 was awarded academician PD Boyer in the U.S. Academy of Sciences for elucidating generation mechanism of ATP—the most important energy molecules. The work was closely related to the energy production and consumption which is required for life activities. According to the modern life science, energy metabolism is the center for life activity, if the energy metabolism is normal, the body can carry out normal vital activities, if no bio-energy is supplied for the body, the life activities cease immediately.

Mitochondria have been described by cytologists since the mid 19th century. According to Scheffler [4], the term mitochondrion was coined by Benda in 1898. However, only in the mid 20th century the role of the mitochondria in oxidative energy metabolism was estab‐ lished in detail [5]. All cells in the body depend on a continuous supply of ATP in order to perform their different physiological and biochemical activities. Mitochondria have a central role in the energy metabolism. Part of the free energy derived from the oxidation of food inside mitochondria is transformed to ATP, energy currency of the cell. This proc‐ ess depends on oxygen. When oxygen is limited, glycolytic products are metabolized di‐ rectly in the cytosol by the less efficient anaerobic respiration that is independent of mitochondria. The following describes the basic processes occurring in a typical normal cell, using glucose as a major source of energy (Figure 1). The breakdown of glucose into water and CO2 includes two steps, namely, glycolysis (the anaerobic phase) taking place in the cytoplasm, and OXPHOS (the aerobic phase) occurring in the mitochondria. Of the total yield of 38 ATP per mole of glucose, two are produced in the glycolysis process and 36 during the OXPHOS. It is important to note that oxygen availability in the mitochond‐

the treatment of mitochondrial-related diseases.

254 Alternative Medicine

Therefore, Qi and bioenergy have identical functions.

**4.1. Energy metabolism in mammalian cells**

**Figure 1.** Overview of the cellular energy metabolic pathways. Mitochondria can metabolize fuels, such as fatty acids, amino acids and pyruvate, derived from glucose. When glucose enters the cell via glucose transporters, it is metabolized by glycolysis to pyruvate. Pyruvate prevalently enters mitochondria through its specific carrier (PC), with only a small amount being metabolized to lactate due to the excess of NADH. In mitochondria, pyruvate de‐ hydrogenase complex (PDH) converts pyruvate into acetyl-CoA, which feeds into the Krebs cycle, of which the net reactive result is to generate NADH and FADH2. The respiratory chain consists of four enzyme complexes (com‐ plexes I–IV) (yellow), and two mobile carriers (coenzyme Q and cytochrome c) along which the electrons liberated by the oxidation of NADH and FADH2 are passed, and ultimately transferred to oxygen. This respiratory process which electrons pass through generates membrane potential (Δψm)– the main driving force for ATP synthesis used by the ATP synthase to phosphorylate ADP and produce ATP, that in turn is carried to the cytosol by adenine nu‐ cleotide translocase (ANT) in exchange for ADP.

The mitochondrial ATP production relies on the ETC, composed of respiratory chain com‐ plexes I–IV, which transfer electrons in a stepwise fashion until they finally reduce oxy‐ gen to form water. The NADH and FADH2 formed in glycolysis, fatty-acid oxidation and the citric acid cycle are energy-rich molecules that donate electrons to the ETC. Electrons move toward compounds with more positive oxidative potentials and the incremental re‐ lease of energy during the electron transfer is used to pump protons (H+ ) into the inter‐ membrane space. Complexes I, III and IV function as H+ pumps that are driven by the free energy of coupled oxidation reactions. During the electron transfer, protons are al‐ ways pumped from the mitochondrial matrix to the intermembrane space, resulting in a potential of ~150–180 mV. Proton gradient generates a chemiosmotic potential, also known as the proton motive force, which drives the ADP phosphorylation via the ATP synthase (FoF1 ATPase i.e., complex V). Fo domain of ATPase couples a proton translocation across the inner mitochondrial membrane (IMM) with the phosphorylation of ADP to ATP [7]. The energy-transducing function is maintained by the mitochondrial inner membrane and over 95% of total cellular ATP is supplied by mitochondrial phosphorylation [8]. Cellular activities can, therefore, be adversely affected by damage to the mitochondrial energytransducing functions [9]. The rate of mitochondrial respiration depends on the phosphor‐ ylation potential expressed as a [ATP]/[ADP] [Pi] ratio across the IMM that is regulated by the adenine nucleotide translocase (ANT). In the case of increased cellular energy de‐ mand when the phosphorylation potential is decreased and more ADP is available, a res‐ piration rate is increased leading to an increased ATP synthesis. There is usually a tight coupling between the electron transport and the ATP synthesis and an inhibition of ATP synthase will therefore also inhibit the electron transport and cellular respiration. Under certain conditions, protons can reenter into mitochondrial matrix without contributing to the ATP synthesis and the energy of proton electrochemical gradient will be released as heat. This process, known as proton leak or mitochondrial uncoupling, could be mediated by protonophores (such as FCCP) and uncoupling proteins (UCPs) [10]. As a conse‐ quence, uncoupling leads to a low ATP production concomitant with high levels of elec‐ tron transfer and high cellular respiration [11].

**4.2. The dysfunction of mitochondrial energy metabolism and human diseases**

biogenesis and function has been an active area of research in recent years [18].

In addition to the mitochondrial role in cellular bioenergetics, the pivotal role of mitochondrial dysfunction in various human diseases has become increasingly clear. For example, the involvement of the mitochondria in tumor cell pathogenesis was initially described by Warburg 80 years ago, and later followed by many studies. The pioneering work of Warburg on the metabolism of tumors led to the hypothesis that the development of cancer may originate when cellular glycolysis increases, while mitochondrial respiration becomes im‐ paired [19-21]. Warburg's hypothesis, termed the "Warburg effect", explains the significance of cellular energy metabolism in the pathophysiology of cancer cells. Since then, a large body of investigations has shown the involvement of the mitochondria in many human diseases. Mitochondrial oxidative damage is a major factor in many human disorders, including mitochondrial hepatopathies, chronic hepatitis C, steatosis, early graft dysfunction after liver transplantation, ischemia–reperfusion injury, ageing and inflammatory damage [22]. Oxida‐ tive damage accumulates more in mitochondria than in the rest of the cells because electrons continually leak from the respiratory chain to form damaging ROS. This oxidative damage may modify mitochondrial proteins, DNA and lipids which may lead to mitochondrial bioenergetics failure leading to necrotic or apoptotic cell death [23]. Despite the collection of vast knowledge on the mitochondrial function and human health, the accumulated informa‐ tion did not translate into practical clinical tools, such as new drugs or medical devices.

Decreased levels of ATP and phosphocreatine were observed in brains of portacaval-shunted rats infused with ammonia [24] as well as in rats with chronic hepatic encephalopathy (HE) [25]. Reduced brain ATP levels were likewise reported in rats with acute hyperammonemia [26]. Further, decreased levels of ATP were observed in cultured astrocytes treated with ammonium chloride [27]. Recent studies have also indicated reduced levels of AMP and ADP in rats with acute hyperammonemia, and such decrease was found to be due to increased activity of AMP deaminase and adenosine deaminase [28,29]. Another possible mechanism for impaired energy metabolism in HE and hyperammonemia is the mitochondrial permea‐

The role of mitochondria in mammalian cells is generally presented as a "central pathway" for energy metabolism, but mitochondria house many additional metabolic pathways and play a key role in apoptosis, free radical production, thermogenesis and calcium signaling. As a consequence, impairment of mitochondrial function is associated with a clinically heteroge‐ neous group of human disorders, often referred to as mitochondrial cytopathies [16]. In recent years, much attention has been attributed to the dysfunction of mitochondrial energy metab‐ olism, which has not only been associated with cardiac failure but also to numerous other disorders, such as cancer, diabetes, obesity and general senescence. The mitochondrion hosts the enzymes of the Krebs cycle and the complexes of the ETC which generate ATP by oxidation of carbohydrates, fatty acids and amino acids. It therefore functions as the foremost supplier of energy substrate to maintain systemic energy balance and homeostasis [17]. Mitochondrial OXPHOS serves a central role for energy homeostasis in mammals. Impaired mitochondrial OXPHOS contributes to the pathogenesis of a wide range of disease conditions, including metabolic disorders, neurodegeneration, and heart failure. Genetic control of mitochondrial

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From its role as the cellular powerhouse, the mitochondrion is emerging as a key participant in cell death. Apoptosis and necrosis are two alternative forms of cell death, with well-defined morphological and biochemical differences [12]. One crucial physiological difference between cells that undergo apoptosis or necrosis is intracellular ATP level. Complex I plays a major role in mitochondrial OXPHOS, include oxidizing NADH in the mitochondrial matrix, reducing ubiquinone to ubiquinol and pumping protons across the inner membrane to drive ATP synthesis [13]. Since its inhibition results in incomplete mitochondrial electron transport and disturbance of mitochondrial energy metabolism, dysfunction of Complex I in the hippocam‐ pus during the initial prolonged epileptic seizure may conceivably lead to necrosis because of a decrease in ATP production [14]. Mitochondrial creatine kinase is an important component of the cellular energy buffering and transport system, connecting oxidative phosphorylation to ATP consumption. The reduced activity of creatine kinase may lead to a decreased cellular ATP/ADP ratio [15].

#### **4.2. The dysfunction of mitochondrial energy metabolism and human diseases**

The mitochondrial ATP production relies on the ETC, composed of respiratory chain com‐ plexes I–IV, which transfer electrons in a stepwise fashion until they finally reduce oxy‐ gen to form water. The NADH and FADH2 formed in glycolysis, fatty-acid oxidation and the citric acid cycle are energy-rich molecules that donate electrons to the ETC. Electrons move toward compounds with more positive oxidative potentials and the incremental re‐

membrane space. Complexes I, III and IV function as H+ pumps that are driven by the free energy of coupled oxidation reactions. During the electron transfer, protons are al‐ ways pumped from the mitochondrial matrix to the intermembrane space, resulting in a potential of ~150–180 mV. Proton gradient generates a chemiosmotic potential, also known as the proton motive force, which drives the ADP phosphorylation via the ATP synthase (FoF1 ATPase i.e., complex V). Fo domain of ATPase couples a proton translocation across the inner mitochondrial membrane (IMM) with the phosphorylation of ADP to ATP [7]. The energy-transducing function is maintained by the mitochondrial inner membrane and over 95% of total cellular ATP is supplied by mitochondrial phosphorylation [8]. Cellular activities can, therefore, be adversely affected by damage to the mitochondrial energytransducing functions [9]. The rate of mitochondrial respiration depends on the phosphor‐ ylation potential expressed as a [ATP]/[ADP] [Pi] ratio across the IMM that is regulated by the adenine nucleotide translocase (ANT). In the case of increased cellular energy de‐ mand when the phosphorylation potential is decreased and more ADP is available, a res‐ piration rate is increased leading to an increased ATP synthesis. There is usually a tight coupling between the electron transport and the ATP synthesis and an inhibition of ATP synthase will therefore also inhibit the electron transport and cellular respiration. Under certain conditions, protons can reenter into mitochondrial matrix without contributing to the ATP synthesis and the energy of proton electrochemical gradient will be released as heat. This process, known as proton leak or mitochondrial uncoupling, could be mediated by protonophores (such as FCCP) and uncoupling proteins (UCPs) [10]. As a conse‐ quence, uncoupling leads to a low ATP production concomitant with high levels of elec‐

From its role as the cellular powerhouse, the mitochondrion is emerging as a key participant in cell death. Apoptosis and necrosis are two alternative forms of cell death, with well-defined morphological and biochemical differences [12]. One crucial physiological difference between cells that undergo apoptosis or necrosis is intracellular ATP level. Complex I plays a major role in mitochondrial OXPHOS, include oxidizing NADH in the mitochondrial matrix, reducing ubiquinone to ubiquinol and pumping protons across the inner membrane to drive ATP synthesis [13]. Since its inhibition results in incomplete mitochondrial electron transport and disturbance of mitochondrial energy metabolism, dysfunction of Complex I in the hippocam‐ pus during the initial prolonged epileptic seizure may conceivably lead to necrosis because of a decrease in ATP production [14]. Mitochondrial creatine kinase is an important component of the cellular energy buffering and transport system, connecting oxidative phosphorylation to ATP consumption. The reduced activity of creatine kinase may lead to a decreased cellular

) into the inter‐

lease of energy during the electron transfer is used to pump protons (H+

tron transfer and high cellular respiration [11].

ATP/ADP ratio [15].

256 Alternative Medicine

The role of mitochondria in mammalian cells is generally presented as a "central pathway" for energy metabolism, but mitochondria house many additional metabolic pathways and play a key role in apoptosis, free radical production, thermogenesis and calcium signaling. As a consequence, impairment of mitochondrial function is associated with a clinically heteroge‐ neous group of human disorders, often referred to as mitochondrial cytopathies [16]. In recent years, much attention has been attributed to the dysfunction of mitochondrial energy metab‐ olism, which has not only been associated with cardiac failure but also to numerous other disorders, such as cancer, diabetes, obesity and general senescence. The mitochondrion hosts the enzymes of the Krebs cycle and the complexes of the ETC which generate ATP by oxidation of carbohydrates, fatty acids and amino acids. It therefore functions as the foremost supplier of energy substrate to maintain systemic energy balance and homeostasis [17]. Mitochondrial OXPHOS serves a central role for energy homeostasis in mammals. Impaired mitochondrial OXPHOS contributes to the pathogenesis of a wide range of disease conditions, including metabolic disorders, neurodegeneration, and heart failure. Genetic control of mitochondrial biogenesis and function has been an active area of research in recent years [18].

In addition to the mitochondrial role in cellular bioenergetics, the pivotal role of mitochondrial dysfunction in various human diseases has become increasingly clear. For example, the involvement of the mitochondria in tumor cell pathogenesis was initially described by Warburg 80 years ago, and later followed by many studies. The pioneering work of Warburg on the metabolism of tumors led to the hypothesis that the development of cancer may originate when cellular glycolysis increases, while mitochondrial respiration becomes im‐ paired [19-21]. Warburg's hypothesis, termed the "Warburg effect", explains the significance of cellular energy metabolism in the pathophysiology of cancer cells. Since then, a large body of investigations has shown the involvement of the mitochondria in many human diseases.

Mitochondrial oxidative damage is a major factor in many human disorders, including mitochondrial hepatopathies, chronic hepatitis C, steatosis, early graft dysfunction after liver transplantation, ischemia–reperfusion injury, ageing and inflammatory damage [22]. Oxida‐ tive damage accumulates more in mitochondria than in the rest of the cells because electrons continually leak from the respiratory chain to form damaging ROS. This oxidative damage may modify mitochondrial proteins, DNA and lipids which may lead to mitochondrial bioenergetics failure leading to necrotic or apoptotic cell death [23]. Despite the collection of vast knowledge on the mitochondrial function and human health, the accumulated informa‐ tion did not translate into practical clinical tools, such as new drugs or medical devices.

Decreased levels of ATP and phosphocreatine were observed in brains of portacaval-shunted rats infused with ammonia [24] as well as in rats with chronic hepatic encephalopathy (HE) [25]. Reduced brain ATP levels were likewise reported in rats with acute hyperammonemia [26]. Further, decreased levels of ATP were observed in cultured astrocytes treated with ammonium chloride [27]. Recent studies have also indicated reduced levels of AMP and ADP in rats with acute hyperammonemia, and such decrease was found to be due to increased activity of AMP deaminase and adenosine deaminase [28,29]. Another possible mechanism for impaired energy metabolism in HE and hyperammonemia is the mitochondrial permea‐ bility transition (MPT). The MPT is characterized by a sudden increase in the permeability of the IMM to small solutes (ions and other molecules <1500 Da). The MPT is due to the opening of the permeability transition pore (PTP) in the IMM, usually in response to an increase in mitochondrial Ca2+ levels. This leads to a collapse of the mitochondrial inner membrane potential that is created by the pumping out of protons by the electron transport chain. Loss of the membrane potential leads to colloid osmotic swelling of the mitochondrial matrix, movement of metabolites across the inner membrane (e.g., Ca2+, Mg2+, glutathione, and NADPH), defective OXPHOS, cessation of ATP synthesis, and the generation of ROS. The latter acts to further aggravate the MPT [30,31]. Ca2+ is a well known inducer of the MPT [32]. Mitochondrial ATP-sensitive K+ channel (mitoKATP) opening results in decreased mitochon‐ drial ROS production. In addition, under energy deprivation conditions, mitoKATP opening inhibits mitochondrial ATP hydrolysis by ATP synthase, which helps to keep the cytosolic ATP/ADP ratio and also to limit mitochondrial Ca2+ uptake, indirectly preventing MPT [15].

long time that respiratory chain-deficient cells are more prone to undergo apoptosis and an increased cell loss is therefore likely of importance in the age-associated mitochondrial dysfunction [42]. In this part, I'd like to point out the link between the mitochondrial en‐ ergy balance and ageing, as well as a possible connection between the mitochondrial me‐

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Mitochondrial theory of ageing: Even though the process of oxidative phosphorylation is efficient, a small percentage of electrons may "leak" from the ETC, particularly from complexes I and III, during normal respiration and prematurely reduce oxygen, forming ROS [43]. Mitochondria is a well known source of cellular ROS; when an electron escapes from the mitochondrial electron transport chain, especially at complex I or III, it may react with molecular oxygen to form superoxide ion. Superoxide ion constantly generated during cellular metabolism gets converted to hydrogen peroxide (H2O2) and other ROS. Under physiological conditions, the maintenance of an appropriate level of intracellular ROS is important in keeping redox balance and signaling cellular proliferation [44]. ROS produced within mito‐ chondria presents almost 90% of the total ROS produced in the cell. The fact that the mito‐ chondrial ETC is the major ROS production site leads to the suggestion that mitochondria are a prime target for oxidative damage and hence the mitochondrial theory of ageing, a correlate to the free radical theory [45]. Over the years, substantial evidence has emerged from mor‐ phological, bioenergetic, biochemical and genetic studies to lend support to this theory [42]. Despite conflicting views concerning the primary role of mitochondrial ROS as a cause of aging [46], the generation of ROS within mitochondria remains the most viable theory to explain the process of aging. Increased levels of ROS within mitochondria are the principal trigger not only for mitochondrial dysfunction, but also for diseases associated with aging in general [47]. The latest results strongly argue that the observed phenotypes in mtDNA mutator mice are a direct consequence of the accumulation of mtDNA point mutations in protein-coding genes, leading to a decreased assembly of mitochondrial ETC complexes, respiratory chain dysfunc‐

On the other hand, the "uncoupling to survive" theory proposes that energy metabolism is in a positive relation with longevity. This theory is also based on the notion that inefficiency in the mitochondrial ATP generation may be necessary to reduce ROS generation in the cell [49]. High proton motive force that drives an efficient ATP synthesis comes with an additional cost, the production of ROS. Because ROS production is highly dependent on the proton motive force, proton leak might help to limit the oxidative damage. There are a number of articles suggesting that UCPs could play an important role in this process. It has been proposed that UCPs have a role in the protection from oxidative damage by lowering a proton motive force thus causing a "mild" uncoupling and the attenuation of superoxide production from electron chain [49]. During "mild" uncoupling, caused by UCPs activation with superoxide and other ROS products derived upon oxidation of membrane phospholipids, ATP is still synthesized,

a respiration rate is increased and in parallel a ROS production is decreased [49,50].

