**Promotion of Blood Fluidity Using Electroacupuncture Stimulation**

Shintaro Ishikawa, Kazuhito Asano and Tadashi Hisamitsu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53036

## **1. Introduction**

Acupuncture is an alternative medicine originating in ancient China that treats patients by manipulating thin, solid needles that have been inserted into acupuncture points in the skin. Current several scientific research supports acupuncture's efficacy in the relief of certain types of pain and post-operative nausea [1,2].

Acupuncture's greatest effectiveness appears to be in symptomatic control of pain and nau‐ sea. The World Health Organization and the United States' National Institutes of Health (NIH) have stated that acupuncture can be effective in the treatment of neurological condi‐ tions and pain [3]. Moreover, it is thought that acupuncture regulates various biological functions. It was presented that acupuncture stimulus influences the cytokine level, hor‐ mone level [4] and leukocyte number [5] as effects for blood.

Blood roles in organisms include waste product removal, body temperature adjustment, as well as oxygen and nutritive supply. Physiologically the first priority driving force of blood circulation is cardiac pressure.

Blood flow is determined by co-action of the cardiovascular system and blood fluidity. However, blood flow is also controlled by a blood hydrodynamic characteristic. It is estab‐ lished that changes in the cardiovascular system will cause changes in blood properties as well [6-9]. Changes of blood cell composition and plasma components may influence blood fluidity in the long term [7], and blood cell activity, such as red blood cell agglutination, leu‐ kocyte adherence, and platelet aggregation, in the short term [8,9].

It is believed that variations in blood fluidity result in disorders of the circulatory system such as arterial sclerosis or embolism, damage to vascular endothelium cells by hyperten‐

sion, glucose tolerance degradation and chronic inflammation, degradation of blood vessel flexibility by hyperlipemia and aging, weakness of blood cells, and degradation of plasma plasticity [10,11].

sanli (ST-36) on the outside crus superior, Sanyinjiao (SP-6) on the inside crus lower part, Hegu (LI-4) between the thumb and the forefinger of the arm, Neiguan (P-6) on the distal inside arm, and Shenshu (BL-23) on the outside dorsum spine. Acupuncture was 5 mm deep and stimulated electrically (3-5V, 30-200μA, rectangular and bi-phasic) at a stimulation fre‐ quency of 1 or 100 Hz to permit the muscle to shrink slightly. An Ohm Palser LFP-4000A

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We determined the blood fluidity using a Micro Channel array Flow Analyzer KH-6 (MC-FAN; MC Laboratory Inc. Japan). Coagulation of blood was blocked by Heparin sodium. We used 100μl of the blood to measure the flow time to the silicon tip of the analyzer (Figure 2)

**Figure 2.** Images of blood flow by MC-FAN MC-FAN measures passing time of blood (100μL).A. Blood flow velocity is

rapid. Blood cells are not observed. B. Blood flow velocity is slow. Platelet agglutination is observed.

(Zen Iryoki Co. Japan) was used as the device of acupuncture stimulus.

**Figure 1.** Locations of the acupoints. Cited from [15], with permission

**2.3. Measurement of blood fluidity**

Recently it is assumed that cerebral infarction, myocardial infarction, and pulmonary infarc‐ tion are caused by an increase of thrombus generation. Common treatments and recurrence prevention for these illnesses include administration of antithrombotic agent such as warfar‐ in and aspirin [12,13].

In the world today Western medicine is sometimes complemented with alternative methods of treatment although this trend is somewhat stalled due to the lack of the scientific evi‐ dence for the efficacy of alternative medicine. Western medicine has shown to have a direct effect on cell and metabolic function.

In oriental medicine, the state in which blood is stagnant is called "oketsu (Yu xie)," meaning preceding state or symptomatic of sickness. Acupuncture stimulation is often clinically used for treatment of "oketsu." The degree of "oketsu" is indicated by tongue color and form, swelling, paroxysmal blushing, and dark circles under the eyes [14]. However, these indexes do not reflect the real hemogram.

"oketsu" is now regarded as physiological blood flow and is studied from the point of view of blood fluidity and vascular resistance. We previously reported [15,16] that (1) various types of stress applied to rats cause blood fluidity to decline drastically, as the platelet adhe‐ sion increases, (2) acupuncture stimulation increases blood fluidity by the depression of pla‐ telet adhesion, and (3) the effects of acupuncture stimulation vary according to the stimulus location (acupoint). Thus, acupuncture stimulus were much effective to improve the de‐ crease of blood fluidity such as "oketsu." The mechanisms that interact between acupuncture stimulus and blood fluidity, however, have not yet been identified.

In this chapter, we first describe our original finding on these topics, and then propose the possible action mechanisms of the improvement of blood fluidity by electroacupuncture.

## **2. Materials & methods**

#### **2.1. Experimental animals**

Specific pathogen-free 7-8 weeks old male Wister rats were purchased from Japan Bio-Sup‐ ply Center (Tokyo, Japan). The animals were maintained at 25 ± 2 ℃, humidity 55 ± 5 %, and a light and dark cycle of 12 hour in our animal facilities. The rats were randomly divided into control group or each experiment group, fed a regular show diet and water during ex‐ periments. Each group has 7 animals..

#### **2.2. Acupuncture stimulus**

The modality of acupuncture needle used was 0.20 × 40 mm (SEIRIN Co. Japan). Punctures were pricked at acupoints to apply the needle equivalency locus of humans (Figure 1): Zu‐ sanli (ST-36) on the outside crus superior, Sanyinjiao (SP-6) on the inside crus lower part, Hegu (LI-4) between the thumb and the forefinger of the arm, Neiguan (P-6) on the distal inside arm, and Shenshu (BL-23) on the outside dorsum spine. Acupuncture was 5 mm deep and stimulated electrically (3-5V, 30-200μA, rectangular and bi-phasic) at a stimulation fre‐ quency of 1 or 100 Hz to permit the muscle to shrink slightly. An Ohm Palser LFP-4000A (Zen Iryoki Co. Japan) was used as the device of acupuncture stimulus.

