Review Article Inti. Chern. Pharm. Med. J. Vol. 2(2), pp.217-229 (2005)

MOLECULAR BIOLOGICAL TECHNIQUES: Application in herbal medicine

Mansoor AhmadI, Zehral, Muniral and Shakeel Ahmad Khan2 J Research institute of Pharmaceutical Sciences, Department of Pharmacognosy, Faculty of

Pharmacy, University of Karachi, Karachi-75270, Pakistan 2 Department of Microbiology, University of Karachi, Karachi-75270, Pakistan

ABSTRACT: Herbal medicine, also called botanical medicine or phytomedicine, are plant deriven medicines that contain, an active ingredients, in the aerial or underground parts of the plants, or other plant materials or combinations thereof, whether in crude state or as plant preparations. Long practiced, outside of conventional medicine, herbalism is becoming more mainstream. As up-to-date analysis and research show their value in the treatment and prevention of diseases. Herbal drugs are obtained from different plant sources, therefore, should be analyzed for their safety, toxicity, usefulness, purity, mechanism of action etc. before their use in humans. Their authentication and standardization by controlling the quality is necessary. Uptill now, this was achieved by traditional methods like plant morphology, histology, various chromatographic techniques and spectrophotometeric analysis. Recently scientists observed that although these traditional methods are very efficient and reproducible for chemoprofiling and analyzing their purity but it cannot determine the absolute chemical characteristics and genetic variation in chemical composition of same species or genotype of medicinal plants. Therefore, for authentication and standardization of medicinal plants, more sophisticated tools are required. Advancements in molecular biology, now provide more standardized and reliable methods in identifying the quality, standard, selection and analysis of genetically pure medicinal plants. The aim of this review is to delineate some molecular biological techniques, which are utilized in the field of herbal medicines. These techniques are useful in authentication of herbal drugs, their standardization, analyzing their effectiveness, usefulness, mechanism of action etc. Although these techniques are quite reliable, there is still a need for better more precised standardization of herbal medicines.

KEY WORDS: Herbal drugs, Molecular biological techniques, DNA markers, chemoprofiling.

INTRODUCTION

Following WHO recommendations of traditional medicines, many developed countries in the world use herbal drugs in the form of nutraceuticals or as the primary source of medicinal compounds. Hence, thus there is an ever increasing use of herbal drugs worldwide. Their safety and efficacy effectiveness must be determined prior to their use.

For quality control and standardization of herbal drugs, traditional methods are used like macroscopic observance (size, shape, color, texture, smell, taste etc.) and microscopic features which are compared with its standard herbal compounds (Blumenthal, M. 1997). These macro and microscopic features are not enough for identification and standardization of herbal drugs because of possibility of substitution of standard herbs with their adulterants. Therefore, for better selection of standard herbal drugs and for their chemoprofiling, chemical and chromatographic techniques are used. These techniques include: Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), High Performance Thin Layer Chromatography (HPTLC), Gas

002-05/11CPMJ@Karachi University Press

Chromatography (GC), Spectrophotometeric methods etc.

HPLC-Mass Spectrometery is used to profile lead substances and to evaluate the pharmaceutical quality of the products. In this method, active constituents can be identified (Tolonen et al 2002).

In TLC/Densitometry, selective separation and simultaneous determination of few drugs can be analyzed even in traces.

Capillary GC with electron captures detection is used for recovery from fortified fragrance products at several concentrations (Wisneski et a12001).

These techniques are routinely used for quality control analysis of herbal medicines. Although these techniques are valuable tools for determination of impurities but it cannot determine the absolute chemical characteristics of medicinal plants for example in-process artifacts and overlaping of certain chemicals like sterols, phenolics. Similarly, in many herbal species, changes in environmental conditions from region to region and growing conditions may

217

Mansoor Ahmad et ai.

