0 RESEARCH ON TRADITIONAL HERBS SHOULD REFER TO THEORIES AND CLINICAL APPLICATION OF TRADITIONAL MEDICINE

1.4 RESEARCH ON TRADITIONAL HERBS SHOULD REFER TO THEORIES AND CLINICAL APPLICATION OF TRADITIONAL MEDICINE

Many traditional herbs are clinically prescribed by practitioners of traditional medicine under the guidance of theories in traditional medicine, such as TCM in China and Ayurveda in India. This aspect has mostly been ignored by scientists in the field of modern research of herbal medicine for product development, particularly in Western countries. Even in Asia, chemists, biologists, and pharmacologists who have been studying herbal medicine with modern knowledge and technology in labs for many years rarely know enough about theories that guide the applications of herbal treatments in the clinic. One of the reasons is that such study is more challenging.

Traditional herbs might treat a disease in a way different from known modern drugs. Take TCM as an example. A disease can be divided into several “zhengs” based on TCM differentiation. “Zheng” is a Chinese word that is similar in meaning to English symptoms or signs. For example, there is “cold zheng,” “hot zheng,” “internal zheng,” “external zheng,” “excessive zheng,” “deficient zheng,” “yin zheng,” “yang zheng,” “damp zheng,” and “bi zheng” (bi means blocked). Different herbs may be used on different patients with same disease but different zhengs. Sometimes, no animal models can be found to match these zhengs for PD study of herbs that are clinically used for treatment of certain types of zhengs. Thus, a new model with a particular zheng has to be established first. To do this, scientists have to be knowledgeable in both traditional and modern medicines. Otherwise, the study results are not reliable. Up to now, Chinese scientists have found out the biological and pathological foundation for most of the zhengs in TCM and established many animal models for pharmacological study of herbal medicine.9

Theories of traditional medicines, such as TCM, cover etiology, pathology, diagnosis, and treatment. Study of these theories can not only help us to explore the mechanisms of herbal treatment, but also help scientists explore possible new etiology and pathology for diseases whose causes are still unknown in modern medicine, thus providing new directions for drug development. For such purposes, a variety of in vitro bioassays on different receptors, enzymes, and other targets and in vivo animal pharmacological tests should be performed on herbs—not only individual ones, but also herbal formulas.

For example, clinical practice has confirmed that Gui Zhi Fu Ling Wan (Cinnamomi and poria composition), a Chinese herbal formula composed of five Chinese herbs, is very effective in decreasing or eliminating uterine fibroids when their diameter is less than 5cm. This has been confirmed by comparing ultrasound exam results before and after the treatment in the clinic and by pharmacological study on rats. The uterine fibroids are usually removed by surgery in modern medicine if they cause severe abnormal bleeding or if they are too big. Quite often, the uterus will be removed together with the fibroids in order to prevent the regrowth of fibroids in the uterus at a later date. The Chinese formula can not only stop abnormal bleeding and decrease and eliminate the fibroid, but also prevent the regrowth of the fibroid because it regulates the imbalance of the hormones, the cause of fibroid growth. Female hormones, particularly estrogen and progesterone, are known to be related to stimulation of fibroids. TCM considers fibroid formation to be related to accumulation of stagnated blood (called “yu zheng”). Therefore, herbs that invigorate blood circulation are added to the formula. Combining the knowledge about formation of uterine fibroids in modern medicine and TCM, the mechanism of herbal treatment can be explained by chemical, biological, and pharmacological study. To study the treatment mechanism of the formula, not only in vitro assays and in vivo animal tests related to hormone regulation should be performed; those involved in blood circulation should also be carried out.

Research on traditional herbal medicine should be performed on the basis of clinical application and reference to the corresponding theories in each system. The main systems of traditional medicine from different countries will be briefly introduced in Section 1.5. TCM is mentioned below only as an example.

The application of traditional Chinese herbs is not as simple as Western drugs in that not all doctors prescribe the same medicines for the same disease. Quite often in TCM, one herbal formula consisting of several Chinese herbs (most often 5 to 15) is used for different diseases. On the other hand, one disease can be treated with different formulas by different doctors. For example, if an herb is unavailable, experienced Chinese doctors can easily modify a formula by replacing one or two herbs to give similar treatment results. This makes research scientists perplexed and frustrated because explanations by clinical doctors using terminology of TCM are sometimes difficult to understand. Due to the current meticulous division of research areas and a limited amount of energy, most scientists focus on indepth study in one field, and have no time to spend on other areas that are not closely related to their research. Even to those familiar with both TCM and modern science, if the knowledge on both sides is not extensive, it is still difficult for them to scientifically explain TCM theories with simple modern medicinal terms.

Many patients turn to TCM treatment after they have tried treatment with Western medicines with no effect. Chinese herbal formulas work better than Western drugs for many diseases, not only chronic ones caused by stress, but also on acute infections such as SARS and the H1N1 flu virus. However, research results show that effects of the components isolated from these herbs are mostly less than those of current Western drugs. Thus the question arises: Why or how are the effects of these formulas better?

According to the experimental results, the answer is definitely not the placebo

effect. The following might explain the reason.

1.    Chinese herbs in a formula can work on different targets, that is, on different receptors and enzymes or other substances in the human body and stimulate the functions of nervous, circulatory, endocrine, immune, digestive, and other systems simultaneously. This is why TCM is a holistic medical system.

2.    TCM emphasizes the protection of the digestive function as well as regulation of qi (pronounced “chee”) and blood (details about the definition and explanation of qi and the importance of regulation of qi and blood in TCM will be given in Chapter 10). TCM believes that a good digestive system will guarantee an effective supply of essential nutrients from foods to the human body. It also believes that blocked qi and blood circulation may cause hundreds of types of diseases. For treatment of chronic diseases with Chinese herbs, there are always herbs that improve blood circulation in the formulas; if the patient has a digestion problem together with other symptoms, herbs that regulate the digestive system are usually given first. These actually emphasize the importance of maintaining cell functions with enough nutrients and excluding metabolites in a timely manner through functional blood circulation.