**Figure 1.** Locations of the acupoints. Cited from [15], with permission

#### **2.3. Measurement of blood fluidity**

sion, glucose tolerance degradation and chronic inflammation, degradation of blood vessel flexibility by hyperlipemia and aging, weakness of blood cells, and degradation of plasma

Recently it is assumed that cerebral infarction, myocardial infarction, and pulmonary infarc‐ tion are caused by an increase of thrombus generation. Common treatments and recurrence prevention for these illnesses include administration of antithrombotic agent such as warfar‐

In the world today Western medicine is sometimes complemented with alternative methods of treatment although this trend is somewhat stalled due to the lack of the scientific evi‐ dence for the efficacy of alternative medicine. Western medicine has shown to have a direct

In oriental medicine, the state in which blood is stagnant is called "oketsu (Yu xie)," meaning preceding state or symptomatic of sickness. Acupuncture stimulation is often clinically used for treatment of "oketsu." The degree of "oketsu" is indicated by tongue color and form, swelling, paroxysmal blushing, and dark circles under the eyes [14]. However, these indexes

"oketsu" is now regarded as physiological blood flow and is studied from the point of view of blood fluidity and vascular resistance. We previously reported [15,16] that (1) various types of stress applied to rats cause blood fluidity to decline drastically, as the platelet adhe‐ sion increases, (2) acupuncture stimulation increases blood fluidity by the depression of pla‐ telet adhesion, and (3) the effects of acupuncture stimulation vary according to the stimulus location (acupoint). Thus, acupuncture stimulus were much effective to improve the de‐ crease of blood fluidity such as "oketsu." The mechanisms that interact between acupuncture

In this chapter, we first describe our original finding on these topics, and then propose the possible action mechanisms of the improvement of blood fluidity by electroacupuncture.

Specific pathogen-free 7-8 weeks old male Wister rats were purchased from Japan Bio-Sup‐ ply Center (Tokyo, Japan). The animals were maintained at 25 ± 2 ℃, humidity 55 ± 5 %, and a light and dark cycle of 12 hour in our animal facilities. The rats were randomly divided into control group or each experiment group, fed a regular show diet and water during ex‐

The modality of acupuncture needle used was 0.20 × 40 mm (SEIRIN Co. Japan). Punctures were pricked at acupoints to apply the needle equivalency locus of humans (Figure 1): Zu‐

stimulus and blood fluidity, however, have not yet been identified.

plasticity [10,11].

204 Alternative Medicine

in and aspirin [12,13].

effect on cell and metabolic function.

do not reflect the real hemogram.

**2. Materials & methods**

periments. Each group has 7 animals..

**2.1. Experimental animals**

**2.2. Acupuncture stimulus**

We determined the blood fluidity using a Micro Channel array Flow Analyzer KH-6 (MC-FAN; MC Laboratory Inc. Japan). Coagulation of blood was blocked by Heparin sodium. We used 100μl of the blood to measure the flow time to the silicon tip of the analyzer (Figure 2)

**Figure 2.** Images of blood flow by MC-FAN MC-FAN measures passing time of blood (100μL).A. Blood flow velocity is rapid. Blood cells are not observed. B. Blood flow velocity is slow. Platelet agglutination is observed.

We assumed that the flow time in the analyzer imitated the capillary blood fluidity index [17]. In an MC-FAN assay, the prolongation of flow time indicates a decrease of blood fluidi‐ ty and a short flow time, an increase of blood fluidity [18].

#### **2.4. Measurement of platelet aggregation ability**

The ability of platelet aggregation was measured with a platelet coagulation measuring sys‐ tem, a platelet aggregometer (PA-20; Kowa Company. Ltd., Tokto, Japan). Blood samples were collected from the inferior vena cava under pentobarbital anesthesia. The blood sample treated with 3.2 % of sodium citrate (2.0 mL) was centrifuged to obtain platelet-rich plasma (PRP) and platelet-poor plasma (PPP).

**Figure 3.** Procedure of study 1-7

*3.1.1. Study 1. The influence of prickle acupuncture locus*

stimulation to Neiguan and Shenshu acupoints.

**Figure 4.** The influence of prickle acupuncture locus

We applied acupuncture stimulation to determine the influence of acupuncture stimulus on blood fluidity for 60 minutes for only 1 day at 1 Hz, 3-5 V [21]. Acupuncture was performed under anesthesia. Blood samples were collected from the abdominal vein after acupuncture stimulus. The blood samples were pretreated with anticoagulant (heparin sodium). The con‐ trol group was anesthetized the same as the experimental groups but did not receive stimu‐ lation. Because the effects of acupuncture vary according to stimulus locus (acupoint), acupuncture was applied to various loci of the trunk, arm and lower extremities. Results showed that blood flow time shortened significantly in the group with stimulation to the Zusanli, Hegu and Sanyingao; however, there were no significant changes in the group with

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PRP in a cuvette was pre-warmed to 37 ℃; adenosine 5'-diphosphate (ADP) was added as an agonist and the aggregation level was measured. This PA-20 device can measure platelet aggregation by the light scattering method. This light scattering method can measure the platelet aggregate size by determining the intensity of scattered light emitted from a parti‐ cle, the light intensity directly corresponding to the particle size. The platelet aggregation curve was separately recorded for each size range as the voltage of light scattering intensity. The aggregates measured were divided into three categories according to size: small (diame‐ ter 9-25 μm), medium (diameter 26-50 μm), and large-sized aggregates (diameter 50-70 μm) [19,20].

#### **2.5. Affirmation of blood cell count**

In blood fluidity experiments, it is important to consider factors which influence blood properties: the number of erythrocytes, leukocytes, platelets and hematocrit.

Therefore, we confirmed that the above-mentioned blood properties showed no significant differences in both the experiment and control groups.

#### **2.6. The experimental profile**

This experiment is divided into three parts. The first is the examination of modality of acu‐ puncture stimulus. The second is the examination of action mechanisms. The third examina‐ tion focuses on the decreasing state of blood fluidity by the restraint stress method. And finally we reviewed whether acupuncture improves blood fluidity.

#### **3. Results & discussion**

#### **3.1. Effects of stimulus approach**

At first, blood fluidity was reviewed in several acupuncture stimulus approaches (Figure 3).

