Effect of Chemical Elicitors on Pentacyclic Triterpenoid Production in In Vitro Cultures of Achyranthes aspera L.

  • L. Sailo
  • Vinayak Upadhya
  • Poornananda M. Naik
  • Neetin Desai
  • Sandeep R. Pai
  • Jameel M. Al-KhayriEmail author


Bioprocess technology for the production of phytochemicals from plant cell cultures mainly depends upon elicitation for enhancing the yields. The application has been successfully demonstrated in various plant species for a number of metabolites of interest. Achyranthes aspera L. is a highly traded medicinal plant known for a wide array of pharmacological properties. In this study, the effect of different concentrations of salicylic acid (SA), methyl jasmonate (MeJA), jasmonic acid (JA), and chitosan (CH) on growth and accumulation of betulinic acid (BA), oleanolic acid (OA), and ursolic acid (UA) in cultures of A. aspera was investigated using RP-UFLC technique. Results for in vitro cultures grown on various concentrations of selected elicitors (50, 100, and 200 μM) were collected and studied at 7-, 15-, and 30-day intervals. Two separate experiments for elicitors with and without plant growth regulators (PGRs), i.e., 6-benzylaminopurine 3.0 mg/L and thidiazuron 0.5 mg/L, were studied. Higher fresh and dry weights were observed in all the treated tubes as compared to control. Optimum cell growth along with higher BA content was observed in MeJA-treated cultures with and without PGRs. Increased OA content was evident in 30-day cultures growing on 100 and 200 μM MeJA supplemented with PGRs. Chitosan both in media supplemented with and without PGRs produced higher content of UA at 30th day. Furthermore, it becomes important to understand the biochemical conversions in light of the metabolic pathways so that we can use this data in maneuvering production of metabolites in A. aspera.


Achyranthes aspera Elicitation Secondary metabolites Triterpenoids 



All the authors are thankful to their higher authorities for their help and support. SRP is indebted to SERB, DST, New Delhi financial assistance (No. SERB/LS-153/2013) for part of work provided during this work.


  1. Aeri, V., Khan, M. I., & Alam, S. (2010). A validated HPLC method for the quantification of oleanolic acid in the roots of Achyranthes aspera Linn. and marketed formulation. International Journal of Pharmacy and Pharmaceutical Sciences, 2, 74–78.Google Scholar
  2. Ali, M. (1993). Chemical investigation of Achyranthes aspera Linn. Oriental Journal of Chemistry, 9, 84–85.Google Scholar
  3. Anonymous. (2007). The wealth of India – Raw materials (pp. 17–18). New Delhi: Council of Scientific and Industrial Research (CSIR).Google Scholar
  4. Anonymous. (2017). The ayurvedic pharmacopoeia of India. (Part 1, Vol. 2). Department of AYUSH, Ministry of Health and Family Welfare. 09. Available via Accessed 2017.