Scientists are currently trying to find out the relationship of mutant genes as causes of diseases, such as Alzheimer’s and Parkinson’s. But what are the main causes of the gene mutations? According to TCM, I would propose that the main cause of such diseases or aging is probably poor capillary blood circulation, which can be caused not only by the fats we eat, but also by the accumulation of metabolites from cells or dead cells. My reasoning is based not only on the above TCM theories and my clinical application of Chinese herbs, but also on the confirmation that the disease of agerelated macular degeneration (AMD) is pathologically related to the accumulation of aging retina in the photoreceptor outer segment membrane,18 part of a research program I performed when I worked as a postdoctoral scientist in the group of Professor Koji Nakanishi from Columbia University. No doubt, further experimentation is required.

Soure: Traditional Herbal Medicine Research Methods, Edited by Willow J.H. Liu Copyright © 2011 John Wiley & Sons, Inc.

REFERENCES

9. Chen, Q. (2006) Pharmacological Research Methodology of Chinese Medicine (2nd ed.), Beijing, People’s Health Publishing House.

10. State Administration of Traditional Chinese Medicine (1999) Zhong Hua Ben Cao. Shanghai, Shanghai Science and Technology Publisher.

11. Arlt, V.M., et al. (2002) Aristolochic acid as a probable human cancer hazard in herbal remedies: a review. Mutagenesis 17(4):265–277.

12. Debelle, F.D., et al. (2008) Aristolochic acid nephropathy: a worldwide problem. Kidney International 74(2):158–169.

13. Schmeiser, H.H., et al. (2009) Chemical and molecular basis of the carcinogenicity of Aristolochia plants. Current Opinion in Drug Discovery & Development 12(1):141–148.

14. Woelkart, K., et al. (2008) Pharmacokinetics of the main alkamides after administration of three different Echinacea purpurea preparations in humans. Planta Medica 74(6):651–656.

15. Bhattaram, V.A., et al. (2002) Pharmacokinetics and bioavailability of herbal medicinal products. Phytomedicine Suppl. 3:1–33.

16. Zhang, L., et al. (2005) Advances in clinical pharmacokinetics of herbal medicines. Journal of US–China Medical Science 2(6):59–72.

17. Clement, Y.N. (2009) Factors affecting the pharmacokinetics of herbal preparations and their impact on the outcome of clinical trials. Focus Alternative Complementary Therapies 14(2):87–91.

18. Liu, J.H., et al. (2000) The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane. Journal of Biological Chemistry 275(38):29354–29360

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0 COMMON MISTAKES SEEN IN RESEARCH ON TRADITIONAL HERBAL MEDICINES

1.3 COMMON MISTAKES SEEN IN RESEARCH ON TRADITIONAL HERBAL MEDICINES

Before starting research on herbal medicines, researchers should carefully search for literature that is related to the study. After reviewing the literature, they should develop a research plan by writing a detailed procedure design. The following common mistakes should be avoided.
1. Starting preparations of samples without identification of herbal materials.

For many reasons, substituted or adulterated herbal medicines are often seen in the markets. Sometimes they are not easily distinguished from the right material with the naked eye.

2. Starting biological or pharmacological experiments without chemical identification and standardization of samples.

I recall that one day an American friend showed me a bag containing an herbal product. The label on the bag said: No chemicals, all natural. This can lead to a popular misconception among consumers. But as scientists, we should know that chemicals are the fundamental substances of biological activities of herbal medicines, and nature is made up of chemicals. Therefore, chemical identification and standardization must be the primary step in the experiment of modern herbal study. Otherwise, the results are not reliable or accepted.

3. Using the wrong extraction method or solvent, such that the bioactive compounds are not extracted.

Make sure the extraction method will extract the corresponding bioactive compounds. For example, if an extract is for a steroid receptor binding assay, the potential ligands will probably be lipophilic, thus a less polar solvent such as chloroform may be selected. If an extract is for an antivirus experiment, the possible bioactive compounds may be large molecular glycoproteins or polyssacharides; lipophilic solvents or alcohol will not extract them out. The best way is to extract the material with different polar solvents in succession and test them separately in the primary test.

4. Using a dosage for the bioassay or animal test that is too low.

Since the efficacies of bioactive compounds in herbs are relatively weaker than the positive modern drug in most cases, and the concentrations of bioactive compounds are very low in the extract, the negative result of a sample in an assay or animal test may become positive if the concentration of sample is increased. Several dosages at different magnitudes are suggested to prepare for the primary test. Sometimes, the concentration of an herbal extract might be 1000 times higher than that of the positive control. For example, when the estrogenic activity was evaluated for red clover, methanol extract of red clover did not show positive results in the estrogen receptor binding assay until its concentration was increased to 20μg/mL.

5. Having a test period in an animal study or clinical trial that is not of sufficient length.

Because the effects of bioactive compounds in herbs are relatively moderate in comparison with the positive modern drug, it usually takes a longer time to see the positive result of an herbal extract in animal tests. For example, an estrogenic test for synthetic drug candidates on ovariectomized rats may only need a week, but positive results of a red clover extract were not observed until the third week of the experiment.
6. Using samples that vary in composition, leading to unrepeatable results.

Ideally, the same batch of herbal sample solution should be used for the same assay or test. If not, chemical analysis should be performed for different batches of samples by HPLC to avoid variable results caused by inconsistent quality or quantity of compounds in samples.

Soure: Traditional Herbal Medicine Research Methods, Edited by Willow J.H. Liu Copyright © 2011 John Wiley & Sons, Inc.

