Traditional Herbal Medicine Research Methods: Identification, Analysis, Bioassay, and Pharmaceutical and Clinical Studies

Contributors

Jia-Tao Feng, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Zhi-jun Huang, The Third Xiangya Hospital in Central South University, Changsha, China

Yu Jin, East China University of Science and Technology, Shanghai, China

Yan-Xiong Ke, East China University of Science and Technology, Shanghai, China.

Chun-Li Li, Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China

Ping Li, Department of Pharmacognosy, China Pharmaceutical University, Nanjing, China.

Xin-Miao Liang, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Hong-Wei Liu, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

Hui-Juan Liu, Department of Pharmacognosy, China Pharmaceutical University, Nanjing, China

Willow J.H Liu, 21st Century Herbs & Health, Inc. CA, U.S.A.

Qi Luo, Changsha National Biomedical Industrial Base, Hunan Engineering Research Center of Botanical Extract, Liuyang, Hunan, China

Xuan Peng, Changsha National Biomedical Industrial Base, Hunan Engineering Research Center of Botanical Extract, Liuyang, Hunan, China

Man-Liang Tan, Changsha National Biomedical Industrial Base, Hunan Engineering Research Center of Botanical Extract, Liuyang, Hunan, China

Fang Wang, Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China

Li-Hui Wang, Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China

Chun-Fu Wu, Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China

Guo-Ping Yang, The Third Xiangya Hospital in Central South University, Changsha, China

Jing-Yu Yang, Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China

Ling Yi, R&D Department, School of Chinese Medicine, Hong Kong Baptist University, Kowloon, Hong Kong

Hong Yuan, The Third Xiangya Hospital in Central South University, Changsha, China