A significant decrease in the mitochondrial bioenergetic capacity with advancing age has been shown in numerous animal models and recently in a study of human volunteers [51]. A study on aged rats showed an increased intra-mitochondrial ROS production and oxidative damage,

tabolism and molecular pathways important for the lifespan extension.

tion and thus to premature ageing [48].

As noted in the above sections dealing with glycolysis, TCA cycle and OXPHOS, various animal models of acute liver failure (ALF) have been used to examine bioenergetic events in ALF. These studies described several abnormalities in cerebral energy metabolism, including glucose utilization [33], reduction in TCA cycle enzyme activity [34], decreased rate of respiratory chain activity [35], inhibition of creatine kinase activity [36], and reduced levels of ATP [37]. Studies showing the induction of the MPT in ammonia-treated cultured astrocytes, as well as in brains of rats with ALF suggest that the MPT plays a crucial role in the bioenergetic failure associated with HE and hyperammonemia. Hypertrigliceridemic liver mitochondria have a higher resting respiration rate but normal OXPHOS efficiency. The mild uncoupling mediated by mitoKATP accelerates respiration rates and reduces ROS generation [38]. Since the mitochondria are involved in a wide range of diseases, a new therapeutic approach was developed 30 years ago, aimed to develop drugs targeting the mitochondria.

#### **4.3. Mitochondrial energy metabolism and ageing**

Ageing is a process characterized by a general decline in physiological functions, and it is also considered as a major risk factor for many age-related diseases, including, but not limited to, neurodegenerative diseases, cardiovascular disorders, and metabolic diseases [39-41]. Ageing can be defined as "a progressive, generalized impairment of function, re‐ sulting in an increased vulnerability to environmental challenge and a growing risk of dis‐ ease and death". Ageing is likely a multifactorial process caused by accumulated damage to a variety of cellular components. During the last 20 years, gerontological studies have revealed different molecular pathways involved in the ageing process and pointed out mi‐ tochondria as one of the key regulators of longevity. Increasing age in mammals corre‐ lates with increased levels of mitochondrial DNA (mtDNA) mutations and a deteriorating respiratory chain function. Experimental evidence in the mouse has linked increased lev‐ els of somatic mtDNA mutations to a variety of ageing phenotypes, such as osteoporosis, hair loss, graying of the hair, weight reduction and decreased fertility. A mosaic respirato‐ ry chain deficiency in a subset of cells in various tissues, such as heart, skeletal muscle, colonic crypts and neurons, is typically found in aged humans. It has been known for a long time that respiratory chain-deficient cells are more prone to undergo apoptosis and an increased cell loss is therefore likely of importance in the age-associated mitochondrial dysfunction [42]. In this part, I'd like to point out the link between the mitochondrial en‐ ergy balance and ageing, as well as a possible connection between the mitochondrial me‐ tabolism and molecular pathways important for the lifespan extension.

bility transition (MPT). The MPT is characterized by a sudden increase in the permeability of the IMM to small solutes (ions and other molecules <1500 Da). The MPT is due to the opening of the permeability transition pore (PTP) in the IMM, usually in response to an increase in mitochondrial Ca2+ levels. This leads to a collapse of the mitochondrial inner membrane potential that is created by the pumping out of protons by the electron transport chain. Loss of the membrane potential leads to colloid osmotic swelling of the mitochondrial matrix, movement of metabolites across the inner membrane (e.g., Ca2+, Mg2+, glutathione, and NADPH), defective OXPHOS, cessation of ATP synthesis, and the generation of ROS. The latter acts to further aggravate the MPT [30,31]. Ca2+ is a well known inducer of the MPT [32].

drial ROS production. In addition, under energy deprivation conditions, mitoKATP opening inhibits mitochondrial ATP hydrolysis by ATP synthase, which helps to keep the cytosolic ATP/ADP ratio and also to limit mitochondrial Ca2+ uptake, indirectly preventing MPT [15].

As noted in the above sections dealing with glycolysis, TCA cycle and OXPHOS, various animal models of acute liver failure (ALF) have been used to examine bioenergetic events in ALF. These studies described several abnormalities in cerebral energy metabolism, including glucose utilization [33], reduction in TCA cycle enzyme activity [34], decreased rate of respiratory chain activity [35], inhibition of creatine kinase activity [36], and reduced levels of ATP [37]. Studies showing the induction of the MPT in ammonia-treated cultured astrocytes, as well as in brains of rats with ALF suggest that the MPT plays a crucial role in the bioenergetic failure associated with HE and hyperammonemia. Hypertrigliceridemic liver mitochondria have a higher resting respiration rate but normal OXPHOS efficiency. The mild uncoupling mediated by mitoKATP accelerates respiration rates and reduces ROS generation [38]. Since the mitochondria are involved in a wide range of diseases, a new therapeutic approach was

Ageing is a process characterized by a general decline in physiological functions, and it is also considered as a major risk factor for many age-related diseases, including, but not limited to, neurodegenerative diseases, cardiovascular disorders, and metabolic diseases [39-41]. Ageing can be defined as "a progressive, generalized impairment of function, re‐ sulting in an increased vulnerability to environmental challenge and a growing risk of dis‐ ease and death". Ageing is likely a multifactorial process caused by accumulated damage to a variety of cellular components. During the last 20 years, gerontological studies have revealed different molecular pathways involved in the ageing process and pointed out mi‐ tochondria as one of the key regulators of longevity. Increasing age in mammals corre‐ lates with increased levels of mitochondrial DNA (mtDNA) mutations and a deteriorating respiratory chain function. Experimental evidence in the mouse has linked increased lev‐ els of somatic mtDNA mutations to a variety of ageing phenotypes, such as osteoporosis, hair loss, graying of the hair, weight reduction and decreased fertility. A mosaic respirato‐ ry chain deficiency in a subset of cells in various tissues, such as heart, skeletal muscle, colonic crypts and neurons, is typically found in aged humans. It has been known for a

developed 30 years ago, aimed to develop drugs targeting the mitochondria.

**4.3. Mitochondrial energy metabolism and ageing**

channel (mitoKATP) opening results in decreased mitochon‐

Mitochondrial ATP-sensitive K+

258 Alternative Medicine

Mitochondrial theory of ageing: Even though the process of oxidative phosphorylation is efficient, a small percentage of electrons may "leak" from the ETC, particularly from complexes I and III, during normal respiration and prematurely reduce oxygen, forming ROS [43]. Mitochondria is a well known source of cellular ROS; when an electron escapes from the mitochondrial electron transport chain, especially at complex I or III, it may react with molecular oxygen to form superoxide ion. Superoxide ion constantly generated during cellular metabolism gets converted to hydrogen peroxide (H2O2) and other ROS. Under physiological conditions, the maintenance of an appropriate level of intracellular ROS is important in keeping redox balance and signaling cellular proliferation [44]. ROS produced within mito‐ chondria presents almost 90% of the total ROS produced in the cell. The fact that the mito‐ chondrial ETC is the major ROS production site leads to the suggestion that mitochondria are a prime target for oxidative damage and hence the mitochondrial theory of ageing, a correlate to the free radical theory [45]. Over the years, substantial evidence has emerged from mor‐ phological, bioenergetic, biochemical and genetic studies to lend support to this theory [42].

Despite conflicting views concerning the primary role of mitochondrial ROS as a cause of aging [46], the generation of ROS within mitochondria remains the most viable theory to explain the process of aging. Increased levels of ROS within mitochondria are the principal trigger not only for mitochondrial dysfunction, but also for diseases associated with aging in general [47]. The latest results strongly argue that the observed phenotypes in mtDNA mutator mice are a direct consequence of the accumulation of mtDNA point mutations in protein-coding genes, leading to a decreased assembly of mitochondrial ETC complexes, respiratory chain dysfunc‐ tion and thus to premature ageing [48].

On the other hand, the "uncoupling to survive" theory proposes that energy metabolism is in a positive relation with longevity. This theory is also based on the notion that inefficiency in the mitochondrial ATP generation may be necessary to reduce ROS generation in the cell [49]. High proton motive force that drives an efficient ATP synthesis comes with an additional cost, the production of ROS. Because ROS production is highly dependent on the proton motive force, proton leak might help to limit the oxidative damage. There are a number of articles suggesting that UCPs could play an important role in this process. It has been proposed that UCPs have a role in the protection from oxidative damage by lowering a proton motive force thus causing a "mild" uncoupling and the attenuation of superoxide production from electron chain [49]. During "mild" uncoupling, caused by UCPs activation with superoxide and other ROS products derived upon oxidation of membrane phospholipids, ATP is still synthesized, a respiration rate is increased and in parallel a ROS production is decreased [49,50].

A significant decrease in the mitochondrial bioenergetic capacity with advancing age has been shown in numerous animal models and recently in a study of human volunteers [51]. A study on aged rats showed an increased intra-mitochondrial ROS production and oxidative damage, increased proton leak rates resulting in a depletion of membrane potential and a reduction of ATPase and complex IV activities. Treatment of aged rats with the insulin-like growth factor 1 (IGF1) corrected these parameters indicating that a cytoprotective effect of IGF1 is closely related to the mitochondrial protection [52]. Caloric restriction is the only dietary intervention that consistently increases median and maximal lifespan in organisms ranging from yeast to mammals. This dietary regime implies 20–50% restriction of the overall caloric intake of animals on *ad libitum* regime [53]. The precise molecular mechanisms of the life-extending actions of caloric restriction still remains unclear, but most likely mitochondrial energy metabolism plays a very important role in this process.

(including Qi, Blood, Yin and Yang deficiencies) caused by the invasion of an exogenous pathogen, excessive physical strain (manual labor, mental labor and sexual intercourse), abnormal emotional states (elation, anger, worry, anxiety, sorrow, fear and terror) or an

Investigation on the Mechanism of Qi-Invigoration from a Perspective of Effects of Sijunzi Decoction on...

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261

Chinese tonic herbs that can produce heath-promoting action are used for the treatment of various patterns of deficiency in body function with respect to Qi, Blood, Yin, or Yang, and their combinations. These types of functional imbalance are viewed as sub-healthy conditions in modern medicine. Chinese tonic herbs are generally classified into four categories on the basis of their health-promoting actions, namely, "Qi-invigorating", "Blood-enriching", "Yinnourishing" and "Yang-invigorating". Of these four types of tonic herbs, the "Qi-invigorating" and "Blood-enriching" herbs are grouped under the "Yang" family and "Yin" family, respec‐ tively. Maintaining Yang and Yin in harmony is akin to attaining the homeostatic state in modern medicine [58]. Yang Qi refers to the body's vital force or functional aspects in general. Unlike the Blood or Body Fluids, Qi is an abstract concept in TCM; it can't be seen and belongs to Yang. Yang Qi sometimes refers to some body qualities and functions like superficial, upward direction, hyper-functioning, stimulating and light. It is the opposite of Yin Qi. Yang deficiency indicates insufficiency of Yang Qi inside the body that fails to provide the functions of warmth, motivation and promotion. Symptoms or signs include aversion to cold, cold limbs, bland taste in the mouth, preference for hot drinks, pale complexion, spontaneous sweating, general swelling, profuse and clear urine or loose stool. The tongue is pale, bulky with a white

slimy coating, and the pulse is deep and slow or thready on examination.

According to TCM theory, Yang is viewed as a manifestation of body function supported by various organs. A "Yang-invigorating" action therefore involves the general up-regulation of cellular activities. As ATP, an energy-rich biomolecule, is universally used for energizing cellular activities, the "Yang-invigorating" action may be mediated by the enhancement of mitochondrial ATP generation [59]. "Yang-invigorating" Chinese tonic herbs have shown to enhance the myocardial mitochondrial ATP generation capacity in mice *ex vivo* and in H9c2 cardiomyocytes [58,60]. All "Yang-invigorating" Chinese tonic herbs dose-dependently enhanced the mitochondrial ATP generation capacity. The stimulation of ATP generation was associated with an increased extent of mitochondrial electron transport [60]. It is believed that the up-regulation of cellular activities by "Yang-invigoration" in Chinese medicine requires an increased supply of ATP, which is in turn largely supported by mitochondrial OXPHOS [58].

Holistically, it is believed that the Yang-invigorating herbs enhance physiological cellular activities, which is in turn critically dependent on mitochondrial ATP generation through the OXPHOS process at the cellular level. A previous study has shown that short-term oral treatment with the methanol extract of Yang-invigorating herbs, including Cortex Eucommiae, Herba Cistanches, Herba Cynomorii, Rhizoma Curculiginis, Herba Epimedii, Radix Dipsaci, Rhizoma Drynariae, Fructus Psoraleae, Semen Cuscutae, Radix Morindae, and Semen Alliion, enhanced myocardial ATP generation and produced significant stimulatory action on pyruvate-supported mitochondrial electron transport in mice [60]. This finding is corroborated by a recent study involving Yang and Yin tonic herbs using a cell-based assay of ATPgenerating capacity, which showed that Yang but not Yin tonic herbs enhanced mitochondrial

improper diet.

The age-related increase in mitochondrial oxidative stress can disrupt mitochondrial structural and functional integrity, thereby triggering a vicious cycle of ROS generation. Experimental findings indicate that the age-related decrease in mitochondrial respiratory efficiency was associated with the significant decline in respiratory complex (I-V) activities, presumably mediated by self-inflicted oxidative damage [54]. In addition, the extent of oxidative damage on key metabolic enzymes increases with age, with consequent decreases in substrate binding affinity and mitochondrial ATP generation capacity [55]. The oxidation of DNA, RNA, protein and lipid molecules in mitochondria and other cellular components can culminate in functional impairment in cells, tissues, and ultimately in vital organs such as brain, heart and liver [56]. Taken together, the capacity to produce ATP and respond to cellular stress decrease as a function of age during the age-associated deterioration of mitochondrial structure and function. The mitochondrial dysfunction results in increased ROS generation, which tilts the cellular environment towards an oxidative state (i.e., impairment of cellular redox balance) and increases the susceptibility to diseases associated with aging [57].

Studies that link mitochondrial respiration/ATP production and longevity are needed to clarify the role of mitochondrial biogenesis, mitochondrial respiration rate and ROS production in different aspects of ageing. However, mitochondria are today in the scientific spotlight and sure hold promises for the future ageing research. That is certainly enough to make mitochon‐ dria a center of our attention.

## **5. Qi-invigoration and Yang-invigoration**

According to TCM theory, in order to have good health you must have sufficient Qi and your internal organs must work in harmony with each other, as long as sufficient Qi flows freely through the meridians and your organs work in harmony your body can remain healthy. If there isn't enough Qi, one or more organs can become imbalanced and develop energy function disorders. TCM frequently references several major Qi states of imbalance. One is an overall "Qi deficiency", which is often described in Western medical terms as chronic fatigue syndrome (CFS), may effect the Lungs with symptoms of shortness of breath, the Stomach/Spleen with symptoms such as poor appetite and the body in general with symptoms of fatigue and weakness. Most treated CFS by invigorating Qi and Yang. For an explanation of TCM, the ultimate reasons for the symptoms described earlier are induced by deficiencies in five organs (including Qi, Blood, Yin and Yang deficiencies) caused by the invasion of an exogenous pathogen, excessive physical strain (manual labor, mental labor and sexual intercourse), abnormal emotional states (elation, anger, worry, anxiety, sorrow, fear and terror) or an improper diet.

increased proton leak rates resulting in a depletion of membrane potential and a reduction of ATPase and complex IV activities. Treatment of aged rats with the insulin-like growth factor 1 (IGF1) corrected these parameters indicating that a cytoprotective effect of IGF1 is closely related to the mitochondrial protection [52]. Caloric restriction is the only dietary intervention that consistently increases median and maximal lifespan in organisms ranging from yeast to mammals. This dietary regime implies 20–50% restriction of the overall caloric intake of animals on *ad libitum* regime [53]. The precise molecular mechanisms of the life-extending actions of caloric restriction still remains unclear, but most likely mitochondrial energy

The age-related increase in mitochondrial oxidative stress can disrupt mitochondrial structural and functional integrity, thereby triggering a vicious cycle of ROS generation. Experimental findings indicate that the age-related decrease in mitochondrial respiratory efficiency was associated with the significant decline in respiratory complex (I-V) activities, presumably mediated by self-inflicted oxidative damage [54]. In addition, the extent of oxidative damage on key metabolic enzymes increases with age, with consequent decreases in substrate binding affinity and mitochondrial ATP generation capacity [55]. The oxidation of DNA, RNA, protein and lipid molecules in mitochondria and other cellular components can culminate in functional impairment in cells, tissues, and ultimately in vital organs such as brain, heart and liver [56]. Taken together, the capacity to produce ATP and respond to cellular stress decrease as a function of age during the age-associated deterioration of mitochondrial structure and function. The mitochondrial dysfunction results in increased ROS generation, which tilts the cellular environment towards an oxidative state (i.e., impairment of cellular redox balance)

Studies that link mitochondrial respiration/ATP production and longevity are needed to clarify the role of mitochondrial biogenesis, mitochondrial respiration rate and ROS production in different aspects of ageing. However, mitochondria are today in the scientific spotlight and sure hold promises for the future ageing research. That is certainly enough to make mitochon‐

According to TCM theory, in order to have good health you must have sufficient Qi and your internal organs must work in harmony with each other, as long as sufficient Qi flows freely through the meridians and your organs work in harmony your body can remain healthy. If there isn't enough Qi, one or more organs can become imbalanced and develop energy function disorders. TCM frequently references several major Qi states of imbalance. One is an overall "Qi deficiency", which is often described in Western medical terms as chronic fatigue syndrome (CFS), may effect the Lungs with symptoms of shortness of breath, the Stomach/Spleen with symptoms such as poor appetite and the body in general with symptoms of fatigue and weakness. Most treated CFS by invigorating Qi and Yang. For an explanation of TCM, the ultimate reasons for the symptoms described earlier are induced by deficiencies in five organs

metabolism plays a very important role in this process.

and increases the susceptibility to diseases associated with aging [57].

dria a center of our attention.

260 Alternative Medicine

**5. Qi-invigoration and Yang-invigoration**

Chinese tonic herbs that can produce heath-promoting action are used for the treatment of various patterns of deficiency in body function with respect to Qi, Blood, Yin, or Yang, and their combinations. These types of functional imbalance are viewed as sub-healthy conditions in modern medicine. Chinese tonic herbs are generally classified into four categories on the basis of their health-promoting actions, namely, "Qi-invigorating", "Blood-enriching", "Yinnourishing" and "Yang-invigorating". Of these four types of tonic herbs, the "Qi-invigorating" and "Blood-enriching" herbs are grouped under the "Yang" family and "Yin" family, respec‐ tively. Maintaining Yang and Yin in harmony is akin to attaining the homeostatic state in modern medicine [58]. Yang Qi refers to the body's vital force or functional aspects in general. Unlike the Blood or Body Fluids, Qi is an abstract concept in TCM; it can't be seen and belongs to Yang. Yang Qi sometimes refers to some body qualities and functions like superficial, upward direction, hyper-functioning, stimulating and light. It is the opposite of Yin Qi. Yang deficiency indicates insufficiency of Yang Qi inside the body that fails to provide the functions of warmth, motivation and promotion. Symptoms or signs include aversion to cold, cold limbs, bland taste in the mouth, preference for hot drinks, pale complexion, spontaneous sweating, general swelling, profuse and clear urine or loose stool. The tongue is pale, bulky with a white slimy coating, and the pulse is deep and slow or thready on examination.