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0 Clinical Studies of Herbal Medicines

1.2.5 Clinical Studies of Herbal Medicines

Anything that exists on the earth has a need for survival. Many traditional herbs have been used on human beings to prevent and treat diseases for hundreds or even thousands of years. The fact should be acknowledged that most of the herbs have been used by countless people. Take Chinese herbal medicine as an example. The efficacies, toxicities, therapeutic and toxic dosages, as well as cautions and contraindications of most herbs have been well recorded in many traditional Chinese herbal books. Although the terminologies used for diagnosis and treatment of diseases in traditional and modern medicines are different, researchers are encouraged to figure out the symptoms described in traditional terminologies for the application of traditional medicine and try to match them to that of modern diseases for scientific clinical trial.

A successful clinical trial depends on accurate scientific design. In comparison to the trial for a single chemical drug, that for an herbal product is more complicated due to the complex composition and difficult quality control of the components. The extract method, the concentrations of the main or bioactive compounds in the products (or the purity of the products), the number and criteria of patients selected, the route and dosage of the administration, the period of the trial, and the method to collect and process the data will all influence the results of the trial.

Unfortunately, many of the reported results of clinical studies on herbal medicine so far are not reliable due to more or less unscientific design. Quite often, the results of clinical trial for one herbal medicine obtained by different research groups vary significantly. A well-known example is St. John’s Wort. Some reported this herb to have an effect on mild depression; others reported no such effect. Possible reasons have been mentioned in the above paragraph. A difference in any step of the experimental design will affect the result.

To obtain reliable clinical trial results for herbal medicines, double-blind experiments should be applied with enough patients selected, ideally using the standard of clinical trial for new drug development. Of course, budgetary constraints are often a hindrance to carrying out such trials.

Soure: Traditional Herbal Medicine Research Methods, Edited by Willow J.H. Liu Copyright © 2011 John Wiley & Sons, Inc.

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0 Chemical Standardization and Quality Control of Herbal Medicines

1.2.4 Chemical Standardization and Quality Control of Herbal Medicines

Substitute or counterfeit herbal materials are often found in the market. Even for the right species, the chemical composition and concentrations of bioactive compounds may vary dramatically with different collection seasons and regions as well as storage. Therefore, it is necessary to chemically standardize the herbal extracts or products for biological, pharmacological, and clinical studies.

The complex composition of herbal medicines makes the quality control of herbal products much more complicated. With the increase in knowledge about the bioactive and main compounds in most of the commonly used herbs and the popular application of various analytical instruments such as HPLC, equipped with UV, MS, and other detectors, fingerprint chromatograms are becoming powerful qualitative and quantitative methods for standardization of herbal medicines. Such standardization is not only necessary for quality control of final herbal products, but also important to guide the species collection and cultivation, as well as the optimization of the processing procedure.

Soure: Traditional Herbal Medicine Research Methods, Edited by Willow J.H. Liu Copyright © 2011 John Wiley & Sons, Inc.

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0 Pharmacological and Toxicological Study of Herbal Medicines

1.2.3 Pharmacological and Toxicological Study of Herbal Medicines

Similar to modern pharmaceutical study, pharmacological study of herbal medicines include pharmacodynamic (PD) and pharmacokinetic (PK) aspects. Broadly, toxicology is also part of the pharmacology.

PD study of traditional herbal medicines is not always easy. Up to now, only the most popularly used herbs, a very small fraction of the total number used, have been well known with respect to pharmacological effects on animals. One reason is that herbs might treat diseases in a way different from known modern drugs. Black cohosh is one example. This herb has long been used in North America for menopause symptoms in women, but in vivo animal study indicated that its extract did not exhibit effects in ovariectomized Sprague–Dawley rats. Further study showed that instead of directly binding to estrogen receptors, extract of black cohosh was reported acting as a mixed competitive ligand and partial agonist of the serotonin and opiate receptor,6,7 which indicates that this herb might treat menopause symp- toms through regulation of the central nervous system.

Chinese scientists have done numerous pharmacological studies on Chinese herbs. Therapeutic mechanisms of the most commonly used Chinese herbs have been known by systematic PD studies.8–10 However, there is another challenge in the pharmacological study of Chinese herbs; that is, in the vast majority of cases, the practitioners prescribe formulas that consist of several (sometimes over 20) herbal ingredients for the treatment. This makes the study difficult not only due to the complex analysis of chemical composition for quality control of the test samples, which is important to keep good reproducibility of the results, but also because of the complex theories of TCM behind the combination of different herbs, which will be mentioned in Chapter 10.

Many people mistakenly believe that herbal products are safe. Although most herbal medicines are relatively safe in comparison with modern drugs, results from toxicological studies show that this is not always true. To a large extent, the safety of herbs depends on dosage and period of administration. It is necessary to mention that purification of some herbal extracts may increase their toxicity. This is because, while the active components are concentrated, the concentration of toxic compounds may also be increased. Sometimes, the active components are toxic. In this case, while the therapeutic effect is enhanced, the toxicity is also increased. Examples include ephedra extract and herbal extracts from the Aristolochia family. Studies of aristolochic acid found in several herbs in Aristolochia family have shown its significant carcinogenic and mutagenic effects and poisoning of the kidney.11–13 In TCM, pro- cessing of raw herbal materials with different methods, such as extended heating with steaming or boiling to decompose the chemical bonds of toxic ester or glycoside compounds in herbs, has been long applied to reduce the toxicity of Chinese herbs. Examples include aconitine in radix Aconiti and sennosides in rhubarb.

PK study of herbal medicines is so far mainly applied to herbs with known active compounds. The concentrations of these active index compounds in the blood, urine, and other body liquids or tissues after a certain period of administration are measured and compared by means of UV, MS, GC-MS, HPLC-MS, and other analytical methods to analyze the distribution of the compounds and change of concentrations with time. To herbs with unclear composition or whose concentration could not be monitored with analytical methods, their efficacies are measured and time-efficacy curves are drawn. In addition, PK–PD models are also applied to the study of herbal PK.