**Figure 3.** Procedure of study 1-7

We assumed that the flow time in the analyzer imitated the capillary blood fluidity index [17]. In an MC-FAN assay, the prolongation of flow time indicates a decrease of blood fluidi‐

The ability of platelet aggregation was measured with a platelet coagulation measuring sys‐ tem, a platelet aggregometer (PA-20; Kowa Company. Ltd., Tokto, Japan). Blood samples were collected from the inferior vena cava under pentobarbital anesthesia. The blood sample treated with 3.2 % of sodium citrate (2.0 mL) was centrifuged to obtain platelet-rich plasma

PRP in a cuvette was pre-warmed to 37 ℃; adenosine 5'-diphosphate (ADP) was added as an agonist and the aggregation level was measured. This PA-20 device can measure platelet aggregation by the light scattering method. This light scattering method can measure the platelet aggregate size by determining the intensity of scattered light emitted from a parti‐ cle, the light intensity directly corresponding to the particle size. The platelet aggregation curve was separately recorded for each size range as the voltage of light scattering intensity. The aggregates measured were divided into three categories according to size: small (diame‐ ter 9-25 μm), medium (diameter 26-50 μm), and large-sized aggregates (diameter 50-70 μm)

In blood fluidity experiments, it is important to consider factors which influence blood

Therefore, we confirmed that the above-mentioned blood properties showed no significant

This experiment is divided into three parts. The first is the examination of modality of acu‐ puncture stimulus. The second is the examination of action mechanisms. The third examina‐ tion focuses on the decreasing state of blood fluidity by the restraint stress method. And

At first, blood fluidity was reviewed in several acupuncture stimulus approaches (Figure 3).

properties: the number of erythrocytes, leukocytes, platelets and hematocrit.

differences in both the experiment and control groups.

finally we reviewed whether acupuncture improves blood fluidity.

ty and a short flow time, an increase of blood fluidity [18].

**2.4. Measurement of platelet aggregation ability**

(PRP) and platelet-poor plasma (PPP).

**2.5. Affirmation of blood cell count**

**2.6. The experimental profile**

**3. Results & discussion**

**3.1. Effects of stimulus approach**

[19,20].

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#### *3.1.1. Study 1. The influence of prickle acupuncture locus*

We applied acupuncture stimulation to determine the influence of acupuncture stimulus on blood fluidity for 60 minutes for only 1 day at 1 Hz, 3-5 V [21]. Acupuncture was performed under anesthesia. Blood samples were collected from the abdominal vein after acupuncture stimulus. The blood samples were pretreated with anticoagulant (heparin sodium). The con‐ trol group was anesthetized the same as the experimental groups but did not receive stimu‐ lation. Because the effects of acupuncture vary according to stimulus locus (acupoint), acupuncture was applied to various loci of the trunk, arm and lower extremities. Results showed that blood flow time shortened significantly in the group with stimulation to the Zusanli, Hegu and Sanyingao; however, there were no significant changes in the group with stimulation to Neiguan and Shenshu acupoints.

**Figure 4.** The influence of prickle acupuncture locus

Figure 4 shows that when acupuncture stimulation was applied to the Hegu, Sanyinjao and Zusanli acupoints, blood fluidity was enhanced in comparison with the control. In addition, the results reveal that blood fluidity is not altered by stimulation of any of the acupoints in this study suggesting specificity of acupoints contributing to blood fluidity. Heparin sodium is combined with antithrombin III and inhibits thrombin activity, coagulation factor Xa and XIIa. In other words heparin sodium does not inhibit agglomeration of platelets directly. When heparin was used as an anticoagulant, MC-FAN blood fluidity observation showed the influence of platelet aggregation ability and erythrocyte deformability.

*3.1.3. Study 3. The influence of stimulus frequency*

does not compete with naloxone administration [23,24,26,27].

**Figure 6.** The influence of stimulus frequency on blood fluidity. Cited from [16], with permission.

The influence of stimulation frequency was investigated (Figure 6). Acupuncture was stimulat‐ ed for for 60 minutes at 1 Hz or 100 Hz, 3-5 V to Zusanli acupoint under anesthesia. When acu‐ puncture stimulus frequency changed, the blood fluidity was enhanced at both 1 Hz and 100 Hz. This result indicates that blood fluidity is not affected by change of stimulus frequency.

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It is known that a low-frequency (1-2 Hz) or a high-frequency (more than 100Hz) stimulus influences mechanisms other than those of acupuncture analgesia [22]. When these two acu‐ puncture analgesic system influences, even an operation is enabled only by acupuncture an‐ esthesia. We think that a decrease of nociception affects blood fluidity. Electric acupuncture of low frequency stimulus (1-5 Hz) secretes arterenol, serotonin and β-endorphin in the cen‐ tral nervous system. In other words, it is thought that the secretion of these transmitters has analgesic and sedative effects on the descending pain modulatory system or the endogenic opioid system [22-24]. In addition, as mechanism through the endogenous opioid system, precedence study shows acupuncture analgesic system of diffuse noxious inhibitory con‐ trols (DNIC) participated in acupuncture and moxibustion induced-analgesia [25]. In addi‐ tion, spinal segment-related analgesia occurs at high-frequency (more than 100Hz) electric acupuncture. This analgesic system produces an analgesic effect in concurrence with the start of the stimulus. It is thought that the gate control theory applies because this system

#### *3.1.2. Study 2. The influence of stimulus time*

This shows the effect of differences of stimulation period (Figure 5). Acupuncture was stimulated for 15 or 60 minutes at 1 Hz, 3-5 V to the Zusanli acupoint under anesthesia. Acu‐ puncture stimulation by Zusanli enhanced the blood fluidity (reduced the blood flow time (Figure 4). Blood samples were collected from the abdominal vein after acupuncture stimu‐ lus. The blood samples were preprocessed with an anticoagulant (heparin sodium).

The control group was anesthetized in the same manner as the experimental groups but did not receive stimulation.

As shown in this result, both stimulation time of 15 minutes and 60 minutes represented similar effect. Even with a short stimulation time, blood fluidity was enhanced suggesting the immediate effect of acupuncture stimulation contributing to blood fluidity.