RAPD marker technique was utilized in combination with two-way pseudo-testcross mapping strategy to differentiate the species of Eucalyptus at genomic level. It was suggested that using this combination of technique, one could construct single individual genetic linkage maps. With crosses of genetically divergent individuals and mitigating the problem of linkage equilibrium between marker and trait loci for the application of marker assisted strategies in tree breeding (Grattapaglia et al. 1994).

With the help of RAPD technique, Jojoba clones were discriminated at the genetic level. The data showed that RAPD technique generated multiple amplified fragments. Some DNA fragments stained as single band contained different DNA species of the same size and that cloned RAPD products of known sequence that do not target repetitive DNA can be used as hybridization probes in RFLP to detect a polymorphism among individuals (Amarger et al. 1995).

Epimedium species including E. Sagittaum, E. Koreanum, E. Grandiflorum var. higoense, E. Trifoliatobinalum, E. Trifoliatobinalum, E. Koreanum, E. Sempervirens and E. Diphyllum were analyzed for genetic characterization and variation using RAPD analysis and RFLP technique (Nakai et al. 1996).

For detecting genetic variation in Neem, which is one of the most useful plants with huge medicinal usage, RAPD technique was employed and the results suggested that Neem might have narrow genetic base (Farooqui et al. 1998).

Researchers also evaluated highly potent antimalarial compound artemisinin, obtained from Artemisia annua L. plant, was sole attention for scientists. Therefore, in order to analyze and select the plant with maximum content of artemisinin, RAPD marker technique was utilized and clearly differentiated genetic variation among these plants and thus could be useful tool for selection of better and superior species (Sangwan et al. 1999).

RAPD analysis was performed for differentiation of 3 species of medicinal plants in genus Scutellaria (S. galericulata, S. Lateriflora and S. baicalensis). RAPD markers were generated by two specific primers that were capable of differentiating members of the 3 species of Scutellaria. Thus, these markers were found

220

Inti. Chern. Pharm. Med. J.

to be useful for identification of the three species of medicinally important Scutellaria plants (Hosokawa et al. 2000).

RAPD analysis was performed on Kenaf (Hibiscus cannabius L.), a is very useful herb. In order to conserve useful Kenaf germplasm, the study of their genetic diversity and to identify different Kenaf va-riety was important. With the help of RAPD analysis and by analyzing Argonomic characters, their different varieties and genetic relationships were identified (Cheng et al. 2002).

RAPD analysis for differentiating adulterants from standard herbs Pharmacognostical identification of American and Oriental ginseng roots was conducted by genomic fingerprinting using arbitrarily primed PCR (APPCR). They amplified DNA from dried or fresh roots of three medicinal Panax species and their adulterants by AP-PCR or RAPD. They found consistent fingerprints for P. ginseng or P. quinquefolius but very different fingerprint from adulterants and thus confirmed that P. ginseng is closely related to P.quinquefolius than to P. notoginseng. For authentication of concerned Panax species, PCR approach might be used (Cheung et al.1994).

RAPD analysis was done to differentiate three medicinal Panax species from their adulterants and this technique was also helpful in authentication of these three Panax species (P. quinquefolius, P. ginseng and P. notoginseng) (Shaw et al. 1995).

DNA fragment obtained by RAPD analysis of Panax quinquefolius was converted to a sequence characterized amplified region (SCAR) marker and primers synthesized on this sequence were used in authentication of Panax species and their adulterants (Wang et al. 2001).

To search high quality Panax ginseng among ginseng populations, RAPD technique was utilized. Along with RAPD analysis, PCR-RFLP analysis was also utilized for authentication of Panax ginseng species (Tochika-Komatsu, Y. et al. 2001).

Genomic fingerprints were also used to differentiate herba elephantopi (Ku-di-dan) from its adulterants. DNA fingerprinting and polymorphism among herba

002-05/IICPMJ@Karachi University Press

Review Article

elephantopi was demonstrated by AP-PCR and RAPD (Cao et al. 1996).

