Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 118, Issue 1, pp 169–177 | Cite as

Effect of plant growth regulators and elicitors on rhinacanthin accumulation in hairy root cultures of Rhinacanthus nasutus (L.) Kurz

Original Paper


Several biologically active secondary metabolites like anthraquinones, sterols, triterpenes, flavonoids and naphthoquinones are present in Rhinacanthus nasutus. Naphthoquinones are important group of compounds generally known as rhinacanthin (RC) consists of 15 derivatives named RC A–D and G–Q of which RC-C, RC-D and RC-N have various medicinal properties. The individual role of two auxins i.e. indole-3-butyric acid (IBA) and α naphthalene acetic acid (NAA) and two elicitors i.e. methyl jasmonate (MJ) and salicylic acid (SA) in Murashige and Skoog medium on hairy root growth and RC (RC-C, RC-D and RC-N) accumulation was investigated in the present study. Time course study revealed that IBA and NAA at 2.5 μM showed maximum fresh weight (FW) and dry weight (DW) 4 weeks after culture. However, RC production was maximum after 6 weeks of culture on both media. A concentration-dependent response was observed when various concentrations of MJ (2.0, 5.0, 10 and 15 μM) and SA (10, 50, 100 and 150 μM) were supplemented in the medium. On MJ and SA media the FW and DW decreased as the concentration of elicitors increased. However, this decrease was more severe in MJ treated cultures. All the MJ and SA treated cultures showed significantly higher amount of RC-C, RC-D and RC-N in hairy roots harvested 7 days after elicitation as compared to control. Of the two elicitors, MJ was more efficient in inducing RC accumulation than SA. The highest RC content (6.3 mg/g DW RC-C; 1.1 mg/g DW RC-D and 0.61 mg/g DW RC-N) was observed after treatment with 10 μM MJ which was about 1.7-, 2.5- and 3.5-fold higher RC-C, RC-D and RC-N respectively than the control.


Auxins Elicitors Hairy root Rhinacanthin Rhinacanthus nasutus Time course 



Dry weight


Fresh weight


High performance liquid chromatography


Indole-3-butyric acid


Methyl jasmonate


Murashige and Skoog medium


α Naphthalene acetic acid


Polymerase chain reaction




Salicylic acid



MKC is thankful to M.G. University, Kottayam, Kerala for the financial assistance in the form of University Junior Research Fellowship and Principal, St. Thomas College, Pala for providing lab facilities. TDT acknowledges the financial support from Indian National Science Academy (INSA, New Delhi) and Polish Academy of Sciences (PAS), Poland in the form of bilateral exchange programme.


