Enhanced production of flavonoids by methyl jasmonate elicitation in cell suspension culture of Hypericum perforatum
© Wang et al.; licensee Springer. 2015
Received: 18 November 2014
Accepted: 22 December 2014
Published: 18 February 2015
Flavonoids of Hypericum perforatum are important secondary metabolites which have been widely utilized in medicine for a range of purposes. The use of methyl jasmonate (MeJA) elicitation for the enhancement of flavonoid production in cell suspension culture of H. perforatum would be an efficient alternative method for the flavonoid production.
MeJA influenced the cells growth and flavonoid production. The optimal elicitation strategy was treatment of the cell cultures with 100 μmol/L MeJA on day 15, which resulted in the highest flavonoid production (280 mg/L) and 2.7 times of control cultures. The activities of catalase (CAT) were inhibited after MeJA treatment in the cell cultures, while the activities of phenylalanine ammonia lyase (PAL) increased, which led to the enhancement of flavonoid production.
MeJA elicitation is a useful method for the enhancement of flavonoid production in cell suspension culture of H. perforatum.
KeywordsMeJA Hypericum perforatum Suspension culture Flavonoids
Hypericum perforatum, commonly known as St. John's wort, is a perennial herb native to Europe and also a traditional medicinal plant which has been utilized in Chinese folk medicine for a range of purposes [1,2]. The medicinal applications of H. perforatum, including skin wounds, eczema, burns, diseases of the alimentary tract, and psychological disorders, have been related to the phenolic composition of the plant, particularly to hypericins, hyperforins, and flavonoids [3,4]. The efficacy of medical constituents of H. perforatum is based on the whole secondary metabolites, rather than the presence of single chemical compound . Flavonoids, specifically quercetin and its glycoside derivatives, comprise the major group of biologically active metabolites in H. perforatum and are important biochemical markers in authenticating the herbal plant materials [4,6].
The consumption of H. perforatum-derived products as pharmaceutical preparations or food additives has increased dramatically, and it is presently one of the most consumed medicinal plants in the world [4,7]. The extracts of H. perforatum currently used in foods and pharmaceutics, mostly composed of flavonoids, are mainly obtained from the top aerial parts collected in the flowering stage [6,8]. However, the quality of the flavonoid-rich extracts derived from field-grown plants may be affected by many environmental factors as well as biological processes . Furthermore, field cultivation of H. perforatum requires a long growth period and plant management, which is a slow and laborious process . Therefore, an alternative method for more efficient and controllable production of flavonoids from H. perforatum is urgently required.
Plant cell, tissue, and organ cultivation technology has been successfully applied to the production of pharmaceutically valuable compounds and other fine chemicals of commercial interest in recent years . For instance, cell suspension cultures of Panax ginseng have been widely used for the production of saponin and other metabolites . Plant-mediated green biomimetic synthesis of silver nanoparticle was considered a widely acceptable technology for rapid production of silver nanoparticles for successfully meeting the excessive need and current market demand and resulting in a reduction in the employment or generation of hazardous substances to human health and the environment . Till now, a number of efforts have been made for the establishment of cell and/or adventitious root cultures of H. perforatum for the production of hypericins, phenolics, and xanthones [12-15]. The information on the factors affecting biomass and aforementioned metabolites of H. perforatum was also investigated through elicitation and culture optimization [1,16-18]. However, there are few reports on the production of flavonoids from cell suspension culture of H. perforatum, and little is known about the strategies for hyperproduction of biomass and flavonoids.
Many cell cultures have been established from plants, but they seldom produce sufficient amounts of the required secondary metabolites. Secondary metabolite biosynthesis in plants depends on environmental stresses; their accumulation can be stimulated by precursors and elicitors . Precursors are intermediate compounds of the secondary metabolite biosynthesis cycles, which would be toxic to the culture if not used at an appropriate stage or concentration . Elicitors are biological (components of microbial cells and poly- and oligosaccharides), chemical (heavy metals, pesticides, and the signaling compounds in plant defense responses), or physical (cold shock, UV, hyperosmotic stress, ultrasound, and pulsed electric field) factors that induce enzymatic activity against stress [19-21]. Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), have been proposed to be important signaling compounds in the process of elicitation leading to the hyperproduction of various secondary metabolites . They have also been reported to play a key role in signal transduction processes that regulate defense responses in plants and shown effective to enhance the production of secondary metabolites in cell cultures [14,19].