By using RAPD analysis, AP-PCR and Genomic fingerprints, Taraxacum mongolicum (species of Compositae) were differentiated from their adulterants (Cao et al. 1997).

RAPD analysis for identification of components in herbal prescriptions The utilization of RAPD technique was studied for determination of components in 3 Chinese herbal prescriptions such as the dried root of Astragalus membranaceus (Fisch.) Bge., the dried root of Ledebouriella seseloides Wolff, and the dried rhizome of Atractylodes macrocephala Koidz and thus concluded that this technique prove the identification of components in Chinese herbal prescriptions (Cheng et al. 1998).

Microsatellites also called short tandem repeats (STR) are simple sequence repeats (SSRs) and are wide spread over the genome of living organisms. Therefore, microsatellite alleles are co-dominant markers. Microsatellite are polymorphic in nature and provide more information per assay than any other technique. Using this technique, one can detect the difference at multiple loci between strains. This technique can be applied for specie and strain identification, genome mapping etc. Internal transcribed spacer regions (ITS-l and ITS-2) are the small sequences of nuclear ribosomal gene utilized extensively in differentiation, examination and diagnosis of taxonomic status of species (Wright et al. 1994, Rong Wen et al.1991, Porter et al. 1991).

This is one of the most effective and reliable techniques for differentiating species of herbal medicines and their adulterants.

Microsatellite technique for differentiating species of herbal medicines and their adulterants Phylogenetic relationship was constructed for 12 species of Panax using internal transcribed spacers (ITS) and the 5.8S coding regions of the nuclear ribosomal DNA repeat and it was found that discrepancy between the sequence divergence pattern and phylogenetic pattern in Panax suggest that sequence divergence data alone is not enough in inferring bio-geographical patterns (Wen et al.1996).

002-05/1ICPMJ@Karachi University Press

Inti. Chern. Pharrn. Med. J.

Studied have been conducted on the molecular differentiation of Atractylodes drugs by using PCR-RFLP and PCR-Selective Restriction analysis (PCRSR) on the 18S-5.8S rDNA Intra-transcribed spacer region 1 (ITS 1) gene. They amplified 18S-5.8S rDNA Intra-transcribed spacer I-gene regions from Atractylodes japonica, A. lancea and A. ovata rhizomes. They made distinction between Atractylodes species by cloning and sequencing of the PCR products. Thus it was concluded that both techniques such as PCR RFLP and PCR- SR analysis on the ITS 1 gene were useful tools for authentication between Atractylodes rhizomes (Cheng et al. 1997).

Researchers also utilized another way, which might be a useful tool for authentication and differentiation of medicinal herbs mainly medicinal Dendrobium species from one another and there adulterants is the sequences of the internal transcribed spacers 2 (ITS 2) of ribosomal DNA and perhaps these regions could be adopted as a molecular marker for differentiating medicinal herbs from adulterants (Lau et al. 2001).

Chemical analysis and differentiation of three different samples of Cannabis sativa L, were classified as tetrahydrocannabinol (THC) and cannabidiol (CBD) chemotypes. HPLC patterns could not differentiate the two samples of CBD but inter-simple sequence repeat (ISSR) fingerprinting clearly differentiated polymorphic DNA patterns of these samples which were undifferentiated by HPLC analysis (Kojoma et al. 2002).

In order to differentiate medicinal Codonopsis species (Codonopsis pilosula, Codonopsis tangshen, Codonopsis modesta and Codonopsis nervosa var. macrantha) from similar adulterants Campanumoea javania and Platycodon grandiflours, the sequence difference between medicinal species from adulterants was evaluated using PCR-RFLP and ITS. These techniques were found to be effective and reliable (Fu et al. 1999).

Other PCR based technique used is ARMS-PCR. This technique is used to detect point mutations, deletions/insertions, heterozygosity in genomic DNA sequence and genotyping. Through this technique, single nucleotide polymorphisms can be analyzed (Newton et al. 1989).

221

Mansoor Ahmad et ai.