**Figure 5.** The influence of stimulus tome on blood fluidity. Cited from [16], with permission.

#### *3.1.3. Study 3. The influence of stimulus frequency*

Figure 4 shows that when acupuncture stimulation was applied to the Hegu, Sanyinjao and Zusanli acupoints, blood fluidity was enhanced in comparison with the control. In addition, the results reveal that blood fluidity is not altered by stimulation of any of the acupoints in this study suggesting specificity of acupoints contributing to blood fluidity. Heparin sodium is combined with antithrombin III and inhibits thrombin activity, coagulation factor Xa and XIIa. In other words heparin sodium does not inhibit agglomeration of platelets directly. When heparin was used as an anticoagulant, MC-FAN blood fluidity observation showed

This shows the effect of differences of stimulation period (Figure 5). Acupuncture was stimulated for 15 or 60 minutes at 1 Hz, 3-5 V to the Zusanli acupoint under anesthesia. Acu‐ puncture stimulation by Zusanli enhanced the blood fluidity (reduced the blood flow time (Figure 4). Blood samples were collected from the abdominal vein after acupuncture stimu‐

The control group was anesthetized in the same manner as the experimental groups but did

As shown in this result, both stimulation time of 15 minutes and 60 minutes represented similar effect. Even with a short stimulation time, blood fluidity was enhanced suggesting

lus. The blood samples were preprocessed with an anticoagulant (heparin sodium).

the immediate effect of acupuncture stimulation contributing to blood fluidity.

**Figure 5.** The influence of stimulus tome on blood fluidity. Cited from [16], with permission.

the influence of platelet aggregation ability and erythrocyte deformability.

*3.1.2. Study 2. The influence of stimulus time*

not receive stimulation.

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The influence of stimulation frequency was investigated (Figure 6). Acupuncture was stimulat‐ ed for for 60 minutes at 1 Hz or 100 Hz, 3-5 V to Zusanli acupoint under anesthesia. When acu‐ puncture stimulus frequency changed, the blood fluidity was enhanced at both 1 Hz and 100 Hz. This result indicates that blood fluidity is not affected by change of stimulus frequency.

It is known that a low-frequency (1-2 Hz) or a high-frequency (more than 100Hz) stimulus influences mechanisms other than those of acupuncture analgesia [22]. When these two acu‐ puncture analgesic system influences, even an operation is enabled only by acupuncture an‐ esthesia. We think that a decrease of nociception affects blood fluidity. Electric acupuncture of low frequency stimulus (1-5 Hz) secretes arterenol, serotonin and β-endorphin in the cen‐ tral nervous system. In other words, it is thought that the secretion of these transmitters has analgesic and sedative effects on the descending pain modulatory system or the endogenic opioid system [22-24]. In addition, as mechanism through the endogenous opioid system, precedence study shows acupuncture analgesic system of diffuse noxious inhibitory con‐ trols (DNIC) participated in acupuncture and moxibustion induced-analgesia [25]. In addi‐ tion, spinal segment-related analgesia occurs at high-frequency (more than 100Hz) electric acupuncture. This analgesic system produces an analgesic effect in concurrence with the start of the stimulus. It is thought that the gate control theory applies because this system does not compete with naloxone administration [23,24,26,27].

**Figure 6.** The influence of stimulus frequency on blood fluidity. Cited from [16], with permission.

#### **3.2. Effects of action mechanism**

In the second set of experiments, we reviewed the mechanism of acupuncture with medicine.

*3.2.2. Study 5. Influence of β antagonist*

administered by i.p. to the control animals.

time was decreased with β-agonist and α-antagonist.

A. The effect of intraperitoneal administered adrenergic drugs on blood fluidity

The influence of sympathetic agonists and antagonists on the blood flow was investigated (Figure 8). We used phenylephrine at 200 μg/kg as an α-agonist, phentolamine at 100 μg/kg as an α-antagonist, isoproterenol at 4 μg/kg as a β-agonist, and propranolol at 40 μg/kg as a β-antagonist. In these experiments, the drugs were dissolved in 1 mL of physiological saline and were administered by intraperitoneal (i.p.) into the rats. Physiological saline (1 mL) was

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The blood flow time was increased with α-agonist and β-antagonist, and the blood flow

**Figure 8.** Influence of sympathetic agonists and antagonists on blood fluidity. Cited from [16], with permission.

Effect of acupuncture together with an adrenergic drug on the blood fluidity was investigat‐ ed to get the insight into the reaction mechanism (Figure 9). β-antagonist, propranolol at 40 μg/kg, was dissolved in 1 mL of physiological saline and administered before the acupunc‐ ture stimulation by i.p into the rats. Acupuncture was stimulated at 1 Hz, 3-5 V for 60 mi‐

B. Effect of β-antagonist on the blood fluidity enhanced by acupuncture

nutes to Zusanli acupoint under anesthesia.

#### *3.2.1. Study 4. Influence of naloxone*

The influence of Naloxone, an antagonist of endogenous opioid was investigated (Figure 7). Acupuncture on the Zusanli was stimulated 60 minutes while injecting naloxone (5 mg/kg, i.p.) into the abdominal cavity every 10 minutes.

Neither acupuncture stimulus nor naloxone was given to the control groups. Physiological saline was injected into the abdominal cavity every 10 minutes of the only acupuncture and the control groups. Results showed that blood flow time decreased significantly in the Zu‐ sanli-stimulated and the Zusanli-stimulated plus naloxone groups, although there was no significant difference between these two groups.

The results of figure 5 show that blood fluidity changes with short time electro acupuncture suggesting the intervention of the nervous system. However, when we consider the fact that blood fluidity was not affected by a difference of stimulus frequency nor naloxone adminis‐ tration, it can be surmised that the endogenic opioid system and the spinal segment system do not contribute to blood fluidity. We speculate that acupuncture stimulus changes blood fluidity by the automatic nervous system and axon reflex, and does not influence the opioid system and the spinal segment analgesia system.

**Figure 7.** Influence of naloxone on blood fluidity. Cited from [16], with permission.

#### *3.2.2. Study 5. Influence of β antagonist*

**3.2. Effects of action mechanism**

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*3.2.1. Study 4. Influence of naloxone*

i.p.) into the abdominal cavity every 10 minutes.

significant difference between these two groups.

system and the spinal segment analgesia system.

**Figure 7.** Influence of naloxone on blood fluidity. Cited from [16], with permission.

In the second set of experiments, we reviewed the mechanism of acupuncture with medicine.