In the present study, the effect of MeJA on cell growth and flavonoid biosynthesis in the H. perforatum cell suspension culture was investigated in small batches. Some parameters, such as elicitation time and MeJA concentration, on biomass and flavonoid production were studied in detail. The activities of key enzymes (catalase and phenylalanine ammonia lyase) related to plant stress responses and secondary metabolite biosynthesis were investigated as well. To the best of our knowledge, this is the first report on the induction of flavonoid production by MeJA in cell suspension culture of H. perforatum.
Materials and methods
MeJA was supplied by Drug Institute of East China University of Science and Technology (Shanghai, China). Salicylic acid (SA), ammonium metavanadate (NH4VO3), and nickel sulfate (NiSO4) were purchased from Shanghai Zhong Lan Chemical Company (Shanghai, China). Acetonitrile of HPLC grade was purchased from TEDIA Company, USA. All other chemicals (analytical grade) were purchased from Shanghai Chemical Co., Ltd. (Shanghai, China).
Plant material and callus induction
H. perforatum plants were provided by Prof. Jie Qian (Tongji University, China) and were cultivated in the greenhouse. The fresh stem explants of H. perforatum were washed thoroughly with tap water, surface-sterilized with 75% ethanol for 60 s, and then soaked in 10% sodium hypochlorite solution for 30 s and rinsed seven times in sterile deionized water. The clean and sterilized stem explants were cut into 5-mm segments and inoculated on solid Murashige and Skoog (MS)  medium supplemented with 1.0 mg/L 2,4-D, 0.2 mg/L 6-benzyladenine (BA), and 25 g/L sucrose to induce callus formation. After 1 month, the successfully induced callus was separated from the explants and cultured separately until used for the establishment of cell suspensions.
Suspension cultures of H. perforatum cells
Cell suspension cultures were initiated from the friable callus and maintained in the liquid MS medium (pH 5.8) supplemented with 1.0 mg/L 2,4-D, 0.2 mg/L 6-benzyladenine (BA), and 25 g/L sucrose. A sterile standard sieve with aperture of 300 μm was used to filter the initial established suspension cells to obtain homogeneous cultures. The pre-weighed cells were cultured in a 250-mL Erlenmeyer flask containing a 50-mL medium; cultures were placed on a rotary shaker shaking at 120 rpm at 25 ± 2°C under continuous illumination. Every 20 days, cells were subcultured to fresh media with 5.0-g fresh weight (FW) in 250-mL flasks.
Elicitation of cell suspension culture
Four elicitors including MeJA, SA, NH4VO3, and NiSO4 were used to study the elicitation effect on flavonoid production by the cell suspension cultures of H. perforatum. MeJA and SA were dissolved in ethanol, NH4VO3 and NiSO4 were dissolved in water, and the four elicitors were filter-sterilized before adding into the suspension cultures. Based on the results of preliminary experiments, the feeding concentration of MeJA, SA, NH4VO3, and NiSO4 was 50, 100, 50, and 15 μM, respectively. As a control, filter-sterilized ethanol was added to the cell suspension culture with a final concentration of 0.2%.
According to the results of the above experiments, MeJA was selected for further study. The MeJA induction time and feeding concentration, known to be crucial to plant cell culture, were investigated by single-factor experimental design by varying a single factor at a time and keeping other factors at a constant value. All experiments were performed in triplicate, and data are expressed as the mean of three samples with standard deviation.
Extraction and HPLC analysis of secondary metabolites
H. perforatum extracts were prepared by soaking 0.5 g of the dried cells in 10 mL of methanol and treated with 1 h of ultrasonic extraction for two times. The extracts were then centrifuged at 10,000 rpm at 4°C. The supernatant was combined and used for HPLC analysis and flavonoid determination.
The HPLC system of Shimadzu LC-10Avp Plus with a PDA detector (SPD-M20A) and a C18 column (4.6 mm × 250 mm, 5 μm, Eclipse XDB C18) was used for the qualitative and quantitative analyses of flavonoids and other metabolites in the methanol extracts. The mobile phase was acetonitrile (A) and 0.1% trifluoroacetic acid (B). A gradient method was used for the separation of the extracted samples: 0-25 min, 15%-40% A; 25-40 min, 40%-100% A. The elution flow rate and detection wavelength were set at 1.0 mL/min and 254 nm, respectively. The identity of peaks separated by HPLC was confirmed by the injection of standard, and UV spectral analysis was carried out to confirm compound identification.
Determination of biomass and flavonoid content
The plant cell biomass is expressed as the gram fresh cell weight and/or dry cell weight per liter. Cell suspension cultures were filtered and washed by deionized water, then cells were collected and weighed to get the FW. The collected cells were dried in the vacuum drying oven at 45°C until constant weight (DW) was attained.