(mouse) were detected by Northern analysis by using 32p-Iabeled cDNA probes. Also Quantitative-PCR analysis was performed for examining gene expression levels (Staudinger et at. 2004).

CONCLUSION

For chemoprofiling and for quality control analysis of herbal drugs, mainly routine pharmacognostic studies are made like evaluating microscopic and macroscopic features of herbal drugs along with HPLC, TLC, HPTLC, GC, Column chromatography, spectrophotometric techniques etc. with the help of these techniques, minor impurities in herbal drug material can be determined and thus ensures better quality of herbal material. Although these techniques are useful tools, they cannot determine the absolute chemical characteristics. and genetic variation in chemical composition of same specie or genotype of medicinal plants. Therefore, there was need for better selection of useful compounds, standard plant specie and analyzing functionality of herbal medicine. Therefore, various molecular biological techniques have provided us better way to authenticate herbal medicines and to study their mechanism of action and effectiveness. In fact, with the help of molecular biological techniques and DNA based markers, significant advances have been made in understanding the mechanism of action, analyzing the usefulness, effectiveness, standard specie detection and confirmation of genetic make up of standard herbal medicine in purified as well as crude form. This advancement is much helpful in quality control analysis and ensures better authentication of herbal drugs.

REFERENCES

Amarger, Y., Mercier, L. Molecular analysis of RAPD DNA based markers: their potential use for the detection of genetic variability in jojoba (Simmondsia chinensis L Schneider). Biochimie., 77(12): 931-936 (1995).

Baum, B.R., Mechanda, S., Livesey, J.F., Binns, S.E., Amason, J.T. Predicting quantitative phytochemical markers in single Echinacea plants or clones from their DNA fingerprints. Phytochemistry., 56(6): 543-549 (2001).

Cai, Z.H., Li, P., Dong, T.T., Tsim, K.W. Molecular diversity of 5S-rRNA spacer domain in FritiIIaria species revealed by PCR analysis. Planta Med., 65(4): 360-364 (1999).

Cao, H., But, P.P., Shaw, P. Authentication of the Chinese drug "ku-di-dan" (herba elephantopi) and its substitutes

226

Inti. Chern. Pharm. Med. J.

using random-primed polymerase chain reaction (PCR). fao Xue Xue Bao., 31(7): 543-553 (1996).

Cao, H., But, P.P., Shaw, P. Identification of herba taraxaci and its adulterants in Hong Kong market by DNA fingerprinting with random primed PCR). Zhongguo Zhong fao Za Zhi., 22(4): 197-200,253 (1997).

Cao, H., Liu, Y., Fushimi, H., Komatsu, K. Identification of notoginseng (Panax notoginseng) and its adulterants using DNA sequencing. Zhong fao Cai, 24(6): 398-402 (2001).

Chan, T.W., But, P.P., Cheng, S.W., Kwok, LM., Lau, F. W., Xu, H.X. Differentiation and authentication of Panax ginseng, Panax quinquefolius, and ginseng products by using HPLC/MS. Anal Chem, 72(6): 12811287 (2000).

Chen, K.T., Su, Y.c., Lin, J.G., Hsin, L.H., Su, Y.P., Su, C.H., Li, S.Y., Cheng, J.H., Mao, SJ. Identification of Atractylodes plants in Chinese herbs and formulations by random amplified polymorphic DNA. Acta. Pharmacol. Sin., 22(6): 493-497 (2001).

Cheng, H.F., Lai, B., Chan S.c., Chou C.P., et ai. Molecular Differentiation of Atractylodes Drugs by PCR

Restriction Fragment Length Poly-morphism and PCR-Selective Restriction Analysis on the 18S-5.8S rDNA Intratranscribed Spacer 1 Gene. Journal of Food and Drug Analysis. 5(4): 319-328 (1997).

Cheng, K.T., Su, B., Chen, CT., Lin, c.c. RAPD analysis of Astragalus medicines marketed in Taiwan. Am. J. Chin. Med., 28(2): 273-278 (2000).

