**2.7 Formulations of TCM**

### *2.7.1 Decoctions*

Decoction is the earliest and most commonly used preparation method in the clinical application of TCM [81]. A TCM decoction is the herbal tea that is made of processed medicinal herbs which are simmered for hours [82]. The preparation of a traditional medicine decoction has multiple steps such as the sequence of boiling the herbal material and different boiling and filtering times. The preparation of a decoction is complex, as it requires experience and often takes considerable time. The herbal ingredients within the formula for decoction can be changed for the individual patient according to the disease. The herbal medicine may include leaves, flowers, roots, bark or fruits, and the procedure of preparation is depending on the part of the herb that is used. It is of concern that in some instances, the quality and stability of herbal material cannot be assured [14]. Also, the unpleasant flavour of a decoction has been a problem for patients for thousands of years. As equipment advances and market demand increases, modern decoctions have become very popular in TCM hospitals, because of convenience [83]. Decoctions have a quick and often complete absorption with good bioavailability [81]. In a modernised decoction method, the medicinal herbs are combined with water and are extracted in TCM decoction machines [83]. Modern formulations also include granules, oral liquids, tablets, capsules and injections. This change in methodology may in fact influence the composition of the final TCM, and this is another area that would benefit from comparative research studies directed at classical and modern decoction methods.

## *2.7.2 Modern granular formulations*

The use of granules has dramatically increased in China since they are easy to handle, store and formulate. The granule made of a single herb is called a single dispensing granule; when made of multiple herbs, it is called a dispensing formula granule [84]. The preparation of granules is included in the extensive process of

**29**

tiple marker assays.

*Traditional Chinese Medicine: From Aqueous Extracts to Therapeutic Formulae*

result in different clinical efficacy between granules and decoction.

method on the therapeutic effect of a final granular TCM product.

Over the past 30 years, chromatographic and spectrophotometric fingerprinting methods have dramatically improved, and the application of hyphenated techniques such as high performance liquid chromatography (HPLC)-mass spectrometry (HPLC-MS), liquid chromatography-nuclear magnetic resonance (LC-NMR) or gas chromatography mass spectrometry (GC-MS) in the analysis of TCM facilitates the determination of the quality in research and in the pharmaceutical industry [4]. However, quality standards are challenging to establish, because the complexity of potential active ingredients in medicinal herbs is used in TCM. Today, commonly applied models for TCM quality control include conventional methods, such as microscopic, and macroscopic identification and comparison with monographs, chemical fingerprint analysis using thin-layer liquid chromatography (TLC), liquid chromatography (LC), HPLC, gas chromatography (GC), GC-MS, etc., and mul-

Chemical fingerprints and bioactivities assay linked with multiple markers is currently recognised to be effective for TCM analysis [88]. The use of known and previously characterised markers for the analysis of constituents is the most popular method for identification and quality control in TCM. For example, the ginsenoside fingerprint profiles are applied for the authentication of Panax species [89]. Moreover, the fingerprint-efficacy relationship of Lycii fructus (Goji 枸杞) was studied by Zhang in 2018. The spectrum of ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) of three batches of Goji was correlated with the biological data using 2,2-diphenyl-

1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays. Compounds relating to the antioxidant activity of Goji, such

**2.8 Fingerprint-efficacy analysis in TCM**

decoction: they are prepared by decoction or aqueous extraction with the aid of suitable excipients. After being dissolved in warm water, they can be ingested by patients. With the increasing development of TCM granules, international research has focused on whether decoction can be replaced by granules with similar effectiveness and safety [15]. During the procedure of granule preparation, the difference of water extraction, concentration, desiccation and granulation may influence the dissolution and chemical profiles of active ingredients [85]. Thus, this may