  1. Bekheet SA, Taha HS, Gabr AMM (2013) Protocol for in vitro morphogenesis and hairy root cultures of Milk thistle (Silybum marianum L. Gaertn). J App Sci Res 9:860–866Google Scholar
  2. Bourgaud F, Gravot A, Milesi S, Gontier E (2001) Production of secondary metabolites: a historical perspective. Plant Sci 161:839–851CrossRefGoogle Scholar
  3. Bulgakov VP (2008) Functions of rol genes in plant secondary metabolism. Biotech Adv 26:318–324CrossRefGoogle Scholar
  4. Das Nilanjana (2006) Propagation prospects of dye yielding plant Rhinacanthus nasutus (Linn.) Kurz. Nat Prod Rad 5:22–23Google Scholar
  5. Dhakulkar S, Ganapathi TR, Bhargava S, Bapat VA (2005) Induction of hairy roots in Gmelina arborea Roxb. and production of verbascoside in hairy roots. Plant Sci 169:812–818CrossRefGoogle Scholar
  6. Dias LLC, Santa-Catarina C, Ribeiro DM, Barros RS, Floh EIS, Otoni WC (2009) Ethylene and polyamine production patterns during in vitro shoot organogenesis of two passion fruit species as affected by polyamines and their inhibitor. Plant Cell Tiss Organ Cult 99:199–208CrossRefGoogle Scholar
  7. Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349PubMedCentralPubMedCrossRefGoogle Scholar
  8. Farnsworth NR, Bunyapraphatsara N (1992) Thai medicinal plant: recommended for primary health care system. Prachachon Company, BangkokGoogle Scholar
  9. Frankfater CR, Dowd MK, Triplett BA (2009) Effect of elicitors on the production of gossypol and methylated gossypol in cotton hairy roots. Plant Cell Tiss Organ Cult 98:341–349CrossRefGoogle Scholar
  10. Georgiev MI, Pavlov AI, Bley T (2007) Hairy root type plant in vitro systems as sources of bioactive substances. Appl Microbiol Biotechnol 74:1175–1185PubMedCrossRefGoogle Scholar
  11. Giri A, Narasu ML (2000) Transgenic hairy roots: recent trends and applications. Biotech Adv 18:1–22CrossRefGoogle Scholar
  12. Gotoh A, Sakaeda T, Kimura T, Shirakawa T, Wada Y, Wada A, Kimachi T, Takemoto Y, Iida A, Iwakawa S, Hirai M, Tomita H, Okamura N, Nakamura T, Okumura K (2004) Antiproliferative activity of Rhinacanthus nasutus (L.) KURZ extracts and the active moiety, Rhinacanthin C. Biol Pharm Bull 27:1070–1074PubMedCrossRefGoogle Scholar
  13. Guillon S, Tremouillaux-Guiller J, Pati P, Rideau M, Gantet P (2006) Harnessing the potential of hairy roots: dawn of a new era. Trends Biotechnol 24:403–409PubMedCrossRefGoogle Scholar
  14. Hwang SJ (2006) Baicalin production in transformed hairy root clones of Scutellaria baicalensis. Biotech Biopro Engg 11:105–109CrossRefGoogle Scholar
  15. Kuzma L, Bruchajzer E, Wysokinska H (2009) Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor. Enzyme Microb Technol 44:406–410CrossRefGoogle Scholar
  16. Li W, Koike K, Asada Y, Yoshikawa T, Nikaido T (2005) Rosmarinic acid production by Coleus forskohlii hairy root. Plant Cell Tiss Organ Cult 80:151–155CrossRefGoogle Scholar
  17. Malarz J, Stojakowska A, Kisiel W (2007) Effect of methyl jasmonate and salicylic acid on sesquiterpene lactone accumulation in hairy roots of Cichorium intybus. Acta Physiol Plant 29:127–132CrossRefGoogle Scholar
  18. Memelink J, Verpoorte R, Kijne W (2001) ORCAnization of jasmonate-responsive gene expression in alkaloid metabolism. Trends Plant Sci 6:212–219PubMedCrossRefGoogle Scholar
  19. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  20. Ozawa R, Arimura G, Takabayashi J, Shimoda T, Nishioka T (2000) Involvement of jasmonate- and salicylate-related signaling pathways for the production of specific herbivore-induced volatiles in plants. Plant Cell Physiol 41:391–398PubMedCrossRefGoogle Scholar
  21. Puttarak P, Charoonratana T, Panichayupakarananta P (2010) Antimicrobial activity and stability of rhinacanthins- rich Rhinacanthus nasutus extract. Phytomed 17:323–327CrossRefGoogle Scholar