Cheng, K.T., Tsay, H.S., Chen, c.F., Chou, T.W. Determination of the components in a Chinese prescription, yu-ping-feng san, by RAPD analysis. Planta Med., 64(6): 563-565 (1998).

Cheng, Z., Lu, B.R., Baldwin, B.S., Sameshima, K., Chen, J.K. Comparative studies of genetic diversity in kenaf (Hibiscus cannabinus L.) varieties based on analysis of agronomic and RAPD data. Hereditas., 136(3): 231239 (2002).

Cheung, K.S., Kwan, H.S., But, P.P., Shaw, P.c. Pharmacognostical identification of American and Oriental ginseng roots by genomic fingerprinting using arbitrarily primed polymerase chain reaction (AP-PCR). J. Ethnopharmacol., 42(1): 67-69 (1994).

Eckert, K.A. and Kunkel, T.A. High fidelity DNA synthesis by the Thermusaquaticus DNA polymerase. Nucleic Acids Res., 18: 3739-3744 (1990).

Ekins R. and Chu, F.W. Microarrays: their origins and applications. Trends in Biotechnology, 17: 217-218 (1999).

Farooqui, N., Ranade, S.A., Sane, P.Y. RAPD profile variation amongst provenances of neem. Biochem. Mol. BioI. Int., 45(5): 931-939 (1998).

Fu, R.Z., Wang, J., Zhang, Y.B., Wang, Z.T., But, P.P., Li, N., Shaw, P.c. Differentiation of medicinal Codonopsis species from adulterants by polymerase chain reaction

Review Article

restriction fragment length polymorphism. Planta Med., 65(7): 648-650 (1999).

Fushimi, H., Komatsu, K., Isobe, M., Namba, T. Application of PCR-RFLP and MASA analyses on 18S ribosomal RNA gene sequence for the identification of three Ginseng drugs. Biol. Pharm. Bull., 20(7): 765-769 (1997).

Grattapaglia, D., Sederoff, R. Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo-testcross: mapping strategy and RAPD markers. Genetics., 137(4): 1121-1137 (1994).

Ha, W.Y., Shaw, P.c., Liu, J., Yau, F.C., Wang, J. Authentication of Panax ginseng and Panax quinquefolius using amplified fragment length polymorphism (AFLP) and directed amplification of minisatellite region DNA (DAMD). J. Agric. Food Chem., 50(7): 1871-1875 (2002).

Hasegawa Y, Takeda S, Ichii S, Koizumi K, Maruyama M, Fujii A, Ohta H, Nakajima T, Okuda M, Baba S, et al. Detection of K-ras mutations in DNAs isolated from feces of patients with colorectal tumors by mutantallele-specific amplification (MASA). Oncogene, 10(7): 1441-1445 (1995).

Hon, C.C., Chow, Y.c., Zeng, F.Y., Leung, F.C.C. Genetic authentication of ginseng and other traditional Chinese medicine. Acta. Pharmacol. Sin., 24(9): 841-846 (2003).

Hosokawa, K., Minami, M., Kawahara, K., Nakamura, I., Shibata, T. Discrimination among three species of medicinal Scutellaria plants using RAPD markers. Planta Med., 66(3): 270-272 (2000).

Hu, c.Y., et al. Effect of freezing of the PCR buffer on the amplification specificity: Allelic exclusion andpreferential amplification of contaminating molecules. CR. Methods Appl., 2: 182-183 (1992).

Jenkins, R.E., Pennington, S.R. Arrays for protein expression profiling: towards a viable alternative to two-dimensional gel electrophoresis? Proteomics, 1(1): 13-29 (2001). Review.

Jia, Y., Yang, Y., Zhou, Y., Song, Y., Liu, L., Song, J., Wang, X., Zhong, L., Yu, X. (Differentiation of rat bone marrow stromal cells into neuron induced by baicalin). Zhonghua Yi Xue Za Zhi., 82(19): 1337-1341 (2002).