The flavonoid content was assayed using UV colorimetric method as reported elsewhere [5,13]. Briefly, 0.5 mL of the methanolic plant cell extract was mixed with 2 mL of distilled water and subsequently with 0.15 mL of a 5% NaNO2 solution. After 6 min, 0.15 mL of a 10% AlCl3 solution was added and the mixture was allowed to stand for a further 6 min before 2 mL of 4% NaOH solution was added. Absorbance of the mixed solution was measured at 510 nm using a UV-Vis spectrophotometer (UV-1650PC, Shimadzu, Japan). Rutin was used as standard compound for the quantification of total flavonoid. Results were expressed as milligram of rutin equivalents per gram of dry cells. Data were expressed as means ± SD for three replications.
Enzyme activity analysis
Suspension culture cells were harvested for evaluation of the catalase (CAT) activity level as described by Georgiew et al . The decomposition of H2O2 was followed by measuring the decrease in absorbance at 240 nm. One unit of CAT is the amount that decomposes 1 μmol of H2O2 for 1 min at pH 7.0 and 25°C. Protein was estimated by the procedure of Lowry et al. , using crystalline bovine albumin as standard. Phenylalanine ammonia lyase (PAL) was extracted from fresh H. perforatum cells with borate buffer (pH 8.8). The cells were ground in the buffer (0.15 g/mL) for 2 min with a pestle and mortar on ice and then centrifuged at 10,000 rpm and 4°C for 20 min to obtain a solid-free extract. The PAL activity was determined based on the conversion of L-phenylalanine to cinnamic acid as described by Wu and Lin .
Results and discussion
Callus induction and cell suspension culture establishment
Selection of MeJA as the elicitor in cell suspension culture of H. perforatum
Some literatures reported the enhancement of metabolite production by NH4VO3 and NiSO4 elicitation . However, the two elicitors revealed negative effects on biomass and flavonoid accumulation in H. perforatum cell cultures (Figure 4). It was probably due to the significant biological toxicity caused by the inorganic salts . Some other elicitors such as fluoro- and hydroxyl-containing derivatives of methyl jasmonate, which were proven more potent than methyl jasmonate in suspension culture of Taxus [31,32], may also serve as possible options for the enhancement of flavonoid biosynthesis in H. perforatum. In addition, some new elicitors have been reported to have significantly promoted secondary metabolite biosynthesis by plant cell cultures . However, elicitation treatments may have varied effects among different cell lines as mentioned above. Therefore, efforts devoted into the enhancement of flavonoid production by H. perforatum cell suspension culture are still required considering its further application. In the present investigation, the addition of MeJA (50 μM) resulted in maximum flavonoid production, which indicated the potential of this abiotic elicitor for the enhancement of flavonoid biosynthesis in cell suspension culture of H. perforatum and was selected for further study.
Effects of MeJA feeding time on cell growth and flavonoid production
Effects of MeJA dosage on cell growth and flavonoid production
Effects of MeJA addition on CAT and PAL activities
PAL is the first enzyme of phenylpropanoid biosynthesis in plants and plays an important role in the biosynthesis of flavonoids, lignins, and many other compounds . An increase in PAL activity after elicitor treatment often results in enhanced secondary metabolism in plant cells . Figure 7B shows the changes of intracellular PAL activity in the suspension cultures after the treatment by MeJA (100 μM). As shown in Figure 7B, MeJA induced the increase of PAL activities and reached maximum (12.1 U/g FW) 4 days after treatment, thereafter decreased. However, cell cultures without MeJA treatment (control) revealed little change of PAL activities. In the presence of MeJA, PAL activity was much higher than that of control (Figure 7B), the elicitor-induced up-regulation of the PAL activity resulted in an improved flavonoid production in the suspension cultures (Figure 6B). Zhao et al.  have observed similar results in Salvia miltiorrhiza cell cultures, which indicated that both biotic and abiotic elicitation treatment resulted in the up-regulation of PAL activity and improved tanshinone accumulation as compared to the control cultures. These results demonstrated that MeJA is an efficient elicitor for the induction of flavonoid production in the suspension cell culture of H. perforatum.
Plant cell suspension culture is an efficient alternative method for the production of useful biochemicals. In this work, MeJA-elicited cell suspension cultures of H. perforatum enhanced the production of flavonoids through the down-regulation of the CAT activity and up-regulation of the PAL activity. The flavonoid production reached 280 mg/L after the optimal elicitation conditions, which was 2.7-fold of the control cultures. These results would be useful for the hyperproduction of flavonoids from H. perforatum and would be useful for the production of valuable compounds from other plant cell cultures.
Dry cell weight
Fresh cell weight
Phenylalanine ammonia lyase
This work was supported by the National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204) and the Fundamental Research Funds for the Central Universities (WF1113010).