According to TCM theory, Yang is viewed as a manifestation of body function supported by various organs. A "Yang-invigorating" action therefore involves the general up-regulation of cellular activities. As ATP, an energy-rich biomolecule, is universally used for energizing cellular activities, the "Yang-invigorating" action may be mediated by the enhancement of mitochondrial ATP generation [59]. "Yang-invigorating" Chinese tonic herbs have shown to enhance the myocardial mitochondrial ATP generation capacity in mice *ex vivo* and in H9c2 cardiomyocytes [58,60]. All "Yang-invigorating" Chinese tonic herbs dose-dependently enhanced the mitochondrial ATP generation capacity. The stimulation of ATP generation was associated with an increased extent of mitochondrial electron transport [60]. It is believed that the up-regulation of cellular activities by "Yang-invigoration" in Chinese medicine requires an increased supply of ATP, which is in turn largely supported by mitochondrial OXPHOS [58].

Holistically, it is believed that the Yang-invigorating herbs enhance physiological cellular activities, which is in turn critically dependent on mitochondrial ATP generation through the OXPHOS process at the cellular level. A previous study has shown that short-term oral treatment with the methanol extract of Yang-invigorating herbs, including Cortex Eucommiae, Herba Cistanches, Herba Cynomorii, Rhizoma Curculiginis, Herba Epimedii, Radix Dipsaci, Rhizoma Drynariae, Fructus Psoraleae, Semen Cuscutae, Radix Morindae, and Semen Alliion, enhanced myocardial ATP generation and produced significant stimulatory action on pyruvate-supported mitochondrial electron transport in mice [60]. This finding is corroborated by a recent study involving Yang and Yin tonic herbs using a cell-based assay of ATPgenerating capacity, which showed that Yang but not Yin tonic herbs enhanced mitochondrial ATP generation capacity in H9c2 cardiomyocytes [58]. Moreover, long-term treatment with a Yang-invigorating Chinese herbal formula (VI-28; composed of Radix Ginseng, Cornu Cervi, Cordyceps, Radix Salviae, Semen Allii, Fructus Cnidii, Fructus Evodiae and Rhizoma Kaemp‐ feriae) was found to enhance mitochondrial ATP generation in brain, heart, liver and skeletal muscle tissues of male and female rats [61].

Yang substance. Due to the popularity and therapeutic values of "Qi-invigorating" herbs, the investigation of biological activities and the underlying mechanisms in relation to "Qiinvigoration" is of great pharmacological interest. In this regard, our earlier study has dem‐ onstrated the relationship between "Qi-invigorating" action and bioenergetic level in skeletal muscle of "Qi-invigorating" herb-treated rats [69]. However, the pharmacological basis of "Qiinvigorating" action has yet to be established. To investigate the mechanism of Qi-invigoration

Investigation on the Mechanism of Qi-Invigoration from a Perspective of Effects of Sijunzi Decoction on...

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263

**6. Research ideas on Qi-invigoration – Mitochondrial energy metabolism**

Sijunzi Decoction (SD), a Chinese recipe issued firstly in the ancient pharmacopeia of the Song Dynasty,"*Taiping Huimin Heji Jufang*", having effects of reinforcing the asthenia Qi, is one of the classic recipes. The recipe consists of ginseng root, white atractylodes rhizome, Poria cocos and honey-fried licorice root. As the traditional Qi-invigorating and spleen-tonifying pre‐ scription, SD experienced repeated clinical validation by the many TCM practitioners for hundreds of years, its prescription is concise, compatibility is decent, the effect is exact, and it is highly regarded. A number of Qi-invigorating prescriptions are derived based on it, and it is a basic prescription for Spleen Qi deficiency syndrome, the series of Qi-invigorating prescriptions derived from SD are widely used for clinical treatment of many diseases, not only for digestive diseases, but also for the treatment of chronic hepatitis, chronic nephritis, and coronary heart disease etc. Pharmacological studies show that SD has anti-aging, antifatigue, anti-hypoxia, antioxidant and immune-improving function. SD can enhance mito‐ chondrial succinate dehydrogenase, cytochrome oxidase activity and relieve the mitochondrial injury of the Spleen Qi deficiency rats. In such case, SD was selected for

Our previous studies show that all the four Qi-invigorating herbal medicines (QIHM) (including ginseng, astragalus root, pilose asiabell root, white atractylodes rhizome) can increase levels of ATP, adenylate energy charge (AEC), total adenylate pool (TAP); on the contrary, all the four Qi-flow regulating herbal medicines (QRHM, including immature bitter orange, magnolia bark, green tangerine and lindera root) can decrease levels of ATP, AEC and TAP in liver cells. In a word, QIHM and QRHM increase and decrease bioenergy level of liver cells respectively *in vivo*. Therefore, Qi is closely related to bioenergy [3]. Previous experimental findings have demonstrated that all "Yang-invigorating" herbs are capable of enhancing mitochondrial ATP generation capacity (ATP-GC) in both cell and animal studies [58,70]. As a subcategory of "Yang-invigorating" herbs, "Qi-invigorating" herbs may also stimulate mitochondrial ATP-GC in various tissues. In a study, using Renshen (*Panax ginseng*), Xiyangsh‐ en (*Panax quinquefolius*) and Dangshen (*Codonopsis pilosulae*), the effect of "Qi-invigorating" Chinese tonic herbs on mitochondrial ATP-GC using *in situ* and *ex vivo* assay systems were investigated. The results showed that the three tested "Qi-invigorating" Shens in Chinese medicine stimulated the ATP-GC *in situ* in the cell-based assay system [71]. Further investi‐

in TCM, the following experiment was performed.

investigating Qi-invigorating role to make it more representative.

**perspective**

Emerging evidence has suggested that in addition to up-regulating mitochondrial functional status, Yang tonic herbs also enhance cellular/mitochondrial antioxidant capacity, and may thus prevent age-related diseases and prolong the healthspan. The proposed biochemical mechanism underlying the antioxidant action of Yang tonic herbs involves a sustained and low level of mitochondrial ROS production, which is secondary to the increased activity of the ETC, with the possible involvement of mitochondrial uncoupling. "Yang invigoration" improves antioxidant defense in the body in the long term and thereby offers a promising prospect for preventing or possibly delaying age-related diseases and the detrimental effects of aging [62]. Studies from various laboratories showed that Yang tonic herbs produced antioxidant actions by free radical-scavenging, inhibition of oxidant production, inhibition of NADPH-dependent lipid peroxidation and increase of antioxidant enzyme activities, with a resultant protection against oxidative tissue damage. These findings were consistent with the earlier study which showed that Yang tonic herbs possessed stronger free radical scavenging activity than that of tonic herbs of other functional categories [63].

A growing body of evidence has revealed the crucial involvement of mitochondrial dysfunc‐ tion and impaired antioxidant status in the pathogenesis of various age-related diseases and the aging process in general [64,65]. Yang tonic herbs/ formulae, which can induce endogenous mitochondrial antioxidant status and functional capacity enhancement [66], may therefore offer a promising prospect for preventing or possibly delaying age-related diseases and the detrimental effects of aging. With respect to Chinese medicine, more than 50% of the elderly people in China were found to show a deficiency of Yang (or Qi) in body function [67], and Yang (or Qi) tonic herbs/formulae are therefore commonly used for retarding the adverse consequences of aging in the practice of Chinese medicine. According to TCM theory, a deficiency of Yang is believed to be one of the causative factors for the development of Parkinson's disease (PD), a common neurodegenerative disease that severely compromises the quality of life in many elderly individuals [68]. Based on the finding that ViNeuro can enhance the mitochondrial ATP generation capacity (a "Yang-invigoration" property), it is plausible that the relief of Parkinsonian symptoms involves an improvement of cellular energy status that eventually leads to an enhancement of neuronal function [62].

TCM frequently references several major Qi, or energy function, problems. One is an overall "Qi deficiency". Qi deficiency is the common cause of a variety of diseases and Qi-invigoration is the basic principle for treatment of Qi deficiency. Doctors of TCM usually compose pre‐ scriptions made up of Qi-invigorating herbal medicines (QIHM) for Qi deficiency, and have accumulated abundant clinical experience for a long time. QIHM is a kind of herbal medicines which can invigorate Qi and treat syndromes of Qi deficiency, they have the effects of invigorating Qi, promoting the production of body fluid and tonifying the Spleen and Lung etc. Within the body, Qi is present in all active aspects of the body, so is considered to be a Yang substance. Due to the popularity and therapeutic values of "Qi-invigorating" herbs, the investigation of biological activities and the underlying mechanisms in relation to "Qiinvigoration" is of great pharmacological interest. In this regard, our earlier study has dem‐ onstrated the relationship between "Qi-invigorating" action and bioenergetic level in skeletal muscle of "Qi-invigorating" herb-treated rats [69]. However, the pharmacological basis of "Qiinvigorating" action has yet to be established. To investigate the mechanism of Qi-invigoration in TCM, the following experiment was performed.

ATP generation capacity in H9c2 cardiomyocytes [58]. Moreover, long-term treatment with a Yang-invigorating Chinese herbal formula (VI-28; composed of Radix Ginseng, Cornu Cervi, Cordyceps, Radix Salviae, Semen Allii, Fructus Cnidii, Fructus Evodiae and Rhizoma Kaemp‐ feriae) was found to enhance mitochondrial ATP generation in brain, heart, liver and skeletal

Emerging evidence has suggested that in addition to up-regulating mitochondrial functional status, Yang tonic herbs also enhance cellular/mitochondrial antioxidant capacity, and may thus prevent age-related diseases and prolong the healthspan. The proposed biochemical mechanism underlying the antioxidant action of Yang tonic herbs involves a sustained and low level of mitochondrial ROS production, which is secondary to the increased activity of the ETC, with the possible involvement of mitochondrial uncoupling. "Yang invigoration" improves antioxidant defense in the body in the long term and thereby offers a promising prospect for preventing or possibly delaying age-related diseases and the detrimental effects of aging [62]. Studies from various laboratories showed that Yang tonic herbs produced antioxidant actions by free radical-scavenging, inhibition of oxidant production, inhibition of NADPH-dependent lipid peroxidation and increase of antioxidant enzyme activities, with a resultant protection against oxidative tissue damage. These findings were consistent with the earlier study which showed that Yang tonic herbs possessed stronger free radical scavenging

A growing body of evidence has revealed the crucial involvement of mitochondrial dysfunc‐ tion and impaired antioxidant status in the pathogenesis of various age-related diseases and the aging process in general [64,65]. Yang tonic herbs/ formulae, which can induce endogenous mitochondrial antioxidant status and functional capacity enhancement [66], may therefore offer a promising prospect for preventing or possibly delaying age-related diseases and the detrimental effects of aging. With respect to Chinese medicine, more than 50% of the elderly people in China were found to show a deficiency of Yang (or Qi) in body function [67], and Yang (or Qi) tonic herbs/formulae are therefore commonly used for retarding the adverse consequences of aging in the practice of Chinese medicine. According to TCM theory, a deficiency of Yang is believed to be one of the causative factors for the development of Parkinson's disease (PD), a common neurodegenerative disease that severely compromises the quality of life in many elderly individuals [68]. Based on the finding that ViNeuro can enhance the mitochondrial ATP generation capacity (a "Yang-invigoration" property), it is plausible that the relief of Parkinsonian symptoms involves an improvement of cellular energy

TCM frequently references several major Qi, or energy function, problems. One is an overall "Qi deficiency". Qi deficiency is the common cause of a variety of diseases and Qi-invigoration is the basic principle for treatment of Qi deficiency. Doctors of TCM usually compose pre‐ scriptions made up of Qi-invigorating herbal medicines (QIHM) for Qi deficiency, and have accumulated abundant clinical experience for a long time. QIHM is a kind of herbal medicines which can invigorate Qi and treat syndromes of Qi deficiency, they have the effects of invigorating Qi, promoting the production of body fluid and tonifying the Spleen and Lung etc. Within the body, Qi is present in all active aspects of the body, so is considered to be a

muscle tissues of male and female rats [61].

262 Alternative Medicine

activity than that of tonic herbs of other functional categories [63].

status that eventually leads to an enhancement of neuronal function [62].

## **6. Research ideas on Qi-invigoration – Mitochondrial energy metabolism perspective**

Sijunzi Decoction (SD), a Chinese recipe issued firstly in the ancient pharmacopeia of the Song Dynasty,"*Taiping Huimin Heji Jufang*", having effects of reinforcing the asthenia Qi, is one of the classic recipes. The recipe consists of ginseng root, white atractylodes rhizome, Poria cocos and honey-fried licorice root. As the traditional Qi-invigorating and spleen-tonifying pre‐ scription, SD experienced repeated clinical validation by the many TCM practitioners for hundreds of years, its prescription is concise, compatibility is decent, the effect is exact, and it is highly regarded. A number of Qi-invigorating prescriptions are derived based on it, and it is a basic prescription for Spleen Qi deficiency syndrome, the series of Qi-invigorating prescriptions derived from SD are widely used for clinical treatment of many diseases, not only for digestive diseases, but also for the treatment of chronic hepatitis, chronic nephritis, and coronary heart disease etc. Pharmacological studies show that SD has anti-aging, antifatigue, anti-hypoxia, antioxidant and immune-improving function. SD can enhance mito‐ chondrial succinate dehydrogenase, cytochrome oxidase activity and relieve the mitochondrial injury of the Spleen Qi deficiency rats. In such case, SD was selected for investigating Qi-invigorating role to make it more representative.

Our previous studies show that all the four Qi-invigorating herbal medicines (QIHM) (including ginseng, astragalus root, pilose asiabell root, white atractylodes rhizome) can increase levels of ATP, adenylate energy charge (AEC), total adenylate pool (TAP); on the contrary, all the four Qi-flow regulating herbal medicines (QRHM, including immature bitter orange, magnolia bark, green tangerine and lindera root) can decrease levels of ATP, AEC and TAP in liver cells. In a word, QIHM and QRHM increase and decrease bioenergy level of liver cells respectively *in vivo*. Therefore, Qi is closely related to bioenergy [3]. Previous experimental findings have demonstrated that all "Yang-invigorating" herbs are capable of enhancing mitochondrial ATP generation capacity (ATP-GC) in both cell and animal studies [58,70]. As a subcategory of "Yang-invigorating" herbs, "Qi-invigorating" herbs may also stimulate mitochondrial ATP-GC in various tissues. In a study, using Renshen (*Panax ginseng*), Xiyangsh‐ en (*Panax quinquefolius*) and Dangshen (*Codonopsis pilosulae*), the effect of "Qi-invigorating" Chinese tonic herbs on mitochondrial ATP-GC using *in situ* and *ex vivo* assay systems were investigated. The results showed that the three tested "Qi-invigorating" Shens in Chinese medicine stimulated the ATP-GC *in situ* in the cell-based assay system [71]. Further investi‐ gations should examine whether representative "Qi-invigorating" herbal formula SD can stimulate mitochondrial ATP-GC *in vivo*.

g/kg/day) for 15 days and was fed with half-full diet once every other day, then the model group mouse was killed for analysis. SDL and SDH mice were given an oral dose of SD (8 and 16 g/kg/day respectively) for 28 days and were killed on forty-fourth day for detection. All the

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Mitochondria were isolated by differential centrifugation using a modified protocol of Fink et al. [72]*.* Protein determinations were carried out by Bradford method using BSA as a standard [73].

Briefly, determination of ATP, ADP, and AMP in cells of skeletal muscle from the thigh of mice, which was carried out with our previous method [74], by gradient RP-HPLC (reversedphase high performance liquid chromatography) with ultraviolet detector at room tempera‐ ture. ATP, ADP and AMP contents in liver cells was calculated by computing the peak area of standard solutions of nucleotides with known concentrations. Total adenylate pool (TAP) and adenylate energy charge (AEC) were calculated by the following formulas respectively: TAP

Respiratory function of liver mitochondria was measured using the Clark-type oxygen electrode method described by Estabrook [75] with slight modifications [3]. Respiratory state 3 and 4 can be calculated according to the OXPHOS curve. Respiration rates were expressed in nanomoles atom O per minute per milligram of protein. Respiratory control ratio (RCR) was the ratio of state 3 to state 4 respiration. P/O ratio is the number of ADP molecules phosphory‐

Data were expressed as means ± SD and statistical differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) *post hoc* multiple comparisons test using the statistical software package SPSS 16.0 for Windows (SPSS Inc., Chicago, Illinois, USA). Results were considered statistically significant at the

*6.2.1. Effects of SD on energy state in skeletal muscle cells of mice under Qi deficiency in vivo*

The Spleen and the Stomach—especially the Spleen—are in charge of producing Qi and blood to nourish the body, particularly the muscles. In Chinese medicine, the Spleen is relat‐ ed to the muscles. Qi is closely related to bioenergy according to the ancient concept of Qi and modern bioenergetics [3]. Therefore, skeletal muscle was used for investigating energy level change of Qi deficiency mice. Impaired mitochondrial ATP formation may be the key

mice were maintained with free access to drinking water.

= [ATP] + [ADP] + [AMP], AEC = ([ATP] + 0.5[ADP])/TAP.

*6.1.6. Measurement of liver mitochondrial respiratory function*

*6.1.5. Measurement of ATP, ADP, and AMP in skeletal muscle cells by HPLC*

*6.1.4. Isolation of liver mitochondria*

lated per oxygen atom reduced.

probability (*P*) values < 0.05 level.

**6.2. Results and discussion**

*6.1.7. Statistical analysis*

Although Qi of TCM is similar to the concept of modern medical bioenergy in some aspects, the mechanism of Qi-invigoration still lacks convincing evidence. Therefore, I take it as my basic point to approach the characteristics of SD on energy metabolism from the production (oxidative phosphorylation) and regulation (adenylate energy charge) of bioenergy (ATP). Since there is no direct detection method on Qi, the Qi-invigorating representative prescrip‐ tions SD were selected to study the effect on dysfunction of energy metabolism caused by Qi deficiency to investigate the mechanism of Qi-invigoration in TCM.

#### **6.1. Materials and methods**

#### *6.1.1. Animals and materials*

Male Kunming mice were purchased from Experimental Animal Center, Dalian University. Spherisorb C18 reversed-phase chromatographic column (4.6 mm×250 mm, 5 μm particle size) was purchased from Dalian Institute of Chemistry and Physics, Chinese Academy of Sciences. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), L-glutamic acid, and DL-malate were from Sigma Chemical (St Louis, MO, USA). Ginseng root, white atractylodes rhizome, Poria cocos, honey-fried licorice root, immature bitter orange, magnolia bark and Rhizoma et Radix Rhei Palmat were purchased from Beijing Tongrentang Drugstore, and identified by professor Li Jiashi at Beijing University of Chinese Medicine. They are *Panax ginseng* C.A. Mey (Tongrentang red ginseng), *Atractylodes macroce‐ phala* Koidz, *Poria cocos*(Schw.) Wolf, *Glycyrrhiza uralensis* Fisch., *Citrus aurantium* L., *Magnolia officinalis* Rehd et Wils, and *Rheum palmatum* L. respectively.