Recently, some cases related to the equivalence of effect between granules and decoction have been reported. Indeed, some differences in chemical consistency were identified, which suggest that the efficacy might not be equivalent between both forms of medicine. Ge-Gen-Qin-Lian-Tang is a classic TCM formula for the treatment of inflammatory bowel disease [86]. The comparison of chemical fingerprints of traditional decoction and granules of Ge-Gen-Qin-Lian-Tang was investigated by liquid chromatography-diode array detector (LC-DAD) to ensure the consistency of efficacy. The fingerprints demonstrated small variations among the 20 peaks, but the peak area of puerain, berberine and baicalein from the granule sample was 50% less than that of the decoction [87]. The TCM formula Da-Cheng-Qi-Tang is commonly used to treat digestive disease. The chemical consistency of decoctions of Da-Cheng-Qi-Tang prepared by traditional and modern methods was investigated. Five compounds were identified as chemical markers, an analysis of which established that the chemical fingerprints were not consistent between these two kinds of decoctions. The study showed that the traditional decoction method had a stronger purgative effect due to higher concentrations of rhein and sennosides [83]. Therefore, it is very important to understand the impact of the formulation

*DOI: http://dx.doi.org/10.5772/intechopen.85733*

#### *Traditional Chinese Medicine: From Aqueous Extracts to Therapeutic Formulae DOI: http://dx.doi.org/10.5772/intechopen.85733*

decoction: they are prepared by decoction or aqueous extraction with the aid of suitable excipients. After being dissolved in warm water, they can be ingested by patients. With the increasing development of TCM granules, international research has focused on whether decoction can be replaced by granules with similar effectiveness and safety [15]. During the procedure of granule preparation, the difference of water extraction, concentration, desiccation and granulation may influence the dissolution and chemical profiles of active ingredients [85]. Thus, this may result in different clinical efficacy between granules and decoction.

Recently, some cases related to the equivalence of effect between granules and decoction have been reported. Indeed, some differences in chemical consistency were identified, which suggest that the efficacy might not be equivalent between both forms of medicine. Ge-Gen-Qin-Lian-Tang is a classic TCM formula for the treatment of inflammatory bowel disease [86]. The comparison of chemical fingerprints of traditional decoction and granules of Ge-Gen-Qin-Lian-Tang was investigated by liquid chromatography-diode array detector (LC-DAD) to ensure the consistency of efficacy. The fingerprints demonstrated small variations among the 20 peaks, but the peak area of puerain, berberine and baicalein from the granule sample was 50% less than that of the decoction [87]. The TCM formula Da-Cheng-Qi-Tang is commonly used to treat digestive disease. The chemical consistency of decoctions of Da-Cheng-Qi-Tang prepared by traditional and modern methods was investigated. Five compounds were identified as chemical markers, an analysis of which established that the chemical fingerprints were not consistent between these two kinds of decoctions. The study showed that the traditional decoction method had a stronger purgative effect due to higher concentrations of rhein and sennosides [83]. Therefore, it is very important to understand the impact of the formulation method on the therapeutic effect of a final granular TCM product.

#### **2.8 Fingerprint-efficacy analysis in TCM**

Over the past 30 years, chromatographic and spectrophotometric fingerprinting methods have dramatically improved, and the application of hyphenated techniques such as high performance liquid chromatography (HPLC)-mass spectrometry (HPLC-MS), liquid chromatography-nuclear magnetic resonance (LC-NMR) or gas chromatography mass spectrometry (GC-MS) in the analysis of TCM facilitates the determination of the quality in research and in the pharmaceutical industry [4]. However, quality standards are challenging to establish, because the complexity of potential active ingredients in medicinal herbs is used in TCM. Today, commonly applied models for TCM quality control include conventional methods, such as microscopic, and macroscopic identification and comparison with monographs, chemical fingerprint analysis using thin-layer liquid chromatography (TLC), liquid chromatography (LC), HPLC, gas chromatography (GC), GC-MS, etc., and multiple marker assays.

Chemical fingerprints and bioactivities assay linked with multiple markers is currently recognised to be effective for TCM analysis [88]. The use of known and previously characterised markers for the analysis of constituents is the most popular method for identification and quality control in TCM. For example, the ginsenoside fingerprint profiles are applied for the authentication of Panax species [89]. Moreover, the fingerprint-efficacy relationship of Lycii fructus (Goji 枸杞) was studied by Zhang in 2018. The spectrum of ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) of three batches of Goji was correlated with the biological data using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays. Compounds relating to the antioxidant activity of Goji, such

*Plant Extracts*

**2.7 Formulations of TCM**

*Composition of herbs and roles in 'Ma-huang-tang' formula.*

modern decoction methods.

