AUTHENTICATION METHODS FOR DRUGS USED IN AYURVEDA, SIDDHA AND UNANI SYSTEMS OF MEDICINE: AN OVERVIEW

AUTHENTICATION METHODS FOR DRUGS USED IN AYURVEDA, SIDDHA AND UNANI SYSTEMS OF MEDICINE: AN OVERVIEW

S. Sudha Revathy*, R. Rathinamala, M. Murugesan

Department of Gunapadam, National Institute of Siddha, Tambaram Sanatorium, Chennai 47, Tamil Nadu, India

ABSTRACT

Traditional systems of medicine in India include Ayurveda,Yoga, Naturopathy, Unani and Siddha. Among them Ayurveda, Siddha and Unani systems (ASU) use plants, minerals and animal products as main drugs to cure various ailments. There has been a boom in the usage of ASU drugs and export is appreciably high in the last two decades. ASU drugs may vary in composition and properties, unlike conventional pharmaceutical products, which are usually prepared from synthetic, chemically pure materials by means of reproducible manufacturing techniques and procedures. Counterfeits and drugs of poor quality degrade the clinical effects of ASU drugs. Thus authentication is a critical step for successful and reliable clinical applications and for further experimental studies on ASU drugs. The authentication methods for herbal, mineral and marine products are discussed in detail which broadly include microscopy, spectroscopy, chromatography, chemometry, immunoassays, DNA fingerprinting etc. A simple method like organoleptic characteristics may hold good to authenticate certain drugs but some may require highly sophisticated techniques too, based on the adulterants and similarity in the chemical constituents. So it is in the hands of the researchers to choose the right method suitable for the drug of interest which would match the reference standard after confirmation from the traditional vaidhyas/practitioners. This article will address about the various common and sophisticated techniques used to authenticate drugs of plant, mineral and animal origin.

Spectroscopy

INTRODUCTION:The traditional systems of medicine have become significantly more popular all over the globe because of the curative property, less toxic and minimal side effects. It is more widely used for the human ailments from time immemorial. It has been estimated that 70-80% of world’s population relies on traditional healthcare. The mode of preparation and plant used in traditional medicine varies from place to place. In addition acceptance of traditional medicines, especially herbal medicines in the developed world is sharply increasing ^{1, 2, 3}.

InASU systems plants, minerals, and animal products are used as main drugs to cure various ailments ⁴. There is a global resurgence in the use of these medicines along with a growing scientific interest in them as a source of new drugs ⁵.There has been a boom in the usage of ASU drugs and export is appreciably high in the last two decades ⁶.

There has been an increase in science based research in ASU drugs for the purpose of globalization. One of the most critical issues involved in any research study is the quality of the test material.

A study cannot be considered scientifically valid if the material tested is not authenticated and characterized such that the material can be reproduced ⁷.

ASU drugs may vary in composition and properties, unlike conventional pharmaceutical products, which are usually prepared from synthetic, chemically pure materials by means of reproducible manufacturing techniques and procedures. Correct identification and quality assurance of the starting material is therefore an essential prerequisite to ensure reproducible quality of these medicines which contribute to its safety and efficacy ^{8, 9}. Counterfeits and drugs of poor quality degrade the clinical effects of ASU drugs. Thus authentication is a critical step for successful and reliable clinical applications and for further experimental studies on ASU drugs.

Authentication is especially useful in cases of drugs that are frequently substituted or adulterated with other varieties which are morphologically and chemically indistinguishable. Several herbal drugs in the market still cannot be identified or authenticated based on their morphological or histological characteristics. Use of wrong drugs may be ineffective or it may worsen the condition ¹⁰.

The method of evaluation of drugs by veteran collectors experience should be confirmed by scientific methods before starting the research work. Evaluation has become even more difficult when several different individual species were powdered and mixed together in a proprietary medicine ¹¹.

1. Authentication of Herbal Drugs: Authenticated raw material is the basic starting point in developing a botanical product. In addition, each step of harvest, storage, processing and formulation may dramatically alter the quality and consistency of final product. Therefore methods to ensure quality control in manufacturing and storage are requisite tools to ensure optimal efficacy and safety of these products. Furthermore, such controls are critical for the evaluation of pharmacological, toxicological or clinical studies involving botanical products.
2. Taxonomic method: The initial step in the identification and authentication of botanical materials entails classical botanical methodologies for collection and documentation of the plant at its source. The botanical origin of the drug is identified and its scientific Latin binomial (i.e. genus species) name is determined based on this method. It is the first step for authentication ¹².

Information such as botanical name, vernacular names, site of collection of plant material, details of collector, habitat, season of collection, altitude and part collected etc. are the essential prerequisites even before authentication ¹².