  5. Aparecida, R. F., de-Andrade-Bareala, C. A., da-Silva-Faria, M. C., & Kato, F. H. (2006). Antimutagenicity of ursolic acid and oleanolic acid against doxorubicin-induced clastogenesis in Balb/c mice. Life Sciences, 79, 1268–1273.CrossRefGoogle Scholar
  6. Aziz, Z. A., Davey, M. R., Power, J. B., Anthony, P., Smith, R. M., & Lowe, K. C. (2007). Production of asiaticoside and madecassocide in Centellaasiatica in vitro and in vivo. Biology in Plants, 51, 34–42.CrossRefGoogle Scholar
  7. Bafna, A. R., & Mishra, S. H. (2004). Effect of methanol extract of Achyranthes aspera Linn. On rifampicin-induced hepatotoxicity in rats. ARS Pharmaceutica, 45, 343–351.Google Scholar
  8. Bhattaraj, N. K. (1992). Folk use of plants in veterinary medicine in Central Nepal. Fitoterapia, 63(6), 497–506.Google Scholar
  9. Bouhouche, N., Solet, J. M., Simon Ramiasa, A., Bonal, J., & Cosson, L. (1998). Conversion of 3-dimethylthiocolchicine into thiocolchicoside by Centella asiatica suspension cultures. Phytochemistry, 47, 743–747.CrossRefGoogle Scholar
  10. Chakraborty, A., Brantner, A., Mukainaka, T., Nobukuni, Y., Kuchide, M., et al. (2002). Cancer chemo preventive activity of Achyranthes aspera leaves on Epstein-Barr virus activation and two stage mouse skin carcinogenesis. Cancer Letters, 177, 1–5.CrossRefPubMedGoogle Scholar
  11. Chao, Z. M., Shang, E. J., He, B., & Zhao, J. (1999). Studies on the chemical constituents of water extract from Achyranthes bidentata. Chinese Pharmaceutical Journal, 34, 587–588.Google Scholar
  12. Cook, T. (1901–1908). The Flora of the presidency of Bombay, 3 vols. London (Reprinted ed. 1958. BSI Calcutta).Google Scholar
  13. Degenhardt, J., Gershenzon, J., Baldwin, I. T., & Kessler, A. (2003). Attracting friend to feast on foes: Engineering terpene emission to make crop plants more attractive to herbivore enemies. Current Opinion in Biotechnology, 14, 169–176.CrossRefPubMedGoogle Scholar
  14. deLange, P. J., Scofield, R. P., & Greene, T. (2004). Achyranthes aspera (Amaranthaceae), a new indigenous addition to the flora of the Kermadec Islands group. New Zealand Journal of Botany, 42, 167–173.CrossRefGoogle Scholar
  15. Dong, Q. Q. (2010). Studies of the constituents of Achyranthes bidentata BL. Master thesis of Yangzhou University, China.Google Scholar
  16. Fontanay, S., Grare, M., Mayer, J., Finance, C., & Duval, R. E. (2008). Ursolic, oleanolic and betulinic acids: Antibacterial spectra and selectivity indexes. Journal of Ethnopharmacology, 120(2), 272–276.CrossRefPubMedGoogle Scholar
  17. Fujii, M., Hirai, Y., Miura, T., Saito, M., Fukumura, M., Hori, Y., Akita, H., Toriizuka, K., & Ida, Y. (2010). Isolation of (S)-N-feruloyl normetanephrine from Achyranthes fauriei and determination of its absolute configuration. Japanese Journal of Pharmacognosy, 64, 26–27.Google Scholar
  18. Gaines, J. L. (2004). Increasing alkaloid production from Catharanthus roseus suspensions through methyl jasmonate elicitation. Pharmaceutical Engineering, 24, 1–6.Google Scholar
  19. Giri, A., & Naraseu, M. L. (2000). Transgenic hairy roots: Recent trends and applications. Biotechnology Advances, 18, 1–22.CrossRefPubMedGoogle Scholar
  20. Gokhale, A. B., Damre, A. S., Kulkami, K. R., & Saraf, M. N. (2002). Preliminary evaluation of anti-inflammatory and anti-arthritic activity of S. lappa, A. speciosa and A. aspera. Phytomedicine, 9(5), 433–437.CrossRefPubMedGoogle Scholar