The influence of Naloxone, an antagonist of endogenous opioid was investigated (Figure 7). Acupuncture on the Zusanli was stimulated 60 minutes while injecting naloxone (5 mg/kg,

Neither acupuncture stimulus nor naloxone was given to the control groups. Physiological saline was injected into the abdominal cavity every 10 minutes of the only acupuncture and the control groups. Results showed that blood flow time decreased significantly in the Zu‐ sanli-stimulated and the Zusanli-stimulated plus naloxone groups, although there was no

The results of figure 5 show that blood fluidity changes with short time electro acupuncture suggesting the intervention of the nervous system. However, when we consider the fact that blood fluidity was not affected by a difference of stimulus frequency nor naloxone adminis‐ tration, it can be surmised that the endogenic opioid system and the spinal segment system do not contribute to blood fluidity. We speculate that acupuncture stimulus changes blood fluidity by the automatic nervous system and axon reflex, and does not influence the opioid

#### A. The effect of intraperitoneal administered adrenergic drugs on blood fluidity

The influence of sympathetic agonists and antagonists on the blood flow was investigated (Figure 8). We used phenylephrine at 200 μg/kg as an α-agonist, phentolamine at 100 μg/kg as an α-antagonist, isoproterenol at 4 μg/kg as a β-agonist, and propranolol at 40 μg/kg as a β-antagonist. In these experiments, the drugs were dissolved in 1 mL of physiological saline and were administered by intraperitoneal (i.p.) into the rats. Physiological saline (1 mL) was administered by i.p. to the control animals.

The blood flow time was increased with α-agonist and β-antagonist, and the blood flow time was decreased with β-agonist and α-antagonist.

**Figure 8.** Influence of sympathetic agonists and antagonists on blood fluidity. Cited from [16], with permission.

B. Effect of β-antagonist on the blood fluidity enhanced by acupuncture

Effect of acupuncture together with an adrenergic drug on the blood fluidity was investigat‐ ed to get the insight into the reaction mechanism (Figure 9). β-antagonist, propranolol at 40 μg/kg, was dissolved in 1 mL of physiological saline and administered before the acupunc‐ ture stimulation by i.p into the rats. Acupuncture was stimulated at 1 Hz, 3-5 V for 60 mi‐ nutes to Zusanli acupoint under anesthesia.

The fluidity of erythrocyte suspension was not altered by acupuncture. (Figure 11), indicat‐ ing that erythrocyte deformability does not contribute to blood fluidity strengthened by acu‐

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**Figure 10.** Observation of platelet aggregation Cited from [16], with permission

**Figure 11.** Examination of erythrocyte suspension fluidity. Cited from [15], with permission.

puncture.

**Figure 9.** The relevance with βantagonist and blood fluidity. Cited from [16], with permission.

As a result, the β-antagonist canceled the enhancement effect of blood fluidity with acu‐ puncture stimulus. There was no significant difference between the β-antagonist plus acu‐ puncture and the control groups. This result indicates that the sympathetic nervous system influences blood fluidity with an acupuncture stimulus.

#### *3.2.3. Study 6. Inhibition of platelet aggregation*

Effect of platelet aggregation was investigated (Figure 10). An ADP an agonist was added in the PRP and the aggregation level was measured by the light scattering method. Acupunc‐ ture decreased the formation of large aggregates. In addition, medium and small-sized ag‐ gregates in the stimulated experimental group increased significantly. However, in animals previously administered with β-antagonist, the decrease of platelet aggregation by acupunc‐ ture was significantly reversed. This result indicates that platelet aggregation may contrib‐ ute to the blood fluidity enhanced by acupuncture.

#### *3.2.4. Study 7. Influence of acupuncture and β-antagonist on the platelet aggregation*

Erythroid deformability was measured by blood fluidity using erythrocyte suspension (Fig‐ ure 11). Physiological saline (2.0 ml) was added to the blood sample treated with heparin sodium (2.0 ml) and centrifuged (400 g × 5 minutes), resulting in a deposition layer of eryth‐ rocyte suspension and removal of the supernatant. Phosphate-buffered saline (PBS) was added to this erythrocyte rich liquid and re-centrifuged twice in the same manner [28]. The layer of erythrocyte was adjusted to 30 % with hematocrit, and this erythrocyte suspension was used for this experiment. The erythrocyte suspension was examined with the MC-FAN.

The fluidity of erythrocyte suspension was not altered by acupuncture. (Figure 11), indicat‐ ing that erythrocyte deformability does not contribute to blood fluidity strengthened by acu‐ puncture.

**Figure 10.** Observation of platelet aggregation Cited from [16], with permission

**Figure 9.** The relevance with βantagonist and blood fluidity. Cited from [16], with permission.

influences blood fluidity with an acupuncture stimulus.

*3.2.3. Study 6. Inhibition of platelet aggregation*

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ute to the blood fluidity enhanced by acupuncture.

As a result, the β-antagonist canceled the enhancement effect of blood fluidity with acu‐ puncture stimulus. There was no significant difference between the β-antagonist plus acu‐ puncture and the control groups. This result indicates that the sympathetic nervous system

Effect of platelet aggregation was investigated (Figure 10). An ADP an agonist was added in the PRP and the aggregation level was measured by the light scattering method. Acupunc‐ ture decreased the formation of large aggregates. In addition, medium and small-sized ag‐ gregates in the stimulated experimental group increased significantly. However, in animals previously administered with β-antagonist, the decrease of platelet aggregation by acupunc‐ ture was significantly reversed. This result indicates that platelet aggregation may contrib‐

Erythroid deformability was measured by blood fluidity using erythrocyte suspension (Fig‐ ure 11). Physiological saline (2.0 ml) was added to the blood sample treated with heparin sodium (2.0 ml) and centrifuged (400 g × 5 minutes), resulting in a deposition layer of eryth‐ rocyte suspension and removal of the supernatant. Phosphate-buffered saline (PBS) was added to this erythrocyte rich liquid and re-centrifuged twice in the same manner [28]. The layer of erythrocyte was adjusted to 30 % with hematocrit, and this erythrocyte suspension was used for this experiment. The erythrocyte suspension was examined with the MC-FAN.