2. Herbarium voucher sample: The sample of collected material should be kept as a voucher sample in a herbarium or a research institute for future references ¹².
3. Macroscopic method: Macroscopic identity of botanical materials is based on parameters like shape, size, color, texture, surface characteristics, fracture characteristics, odor, taste and such organoleptic properties that are compared to a standard reference material ¹³.
4. Microscopic method: Microscopy is used to determine the structural, cellular and internal tissue features of botanicals. It is usually used to identify and differentiate two herbals that are similar ^{14, 15}. This is the commonly used technique, convenient, quick and can be applied to proprietary medicines too ¹⁶.An example of a botanical that can utilize microscopic techniques to aid in its identification is star anise (Illicium verum Hook.f). As the name suggests, star anise is star shaped fruits that taste like anise; originally a native of southern China, it has now been introduced throughout the tropics and subtropical Eastern Asia.

The fruit is used principally as an aromatic spice in China and India to flavor food and confectionary items ¹⁷. It is known for its therapeutic value in traditional Chinese medicine for treating rheumatism, back pain and hernias. Unfortunately, an increasing number of cases of infants, suffering from acute neurological effects- such as seizure, vomiting and rapid eyeball movement have been reported in western countries and United States after the consumption of star anise herbal tea ¹⁸.

These diverse events were suspected to be due to adulteration of Chinese star anise with Japanese or “Bastard” anise ¹⁹. Japanese star anise (Illicium anisatum) is well known to contain the toxic sesquisterpenes ^20-22.

1. Fluorescence Microscope: Using the microscope to determine the identity of herbal medicines, namely, microscopic authentication refers to observing cell structure and internal features using a microscope and its derivatives. Besides the ordinary light microscope, polarized and fluorescence microscopes can also be used to enhance the accuracy of authentication. Use of these microscopes expands the number of features available for use in identification. For example, it has been found that starch grains, crystals of calcium oxalate, stone cells, vessels and fibers have stable and special polariscopic characteristics ^{23, 24}.

The fluorescence microscope reveals the fluorescence emitted from herbal tissues under illumination. Many herbal tissues, by virtue of their chemical structures or secondary metabolites, have the ability to emit light of a specific wavelength following the absorption of light with a shorter wavelength and higher energy ²⁵. For example, in recent years, the fluorescence microscope has been applied to distinguish the medicinal herb Oldenlandia diffusa from other species of the same genus which are confused with it, in herbal markets ²⁶. The fluorescence microscope and micro spectrometer can be used to authenticate powdered ASU drugs and measure the distribution of chemicals in the cross section of these drugs.

5. Physicochemical methods: Physicochemical parameters include total ash, water soluble ash, acid insoluble ash and sulphated ash. These values of the individual drugs or the proprietary medicines can be compared with the standard values of Indian pharmacopoeia and thus the identity can be ascertained ²⁷.
6. Chemometric and Spectral methods: Initially the use of infrared (IR) spectroscopic method is restricted only for structural elucidation of isolated compounds from the herbal matrices. It is also found useful in phytochemical studies as a “fingerprinting” device, for comparing a natural with synthetic sample ²⁸. With the advance of computer technology, chemometric method has become a leading tool among the scientific communities towards faster analysis and shorter product development time ²⁹. Among others, an unsupervised pattern recognition technique such as Principal Component Analysis (PCA) is the most often used method for handling multivariate data without prior knowledge about the study samples ³⁰.

While the supervised classification procedure using Soft Independent Modeling of Class Analogy (SIMCA) based on making a PCA model to assign unknown samples into the predefine class model has also been applied to the analysis of infrared spectra ³¹.A study using FTIR transmission spectroscopy, associated with the appropriate chemometric methods (PCA and SIMCA) was done to classify Orthosiphon staminens Benth (well known as Java tea for treating infection of the urinary tract, kidney and bladder stone disease) based on its geographical origin and varieties from the obtained characteristics infrared spectrum. Chemometric analysis of spectra is rapid and simple since no chemical treatment of samples is required ³².

7. Chromatographic methods: High Performance Liquid Chromatography (HPLC), Capillary Electrophoresis (CE) and Thin Layer Chromatography (TLC) are the most commonly used analytical methods for herbal products.³³The analysis of volatile compounds by gas chromatography is very important in chemical analysis of herbal medicines ³⁴.

a)      Thin Layer Chromatography (TLC): TLC was the common choice for the analysis of herbs before instrumental chromatography methods like GC and HPLC were established. Even nowadays, TLC is still frequently used for the analysis of herbal medicines since various pharmacopoeias still use TLC to provide first characteristic fingerprints of herbs ^{35, 36}. TLC has the advantages of many fold possibilities of detection in analyzing herbal medicines. In addition, TLC is rather simple and can be employed for multiple sample analysis.