Jian-Guo Zeng, Hunan Agricultural University, Changsha, China

Abbreviations

AAS

atomic absorption spectrometry

AEs

adverse effects

AFLP

amplified fragment length polymorphism

AFS

atomic fluorescence spectrometry

AIC

Akaike Information Criterion

ALP

alkaline phosphatase

AMD

age-related macular degenerative disease

ANOVA

analysis of variance

AP

alkaline phosphatase

APCI

atmospheric pressure chemical ionization

API

atmospheric pressure ionization

AP-PCR

arbitrarily primed polymerase chain reaction

APPI

atmospheric pressure photoionization

ASTM

American Society for Testing and Materials

BMD

bone mineral density

CAG

coronary angiography

CAM

complementary and alternative medicine

CCCD

China Certification Committee for Drugs

CD

circular dichroism

CE

capillary electrophoresis

CHD

coronary heart disease

CHL

Chinese hamster lung cell

CI

chemical ionization

CID

collision-induced dissociation

CIOMS

Council for International Organizations of Medical Sciences

CNPIC

China National Pharmaceutical Industry Corporation Limited

CNS

central nervous system

COSY

chemical shift correlation spectroscopy

COX

cyclooxygenase

CP

cyclophosphamide

CPC

centrifugal partition chromatography

CQS

comprehensive quality systems

DAD

diode array detector

DCC

droplet countercurrent

DEPT

distortionless enhancement by polarization transfer

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

dimethyl sulfoxide

DOPAC

3,4-dihydroxyphenylacetic acid

DPD

deoxypyridinoline

DPPH

2,2-diphenyl-1-picrylhydrazyl

E

enzyme

E2

estradiol

ECD

electrochemical detector

ECG

electrocardiogram

ECL

enhanced chemiluminescence

EFPIA

European Federation of Pharmaceutical Industries Associations

EI

electron ionization

EIA

enzyme immunoassay

EIS

enzyme-inhibitor-substrate complex

ELISA

enzyme-linked immunosorbent assay

ELS

evaporative light scattering

ELSD

evaporative light scattering detector

EMEA

European Medicines Agency

ER

estrogenic receptor

ERB

Ethical Review Board

ERE

estrogen-responsive element

ERT

estrogen replacement therapy

ES

enzyme-substrate complex

ESI

electrospray ionization

EU

European Union

FAB

fast atom bombardment

FBS

fetal bovine serum

FC

flash chromatography

FCPC

fast centrifugal partition chromatography

FD

field desorption

FDA

Food and Drug Administration

FDCA

Federal Food, Drug, and Cosmetic Act

FI

field ionization

FLARE

fragment length associated repair enzyme

FOB

functional observatory battery

FT

Fourier transform

FT-ICR

Fourier transform ion cyclotron resonance

FTMS

Fourier transform mass spectrometry

GAP

good agriculture practice

GABA

γ-aminobutyric acid

GC

gas chromatography

GCP

good clinical practice

GE

gel electrophoresis

GEP

good extracting practice

GLP

good laboratory practice

GMP

good manufacturing practice

GOT

glutamate oxaloacetate transaminase

GPT

glutamate pyruvate transaminase

GSLS

Ginseng stem and leaf saponins

GTP

guanosine triphosphate

HBV

anti-hepatitis B virus

HHS

Department of Health and Human Services

5-HIAA

5-hydroxyindoleacetic acid

HILIC

hydrophilic interaction liquid chromatography

HMBC

heteronuclear multiple bond correlation

HMQC

heteronuclear multiple quantum coherence

HPLC

high-performance liquid chromatography

HRT

hormone replacement therapies

HSCC

high-speed countercurrent

HSQC

heteronuclear single quantum coherence

HT

serotonin

HTS

high-throughput screening

HVA

homovanillic acid

IBS

irritable bowel syndrome

ICH

International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use

ICP-MS

inductively coupled plasma mass spectroscopy

IEC

independent ethics committee

IND

investigational new drug

IR

infrared

IRB

institutional review board

ISSR

inter-simple sequence repeats

ITS

internally transcribed spacer

JPMA

Japan Pharmaceutical Manufacturers Association

LC

liquid chromatography

LD

lethal dose

LDL

low-density lipoprotein

LH

luteinizing hormone

LhRh

luteinizing hormone releasing hormone

LIT

linear ion trap

LOD

limit of detection

LOQ

limit of quantitation

LPH

lipotropic hormone

LPLC

low-pressure liquid chromatography

LS

light scattering

LSD

least significant difference

MAE

microwave-assisted extraction

MALDI

matrix-assisted laser desorption

MBC

metastatic breast cancer

MEM

minimum essential medium

MHLW

Ministry of Health, Labor, and Welfare

MOH

Ministry of Health

MPLC

medium-pressure liquid chromatography

MRM

multiple-reaction monitoring

MS

mass spectrum; mass spectrometer; mass spectrometry

MTD

maximum tolerated dose

MTS

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MW

molecular weight

NAD+

nicotinamide adenine dinucleotide

NADH

reduced form of nicotinamide adenine dinucleotide

NADP+

nicotinamide adenine dinucleotide phosphate

NADPH

reduced form of nicotinamide adenine dinucleotide phosphate

NCCAM

National Center for Complementary and Alternative Medicine

NCI

National Cancer Institute

NDA

new drug application

NEI

neuroendocrine-immune network

NIH

National Institutes of Health

NIR

near-infrared

NIRS

near infrared spectrometer; near infrared spectrometry

NLM

National Library of Medicine

NMR

nuclear magnetic resonance

NOAEL

no-observed-adverse-effect level

NOE

nuclear Overhauser effect

NOESY

nuclear Overhauser effect spectroscopy

NP–LC

normal phase liquid chromatography

ODS

octadecasilica

OHRP

Office for Human Research Protections

ORAC

oxygen radical absorbance capacity

ORD

optical rotatory dispersion

ORR

objective response rate

OVX

ovariectomized rat model

PB

particle beam

PBS

phosphate buffer saline

PC

paper chromatography

PCR

polymerase chain reaction

PD

pharmacodynamics

PE

phosphatidylethanolamine

PK

pharmacokinetics

PLE

pressurized liquid extraction

PMS

phenazine methosulfate

pQCT

peripheral quantitative computed tomography

PQR

product quality review

PR

progestin receptor

PRMA

Pharmaceutical Research and Manufacturers of America

PTLC

preparative thin layer chromatography

QA

quality assurance

QC

quality control

QOL

quality-of-life

QRM

quality risk management

QT

Q wave and T wave in ECG

QU

quality unit

RACE

rapid amplification of cDNA ends

RAPD

random amplified polymorphic DNA

RDA

retro-Diels–Alder

RFLP

restriction fragment length polymorphism

RI

refractive index

RP–LC

reversed phase liquid chromatography

RT-PCR

reverse transcriptase-polymerase chain reaction

SATCM

State Administration of Traditional Chinese Medicine