Kalpana, J., Preeti, C., Dnyaneshwar, W., and Bhushan, P. Molecular markers in herbal drug technology. Current Science, 87(2): 159-165 (2004).

Kapteyn, J., Goldsbrough, B., Simon, E. Genetic relationships and diversity of commercially relevant Echinacea species. Theor. Appl. Genet., 105(2-3): 369-376 (2002).

Kojoma, M., Iida, 0., Makino, Y., Sekita, S., Satake, M. DNA fingerprinting of Cannabis sativa using intersimple sequence repeat (ISSR) amplification. Planta Med., 68(1): 60-63 (2002).

002-0SIIICPMJ@Karachi University Press

IntI. Chern. Pharrn. Med. J.

Kwok, S. Procedures to minimize PCR-Product carry-over. In: PCR protocols. A guide to methods and

applications, eds. Innis, M.A., Gelfand, D.H., Sninsky, U., and White, T.J. Academic Press, Inc., San Diego, CA., pp. 142-145 (1990).

Lau, D.T., Shaw, P.c., Wang, J., But, P.P. Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA. Planta Med., 67(5): 456-460 (2001).

Lawyer, F.C., Stoffel, S., Saiki, R.K., Myambo, K.B., Drummond, R., and Gelfand, D.H. Isolation, characterization and expression in Kcoli of the DNA polymerase gene from the extreme thermophile, Thermus aquaticus. J. Biol. Chem., 264: 6427-6437 (1989).

Lee, S.M., Li, M.L., Tse, Y.c., Leung, S.c., Lee, M.M., Tsui, S.K., Fung,. K.P., Lee, c.Y., Waye, M.M. Paeoniae radix, a Chinese herbal extract, inhibit hepatoma cells growth by inducing apoptosis in a p53 independent pathway. J. Life Sci., 71(19): 2267-2277 (2002).

Li, Y.F., Li, y.x., Lin, J., Xu, Y., Wan, F., Tang, L., Chen, F. Identification of bulb from FritiIlaria cirrhosa by PCR with specific primers. Planta Med., 69(2): 186 188 (2003).

Mark Blumenthal. Guidelines for the assessment of herbal medicines are provided in Quality assurance of pharmaceuticals: a compendium of guidelines and related materials. Vol. 1. Geneva, World Health Organization, 31-37 (1997).

Messmer, M., Berger, K., Simmen, D., Parler, N., Hasler, c., Schaffiner, W., Buter, B. Oral Presentation, WOCMAP II Conference in Budapest, Hungary (2001).

Mizukami, H., Okabe, Y., Kohda, H.,c Hiraoka, N. Identification of the crude drug atractylodes rhizome (Byaku-jutsu) and atractylodes lancea rhizome (Sojutsu) using chloroplast TrnK sequence as a molecular marker. Biol. Pharm. Bull., 23(5): 589-594 (2000).

Mullis, K., et al. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold

Spring Harb. Symp. Quant. Biol., 51(1): 263-273 (1986). Nakai, R. Shoyama, Y., Shiraishi, S. Genetic characterization

of Epimedium species using random amplified polymorphic DNA (RAPD) and PCRrestriction fragment length polymorphism (RFLP) diagnosis. Biol. Pharm. Bull., 19( 1): 67-70 (1996).

Negi, M.S., Singh, A., Lakshmikumaran, M. Genetic variation and relationship among and within Withania species as revealed by AFLP markers. Genome, 43(6): 975-980 (2000).

Nei, M. Genetic distances between populations. Am. Nature, 106: 83-92 (1992). Newton, c.R., Graham, A., Heptinstall, L.E., Powell, S.1., Summers, C., Kalsheker, N., Smith, J.c., Markham, A.F. Nucleic Acids Res., 17(7): 2503-2516 (1989).

227

Mansoor Ahmad et al.