For each plate, more than 30 spots of samples can be studied simultaneously in one time ³⁷. In summary, the advantages of using TLC to construct the fingerprint of herbal medicines are its simplicity, versatility, high velocity, specific sensitivity, simple sample preparation and its economy. Thus TLC is a convenient method to determine the quality and possible adulteration of herbal products ³⁸.

b)      High Performance Liquid Chromatography (HPLC): HPLC is a popular method for the analysis of herbal medicines, because it is easy to learn and use and is not limited by the volatility or stability of the sample compound. In general HPLC can be used to analyze almost all the compounds in herbal medicines ³⁹.

c)      Gas Chromatography (GC): The GC of the volatile oil gives a reasonable fingerprint and can be used to identify the plant. The extraction of the volatile oil is relatively straight forward and can be standardized and the components can be readily identified using GC-MS analysis. The advantages of GC clearly lie in its high sensitivity of detection for almost all the volatile chemical compounds ³⁴.

d)      Capillary Electrophoresis (CE): Capillary electrophoresis (CE) allows an efficient way to document the purity/complexity of a sample and can handle virtually every link of charged sample components ranging from simple inorganic ions to DNA. CE is promising for the separation and analysis of active ingredients in herbal medicines, since it needs only small amounts of standards and can analyze samples rapidly with a very good separation activity ³⁴.

8. Chemical Fingerprinting: A chemical fingerprinting is a unique pattern that indicates the multiple chemical markers within a sample ⁴⁰.The European Medicines Agency (EMEA) defines chemical markers as chemically defined constituents, or group of constituents of herbal medicinal product which are of interest, regardless whether they posses any therapeutic activity ⁴¹. The quantity of a chemical marker can be an indicator of the quality of herbal medicine.

The study of chemical markers is applicable to many research areas, including authentication of genuine species, search for new resources or substitutes of raw materials, optimization of extraction and purification methods, structure elucidation and purity determination.

9. Molecular markers: Molecular markers generally refer to biochemical constituents, including primary and secondary metabolites and other macromolecules such as nucleic acids. DNA markers are reliable for informative polymorphisms as the genetic composition is unique for each species and is not affected by age, physiological conditions as well as environmental factors ⁴². DNA can be extracted from fresh or dried organic tissue of the botanical material and hence the physiological form of the sample for assessment does not restrict detection ^{43, 44}.      Various types of DNA based molecular techniques are utilized to evaluate DNA polymorphisms. These are hybridization based methods, polymerase chain reaction (PCR) based methods and sequencing based methods ^{45, 46, 47}.

a)      Hybridization based methods: Hybridization based methods include Restricted Fragment Length Polymorphism (RFLP) and variable number tandem repeats.⁴⁸ Labeled probes such as random genomic clones, cDNA clones, probes for micro satellite⁴⁹ and mini satellite⁵⁰ sequences are hybridized to filters containing DNA, which has been digested with restriction enzymes. Polymorphisms are detected by presence or absence of bands upon hybridization.

b)      PCR based methods: PCR based methods involve in vitro amplification of particular DNA sequences or loci, with the help of specific or arbitrary oligonucleotide primers and the thermo stable DNA polymerase enzyme. PCR based techniques where random primers are used include Randomly Amplified Polymorphic DNA (RAPD) ^{51, 52}, Arbitrarily Primed PCR (AP-PCR) ⁵³ and DNA amplification fingerprinting ^{54, 55}. A recent approach known as Amplified Fragment Length Polymorphism (AFLP) ^{56, 57} is a technique that is based on the detection of genomic restriction fragments by PCR amplification.

c)      Microchip method: A DNA micro array is a multiplex technology used in molecular biology and in medicine. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing Pico moles (10-12 moles) of a specific DNA sequence, known as probes. This can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (called target) under high stringency conditions. Probe-target hybridization is usually detected and quantified by detection of flurophore, silver, or chemiluminescence’s labeled targets to determine relative abundance of nucleic acid sequences in the target.

Since an array can contain tens to thousands of probes a micro array experiment can accomplish many genetic tests in parallel. ⁵⁸. DNA based techniques have widely used for authentication of plant species of medicinal importance. This is especially useful in case of those that are frequently substituted or adulterated with other species or varieties that are morphologically or phytochemically indistinguishable ⁵⁹.

Dried fruit samples of Lycium barbarum were differentiated from its related species using RAPD markers ⁶⁰. The RAPD technique has also been used for determining the components of a Chinese herbal prescription Yu-feng-san. In this study, the presence of three herbs (Astragalus membranaceous, Ledebouriella seseloides and Atractylodis macrocephala) in the formulation has been detected using a single RAPD primer ⁶¹.