  22. Rao SR, Ravishankar GA (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotech Adv 20:101–153CrossRefGoogle Scholar
  23. Reanmongkol W, Subhadhirasakul S, Panichayupakaranant P, Kim KM (2003) Antiallergic and anti-oxidant activities of some compounds from Thai medicinal plants. Pharmal Biol 41:592–597CrossRefGoogle Scholar
  24. Rhee HS, Cho HY, Son SY, Yoon SYH, Park JM (2010) Enhanced accumulation of decursin and decursinol angelate in root cultures and intact roots of Angelica gigas Nakai following elicitation. Plant Cell Tissue Organ Cult 101:295–302CrossRefGoogle Scholar
  25. Rojanapo W, Tepsuwan A, Siripong P (1990) Mutagenicity and antimutagenicity of Thai medicinal plants. Basic Life Sci 52:447–452PubMedGoogle Scholar
  26. Satdive RK, Fulzele DP, Eapen S (2007) Enhanced production of azadirachtin by hairy root cultures of Azadirachta indica A. Juss by elicitation and media optimization. J Biotechnol 128:281–289PubMedCrossRefGoogle Scholar
  27. Schenk PM, Kazan K, Wilson I, Anderson JP, Richmond T, Somerville SC, Manners JM (2000) Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proc Nat Acad Sci USA 97:11655–11660PubMedCentralPubMedCrossRefGoogle Scholar
  28. Sendl A, Chen JL, Jolad SD, Stoddart C, Rozhon E, Kernan M (1996) Two new naphthoquinones with antiviral activity from Rhinacanthus nasutus. J Nat Prod 59:808–811PubMedCrossRefGoogle Scholar
  29. Sharma M, Sharma A, Kumar A, Basu SK (2011) Enhancement of secondary metabolites in cultured plant cells through stress stimulus. Am J Plant Physiol 6:50–71CrossRefGoogle Scholar
  30. Shinde AN, Malpathak N, Fulzele DP (2009) Enhanced production of phytoestrogenic isoflavones from hairy root cultures of Psoralea corylifolia L. using elicitation and precursor feeding. Biotechnol Bioprocess Eng 14:288–294CrossRefGoogle Scholar
  31. Siripong P, Kanokmedhakul K, Piyaviruyakul S, Yahuafai J, Chanpai R, Ruchirawat S, Oku N (2006a) Antiproliferative naphthoquinone esters from Rhinacanthus nasutus KURZ. roots on various cancer cells. J Trad Med 23:166–172Google Scholar
  32. Siripong P, Yahuafai J, Shimizu K, Ichikawa K, Yonezawa S, Asai T, Kwanjai K, Somsak R, Natoto O (2006b) Antitumor activity of liposomal naphthoquinone esters isolated from Thai medicinal plant: Rhinacanthus nasutus Kurz. Biol Pharm Bull 29:2279–2283PubMedCrossRefGoogle Scholar
  33. Siripong P, Yahuafai J, Piyaviriyakul S, Kanokmedhakul K, Koide H, Ishii T, Shimizu K, Ruchirawat S, Oku N (2012) Inhibitory effect of liposomal rhinacanthin-N isolated from Rhinacanthus nasutus on pulmonary metastasis in mice. Biol Pharm Bull 35:1197–1200PubMedCrossRefGoogle Scholar
  34. Siriwatanametanon N, Fiebich BL, Efferth T, Prieto JM, Heinrich M (2010) Traditionally used Thai medicinal plants: in vitro anti-inflammatory, anticancer and antioxidant activities. J Ethnopharmacol 130:196–207PubMedCrossRefGoogle Scholar
  35. Sivanandhan G, Arun M, Mayavan S, Rajesh M, Jeyaraj M, Kapil Dev G, Manickavasagam M, Selvaraj N, Ganapathi A (2012) Optimization of elicitation conditions with methyl jasmonate and salicylic acid to improve the productivity of withanolides in the adventitious root culture of Withania somnifera (L.) Dunal. Appl Biochem Biotechnol 168:681–696PubMedCrossRefGoogle Scholar
  36. Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Arjunan A, Muthuselvam M, Manickavasagam M, Selvaraj N, Ganapathi A (2013) Increased production of withanolide A, withanone, and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell Tiss Organ Cult. doi: 10.1007/s11240-013-0297-z
  37. Sudha CG, Reddy OB, Ravishankar GA, Seeni S (2003) Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook f., a rare and endemic medicinal plant. Biotech Lett 25:631–636CrossRefGoogle Scholar