Ngan, E, Shaw, P., But, P., Wang, J. Molecular authentication of Panax species. Phytochemistry, 50(5): 787-791 (1999).

Nuwaysir, E.F., Bittner, M., Trent, J., Barrett, J.c., and Afshari, C.A. Microarray and Toxicology: The Advent of Toxicogenomics. Molecular Carcino-genesis, 24:153-159 (1999).

Porter, C.H., Collins, F.H. Species-diagnostic difference in a ribosomal NA internal transcribed spacer from the sibling species Anopheles freeborni and Anopheles hermsi (Diptera: Culicidae). American Journal of Tropical Medicine and Hygiene, 45: 271-279 (1991).

Rongwen, J. et al. The use of microsatellite DNA markers for soybean genotype identification. Theor Appl Genet, 90: 43-48 (1995).

Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.1., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239: 487-491 (1988).

Sangwan, R.S., Sangwan, N.S., Jain, D.C., Kumar, S., Ranade, S.A. RAPD profile based genetic characterization of chemotypic variants of Artemisia annua L. Biochem. Mol. BioI. Int., 47(6): 935-944 (1999).

Sasaki, Y., Fushimi, H., Cao, H., Cai, S.Q., Komatsu, K. Sequence analysis of Chinese and Japanese Curcuma drugs on the 18S rRNA gene and trnK gene and the application of amplification-refractory mutation system analysis for their authentication. Bioi. Pharm. Bull., 25(12): 1593-1599 (2002).

Schena, M., Shalon, D., Davis, R.W., Brown, P.O. (Stanford) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 20, 270(5235): 467-70 (1995).

Shaw, P.c., But, P.P. Authentication of Panax species and their adulterants by random-primed polymerase chain reaction. Planta Med., 61(5): 466-469 (1995).

Shibata K, Mori M, Kitano S, Akiyoshi T. Detection of ras gene mutations in peripheral blood of carcinoma patients using CD45 immunomagnetic separation and nested mutant allele specific amplification. Int. J.

Oncol., 12: 1333-1338 (1998). . Shibing SU et al. Suppressive effect of herbal medicine TJ-

10 on acinar cell apoptosis in rat spontaneous chronic pancreatitis. American J. Traditional Chinese Medicine (2001).

Staudinger, J.L. Guggulsterones activate ER, PR, and PXR 535. J. Pharmacol. Experimantal Therapeutics, 310(2): 528-535 (2004).

Suggs, S.V., et al. Use of synthetic oligodeoxyribonucleotides for the isolation of specific cloned DNA sequences. ICN-uCLA Symp. Mol. Cell Bioi., 23: 683 (1981).

Sugimoto, N., Kiuchi, E, Mikage, M., Mori, M., Mizukami, H., Tsuda, Y. DNA profiling of Acorus calamus

228

IntI. Chern. Pharm. Med. J.

chemotypes differing in essential oil composition. Bioi. Pharm. Bull., 22(5): 481-485 (1999).

Tochika-Komatsu, Y., Asaka, I., Ii, I. A random amplified polymorphic DNA (RAPD) primer to assist the identification of a selected strain, aizu K -111 of Panax ginseng and the sequence amplified. BioI. Pharm. Bull., 24(10): 1210-1213 (2001).

Tolonen, A., Uusitato, J., Hohtola, A., and Jalonen, J. The determination of Naphthdianthrones and Phloroglucinols from Hypericum Perforatum extract by HPLCffandem MS. RAPD Comm. In MS., 16: 396-402 (2002).

.Um, J.Y., Chung, H.S., Kim, M.S., Na, H.1., Kwon, H.1., Kim, J.1., Lee, K.M. et al. Molecular authentication of Pan ax ginseng species by RAPD analysis and PCR-RFLP. Bioi., Pharm. Bull., 24(8): 872-875 (2001).