1. Authentication of Minerals:       The use of minerals as source of drugs is largely used in Indian systems of medicine next to herbals, especially in Siddha medicine. They are processed with herbal juices and made into their calcified or oxide forms and administered as drugs. Hence their identification is equally important to herbal drugs.
2. Physical properties: The physical properties of individual minerals will be a powerful tool for identification. These properties include nature, colour, streak, tenacity, transparency, luster, hardness, fracture, cleavage or parting, magnetism and specific gravity. These unique physical characteristics of minerals can be used for the authentication.
3. Optical properties: Optical properties include application of optical crystallography in identification of minerals that are crystalline and transparent irrespective of their chemical composition. This can be determined within limits by means of polarizing microscope.
4. Refractive Index (RI):       It is defined as ratio of velocity of light in media to that in vacuum. It varies with wavelength. Immersion method is used to determine the RI. For example Alum and Garnet are isotopic, Calcite, Quartz, Sapphire and Ruby are anisotropic – uniaxial, and Mica and Gypsum are anisotropic – biaxial.
5. Chemical properties:       The Chemical properties include effect on heat, solubility, reaction with acids like HCl, HNO_3, H₂ SO₄, assays which include Volumetric/ Gravimetric/ AAS/ ICPA/ Flame photometry for Na, K, qualitative test for carbonate and sulphate, analysis of heavy metals like Arsenic and other elements.
6. Microscopic methods: It is a simple, inexpensive and widely used method for the authentication of minerals ¹². Light microscopy and polarized microscopy are the common microscopes used for authentication. Light microscopes have a wide application in mineralogy ⁶². A polarizing microscope is a microscope that is mainly used in geological studies to study geological specimens. For this reason, it is also known as a petrography microscope. A comparative study was carried out with microscopy for the Chinese patent medicine Bo Ying Compound.

The micro morphological characteristics of its 22 components and in crude constituents have been documented and compared with each other. Their corresponding features were described and documented with color digital micrographs, so as to authenticate the presence of genuine crude constituents in the medicine. Another study focused on the authentication of four kinds of mineral arsenicals, including orpiment, realgar, arsenolite and arsenic trioxide.

The macroscopic and microscopic characteristics of the minerals were examined and they found that the all can be easily identified and authenticated by using light microscopy coupled with polarized microscopy ⁶³.

6. Spectroscopic methods:
   1. Near Infrared Spectroscopy (NIRS): Near Infrared Spectroscopy has received much attention for chemical quality and process control because of its speed and attribute of requiring little or no sample preparation ⁶⁴. NIRS uses the near infrared region of the electromagnetic spectrum(from about 800 nm to 2500nm).
   2. Electron Spectroscopy for Chemical Analysis (ESCA): ESCA otherwise known as X- ray Photoelectron Spectroscopy (XPS) involves only the top 20-50A° of the sample, making it an extremely surface sensitive technique. ESCA spectra can also provide information about an element’s chemical environment or oxidation state. The chemical environment of an atom affects the strength with which electrons are bound to it. Atoms associated with different chemical environments produce peaks with slightly different binding energies which are referred to as chemical shift.
   3. Inductively Coupled Plasma Mass Spectrometry (ICP-MS): ICP-MS is a type of mass spectrometry that is highly sensitive and capable of the determination of a range of metals and several non metals at concentration below one part in 10¹² (parts per trillion). Samples are decomposed to neutral elements in high temperature argon plasma and analyzed based on their mass to charge ratios. It is an automated, simple and unique quantitative and qualitative analysis. It measures elemental isotopes ratio.
   4. Atomic Absorption Spectrometry (AAS): AAS is based on the light absorption of elements. Nearly 30-40 elements can be detected. AAS is used for the quantitative estimation of inorganic minerals in plant drugs/poly herbal formulations, drugs of mineral/ metals and animal origin. The estimation can be made at ppm (parts per million) level and still lower levels by graphite furnace method.
   5. X-ray Diffraction Analysis (XRD): The X-ray diffraction pattern of a pure substance is like a fingerprint of the substance. The powder diffraction method is ideally suitable for characterization and identification of polycrystalline phases. Today about 50,000 inorganic and 25,000 organic single components, crystalline phases, and diffraction patterns have been collected and stored on magnetic or optical media as standards. The main use of powder diffraction is to identify components in a sample by a search/match procedure. Further more, the areas under the peak are related to the amount of each phase present in the sample.
   6. X-ray Fluorescence Analysis (XRF): X-ray fluorescence (XRF) is the emission of characteristic “secondary” (or fluorescent) X- rays from a material that has been excited by bombarding with high energy X- rays or gamma rays. The phenomenon is widely used for elemental analysis and chemical analysis particularly in the investigation of metals, and for research in geochemistry, forensic science and archeology.
7. Authentication of Marine and Animal Products: Animal and marine products have constituted part of the inventory of medicinal substances used in various cultures since ancient times. In Indian systems of medicine nearly 15-20 percent is based on animal derived substances ⁶⁵. Although research on marine natural products started only about 50 years ago, marine organisms have been used in traditional systems of medicine in India much before that ⁶⁶. The following techniques would help in the authentication methods of these products.
8. Chromatographic methods:
   1. Gas Chromatography /Mass Spectrometry (GC-MS): GC-MS is the marriage of two analytical methods into a versatile technique for the identification of complex volatile materials. Gas Chromatography (GC) effectively separates the constituents of the sample for subsequent analysis and identification by Mass spectrometry (MS). The first result obtained is the compiled data of total ion chromatogram (TIC), which is a plot of the total mass eluting from the GC and detected by MS as a function of time. In a study, effective extraction and GC/MS protocols were established for the detection of authentic and counterfeit components found in allegedly musk containing samples collected from various sources in Taiwan ⁶⁷.
   2. Liquid Chromatography-Mass Spectrometry (LC-MS): Liquid chromatography mass spectrometry (LC-MS or alternatively HPLC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high sensitivity and specificity.