#### *6.1.2. Preparation of Sijunzi Decoction (SD) and Xiaochengqi Decoction (XD)*

SD and XD were prepared by hot-water extraction. Powdered dry Ginseng root, white atractylodes rhizome, Poria cocos and honey-fried licorice root (1:1:1:1) were immersed in distilled water (the ratio of the drug and distilled water was 1:10) for 2 hours and extracted thrice for 0.5 hour each in a boiling water bath. The filtrate was collected after filtration with gauze, mixed and condensed to 0.5 g crude drug/mL under a reduced pressure and then centrifuged at 3000 rpm for 10 min. The supernatant (SD) was collected and stored at 4°C. XD [Rhizoma et Radix Rhei Palmat, magnolia bark and immature bitter orange (4:5:3)] was prepared by the same way as SD and condensed to 2.5 g crude drug/mL.

#### *6.1.3. Spleen Qi deficiency model*

Spleen Qi deficiency model was established by exhaustion, dissipating stagnant Qi and irregular diet which was induced by XD and semi-starvation. Forty mice were randomly divided into four groups: Normal group, model group, SD low dose group (SDL) and SD high dose group (SDH). Normal group mouse was administered normal saline (10 mL/kg/day) for 43 days by oral gavage. All the other group mouse was administered an oral dose of XD (60 g/kg/day) for 15 days and was fed with half-full diet once every other day, then the model group mouse was killed for analysis. SDL and SDH mice were given an oral dose of SD (8 and 16 g/kg/day respectively) for 28 days and were killed on forty-fourth day for detection. All the mice were maintained with free access to drinking water.

## *6.1.4. Isolation of liver mitochondria*

gations should examine whether representative "Qi-invigorating" herbal formula SD can

Although Qi of TCM is similar to the concept of modern medical bioenergy in some aspects, the mechanism of Qi-invigoration still lacks convincing evidence. Therefore, I take it as my basic point to approach the characteristics of SD on energy metabolism from the production (oxidative phosphorylation) and regulation (adenylate energy charge) of bioenergy (ATP). Since there is no direct detection method on Qi, the Qi-invigorating representative prescrip‐ tions SD were selected to study the effect on dysfunction of energy metabolism caused by Qi

Male Kunming mice were purchased from Experimental Animal Center, Dalian University. Spherisorb C18 reversed-phase chromatographic column (4.6 mm×250 mm, 5 μm particle size) was purchased from Dalian Institute of Chemistry and Physics, Chinese Academy of Sciences. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), L-glutamic acid, and DL-malate were from Sigma Chemical (St Louis, MO, USA). Ginseng root, white atractylodes rhizome, Poria cocos, honey-fried licorice root, immature bitter orange, magnolia bark and Rhizoma et Radix Rhei Palmat were purchased from Beijing Tongrentang Drugstore, and identified by professor Li Jiashi at Beijing University of Chinese Medicine. They are *Panax ginseng* C.A. Mey (Tongrentang red ginseng), *Atractylodes macroce‐ phala* Koidz, *Poria cocos*(Schw.) Wolf, *Glycyrrhiza uralensis* Fisch., *Citrus aurantium* L., *Magnolia*

SD and XD were prepared by hot-water extraction. Powdered dry Ginseng root, white atractylodes rhizome, Poria cocos and honey-fried licorice root (1:1:1:1) were immersed in distilled water (the ratio of the drug and distilled water was 1:10) for 2 hours and extracted thrice for 0.5 hour each in a boiling water bath. The filtrate was collected after filtration with gauze, mixed and condensed to 0.5 g crude drug/mL under a reduced pressure and then centrifuged at 3000 rpm for 10 min. The supernatant (SD) was collected and stored at 4°C. XD [Rhizoma et Radix Rhei Palmat, magnolia bark and immature bitter orange (4:5:3)] was

Spleen Qi deficiency model was established by exhaustion, dissipating stagnant Qi and irregular diet which was induced by XD and semi-starvation. Forty mice were randomly divided into four groups: Normal group, model group, SD low dose group (SDL) and SD high dose group (SDH). Normal group mouse was administered normal saline (10 mL/kg/day) for 43 days by oral gavage. All the other group mouse was administered an oral dose of XD (60

deficiency to investigate the mechanism of Qi-invigoration in TCM.

*officinalis* Rehd et Wils, and *Rheum palmatum* L. respectively.

*6.1.2. Preparation of Sijunzi Decoction (SD) and Xiaochengqi Decoction (XD)*

prepared by the same way as SD and condensed to 2.5 g crude drug/mL.

stimulate mitochondrial ATP-GC *in vivo*.

**6.1. Materials and methods**

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*6.1.1. Animals and materials*

*6.1.3. Spleen Qi deficiency model*

Mitochondria were isolated by differential centrifugation using a modified protocol of Fink et al. [72]*.* Protein determinations were carried out by Bradford method using BSA as a standard [73].

## *6.1.5. Measurement of ATP, ADP, and AMP in skeletal muscle cells by HPLC*

Briefly, determination of ATP, ADP, and AMP in cells of skeletal muscle from the thigh of mice, which was carried out with our previous method [74], by gradient RP-HPLC (reversedphase high performance liquid chromatography) with ultraviolet detector at room tempera‐ ture. ATP, ADP and AMP contents in liver cells was calculated by computing the peak area of standard solutions of nucleotides with known concentrations. Total adenylate pool (TAP) and adenylate energy charge (AEC) were calculated by the following formulas respectively: TAP = [ATP] + [ADP] + [AMP], AEC = ([ATP] + 0.5[ADP])/TAP.

#### *6.1.6. Measurement of liver mitochondrial respiratory function*

Respiratory function of liver mitochondria was measured using the Clark-type oxygen electrode method described by Estabrook [75] with slight modifications [3]. Respiratory state 3 and 4 can be calculated according to the OXPHOS curve. Respiration rates were expressed in nanomoles atom O per minute per milligram of protein. Respiratory control ratio (RCR) was the ratio of state 3 to state 4 respiration. P/O ratio is the number of ADP molecules phosphory‐ lated per oxygen atom reduced.

#### *6.1.7. Statistical analysis*

Data were expressed as means ± SD and statistical differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) *post hoc* multiple comparisons test using the statistical software package SPSS 16.0 for Windows (SPSS Inc., Chicago, Illinois, USA). Results were considered statistically significant at the probability (*P*) values < 0.05 level.

#### **6.2. Results and discussion**

#### *6.2.1. Effects of SD on energy state in skeletal muscle cells of mice under Qi deficiency in vivo*

The Spleen and the Stomach—especially the Spleen—are in charge of producing Qi and blood to nourish the body, particularly the muscles. In Chinese medicine, the Spleen is relat‐ ed to the muscles. Qi is closely related to bioenergy according to the ancient concept of Qi and modern bioenergetics [3]. Therefore, skeletal muscle was used for investigating energy level change of Qi deficiency mice. Impaired mitochondrial ATP formation may be the key characteristic of Qi deficiency. I found that Qi deficiency led to a marked fall in cellular ATP, and a rise in cellular AMP associated with decreases in ATP/ADP and ATP/AMP ratios. The changes in ATP/ADP ratio might significantly influence mitochondrial membrane potential (∆ψm) [76]. The cellular AMP/ATP ratio was monitored as an index of metabolic stress [77]. Through the action of adenylate kinase (AK), any decrease in the cellular ATP/ADP ratio is converted into a decrease in the ATP/AMP ratio [78]. Qi deficiency elicits a marked decrease in the ATP/AMP ratio. The ATP/AMP ratio reduced from 27.4 of normal group to 6.25 under Qi deficiency conditions, whereas the ATP/ADP ratio reduced from 4.95 to 3.38. Qi deficien‐ cy has altered cellular energy state.

Qi deficiency induced anti-ATP circumstance. ATP levels were drastically lowered by Qi

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The Spleen Qi-deficiency mice are characterized by lassitude of the limbs and poor appetite etc. In short, the TCM therapeutic approach of invigorating Qi and tonifying the spleen by SD can improve the mitochondrial energy metabolism of muscle cells as well as symptoms for the

Liver plays important role in metabolism to maintain energy level and structural stability of body. It is also site of biotransformation by which toxic compounds get transformed into less harmful products to reduce toxicity [80]. The state 3 respiration (oxygen consumption), the respiratory control ratio (RCR) values and P/O ratio of liver mitochondria of model mice driven by complex I substrates were all significantly decreased compared with the normal. Liver mitochondria isolated from SD treated rats showed significant decrease in state 3 respiration, RCR and P/O ratio, compared to the rates in mitochondria from models (Table 2). State 4 respiration was not significantly altered in SD treated rats. It showed that the efficiency of ATP production via ADP phosphorylation was decreased. Qi deficiency allows tissues to minimize their energy needs. In perfectly coupled mitochondria, there would be no proton leak across the IMM, and the entire gradient generated by the respiratory chain would be used to generate ATP [81]. Control of OXPHOS allows a cell to produce only the precise amount of ATP required to sustain its activities. Recall that under normal circumstances, electron transport and ATP synthesis are tightly coupled. The value of P/O ratio (the number of moles of Pi consumed for each oxygen atom reduced to H2O) reflects the degree of coupling observed between electron transport and ATP synthesis [82]. Oxygen consumption increase dramatically when ADP is supplied. The control of aerobic respiration by ADP is referred to as respiratory control. Substrate oxidation accelerates only when an increase in the concentration of ADP signals that the ATP pool needs to be replenished. This regulation matches the rates of phosphorylation of ADP and of cellular oxidations via glycolysis, the citric acid cycle, and the electron transport

deficiency but SD stimulated an increased output of ATP.

*6.2.2. The effects of SD on liver mitochondrial respiratory function in vivo*

Spleen Qi-deficiency of experimental animals.

chain to the requirement for ATP [79].

**Dose (g/kg/day)**

**State 3 (nmol/min/mg) <sup>d</sup>**

d nanomole O2 per minute per milligram protein (nmol O2 min-1 mg protein-1).

RCR: respiratory control ratio; SDL: SD low dose group; SDH: SD high dose group.

**Table 2.** Effects of SD on liver mitochondrial respiratory function *in vivo.*

Model - 66±10 18.8±2.5 3.5±0.5 2.08±0.33 Normal - 82±12b 19.4±1.8 4.2±0.6a 2.59±0.28b SDL 8 60±14 18.7±2.3 3.2±0.7 1.90±0.24 SDH 16 56±11a 18.3±2.2 2.9±0.6a 1.78±0.22a

*P* < 0.05, b*P* < 0.01 compared to model group.

**State 4**

**(nmol/min/mg) <sup>d</sup> RCR P/O ratio**

**Group**

All values are mean±SD (n=10). a

Adenylate energy charge (AEC) represents a linear measure of the metabolic energy stored in the adenine nucleotide system. Energy metabolism would be regulated by the relative amount of ATP available, as described by AEC. ATP has been called the "energy currency" of the cell, TAP is a measure of the cell energy status. TAP levels and AEC in muscle cells of model group were decreased compared with normal group. The AMP level in model group remained twofold higher than in normal group. SD treatment could increase ATP, TAP levels and ATP/ ADP, ATP/AMP ratio, AEC in muscle cells in a dose-dependent manner. ATP/AMP ratio in SDH (16 g/ kg/day) group increased over fivefold than in model group (Table 1).


All values are mean±SD (n=10). a *P*<0.05, b*P*<0.01, c *P* < 0.001 compared to model group.

Each value expressed in mM (ATP, ADP, AMP, TAP) or as a ratio (AEC, ATP/ADP, ATP/AMP).

SDL: SD low dose group; SDH: SD high dose group; ATP: adenosine triphosphate; ADP: adenosine diphosphate; AMP: adenosine monophosphate; TAP: total adenylate pool; AEC: adenylate energy charge.

**Table 1.** Effects of Sijunzi Decoction on adenylates level in skeletal muscle cells of mice *in vivo*.

Recently, a second mechanism of respiratory control has been found in eukaryotes. This control is based on the intramitochondrial ATP/ADP ratio, with a high ratio inhibiting oxidative phosphorylation through allosteric binding of ATP to a subunit of Complex IV. This inhibition is reversed when the concentration of ADP increases [79]. Energy metabolism would be regulated by AEC. In this study, we found that Qi deficiency significantly decreased AEC, which was reversed by SD accompanied by an increase in ATP. Thus, stimulation of ATP production by SD may be achieved through the regulation of the mitochondria by affecting the AEC response. This is consistent with the ability of *P. ginseng* in increasing ATP [75]. SD was able to enhance ATP production, cellular ATP levels are closely linked to mitochondrial function, which is regulated perhaps by AEC. SD was able to regulate AEC, possibly linked to mitochondrial ATP production. Data showed SD to be an enhancer of ATP production under Qi deficiency induced anti-ATP circumstance. ATP levels were drastically lowered by Qi deficiency but SD stimulated an increased output of ATP.

The Spleen Qi-deficiency mice are characterized by lassitude of the limbs and poor appetite etc. In short, the TCM therapeutic approach of invigorating Qi and tonifying the spleen by SD can improve the mitochondrial energy metabolism of muscle cells as well as symptoms for the Spleen Qi-deficiency of experimental animals.

#### *6.2.2. The effects of SD on liver mitochondrial respiratory function in vivo*

characteristic of Qi deficiency. I found that Qi deficiency led to a marked fall in cellular ATP, and a rise in cellular AMP associated with decreases in ATP/ADP and ATP/AMP ratios. The changes in ATP/ADP ratio might significantly influence mitochondrial membrane potential (∆ψm) [76]. The cellular AMP/ATP ratio was monitored as an index of metabolic stress [77]. Through the action of adenylate kinase (AK), any decrease in the cellular ATP/ADP ratio is converted into a decrease in the ATP/AMP ratio [78]. Qi deficiency elicits a marked decrease in the ATP/AMP ratio. The ATP/AMP ratio reduced from 27.4 of normal group to 6.25 under Qi deficiency conditions, whereas the ATP/ADP ratio reduced from 4.95 to 3.38. Qi deficien‐

Adenylate energy charge (AEC) represents a linear measure of the metabolic energy stored in the adenine nucleotide system. Energy metabolism would be regulated by the relative amount of ATP available, as described by AEC. ATP has been called the "energy currency" of the cell, TAP is a measure of the cell energy status. TAP levels and AEC in muscle cells of model group were decreased compared with normal group. The AMP level in model group remained twofold higher than in normal group. SD treatment could increase ATP, TAP levels and ATP/ ADP, ATP/AMP ratio, AEC in muscle cells in a dose-dependent manner. ATP/AMP ratio in

SDH (16 g/ kg/day) group increased over fivefold than in model group (Table 1).

**AMP (mM)**

Model - 1.01±0.21 0.29±0.13 0.16±0.08 1.46±0.29 0.78±0.07 3.38±0.84 6.25±4.3 Normal - 1.66±0.31c 0.34±0.11 0.06±0.04b 2.06±0.38b 0.89±0.08b 4.95±0.69c 27.4±7.8c SDL 8 1.35±0.23b 0.32±0.12 0.09±0.06a 1.76±0.34a 0.85±0.05a 4.26±0.74a 14.8±5.7b SDH 16 1.68±0.28c 0.35±0.15 0.05±0.05b 2.08±0.40b 0.90±0.07b 4.82±0.85b 33.8±8.3c

SDL: SD low dose group; SDH: SD high dose group; ATP: adenosine triphosphate; ADP: adenosine diphosphate; AMP:

Recently, a second mechanism of respiratory control has been found in eukaryotes. This control is based on the intramitochondrial ATP/ADP ratio, with a high ratio inhibiting oxidative phosphorylation through allosteric binding of ATP to a subunit of Complex IV. This inhibition is reversed when the concentration of ADP increases [79]. Energy metabolism would be regulated by AEC. In this study, we found that Qi deficiency significantly decreased AEC, which was reversed by SD accompanied by an increase in ATP. Thus, stimulation of ATP production by SD may be achieved through the regulation of the mitochondria by affecting the AEC response. This is consistent with the ability of *P. ginseng* in increasing ATP [75]. SD was able to enhance ATP production, cellular ATP levels are closely linked to mitochondrial function, which is regulated perhaps by AEC. SD was able to regulate AEC, possibly linked to mitochondrial ATP production. Data showed SD to be an enhancer of ATP production under

**TAP (mM)**

*P* < 0.001 compared to model group.

**AEC ATP/ADP ATP/AMP**

**ADP (mM)**

*P*<0.05, b*P*<0.01, c

Each value expressed in mM (ATP, ADP, AMP, TAP) or as a ratio (AEC, ATP/ADP, ATP/AMP).

**Table 1.** Effects of Sijunzi Decoction on adenylates level in skeletal muscle cells of mice *in vivo*.

adenosine monophosphate; TAP: total adenylate pool; AEC: adenylate energy charge.

cy has altered cellular energy state.

**Group Dose**

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**(g/kg/day)**

All values are mean±SD (n=10). a

**ATP (mM)** Liver plays important role in metabolism to maintain energy level and structural stability of body. It is also site of biotransformation by which toxic compounds get transformed into less harmful products to reduce toxicity [80]. The state 3 respiration (oxygen consumption), the respiratory control ratio (RCR) values and P/O ratio of liver mitochondria of model mice driven by complex I substrates were all significantly decreased compared with the normal. Liver mitochondria isolated from SD treated rats showed significant decrease in state 3 respiration, RCR and P/O ratio, compared to the rates in mitochondria from models (Table 2). State 4 respiration was not significantly altered in SD treated rats. It showed that the efficiency of ATP production via ADP phosphorylation was decreased. Qi deficiency allows tissues to minimize their energy needs. In perfectly coupled mitochondria, there would be no proton leak across the IMM, and the entire gradient generated by the respiratory chain would be used to generate ATP [81]. Control of OXPHOS allows a cell to produce only the precise amount of ATP required to sustain its activities. Recall that under normal circumstances, electron transport and ATP synthesis are tightly coupled. The value of P/O ratio (the number of moles of Pi consumed for each oxygen atom reduced to H2O) reflects the degree of coupling observed between electron transport and ATP synthesis [82]. Oxygen consumption increase dramatically when ADP is supplied. The control of aerobic respiration by ADP is referred to as respiratory control. Substrate oxidation accelerates only when an increase in the concentration of ADP signals that the ATP pool needs to be replenished. This regulation matches the rates of phosphorylation of ADP and of cellular oxidations via glycolysis, the citric acid cycle, and the electron transport chain to the requirement for ATP [79].


d nanomole O2 per minute per milligram protein (nmol O2 min-1 mg protein-1).

All values are mean±SD (n=10). a *P* < 0.05, b*P* < 0.01 compared to model group.

RCR: respiratory control ratio; SDL: SD low dose group; SDH: SD high dose group.