SAGE

serial analysis of gene expression

SCE

supercritical CO2 extraction

SCGE

single cell gel electrophoresis

SDA

State Drug Administration

SEC

size exclusion chromatography

SFC

supercritical fluid chromatography

SFDA

State Food and Drug Administration

SFE

supercritical fluid extraction

SOP

standard operation procedure

SPE

solid phase extraction

SRM

selected-reaction monitoring

SSR

simple sequence repeats

SSRI

serotonin selective reuptake inhibitors

SWH

Sambucus williamsii HANCE extract

TCM

traditional Chinese medicine

TFA

trifluoroacetic acid

THF

tetrahydrofuran

TLC

thin layer chromatography

TMS

trimethylsyl

TOCSY

total correlation spectroscopy

TOF

time-of-flight

TQS

total quality systems

TSP

thermospray ionization

TTP

time to progression

UV

ultraviolet

UV-Vis

ultraviolet-visible spectrometry

VLC

vacuum liquid chromatography

WET

water suppression enhanced through T1 effects

WHO

World Health Organization

WMA

World Medical Assembly

Chapter 1

Introduction to Traditional Herbal Medicines and Their Study

Willow J.H. Liu

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 maintained 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 transmission 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 practiced 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.

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 teamwork 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.

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 pharmacists 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 antipyretic drug in the world.

Since the 1950s, chromatography, including medium-pressure liquid chromatography (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, 28 kg 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 identification can all be applied to identify the raw materials. The availability of HPLC chromatogram or gene fingerprints makes identification of species highly accurate.

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 compounds 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

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 symptoms 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, processing 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 medicine 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

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.

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.

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 20 µg/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.

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 5 cm. 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 in-depth 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 age-related 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.

1.5 BRIEF INTRODUCTION OF DIFFERENT SYSTEMS OF TRADITIONAL MEDICINE

The use of plants for prevention and treatment of diseases is the earliest type of medicine on earth. The practice of traditional medicine developed along with the cultures of ancient China, India, Egypt, and other places. Different species of plants are used as medicines for treatment in different countries because of the different ecological environments. In countries with long histories and cultures, theories of etiology and pathology, methods for diagnosis, and treatment with herbal medicines or other methods under these theories were gradually formed along with the understanding of diseases and accumulated therapeutic experiences, and their own complete medical systems finally established. To fully explore the preventative and therapeutic mechanisms of traditional herbal medicines, it is necessary to have a deep understanding of the theories in their corresponding medical systems. The following are brief introductions of some ancient but still currently popular traditional medical systems in the world. The systems in which herbal medicine is not a key therapeutic tool are not covered here.

As a summary, holistic is one of the most common characteristics of these major popular medical systems and their biggest difference from conventional or allopathic medicine. It is not only reflected in the beliefs of importance of interaction and harmonization between the human body and environment and among organs and tissues, but also implemented in treatment with different herbal components.

1.5.1 Traditional Chinese Medicine (TCM)

TCM originated in China thousands of years ago through meticulous observation of nature, the cosmos, and the human body. Today, it not only remains as a form of primary care in health systems throughout most Asian countries, but also as the most popular complementary or alternative form of medicine in most of the Western countries. It has an extremely complex theory system established mainly on the basis of two philosophical views, the integral and dialectical concepts. The major theories include yin and yang, the five elements, Zang–Fu theory, qi and blood theory, meridians, collaterals, etiology and pathology, and prevention.¹⁹

Yin and Yang

Yin and yang reflect all the forms and characteristics existing in the universe. They may represent two separate phenomena with opposite natures, as well as different and opposite aspects within the same phenomenon. While yin is dark, passive, downward, cold, contracting, and weak, yang is bright, active, upward, hot, expanding, and strong.

The basic theory of yin and yang is about their relationship. They are opposing, interdependent, inter-transforming (in a state of constant change), and balanced. The yin and yang theory is applied in TCM for diagnosis and as the principles of treatment. The imbalance and fluctuation of yin and yang are considered the basic causative factors of disease occurrence and development. The goal of clinical treatment is to restore yin–yang balance in the patient. For example, heat syndromes are treated with cold nature herbs, while cold syndromes are treated with hot nature ones.

The Five Elements

In this theory, nature is divided into five elements: wood, fire, earth, metal, and water. Color, taste, emotion, sense, season, organs in human body, and others can all be classified into the five elements. The laws of movement of the five elements are as follows: inter-promoting, interacting, counteracting, and mutual relation. The five elements theory is applied in TCM to explain the physiological and pathological interrelationship among Zang–Fu organs and guide diagnosis and treatment of diseases.