  21. Gupta, S. S., Bhagwat, A. W., & Ram, A. K. (1972). Cardiac stimulant activity of the saponin of Achyranthes aspera L. The Indian Journal of Medical Research, 60, 462–471.PubMedGoogle Scholar
  22. Harsha, V. H., Hebbar, S. S., Hegde, G. R., & Shripathi, V. (2004). Ethnomedicobotany of Uttara Kannada District, Karnataka state. Bulletin of the BotanicalSurvey of India, 46, 330–336.Google Scholar
  23. Hayashi, H., Huang, P., & Inoue, K. (2003). Up-regulation of soya saponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra. Plant and Cell Physiology, 44, 404–411.CrossRefPubMedGoogle Scholar
  24. He, X., Wangb, X., Fang, J., Chang, Y., Ning, N., Guo, H., Huang, L., & Huang, X. (2017). The genus Achyranthes: A review on traditional uses, phytochemistry, and pharmacological activities. Journal of Ethnopharmacology, 203, 260–278.CrossRefPubMedGoogle Scholar
  25. Hebbar, S. S., Harsha, H. V., Shripathi, V., & Hegde, G. R. (2004). Ethnomedicine of Dharwad District in Karnataka, India- plants in oral healthcare. Journal of Ethnopharmacology, 94, 261–266.CrossRefPubMedGoogle Scholar
  26. Hsu, H. Y., Yang, J. J., & Lin, C. C. (1997). Effects of oleanolic acid and ursolic acid on inhibiting tumor growth and enhancing the recovery of hematopoietic system post irradiation in mice. Cancer Letters, 111, 7–13.CrossRefPubMedGoogle Scholar
  27. Jeong, H. G. (1999). Inhibition of cytochrome P450 2E1 expression by oleanolic acid: Hepatoprotective effects against carbon tetrachloride induced hepatic injury. Toxicology Letters, 105, 215–222.CrossRefPubMedGoogle Scholar
  28. Khare, C. P. (2007). Indian medicinal plants (pp. 11–13). Berlin: Springer.Google Scholar
  29. Kim, Y., Wyslouzil, B. E., & Weathers, P. J. (2002). Secondary metabolism of hairy root cultures inbioreactors. In Vitro Cellular & Developmental Biology. Plant, 38, 1–10.CrossRefGoogle Scholar
  30. Kumar, S. V., Sankar, P., & Varatharajan, R. (2009). Anti-inflammatory activity of roots of Achyranthes aspera. Pharmaceutical Biology, 47(10), 973–975.CrossRefGoogle Scholar
  31. Liu, J. (1997). Pharmacology of oleanolic acid and ursolic acid. Journal of Ethnopharmacology, 49, 57–68.CrossRefGoogle Scholar
  32. Meng, D. L. (2004). Studies of the constituents and biological activities of Achytanthes bidentata BL. Dr. thesis of Shenyang Pharmaceutical Univiversity, China.Google Scholar
  33. Meng, D. L., Li, X., Xiong, Y. H., & Wang, J. H. (2002). Study on the chemical constituents of Achyranthes bidentata Bl. Journal of Shenyang Pharmaceutical University, 19, 27–30.Google Scholar
  34. Mishra, D. K., & Singh, N. P. (2001). Endemic and threatened flowering plants of Maharashtra (pp. 196–197). Calcutta: Botanical Survey of India.Google Scholar
  35. Misra, T. N., Singh, R. S., Pandey, H. S., & Prasad, C. (1991). An aliphatic dihydroxyketone from Achyranthes aspera. Phytochemistry, 30, 2076–2078.CrossRefGoogle Scholar
  36. Misra, T. N., Singh, R. S., Pandey, H. S., Prasad, C., & Singh, B. P. (1993). Two long chain compounds from Achyranthes aspera. Phytochemistry, 33, 221–223.CrossRefGoogle Scholar
  37. Misra, T. N., Singh, R. S., Pandey, H. S., Prasad, C., & Singh, S. (1996). Isolation and characterization of two new compounds from Achyranthes aspera Linn. Indian Journal of Chemistry (Section B), 35, 637–639.Google Scholar
  38. Nadkarni, K. M. (2009). Indian materia medica (Vol. 1). Mumbai: Bombay Popular Prakashan pp. 21.Google Scholar
