Qualitative and Quantitative Determination of Tryptanthrin, Indirubin, Indican and Isatin Indole Alkaloids During Vegetative and Flowering Stages in the Roots and Leaves of Isatis constricta P. H. Davis

Öz

Isatis genus more known as the indigo plant is a plant belonging to the Brassicaceae family in Turkey. Turkey has 31 types and 15 subtypes in various provinces.18 of these species and 3 of the subspecies are assumed as endemic. All taxa of the genus are extensively used in dyeing and treatment of various infections. They contain large number of secondary compounds like phenolics, flavonoids and alkaloids in root, leaf, flower and fruit parts. Among these indole alkaloids like triptanthrin, indirubin, indican and isatin are used in treatment of many diseases. These are obtained from the leaves and roots are pharmacologically active compounds. This study revealed that this biannual herbaceous plant grows 30 - 50 cm in length on volcanic slopes, at 1200 - 1300 meters above sea level. It generally grows in Eastern Anatolia and the Mediterranean region of Turkey. The study aimed to determine optimum ontogenetic stage of Isatis constricta to extract natural bio compounds like indirubin, isatin, indican, tryptanthrin. The leaf and root samples used in this study were collected from Maden county of Elazığ province. They were tested for the presence of these compounds during vegetative and flowering period of the plant. The HPLC (HighPerformance Liquid Chromatography) analysis results showed that tryptanthrin 0.482 (±0.0072) µg/g and indirubin 0.029 (±0.00007) μg/g has the highest concentrations in the leaf samples obtained during the vegetative period. However, isatin 4.358 (± 0.0985) μg/g and indican 21.330 (± 0.114) μg/g were found to have the highest concentration in leaf samples obtained during flowering period. When both results are compared, it can be seen that the amount of tryptanthrin, isatin, indirubin and indican alkaloids were found in the leaves of I. constricta higher than to the roots. This research idicate that the most appropriate development stage of I. constricta to obtain triptanthrin indirubin, indican and isatin compounds.

Anahtar Kelimeler

• Tryptanthrin
• indirubin
• indican
• isatin
• indole alkaloids

Isatis Constricta P. H. Davis’in Köklerinde ve Yapraklarında Vejetatif ve Çiçeklenme Dönemlerinde Triptanthrin İndirubin, İndican ve İsatin İndol Alkaloidlerinin Kalitatif ve Kantitatif Tayini

Öz

Türkiye’de daha çok çivit otu olarak bilinen İsatis cinsi Brassicaceae familyasına ait bir bitkidir. Türkiye’nin çeşitli yerlerinde bu bitkiye ait 31 tür ve 15 alt türü bulunmaktadır. Bu türlerin 18 tanesi ve alttürlerin 3 tanesi endemiktir. Cinsin bütün taksonları çeşitli enfeksiyonların tedavisinde ve boyamada yaygın olarak kullanılmaktadır. Kök, yaprak, çiçek ve meyve kısımlarında fenolikler, flavonoidler ve alkaloidler gibi çok sayıda ikincil bileşik bulunmaktadır. Bunlar arasında triptanthrin, indirubin, indican ve isatin gibi alkaloidler birçok hastalığın tedavisinde kullanılmaktadır. Bunlar hem yapraklar hem de köklerden elde edilen farmakolojik olarak aktif bileşiklerdir. Bu çalışmada kullanılan bitkisi 30 - 50 cm kadar boylanan, deniz seviyesinden 1200 - 1300 metre yüksekte volkanik yamaçlarda bulunan iki yıllık önemli otsu bir bitkidir. Bu bitki genellikle Doğu Anadolu ve Türkiye’nin Akdeniz bölgesinde doğal olarak yetişmektedir. Bu çalışmanın amacı, Isatis constricta ’da bulunan indirubin, isatin, indican, triptanthrin gibi doğal bileşikleri elde etmek için bitkinin en uygun gelişim dönemini belirlemek ve bu bileşiklerin bitkinin hangi kısımlarında daha fazla miktarda bulunduklarını tespit etmektir. Bu çalışmada kullanılan yaprak ve kök örnekleri Elazığ ili Maden ilçesinden toplanmıştır. Test edilen bu örnekler bitkinin vejetatif ve çiçeklenme dönemlerinde toplanılmıştır. HPLC (Yüksek Performanslı Sıvı Kromatografisi) analiz sonuçlarıyla, triptanthrin 0.482 (±0.0072) µg/g ve indirubinin 0.029 (±0.00007) μg/g bileşikleri vejetatif döneminde elde edilen yaprak örneklerinde en yüksek konsantrasyonlara sahip olduğu tespit edilmiştir. Ancak isatin 4.358 (± 0.0985) μg/g ve indican 21.330 (± 0.114) μg/g çiçeklenme döneminde elde edilen yaprak örneklerinde en yüksek konsantrasyona sahip oldukları tespit edilmiştir. Her iki sonuca da bakıldığında I. constricta’nın yapraklarında bulunan triptantrin, Isatin, indirubin ve indican alkaloidlerinin miktarı, köklerine oranla daha yüksek olduğu görülmektedir Bu çalışma sonucunda triptanthrin, indirubin, indican ve isatini elde etmek için I. constricta’nın en uygun gelişim dönemi ile bitki kısımları belirlenmiştir. 