Generally its application is oriented towards the specific detection and potential identification of chemicals in the presence of other chemicals. Raw materials like honey and animal fats can be authenticated by this technique and the spectral fingerprints of them can be generated. A qualitative analysis of carotenoid composition was performed by HPLC/UV on samples of Corallium rubrum to generate a chromatogram profile ⁶⁸.

2. Immunoassays:
   1. Enzyme linked Immunosorbent Assay (ELISA): The use of immunological methods for the detection of adulteration in milk and milk products resulted in the development of sensitive, reliable assays capable of detecting low levels of adulteration in milk and milk products. The use of monoclonal antibodies, careful selection of target antigen, and suitable ELISA format has greatly increased the good analysis ability to distinguish between species in milk and milk products ⁶⁹.
3. Non Immunological protein based methods:
   1. Poly Acrylamide Gel Electrophoresis (PAGE): Polyacrylamide gel electrophoresis (PAGE) provides a versatile, gentle and high resolution method for fractionation and physical-chemical characterization of molecules on the basis of size, conformation and net charge. The polymerization reaction can be rigorously controlled to provide uniform gels of reproducible, measurable pore size over a wide range.

This makes it possible to obtain reproducible relative mobility (R_f) values as physical-chemical constants ⁷⁰.

1. Isoelectric Focusing (IEF): Isoelectric focusing (IEF), also known as electro focusing, is a technique for separating different molecules by their electric charge differences. It is a type of zone electrophoresis, usually performed on  proteins in a gel, that take advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings. The above said non immunological protein based methods is focused for milk speciation especially for milk caseins and whey proteins ⁷⁰.
2. DNA Finger Printing Methods: Genetic fingerprinting, DNA testing or DNA profiling is a technique to distinguish between individuals of the same samples using only samples of their DNA. DNA fingerprints depend on the genetic differences between individuals, the so called DNA markers. DNA fingerprinting is a powerful tool in poultry for investigating genetic diversity within stocks and establishing relationship among stocks and characterizing individuals or populations genotypically. The different techniques of DNA profiling include Restricted Fragment Length Polymorphism (RFLP), PCR- Based techniques; Random amplified polymorphic DNA (RAPD), Amplified fragment length polymorphism (AFLP) and Real time PCR (RT-PCR) ⁷¹.

4. Spectral methods: The spectral methods used to authenticate animal include UV-Vis spectrophotometer, Near Infrared and Fourier Transform Infrared spectrometer etc. These methods are specifically employed in the authentication of honey, which is one of the most common adjuvant used in Indian system of medicine.

CONCLUSION: A significant number of methods to authenticate ASU drugs have been addressed here. A simple method like organoleptic characteristics may hold good to authenticate certain drugs but some may require highly sophisticated techniques too, based on the adulterants and similarity in the chemical constituents.

So, it is in the hands of the researchers to choose the right method suitable for the drug of interest which would match the reference standard. Even before choosing the methods the raw materials should be confirmed with traditional practitioners/ vaidhyas.

In addition, while utilizing chemical methods and other analytical tools it is mandatory to stick on to the latest validated techniques that suit the study. One needs to understand what type of raw material/formulation one is dealing with and the type of preparation to be evaluated. The evaluation of the adulterants simultaneously along with study drug will pave way for the researchers working on the same drug to eliminate the adulterants and identify the standard and pure raw material.