This book covers the PD and toxicology studies of herbal medicines, but not the PK. The reason is that the methods of sample collection for PK study of herbal medi- cine are the same as those for modern drugs. The analytical methods for absorbed and metabolized known compounds in herbs can refer to the qualitative and quantitative analysis of herbal medicines in Chapter 9. Keep in mind that the complex chemical composition of herbal preparations always makes the analysis relatively difficult.

In comparison with so many PD study reports of herbal medicines, only a few systematic PK studies for herbal preparations have been reported; one example is the PK of alkamides in Echinacea purpurea.14 Progress of the PK study is covered in recent review articles.15–17

Soure: Traditional Herbal Medicine Research Methods, Edited by Willow J.H. Liu Copyright © 2011 John Wiley & Sons, Inc.

REFERENCES

6. Burdette, J.E., et al. (2003) Black cohosh acts as a mixed competitive ligand and partial agonist of the serotonin receptor. Journal of Agricultural and Food Chemistry 51(19):5661–5670.

7. Rhyu, M.R., et al. (2006) Black cohosh (Actaea racemosa, Cimicifuga racemosa) behaves as a mixed competitive ligand and partial agonist at the human mu opiate receptor. Journal of Agricultural and Food Chemistry 54(26):9852–9857.

8. Shen, Y.J. and Chen, C.X. (2008) Traditional Chinese Pharmacology (5th ed.), Shanghai, Shanghai Science and Technology Publisher.

9. Chen, Q. (2006) Pharmacological Research Methodology of Chinese Medicine (2nd ed.), Beijing, People’s Health Publishing House.

10. State Administration of Traditional Chinese Medicine (1999) Zhong Hua Ben Cao. Shanghai, Shanghai Science and Technology Publisher.

11. Arlt, V.M., et al. (2002) Aristolochic acid as a probable human cancer hazard in herbal remedies: a review. Mutagenesis 17(4):265–277.

12. Debelle, F.D., et al. (2008) Aristolochic acid nephropathy: a worldwide problem. Kidney International 74(2):158–169.

13. Schmeiser, H.H., et al. (2009) Chemical and molecular basis of the carcinogenicity of Aristolochia plants. Current Opinion in Drug Discovery & Development 12(1):141–148.

14. Woelkart, K., et al. (2008) Pharmacokinetics of the main alkamides after administration of three different Echinacea purpurea preparations in humans. Planta Medica 74(6):651–656.

15. Bhattaram, V.A., et al. (2002) Pharmacokinetics and bioavailability of herbal medicinal products. Phytomedicine Suppl. 3:1–33.

16. Zhang, L., et al. (2005) Advances in clinical pharmacokinetics of herbal medicines. Journal of US–China Medical Science 2(6):59–72.

17. Clement, Y.N. (2009) Factors affecting the pharmacokinetics of herbal preparations and their impact on the outcome of clinical trials. Focus Alternative Complementary Therapies 14(2):87–91.

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0 Bioassay Screening and Mechanism Study of Herbal Medicines

1.2.2 Bioassay Screening and Mechanism Study of Herbal Medicines

Scientists have spent over a hundred years trying to screen new drug candidates from herbal medicines. Recently, due to the rapid growth of products of herbal medicine or alternative medicine all over the world, their efficacy and safety have become more and more important. More attention has been drawn to the preventative and therapeutic mechanism study of herbal medicines. For both reasons, bioassay study on herbs is indispensible. Thanks to the advancement of biological technologies, more and more bioassays are available for mechanism study. The mechanism of many effective herbal medicines has been elucidated, such as the well-known ginkgo, Echinacea, red clover, black cohosh, ginseng, and many Chinese and other traditional herbs. Bioassays in vitro are usually followed by in vivo animal tests to further confirm the functional mechanism and understand the absorption, metabolism, and toxicity in living bodies.

Bioassay is commonly performed using enzymes, receptors, genes, cells, and sometimes tissues. In comparison to screening for new drug candidates of single compounds, screening herbal extracts or fractions is relatively difficult due to the solubility or complex composition in herbal samples. Compounds in an extract might interfere with each other, or more specifically, the activity of one compound might be masked by another in the mixture due to the adverse effect or toxicity of the latter. So, the bioassay result of an herbal extract should be carefully evaluated, particularly when a high-throughput method is applied, not only due to the mentioned interference, but also because of the dramatically varied concentrations of bioactive components in different samples prepared under the same conditions.

Mechanism study for herbal medicine does not necessarily use high-technology equipment. The most important thing is to select the right targets. Different enzymes, receptors, or genes should be tested for mechanism of an herbal extract. Assays at different levels should be applied to ensure the positive or negative research results. Evaluation of estrogenic activity of red clover and black cohosh extracts using different bioassays can be used as an example.2

In many cases, the corresponding bioactive components for the functional mechanism of herbal medicines are common or universally distributed compounds. Such results may disappoint researchers looking for new drug development, but they are very helpful to scientists who are dedicated to explaining the functions of herbs or willing to understand more about physiological functions of these common com- pounds in the human body. Examples include linolic acid, a cyclooxygenase (COX) inhibitor in Angelica pubescens3 and an estrogenic agonist in Vitex agnus-castus L. (chaste berry),4 and Nω-methylserotonin, a serotonin agonist in black cohosh.5

Soure: Traditional Herbal Medicine Research Methods, Edited by Willow J.H. Liu Copyright © 2011 John Wiley & Sons, Inc.

REFERENCES

2. Liu, J.H., et al. (2001) Evaluation of estrogenic activities of plant extracts on the potential treatment of menopausal symptoms. Journal of Agricultural and Food Chemistry 49(5):2472–2479.

3.  Liu, J.H., et al. (1998) Inhibitory effect of Angelica pubescens f. biserrata on 5-lipooxygenase and cyclooxygenase. Planta Medica 64(6):525–529.