*3.2.4. Study 7. Influence of acupuncture and β-antagonist on the platelet aggregation*

**Figure 11.** Examination of erythrocyte suspension fluidity. Cited from [15], with permission.

#### **3.3. Influence of restraint stress**

Finally, we also focused on the acupuncture stimulus mechanism with the restraint stress method. As preliminary research, we hypothesized that various stressors decrease blood flu‐ idity. Restraint in a rectangular acrylic box for six hours was used as the stressor. Acupunc‐ ture stimulation was given for 1 hour (after 5 hours of restriction) at 1 Hz, 3-5V to the Zusanli acupoint with arousal (Figure 12).

*3.3.2. Study 9. Affecters that are involved in platelet aggregation*

Changes in the blood noradrenaline level after restraing stress and acupuncture was moni‐ tored by 2-CAT (A-N) Research ELISA (Labor Diagnostika Nord GmbH & Co. KG) (Figure 14). In the restraint group, the blood noradrenalin level increased. In the restraint and acupunc‐ ture group, the adrenalin level decreased. These results suggest that acupuncture stimulus

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Changes in the blood ATP level after restraint and acupuncture was monitored by AMERIC

In the restraint group, the blood ATP level increased. In the restraint and acupuncture group, the ATP level decreased, indicating that acupuncture stimulus most likely inhibits

ATP kit (Applied Medical Enzyme Research Ins. Co.) (Figure 15).

(a) Blood noradrenalin level

inhibits noradrenalin release.

**Figure 14.** Blood noradrenalin level

(b) Blood ATP level

ATP release.

**Figure 12.** Experimental procedures for the study 8 and 9

#### *3.3.1. Study 8. Observation of blood fluidity and platelet aggregation*

Effect on the blood fluidity and platelet aggregation was investigated (Figure 13). In the re‐ straint group, the blood fluidity decreased and the platelet aggregation increased. However, the acupuncture stimulus reversed these changes.

**Figure 13.** Influence of restraint stress on the bloody fluidity (A) and platelet aggregation (B)

#### *3.3.2. Study 9. Affecters that are involved in platelet aggregation*

#### (a) Blood noradrenalin level

**3.3. Influence of restraint stress**

214 Alternative Medicine

Zusanli acupoint with arousal (Figure 12).

**Figure 12.** Experimental procedures for the study 8 and 9

the acupuncture stimulus reversed these changes.

*3.3.1. Study 8. Observation of blood fluidity and platelet aggregation*

**Figure 13.** Influence of restraint stress on the bloody fluidity (A) and platelet aggregation (B)

Finally, we also focused on the acupuncture stimulus mechanism with the restraint stress method. As preliminary research, we hypothesized that various stressors decrease blood flu‐ idity. Restraint in a rectangular acrylic box for six hours was used as the stressor. Acupunc‐ ture stimulation was given for 1 hour (after 5 hours of restriction) at 1 Hz, 3-5V to the

Effect on the blood fluidity and platelet aggregation was investigated (Figure 13). In the re‐ straint group, the blood fluidity decreased and the platelet aggregation increased. However,

Changes in the blood noradrenaline level after restraing stress and acupuncture was moni‐ tored by 2-CAT (A-N) Research ELISA (Labor Diagnostika Nord GmbH & Co. KG) (Figure 14).

In the restraint group, the blood noradrenalin level increased. In the restraint and acupunc‐ ture group, the adrenalin level decreased. These results suggest that acupuncture stimulus inhibits noradrenalin release.

**Figure 14.** Blood noradrenalin level

#### (b) Blood ATP level

Changes in the blood ATP level after restraint and acupuncture was monitored by AMERIC ATP kit (Applied Medical Enzyme Research Ins. Co.) (Figure 15).

In the restraint group, the blood ATP level increased. In the restraint and acupuncture group, the ATP level decreased, indicating that acupuncture stimulus most likely inhibits ATP release.

**Figure 16.** Blood NO2 level

lar Ca2+.

and vasucular smooth muscle generally.

inhibits the nerve action of pain.

There is widespread appreciation that ATP also acts as an extracellular mediator. The extracel‐ lular ATP is released from nerve endings and various secretory cells in an exocytotic manner, from non-excitable cells by mechanical shear stress and from the cytosol of damaged cells. Ex‐ tracellular ATP activates multiple cell surface P2 receptors and increases intracellular Ca2+, thereby inducing diverse physiological effects, such as regulation of neurotransmitter release,

Promotion of Blood Fluidity Using Electroacupuncture Stimulation

http://dx.doi.org/10.5772/53036

217

Nitric oxide (NO) which decreases intracellular Ca2+ plays an important role in the regula‐ tion of vasoconstriction, inhibition of platelet aggregation, and suppression of smooth mus‐ cle cell proliferation. Noradrenalin and ATP increase intracellular Ca2+ in vasucular smooth muscle and platelets [33]. However NO inhibits these functions by depression of intracellu‐

We think that acupuncture stimulus may control intracellular Ca2+ level of a blood platelet

The cardiovascular system are controlled at least in part by blood fluidity. In addition, it is important that blood circulates smoothly for the removal of reactive oxygen and waste products. When pain results from a bruise, distortion and muscle ache, it is known that acu‐ puncture desensitizes the pain. Many preliminary researches have shown that acupuncture

The present study suggests that acupuncture stimulus changes blood fluidity separately by the cardiovascular system and a new blood flow improvement system that removes pain in re‐ sponse to a lesion, although the precise mechanisms are still a question for future investigation.

smooth muscle contraction, prostaglandin formation and platelet aggregation [32].

**Figure 15.** Blood ATP level

#### (c) Blood NO level

Changes in blood nitric oxide (NO) level after restraint and acupuncture was monitored by Greies test (NO2/NO3 assay kit, Dojindo Molecular Technologies, Inc) (Figure 16). NO is produced by a vascular endothelial cells, and regulates a vascular endothelium function. NO is a molecular mediator of many physiological processes, including vasodilation, in‐ flammation, thrombosis, immunity and neurotransmission [29,30]. Half-life of NO is very short (for 3-6 seconds). It is thought that NO2 - reflects production of NO. Therefore a level of NO2 - , which occurred by the oxidation of NO, was examined by the Griess test (NO2 /NO3 Assay Kit, Dojindo Molecular Technologies, Inc.).