**Table 2.** Effects of SD on liver mitochondrial respiratory function *in vivo.*

Mitochondria produce significant amounts of cellular ROS via aberrant O2 reaction during electron transport. This process in physiological conditions is tightly controlled with majority of ROS produced remaining inside intact mitochondria. The rate of mitochondrial respiration and ROS formation is largely influenced by the coupling state of the mitochondria [83]. SD decrease oxygen consuming rate and RCR of liver mitochondria maybe by improving of mitochondrial energy status (Figure 2), therefore, reduce mitochondrial ROS production. We consider this is appearance of lowering standard metabolic rate and is a kind of protective adaptation. Qi deficiency patients need nutritional supplements, adequate rest, and should reduce energy consumption, SD can just achieve this goal. It is conceivable that impairment of mitochondrial ATP production and the resulting energy depletion can lead to apoptosis. Aging-associated declines in mitochondrial respiratory function can lead to lower ATP production and higher oxidative stress. Lower ATP levels can decrease the efficiency of energydependent processes and ATP-mediated signal transductions [84]. An explanation of the protective effects of SD on mitochondria is based on the improvement of cellular energy status.

ficiency. However, the mechanisms for Qi-invigoration in TCM remain elusive. We pro‐ pose a hypothesis that Qi is closely related to bioenergy according to the ancient concept of Qi and modern bioenergetics [3], which is the entry point; all the QIHM have the regu‐ larity of same pharmacological effects, such as exercise capacity improvement, anti-fati‐ gue, anti-oxidation and anti-apoptosis, etc. which are all closely related to mitochondrial function, which is the basis of study; Qi-invigorating prescriptions and QIHM have a good effect in improving the energy metabolism and for treatment of mitochondria-relat‐ ed diseases, and the Qi-invigorating representative prescriptions Sijunzi Decoction (SD) was used for treatment of Qi deficiency, which is the object of study. The mechanism of energy metabolism improvement has been explored from mitochondrial oxidative phos‐ phorylation, intracellular adenylates levels, and the mechanism of mitochondrial protec‐ tion of SD were investigated. In summary, SD was able to improve mitochondrial function by enhancing cellular bioenergetics and had the pharmaceutical activities of mito‐ chondrial protection. The study provides scientific evidence for the mechanism of Qi-in‐ vigoration in TCM which is achieved by improving mitochondrial energy metabolism.

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This work was supported by the Fundamental Research Funds for the Central Universities in China (grant number: DC12010210); the Post-doctoral Research Station of Daxing'anling Beiqishen Green Industry Group, Doctoral Research Center of Heilongjiang University of Chinese Medicine (No.LRB10-316); and the Talents Project of Dalian Nationalities University

[1] Zhang YS. Mathematical Reasoning of Treatment Principle Based on "Yin Yang Wu Xing" Theory in Traditional Chinese Medicine (II). Chinese Medicine 2011; 2: 158-170.

[2] Low PKC, Ang S-L. The Foundation of Traditional Chinese Medicine. Chinese Medi‐

College of Life Science, Dalian Nationalities University, Dalian, China

**Acknowledgements**

(No.20116126).

**Author details**

Xing-Tai Li

**References**

cine 2010; 1: 84-90.

**Figure 2.** The action site of mitochondria as potential targets for SD therapy. SD treatment could increase ATP and TAP, decrease AMP levels and increase ATP/ADP, ATP/AMP ratio, AEC in muscle cells which feedback inhibit OXPHOS by decreasing RCR, state 3 respiration and P/O ratio of liver mitochondria. This is the result of improved mitochondrial energy metabolism and bioenergetic level and the potential Qi-invigoration mechanism of SD.

#### **7. Conclusion**

Qi is the hub from basic theory to clinical practice and health longevity in TCM. The true foundation of TCM is Qi. All kinds of diseases and ailments are born from Qi, Qi defi‐ ciency is the common cause of a variety of diseases and can lead to mitochondrial energy metabolism dysfunction, and Qi-invigoration is the basic principle for treatment of Qi de‐ ficiency. However, the mechanisms for Qi-invigoration in TCM remain elusive. We pro‐ pose a hypothesis that Qi is closely related to bioenergy according to the ancient concept of Qi and modern bioenergetics [3], which is the entry point; all the QIHM have the regu‐ larity of same pharmacological effects, such as exercise capacity improvement, anti-fati‐ gue, anti-oxidation and anti-apoptosis, etc. which are all closely related to mitochondrial function, which is the basis of study; Qi-invigorating prescriptions and QIHM have a good effect in improving the energy metabolism and for treatment of mitochondria-relat‐ ed diseases, and the Qi-invigorating representative prescriptions Sijunzi Decoction (SD) was used for treatment of Qi deficiency, which is the object of study. The mechanism of energy metabolism improvement has been explored from mitochondrial oxidative phos‐ phorylation, intracellular adenylates levels, and the mechanism of mitochondrial protec‐ tion of SD were investigated. In summary, SD was able to improve mitochondrial function by enhancing cellular bioenergetics and had the pharmaceutical activities of mito‐ chondrial protection. The study provides scientific evidence for the mechanism of Qi-in‐ vigoration in TCM which is achieved by improving mitochondrial energy metabolism.

## **Acknowledgements**

Mitochondria produce significant amounts of cellular ROS via aberrant O2 reaction during electron transport. This process in physiological conditions is tightly controlled with majority of ROS produced remaining inside intact mitochondria. The rate of mitochondrial respiration and ROS formation is largely influenced by the coupling state of the mitochondria [83]. SD decrease oxygen consuming rate and RCR of liver mitochondria maybe by improving of mitochondrial energy status (Figure 2), therefore, reduce mitochondrial ROS production. We consider this is appearance of lowering standard metabolic rate and is a kind of protective adaptation. Qi deficiency patients need nutritional supplements, adequate rest, and should reduce energy consumption, SD can just achieve this goal. It is conceivable that impairment of mitochondrial ATP production and the resulting energy depletion can lead to apoptosis. Aging-associated declines in mitochondrial respiratory function can lead to lower ATP production and higher oxidative stress. Lower ATP levels can decrease the efficiency of energydependent processes and ATP-mediated signal transductions [84]. An explanation of the protective effects of SD on mitochondria is based on the improvement of cellular energy status.

**Figure 2.** The action site of mitochondria as potential targets for SD therapy. SD treatment could increase ATP and TAP, decrease AMP levels and increase ATP/ADP, ATP/AMP ratio, AEC in muscle cells which feedback inhibit OXPHOS by decreasing RCR, state 3 respiration and P/O ratio of liver mitochondria. This is the result of improved mitochondrial

Qi is the hub from basic theory to clinical practice and health longevity in TCM. The true foundation of TCM is Qi. All kinds of diseases and ailments are born from Qi, Qi defi‐ ciency is the common cause of a variety of diseases and can lead to mitochondrial energy metabolism dysfunction, and Qi-invigoration is the basic principle for treatment of Qi de‐

energy metabolism and bioenergetic level and the potential Qi-invigoration mechanism of SD.

**7. Conclusion**

268 Alternative Medicine

This work was supported by the Fundamental Research Funds for the Central Universities in China (grant number: DC12010210); the Post-doctoral Research Station of Daxing'anling Beiqishen Green Industry Group, Doctoral Research Center of Heilongjiang University of Chinese Medicine (No.LRB10-316); and the Talents Project of Dalian Nationalities University (No.20116126).

## **Author details**

Xing-Tai Li

College of Life Science, Dalian Nationalities University, Dalian, China

## **References**


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**Chapter 12**

**Network Pharmacology and**

**Traditional Chinese Medicine**

Qihe Xu, Fan Qu and Olavi Pelkonen

http://dx.doi.org/10.5772/53868

**1. Introduction**

Additional information is available at the end of the chapter

Traditional Chinese medicine (TCM), an age-old healthcare system derived from China, is a mainstream medicine in China and is also popular in many other parts of the world [1-3]. Due to historic reasons, the scientific base of TCM awaits consolidation but emerging evidence has be‐ gun to illustrate TCM as an area of important medical rediscoveries. For example, the 2011 Lask‐ er-DeBakey Clinical Medical Research Award was awarded to Youyou Tu for the discovery of Chinese herb-derived artemisinin, a drug for malaria that has saved millions of lives across the globe [4,5] and the 7th Annual Szent-Györgyi Prize was awarded to Zhen-Yi Wang and Zhu Chen for their TCM research that led to the successful development of a new therapeutic approach to acute promyelocytic leukaemia. These award-winning projects were both conducted well be‐ fore the human genome was decoded and when information technology was in infancy. What has TCM to offer in the post-genomic era and the Information Age? To address this important question, the GP-TCM project kicked in as the 1st EU-funded EU-China collaboration dedicated to applying emerging technologies to TCM research [6,7]. Besides the consensus that omics and systems biology approaches will likely play major roles in addressing the complexity of TCM [7-9], more than half GP-TCM consortium members who responded to a consortium survey also cast votes of confidence in network pharmacology in TCM research [7]. Then, what is network pharmacology? What is the state of the art of this technology in modern pharmacological and

toxicological studies, and finally, what are its possible roles in TCM research?

Network could be used to refer to any interconnected things or people in a virtual or actual net-like structure. For example, in information technology, anatomy, systems biology and

and reproduction in any medium, provided the original work is properly cited.

© 2012 Xu et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

**2. What is network pharmacology?**

## **Chapter 12**

## **Network Pharmacology and Traditional Chinese Medicine**

Qihe Xu, Fan Qu and Olavi Pelkonen

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53868

## **1. Introduction**

Traditional Chinese medicine (TCM), an age-old healthcare system derived from China, is a mainstream medicine in China and is also popular in many other parts of the world [1-3]. Due to historic reasons, the scientific base of TCM awaits consolidation but emerging evidence has be‐ gun to illustrate TCM as an area of important medical rediscoveries. For example, the 2011 Lask‐ er-DeBakey Clinical Medical Research Award was awarded to Youyou Tu for the discovery of Chinese herb-derived artemisinin, a drug for malaria that has saved millions of lives across the globe [4,5] and the 7th Annual Szent-Györgyi Prize was awarded to Zhen-Yi Wang and Zhu Chen for their TCM research that led to the successful development of a new therapeutic approach to acute promyelocytic leukaemia. These award-winning projects were both conducted well be‐ fore the human genome was decoded and when information technology was in infancy. What has TCM to offer in the post-genomic era and the Information Age? To address this important question, the GP-TCM project kicked in as the 1st EU-funded EU-China collaboration dedicated to applying emerging technologies to TCM research [6,7]. Besides the consensus that omics and systems biology approaches will likely play major roles in addressing the complexity of TCM [7-9], more than half GP-TCM consortium members who responded to a consortium survey also cast votes of confidence in network pharmacology in TCM research [7]. Then, what is network pharmacology? What is the state of the art of this technology in modern pharmacological and toxicological studies, and finally, what are its possible roles in TCM research?

## **2. What is network pharmacology?**

Network could be used to refer to any interconnected things or people in a virtual or actual net-like structure. For example, in information technology, anatomy, systems biology and

© 2012 Xu et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

social science, it could refer to interconnect computers (e.g. intranet or internet), bodily structures (e.g. neurons and vessels), molecules (e.g. genes, mRNAs, proteins, metabolites), or an association of individuals having a common interest, formed to provide mutual assis‐ tance, helpful information, or the like (e.g. the FP7 GP-TCM consortium) [6,7], respectively. In network pharmacology, "network" doesn't mean that a group of scientists who share similar interests are interconnected, as the FP7 GP-TCM consortium and the famous Poly‐ math Project of mathematicians do [7,10], nor does it refer to interconnected anatomical structures or computers. Instead, the concept is built on the belief that targeting multiple no‐ des in interconnected molecular systems, rather than individual molecules, could lead to better efficacy and fewer adverse effects [11,12]. It integrates polypharmacology [13,14] and computational pharmacology or *in silico* pharmacology [15] and is based on the principles and objectives of systems pharmacology [16,17]. Thus, network pharmacology could be re‐ garded as the technical route to the ultimate ideal of systems pharmacology, in which drugs are designed to benefit a human being as an integrative system, taking into consideration the complex dynamics of interconnected organic and molecular systems.

**3. Principles of systems biology and network pharmacology**

theory; Barabasi & Oltvai also referred to them as 'scale-rich' [21].

Node Basic component interacting (pair-wise) with other node(s)

Node degree or connectivity Number of links to other nodes; "hubs" are nodes with a

them in any network

Bridging node A node bridging the shortest path between two other

Bridging centrality Measure for connectivity within a network for the measured node

nodes or modules within a network

**Table 1.** Important network characteristics in biological and pharmacological networks [18,19,21]

Path length The average separation between arbitrarily chosen nodes

Detailed descriptions of principles of networks in systems biology can be found in several arti‐ cles and reviews [20-22]. Herein, only a short presentation of the most important features is provided. Some most important characteristics and their biological examples are shown in Ta‐ ble 1. Network is formed by nodes (basic building blocks), their connections ('edges') and mod‐ ules (a collection of nodes with a higher number of connections with each other in comparison with the rest of the network), and is characterised by a number of topological features defining relationships between network objects. There is a hierarchy in the properties of nodes in that some of them ("hubs") are more central with a high number of connections to other nodes whereas the majority of nodes have only one or a few connections at the most with other nodes. Bridging nodes connect two other nodes or modules in the network. As a consequence of nonrandom nature of biological networks, these networks are called "scale-free" in the network

**Network characteristics Definition and explanation Biological entities and functions**

Edge (link, connection) Connection between two nodes • Connection may be physical, regulatory,

large number of connections, but there are only a few of

Clustering coefficient A measure of grouping tendency of the nodes • Points to a motif and/or module Motif and motif clusters Recurring, significant patterns of interconnections • Elementary building blocks (sub-

Network module A set of nodes with high internal connectivity • Subunits of a protein complex; dynamic

**(examples)**

Network Pharmacology and Traditional Chinese Medicine

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network)

reactions

data

module

etc.

genetic interaction;

• Small-molecular substrates (metabolic

•Genes (genetic regulatory network) •Proteins (protein-protein network)

• Metabolic network: enzyme-catalysed

• Genetic regulatory network: expression

•Associated with topological robustness of biological networks, i.e. small degree nodes are more "disposable" than hubs

• Proximal and distal nodes in a functional

networks) of biological networks

signalling cascade

functional unit, e.g. metabolic pathway,

• A node linking two functional units ("crosstalk" point; a potential drug target),

In brief, network pharmacology is based on the principles of network theory and systems biolo‐ gy. Graph or network theory is a branch of mathematics, which is concerned with characteristics of networks ("webs") of interacting objects. Systems biology, as the name implies, deals with complex and comprehensive living systems involving a finite number of hierarchically ordered components, which form interacting networks affected by, and responding to, various pertur‐ bations within the system itself and from the environment [18]. Typical for the network's re‐ sponse to perturbations is the return of a system to a previous state or the adoption of a new homeostasis. 'Systems biology is an analytical approach to investigating relationships among system's components in order to understand its emergent, i.e. network-level properties' [19]. Emergent properties, e.g. homeostasis, are higher-level characteristics of complex systems, which are difficult to understand and predict just by studying a few components at a time in iso‐ lation. In medicine and pharmacology, when traditional approaches are mostly concerned with individual molecules or pathways, systems biology aims at integration of biological complexity at all levels of biological organisation, be it cell, organ, organism, or population.

Although polypharmacology and computational pharmacology have a relatively long histo‐ ry, network pharmacology and systems pharmacology are emerging new concepts that were only developed in the past 5-7 years. In October 2011, the Quantitative and Systems Pharma‐ cology Working Group of the US National Institutes of Health published a white paper enti‐ tled *Quantitative and Systems Pharmacology in the Post-Genomic Era: New Approaches to Discovering Drugs and Understanding Therapeutic Mechanisms*, which provided a general re‐ port-level overview of the field from the perspectives of drug development and therapy and listed a number of important research goals for the future. It may be of interest to recapitu‐ late one of the working definitions of the report:

*lar networks in space and time and how they impact human pathophysiology."*

*<sup>&</sup>quot;The goal of Quantitative and Systems Pharmacology is to understand, in a precise, predictive manner, how drugs modulate cellu‐*

## **3. Principles of systems biology and network pharmacology**

social science, it could refer to interconnect computers (e.g. intranet or internet), bodily structures (e.g. neurons and vessels), molecules (e.g. genes, mRNAs, proteins, metabolites), or an association of individuals having a common interest, formed to provide mutual assis‐ tance, helpful information, or the like (e.g. the FP7 GP-TCM consortium) [6,7], respectively. In network pharmacology, "network" doesn't mean that a group of scientists who share similar interests are interconnected, as the FP7 GP-TCM consortium and the famous Poly‐ math Project of mathematicians do [7,10], nor does it refer to interconnected anatomical structures or computers. Instead, the concept is built on the belief that targeting multiple no‐ des in interconnected molecular systems, rather than individual molecules, could lead to better efficacy and fewer adverse effects [11,12]. It integrates polypharmacology [13,14] and computational pharmacology or *in silico* pharmacology [15] and is based on the principles and objectives of systems pharmacology [16,17]. Thus, network pharmacology could be re‐ garded as the technical route to the ultimate ideal of systems pharmacology, in which drugs are designed to benefit a human being as an integrative system, taking into consideration

In brief, network pharmacology is based on the principles of network theory and systems biolo‐ gy. Graph or network theory is a branch of mathematics, which is concerned with characteristics of networks ("webs") of interacting objects. Systems biology, as the name implies, deals with complex and comprehensive living systems involving a finite number of hierarchically ordered components, which form interacting networks affected by, and responding to, various pertur‐ bations within the system itself and from the environment [18]. Typical for the network's re‐ sponse to perturbations is the return of a system to a previous state or the adoption of a new homeostasis. 'Systems biology is an analytical approach to investigating relationships among system's components in order to understand its emergent, i.e. network-level properties' [19]. Emergent properties, e.g. homeostasis, are higher-level characteristics of complex systems, which are difficult to understand and predict just by studying a few components at a time in iso‐ lation. In medicine and pharmacology, when traditional approaches are mostly concerned with individual molecules or pathways, systems biology aims at integration of biological complexity

Although polypharmacology and computational pharmacology have a relatively long histo‐ ry, network pharmacology and systems pharmacology are emerging new concepts that were only developed in the past 5-7 years. In October 2011, the Quantitative and Systems Pharma‐ cology Working Group of the US National Institutes of Health published a white paper enti‐ tled *Quantitative and Systems Pharmacology in the Post-Genomic Era: New Approaches to Discovering Drugs and Understanding Therapeutic Mechanisms*, which provided a general re‐ port-level overview of the field from the perspectives of drug development and therapy and listed a number of important research goals for the future. It may be of interest to recapitu‐

*"The goal of Quantitative and Systems Pharmacology is to understand, in a precise, predictive manner, how drugs modulate cellu‐*

the complex dynamics of interconnected organic and molecular systems.

278 Alternative Medicine

at all levels of biological organisation, be it cell, organ, organism, or population.

late one of the working definitions of the report:

*lar networks in space and time and how they impact human pathophysiology."*

Detailed descriptions of principles of networks in systems biology can be found in several arti‐ cles and reviews [20-22]. Herein, only a short presentation of the most important features is provided. Some most important characteristics and their biological examples are shown in Ta‐ ble 1. Network is formed by nodes (basic building blocks), their connections ('edges') and mod‐ ules (a collection of nodes with a higher number of connections with each other in comparison with the rest of the network), and is characterised by a number of topological features defining relationships between network objects. There is a hierarchy in the properties of nodes in that some of them ("hubs") are more central with a high number of connections to other nodes whereas the majority of nodes have only one or a few connections at the most with other nodes. Bridging nodes connect two other nodes or modules in the network. As a consequence of nonrandom nature of biological networks, these networks are called "scale-free" in the network theory; Barabasi & Oltvai also referred to them as 'scale-rich' [21].