4.  Liu, J.H., et al. (2004) Isolation of linoleic acid as an estrogenic compound from the fruits of Vitex agnus-castus L. (chaste berry). Phytochemistry 11(1):18–23.

5. Powell, S.L., et al. (2008) In vitro serotonergic activity of black cohosh and identification of Nω- methylserotonin as a potential active constituent. Journal of Agricultural and Food Chemistry 56(24):11718–11726.

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0 Extraction, Isolation, and Identification of Compounds in Herbal Medicines

1.2.1 Extraction, Isolation, and Identification of Compounds in Herbal Medicines

All the substances in the universe, including plants, are composed of chemical compounds. To study herbal medicine, the major bioactive chemical components should be first known. Only after the biological compounds in herbs are correctly extracted, isolated, and identified can biochemical, biological, or pharmacological studies be performed scientifically.

Chemical studies of herbal medicines provide fundamental substances for further studies of biological and pharmacological activity. During the earlier decades of the 1800s, chemical studies in plants could only be performed on active compounds that were highly concentrated and isolated into a relatively pure form by techniques such as distillation or extraction with water, acid, base, or alcohol. Their structures were mainly determined by chemical degradation and proven by synthesis in an unambiguous manner. Scientists were unable to determine the stereochemistry of compounds.

The well-known example is the story of aspirin. According to records about willow leaves as an antipyretic treatment in Ebers papyrus, and following the same application of teas made from willow bark as an English herb, chemists and phar- macists successfully isolated salicin from the bark of the white willow, Salix alba, between 1825 and 1826. The compound responsible for the remedy was subsequently converted to salicylic acid via hydrolysis and oxidation, and proved as such a successful antipyretic (fever reducer) that it was actively manufactured and used worldwide. Due to severe gastrointestinal toxicity, salicylic acid was converted into acetylsalicylic acid via acetylation by scientists at Bayer. It was given its trade name of aspirin in 1899. Today, aspirin is still the most widely used analgesic and anti- pyretic drug in the world.

Since the 1950s, chromatography, including medium-pressure liquid chroma- tography (MPLC) and high-performance liquid chromatography (HPLC), and other methods such as supercritical fluid extraction (SFE), droplet countercurrent (DCC), and high-speed countercurrent (HSCC) have been popularly applied for isolation of natural products, while different types of spectral equipment such as infrared (IR), ultraviolet (UV), nuclear magnetic resonance (NMR), circular dichroism (CD), and mass spectrometer (MS), as well as MS coupled with gas chromatography (GC), have been commonly used for structure identification. Later on, LC-MS and LC-NMR also became available and gradually more popular in the last few decades. These advances have made the time for extraction, isolation, and identification of compounds from herbal medicines much shorter than that of a century ago. Modern extraction and isolation techniques, combined with all types of chromatography, are often guided by bioassays to isolate the active compounds. High-throughput screening with robots also dramatically lowers the screening times. Thus, structure-efficacy elucidation of newly isolated bioactive compounds is no longer a time-consuming and difficult process.

However, the process of finding new drug candidates from herbs for drug development is no longer as easy as the story of aspirin. The story of taxol is that of a difficult journey of a trace compound from a plant becoming a powerful new drug. Taxol is one of the most well-known diterpenes with a very complex steroid structure and anticancer activity. The extract of the bark of Pacific yew (Taxus brevifolia) was first found to be cytotoxic in a cellular assay in 1964. The active ingredient was isolated in 1966 with a very low amount, and the structure was published in 1971. By 1969, 28kg of crude extract had been isolated from almost 1200 kg of bark, but yielded only 10 g of pure material. The research result showed that it acts to stabilize the mitotic apparatus in cells, causing them to act as normal cells rather than undergo rapid proliferation as they do in cancer. But it was not until the late 1980s that its value as an anticancer drug was confirmed.1

Current modern methods and techniques such as all kinds of chromatography and spectrometry, and their combined application make the extraction, isolation, and structure identification of bioactive compounds from herbs dramatically faster than half a century ago. Highly accurate analytical equipment, such as HPLC coupled with UV and/or MS and other detectors, makes the quality control and standardization of herbal products more reliable for pharmacological and clinical studies. Advanced biochemical and biological technologies, such as microarray, allow scientists to easily explore the mechanism study at the enzyme, receptor, and gene levels quantitatively using only small amounts of samples. These advanced technologies and their applications to herbal study will be introduced in the following chapters. With all these available high technologies, time for isolation and identification of compounds from herbs is becoming shorter and trace bioactive compounds are more easily obtained. With the popularity of various spectroscopy methods, identification of isolated compounds is becoming much easier than it was decades ago. Application of hyphenated LC-UV/MS and LC-NMR techniques greatly accelerates the systematic identification of compounds in an herbal extract.

To perform any herbal study, identification of the herbal materials used for study should never be neglected. Morphological, microscopic, physical, or chemical iden- tification can all be applied to identify the raw materials. The availability of HPLC chromatogram or gene fingerprints makes identification of species highly accurate.

Soure: Traditional Herbal Medicine Research Methods, Edited by Willow J.H. Liu Copyright © 2011 John Wiley & Sons, Inc.

REFERENCES

1. Goodman, J. and Walsh, V. (2001) The Story of Taxol: Nature and Politics in the Pursuit of An Anti-Cancer Drug. Cambridge, Cambridge University Press.

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0 RESEARCH AND DEVELOPMENT OF HERBAL MEDICINES

1.2 RESEARCH AND DEVELOPMENT OF HERBAL MEDICINES

The use of herbal medicines for treatment of diseases was documented several thousand years ago. As seen from journals, studies on herbal medicines have been encompassed under several different names, such as plant medicine, phytomedicine, pharmacognosy, and natural products. “Natural products” usually refer to products processed or derived from living organisms, including plants, animals, insects, microorganisms, and marine organisms.

