Journal of Plant Growth Regulation

, Volume 38, Issue 2, pp 373–384 | Cite as

Elicitation Enhanced the Yield of Glycyrrhizin and Antioxidant Activities in Hairy Root Cultures of Glycyrrhiza glabra L.

  • Mrinalini Srivastava
  • Gaurav Singh
  • Swati Sharma
  • Sudhir Shukla
  • Pratibha MisraEmail author


Glycyrrhiza glabra L. has become an endangered medicinal plant due to the unabated extraction of glycyrrhizin. Glycyrrhizin is a triterpenoid saponin that is a root centric secondary metabolite having numerous pharmacological properties, such as anti-inflammatory, immunomodulatory, antiallergic, antiulcer, and is found to be effective even against HIV. Harvesting of the roots for high value glycyrrhizin destroys the whole plant causing existential threat to the plant itself and consequent damage to biodiversity. The present study establishes that hairy root cultures of G. glabra, using an optimized elicitor, can dramatically enhance focused production of glycyrrhizin at a much faster pace year-round without causing destruction of the plant. Hairy root cultures of G. glabra were developed using the Agrobacterium rhizogenes A4 strain. The glycyrrhizin content was enhanced using different biotic and abiotic elicitors, for example, PEG (polyethylene glycol), CdCl2, cellulase, and mannan at different concentrations and durations. PEG at 1% concentration enhanced the yield of glycyrrhizin up to 5.4-fold after 24 h of exposure, whereas 200 µg mL−1 cellulase enhanced glycyrrhizin yield to 8.6-fold after 7 days of treatment. Mannan at 10 mg L−1 concentration enhanced the production of glycyrrhizin up to 7.8-fold after 10 days of stress. Among different antioxidant enzymes, SOD activity was significantly enhanced under drought, cellulase and mannan stress. This identification of elicitors can result in abundant supply of valuable glycyrrhizin to meet broad spectrum demand through commercial production without endangering G. glabra L.