- Wu SQ, Yu XK, Lian ML, Park SY, Piao XC (2014) Several factors affecting hypericin production of Hypericum perforatum during adventitious root culture in airlift bioreactors. Acta Physiol Plant 36:975–981View ArticleGoogle Scholar
- Zou Y, Lu Y, Wei D (2004) Antioxidant activity of a flavonoid-rich extract of Hypericum perforatum L. in vitro. J Agric Food Chem 52:5032–5039View ArticleGoogle Scholar
- Cui XH, Murthy HN, Wu CH, Paek KY (2010) Adventitious root suspension cultures of Hypericum perforatum: effect of nitrogen source on production of biomass and secondary metabolites. In Vitro Cell De-Pl 46:3728–3741Google Scholar
- Saddiqe Z, Naeem I, Maimoona A (2010) A review of the antibacterial activity of Hypericum perforatum L. J Ethnopharmacol 131:511–521View ArticleGoogle Scholar
- Cui XH, Murthy HN, Jin YX, Yim YH, Kim JY, Paek KY (2011) Production of adventitious root biomass and secondary metabolites of Hypericum perforatum L. in a balloon type airlift reactor. Bioresour Technol 102:10072–10079View ArticleGoogle Scholar
- Bagdonaitė E, Mártonfi P, Repčák M, Labokas J (2012) Variation in concentrations of major bioactive compounds in Hypericum perforatum L. from Lithuania. Ind Crops Prod 35:302–308View ArticleGoogle Scholar
- Savio LEB, Astarita LV, Santarém ER (2011) Secondary metabolism in micropropagated Hypericum perforatum L. grown in non-aerated liquid medium. Plant Cell Tiss Org 108:465–472View ArticleGoogle Scholar
- Zou YP, Lu YH, Wei DZ (2010) Protective effects of a flavonoid-rich extract of Hypericum perforatum L. against hydrogen peroxide-induced apoptosis in PC12 cells. Phytother Res 24:6–10View ArticleGoogle Scholar
- Baque MA, Moh SH, Lee EJ, Zhong JJ, Paek KY (2012) Production of biomass and useful compounds from adventitious roots of high-value added medicinal plants using bioreactor. Biotechnol Adv 30:1255–1264View ArticleGoogle Scholar
- Wu J, Zhong JJ (1999) Production of ginseng and its bioactive components in plant cell culture: current technological and applied aspects. J Biotechnol 68:89–99View ArticleGoogle Scholar
- Priya B, Mantosh S, Aniruddha M (2014) Leaf extract mediated green synthesis of silver nanoparticles from widely available Indian plants: synthesis, characterization, antimicrobial property and toxicity analysis. Bioresour Bioprocess 1:3View ArticleGoogle Scholar
- Xu M, Yang B, Dong J, Lu D, Jin H, Sun L, Xu X (2011) Enhancing hypericin production of Hypericum perforatum cell suspension culture by ozone exposure. Biotechnol Prog 27:1101–1116View ArticleGoogle Scholar
- Cui XH, Chakrabarty D, Lee EJ, Paek KY (2010) Production of adventitious roots and secondary metabolites by Hypericum perforatum L. in a bioreactor. Bioresour Technol 101:4708–4716View ArticleGoogle Scholar
- Walker TS, Pal Bais H, Vivanco JM (2002) Jasmonic acid-induced hypericin production in cell suspension cultures of Hypericum perforatum L. (St. John's wort). Phytochemistry 60:289–293View ArticleGoogle Scholar
- Tocci N, D'Auria FD, Simonetti G, Panella S, Palamara AT, Pasqua G (2012) A three-step culture system to increase the xanthone production and antifungal activity of Hypericum perforatum subsp. angustifolium in vitro roots. Plant Physiol Biochem 57:54–58View ArticleGoogle Scholar
- Tocci N, Simonetti G, D'Auria FD, Panella S, Palamara AT, Valletta A, Pasqua G (2011) Root cultures of Hypericum perforatum subsp. angustifolium elicited with chitosan and production of xanthone-rich extracts with antifungal activity. Appl Microbiol Biotechnol 91:977–987View ArticleGoogle Scholar
- Cui XH, Murthy HN, Paek KY (2014) Pilot-scale culture of Hypericum Perforatum L. adventitious roots in airlift bioreactors for the production of bioactive compounds. Appl Biochem Biotechnol 14:1123–1128Google Scholar
- Gadzovska S, Maury S, Delaunay A, Spasenoski M, Hagège D, Courtois D, Joseph C (2012) The influence of salicylic acid elicitation of shoots, callus, and cell suspension cultures on production of naphtodianthrones and phenylpropanoids in Hypericum perforatum L. Plant Cell Tiss Org 113:25–39View ArticleGoogle Scholar