Data from the WHO show that 25% of modern medicines are made from plants that were first used traditionally. Examples include atropine, morphine, quinine, ephedrine, warfarin, aspirin, digoxin, vincristine, taxol, and hyoscine.

Traditional medicine needs to be modernized in the twenty-first century. However, modernization of traditional medicine should not be simply Westernization. For herbal medicines, the purpose of a study is not only to screen out bioactive compounds from herbal extracts for new drug development, but also to standardize and control the quality of raw herbal materials and their products to ensure the safety and efficacy; and more importantly, to reveal their preventative and therapeutic mechanisms. So far, only a relatively small number of herbal medicines have been well studied from all of these aspects; these herbs include Echinacea, ginkgo, ginseng, and licorice.

To a large extent, the depth and progress of research on herbal medicines depend on the development of related technology and equipment, as well as the in-depth understanding of the human body and diseases. Mechanism study and functional evaluation of herbal medicine involve the fields of chemistry, biochemistry, biology, pharmacology, toxicology, and clinical study. Thus, organized and consistent team- work is absolutely vital.

Researchers from different labs need to work closely together, discuss problems frequently, and analyze the results instantly. A scientist for extraction and isolation of herbal medicines in the chemistry lab should have enough knowledge of biology and pharmacology to provide an appropriate sample because an improperly extracted or isolated sample provided from his or her lab for biological and pharmacological study could lead to wrong results in the bioassay or animal test. The scientist in the bioassay or animal lab for screening or mechanism study of herbal medicines should make sure that the sample to be tested is correctly extracted, that the concentrations of tested samples are within a proper range, and that the design of the experiment is scientific enough to provide a true result. And to reach such a goal, an adequate understanding of the research target, the functions and indications, as well as clinic applications of the study herb is necessary. The following are several main aspects of herbal medicine research.

Soure: Traditional Herbal Medicine Research Methods, Edited by Willow J.H. Liu Copyright © 2011 John Wiley & Sons, Inc.

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0 DEFINITION AND TRENDS OF TRADITIONAL HERBAL MEDICINES

1.1 DEFINITION AND TRENDS OF TRADITIONAL HERBAL MEDICINES

According to the World Health Organization (WHO), traditional medicine refers to health practices, approaches, knowledge, and beliefs incorporating plant, animal, and mineral-based medicines, spiritual therapies, manual techniques, and exercises, applied singularly or in combination to treat, diagnose, and prevent illnesses or to maintain well-being. If the material being used is of plant origin, then it is called traditional herbal medicine.

Different types of traditional medicines are widely applied in Asia, Africa, and Latin America to meet primary health-care needs. Traditional medicine has main-tained its popularity in most regions of the developing world. The application is also rapidly spreading in industrialized countries, where adaptations of traditional medicines are often termed “complementary” or “alternative.” In the United States, the National Institutes of Health (NIH) uses the name complementary and alternative medicine (CAM) to cover health systems, practices, and products that are not considered part of conventional medicine. Worldwide, among all the different traditional medicine systems, traditional Chinese medicine (TCM) is currently the most popular, followed by Indian medicine. In Western terminology, the name “Oriental medicine” covers Chinese, Japanese, and Korean medicines preferred by immigrants from Korea, while “Asian medicine” is often used to include TCM, Indian (Ayurveda), and Tibetan medicine. Among all treatment methods in traditional medicine systems, medicinal herbs are the most widely applied.

Medicine has been revolutionized in Europe by advances in chemistry, laboratory techniques, and equipment since Robert Koch discovered the transmis- sion of disease by bacteria, followed by the discovery of antibiotics in the early 1900s. Thus, modern medicine is commonly called Western medicine even though there are also traditional medicines in Western countries. It is also called conventional medicine.

Webster’s medical dictionary defines conventional medicine as medicine prac- ticed by holders of medical doctor (M.D.) or doctor of osteopathy (D.O.) degrees and by their allied health professionals, such as physical therapists, psychologists, and registered nurses. Other terms for Western medicine or conventional medicine include allopathy and allopathic medicine, mainstream medicine, orthodox medicine, regular medicine, and biomedicine.

Although conventional medicine is the mainstream medicine in Western countries, application of traditional medicine, including herbal medicines, is growing worldwide for many reasons, in particular, the side effects or inefficacy of modern drugs. The following data are provided by the WHO.

- In Africa, up to 80% of the population uses traditional medicine for primary health care.

- In China, traditional herbal preparations account for 30–50% of the total medicinal consumption.

- In Europe, North America, and other industrialized regions, over 50% of the population has used complementary or alternative medicine at least once.

- In Germany, 90% of the population has used a natural remedy at some point in their lives.

- The global market for herbal medicines currently stands at over USD$60 billion annually, and is growing steadily.

Since the last century, scientists all over the world have studied herbal medicines from the fields of chemistry, biology, pharmacology, toxicology, and clinical trials. Recently, in addition to screening out new drug candidates, investigators also expect to explore the preventative and therapeutic mechanism of herbal medicines that play very important roles in most of the traditional medicine systems, such as TCM and Ayurveda medicine.

Soure: Traditional Herbal Medicine Research Methods, Edited by Willow J.H. Liu Copyright © 2011 John Wiley & Sons, Inc.

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0 Flavonoids-Definition and Distribution in Plants

3.1.2 Flavonoids

Definition and Distribution in Plants

Flavonoids are polyphenolic compounds that are ubiquitous in nature. It is one of the most important groups of secondary plant metabolites. The chemical structure of flavonoids is based on a C15 skeleton with a chromane ring bearing a second aromatic ring B in position 2, 3, or 4 (see Fig. 3.5).

More than 6000 flavonoid compounds have been purified and identified, many of which occur in fruits, vegetables, and beverages. Flavonoids constitute one of the most characteristic classes of compounds in higher plants, and are responsible for many of the plant colors that dazzle us with their brilliant shades of yellow, orange, or red.