Glycyrrhiza glabra Glycyrrhizin Cellulase Mannan PEG Stress Hairy root culture 



The authors are thankful to the Director, CSIR-National Botanical Research Institute, Lucknow, for providing the facilities. The authors are also thankful to Indian Institute of Integrated Medicine, Jammu, for providing elite material of G. glabra (rhizome). MS and GS are thankful to CSIR, New Delhi, for providing Senior Research Fellowship.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abedi T, Pakniyat H (2010) Antioxidant enzyme changes in response to drought stress in ten cultivars of oilseed rape (Brassica napus L.). Czech J Genet Plant Breed 46:27–34CrossRefGoogle Scholar
  2. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126CrossRefGoogle Scholar
  3. Agostini E, Talano MA, Gonzalez PS, Oller AL, Medina MI (2013) Application of hairy roots for phytoremediation: what makes them an interesting tool for this purpose? Appl Microbiol Biotechnol 97:1017–1030CrossRefGoogle Scholar
  4. Alam P, Foudah AI, Zaatout HH, Kamal YT, Abdel-Kader MS (2017) Quantification of glycyrrhizin biomarker in Glycyrrhiza glabra rhizome and baby herbal formulations by validated RP-HPTLC methods. Afr J Tradit Complement Altern Med 14(2):198–205CrossRefGoogle Scholar
  5. Baíza AM, Quiroz-Moreno A, Ruíz JA, Loyola-Vargas VM (1999) Genetic stability of hairy root cultures of Datura stramonium. Plant Cell Tissue Organ Cult 59:9–17CrossRefGoogle Scholar
  6. Bakalova S, Nikolova A, Nedeva D (2004) Isoenzyme profiles of peroxidase, catalase and superoxide dismutase as affected by dehydration stress and ABA during germination of wheat seeds. J Plant Physiol 30:64–77Google Scholar
  7. Beyer WF Jr, Fridovich I (1987) Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal Biochem 161:559–566CrossRefGoogle Scholar
  8. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  9. Bulgakov VP (2008) Functions of rol genes in plant secondary metabolism. Biotechnol Adv 26:318–324CrossRefGoogle Scholar
  10. Bulgakov VP, Shkryl YN, Veremeichik GN, Gorpenchenko TY, Vereshchagina YV (2013) Recent advances in the understanding of Agrobacterium rhizogenes-derived genes and their effects on stress resistance and plant metabolism. Adv Biochem Eng Biotechnol 134:1–22Google Scholar
  11. Chandra S, Chakraborty N, Chakraborty A, Rai R, Bera B, Acharya K (2014) Abiotic elicitor-mediated improvement of innate immunity in Camellia sinensis. J Plant Growth Regul 33:849–859CrossRefGoogle Scholar
  12. Chen YE, Su YQ, Zhang CM, Ma J, Mao HT, Yang ZH, Yuan M, Zhang ZW, Yuan S, Zhang HY (2017a) Comparison of photosynthetic characteristics and antioxidant systems in different wheat strains. J Plant Growth Regul 37:347–359CrossRefGoogle Scholar
  13. Chen Z, Yang B, Hao Z, Zhu J, Zhang Y, Xu T (2017b) Exogenous hydrogen sulfide ameliorates seed germination and seedling growth of Cauliflower under Lead stress and its antioxidant role. J Plant Growth Regul 37:5–15CrossRefGoogle Scholar
  14. Gantait A, Pandit S, Nema NK, Mukjerjee PK (2010) Quantification of glycyrrhizin in Glycyrrhiza glabra extract by validated HPTLC densitometry. J AOAC Int 93:492–495Google Scholar
  15. Georgiev VG, Weber J, Kneschke EM, Denev PN, Bley T, Pavlov AI (2010) Antioxidant activity and phenolic content of betalain extracts from intact plants and hairy root cultures of the red beetroot Beta vulgaris cv. Detroit dark red. Plant Foods Hum Nutr 65:105–111CrossRefGoogle Scholar
  16. Gunes A, Pilbeam DJ, Inal A, Coban S (2008) Influence of silicon on sunflower cultivars under drought stress, I: growth, antioxidant mechanisms, and lipid peroxidation. Commun Soil Sci Plant Anal 39:1885–1903CrossRefGoogle Scholar
  17. Hatami M, Ghorbanpour M (2014) Defense enzymes activity and biochemical variations of Pelargonium zonale in response to nanosilver particles and dark storage. Turk J Biol 38:130–139CrossRefGoogle Scholar
  18. Hayashi H, Hirota A, Hiraoka N, Ikeshiro Y (1999) Molecular cloning and characterization of two cDNAs for Glycyrrhiza glabra squalene synthase. Biol Pharm Bull 22:947–950CrossRefGoogle Scholar
  19. Hayashi H, Huang P, Kirakosyan A, Inoue K, Hiraoka N, Ikeshiro Y, Kushiro T, Shibuya M, Ebizuka Y (2001) Cloning and characterization of a cDNA encoding beta-amyrin synthase involved in glycyrrhizin and soyasaponin biosyntheses in licorice. Biol Pharm Bull 24:912–916CrossRefGoogle Scholar
  20. Hirayama T, Shinozaki K (2010) Research on plant abiotic stress responses in the post-genome era: past, present and future. Plant J 61:1041–1052CrossRefGoogle Scholar
  21. Hussain MS, Fareed S, Ansari S, Rahman MA, Ahmad IZ, Saeed M (2012) Current approaches toward production of secondary plant metabolites. J Pharm Bioallied Sci 4:10–20CrossRefGoogle Scholar