**Table 1.** Important network characteristics in biological and pharmacological networks [18,19,21]

Complex networks possess characteristics that are of considerable importance for the inves‐ tigation of drug discovery and drug treatment. Emergent properties of networks have al‐ ready been mentioned earlier. Recently, there has been some theoretical and experimental work on strong and weak emergent features of networks [23]. Network robustness is a very important feature, which refers to the ability of a network to respond to external or internal perturbations [21]. Biological networks demonstrate remarkable robustness, which is at least partially based on a scale-free assembly: failure of nodes with few connections (small degree nodes), which form the majority of nodes, does not affect the integrity of the network, whereas failure of a few key hubs disintegrates the network. This latter phenomenon also is the basis of vulnerability of a network, if key hubs are targets of disruptive influences.

sary raw material for building networks which encompass biologically relevant nodes (genes, proteins, metabolites), their connections (biochemical, regulatory), and modules (pathways, functional units), which through iterative process can become an increasingly relevant representation of real biological phenomena. On the other hand, the network analy‐ sis, once developed to a sufficient extent, offers a framework for data inclusion and interpre‐ tation by incorporating all pieces of information coming from earlier studies, current omics, high-content and high-throughput screening experiments, expected or unspecific findings, and these interpretations may lead to new experimental designs, both virtual and real.

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Some experts envisage as a final goal the building of a virtual or *in silico* human [23]. Actual‐ ly leading systems biologists signed the so-called Tokyo declaration in February 2008, with the aim for an *in silico* replica of a whole human body to be 90% complete by 2038. At the present, there are quite a number of simulation packages as spatiotemporal representations

Many diseases, especially chronic ones, are initiated and perpetrated via dysregulation of multiple pathways, even if the primary reason is the mutation in a central gene associated with an endogenous or exogenous insult. The application of network analysis on human diseases, especially on those associated with polymorphisms, but increasingly also on dis‐ eases not primarily associated with structural mutations, has made it increasingly clear that chronic diseases demonstrate changes in expression of a large number of genes, proteins and metabolites, involve a large number of modules or functional units and show considera‐ ble overlap of important genes and network modules [27-31]. Obvious implications of this complexity are that single-target drugs may be completely inadequate to remedy a complex situation, and efficiency of any drug could be highly dependent on importance (centrality) of the target ("node" or "edge") in the disease network. In this respect, studies on drug-tar‐ get networks suggest that many drugs developed earlier have rather peripheral targets in the disease-associated networks whereas many more novel drugs are interacting with tar‐

of various cellular functions [18].

**5. Diseases as perturbations in biological networks**

gets closer to disease aetiology-linked components [32].

**6. How to use network pharmacology in drug development?**

Since the beginning of the genomic era, drug discovery process turned towards target based approaches for a deceptively simple reason: an ever efficient identification of a large number of potential targets for small and large molecules by the application of molecular biological and pharmacogenomics tools. Expectations have been large, but still costs have increased and the number of new medicinal entities stalled or decreased. Variable reasons for the in‐ creasing costs and a huge attrition even during clinical trials have been suggested, but many experts have begun to claim that the currently popular target-based approach is basically

It is perhaps fair to mention and emphasise that many network-level emergent properties are important concepts in physiology, which is a system-level discipline. Concepts such as homeostasis, set-points, regulation, feedback control and redundancy have been in physiol‐ ogy for a long time to explain and model the interactions between cells, organs, systems and organisms [24]. Many of these system-level concepts have direct correspondences or rela‐ tives in network systems biology.

## **4. How to build a network?**

Building a network involves two opposite approaches: a bottom-up approach on the basis of established biological knowledge and a top-down approach starting from the statistical analysis of available data [18]. In a more detailed level, there are several ways to build and illustrate a biological network [25]. Perhaps the most versatile and general way is the *de novo* assembly of a network from direct experimental or computational interactions, e.g. chemi‐ cal/gene/protein screens. For the broad screening, the application of known interactions to an omic data set either manually or by using pathway-analysis software (Ingenuity Pathway Analysis, MetaCore, etc) has been widely used for hypothesis building and for identifying crucial network components. The most direct way to employ time-honoured modelling and simulation practices and more restricted and focused experimental datasets is by reverse en‐ gineering to generate a subset of networks *ab initio*. Most biochemical and regulatory path‐ ways have been built in the past via painstaking experimental work on a single or a few components of a system, which has become understandable *in toto* only later in the research process. Likewise, it has to be realised that the first assembly of a network is just the begin‐ ning of an iterative modelling-simulation-experimentation cycle and the final outcome may be quite different from the original network.

Building a biologically relevant network needs a lot of relevant information. Indeed, emer‐ gence of systems biology and network analysis has occurred alongside with, and made pos‐ sible to a considerable extent by, the developments in various omic technologies, highthroughput platforms, high-content screens, bioinformatics, and large-scale data handling and storage [26]. Production of data on genes (genomics), transcripts (transcriptomics), pro‐ teins (proteomics), epigenetic changes, metabolites (metabolomics) has put forth the neces‐ sary raw material for building networks which encompass biologically relevant nodes (genes, proteins, metabolites), their connections (biochemical, regulatory), and modules (pathways, functional units), which through iterative process can become an increasingly relevant representation of real biological phenomena. On the other hand, the network analy‐ sis, once developed to a sufficient extent, offers a framework for data inclusion and interpre‐ tation by incorporating all pieces of information coming from earlier studies, current omics, high-content and high-throughput screening experiments, expected or unspecific findings, and these interpretations may lead to new experimental designs, both virtual and real.

Some experts envisage as a final goal the building of a virtual or *in silico* human [23]. Actual‐ ly leading systems biologists signed the so-called Tokyo declaration in February 2008, with the aim for an *in silico* replica of a whole human body to be 90% complete by 2038. At the present, there are quite a number of simulation packages as spatiotemporal representations of various cellular functions [18].

## **5. Diseases as perturbations in biological networks**

Complex networks possess characteristics that are of considerable importance for the inves‐ tigation of drug discovery and drug treatment. Emergent properties of networks have al‐ ready been mentioned earlier. Recently, there has been some theoretical and experimental work on strong and weak emergent features of networks [23]. Network robustness is a very important feature, which refers to the ability of a network to respond to external or internal perturbations [21]. Biological networks demonstrate remarkable robustness, which is at least partially based on a scale-free assembly: failure of nodes with few connections (small degree nodes), which form the majority of nodes, does not affect the integrity of the network, whereas failure of a few key hubs disintegrates the network. This latter phenomenon also is the basis of vulnerability of a network, if key hubs are targets of disruptive influences.

It is perhaps fair to mention and emphasise that many network-level emergent properties are important concepts in physiology, which is a system-level discipline. Concepts such as homeostasis, set-points, regulation, feedback control and redundancy have been in physiol‐ ogy for a long time to explain and model the interactions between cells, organs, systems and organisms [24]. Many of these system-level concepts have direct correspondences or rela‐

Building a network involves two opposite approaches: a bottom-up approach on the basis of established biological knowledge and a top-down approach starting from the statistical analysis of available data [18]. In a more detailed level, there are several ways to build and illustrate a biological network [25]. Perhaps the most versatile and general way is the *de novo* assembly of a network from direct experimental or computational interactions, e.g. chemi‐ cal/gene/protein screens. For the broad screening, the application of known interactions to an omic data set either manually or by using pathway-analysis software (Ingenuity Pathway Analysis, MetaCore, etc) has been widely used for hypothesis building and for identifying crucial network components. The most direct way to employ time-honoured modelling and simulation practices and more restricted and focused experimental datasets is by reverse en‐ gineering to generate a subset of networks *ab initio*. Most biochemical and regulatory path‐ ways have been built in the past via painstaking experimental work on a single or a few components of a system, which has become understandable *in toto* only later in the research process. Likewise, it has to be realised that the first assembly of a network is just the begin‐ ning of an iterative modelling-simulation-experimentation cycle and the final outcome may

Building a biologically relevant network needs a lot of relevant information. Indeed, emer‐ gence of systems biology and network analysis has occurred alongside with, and made pos‐ sible to a considerable extent by, the developments in various omic technologies, highthroughput platforms, high-content screens, bioinformatics, and large-scale data handling and storage [26]. Production of data on genes (genomics), transcripts (transcriptomics), pro‐ teins (proteomics), epigenetic changes, metabolites (metabolomics) has put forth the neces‐

tives in network systems biology.

280 Alternative Medicine

**4. How to build a network?**

be quite different from the original network.

Many diseases, especially chronic ones, are initiated and perpetrated via dysregulation of multiple pathways, even if the primary reason is the mutation in a central gene associated with an endogenous or exogenous insult. The application of network analysis on human diseases, especially on those associated with polymorphisms, but increasingly also on dis‐ eases not primarily associated with structural mutations, has made it increasingly clear that chronic diseases demonstrate changes in expression of a large number of genes, proteins and metabolites, involve a large number of modules or functional units and show considera‐ ble overlap of important genes and network modules [27-31]. Obvious implications of this complexity are that single-target drugs may be completely inadequate to remedy a complex situation, and efficiency of any drug could be highly dependent on importance (centrality) of the target ("node" or "edge") in the disease network. In this respect, studies on drug-tar‐ get networks suggest that many drugs developed earlier have rather peripheral targets in the disease-associated networks whereas many more novel drugs are interacting with tar‐ gets closer to disease aetiology-linked components [32].

## **6. How to use network pharmacology in drug development?**

Since the beginning of the genomic era, drug discovery process turned towards target based approaches for a deceptively simple reason: an ever efficient identification of a large number of potential targets for small and large molecules by the application of molecular biological and pharmacogenomics tools. Expectations have been large, but still costs have increased and the number of new medicinal entities stalled or decreased. Variable reasons for the in‐ creasing costs and a huge attrition even during clinical trials have been suggested, but many experts have begun to claim that the currently popular target-based approach is basically flawed as a guide for drug discovery process. Instead, many authors have argued that sys‐ tems biology and polypharmacology encompassing network thinking should be adopted to remedy the current difficulties in drug discovery and development. However, because net‐ work pharmacology is a relatively new concept, there is not too much robust data to demon‐ strate its superiority in drug development process. Yet some pieces of information seem to point out that indeed network pharmacology is providing a new paradigm [12,33]. Some of the current suggestions based on network pharmacology are compiled in Table 2.

in-class drugs. Actually the analysis of Swinney & Anthony concurs in many ways to the findings of Yildirim et al [32], in that many recent new drugs are interacting with novel tar‐ gets thought to be more central in a corresponding disease aetiology, whereas follow-on molecules tend to stick to well-known, often more peripheral targets, which are more distal

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Although Swinney & Anthony did not specially mention network pharmacology (or corre‐ sponding) in their analysis, they refer to many crucial papers on network pharmacology. In their analysis phenotypic screening means the use of functional assays, which usually in‐ form physiological parameters closer to real-life *in vivo* goals of drug therapy. Functional as‐ says associated with the elucidation of the molecular mechanisms of action are much closer to the network analysis than the target-based screening. Intuitively it seems clear that func‐ tional assays are superior, at least from the drug discovery and development point of view, than target-based assays. However, in reality target-centred thinking has been dominant for

**7. Properties of currently used drugs: Polypharmacology meets network**

Even if the current paradigm has been 'one target (or disease/symptom)-one drug', practis‐ ing pharmacologists have always known that practically all drugs have multiple effects based on various known or unknown mechanisms, some desirable and others indifferent or harmful. A very good example is anti-psychotic drugs interacting with a large number of re‐ ceptors and other targets. One target-one drug paradigm created a vision of a "magic bul‐ let", which was eagerly adopted, although some scientists pointed out that even such "magic bullets" have pharmacokinetics-associated problems, e.g. potential drug-drug inter‐ actions, as well as structure-related problems such as allergic reactions. Now it is becoming increasingly apparent that biological systems are complex, redundant, homeostatic and re‐ silient to perturbations and, consequently, most diseases are exhibiting much wider pertur‐ bations and variations than once thought. A new discipline, termed loosely as

polypharmacology, has been gaining ground both conceptually and experimentally.

It seems highly likely that most current drugs are interacting with multiple targets. Current drug-protein interaction and chemogenomic studies have indicated that many drugs are in‐ teracting with two or more targets at reasonably close affinities. In these studies especially, the database of the FDA-approved drugs and their targets (effects) have been employed to create networks of drug-protein interactions [32,36] or to model similarities in chemical structure between drugs and potential ligands for the prediction of drug-target interactions [12, 37-38]. In Figure 1, a general approach to make use of the polypharmacology network is outlined [11]. In this approach the polypharmacology network is mapped onto the biological network, for example human disease-gene network, to reveal multiple actions of drugs on

from core components of disease networks.

multiple targets and multiple diseases [30].

more than a decade.

**pharmacology**


**Table 2.** Drug design, discovery, and repurposing potentialities of network pharmacology

Recently, Swinney & Anthony analysed preclinical discovery strategies that were used to identify potential drug candidates, which were ultimately approved by the US Food and Drug Administration (FDA) between 1999 and 2008 [35]. They classified strategies to targetbased screening, phenotypic screening, modification of natural substances and biologicbased approaches, with an additional consideration on molecular mechanisms of action (MMOA). Out of the 259 agents that were approved, 75 were first-in-class drugs with new MMOAs, and out of these, 50 (67%) were small molecules and 25 (33%) were biologics. They claimed that the contribution of phenotypic screening to the discovery of first-in-class smallmolecule drugs exceeded that of target-based approaches — with 28 and 17 of these drugs coming from the two approaches, respectively — in an era when the major focus was on tar‐ get-based approaches. They postulated that a target-centric approach for first-in-class drugs, without consideration of an optimal MMOA, might contribute to the current high attrition rates and low productivity in pharmaceutical research and development. Instead, among follow-on drugs a vast majority were the outcomes of target-based approaches, which seem rather natural considering that for these drugs mechanism of action and many other crucial pieces of information could come much earlier and in more useful manner than for the firstin-class drugs. Actually the analysis of Swinney & Anthony concurs in many ways to the findings of Yildirim et al [32], in that many recent new drugs are interacting with novel tar‐ gets thought to be more central in a corresponding disease aetiology, whereas follow-on molecules tend to stick to well-known, often more peripheral targets, which are more distal from core components of disease networks.

flawed as a guide for drug discovery process. Instead, many authors have argued that sys‐ tems biology and polypharmacology encompassing network thinking should be adopted to remedy the current difficulties in drug discovery and development. However, because net‐ work pharmacology is a relatively new concept, there is not too much robust data to demon‐ strate its superiority in drug development process. Yet some pieces of information seem to point out that indeed network pharmacology is providing a new paradigm [12,33]. Some of

the current suggestions based on network pharmacology are compiled in Table 2.

Magic bullet aimed at target; if a target is a hub, the

functional unit of importance to disease (symptom

between nodules, but not vital to cell function

Recently, Swinney & Anthony analysed preclinical discovery strategies that were used to identify potential drug candidates, which were ultimately approved by the US Food and Drug Administration (FDA) between 1999 and 2008 [35]. They classified strategies to targetbased screening, phenotypic screening, modification of natural substances and biologicbased approaches, with an additional consideration on molecular mechanisms of action (MMOA). Out of the 259 agents that were approved, 75 were first-in-class drugs with new MMOAs, and out of these, 50 (67%) were small molecules and 25 (33%) were biologics. They claimed that the contribution of phenotypic screening to the discovery of first-in-class smallmolecule drugs exceeded that of target-based approaches — with 28 and 17 of these drugs coming from the two approaches, respectively — in an era when the major focus was on tar‐ get-based approaches. They postulated that a target-centric approach for first-in-class drugs, without consideration of an optimal MMOA, might contribute to the current high attrition rates and low productivity in pharmaceutical research and development. Instead, among follow-on drugs a vast majority were the outcomes of target-based approaches, which seem rather natural considering that for these drugs mechanism of action and many other crucial pieces of information could come much earlier and in more useful manner than for the first-

affected in an optimal manner without compromising vital cellular functions

**Table 2.** Drug design, discovery, and repurposing potentialities of network pharmacology

Current paradigm

specific variants [34]

No good example

Anti-psychotics on multiple transmitter-associated receptors

active site

Inherited disorders seem to separate into node removal and edgetic-

Inhibitors of protein kinases with common structural motifs in the

**Target Rationale Example**

consequence may be too much toxicity

Edgetic perturbation Drug targeting towards a certain edge (connection) of an intended target

Motifs, modules Drug targeting towards a common feature or a

Bridging nodes A target resulting in a modulation of crosstalk

Multi-targets Multiple disease-associated nodes, which can be

or aetiology)

Molecular target identification

282 Alternative Medicine

Although Swinney & Anthony did not specially mention network pharmacology (or corre‐ sponding) in their analysis, they refer to many crucial papers on network pharmacology. In their analysis phenotypic screening means the use of functional assays, which usually in‐ form physiological parameters closer to real-life *in vivo* goals of drug therapy. Functional as‐ says associated with the elucidation of the molecular mechanisms of action are much closer to the network analysis than the target-based screening. Intuitively it seems clear that func‐ tional assays are superior, at least from the drug discovery and development point of view, than target-based assays. However, in reality target-centred thinking has been dominant for more than a decade.

## **7. Properties of currently used drugs: Polypharmacology meets network pharmacology**

Even if the current paradigm has been 'one target (or disease/symptom)-one drug', practis‐ ing pharmacologists have always known that practically all drugs have multiple effects based on various known or unknown mechanisms, some desirable and others indifferent or harmful. A very good example is anti-psychotic drugs interacting with a large number of re‐ ceptors and other targets. One target-one drug paradigm created a vision of a "magic bul‐ let", which was eagerly adopted, although some scientists pointed out that even such "magic bullets" have pharmacokinetics-associated problems, e.g. potential drug-drug inter‐ actions, as well as structure-related problems such as allergic reactions. Now it is becoming increasingly apparent that biological systems are complex, redundant, homeostatic and re‐ silient to perturbations and, consequently, most diseases are exhibiting much wider pertur‐ bations and variations than once thought. A new discipline, termed loosely as polypharmacology, has been gaining ground both conceptually and experimentally.

It seems highly likely that most current drugs are interacting with multiple targets. Current drug-protein interaction and chemogenomic studies have indicated that many drugs are in‐ teracting with two or more targets at reasonably close affinities. In these studies especially, the database of the FDA-approved drugs and their targets (effects) have been employed to create networks of drug-protein interactions [32,36] or to model similarities in chemical structure between drugs and potential ligands for the prediction of drug-target interactions [12, 37-38]. In Figure 1, a general approach to make use of the polypharmacology network is outlined [11]. In this approach the polypharmacology network is mapped onto the biological network, for example human disease-gene network, to reveal multiple actions of drugs on multiple targets and multiple diseases [30].