Anahtar Kelimeler

• Tryptanthrin
• indirubin
• indican
• isatin
• indol alkaloidler

Kaynakça

1. Alex, D., Lam, I. K., Lin, Z. X., Lee, S. M. Y. (2010). Indirubin Shows Anti-Angiogenic Activity in an In Vivo Zebrafish Model and an In Vitro HUVEC Model. Journal of Ethnopharmacology, 131, 242–247.
2. Anonymous. (2016a). https://en.wikipedia.org/wiki/ındic that. Sirnak. Accessed on 27.11.2016.
3. Anonymous. (2016b) The https://en.wikipedia.org/wiki/ısat. Sirnak. Accessed on 27.11.2016.
4. Bown, D. (1995). Encyclopedia of Herbs and Their Uses. London: Dorling Kindersley, 361-365.
5. Chang, Y. S., Ho, Y. L. (2001). Studies on the Homonymic Chinese Crude Drug Species in Taiwan. Evaluation of the Quality of DA-Ching-Yeh and Ching-Dai. Analytical Sciences, 17, 423-426.
6. Davis, P.H. (ed.). 1965-1985. Flora of Turkey and the East Aegean Islands, vol.1-9, Edinburgh.
7. Ensley, B. D., Ratzkin B. J., Osslund T. D., Simon M. J., Wackett L. P., Gibson D. T. (1983). Expression of Naphthalene Oxidation Genes in Escherichia Coli Results in the Biosynthesis of Indigo. Science, 222, 167-169.
8. Guo, Y., Chen, F. (1986). TLC-UV-Spectrophotometric and TLC-Scanning Determination of Isatin TLC-UV-Spectrophotometric and TLC-Scanning Determination of Isatin in Leaf of Isatis L., Zhongcaoyao, 17: 8-11.