*2.7.2 Modern granular formulations*

Decoction is the earliest and most commonly used preparation method in the clinical application of TCM [81]. A TCM decoction is the herbal tea that is made of processed medicinal herbs which are simmered for hours [82]. The preparation of a traditional medicine decoction has multiple steps such as the sequence of boiling the herbal material and different boiling and filtering times. The preparation of a decoction is complex, as it requires experience and often takes considerable time. The herbal ingredients within the formula for decoction can be changed for the individual patient according to the disease. The herbal medicine may include leaves, flowers, roots, bark or fruits, and the procedure of preparation is depending on the part of the herb that is used. It is of concern that in some instances, the quality and stability of herbal material cannot be assured [14]. Also, the unpleasant flavour of a decoction has been a problem for patients for thousands of years. As equipment advances and market demand increases, modern decoctions have become very popular in TCM hospitals, because of convenience [83]. Decoctions have a quick and often complete absorption with good bioavailability [81]. In a modernised decoction method, the medicinal herbs are combined with water and are extracted in TCM decoction machines [83]. Modern formulations also include granules, oral liquids, tablets, capsules and injections. This change in methodology may in fact influence the composition of the final TCM, and this is another area that would benefit from comparative research studies directed at classical and

The use of granules has dramatically increased in China since they are easy to handle, store and formulate. The granule made of a single herb is called a single dispensing granule; when made of multiple herbs, it is called a dispensing formula granule [84]. The preparation of granules is included in the extensive process of

*2.7.1 Decoctions*

**Figure 13.**

**28**

as chlorogenic acid, quercetin and kaempferol, were subsequently identified by analysis of UPLC-MS/MS data [90].

Because of TCM's complexity, it is rare that any single ingredient provides the overall evidence for medical efficacy. Chemometric methods, such as similarity evaluation or principal component analysis, are often used to investigate the fingerprint and bioactivity relationship within the TCM [91]. GC-MS is usually applied for the analysis of volatile metabolites, whereas LC-MS is often used to identify and quantify non-volatile components. Hyphenated methods, such as LC-MS can provide information on fragments of constituents, which can help in the elucidation and identification of chemical structures. In recent years, LC-MS analysis has been widely used in TCM due to its high sensitivity, selectivity and generation of specific information [92]. NMR metabolomic profiling is currently recognised as a quick and generic method in the study and quality control of TCM material. For instance, it was reported that the quality control of *Radix Angelica Sinensis* was studied by NMR profiling [93], which resulted in observed differences between samples prepared by different methods.

To ensure the consistency of herbal medicine, the concept of phyto-equivalence was developed. Phyto-equivalence is a comparison of the chromatographic fingerprint of a herbal medicine with the profile of a standard reference product. The fingerprint is defined as a characteristic profile, which reflects the overall complex chemical composition of the sample analysed by chromatographic or electrophoretic techniques. Chromatographic fingerprints are accepted by the US Food and Drug Administration (FDA) in applications for new product approvals [94]. In TCM studies, fingerprints could provide a complete set of information of chemical components including the relative quantity of all detectable analytes [95]. Fingerprinting is widely used to authenticate or differentiate between species, geographical regions or processing methods in TCM.

The 'fingerprint-efficacy relationship' is a method that associates TCM fingerprints with a specific pharmacological effect [96]. Multiple methods relate chemical fingerprints, such as characteristic peaks, to bioactivities. The 'fingerprint-efficacy relationship' investigation procedure (**Figure 14**) involves finding the appropriate analytical methods for fingerprints, identifying assays for the various bioactivities, using statistical methods to find the fingerprint-efficacy relationship, select candidate components, and validation of the bioactivities of the identified candidate

**31**

**Acknowledgements**

**3. Conclusion**

*Traditional Chinese Medicine: From Aqueous Extracts to Therapeutic Formulae*

components [88]. For instance, *Valeriana jatamansi* Jones (Zhi zhu xiangI 蜘蛛香) has a long history of use in TCM to treat mood disorders like anxiety. The 'fingerprint-efficacy relationship' was studied by correlating HPLC fingerprints with *in vitro* and *in vivo* tests. Four chemical components, hesperidin, isochlorogenic acid A, isochlorogenic B and isochlorogenic C are regarded as multiple biological mark-