Biological Activities of Flavonoids

Flavonoids have attracted considerable interest recently because of their potential beneficial effects on human health. Flavonoids are most commonly known for their antioxidant activity. The capacity of flavonoids acting as antioxidants depends upon their molecular structures. The positions of hydroxyl groups and other features in the chemical structure of flavonoids are important for their antioxidant and free radical scavenging activities. Quercetin, the most abundant dietary flavonol, is a potent antioxidant because of its proper structural features for free radical scavenging activity. It has been discovered that flavonoids also provide other important biological activities such as antibacterial, antiviral, antiallergic, antiplatelet, antiinflammatory, and antitumor activities. The intake of certain subclasses of flavonoids is demonstrated to be associated with lower occurrence of coronary heart disease. The antiviral function of flavonoids has been demonstrated with the HIV virus, and also with HSV-1, a herpes simplex virus.

Properties of Flavonoids

Most flavonoids are crystalline solids, and only a few are amorphous powders. The colors of flavonoids are dependent on the conjugated system and the number and position of substituents of auxochromes. For example, the hydroxyl and methoxy at 7- or 4′- position will deepen the color of compounds due to their acceleration to the electron rearrangement. In general, flavone, flavanol, and their glycosides are grayish yellow or yellow solids, while flavanone and flavanonol have no color due to lack of conjugated system. But with the treatment of aqueous ammonia, they feature charac- teristic color or fluorescence and change color. The color of isoflavone is pale yellow due to short conjugated structure. The color of chalcone ranges from yellow to orange. There is a strong relationship between the color of anthocyanins and pH value. The color is red, purple, and blue at pH < 7, pH = 8.5, and pH > 8.5, respectively.

The solubilities of flavonoids in solvents depend on their existing forms. Aglycones of flavonoids are less soluble in water, but easily soluble in methanol, ethanol, trichloromethane, and other organic solvents. Flavonoid glycosides are easily dissolved in hot water, methanol, and other polar solvents, but they are solid in benzol, trichloromethane, and other organic solvents. The more sugars connected to the aglycone, the more soluble the glycoside is in the water. Most flavonoids containing phenolic hydroxyl groups are soluble in alkaline aqueous solution (such as sodium carbonate solution) and alkaline organic solvents (such as picolinamide and dimethylformamide).

Flavonoids are usually weakly acidic due to the presence of phenolic hydroxyl groups, and can dissolve in basic solutions. Because of the presence of phenolic hydroxyl groups and γ- pyrone, flavonoid compounds have the capacity to produce various colors when reacting with some reagents, as listed in Table 3.1.

Structures and Classification

Based on chemical structures, flavonoids are categorized into flavones, flavonols, flavanones, isoflavones, chalcones, catechins, anthocyanidins, xanthones, and aurones.

1. Flavones: refer to flavonoids that share structural features, which include the B ring substituted at C2, a double bond at C2-C3, and carbonyl group at C4 (see Fig. 3.6 A).

2. Flavonols: refer to flavonoids that share structural features with flavones including the B ring substituted at C2, a double bond at C2-C3, and a car- bonyl group at C4, but with an addition of a hydroxyl group substituted at C3 (see Fig. 3.6B).

3.  Flavanones: refer to flavonoids that share structural features with flavones including the B ring substituted at C2, and a carbonyl group at C4, but without a double bond at C2-C3 (see Fig. 3.6C).

4.    Flavanonols: refer to flavonoids that share structural features with flava- nones including the B ring substituted at C2 and a carbonyl group at C4, and without a double bond at C2-C3, but with an addition of a hydroxyl group substituted at C3 (see Fig. 3.6D).

5.    Isoflavones: refer to flavonoids that share structural features of flavones including a double bond at C2- C3 and a carbonyl group at C 4, but the B ring is attached at C3 (see Fig. 3.7 A).

6.    Isoflavanones: refer to flavonoids that share structural features of isoflavones including the B ring substituted at C3 and a carbonyl group at C4, but without a double bond at C2-C3 (see Fig. 3.7B).

7.    Chalcones: refer to flavonoids that share structural features of flavones including a double bond at C2- C3 and a carbonyl group at C 4, but the C ring is opening at position 1 (see Fig. 3.7C).

8.    Dihydrochalcones: refer to flavonoids that share structural features of chal- cones including the opening of the C ring at position 1 and a carbonyl group at C4, but without a double bond at C2-C3 (see Fig. 3.7D).

9.    Catechins: refer to flavonoids that share structural features of flavones including the B ring substituted at C2, hydroxyl groups substituted at C3 or/ and C4, but without a carbonyl group at C4 (see Fig. 3.8A).

10. Anthocyanidins: refer to flavonoids that have 2-phenylbenzopyrylium salts

structures (see Fig. 3.8B).

11. Xanthones: refer to flavonoids that have benzochromnone skeleton xantho- nes (see Fig. 3.8C).

12. Aurones: refer to flavonoids that have the 2-benzylidene coumaranone skeleton; its C ring is a five-member ring (see Fig. 3.8D).

Soure: Traditional Herbal Medicine Research Methods; Edited by Willow J.H. Liu; A John Wiley & Sons, Inc., Publication

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0 Alkaloids

3.1.1 Alkaloids

Definition and Distribution in Plants

Alkaloids are a large class of nitrogen-containing secondary metabolites of plants, microbes, or mammals. Alkaloids are famous for their manifold pharmacological activities. Since the discovery of morphine from the opium poppy, Papaver som- niferum, in 1806, more than 10,000 alkaloids have been purified and identified from natural resources. Many modern drugs are produced from naturally occurring alkaloids or their synthetic analogs.

Alkaloids are mainly distributed in higher plants such as the Taxus genus, the Pinus genus, the Picea genus, the Ketelearia genus, the Ephedra genus, the Cephalotaxus genus, the Ranunculaceae family, the Berberidaceae family, the Menispermaceae family, the Magnoliaceae family, and the Liliaceae family, but rarely found in lower plants.