In spite of all these factors, the microbial contamination, pesticide residue and heavy metal analysis should be considered before processing the raw material for drug preparation. This is regarding the safety issue of drugs. This type of analysis is required when evaluating the authenticity of botanicals because these extraneous contaminants can cause undesired physiological effects.

To conclude, more basic research should be carried out and many individuals should be trained with these authentication techniques to address this issue prevailing in ASU drugs.

ACKNOWLEDGEMENT: We extend our sincere thanks to Prof. Dr. K. Manickavasagam MD(s), Director, National Institute of Siddha, Tambaram Sanatorium and Ms. Savitha Satakopan, Former chair person, Member, Ayurvedic Pharmacopoeia Committee, Ministry of Health, Government of India, for their encouragement and suggestions towards the work.

REFERENCES

1. Meena Devi VN, Nagendra Prasad P, Kalirajan K, Infrared spectral studies on Siddha drug- Pavala parpam, International journal of pharma and biosciences, 2010; 1(4):474-483
2. Subrat N, Ayurvedic and herbal products industry an overview.  In:  Paper presented at workshop on wise practices and experimental learning in the conservation and management of Himalayan medicinal plants, Katmandu, Nepal: 2002; 15-20.
3. Binu S, Uses of pteridophytes among the tribals in pathanamthitta district, Kerala, India. Journal of non timber forest products, 2008; 5(2): 129-131.
4. Bose DM, Sen SN and Subbarayappa BV(Ed) Subbarayappa BV (Au), Chemical practices and alchemy: In a Concise History of Science in India, Indian National Science Academy, New Delhi, 1971: 315-335.
5. Urmila Thatte, Clinical Research Ayurvedic Medicines. Pharma Times, 2005; 37(7): 9-12.
6. Sathiyarajeswaran P et al., Powder Diffraction fingerprints on cinnabar and its preparations. Journal of Siddha, 2009; 2(1): 29- 33.
7. Wolsko PM, Solond A, Phillips RS, Schachter SC and Eisenberg DM. Lack of herbal supplement characterization in published randomized controlled trials. Am. J. Med 2005; 118: 1087-1093.
8. Strans SE, Herbal Medicines - What's in the Bottle? N Engl J Med 2002; 347:1997-1998
9. Peter A.G.M De Smet, Herbal Remedies, N Engl J Med 2002; 347: 2046-2056.
10. Cosyns JP, Jadoul M, Squifflet JP,Wese FX, Van Yersele DS, Urothelial lesions in Chinese herb Nephropathy. Am. Kidney Dis 1999; 33: 1011-1017.
11. Zhong Zhen Zhao et al., Application of microscopy in Authentication of Chinese latent  Medicine – Bo Ying Compound, Microscopy research and Technique 2005; 67:305-311.
12. Smille TJ and Khan IA, A Comprehensive approach to Identifying and authenticating Botanical Products, Clinical Pharmacology and therapeutics 2010; 87(2): 175-186.
13. Quality control methods for Medicinal Plant materials, WHO, Geneva, 1998
14. Cheng XX, Kang TG and Zhao ZZ. Studies on microscopic identifying of animal drugs remnant hair (3): identification of several species of Cauda cervi. J. Nat. Med 2007; 57:163-171.
15. Lau. PW, Peng Y and Zhao ZZ. Microscopic identification of Chinese patent medicine- Wu Zi Yan Zong. Wan J.Nat. Med 2004; 58:258-265.
16. The Ayurvedic Pharmacopoeia of India. The controller of Publications Civil lines, Ministry of Health and Family welfare of India. Delhi (India): 2001; 3.
17. Duke JA, Bogenschutz-Godwin MJ, ducellier J and Duke PK.CRC Handbook of Medicinal Spices Boca Raton (F.L) CRC Press;2003;187-189.
18. Ize-Ludlow,Rafone D, Bruck S, Berstein IS, Duchonomy JN and Pena BM. Neurotoxicities in infants seen with the consumption of star anise tea. Peadiatrics 2004; 114: e 653-e656.
19. US Food and Drug Administration. Inspections compliance, enforcement and criminal Investigations<http:/www.fda. gov/ICECI/EnforcementActions/EnforcementStory/EnforcementStory Archive/ucm 095929.htm> (2009).
20. Kudo Y,OKa JI, Yamada K Anisatin a potent GABA antagonist, isolated from Illicium cristatum.  Neuro sci Lett 1981; 25: 83-88.