  38. Sun J, Xiao J, Wang X, Yuan X, Zhao B (2012) Improved cardenolide production in Calotropis gigantean hairy roots using mechanical wounding and elicitation. Biotech Lett 34:563–569CrossRefGoogle Scholar
  39. Tewtrakul S, Tansakul P, Panichayupakaranant P (2009) Antiallergic principles of Rhinacanthus nasutus leaves. Phytomed 16:929–934CrossRefGoogle Scholar
  40. Thimmaraju R, Venkatachalam L, Bhagyalakshmi N (2008) Morphometric and biochemical characterization of red beet (Beta vulgaris L.) hairy roots obtained after single and double transformations. Plant Cell Rep 27:1039–1052PubMedCrossRefGoogle Scholar
  41. Triplett BA, Stephanie C, Moss SC, Bland JM, Dowd MK (2008) Induction of hairy root cultures from Gossypium hirsutum and Gossypium barbadense to produce gossypol and related compounds. In Vitro Cell Dev Biol Plant 44:508–517CrossRefGoogle Scholar
  42. Udomsuk L, Jarukamjorn K, Tanaka H, Putalun W (2011) Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation. Biotech Lett 33:369–374CrossRefGoogle Scholar
  43. Van der Fits L, Memelink J (2000) ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289:295–297PubMedCrossRefGoogle Scholar
  44. Verpoorte R, Heijden RVD, Hoopen HJGT, Memelink J (1999) Metabolic engineering of plant secondary metabolite path-ways for the production of fine chemicals. Biotech Lett 21:467–479CrossRefGoogle Scholar
  45. Vervliet G, Holsters M, Teuchy H, Montagu MV, Schell J (1975) Characterization of different plaque-forming and defective temperate phages in Agrobacterium strains. J Gen Virol 26:33–48PubMedCrossRefGoogle Scholar
  46. Washida D, Shimomura K, Takido M, Kitanaka S (2004) Auxins affected ginsenoside production and growth of hairy roots in Panax Hybrid. Biol Pharm Bull 27:657–660PubMedCrossRefGoogle Scholar
  47. Wongwattanasathien O, Kangsadalampai K, Tongyonk L (2010) Antimutagenicity of some flowers grown in Thailand. Food Chem Toxicol 48:1045–1051PubMedCrossRefGoogle Scholar
  48. Wu TS, Yang CC, Wu P, Liu L (1995) A Quinol and steroids from the leaves and stems of Rhinacanthus nasutus. Phytochem 40:1247–1249CrossRefGoogle Scholar
  49. Wu TS, Hsu HC, Wu PL, Teng CM, Wu YC (1998a) Naphthoquinone esters from the root of Rhinacanthus nasutus. Chem Pharma Bull 46:413–418CrossRefGoogle Scholar
  50. Wu TS, Tien HJ, Yeh MY, Lee KH (1998b) Rhinacanthin Q, A naphthoquinone from Rhinacanthus nasutus and its biological activity. Phytochem 27:3787–3788CrossRefGoogle Scholar
  51. Xu T, Zhang L, Sun X, Zhang H, Tang K (2004) Production and analysis of organic acids in hairy-root cultures of Isatis indigotica Fort. (indigo woad). Biotech App Biochem 39:123–128CrossRefGoogle Scholar
  52. Zhao J, Zhu WH, Hu Q (2001) Selection of fungal elicitors to increase indole alkaloid accumulation in Catharanthus roseus suspension cell culture. Enzyme Microb Technol 28:666–672PubMedCrossRefGoogle Scholar
  53. Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–333PubMedCrossRefGoogle Scholar
  54. Zhao JL, Zhou LG, Wu JY (2010) Promotion of Salvia miltiorrhiza hairy root growth and tanshinone production by polysaccharideprotein fractions of plant growth-promoting rhizobacterium Bacillus cereus. Process Biochem 45:1517–1522CrossRefGoogle Scholar
  55. Zhou ML, Zhu XM, Shao JR, Tang YX, Wu YM (2011) Production and metabolic engineering of bioactive substances in plant hairy root culture. Appl Microbiol Biotechnol 90:1229–1239PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.Post Graduate and Research Department of BotanySt. Thomas CollegePala, KottayamIndia
  2. 2.Department of Pharmaceutical Biology and BotanyMedical University in WroclawWroclawPoland

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