Van Droogenbroeck, B., Breyne, P., Goetghebeur, P., Romeijn-Peeters, E., Kyndt, T., Gheysen, G. AFLP analysis of genetic relationships among papaya and its wild relatives (Caricaceae) from Ecuador. Theor. Appl. Genet., 105(2-3): 289-297 (2002).

Vieira, R.F., Grayer, R.J., Paton, A., Simon, J.E. Genetic diversity of Ocimum gratissimum L. based on volatile oil constituents, flavonoids and RAPD markers. Biochem. Syst. Ecol., 29(3): 287-304 (2001).

Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van de Lee, T., Homes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., et al. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res., 23(21): 4407-4414 (1995).

Wang, C.Z., Li, P., Ding, J.Y., Jin, G.Q., Yuan,C.S. Identification of Fritillaria pallidiflora Using Diagnostic PCR and PCR-RFLP Based on Nuclear Ribosomal DNA Internal Transcribed Spacer Sequences. Planta Med., 71(4): 384-386 (2005).

Wang, J., Ha, W.Y., Ngan, EN., But, P.P., Shaw, P.C. Application of sequence characteriied amplified region (SCAR) analysis to authenticate Pan ax species and their adulterants. Planta Med., 67(8): 781-783 (2001).

Welsh, J., McClelland, M. Fingerprinting genomes using PCR and arbitrary primers. Nucleic acids Res., 18: 7213-7218 (1990).

Wen, J., Zimmer, E.A. Phylogeny and biogeography of Pan ax L. (the ginseng genus, araliaceae): inferences from ITS sequences of nuclear ribosomal DNA. Mol. Phylogenet. Evol., 6(2): 167-177 (1996).

Wilcox, J.N. Fundamental Principles of in situ hybridization. J. Histochem. Cytochem., 41: 1725-1733 (1993).

Wisneski, H.H. Determination of Coumarin in Fragrance Products by Capillary Gas Chromato-graphy with Electron Capture Detection. J. AOAC Int., 84(3): 689692 (2001).

Wright, J.M. and P. Bentzen. Microsatellites: genetic markers for the future. Reviews in Fish Biology and Fisheries., 4: 384-388 (1994).

002-0SIIICPMJ@Karachi University Press

Review Article

Yang, D.Y., Fushimi, H., Cai, S.Q., Komatsu, K. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) analyses of medicinally used Rheum species and their application for identification of Rhei Rhizoma. BioI. Pharm. Bull., 27(5): 661-669 (2004).

Zhang, K.Y., Leung, H.W., Yeung, H.W., Wong, R.N. Differentiation of Lycium barbarum from its related Lycium species using random amplified polymorphic DNA. Planta Med., 67(4): 379-381 (2001).

Zhang, Y.B., Wang, J., Wang, Z.T., But, P.P., Shaw, P.c. DNA microarray for identification of the herb of dendrobium species from Chinese medicinal formulations. Planta Med., 69(12): 1172-1174 (2003).

Zhao, A.G., Zhao, H.L., Jin, X.J., Yang, J.K., Tang, L.D. Effects of Chinese Jianpi herbs on cell apoptosis and related gene expression in human gastric cancer grafted

002-05IIICPMJ@Karachi University Press 229

Inti. Chern. Pharm. Med. J.

onto nude mice. World J. Gastroenterol., 8(5): 792-796 (2002).

Zhao, K.J., Dong, T.T., Cui, X.M., Tu, P.F., Tsim, K.W. Genetic distinction of radix adenophorae from its adulterants by the DNA sequence of 5S-rRNA spacer domains. Am. J. Chin. Med., 31(6): 919-926 (2003).

Zhu, S., Fushimi, H., Cai, S., Komatsu, K. Species identification from Ginseng drugs by multiplex amplification refractory mutation system (MARMS). Planta Med., 70(2): 189-192 (2004). Zurer, P. DNA profiling fast becoming accepted tool for

identification. Chemical and Engineering News, pp.8 15 (1994).

Manuscript received 03 - 05 - 2005 Accepted for publication 02 - 07 - 2005