Authentication of shankhpushpi by RAPD markers

Giriş: Çok eski bir Hint tıb sistemi olan Ayurveda’da önemli bir yerel ilaç olarak kullanılan “Shankhpushpi”, hafıza gücünü ve zekayı artırmaktadır. Uykusuzluk, sara, halüsinasyon ve anksiyete rahatsızlıkları için, tek başına veya başka otlarla kombinasyon halinde bir çok Ayurvedik formülde kullanılmaktadır. Ülkenin değişik bölgelerinde, en azından üç farklı bitki türü, yani Clitoria ternatea, Convolvulus pluricaulis ve Evolvulus alsinoides, bu ilacın kaynağı olarak kullanılmaktadır. Yüksek talep ve artan fiyatlar nedeniyle, shankhpushpi’ye diğer benzer türler karıştırılmaktadır. Bu nedenle, hakiki malzemeyi karışım için kullanılanlardan ayırt etmek için güvenilir bir doğrulama metoduna ihtiyaç vardır. Bu çalışma, C. pluricaulis, E. alsinoides ve C. ternatea’nın tanımlanması ve ilacın pazardan alınan örneklerinin hakikiliğini saptanması için RAPD tabanlı markörler geliştirmeyi amaçlamaktadır. Materyal ve Metot: Ghaziabad ve Delhi’den, Shankhpushpi’nin kaynak bitkilerinin taze örnekleri toplandı. Pazar örnekleri, Hindistan’ın farklı coğrafi bölgelerindeki ham-ilaç pazarlarından elde edildi. Hakiki ve pazar örneklerinin karakterizasyonu için RAPD tekniği kullanıldı. İzole edilen DNA’nın çoğaltılması için, yirmi beş 11-mer oligonükleotit primeri kullanıldı. Bulgular: 25 primerden sadece dört tanesi (OPN-03, OPN-04, OPN-05 ve OPN-06), pazar örneklerinin karakterizasyonu için kullanılan temiz ve tekrarlanabilir bantlar oluşturan çoğaltım ürünü verdi. Pazar ürünlerinden bazılarının RAPD profili, hakiki örneklerle eşleşmedi ve bu durum, bu örneklerin ya karışımlı veya sahte olduğunu göstermektedir. Sonuç: Bu çalışmada geliştirilen RAPD markörleri, shankhpushpi olarak satılan bitki materyallerinin doğrulanmasında yol gösterici olabilir.

RAPD markörleri ile shankhpushpi’nin doğrulanması

Background: “Shankhpushpi”, an important indigenous drug of Ayurveda, an ancient system of Indian medicine, improves memory power and intellect. It is used in many Ayurvedic formulations, either singly or in combination with other herbs, meant for sleeplessness, epilepsy, hallucinations and anxiety. At least three different plant species viz., Clitoria ternatea, Convolvulus pluricaulis and Evolvulus alsinoides are used as the source of this drug in the different parts of the country. Because of increased demand and high price, shankhpushpi is often adulterated in the trade by other related species. Therefore, a reliable authentication method is needed to facilitate differentiation/ identification of the genuine material from its adulterants. The present study was aimed at developing RAPD-based markers for identification of C. pluricaulis, E. alsinoides and C. ternatea, and analyzing the market samples of the drug to ascertain their authenticity. Materials and Methods: Fresh samples of source plants of shankhpushpi were collected from Ghaziabad and Delhi. The market samples were procured from the crude-drug markets of different geographical regions of India. The amplified polymorphic DNA (RAPD) technique was employed for characterization of genuine and market samples. Twenty-five 11-mer oligonucleotide primers were used to amplify the DNA isolated. Results: Out of 25 primers, only four (OPN-03, OPN-04, OPN-05 and OPN-06) yielded amplification products that produced clear and reproducible bands, which were used to characterize the market samples. RAPD profile of some market samples did not match with the authentic samples, indicating that these samples were either adulterated or spurious. Conclusions: The RAPD markers developed in this study may provide guidance for the authentication of plant materials traded as shankhpushpi.

Kaynakça

Cao H, But PP, Shaw PC (1996) Authentication of the Chinese drug “Ku-di-dan” (Herba elephantopi) and its substitutes using random primed polymerase reaction (PCR). Acta Pharmaceutica Sinica 31: 543-553.

Chan K (2003) Some aspects of toxic components in herbal medicine. Chemosphere 52: 1361-1371.

Chen KT, Su YC Lin JG Hsin LH Su YP Su CH, Li SY, Cheng JH, Mao SJT (2001) Identification of Atractylodes plants in Chinese herbs and formulations by random amplified polymorphic DNA. Acta Pharmacologica Sinica 22: 493-497.

Cheng KT, Chang HC, Su CH, Hsu FL (1997) Identification of dried rhizomes of Coptis species using random amplified polymorphic DNA. Botanical Bulletin of Academia Sinica 38: 241-244.

Chuneker KC (1982) Bhavaprakasa Nighantu. Chaukhambha Bharti Academy, Varanasi, India. Daniel M (2004) Impediments preventing India from becoming an herbal giant. Current Science 87: 275-276.