Zang–Fu Theory

In TCM, the heart, lung, spleen, liver, and kidney are known as the five Zang organs, while the gallbladder, stomach, small intestine, large intestine, bladder, and triple energizer are the six Fu organs. The pericardium is a protective membrane of the heart, so it is also considered an organ. The triple energizer is the central body cavity that is connected to Zang organs. There is no biomedical equivalent of the triple energizer in modern medicine. Its function includes transformation, purification, and distribution of air, food, and water. It can be further divided into three parts. The upper part regulates respiration and the circulation of protective qi, the middle part governs the qi of the various digestive system functions, and the lower part controls the qi of the absorption of fluids/nutrients, waste disposal, and sexuality/reproduction. The triple energizer is the central energetic structure and strength of human health and well-being.

The Zang organs are solid and yin in nature. Their physiological functions are to manufacture and store essential substances, including vital essence, qi, blood, and body fluid. They are connected with meridians for the transmission of qi and blood. The Fu organs are hollow and yang in nature. Their physiological functions are to receive and digest food, and transmit and excrete the wastes. Fu organs are also connected with meridians. Interconnected by the meridian system, the Zang and Fu organs have an internally–externally linked relationship.

Qi, Blood, and Body Fluid

They are considered fundamental substances that maintain the normal vital activities of the human body and physiological functions of the Zang–Fu, tissue, and meridians.

Qi has such a special meaning in TCM for which no English word exists for translation. It denotes both the essential substances of the human body and the functional activities of the Zang–Fu and tissues. Blood is a red liquid circulating in the vessels, similar to blood in modern terminology. Body fluid is a collective term for all the normal fluids of the body, which include saliva, tears, nasal discharge, sweat, and urine, as well as liquids in stomach, intestines, joint, and other cavities.

Meridians and Collaterals

They are pathways in which the qi and blood of the human body are circulated. Meridians constitute the main trunks and run longitudinally and interiorly within the body, while collaterals represent branches of the meridians and run transversely and superficially from the meridians.

The functions of the meridians and collaterals include transporting qi and blood, regulating yin and yang, resisting pathogens, reflecting symptoms and signs, and transmitting needling sensation to regulate deficiency and excess condition when acupuncture and moxibustion are applied.

Causes of Diseases

TCM believes that the causes of disease include the six exogenous factors (wind, cold, summer heat, damp, dryness, and fire), the seven emotional factors (joy, anger, melancholy, worry, grief, fear, and fright), improper diet, overstrain, lack of physical exercise, traumatic injuries, bites by insects or wild animals, as well as stagnant blood and phlegm fluid.

Diagnostic Methods

TCM diagnosis includes inspection, auscultation and olfaction, inquiry, and palpation.

Treatment Methods

The TCM treatment methods include herbal medicine, acupuncture, dietary therapy, Tui na, and massage. Qi gong and Tai ji are also strongly affiliated with TCM.

Information about properties, current researches, and modern pharmacology of Chinese herbal medicines, and the understanding of TCM theories with modern medical terminology will be given in Chapter 10.

1.5.2 Kampo Medicine

Kampo is the Japanese study and adaptation of Chinese medicine. The first Chinese medical works were introduced to Japan around the fourth or fifth century AD. Since then, the Japanese have established their own herbal medical system and diagnosis based on TCM. Kampo utilizes most of the TCM treatment methods, including herbs, acupuncture, and moxibustion.

Kampo is currently integrated into the national health-care system in Japan. Different from modifying formulas applied in TCM clinics, the Japanese Kampo uses standardized, fixed, and precise combinations of herbs. Today, about 75% of Japanese physicians prescribe Kampo formulas. Since 1967, the Japanese Ministry of Health, Labor, and Welfare has approved 148 Kampo formulas for coverage and reimbursement in the national health insurance plan. The formulas are prepared under strict manufacturing conditions with the Ministry’s standardization methodology.

1.5.3 Indian Medicines

Indian medicines include three different systems: Ayurveda, Siddha, and Unani. They are different in origin and practice areas, as well as the theory and application.