21. Yamada K, Takada S, Nakamura S and Hirata Y, Structures of anisatin and neoanisatin toxic sesquiterpenes from Illicium anisatum. Tetrahedron 1968; 24: 199- 220.
22. Kouno I, Mori K, Akiyama T and Hashimoto M. Two sesquiterpene lactones from Illicium anisatum. Phytochemistry 1991; 30:351-353.
23. Zhao ZZ, Shimomura H, Sashida Y, Tujino R, Ohamoto T, Kazami T. Identification of traditional Chinese patent medicine by polariscope (1): Polariscopic Characteristics of starch grains and calcium oxalate crystals. Natural Medicine 1996; 50: 389-398.
24. Zhao ZZ et al., Identification of crude drugs in traditional Chinese patent medicines by means of microscope and polariscope (2): Polariscopic characteristics of stone cells, vessels and fibres. Natural medicine 1997; 51: 504-511.
25. Wang YQ, Liang ZT, Li Qin, Vang H, Chen HB, Zhao Z, Li P. Identification of powdered Chinese herbal Medicines (CHMS) by Fluorescence Microscopy, Part I : Fluorescent Characteristics of Mechanical Tissues, Conducting Tissues and Ergastic substances, Microscopy Research and Technique 2006; 69:277-282.
26. Liang ZT, Jiang ZH, Leing KSY, Peng Y, Zhao ZL. Distinguishing the medicinal herb Oldenlandia diffusa from similar species of the same genus using fluorescence microscopy. Microscopy Research and Technique 2006; 69: 277-282.
27. Indian pharmacopoeia, Controller of Publications, New Delhi, India; 1995: 2: A 54.
28. Horborne JB, Phytochemical methods, A guide to Modern techniques of Plant Analysis, Chapman and Hall, UK, Edition 3,1998.
29. Seashottz MB, Chemometrics and Intelligent Laboratory systems, 45; 1999:55.
30. Miller JN and Miller JC. Statistics and Chemometrics for Analytical Chemistry, Prentie Hall Edition 4; 2000.
31. Lotrim AR, Ferraz A, Gancalves AR, Silva FT and Bruns RE, Identifying the origin of lignans and monitoring their structural changes by means of FTIR – PCA, SIMCA Bioresource Technology; 1999; 68: 29- 34.
32. Chow Oon Sim et al., Assessment of Herbal Medicines of chemometrics–Assisted FTIR spectra, Analytica clinica Acta 2004;
33. Baralcat HH, Sahar Hussin AM, Mohamed Marzouk S, Merfort I, Linscheid M, Nawwar Mohamed AM. Polyphenolic metabolities of Epilobium hirsutisam. Phytochemistry 1997; 46: 935-941.
34. Yi-Zeng Liang et al., Quality control of herbal medicines, Journal of Chromatography B 2004; 812: 53-70.
35. Li N, Lin G, Kwan YW, Min ZD, Simultaneous quantification of five major biologically active ingredients of saffron by high-performance liquid chromatography. J Chromatogr A 1990; 849(2):349-55.
36. Wagner A, Bladt S, Rickl V, Plant Drug Analysis: A thin Layer Chromatography Atlas, Spinger Berlin, Edition 2,1996.
37. Baercheim Svendsen A, Modern TLC 2. J. Planar chromatogr.1989; 8.
38. Chau FT, Chan TP, Wang J. TLCQA: quantitative study of thin-layer chromatography. Journal of Bioinformatics 1998; 14(6): 540-541.
39. Lin G, Li P, Li SL, Chan SW, Chromatographic analysis of Fritillaria isosteroidal alkaloids, the active ingredients of Beimu, the antitussive traditional Chinese medicinal herb. J.Chromatogr.A 2001; 935: 321-38
40. Songlin Li et al., Chemical markers for the quality control of herbal medicine: an overview, Chinese medicine 2008; 3:7.
41. The European Medicines Agency: Reflection paper on markers used for quantitative and qualitative analysis of herbal medicinal products and traditional herbal medicinal products.(http://www.emea.europa.eu/pdfs/human/hmpc/25362907en.pdf)
42. Chan K. Some aspects of toxic contaminants in herbal medicines. Chemosphere 2003; 52:1361-1371
43. Warude D, Chavan P, Kalpana J and Pat wardhan B, DNA Isolation from Fresh and Dry Plant Samples with highly Acidic Tissue Extracts. Plant Mol Biol. Rep 2000; 21:1.
44. Singh M, Bandana and Ahuja PS, Plant Mol. Biol. Rep.1999; 17:171.
45. Joshi SP, Ranjekar PK, Gupta VS.  Molecular markers in Plant genome analaysis. Curr. Sci. 1999;77: 230-240.
46. Powell W, Morganite M, Andre C, Hanafly M, Vogel J, Tingey S and Rafalski A, Mol. Breed 1996; 2: 225-238.