In the restraint group, the blood NO2 level decreased. In the restraint and acupuncture group, the NO2 level increased. indicating that acupuncture stimulus most likely improves NO2 release.

Stimulation of α1-adrenoceptor with noradrenaline elicits increases in intracellular Ca2+ con‐ centration and induces platelet aggregation and vasoconstriction. Furthermore, high-shearrate conditions activates von Willebrand factor (vWF) in plasma and vascular sub endothelium and produces platelet aggregation [31]. Vascular smooth muscle is controlled between contraction (induced via α1-adrenoceptor) and relaxation (induced via β2-aderno‐ ceptor). It seems that platelet aggregation is enhanced as α1-adrenoceptor function is more remarkable than β2-adrenoceptor function. Therefore acupuncture stimulus down-regulates catecholamine secretion from adrenal medulla and may relax with blood vessels.

**Figure 16.** Blood NO2 level

**Figure 15.** Blood ATP level

short (for 3-6 seconds). It is thought that NO2

In the restraint group, the blood NO2


Assay Kit, Dojindo Molecular Technologies, Inc.).

Changes in blood nitric oxide (NO) level after restraint and acupuncture was monitored by Greies test (NO2/NO3 assay kit, Dojindo Molecular Technologies, Inc) (Figure 16). NO is produced by a vascular endothelial cells, and regulates a vascular endothelium function. NO is a molecular mediator of many physiological processes, including vasodilation, in‐ flammation, thrombosis, immunity and neurotransmission [29,30]. Half-life of NO is very

, which occurred by the oxidation of NO, was examined by the Griess test (NO2 /NO3

Stimulation of α1-adrenoceptor with noradrenaline elicits increases in intracellular Ca2+ con‐ centration and induces platelet aggregation and vasoconstriction. Furthermore, high-shearrate conditions activates von Willebrand factor (vWF) in plasma and vascular sub endothelium and produces platelet aggregation [31]. Vascular smooth muscle is controlled between contraction (induced via α1-adrenoceptor) and relaxation (induced via β2-aderno‐ ceptor). It seems that platelet aggregation is enhanced as α1-adrenoceptor function is more remarkable than β2-adrenoceptor function. Therefore acupuncture stimulus down-regulates

level increased. indicating that acupuncture stimulus most likely improves


catecholamine secretion from adrenal medulla and may relax with blood vessels.


level decreased. In the restraint and acupuncture

(c) Blood NO level

216 Alternative Medicine

NO2 -

NO2 release.

group, the NO2

There is widespread appreciation that ATP also acts as an extracellular mediator. The extracel‐ lular ATP is released from nerve endings and various secretory cells in an exocytotic manner, from non-excitable cells by mechanical shear stress and from the cytosol of damaged cells. Ex‐ tracellular ATP activates multiple cell surface P2 receptors and increases intracellular Ca2+, thereby inducing diverse physiological effects, such as regulation of neurotransmitter release, smooth muscle contraction, prostaglandin formation and platelet aggregation [32].

Nitric oxide (NO) which decreases intracellular Ca2+ plays an important role in the regula‐ tion of vasoconstriction, inhibition of platelet aggregation, and suppression of smooth mus‐ cle cell proliferation. Noradrenalin and ATP increase intracellular Ca2+ in vasucular smooth muscle and platelets [33]. However NO inhibits these functions by depression of intracellu‐ lar Ca2+.

We think that acupuncture stimulus may control intracellular Ca2+ level of a blood platelet and vasucular smooth muscle generally.

The cardiovascular system are controlled at least in part by blood fluidity. In addition, it is important that blood circulates smoothly for the removal of reactive oxygen and waste products. When pain results from a bruise, distortion and muscle ache, it is known that acu‐ puncture desensitizes the pain. Many preliminary researches have shown that acupuncture inhibits the nerve action of pain.

The present study suggests that acupuncture stimulus changes blood fluidity separately by the cardiovascular system and a new blood flow improvement system that removes pain in re‐ sponse to a lesion, although the precise mechanisms are still a question for future investigation.

## **4. Conclusion**

We conclude that acupuncture stimulation increases blood fluidity. And several points as follows seem to be important to understand the mechanism.

[5] Yamaguchi N, Hashimoto H, Arai M, Takeda S, Kawada N, Taru A, Li AL, Izumi H, Sugiyama K. Effect of Acupuncture on Leukocyte and Lymphocyte Subpopulation in

Promotion of Blood Fluidity Using Electroacupuncture Stimulation

http://dx.doi.org/10.5772/53036

219

[8] Fusegawa Y, Hashizume H, Okumura T, Deguchi Y, Shina Y, Ikari Y, Tanabe T. Hy‐ pertensive patients with carotid artery plaque exhibit increased platelet aggregabili‐

[9] Lee CY, Kim KC, Park HW, Song JH, Lee CH. Rheological properties of erythrocytes

[10] Ohno H, Kimu CK, Kim JH, Jung YS, Bae SY, Toshinai K, Miyazaki H, Esaki K, Take‐ masa T, Kinugasa T, Kizaki T, Hitomi Y, Ookawara T, Oh-Ishi S, Haga S. Hematolog‐ ical Response in Juveniles after Training at Moderate Altitude. Advances in Exercise

[11] Kikuchi Y. Effect of leukocytes and platelets on blood flow through a parallel array of microchannels: micro- and macroflow relation and rheological measures of leuko‐

[12] Horn NA, Anastase DM, Hecker KE, Baumert JH, Robitzsch T, Rossaint R. Epinephr‐ ine enhances platelet-neutrophil adhesion in whole blood in vitro. Anesth Analg.,

[13] Ochi N, Tabara Y, Igase M, Nagai T, Kido T, Miki T, Kohara K. Silent cerebral micro‐ bleeds associated with arterial stiffness in an apparently healthy subject. Hyperten‐

[14] Horibe Y, Ishino S, Hisamitsu N, Thein Lain, Ishikawa S, Sato T, Hisamitsu T. "Oket‐ su" and Hemorheological Changes : Examination by Micro Channel Array Flow Ana‐

[15] Ishikawa S, Murai M, Sato T, Sunagawa M, Tokita E, Aung SK, Asano K, Hisamitsu T. Promotion of blood fluidity by inhibition of platelet adhesion using electroacu‐