Principal component analysis (PCA) is a statistical method, which can reduce the number of variables and dimensionality and create principal components (PCs) to explain the variables in original data. PCA is usually combined with cluster analysis, correlation analysis or regression analysis to determine the relationship of fingerprints and efficacy. It extracts data and can remove redundant information to focus on the main factors. PCA is often used together with other chemometric methods due to the lack of a specific quantification mathematical model for variables [98]. For example, *Andrographis herba* (Chuan xin lian 穿心莲) is well-known in China because of its bitter taste, which is one of the five flavours in TCM theory. The bitter flavour is recognised as relating to the pharmacological effects of *Andrographis herba*. In Zhang's group, the chemical components of 30 different types of *Andrographis herba* and fingerprint spectrum were determined by HPLC, and the PCA was applied to analyse the chemical components relating to bitter taste relating. According to the results from PCA, andrographolide, neoandrographolide, 1,4-deoxyandrographolide and dehyandrographlide were determined as substances responsible for the bitter flavour of Chuan xin lian [99]. *Ephedra sinica* Stapf (Ma huang 麻黄) is commonly applied for rheumatism, asthma, fever and rheumatoid arthritis in China. The significance of the inorganic elements of Ma huang from different geographical regions was studied by using elemental plasma mass spectroscopy fingerprints and PCA, and the study showed that this is an effective strategy to discriminate TCM samples [100].

The therapeutic formulae used for health maintenance and disease treatment in TCM are often complex mixtures whose chemical fingerprints are influenced by many factors, by the ancient practices of Daodi cultivation, Paozhi processing and Fangji combinations. As TCM undergoes modernisation and seeks increased entry to international markets which present rigorous legislative barriers, the quality,

In addition, there are increasing demands on supply, and attention needs to be focused on sustainable production, including mass cultivation and appropriately structured wild harvesting. It is obvious from ongoing research that the ancient processes, which vary across the land mass of China, influence the chemical fingerprint of the resulting therapeutic formulae, and thus render TCMs with the same

Going forward, it is important for the scientific community to continue to apply sophisticated methodologies and chemometric analyses toward understanding how the ancient TCM processes impact on the final product and how sustainable practices can be implemented that will lead to standardised therapeutic formulae which

The authors acknowledge The Chinese Scholarship Council and Trinity College

safety and efficacy are under increasing scrutiny.

name, from different regions, to have different potency.

reach the international standards of quality, safety and efficacy.

Dublin for the co-funding of Jinfan Wang's postgraduate research.

*DOI: http://dx.doi.org/10.5772/intechopen.85733*

ers for this anti-anxiety effect [97].

**Figure 14.**

*The method of 'fingerprint-efficacy relationship' in TCM [88].*

*Traditional Chinese Medicine: From Aqueous Extracts to Therapeutic Formulae DOI: http://dx.doi.org/10.5772/intechopen.85733*

components [88]. For instance, *Valeriana jatamansi* Jones (Zhi zhu xiangI 蜘蛛香) has a long history of use in TCM to treat mood disorders like anxiety. The 'fingerprint-efficacy relationship' was studied by correlating HPLC fingerprints with *in vitro* and *in vivo* tests. Four chemical components, hesperidin, isochlorogenic acid A, isochlorogenic B and isochlorogenic C are regarded as multiple biological markers for this anti-anxiety effect [97].