- Zhao JL, Zhou LG, Wu JY (2010) Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures. Appl Microbiol Biotechnol 87:137–144View ArticleGoogle Scholar
- Gueven A, Knorr D (2011) Isoflavonoid production by soy plant callus suspension culture. J Food Eng 103:237–243View ArticleGoogle Scholar
- Lin L, Wu J (2002) Enhancement of shikonin production in single- and two-phase suspension cultures of Lithospermum erythrorhizon cells using low-energy ultrasound. Biotechnol Bioeng 5:78–81Google Scholar
- Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–485View ArticleGoogle Scholar
- Georgiev M, Abrashev R, Krumova E, Demirevska K, Ilieva M, Angelova M (2009) Rosmarinic acid and antioxidant enzyme activities in Lavandula vera MM cell suspension culture: a comparative study. Appl Biochem Biotechnol 159:415–423View ArticleGoogle Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–271Google Scholar
- Dong HD, Zhong JJ (2001) Significant improvement of taxane production in suspension cultures of Taxus chinensis by combining elicitation with sucrose feed. Biochem Eng J 8:145–162View ArticleGoogle Scholar
- Cai Z, Riedel H, Saw NMMT, Mewis I, Reineke K, Knorr D, Smetanska I (2011) Effects of elicitors and high hydrostatic pressure on secondary metabolism of Vitis vinifera suspension culture. Process Biochem 46:1411–1422View ArticleGoogle Scholar
- Ketchum RE, Tandon M, Gibson DM, Begley T, Shuler M (1999) Isolation of labeled 9-dihydrobaccatin III and related taxoids from cell cultures of Taxus canadensis elicited with methyl jasmonate. J Nat Prod 62:1395–1406View ArticleGoogle Scholar
- Saniewski M, Miszczak A, Kawa-Miszczak L, Wegrzynowicz-Lesiak E, Miyamoto K, Ueda J (1998) Effects of methyl jasmonate on anthocyanin accumulation, ethylene production, and CO2 evolution in uncooled and cooled tulip bulbs. J Plant Growth Regul 17:33–42View ArticleGoogle Scholar
- Veerashree V, Anuradha C, Kumar V (2012) Elicitor-enhanced production of gymnemic acid in cell suspension cultures of Gymnema sylvestre R. Br. Plant Cell Tiss Org 108:27–33View ArticleGoogle Scholar
- Huang C, Zhong JJ (2013) Elicitation of ginsenoside biosynthesis in cell cultures of Panax ginseng by vanadate. Process Biochem 48:1227–1245View ArticleGoogle Scholar
- Qian ZG, Zhao ZJ, Xu YF, Qian XH, Zhong JJ (2005) A novel synthetic fluoro-containing jasmonate derivative acts as a chemical inducing signal for plant secondary metabolism. Appl Microbiol Biotechnol 68:98–103View ArticleGoogle Scholar
- Qian ZG, Zhao ZJ, Xu YF, Qian XH, Zhong JJ (2004) Novel chemically synthesized hydroxyl-containing jasmonates as powerful inducing signals for plant secondary metabolism. Biotechnol Bioeng 86:809–816View ArticleGoogle Scholar
- Huang C, Zhong JJ (2013) Enhancement of ginsenoside biosynthesis in cell cultures of Panax ginseng by N,N'-dicyclohexylcarbodiimide elicitation. J Biotechnol 165:30–36View ArticleGoogle Scholar
- Vasconsuelo A, Boland R (2007) Molecular aspects of the early stages of elicitation of secondary metabolites in plants. Plant Sci 172:861–877View ArticleGoogle Scholar
- Xu C, Zhao B, Ou Y, Wang X, Yuan X, Wang Y (2006) Elicitor-enhanced syringin production in suspension cultures of Saussurea medusa. World J Microb Biotechnol 23:965–979View ArticleGoogle Scholar
- Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–294View ArticleGoogle Scholar
- Kim YH, Kim Y, Cho E, Kwak S, Kwon S, Bae J, Huh GH (2004) Alterations in intracellular and extracellular activities of antioxidant enzymes during suspension culture of sweetpotato. Phytochemistry 65:2471–2484View ArticleGoogle Scholar
- Guo ZJ, Lamb C, Dixon RA (1998) Potentiation of the oxidative burst and isoflavonoid phytoalexin accumulation by serine protease inhibitors. Plant Physiol 118:1487–1495View ArticleGoogle Scholar
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