Biological Activities of Alkaloids

Alkaloids are a group of substances possessing various remarkable biological activi- ties, such as antibacterial (e.g., berberine), anti-malaria (e.g., quinine), analgesia (e.g., morphine), anesthesia (e.g., cocaine), anticancer (e.g., vincrinstine), cardiant (e.g., dl-demethylcoclaurine), antihypertention (e.g., resepine), cholinomimeric action (e.g., galatamine), relieving cough (e.g., codeine), spasmolysis (e.g., atro- pine), vasodilatation (e.g., vincamine), anti-arhythmia (e.g., quinidine), and anti- asthma (e.g., ephedrine). For example, the morphine alkaloids are powerful pain relievers and narcotics, and vincristine, isolated from Vinca rosea (now Catharanthus roseus), is one of the most potent anti-leukemic drugs in use today.

Properties of Alkaloids

Alkaloids in plants exist as free states, as salts, or as N-oxides. The nitrogen com- pounds may exist as a primary amine, as a secondary amine, as a tertiary amine, or as a quaternary amine. They are generally white or yellowish solids with a few exceptions (i.e., nicotine is a brown liquid) and have bitter taste. They can produce a precipitate when reacted with heavy metal iodides. Most alkaloids are able to form cream-colored precipitate with Mayer’s reagent (potassiomercuric iodide solution). The reaction of Dragendorff’s reagent (solution of potassium bismuth iodide) with alkaloids gives an orange-colored precipitate, which is sensitive and can be used for the detection of alkaloids on thin layer chromatography (TLC).

Structures and Classification

Alkaloids are commonly subclassified according to their biosynthetic origin. Most alkaloids can be covered in the following subgroups.

1. Alkaloids derived from the nonprotein amino acid L-ornithine including pyrrolidine/tropane and pyrrolizidine alkaloids (see Fig. 3.1A).

2. Alkaloids derived from the amino acid L-lysine including piperidine, quino- lizidine, and indolizidine alkaloids (see Fig. 3.1B)

3. Alkaloids derived from nicotinic acid including pyridine alkaloids (see Fig. 3.1C).

4. Alkaloids derived from the amino acid L-phenylalanine and L-tyrosine including phenyethylamines, tetrahydroisoquinoline, benzyltetrahydroiso- quinoline, phenethylisoquinoline, and terpenoid tetrahydroisoquinoline alkaloids (see Fig. 3.2 A).

5. Alkaloids derived from the amino acid L-tryptophan including indole, ter- penoid indole, quinoline, pyrroloindole, and ergot alkaloids (see Fig. 3.2B).

6. Alkaloids derived from the origin of terpenoids (see Fig. 3.3A).

7. Alkaloids derived from the origin of steroids (see Fig. 3.3B).

8. Alkaloids derived from anthranilic acid (see Fig. 3.4A).

9. Alkaloids derived from histidine (see Fig. 3.4B).

10. Alkaloids derived from purine derivatives (see Fig. 3.4C).

 Soure: Traditional Herbal Medicine Research Methods; Edited by Willow J.H. Liu; A John Wiley & Sons, Inc., Publication

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0 Extraction and Isolation of Compounds from Herbal Medicines

Extraction and Isolation of Compounds from Herbal Medicines

Hong-Wei Liu

Plants, particularly medicinal herbs, constitute the foundation of traditional pharma- copeias, and have produced many currently important pharmaceutical drugs, for example, taxol from Taxus brevifolia, vinblastin and vincristine from Catharanthus roseus, and huperzine A from Huperzia serrata. There are at least 250,000 species of higher plants on earth, but only about 5–10% of them have been investigated so far. With the development of modern molecular biotechnology, there is an increasing demand for extraction and isolation of compounds from herbs for the purpose of screening bioactive chemical molecules for new drug development, exploring thera- peutic and preventative mechanism of herbs, as well as establishing quality control and standardization of herbs and herbal products.

The chemicals in plants can be divided into primary metabolites and secondary metabolites based on the range of molecular weight, distributions in species, and biological roles to plants. The ubiquitous macromolecules (mol wt > 2000 amu) of primary metabolism in plants, for example, polysaccharides, proteins, lipids, and nucleic acids, are called primary metabolites. They provide nutrients, and thus are essential for growth and survival. In contrast to primary metabolites, small organic compounds (mol wt < 2000 amu) are produced often by a particular species, usually possessing important biological activity. They are not necessary for growth and survival and are called secondary metabolites.

A number of secondary metabolites in plants serve as chemical messengers and defensive chemicals, and play significant biological and ecological roles. There is growing interest in the study of plant secondary metabolites as they represent a tremendous library of potentially useful leading compounds for new drug develop- ment. The secondary metabolites comprise of a range of chemically diverse compounds. Based on the chemical structure features, they are classified as alkaloids, flavonoids, coumarins, lignans, quinones, terpenoids, and so on.1–5

Isolation of chemical compounds from herbs is an important step for a systematic study of herbal medicine. It provides compounds not only for structural identification or elucidation and standards for quantitative and qualitative analysis for quality control of herbal extracts or products, but also for in vitro bioassay screen and in vivo pharmacological and toxicological study and clinical trials. Before isolation of an herbal medicine, the material should first be identified using methods introduced in Chapter 2, to ensure it is the right species and was collected and stored properly. Extraction and isolation should be carried out with a well-planned scheme based on knowledge of the sample. Different extraction and isolation methods are suitable for different types of compounds. Thus, it is important to do a complete literature search and know the characteristics of compounds in the sample before making the plan. This chapter will mainly introduce the characteristics of major types of secondary metabolites in plants and commonly used methods for extraction and isolation.

Soure: Traditional Herbal Medicine Research Methods; Edited by Willow J.H. Liu; A John Wiley & Sons, Inc., Publication

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