Principal component analysis (PCA) is a statistical method, which can reduce the number of variables and dimensionality and create principal components (PCs) to explain the variables in original data. PCA is usually combined with cluster analysis, correlation analysis or regression analysis to determine the relationship of fingerprints and efficacy. It extracts data and can remove redundant information to focus on the main factors. PCA is often used together with other chemometric methods due to the lack of a specific quantification mathematical model for variables [98]. For example, *Andrographis herba* (Chuan xin lian 穿心莲) is well-known in China because of its bitter taste, which is one of the five flavours in TCM theory. The bitter flavour is recognised as relating to the pharmacological effects of *Andrographis herba*. In Zhang's group, the chemical components of 30 different types of *Andrographis herba* and fingerprint spectrum were determined by HPLC, and the PCA was applied to analyse the chemical components relating to bitter taste relating. According to the results from PCA, andrographolide, neoandrographolide, 1,4-deoxyandrographolide and dehyandrographlide were determined as substances responsible for the bitter flavour of Chuan xin lian [99]. *Ephedra sinica* Stapf (Ma huang 麻黄) is commonly applied for rheumatism, asthma, fever and rheumatoid arthritis in China. The significance of the inorganic elements of Ma huang from different geographical regions was studied by using elemental plasma mass spectroscopy fingerprints and PCA, and the study showed that this is an effective strategy to discriminate TCM samples [100].

### **3. Conclusion**

*Plant Extracts*

analysis of UPLC-MS/MS data [90].

as chlorogenic acid, quercetin and kaempferol, were subsequently identified by

Because of TCM's complexity, it is rare that any single ingredient provides the overall evidence for medical efficacy. Chemometric methods, such as similarity evaluation or principal component analysis, are often used to investigate the fingerprint and bioactivity relationship within the TCM [91]. GC-MS is usually applied for the analysis of volatile metabolites, whereas LC-MS is often used to identify and quantify non-volatile components. Hyphenated methods, such as LC-MS can provide information on fragments of constituents, which can help in the elucidation and identification of chemical structures. In recent years, LC-MS analysis has been widely used in TCM due to its high sensitivity, selectivity and generation of specific information [92]. NMR metabolomic profiling is currently recognised as a quick and generic method in the study and quality control of TCM material. For instance, it was reported that the quality control of *Radix Angelica Sinensis* was studied by NMR profiling [93], which resulted in observed differences between samples prepared by different methods.

To ensure the consistency of herbal medicine, the concept of phyto-equivalence

The 'fingerprint-efficacy relationship' is a method that associates TCM fingerprints with a specific pharmacological effect [96]. Multiple methods relate chemical fingerprints, such as characteristic peaks, to bioactivities. The 'fingerprint-efficacy relationship' investigation procedure (**Figure 14**) involves finding the appropriate analytical methods for fingerprints, identifying assays for the various bioactivities, using statistical methods to find the fingerprint-efficacy relationship, select candidate components, and validation of the bioactivities of the identified candidate

was developed. Phyto-equivalence is a comparison of the chromatographic fingerprint of a herbal medicine with the profile of a standard reference product. The fingerprint is defined as a characteristic profile, which reflects the overall complex chemical composition of the sample analysed by chromatographic or electrophoretic techniques. Chromatographic fingerprints are accepted by the US Food and Drug Administration (FDA) in applications for new product approvals [94]. In TCM studies, fingerprints could provide a complete set of information of chemical components including the relative quantity of all detectable analytes [95]. Fingerprinting is widely used to authenticate or differentiate between species,

**30**

**Figure 14.**

*The method of 'fingerprint-efficacy relationship' in TCM [88].*

geographical regions or processing methods in TCM.

The therapeutic formulae used for health maintenance and disease treatment in TCM are often complex mixtures whose chemical fingerprints are influenced by many factors, by the ancient practices of Daodi cultivation, Paozhi processing and Fangji combinations. As TCM undergoes modernisation and seeks increased entry to international markets which present rigorous legislative barriers, the quality, safety and efficacy are under increasing scrutiny.

In addition, there are increasing demands on supply, and attention needs to be focused on sustainable production, including mass cultivation and appropriately structured wild harvesting. It is obvious from ongoing research that the ancient processes, which vary across the land mass of China, influence the chemical fingerprint of the resulting therapeutic formulae, and thus render TCMs with the same name, from different regions, to have different potency.

Going forward, it is important for the scientific community to continue to apply sophisticated methodologies and chemometric analyses toward understanding how the ancient TCM processes impact on the final product and how sustainable practices can be implemented that will lead to standardised therapeutic formulae which reach the international standards of quality, safety and efficacy.

### **Acknowledgements**

The authors acknowledge The Chinese Scholarship Council and Trinity College Dublin for the co-funding of Jinfan Wang's postgraduate research.

*Plant Extracts*