Dey AC (1980) Indian Medical Plants used in Ayurvedic Preperations. Bishen Singh-Mahendra Pal Singh, Dehradun.

Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12: 13-15. Ellsworth DL, Rittenhouse D, Honeycutt RL (1993) Artifactual variation in random amplified polymorphic DNA banding patterns. Biotechniques 14: 214-217.

Feng T, Liu S, He X-J (2010) Molecular authentication of the traditional Chinese medicinal plant Angelica sinensis based on internal transcribed spacer of nrDNA. Electronic Journal of Biotechnology 13, 1. doi: 10.2225/vol13-issue1- fulltext-13

Hammad I, Qari SH (2010) Genetic diversity among Zygophyllum (Zygophyllaceae) populations based on RAPD analysis. Genetics and Molecular Research 9: 2412-2420. doi: 10.4238/vol9-4gmr1144

Harini SS, Leelambika M, Kameshwari MN, Sathyanarayana N (2008) Optimization of DNA isolation and PCR-RAPD methods for molecular analysis of Urginea indica Kunth. Journal of Integrative Biology 2: 138-144.

Heubl G (2010) New aspects of DNA-based authentication of Chinese medicinal plants by molecular biological techniques. Planta Medica 76: 1963-74.

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

Khan S, Mirza KJ, Abdin MZ (2010) Development of RAPD markers for authentication of medicinal plant Cuscuta reflexa. EurAsian Journal of Biosciences 4: 1-7. doi:10.5053/ejobios.2010.4.0.1

Khan S, Mirza KJ, Abdin MZ (2011) DNA fingerprinting for the authentication of Ruta graveolens. African Journal of Biotechnology 10(44): 8709-8715.

Khan S, Mirza KJ, Tayyab, Abdin MZ (2009) RAPD profile for authentication of medicinal plant Glycirrhiza glabra L. Medicinal and Aromatic Plant Science and Biotechnology 3: 48-51.

Kumble KD (2003) Protien microarrays, new tools for pharmaceutical development. Analytical and Bio Analytical Chemistry 377: 812-819.

Li YF, Li XY, Lin J, Xu Y, Yan L, Tang F, Chen F (2003) Identification of bulb from Frittillaria cirrhosa by PCR with specific primers. Planta Medica 69: 186-188.

Liu P, Yang YS, Hao CY, Guo WD (2007) Ecological risk assessment using RAPD and distribution pattern of a rare and endangered species. Chemosphere 68(8): 1497-1505. doi: 10.1016/j.chemosphere.2007.03.018

Micheli MR, Bova R, Pascale E, D’Ambrosio E (1994) Reproducible DNA fingerprinting with the random amplified polymorphic DNA (RAPD) method. Nucleic Acids Research 22: 1921-1922.

Moose NS (1978) Ayurvedic Flora Medica. Vaidyasarathy Press, Kottayam, Kerala.

Nadkarni KM (1976) Indian Materia Medica. Popular Prakashan Pvt. Ltd, Bombay.

Nair KV, Nair AR, Nair CPR (1997) Standardization of Ayurvedic drugs. Bulletin of Medico-Ethno Botanical Research 18: 151-156.

Rivera-Arce E, Gattuso M, Alvarado R, Zarate E, Aguero J, Feria I, Lozoya X (2007) Pharmacognostical studies of the plant drug Mimosa tenuiflorae cortex. Journal of Ethnopharmacology 113(3): 400-408. doi: 10.1016/j.jep.2007.06.023

Shaw PC, But PPH (1995) Authentication of Panax species and their adulterants by random primed polymerase chain reaction. Planta Medica 61: 466-469.

Shinde M, Dhalwal K, Mahadik KR, Joshi KS, Patwardhan BK (2007) RAPD Analysis for Determination of Components in Herbal Medicine. Evidence Based Complementary and Alternative Medicine 4: 21-23. doi: 10.1093/ecam/nem109

Shioda H, Satoh K, Nagai F, Okubo T, Seto T, Hamano T, Kamimura H, Kano I (2003) Identification of Aloe species by random amplified polymorphic DNA (RAPD) analysis. Journal of the Food Hygienic Society of Japan 44: 203-207.

Singh TB, Chuneker KC (1972) Glossary of Vegetable Drugs. Brhattrayi, Chowkhamba Sanskrit Series Varanasi.

Yip PY, Chau CF, Mak CY, Kwan HS (2007) DNA methods for identification of Chinese medicinal materials. Chinese Medicine 2, 9. doi: 10.1186/1749-8546-2-9

Wolf HT, Zundorf I, Winckleer T, Bauer R, Dingermann T (1999) Characterization of Echinacea species and detection of possible adulterations by RAPD analysis. Planta Medica 65: 773-774.

Zhang HY, Li FS, Liu XZ, He LL, Yang QH, He SC (2008) Analysis of genetic variation in Erianthus arundinaceum by random amplified polymorphic DNA markers. African Journal of Biotechnology 7(19): 3414-3418.

Zhang KYB, Leung HW, Yeung HW, Wong RNS (2001) Differentiation of Lycium barbarum from its related Lycium species using random amplified polymorphic DNA. Planta Medica 67: 379-381.

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