9. Güner A, Aslan S, Ekim T, Vural M, Babaç MT (2012). Türkiye Bitkileri Listesi (Damarlı Bitkiler). İstanbul, Turkey: Flora Araştırmaları Derneği ve Nezahat Gökyiğit Botanik Bahçesi Yayını (in Turkish).
10. Hamburger, M. (2002). Isatis tinctoria - From the Rediscovery of an Ancient Medicinal Plant Towards a Novel Anti-Inflamatory Phytopharmaceutical. Phytochemistry Reviews, 1: 333-344.
11. Hoessel, R., Leclerc, S., Endicott, J. A., Nobel M.E.M., Laurie, A., Tunnah, P., … Meijer, L. (1999). Indirubin, the Active Constituent of a Chinese Antileukemia Medicine, Inhibits Cyclin-Dependent Kinases. Nature Cell Biology, 1: 60–67.
12. Kimoto, T., Hino, K., Koya-Miyata, S., Yamamoto, Y., Takeuchi, M., Nishizaki, Y., Kurimoto, M., (2001). Cell Differentiation and Apoptosis of Monocytic and Promyelocytic Leukemia Cells (U-937 and HL-60) By Tryptanthrin, an Active Ingredient of Polygonum Tinctorium Lour. Pathology International, 51: 315-325
13. Kırıcı, S. (1998). Dyestuffs Obtained from Natural Waters. Ç.Ü.Z.F. Magazine, 9-11.
14. Liau, B. C., Jong, T. T., Lee, M. R., Chen, S. S. (2007). LC-APCI-MS Method for Detection and Analysis of Tryptanthrin, Indigo, and Indirubin in Daqingye and Banlangen. Journal of Pharmaceutical and Biomedical Analysis, 43: 346-351.
15. Mısırdalı, H. (1985). Taxonomic and Cytological Investigations on the Species of Isatis L., grown in the Eastern and South Eastern Anatolia and Over the Regions of Eastern Mediterranean. TÜBİTAK Scientific Research Group, Project No: TBAG-535, Eskişehir, 139.
16. Medvedev, A., Buneeva, A., Glover, V. (2007). Biological Targets for Isatin and Its Analogues, Implications for Therapy, Biologics. 1(2): 151–162.
17. Oberthür, C., Schneider, B., Graf, H., Hamburger, M. (2004). The Elusive Indigo Precursors in Woad (I. tinctoria L.) Identification of the Major Indigo Precursor, Isatan A, and A Structure Revision of Isatan B. Chemistry & Biodiversity, 1: 174-182.
18. Oksay, D., Oksay, M. (2009). Bitki Sekonder Metabolitlerinin Biyoteknolojik Önemi e-Journal of New World Sciences Academy Ecological Life Sciences, 4: 31-41.
19. O’Neill, C. (1892). Products from Indigo-Blue. Chemistry News, 65: 124.
20. Ozgen, M., Ertunc, F., Kinaci, G., Yildiz, M., Birsin, M., Ulukan, H., Sancak, C. (2005). New in Agricultural Technologies Approaches and Applications, Plant Biotechnology. Turkey Agricultural Engineering VI. Technical Congress, Ankara, Vol. 1, 315–346.
21. Recio, M. C., Cerdá-Nicolás, M., Potterat, O., Hamburger, M., Ríos, J. L. (2006). Anti-Inflammatory and Antiallergic Activity in Vivo of Lipophilic Isatis tinctoria Extracts and Tryptanthrin. Planta Medica,72: 539-546.
22. Spink, B. C., Hussain, M. M., Katz, B. H., Eisele, L., Spink, D. C. (2003). Transient Induction of Cytochromes P450 1A1 and 1B1 in MCF-7 Human Breast Cancer Cells by Indirubin. Biochemical Pharmacology, 66: 2313–2321.
23. Takei, Y., Kunikata, T., Aga, M., Ushio, S., Iwaki, K., Ikeda, M., Kurimoto, M. (2003). Tryptanthrin Inhibits Interferon-Gamma Production by Peyer’s Patch Lymphocytes Derived from Mice That Had Been Orally Administered Staphylococcal Enterotoxin. Biological and Pharmaceutical Bulletin, 3: 365-367.
24. Vanisree, M., Lee, C.Y., Lo, S.F., Nalawade, S. M, Lin, C. Y., Tsay, H. S. (2004). Studies on the Production of Some Important Secondary Metabolites from Medicinal Plants by Plant Tissue Cultures. Botanical Bulletin of Academia Sinica, 45: 1-22.
25. Yu, S. T., Chen, T. M., Chern, J. W., Tseng, S. Y., Chen, Y. H. (2009). Downregulation of Gstpi Expression by Tryptanthrin Contributing to Sensitization of Doxorubicin-Resistant MCF-7 Cells Through C-Jun NH2-Terminal Kinase-Mediated Apoptosis. Anticancer Drugs, 20: 382-388.
26. Wenying Z; Guijie T; Rongduo Y (2000). TLC Determination of Indican in the Radix Isatidis, Folium Isatidis and Granules of Banlangen. Chinese Journal of Pharmaceutical Analysis, 5, (1):322-323.
27. Zou, P., Koh, H. L. (2007). Determination of Indican, Isatin, Indirubin and Indigotin in Isatis indigotica by Liquid Chromatography/Electrospray İonization Tandem Mass Spectrometry. Rapid Communications in Mass Spectrometry, 21(7): 1239-1246.