  39. Naik, P. M., & Al-Khayri, J. M. (2016). Abiotic and biotic elicitors-role in secondary metabolites production through in vitro culture of medicinal plants. In A. K. Shanker & C. Shankar (Eds.), Abiotic and biotic stress in plants – Recent advances and future perspectives (pp. 247–277). Rijeka: In Tech.Google Scholar
  40. Neogi, L. G., Garg, D., & Rathor, R. S. (1970). Preliminary pharmacological studies on achyranthine. The Indian Journal of Pharmacy, 32, 43–46.Google Scholar
  41. Ovesna, Z., Kozics, K., & Slamenova, D. (2006). Protective effects of ursolic acid and oleanolic acid in leukemic cells. Mutation Research, 600, 131–137.CrossRefPubMedGoogle Scholar
  42. Pai, S. R., Upadhya, V., Hegde, H. V., Joshi, R. K., & Kholkute, S. D. (2014). New report of triterpenoid betulinic acid (BA) along with oleanolic acid (OA) from Achyranthes aspera by RP-UFLC analysis and confirmation using HPTLC and FTIR techniques. Journal of Planar Chromatography--Modern TLC, 27(1), 38–41.CrossRefGoogle Scholar
  43. Pai, S. R., Upadhya, V., Hegde, H. V., Joshi, R. K., & Kholkute, S. D. (2016). Determination of betulinic acid, oleanolic acid and ursolic acid from Achyranthes aspera L. using RP-UFLC-DAD analysis and evaluation of various parameters for their optimum yield. Indian Journal of Experimental Biology, 54(3), 196–202.PubMedGoogle Scholar
  44. Prain, D. (1963). Bengal plants (pp 646–655). Calcutta: Botanical Survey of IndiaGoogle Scholar
  45. Punekar, S. A., & Lakshminarasimhan, P. (2011). Flora of Anshi National Park: Western Ghats-Karnataka (pp. 672), Pune: Biospheres Publication.Google Scholar
  46. Ram, A. K., Bhagwat, A. W., & Gupta, S. S. (1971). Effect of the saponin of Achyranthes aspera on the phosphorylase activity of rat heart. Indian Journal of Physiology and Pharmacology, 15, 107–110.PubMedGoogle Scholar
  47. Rameswar, R. D. (2007). Essential oil constituents of Achyranthes aspera leaves. Indian Perfumer, 51(1), 33–34.Google Scholar
  48. Roberts, S. C. (2007). Production and engineering of terpenoids in plant cell culture. Nature Chemical Biology, 3, 387–395.CrossRefPubMedGoogle Scholar
  49. Sharma, V., & Chaudhary, U. (2015). An overview on indigenous knowledge of Achyranthes aspera. Journal of Critical Reviews, 2(1), 7–19.Google Scholar
  50. Sharma, S. K., Vasudeva, N., & Ali, M. (2009). A new aliphatic acid from Achyranthes aspera Linn roots. Indian Journal of Chemistry Section B, 48, 1164–1169.Google Scholar
  51. Shu, N. X. (2003). Flora of China, 5, 424–426. Downloaded on 01 January 2011.
  52. Singh, V. K., Ali, Z. A., & Zaidi, S. T. H. (1996). Siddiqui MK Ehnomedicinal uses of plants from gonad district forests of Uttar Pradesh, India. Fitoterapia, 67(2), 129–139.Google Scholar
  53. Singh, N. P., Lakshminarasimhan, P., Karthikeyan, S., & Prasanna, P. V. (2000). Flora of Maharashtra state (Vol. 2, pp. 777–779). Calcutta: Botanical Survey of India.Google Scholar
  54. Srivastav, S., Singh, P., Mishra, G., Jha, K. K., & Khosa, R. L. (2011). Achyranthes aspera-An important medicinal plant: A review. Journal of Natural Product and Plant Resources, 1, 1–14.Google Scholar
  55. Subbarayana, P. R., Sarkarb, M., Impellizzeric, S., Raymoc, F., Lokeshward, B. L., et al. (2010). Antiproliferative and anticancerous properties of Achyranthes aspera: Specific inhibitory activity against pancreatic cancer cells. Journal of Ethnopharmacology, 131, 78–82.CrossRefGoogle Scholar
  56. Tang, X., Pei, G., Zhou, Z. Y., & Tan, J. W. (2013). Chemical constituents from roots of Achyranthes bidentata. Journal of Tropical and Subtropical Botany, 21, 57–62.Google Scholar