**Author(s) Name(s):** Qihe Xu, Fan Qu and Olavi Pelkonen

**Page No.** 

**3** 16 (line 3 of Table 1)

**5** Reverse line 3

**9** Reverse line 3

**8** Figure 1 (quality was poor)

However, most of the studies on polypharmacology are based on computational and statisti‐ cal associations, although some of the major findings have been studied further experimen‐ tally [37,38]. For example, a recent study demonstrated that unknown and unexpected "offtarget" effects of many marketed drugs can be predicted by the computational analysis of ligand-target interaction; some predictions were experimentally confirmed [39]. Especially chemogenomic and chemoproteomic studies are based on direct or calculated affinities. It should be pointed out that affinity is not a reaction or other immediate outcome, e.g. antago‐ nism, of an interaction and more distal functional or physiological consequences may or may not occur for various reasons even if a primary interaction has been demonstrated or predicted. Still clear evidence on functional consequences is required to be sure that an ac‐ tual pharmacological significance is demonstrated for a substance. **Chapter Title:** Network Pharmacology and Traditional Chinese Medicine **PROOF CORRECTIONS FORM Line No. Delete Replace with 2** 5 does'nt mean a group of Does'nt mean that a group of **3** 5 (basic building cellular blocks) (basic building blocks) (cellular entity) A number of new medicinal entities the number of new medicinal entities

Recently, a polypharmacological approach has been extended to include functional consid‐ erations. Simon et al [40] employed 1177 FDA-approved small molecular drugs by investi‐ gating interaction profiles based on *in silico* docking/scoring methods to a series of virtual non-target protein binding sites and contrasting these profiles with 177 major drug catego‐ ries of the same series of FDA-approved drugs. Statistical analyses confirmed a close rela‐ tionship between the studied effect categories and interaction profiles of small molecule drugs. On the basis of this relationship, the comprehensive effect profiles of drugs were ap‐ parent and furthermore, effects not previously associated with particular drugs could be predicted. A rather curious finding – which is not easily explained by classical pharmacolog‐ ical concepts – was that the prediction power was independent of the composition of the protein set used for interaction profile generation. Perhaps general chemical and physico‐ chemical properties of molecules are of importance for potential interactions in general, whereas pharmacophores, i.e. specific stereochemical groups, are crucial for specific high-

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Network approach helps to understand and reveal on-target and off-target toxicity of phar‐ maceuticals, but it also helps to delineate the toxicities of any chemicals, be they industrial chemicals, agrochemicals, cosmetics, environmental pollutants, etc. Omic approaches pro‐ vide voluminous information about time-dependent changes at various levels of biological complexity after the administration of a chemical and provide the so-called signatures of toxicity. On the other hand, the application of known and characterised toxicants has de‐ lineated a finite number of pathways of toxicity. Bringing this information together at the established network and systems level would create a 'systems toxicology' approach, analo‐

A relatively isolated area in pharmacology and toxicology is the model building to describe the behaviour and disposition of drugs and other chemicals in the body [42]. Especially those models that make use of physiological principles resemble in many ways network pharmacology. Whole body PBPK models consist of absorption sites and manners, tissues with membranes drugs and their metabolites have to cross, with special reference to tissues which metabolise and excrete drugs and their metabolites, and so on. Concentrations of a studied chemical (and its important metabolites) in these various compartments could be equated to nodes. Connections are permeation and corresponding constants (for passive and active processes in the membranes and other cellular barriers, distribution coefficients, enzy‐ matic reactions (clearance), and so on. Although the number of building blocks in pharma‐ cokinetic models is finite and certainly much less than in most systems pharmacology networks, models have become quite complex, but still useful for predicting the behaviour

gous to systems pharmacology and polypharmacology [41].

**9. Physiologically based pharmacokinetic (PBPK) modelling**

affinity interactions.

**8. Systems toxicology**

Building blocs Building blocks **10** 16 10. TCM network pharmacology & "Network targets" for TCM drugs 10. TCM network pharmacology & "network targets" for TCM drugs **Figure 1. A network-centric view of drug action**. Primary building blocks of network pharmacology are the drug-target network (above) and the biological network (below). The network in the centre is a part of the biological network in which proteins (nodes) targeted by the same drug are represented in the same colour. Consequently drug efficacy and toxicity can be understood by action at specific nodes and hubs. For the definition of nodes and hubs, see Table 1. The figure is re‐ printed by permission from Macmillan Publishers Ltd: [Nature Biotechnology] (11), copyright (2007).

1

Recently, a polypharmacological approach has been extended to include functional consid‐ erations. Simon et al [40] employed 1177 FDA-approved small molecular drugs by investi‐ gating interaction profiles based on *in silico* docking/scoring methods to a series of virtual non-target protein binding sites and contrasting these profiles with 177 major drug catego‐ ries of the same series of FDA-approved drugs. Statistical analyses confirmed a close rela‐ tionship between the studied effect categories and interaction profiles of small molecule drugs. On the basis of this relationship, the comprehensive effect profiles of drugs were ap‐ parent and furthermore, effects not previously associated with particular drugs could be predicted. A rather curious finding – which is not easily explained by classical pharmacolog‐ ical concepts – was that the prediction power was independent of the composition of the protein set used for interaction profile generation. Perhaps general chemical and physico‐ chemical properties of molecules are of importance for potential interactions in general, whereas pharmacophores, i.e. specific stereochemical groups, are crucial for specific highaffinity interactions.

## **8. Systems toxicology**

However, most of the studies on polypharmacology are based on computational and statisti‐ cal associations, although some of the major findings have been studied further experimen‐ tally [37,38]. For example, a recent study demonstrated that unknown and unexpected "offtarget" effects of many marketed drugs can be predicted by the computational analysis of ligand-target interaction; some predictions were experimentally confirmed [39]. Especially chemogenomic and chemoproteomic studies are based on direct or calculated affinities. It should be pointed out that affinity is not a reaction or other immediate outcome, e.g. antago‐ nism, of an interaction and more distal functional or physiological consequences may or may not occur for various reasons even if a primary interaction has been demonstrated or predicted. Still clear evidence on functional consequences is required to be sure that an ac‐

1

10. TCM network pharmacology & "network

**Figure 1. A network-centric view of drug action**. Primary building blocks of network pharmacology are the drug-target network (above) and the biological network (below). The network in the centre is a part of the biological network in which proteins (nodes) targeted by the same drug are represented in the same colour. Consequently drug efficacy and toxicity can be understood by action at specific nodes and hubs. For the definition of nodes and hubs, see Table 1. The figure is re‐

targets" for TCM drugs

printed by permission from Macmillan Publishers Ltd: [Nature Biotechnology] (11), copyright (2007).

tual pharmacological significance is demonstrated for a substance.

A number of new medicinal entities the number of new medicinal entities

**Proof Corrections Form** 

**PROOF CORRECTIONS FORM** 

**Line No. Delete Replace with** 

**2** 5 does'nt mean a group of Does'nt mean that a group of

Building blocs Building blocks

**3** 5 (basic building cellular blocks) (basic building blocks)

**Author(s) Name(s):** Qihe Xu, Fan Qu and Olavi Pelkonen

284 Alternative Medicine

(cellular entity)

**8** Figure 1 (quality was poor)

**Page No.** 

**3** 16 (line 3 of Table 1)

**5** Reverse line 3

**9** Reverse line 3

**10** 16 10. TCM network pharmacology &

"Network targets" for TCM drugs

**Chapter Title:** Network Pharmacology and Traditional Chinese Medicine

Network approach helps to understand and reveal on-target and off-target toxicity of phar‐ maceuticals, but it also helps to delineate the toxicities of any chemicals, be they industrial chemicals, agrochemicals, cosmetics, environmental pollutants, etc. Omic approaches pro‐ vide voluminous information about time-dependent changes at various levels of biological complexity after the administration of a chemical and provide the so-called signatures of toxicity. On the other hand, the application of known and characterised toxicants has de‐ lineated a finite number of pathways of toxicity. Bringing this information together at the established network and systems level would create a 'systems toxicology' approach, analo‐ gous to systems pharmacology and polypharmacology [41].

## **9. Physiologically based pharmacokinetic (PBPK) modelling**

A relatively isolated area in pharmacology and toxicology is the model building to describe the behaviour and disposition of drugs and other chemicals in the body [42]. Especially those models that make use of physiological principles resemble in many ways network pharmacology. Whole body PBPK models consist of absorption sites and manners, tissues with membranes drugs and their metabolites have to cross, with special reference to tissues which metabolise and excrete drugs and their metabolites, and so on. Concentrations of a studied chemical (and its important metabolites) in these various compartments could be equated to nodes. Connections are permeation and corresponding constants (for passive and active processes in the membranes and other cellular barriers, distribution coefficients, enzy‐ matic reactions (clearance), and so on. Although the number of building blocks in pharma‐ cokinetic models is finite and certainly much less than in most systems pharmacology networks, models have become quite complex, but still useful for predicting the behaviour of a drug in the body under various circumstances. Efforts to link PBPK models with *in vi‐ tro*-*in vivo* extrapolations under the systems pharmacology umbrella are underway [43].

(TCM-ID: http://tcm.cz3.nus.edu.sg/group/tcm-id/tcmid\_ns.asp) have been developed by

Network Pharmacology and Traditional Chinese Medicine

http://dx.doi.org/10.5772/53868

287

In TCM, formulae are usually prescribed based on TCM syndrome patterns of a given pa‐ tient, rather than a disease as defined in Western medicine. Thus, an important part of TCM network pharmacology is to establish links between network molecular targets and TCM syndrome patterns. Ma et al surveyed 4575 cases of Cold Syndrome patients and examined gene expression information of a typical Cold Syndrome pedigree by microarray. Results in‐ dicated that Cold Syndrome related genes played an essential role in energy metabolism, which were tightly correlated with the genes of neurotransmitters, hormones and cytokines in the neuro-endocrine-immune interaction network [52]. In TCM clinics, Cold Syndrome is treated by Warm formulae and Hot Syndrome is often treated by Cold formulae. Identifica‐ tion of the gene networks of Cold and Hot Syndromes [52, 53-55] should help understand

nature of a condition and unravel mechanisms of its related TCM treatment [56].

**11.1. "Disease-gene-target" network-based studies to identify targets and pathways affected by TCM drugs and to obtain fuller pictures of the efficacy and mechanisms of**

In addition, Wang and colleagues (2011) proposed that network pharmacology could be ap‐

Sun et al performed bioinformatic analysis of anti-Alzheimer's herbal medicines and found that ingredients of anti-Alzheimer's herbs not only bound symptom-relieving tar‐ gets, but also interact closely with a variety of successful therapeutic targets related to other diseases, such as inflammation, cancer and diabetes, suggesting the possible cross‐ talk between these complicated diseases. Furthermore, the anti-Alzheimer's herbal ingre‐ dients densely targeted pathways of Ca2+ equilibrium maintaining upstream of cell

Wen et al used microarray and network analysis to establish that Si-Wu-Tang is an Nrf2 ac‐ tivator and phytoestrogen, thus suggesting its use as a nontoxic chemopreventive agent [58]. In fact, network analysis of all sorts of omic data can be used to explore the molecular tar‐ gets and mechanisms of action of TCM drugs [59,60], as recently reviewed by Buriani et al [8]. In network pharmacology, roles for functional genes and proteins might vary in differ‐ ent stages of the same disease, thus the same disease could be treated differently, as em‐ phasised in TCM; on the other hand, some functional proteins are "hubs" in the disease networks of more than one disease, thus different diseases could be treated similarly by tar‐ geting the same hubs [45]. Based on gene and phenotype information associated with the ingredient herbs of the classical Liu-wei-di-huang (LWDH) formula and LWDH-treated dis‐ eases, it was found that LWDH-treated diseases showed high phenotype similarity and identified certain "co-modules" enriched in cancer pathways and neuro-endocrine-immune

academics based in China and Singapore [50,51].

plied to the following aspects of TCM studies:

proliferation and inflammation [57].

**action of TCM drugs**

**11. Applications of TCM network pharmacology**

Whole-body PBPK models illustrate also important challenges to, and potentialities of, net‐ work pharmacology. First of all, the framework for modelling is multi-scale [44], starting from enzymes and transporters (dealing with transformation and movement of drugs) and their quantitative functions (clearance, metabolite formation, membrane penetration) and their regulation and functions in the cells and tissues, kinetics of drugs and their metabolites throughout the body via circulation, distribution to different organs, elimination in urine, in bile, and integration of all processes into a dynamic model representing an individual (*in sil‐ ico* human or animal, for that matter) and extending the modelling to evolution, develop‐ ment, environment, populations, diseases, etc. PBPK modelling is increasingly used in drug development and toxicity risk assessment with considerable success, probably because it is a rather restricted in dealing with behaviour of a single substance in the framework of a finite number of active players. On the other hand, pharmacodynamic models that have been de‐ veloped for at least a couple of decades are closer to network building (*ab initio* models).

## **10. TCM network pharmacology & "network targets" for TCM drugs**

In 2010, Liu & Du raised the concept of "TCM network pharmacology", linking the multiple components that play principal, complementary and assistant therapeutic roles in TCM for‐ mulae to principal, complementary and assistant targets in a disease network. They believed that such an approach to projecting a TCM drug component network onto a disease network offers a novel philosophical guide and technological route to designing and understanding mechanisms of action of TCM drugs and is thus likely proven important in modernisation of TCM [45]. Similarly, Li emphasised "network targets" of systems, connectivity and predic‐ tiveness features in studying TCM formulae and syndromes and the work of his team showed that the "network target" approach could facilitate discovery of effective com‐ pounds, understanding their interrelation, elucidating relationship between TCM formulae and diseases or TCM syndrome, developing rational TCM drug, as well as guiding integrat‐ ed use of TCM and conventional drugs [46].

TCM network pharmacology heavily relies on omic platforms as well as algorithm- and net‐ work-based computational tools, which are elegantly summarised most recently by Leung et al [47]. In addition, TCM network pharmacology heavily relies on ever updating omics, pharmacological and TCM-related databases. While concerns about duplication of efforts, poor standardisation and low sustainability remain, many TCM databases have been devel‐ oped, as recently reviewed by Barlow et al [48]. To mention a few, the Chem-TCM database developed by King's College London [49] has now been commercialised by TimTec LLC (http://www.chemtcm.com); the trial version of World Traditional & Natural Medicine Pat‐ ent Database is currently being developed by Beijing East Linden Co. Ltd (http://www.east‐ linden.net/NewsShow.aspx?news\_id=20081127102018850246); and the Herbal Ingredient Target database (HIT: http://lifecenter.sgst.cn/hit/) and the TCM Information Database (TCM-ID: http://tcm.cz3.nus.edu.sg/group/tcm-id/tcmid\_ns.asp) have been developed by academics based in China and Singapore [50,51].

## **11. Applications of TCM network pharmacology**

of a drug in the body under various circumstances. Efforts to link PBPK models with *in vi‐ tro*-*in vivo* extrapolations under the systems pharmacology umbrella are underway [43].

Whole-body PBPK models illustrate also important challenges to, and potentialities of, net‐ work pharmacology. First of all, the framework for modelling is multi-scale [44], starting from enzymes and transporters (dealing with transformation and movement of drugs) and their quantitative functions (clearance, metabolite formation, membrane penetration) and their regulation and functions in the cells and tissues, kinetics of drugs and their metabolites throughout the body via circulation, distribution to different organs, elimination in urine, in bile, and integration of all processes into a dynamic model representing an individual (*in sil‐ ico* human or animal, for that matter) and extending the modelling to evolution, develop‐ ment, environment, populations, diseases, etc. PBPK modelling is increasingly used in drug development and toxicity risk assessment with considerable success, probably because it is a rather restricted in dealing with behaviour of a single substance in the framework of a finite number of active players. On the other hand, pharmacodynamic models that have been de‐ veloped for at least a couple of decades are closer to network building (*ab initio* models).

**10. TCM network pharmacology & "network targets" for TCM drugs**

ed use of TCM and conventional drugs [46].

286 Alternative Medicine

In 2010, Liu & Du raised the concept of "TCM network pharmacology", linking the multiple components that play principal, complementary and assistant therapeutic roles in TCM for‐ mulae to principal, complementary and assistant targets in a disease network. They believed that such an approach to projecting a TCM drug component network onto a disease network offers a novel philosophical guide and technological route to designing and understanding mechanisms of action of TCM drugs and is thus likely proven important in modernisation of TCM [45]. Similarly, Li emphasised "network targets" of systems, connectivity and predic‐ tiveness features in studying TCM formulae and syndromes and the work of his team showed that the "network target" approach could facilitate discovery of effective com‐ pounds, understanding their interrelation, elucidating relationship between TCM formulae and diseases or TCM syndrome, developing rational TCM drug, as well as guiding integrat‐

TCM network pharmacology heavily relies on omic platforms as well as algorithm- and net‐ work-based computational tools, which are elegantly summarised most recently by Leung et al [47]. In addition, TCM network pharmacology heavily relies on ever updating omics, pharmacological and TCM-related databases. While concerns about duplication of efforts, poor standardisation and low sustainability remain, many TCM databases have been devel‐ oped, as recently reviewed by Barlow et al [48]. To mention a few, the Chem-TCM database developed by King's College London [49] has now been commercialised by TimTec LLC (http://www.chemtcm.com); the trial version of World Traditional & Natural Medicine Pat‐ ent Database is currently being developed by Beijing East Linden Co. Ltd (http://www.east‐ linden.net/NewsShow.aspx?news\_id=20081127102018850246); and the Herbal Ingredient Target database (HIT: http://lifecenter.sgst.cn/hit/) and the TCM Information Database

In TCM, formulae are usually prescribed based on TCM syndrome patterns of a given pa‐ tient, rather than a disease as defined in Western medicine. Thus, an important part of TCM network pharmacology is to establish links between network molecular targets and TCM syndrome patterns. Ma et al surveyed 4575 cases of Cold Syndrome patients and examined gene expression information of a typical Cold Syndrome pedigree by microarray. Results in‐ dicated that Cold Syndrome related genes played an essential role in energy metabolism, which were tightly correlated with the genes of neurotransmitters, hormones and cytokines in the neuro-endocrine-immune interaction network [52]. In TCM clinics, Cold Syndrome is treated by Warm formulae and Hot Syndrome is often treated by Cold formulae. Identifica‐ tion of the gene networks of Cold and Hot Syndromes [52, 53-55] should help understand nature of a condition and unravel mechanisms of its related TCM treatment [56].

In addition, Wang and colleagues (2011) proposed that network pharmacology could be ap‐ plied to the following aspects of TCM studies:

#### **11.1. "Disease-gene-target" network-based studies to identify targets and pathways affected by TCM drugs and to obtain fuller pictures of the efficacy and mechanisms of action of TCM drugs**

Sun et al performed bioinformatic analysis of anti-Alzheimer's herbal medicines and found that ingredients of anti-Alzheimer's herbs not only bound symptom-relieving tar‐ gets, but also interact closely with a variety of successful therapeutic targets related to other diseases, such as inflammation, cancer and diabetes, suggesting the possible cross‐ talk between these complicated diseases. Furthermore, the anti-Alzheimer's herbal ingre‐ dients densely targeted pathways of Ca2+ equilibrium maintaining upstream of cell proliferation and inflammation [57].

Wen et al used microarray and network analysis to establish that Si-Wu-Tang is an Nrf2 ac‐ tivator and phytoestrogen, thus suggesting its use as a nontoxic chemopreventive agent [58]. In fact, network analysis of all sorts of omic data can be used to explore the molecular tar‐ gets and mechanisms of action of TCM drugs [59,60], as recently reviewed by Buriani et al [8]. In network pharmacology, roles for functional genes and proteins might vary in differ‐ ent stages of the same disease, thus the same disease could be treated differently, as em‐ phasised in TCM; on the other hand, some functional proteins are "hubs" in the disease networks of more than one disease, thus different diseases could be treated similarly by tar‐ geting the same hubs [45]. Based on gene and phenotype information associated with the ingredient herbs of the classical Liu-wei-di-huang (LWDH) formula and LWDH-treated dis‐ eases, it was found that LWDH-treated diseases showed high phenotype similarity and identified certain "co-modules" enriched in cancer pathways and neuro-endocrine-immune pathways, which may be responsible for the action of treating different diseases by the same LWDH formula [61].