Ayurveda

The term means the science of life. It is another one of the oldest systems of medicine in the world. According to the web site of the U.S. National Center for Complementary and Alternative Medicine (NCCAM) in NIH (http://nccam.nih.gov/health/ayurveda/introduction.htm), Ayurveda medicine originated in India several thousand years ago and continues to be practiced in India, where nearly 80% of the population uses it exclusively or in combination with Western medicine. It is also practiced in Bangladesh, Sri Lanka, Nepal, and Pakistan. Two ancient books, Caraka Samhita and Sushruta Samhita, written in Sanskrit more than 2000 years ago, are considered the main texts on Ayurvedic medicine.

Ayurvedic medicine aims to integrate and balance the body, mind, and spirit; thus, it is also viewed as holistic. This balance is believed to lead to happiness and health, and to help prevent illness. A chief aim of Ayurvedic practices is to cleanse the body of substances that can cause disease, thus helping to reestablish harmony and balance. Ayurvedic medicine has several key foundations that pertain to health and disease. These concepts have to do with universal interconnectedness, the body’s constitution (prakriti), and life forces (doshas).

Interconnectedness

This is about the relationships among people, their health, and the universe as the basis for how Ayurvedic practitioners think about problems that affect health. It believes that disease arises when a person is out of harmony with the universe. Disruptions can be physical, emotional, spiritual, or a combination of these.

Constitution (Prakriti)

This refers to a person’s general health, the likelihood of becoming out of balance, and the ability to resist and recover from disease or other health problems. It is called the prakriti, which means a person’s unique combination of physical and psychological characteristics and the way the body functions to maintain health. It is believed to be unchanged over a person’s lifetime and influenced by such factors as digestion and how the body deals with waste products.

Life Forces or Energies (Doshas)

Different from TCM, the five fundamental elements in Ayurveda that make up the universe and also human physiology are space, air, fire, water, and earth. Ayurveda believes that health is maintained by the balancing of three subtle energies known as doshas. There are three doshas, called Vata, Pitta and Kapha, and each is mainly a combination of two elements. Vata dosha is made up of space and air. Pitta dosha is a combination of fire and water. Kapha dosha is made up of water and earth. Together, the doshas orchestrate all the activities that occur within us. A person’s chances of developing certain types of diseases are thought to be related to the way doshas are balanced, the state of the physical body, and mental or lifestyle factors.

Ayurvedic practitioners first determine the patient’s primary dosha and the balance among the three doshas by Asking, Observing, and Checking a pulse (each dosha is thought to make a particular kind of pulse). The goals of treatment include eliminating impurities, reducing symptoms, increasing resistance to disease, and reducing worry and increasing harmony in the patient’s life.

Ayurvedic treatments rely heavily on plants, including herbs, oils, and common spices. Currently, more than 600 herbal formulas and 250 single-plant drugs are included in the pharmacy of Ayurvedic treatments. According to their effects, for example, healing, promoting vitality, or relieving pain, Ayurvedic medicines have been divided into categories.

Siddha

Siddha is mainly practiced in south India. This system of medicine is believed to be developed by the Siddhars, the ancient supernatural spiritual saints of India, who developed methods and medications that are believed to strengthen their physical body and thereby their soul, including intense yogic practices and years of fasting and meditation.

Unani

Unani means Greek. Unani medicine originated around 980 AD in Persia. The basic knowledge as a healing system was collected by Hakim Ibn Sina. This system is based on the theory of the presence of the elements, which are fire, water, earth, and air. These elements are present in different fluids (phlegm, blood, yellow bile, and black bile). The balance of these element leads to health, and imbalance leads to illness. Most medicines and remedies used in Unani are also used in Ayurveda. The base used in Unani medicine is often honey. Real pearls and metal are also used in making Unani medicine, based on the kind of ailment it is aimed to heal. In India, Unani practitioners can practice as qualified doctors.

1.5.4 Tibetan Medicine

Tibetan medicine combines elements of Indian, Chinese, and Greek medical traditions. Dietary modification, medicines composed of herbs and minerals, acupuncture, and moxabustion are applied for the treatment of illness. Tibetan medicine is currently practiced in Tibet, India, Nepal, Bhutan, China, and Mongolia, and is spreading to North America and Europe.

1.5.5 Muti

Muti is a term for traditional medicine in Southern Africa. The word means tree. African traditional medicine makes use of various natural products, many of which are derived from trees. For this reason, medicine generally is known as Muti. In Southern Africa, the word muti is in widespread use in most indigenous African languages, as well as in South African English and Afrikaans, where it is sometimes used as a slang word for medicine in general.

1.5.6 Islamic Medicine (Arabic Medicine)

Islamic medicine (Arabic medicine) refers to medicine developed