47. Botstein D, White RL, Skolnick M and Davis RW, Construction of a Genetic Linkage Map in Man Using Restriction Fragment Length Polymorphisms. Amt. J. hum. Genet 1980; 32: 314-331.
48. Nakamura Y, Julier C, Wolff R, Holm T, O’ connell P, Lepert M and White R, Nucleic Acids Res 1987; 15 :2537-2547.
49. Litt. M and Luty JA, A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet 1989; 44:  397-401.
50. Jeffrey AJ. Wilson V and Thein SL, Hypervariable ‘minisatellite’ regions in human DNA. Nature 1985; 314:  67-73.
51. Williams JGK, Kubelik AR, Livak J, Rafalski JA and Tingey SV, DNA Polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic acids 1990; 18: 6531-6535.
52. Welsh J and Mcchelland M, Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 1990; 18(24): 7213-7218.
53. Welsh J and Mcchelland M, Genomic fingerprinting using AP-PCR and a matrix of pair wise combination of primers. Nucleic Acids Res, 1991; 19:5275-5279.
54. Caetano- Anolles G, Bassam BJ and Gresshof PM, DNA amplification fingerprinting using very short arbitrary oligo nucleotide primers.  Biotechnology (NY) 1991; 9:553-557.
55. Caetano- Anolens G, Bassam BJ. DNA amplification fingerprinting using arbitrary oligo nucleotide primers.   Appl. Biochem. Biotechnol 1993; 42:189-200.
56. Zabeau M, European Patent Application, Publication no 0534858A 1, 1993
57. Vos. P, Hoggers R, Blecker M, Reijans M, Vande Lee T, Hornes M et al., AFLP :  a new technique  for DNA fingerprinting. Nucleic Acids Res 1995 ; 23(11) : 4407-4414.
58. Hao Da – Chang, CHEN shi-lin, XIAO pei-gen, PENG Yong, Authentication of medicinal plants by DNA based markers and Genomics.Chinese Herbal Medicines 2010; 2(4): 250-261.
59. Kalpana Joshi et al., Molecular markers in herbal drug technology, Current Science 2004; 87(2): 159-165.
60. Zhang KY, Laung HW, Yeung HW and Wong RN, Differention of Lycium barbarum from its related Lycium species using random amplified polymorphic DNA. Planta Med 2001; 67:379-381.
61. Chang KT, Tsay HS, Chan CF and Chou TW, Determination of the components in a Chinese prescription, YuPing-Feng San, by RAPD analysis. Planta Medica, 64: 563-565.
62. Katarina Logy, Chalmers Dept.  Optical microscopy; Dept. Applied physics. 2006-01-20
63. Qin.LI et al., Authentication of the 31 species of Toxic and Potent Chinese Materia Medica by microcopic technique assisted by ICP-MS analysis, Part 4 : Four kinds of Toxic and Potent Mineral Arsenical CMMS, Microscopy research and technique 2011; 74 : 1-8.
64. Siesler HW, Ozaki Y, Karvata S, Heise HM. (Ed) Near-Infrared Spectroscopy – Principles, Instruments, Applications. Wiley-VCH verlag: 2002
65. Romulo RN Alves Lerece L. Rosa, Why study the use of animal products in traditional medicines? J Ehnobiol Ethnomed 2005; 1: 5.
66. Radhika Gopal et al., Marine organism in Indian medicine and their future prospects, Natural Products Radiance 2008; 7(2): 139-145.
67. Dong – Liang Lin, Hsien-Cheh Chang, Su-Hui Huang Characterization of Allegedly Musk – containing Medicinal products Taiwan, J Forensic Sci 2004; 49(6): 1-6
68. Jelena Cvejic et al., Determination of cathaxanthin in the red coral (Corallium rubrum) from Marseille by HPLC combined with UV and MS detection, Mar Biol 2007; 152: 855-862.
69. Ian. P. Hurley et al., Application of Immunological methods for the detection of species adulteration in dairy products, International Journal of food Science and Technology 2004;39: 873-878.
70. Chrambach A, Rod bard D, Polyacrylamide gel electrophoresis, Science 1971; 172 (982): 440-51.
71. Farrag SA, Tanatarov AB  and  Sottan ME, Using of DNA fingerprinting in poultry Research, International Journal of poultry science; 2010: 9 (5) : 406-416.
    

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    How to cite this article:

    Revathy SS, Rathinamala R and Murugesan M: Authentication Methods For Drugs Used In Ayurveda, Siddha And Unani Systems Of Medicine: An Overview. Int J Pharm Sci Res, 2012; Vol. 3(8): 2352-2361.

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