[16] Ishikawa S, Suga H, Fukushima M, Yoshida A, Yoshida Y, Sunagawa M, Hisamitsu T. Blood fluidity enhancement by electrical acupuncture stimulation is related to an

[17] Kikuchi Y, Sato K, Ohki H. Optically accessible microchannels fomed in a single crystal silicon substrate for studics of blood rhcology.Microvasc Res., 44: 226-40, 1992

[18] Seki K, Sumino H, Murakami M. Study on blood rheology measured by MC-FAN.

lyzer. Japanese Journal of Oriental Medicine, 55(5): 645-8, 2004 (in Japanese)

puncture stimulation., J Acupunct Meridian Stud., 4(1): 44-53, 2011

adrenergic mechanism, J Acupunct Meridian Stud., 5(1): 21-28, 2012

Rinsho Byori, 51(8): 770-5, 2003 (in Japanese)

cyte and platelet activities. Microvasc Res., 50(2): 288-300, 1995

from male hypercholesterolemia. Microvascular Research, 67: 133–8, 2004

Human Peripheral Blood: Quantitative discussion. BCPM, 65(4): 199-206, 2002 [6] Hirata C, Kobayashi H, Mizuno N, Kutsuna H, Ishina K, Ishii M. Effect of Normal Human Erythrocytes on Blood Rheology in Microcirculation. Osaka City Medical

Journal, 53(2): 73-85, 2007

[7] George JN. Platelets. Lancet, 355: 1531–9, 2000

ty. Thrombosis Research, 117: 615-22, 2006

and Sports Physiology, 10(2): 31-5, 2004

100(2): 520-6, 2005

sion Research, 32(4): 255-60, 2009


It is thought that the acupuncture stimulation changes platelet aggregation by various fac‐ tors, including the presence of catecholamine, ATP and NO.

## **Author details**

Shintaro Ishikawa1 , Kazuhito Asano1,2 and Tadashi Hisamitsu1

1 Department of Physiology, School of Medicine, Showa University, Tokyo, Japan

2 Division of Physiology, School of Nursing and Rehabilitation Sciences, Showa University, Kanagawa, Japan

## **References**


**4. Conclusion**

218 Alternative Medicine

**Author details**

Shintaro Ishikawa1

Kanagawa, Japan

**References**

2005.

20(1): 41-8, 2010 (in Japanese)

We conclude that acupuncture stimulation increases blood fluidity. And several points as

**2.** The acupuncture analgesic system seems to be independent from this mechanism that

**3.** Inhibition of the sympathetic nervous activity by the acupuncture stimulation is sug‐

**4.** It seems that acupuncture stimulus has a stronger influence on platelets than erythro‐

It is thought that the acupuncture stimulation changes platelet aggregation by various fac‐

, Kazuhito Asano1,2 and Tadashi Hisamitsu1

1 Department of Physiology, School of Medicine, Showa University, Tokyo, Japan

2 Division of Physiology, School of Nursing and Rehabilitation Sciences, Showa University,

[1] Lee A, Fan, LTY. Lee, Anna. Stimulation of the wrist acupuncture point P6 for pre‐ venting postoperative nausea and vomiting. Cochrane Database of Systematic Re‐

[2] Ernst E, Lee MS, Choi TY. Acupuncture: does it alleviate pain and are there serious

[3] McCarthy M. Critics slam draft WHO report on homoeopathy. Lancet, 366: 705-6.

[4] Sakuma M, Arai M, Matsuba S, Yamaguchi N. Effects of Acupuncture on Intracellu‐ lar Cytokine and Hormone Levels in Human Peripheral Blood. Cytometry research,

views (Online) (2): CD003281. doi:10.1002/14651858.CD003281.pub3. 2009

risks? A review of reviews. Pain. 152(4):755-64. 2011

follows seem to be important to understand the mechanism.

gested to have important role in the mechanism.

tors, including the presence of catecholamine, ATP and NO.

**1.** There is specificity of stimulus locus.

acupuncture increase blood fluidity.

cytes, and changes blood fluidity.


[19] Matsuno H, Tokuda H, Ishisaki A, Zhou Y, Kitajima Y, Kozawa O. P2Y12 receptors play a significant role in the development of platelet microaggregation in patients with diabetes. J Clin Endocrinol Metab., 90(2): 920-7, 2005:

**Chapter 10**

**Enormous Potential for**

Additional information is available at the end of the chapter

Dae Youn Hwang

**1. Introduction**

those of *L. spicata* [3,4].

http://dx.doi.org/10.5772/53520

**Development** *Liriope platyphylla Wang et Tang* **as a**

*Liriope platyphylla Wang et Tang* (*L. platyphylla*) is named *Liriope muscari* with Binomial name and called big blue lilyturf, lilyturf, border grass and monkey grass with common name [1]. It is widely used as one of the 50 fundamental herbs in traditional Oriental medicine; the species is one member of low, herbaceous flowing plats, which grows commonly in the shady forests of East Asia including China, Korea and Japan at elevation of 100 to 1400 m. Also, it typically grows 30-45 cm tall and have grass-like evergreen foliage and lilac-purple flowers which produce single seeded berries on spike in the fall [1,2]. Their roots are long fibrous with terminal tubers. Its flower is showy form which an erect spikes with tiered whorls of dense, white to violet-purple flowers rising above the leaves as like grape hya‐ cinth. This flower differents into blackish berries which can maintain their status into winter season (Fig. 1A). Furthermore, *L. platyphylla* bear a strong likeness to *L. spicata* (creeping lily‐ turf) which is the most common species in this genus. Although the prominent flower spike extending above the leaves is alike, the leaves of *L. platyphylla* were wider and longer than

*L. platyphylla* is easily well grown in most condition of soil including average, medium, welldrained type in full sun shines to partial shade although the ideal condition for it growth are fertile and moist soils with partial shade. Furthermore, it has a wide range of tolerance for light, heat, humidity, drought and soil condition. Because of these advantages, they widely used as one of the most popular border plant and groundcover in southeastern USA [1].

Of several parts in *L. platyphylla,* only roots have generally been used for a variety of purposes, in‐ cluding as a therapeutic drug and in teas (Fig. 1B and C) [1]. However, there are a few studies

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

© 2012 Hwang; 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.

**Therapeutic Drug on the Human Chronic Disease**