  57. Tokuda, H., Ohigashi, H., Koshimizu, K., & Ito, Y. (1986). Inhibitory effects of ursolic and oleanolic acid on skin tumor promotion b 12-otetradecanoylphorbol-13-acetate. Cancer Letters, 33, 279–285.CrossRefPubMedGoogle Scholar
  58. Tondon, N. (2011). Quality standards of Indian medicinal plants (Vol. IX, pp. 18–31). New Delhi: Indian Council of Medical Research.Google Scholar
  59. Tripathy, S., Seth, P., & Kushtwar, R. S. (2017). Achyranthes aspera one of important medicinal plant of Indian Flora. Innovative International Journal of Medical Pharmaceutical Sciences, 2(3), 22–26.Google Scholar
  60. Upadhya, V. (2015). Ethnomedicobotany and development of quality control parameters for selected medicinal plants of Belgaum region. Ph. D. thesis of KLE University, Belagavi, India.Google Scholar
  61. Upadhya, V., Mesta, D., Hegde, H. V., Bhat, S., & Kholkute, S. D. (2009). Ethnomedicinal plants of Belgaum region, Karnataka. Journal of Economic and Taxonomic Botany, 33, 300–308.Google Scholar
  62. Upadhya, V., Ankad, G. M., Pai, S. R., Hegde, H. V., & Kholkute, S. D. (2014). Accumulation and trends in distribution of three triterpenoids in various parts of Achyranthes coynei determined using RP-UFLC analysis. Pharmacognosy Magazine, 10, 398–401.CrossRefPubMedPubMedCentralGoogle Scholar
  63. Upadhya, V., Pai, S. R., & Hegde, H. V. (2015). Effect of method and time of extraction on total phenolic content in comparison with antioxidant activities in different parts of Achyranthes aspera. Journal of King Saud University – Science, 27(3), 204–208.CrossRefGoogle Scholar
  64. Varuna, K. M., Khan, M. U., & Sharma, P. K. (2010). Review on Achyranthes aspera. Journal of Pharmacy Research, 3, 714–717.Google Scholar
  65. Ved, D. K., & Goraya, G. S. (2007). Demand and supply of medicinal plants in India. New Delhi/Bangalore: NMPB/FRLHT.Google Scholar
  66. Verpoorte, R., Contin, A., & Memelink, J. (2002). Biotechnology for the production of plant secondary metabolites. Phytochemistry Reviews, 1, 13–25.CrossRefGoogle Scholar
  67. Vetrichelvan, T., & Jegadeesan, M. (2003). Effect of alcoholic extract of Achyranthes aspera Linn. on acute and sub-acute inflammation. Phytotherapy Research, 17, 77–79.CrossRefPubMedGoogle Scholar
  68. Wei, S., Liang, H., Zhao, Y. Y., & Zhang, R. Y. (1997). Separation and identification of the compounds from Achyranthes bidentata Bl. China Journal of Chinese Materia Medica, 22, 293–295.PubMedGoogle Scholar
  69. Woźniak, L., Skąpska, S., & Marszałek, K. (2015). Ursolic acid—A pentacyclic triterpenoid with a wide spectrum of pharmacological activities. Molecules, 20(11), 20614–20641.CrossRefPubMedGoogle Scholar
  70. Yadav, S. R., & Sardesai, M. M. (2002). Flora of Kolhapur Flora of Kolhapur District. Kolhapur: Shivaji University Press.Google Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • L. Sailo
    • 1
  • Vinayak Upadhya
    • 2
  • Poornananda M. Naik
    • 3
  • Neetin Desai
    • 4
  • Sandeep R. Pai
    • 4
  • Jameel M. Al-Khayri
    • 3
    Email author
  1. 1.Regional Institute of Paramedical and Nursing Sciences (RIPANS)AizawlIndia
  2. 2.Department of Forest Products and UtilizationCollege of ForestrySirsiIndia
  3. 3.Department of Agricultural BiotechnologyCollege of Agriculture and Food Sciences, King Faisal UniversityAl-HassaSaudi Arabia
  4. 4.Amity Institute of Biotechnology (AIB), Amity UniversityMumbaiIndia

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