**11.3. Building "TCM drug properties-clinical indications-adverse effects" networks and**

Network Pharmacology and Traditional Chinese Medicine

http://dx.doi.org/10.5772/53868

289

Jia et al analysed 117 drug combinations and identified general and specific modes of action and highlighted the potential value of molecular interaction profiles in the discovery of nov‐

Zhu et al performed network analysis of 2215 chemicals identified in 62 Chinese herbs indi‐ cated for patients with chronic kidney diseases, including 836 chemicals contained in 22 to‐ nifying herbs and 1379 chemicals contained in 40 evil-expelling herbs, according to TCM theory, in comparison with 99 drugs used in conventional medicine. Interaction networks of tonifying herbs, evil-expelling herbs and drugs showed different patterns, regarding net‐ work parameters, especially network degree, average number of neighbours and character‐

Wu et al constructed a relational network of TCM decoction slices to discover and interpret the correlations between the natures and functions of decoction slices and their clinically in‐ dicated symptoms and channel tropism as defined in TCM. 3016 pairs of decoction slice-

Emerging studies have supported the potential for network pharmacology in quality control of TCM drugs [69], which can well interpret the mechanisms of action of TCM drugs [70], help understand how different constituents of a TCM formulation and how TCM and chem‐ ical drugs synergised through targeting different nodes in disease-related networks [71,72]. There is no regulatory requirement of omics-based data in any submitted dossier to any reg‐ ulatory agency, including for TCM products. However, it has been acknowledged that such studies are being increasingly performed, and almost surely will eventually be included into regulatory submission dossiers, possibly initially as supplementary materials [9]. Such a

symptom correlation associated with 646 decoction slices were discovered [68].

**11.4. Evaluation of the safety, efficacy and stability of TCM products through**

prospect is likely also shared with systems and network pharmacology.

**12. Inspirations and challenges that TCM has to offer to network**

In network pharmacology, "network" refers to the molecular network in a targeted organ‐ ism, for instance the "network targets" in patients. In TCM network pharmacology, howev‐ er, complex TCM drugs themselves become another important molecular network, which might be called "network bullets" that interact with "network targets" in order to help the body to regain balance. Importantly, some components of TCM drugs are not to target "net‐ work targets", but to target other drug components, so as to alleviate their side-effects, im‐

**illustrating the relationship between TCM drug properties and efficacy**

el multicomponent therapies [66].

istic path lengths and shortest paths [67].

**pharmacology**

**12.1. More networks**

**constructing network models and network analysis**

#### **11.2. Construction of "TCM drugs-targets-diseases" network and elucidation of the scientific base of TCM drug formulation through network analysis**

Zheng et al studied the interactions between 514 compounds contained in a Chinese herbal formula Jingzhi Tougu Xiaotong Granule (JZTGXTG) and 35 drug targets of relevance to os‐ teoarthritis and the distribution of 514 compounds in drug-target space. By analysing pa‐ rameters of the JZTGXTG compound-target interaction network and the drug-target interaction network including network heterogeneity and characteristic path length, the re‐ sults illustrated the possible molecular mechanisms of JZTGXTG in the prevention and treatment of osteoarthritis at the network pharmacology level [62].

To predict multi-targets by multi-compounds found in Aconiti Lateralis Radix Praeparata, Wu et al constructed the corresponding multi-compound-multi-target network based on the drug-target relationship of FDA approved drugs. The predicted targets of 22 compounds of Aconiti Lateralis Radix Praeparata were validated by literature. Each compound in the es‐ tablished network was correlated with 16.3 targets on average, while each target was corre‐ lated with 4.77 compounds on average, which reflected the "multi-compound and multitarget" characteristic of TCM drugs [63].

A "network target" approach has been applied to virtual screening and established an algo‐ rithm known as network target-based identification of multicomponent synergy (NIMS) to prioritise synergistic combinations of agents in a high-throughput manner [64]. From a "net‐ work target" perspective, a method called distance-based mutual information model (DMIM) was established to identify useful relations among herbs in numerous herbal for‐ mulae and a novel concept of "co-module" across herb-biomolecule-disease multilayer net‐ works was proposed to explore the potential mechanisms of herbal formulations [61]. DMIM, when used for retrieving herb pairs, achieved a good balance among the herb's fre‐ quency, independence, and distance in herbal formulae. A herb network constructed by DMIM from 3865 collaterals-related herbal formulae not only recovered traditionally de‐ fined herb pairs and formulae, but also generated novel anti-angiogenic herb ingredients and herb pairs with synergistic or antagonistic effects [61].

Li et al constructed a network of nine major active compounds from Fufang Danshen formu‐ la, their multi-targets and multiple related diseases. The nine compounds were tanshinone II A, salvianolic acid B, protocatechuic aldehyde, danshensu, cryptotanshinone, notoginseno‐ side R1, ginsenoside Rg1, ginsenoside Rb1 and borneol. Network analysis showed that these compounds could modulate 42 genes associated with cardiovascular diseases (e.g. PPARG, ACE, KCNJ11, KCNQ1 and ABCC8), which were related to 30 clinical conditions, including non-insulin-dependent diabetes mellitus, hyperinsulinaemic hypoglycaemia, hypertension and coronary heart disease [65].

#### **11.3. Building "TCM drug properties-clinical indications-adverse effects" networks and illustrating the relationship between TCM drug properties and efficacy**

Jia et al analysed 117 drug combinations and identified general and specific modes of action and highlighted the potential value of molecular interaction profiles in the discovery of nov‐ el multicomponent therapies [66].

Zhu et al performed network analysis of 2215 chemicals identified in 62 Chinese herbs indi‐ cated for patients with chronic kidney diseases, including 836 chemicals contained in 22 to‐ nifying herbs and 1379 chemicals contained in 40 evil-expelling herbs, according to TCM theory, in comparison with 99 drugs used in conventional medicine. Interaction networks of tonifying herbs, evil-expelling herbs and drugs showed different patterns, regarding net‐ work parameters, especially network degree, average number of neighbours and character‐ istic path lengths and shortest paths [67].

Wu et al constructed a relational network of TCM decoction slices to discover and interpret the correlations between the natures and functions of decoction slices and their clinically in‐ dicated symptoms and channel tropism as defined in TCM. 3016 pairs of decoction slicesymptom correlation associated with 646 decoction slices were discovered [68].

#### **11.4. Evaluation of the safety, efficacy and stability of TCM products through constructing network models and network analysis**

Emerging studies have supported the potential for network pharmacology in quality control of TCM drugs [69], which can well interpret the mechanisms of action of TCM drugs [70], help understand how different constituents of a TCM formulation and how TCM and chem‐ ical drugs synergised through targeting different nodes in disease-related networks [71,72]. There is no regulatory requirement of omics-based data in any submitted dossier to any reg‐ ulatory agency, including for TCM products. However, it has been acknowledged that such studies are being increasingly performed, and almost surely will eventually be included into regulatory submission dossiers, possibly initially as supplementary materials [9]. Such a prospect is likely also shared with systems and network pharmacology.

## **12. Inspirations and challenges that TCM has to offer to network pharmacology**

#### **12.1. More networks**

pathways, which may be responsible for the action of treating different diseases by the same

Zheng et al studied the interactions between 514 compounds contained in a Chinese herbal formula Jingzhi Tougu Xiaotong Granule (JZTGXTG) and 35 drug targets of relevance to os‐ teoarthritis and the distribution of 514 compounds in drug-target space. By analysing pa‐ rameters of the JZTGXTG compound-target interaction network and the drug-target interaction network including network heterogeneity and characteristic path length, the re‐ sults illustrated the possible molecular mechanisms of JZTGXTG in the prevention and

To predict multi-targets by multi-compounds found in Aconiti Lateralis Radix Praeparata, Wu et al constructed the corresponding multi-compound-multi-target network based on the drug-target relationship of FDA approved drugs. The predicted targets of 22 compounds of Aconiti Lateralis Radix Praeparata were validated by literature. Each compound in the es‐ tablished network was correlated with 16.3 targets on average, while each target was corre‐ lated with 4.77 compounds on average, which reflected the "multi-compound and multi-

A "network target" approach has been applied to virtual screening and established an algo‐ rithm known as network target-based identification of multicomponent synergy (NIMS) to prioritise synergistic combinations of agents in a high-throughput manner [64]. From a "net‐ work target" perspective, a method called distance-based mutual information model (DMIM) was established to identify useful relations among herbs in numerous herbal for‐ mulae and a novel concept of "co-module" across herb-biomolecule-disease multilayer net‐ works was proposed to explore the potential mechanisms of herbal formulations [61]. DMIM, when used for retrieving herb pairs, achieved a good balance among the herb's fre‐ quency, independence, and distance in herbal formulae. A herb network constructed by DMIM from 3865 collaterals-related herbal formulae not only recovered traditionally de‐ fined herb pairs and formulae, but also generated novel anti-angiogenic herb ingredients

Li et al constructed a network of nine major active compounds from Fufang Danshen formu‐ la, their multi-targets and multiple related diseases. The nine compounds were tanshinone II A, salvianolic acid B, protocatechuic aldehyde, danshensu, cryptotanshinone, notoginseno‐ side R1, ginsenoside Rg1, ginsenoside Rb1 and borneol. Network analysis showed that these compounds could modulate 42 genes associated with cardiovascular diseases (e.g. PPARG, ACE, KCNJ11, KCNQ1 and ABCC8), which were related to 30 clinical conditions, including non-insulin-dependent diabetes mellitus, hyperinsulinaemic hypoglycaemia, hypertension

**11.2. Construction of "TCM drugs-targets-diseases" network and elucidation of the**

**scientific base of TCM drug formulation through network analysis**

treatment of osteoarthritis at the network pharmacology level [62].

and herb pairs with synergistic or antagonistic effects [61].

target" characteristic of TCM drugs [63].

and coronary heart disease [65].

LWDH formula [61].

288 Alternative Medicine

In network pharmacology, "network" refers to the molecular network in a targeted organ‐ ism, for instance the "network targets" in patients. In TCM network pharmacology, howev‐ er, complex TCM drugs themselves become another important molecular network, which might be called "network bullets" that interact with "network targets" in order to help the body to regain balance. Importantly, some components of TCM drugs are not to target "net‐ work targets", but to target other drug components, so as to alleviate their side-effects, im‐ prove activity of the principal drug component, improve absorption and/or facilitate delivery of the principal drug to the targeted disease areas. Thus, TCM network pharmacol‐ ogy involves at least two networks to be considered in modelling and analysis.

ancient Europe relationist science was soon marginalised and analytical science became the approach of choice, in ancient China the beginnings of an analytical perspective were not pursued further and relationist science became the approach of choice [76]. Nowadays, the post-genomic era is characterised by globalisation and digitalisation. While omic data repre‐ sent the state of the art of the analytical power of Western science, these data could be mean‐ ingless "sacs of data" unless they are linked up functionally using a relationist approach. At this point, the dominant approaches in the West and East are integrated to relate pieces of fragmented omic data and their functions and this might well serve as a bridge of both med‐ ical traditions. Analyses of state-of-the-art modern and TCM pharmacological and toxicolog‐ ical research data appear to support the concept of network pharmacology, i.e. a systems network-based model can help better understand health, disease and how Western medi‐ cine, TCM drugs or integrated TCM and Western medicine work. It can be expected that this approach could play a more important role in research and development of new drugs and

Network Pharmacology and Traditional Chinese Medicine

http://dx.doi.org/10.5772/53868

291

This manuscript is supported by the GP-TCM project funded by the European Commission under the FP7 grant agreement No. 223154. Dr. Qihe Xu is the Coordinator of the project; Professor Olavi Pelkonen is a member of the management and science committee. Both Pro‐

in helping understand the mechanisms of action of drugs, especially in TCM.

fessor Olavi Pelkonen and Dr. Fan Qu are non-beneficiary members.

\*Address all correspondence to: qihe.xu@kcl.ac.uk and olavi.pelkonen@oulu.fi

3 Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland

[1] Normile D. Asian medicine. The new face of traditional Chinese medicine. Science

1 Department of Renal Medicine, King's College London, London, UK

2 Women's Hospital, Zhejiang University, Hangzhou, Zhejiang, China

and Olavi Pelkonen3\*

\*Address all correspondence to: qihe.xu@kcl.ac.uk

**Acknowledgements**

**Author details**

Qihe Xu1\*, Fan Qu2

**References**

2003;299(5604): 188-90.

In TCM, as guided by TCM theories, it is of paramount importance to choose a number of herbs (sometimes also zoological or mineral components) based on particular symptoms and characteristics of a patient. To assemble a formula or *fangji*, principal and enabling herbs are combined in order to optimise the effectiveness and minimise adverse effects. The prin‐ cipal herbs are known as the *jun* herbs, which treat the main cause or primary symptoms of a disease. The enabling herbs include the *chen* herbs, which serve to augment or broaden the effects of *jun* herbs and to relieve secondary symptoms; *zuo* herbs, which modulate the ef‐ fects of *jun* and *chen* herbs and to counteract the toxic or side effects of these herbs; and the *shi* herbs, which function to facilitate absorption and delivery of active herbal components to the target organs. Thus, the combination of principal (*Jun*) and enabling (*Chen*, *Zuo* and/or *Shi*) components to form a drug network could form the basis for designing novel "network bullets" in the future application of network pharmacology.

#### **12.2. More holistic**

Network pharmacology aims to research and develop drugs holistically. However, the current model of network pharmacology that focuses on psychological and somatic diseases separate‐ ly could be improved if it is to meet requirements of the psychosomatic model of health and ail‐ ments. Specifically, in addition to the well-known placebo effects of any interventions, the pathological damage of emotions to the internal organs is of primary concern of TCM practi‐ tioners. We propose that psychosomatic factors should be linked up in the next generation of network pharmacology and emotions should be included in the equations of future network analysis. By doing so, research might eventually help unravel and harness placebo effects and tackle psychosomatic ailments in a network pharmacological perspective.

#### **12.3. More individualised**

Personalised medicine is gaining momentum [73-75]. Network pharmacology needs to catch up with this trend as well. In TCM, individualisation goes beyond personalisation, because the same person at different ages, on different diets or living style, under different weather condition and at the different phases of the same disease could be diagnosed and treated differently. Can network pharmacology not only be personalised but also individualised, taking all the above variations into account?

## **13. Conclusions and perspectives**

According to Paul Unschuld, a renowned German sinologist, cultural background has a great impact on the preferred directions of medical science. For example, both in ancient Greece and China philosophers came up with the idea of "relationist science" or "science of systematic correspondences" on the one hand and "analytical science" on the other. While in ancient Europe relationist science was soon marginalised and analytical science became the approach of choice, in ancient China the beginnings of an analytical perspective were not pursued further and relationist science became the approach of choice [76]. Nowadays, the post-genomic era is characterised by globalisation and digitalisation. While omic data repre‐ sent the state of the art of the analytical power of Western science, these data could be mean‐ ingless "sacs of data" unless they are linked up functionally using a relationist approach. At this point, the dominant approaches in the West and East are integrated to relate pieces of fragmented omic data and their functions and this might well serve as a bridge of both med‐ ical traditions. Analyses of state-of-the-art modern and TCM pharmacological and toxicolog‐ ical research data appear to support the concept of network pharmacology, i.e. a systems network-based model can help better understand health, disease and how Western medi‐ cine, TCM drugs or integrated TCM and Western medicine work. It can be expected that this approach could play a more important role in research and development of new drugs and in helping understand the mechanisms of action of drugs, especially in TCM.

## **Acknowledgements**

prove activity of the principal drug component, improve absorption and/or facilitate delivery of the principal drug to the targeted disease areas. Thus, TCM network pharmacol‐

In TCM, as guided by TCM theories, it is of paramount importance to choose a number of herbs (sometimes also zoological or mineral components) based on particular symptoms and characteristics of a patient. To assemble a formula or *fangji*, principal and enabling herbs are combined in order to optimise the effectiveness and minimise adverse effects. The prin‐ cipal herbs are known as the *jun* herbs, which treat the main cause or primary symptoms of a disease. The enabling herbs include the *chen* herbs, which serve to augment or broaden the effects of *jun* herbs and to relieve secondary symptoms; *zuo* herbs, which modulate the ef‐ fects of *jun* and *chen* herbs and to counteract the toxic or side effects of these herbs; and the *shi* herbs, which function to facilitate absorption and delivery of active herbal components to the target organs. Thus, the combination of principal (*Jun*) and enabling (*Chen*, *Zuo* and/or *Shi*) components to form a drug network could form the basis for designing novel "network

Network pharmacology aims to research and develop drugs holistically. However, the current model of network pharmacology that focuses on psychological and somatic diseases separate‐ ly could be improved if it is to meet requirements of the psychosomatic model of health and ail‐ ments. Specifically, in addition to the well-known placebo effects of any interventions, the pathological damage of emotions to the internal organs is of primary concern of TCM practi‐ tioners. We propose that psychosomatic factors should be linked up in the next generation of network pharmacology and emotions should be included in the equations of future network analysis. By doing so, research might eventually help unravel and harness placebo effects and

Personalised medicine is gaining momentum [73-75]. Network pharmacology needs to catch up with this trend as well. In TCM, individualisation goes beyond personalisation, because the same person at different ages, on different diets or living style, under different weather condition and at the different phases of the same disease could be diagnosed and treated differently. Can network pharmacology not only be personalised but also individualised,

According to Paul Unschuld, a renowned German sinologist, cultural background has a great impact on the preferred directions of medical science. For example, both in ancient Greece and China philosophers came up with the idea of "relationist science" or "science of systematic correspondences" on the one hand and "analytical science" on the other. While in

ogy involves at least two networks to be considered in modelling and analysis.

bullets" in the future application of network pharmacology.

tackle psychosomatic ailments in a network pharmacological perspective.

**12.2. More holistic**

290 Alternative Medicine

**12.3. More individualised**

taking all the above variations into account?

**13. Conclusions and perspectives**

This manuscript is supported by the GP-TCM project funded by the European Commission under the FP7 grant agreement No. 223154. Dr. Qihe Xu is the Coordinator of the project; Professor Olavi Pelkonen is a member of the management and science committee. Both Pro‐ fessor Olavi Pelkonen and Dr. Fan Qu are non-beneficiary members.

## **Author details**

Qihe Xu1\*, Fan Qu2 and Olavi Pelkonen3\*

\*Address all correspondence to: qihe.xu@kcl.ac.uk

\*Address all correspondence to: qihe.xu@kcl.ac.uk and olavi.pelkonen@oulu.fi

1 Department of Renal Medicine, King's College London, London, UK

2 Women's Hospital, Zhejiang University, Hangzhou, Zhejiang, China

3 Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland

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## *Edited by Hiroshi Sakagami*

Alternative medicine is recognized as medical products and practices that do not belong to the standard cares taken by medical doctors, doctors of osteopathy and allied health professionals. It has developed into a multitude of medical products and practices that significantly improve the body condition and show disease prevention actions. The content of this book does not cover all areas of alternative medicine, but provides the reader with insights into selected aspects of established and new therapies. It consists of 12 chapters that are separated into 4 parts: (1) Historical and Cultural Perception, (2) Compositional Analysis, (3) Therapeutic Potential, and (4) Action Mechanism and Future Direction, written by world experts who are reviewing their original and others' research. The book will be useful to students, clinicians, teachers and researchers who have interest in advances in alternative medicines.

Alternative Medicine

Alternative Medicine

*Edited by Hiroshi Sakagami*

Photo by YakubovAlim / iStock