  22. Ito M, Nakashima H, Baba M, Pauwels R, De Clercq E, Shigeta S, Yamamoto N (1987) Inhibitory effect of glycyrrhizin on the in vitro infectivity and cytopathic activity of the human immunodeficiency virus [HIV (HTLV-III/LAV)]. Antivir Res 7:127–137CrossRefGoogle Scholar
  23. Ito M, Sato A, Hirabayashi K, Tanabe F, Shigeta S, Baba M, De Clercq E, Nakashima H, Yamamoto N (1988) Mechanism of inhibitory effect of glycyrrhizin on replication of human immuno deficiency virus (HIV). Antivir Res 10:289–298CrossRefGoogle Scholar
  24. Kim YJ, Lee CS (2008) Glycyrrhizin attenuates MPTP neurotoxicity in Mouse and MPP-induced cell death in PC12 Cells. Korean J Physiol Pharmacol 12:65–71CrossRefGoogle Scholar
  25. Kim OT, Kim SH, Ohyama K, Muranaka T, Choi YE, Lee HY, Kim MY, Hwang B (2010) Upregulation of phytosterol and triterpene biosynthesis in Centella asiatica hairy roots overexpressed ginseng farnesyl diphosphate synthase. Plant Cell Rep 29:403–411CrossRefGoogle Scholar
  26. Klusener B, Weiler EW (1999) Pore-forming properties of elicitors of plant defense reactions and cellulolytic enzymes. FEBS Lett 459:263–266CrossRefGoogle Scholar
  27. Kovalenko P, Antonjuk V, Maliuta S (2004) Secondary metabolites synthesis in transformed cells of Glycyrrhiza glabra L. and Potentilla alba L. as producents of radioprotective compounds. Ukr Bioorg Acta 1:13–22Google Scholar
  28. Krasensky J, Jonak C (2012) Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. J Exp Bot 63:1593–1608CrossRefGoogle Scholar
  29. Lu HY, Liu JM, Zhang HC, Yin T, Gao SL (2008) Ri-mediated transformation of Glycyrrhiza uralensis with a squalene synthase gene (GuSQS1) for production of glycyrrhizin. Plant Mol Biol Rep 26:1–11CrossRefGoogle Scholar
  30. Ma CJ (2008) Cellulase elicitor induced accumulation of capsidiol in Capsicum annuum L. suspension cultures. Biotechnol Lett 30:961–965CrossRefGoogle Scholar
  31. Matsuda F, Hirai MY, Sasaki E, Akiyama K, Yonekura-Sakakibara K, Provart NJ, Sakurai T, Shimada Y, Saito K (2010) AtMetExpress development: a phytochemical atlas of Arabidopsis development. Plant Physiol 152:566–578CrossRefGoogle Scholar
  32. Mehrotra S, Kukreja AK, Khanuja SPS, Mishra BN (2008) Genetic transformation studies and scale up of hairy root culture of Glycyrrhiza glabra in bioreactor. Electron J Biotechnol 11:69–75CrossRefGoogle Scholar
  33. Moharrami F, Hosseini B, Sharafi A, Farjaminezhad M (2017) Enhanced production of hyoscyamine and scopolamine from genetically transformed root culture of Hyoscyamus reticulatus L. elicited by iron oxide nanoparticles. In Vitro Cell Dev Biol Plant 53:104–111CrossRefGoogle Scholar
  34. Munoz-Bertomeu J, Sales E, Ros R, Arrillaga I, Segura J (2007) Up-regulation of an N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase enhances production of essential oils and sterols in transgenic Lavandula latifolia. Plant Biotechnol J 5:746–758CrossRefGoogle Scholar
  35. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with Tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  36. Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880Google Scholar
  37. Nasrollahi V, Mirzaie-asl A, Piri K, Nazeri S, Mehrabi R (2014) The effect of drought stress on the expression of key genes involved in the biosynthesis of triterpenoid saponins in liquorice (Glycyrrhiza glabra). Phytochemistry 103:32–37CrossRefGoogle Scholar
  38. Nieto B, Fores O, Arro M, Ferrer A (2009) Arabidopsis 3-hydroxy-3-methylglutaryl-CoA reductase is regulated at the post-translational level in response to alterations of the sphingolipid and the sterol biosynthetic pathways. Phytochemistry 70:53–59CrossRefGoogle Scholar
  39. Okubo K, Yoshiki Y (2000) The role of triterpenoid on reactive oxygen scavenging system: approach from the new chemiluminescence system (XYZ system). Biofactors 13:219–223CrossRefGoogle Scholar
  40. Ozkur O, Ozdemir F, Bor M, Turkan I (2009) Physiochemical and antioxidant responses of the perennial xerophyte Capparis ovata Desf. to drought. Environ Exp Bot 66:487–492CrossRefGoogle Scholar
  41. Pan Y, Wu LJ, Yu ZL (2006) Effect of salt and drought stress on antioxidant enzymes activities and SOD isoenzymes of liquorice (Glycyrrhiza uralensis Fisch). Plant Growth Regul 49:157–165CrossRefGoogle Scholar
  42. 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 128:281–289CrossRefGoogle Scholar
  43. Sawai S, Saito K (2011) Triterpenoid biosynthesis and engineering in plants. Front Plant Sci 2:1–8CrossRefGoogle Scholar
  44. Seki H, Ohyama K, Sawai S, Mizutani M, Ohnishi T, Sudo H, Akashi T, Aoki T, Saito K, Muranaka T (2008) Licorice beta-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin. Proc Natl Acad Sci USA 105:14204–14209CrossRefGoogle Scholar
  45. Seki H, Sawai S, Ohyama K, Mizutani M, Ohnishi T, Sudo H, Fukushima EO, Akashi T, Aoki T, Saito K, Muranaka T (2011) Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin. Plant Cell 23:4112–4123CrossRefGoogle Scholar
  46. Selmar D, Kleinwächter M (2013) Influencing the product quality by deliberately applying drought stress during the cultivation of medicinal plants. Ind Crops Prod 42:558–566CrossRefGoogle Scholar
  47. Seo JW, Jeong JH, Shin CG, Lo SC, Han SS, Yu KW, Harada E, Han JY, Choi YE (2005) Overexpression of squalene synthase in Eleutherococcus senticosus increases phytosterol and triterpene accumulation. Phytochemistry 66:869–877CrossRefGoogle Scholar
  48. Shabani L, Ehsanpour A, Asghari G, Emami J (2009) Glycyrrhizin production by in vitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid. Russ J Plant Physiol 56:621–626CrossRefGoogle Scholar
  49. Sharma S, Thokchom R (2014) A review on endangered medicinal plants of India and their conservation. J Crop Weed 10(2):205–218Google Scholar
  50. Shirazi Z, Piri K, Asl AM, Hasanloo T (2012) Glycyrrhizin and isoliquiritigenin production by hairy root culture of Glycyrrhiza glabra. J Med Plant Res 6:4640–4646CrossRefGoogle Scholar
  51. Shkryl YN, Veremeichik GN, Bulgakov VP, Tchernoded GK, Mischenko NP, Fedoreyev SA, Zhuravlev YN (2008) Individual and combined effects of the rolA, B, and C genes on anthraquinone production in Rubia cordifolia transformed calli. Biotechnol Bioeng 100:118–125CrossRefGoogle Scholar
  52. Shkryl YN, Veremeichik GN, Bulgakov VP, Gorpenchenko TY, Aminin DL, Zhuravlev YN (2010) Decreased ROS level and activation of antioxidant gene expression in Agrobacterium rhizogenes pRiA4-transformed calli of Rubia cordifolia. Planta 232:1023–1032CrossRefGoogle Scholar
  53. Singh G, Tiwari M, Singh SP, Singh S, Trivedi PK, Misra P (2016) Silencing of sterol glycosyltransferases modulates the withanolide biosynthesis and leads to compromised basal immunity of Withania somnifera. Sci Rep 6:25562CrossRefGoogle Scholar
  54. Srivastava M, Purshottam DK, Srivastava AK, Misra P (2013) In vitro conservation of Glycyrrhiza glabra by slow growth culture. Int J Biotechnol Res 3:49–58Google Scholar
  55. Srivastava M, Sharma S, Misra P (2016) Elicitation based enhancement of secondary metabolites in Rauwolfia serpentina and Solanum khasianum hairy root cultures. Pharmacogn Mag 12(46):315–320CrossRefGoogle Scholar
  56. Tamura K, Seki H, Suzuki H, Kojoma M, Saito K, Muranaka T (2017) CYP716A179 functions as a triterpene C-28 oxidase in tissue-cultured stolons of Glycyrrhiza uralensis. Plant Cell Rep 36:437–445CrossRefGoogle Scholar
  57. Tenea GN, Calin A, Gavrila L, Cucu N (2008) Manipulation of root biomass and biosynthetic potential of Glycyrrhiza glabra L. plants by Agrobacterium rhizogenes mediated transformation. Roum Biotechnol Lett 13:3922–3932Google Scholar
  58. Theboral J, Sivanandhan G, Subramanyam K, Arun M, Selvaraj N, Manickavasagam M, Ganapathi A (2014) Enhanced production of isoflavones by elicitation in hairy root cultures of Soybean. Plant Cell Tissues Organ 117:477–481CrossRefGoogle Scholar
  59. Thwe A, Arasu M, Li X, Park CH, Kim SJ, Al-Dhabi NA, Park SU (2016) Effect of different Agrobacterium rhizogenes strains on hairy root induction and phenylpropanoid biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn). Front Microbiol 7:1–10CrossRefGoogle Scholar
  60. Tohma HS, Gulçin I (2010) Antioxidant and radical scavenging activity of aerial parts and roots of Turkish liquorice (Glycyrrhiza glabra L.). Int J Food Prop 13:657–671CrossRefGoogle Scholar
  61. Torkamani M, Jafari M, Abbaspour N, Heidary R, Safaie N (2014) Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation. Open Life Sci 9:853–863Google Scholar
  62. Turtola S, Manninen AM, Rikala R, Kainulainen P (2003) Drought stress alters the concentration of wood terpenoids in Scots pine and Norway spruce seedlings. J Chem Ecol 29:1981–1995CrossRefGoogle Scholar
  63. Wongwicha W, Tanaka H, Shoyama Y, Putalun W (2011) Methyl jasmonate elicitation enhances glycyrrhizin production in Glycyrrhiza inflata hairy roots cultures. Z Naturforsch C 66:423–428CrossRefGoogle Scholar
  64. Yamaner Ö, Erdağ B, Gökbulut C (2013) Stimulation of the production of hypericins in in vitro seedlings of Hypericum adenotrichum by some biotic elicitors. Turk J Bot 37:153–159Google Scholar
  65. Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–333CrossRefGoogle Scholar
  66. Zheng X, Van Huystee R (1992) Peroxidase-regulated elongation of segments from peanut hypocotyls. Plant Sci 81:47–56CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.CSIR- National Botanical Research InstituteLucknowIndia
  2. 2.Integral UniversityLucknowIndia

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