Elicitor-enhanced production of gymnemic acid in cell suspension cultures of Gymnema sylvestre R. Br.
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Cell suspension cultures of Gymnema sylvestre treated with four different elicitors, methyl jasmonate (MJ), yeast extract, chitin and pectin were studied for the production of gymnemic acid as gymnemagenin equivalent, that was analyzed by high performance liquid chromatography (HPLC). All the four tested elicitors induced gymnemic acid production in cell suspension cultures. Highest gymnemic acid content was achieved following treatment with yeast extract (100.47 ± 0.28 mg/l), this was followed by MJ (70.43 ± 0.26 mg/l), pectin (64.19 ± 0.23 mg/l) and chitin (62.72 ± 0.13 mg/l). The addition of elicitors has shown a significant influence on cell growth that affected cell growth compared to respective controls. The highest gymnemic acid production was obtained after 20 days of elicitation in cultures treated with 0.5 g l−l yeast extract, it was 5.25-folds greater than in control. These results suggest that the addition of an elicitor to Gymnema sylvestre cell suspension cultures could stimulate and enhance gymnemic acid production. In our present study we could able to overproduce gymnemic acid up to 51.97 ± 0.26 mg l−l (dry weight basis) in yeast extract treated cell suspension cultures.
KeywordsGymnema sylvestre Elicitor Methyl jasmonate Gymnemagenin HPLC
Gymnema sylvestre R. Br. (Aslepiadaceae) is a woody, vine-like plant that climbs on bushes and trees, native to South Asia from India to the southern part of China. The leaves of this plant, popularly known as “Gur-mar” in India, are renowned for the distinctive property of suppressiing the taste of sweetness. Leaves of G. sylvestre have been used in India for the treatment of diabetes for over 2,000 years (Nadakarni 1992). Several investigators reported antidiabetic properties of G. sylvestre leaf extract, not only in rats (Choudhury 1988; Chattopadhaya 1999; Okabayashi et al. 1990; Shanmugasundaram et al. 1990a) but also in humans (Shanmugasundaram et al. 1990b; Hirata et al. 1992). The mechanisms by which G. sylvestre produces antidiabetic effects include: recovery of pancreatic β-ells (Baskaran et al. 1990; Shanmugasundaram et al. 1990a, b), inhibition of glucose absorption (Hirata et al. 1992; Shimizu et al. 1997), stimulation of insulin release (Persaud et al. 1999; Sugihara et al. 2000) and increased glucose tolerance (Kar et al. 1999). Apart from this, the lipid lowering properties (Ahmed et al. 2008; Rachh et al. 2010; Shigematsu et al. 2001; Wang et al. 1998), glucose homeostasis (Rafiullah et al. 2006), antihyperglycemic effect (Gholap and Kar 2005) have been reported. It is also used in the treatment of asthma, cough, eye complaints, inflammations, family planning and snakebite (Selvanayagam et al. 1995; Uniyal 1993), besides possessing antimicrobial, diuretic, stomachic, antihypercholestremic, hepatoprotective and anti-saccharine activities (Nadkarni and Nadkarni 1976; Liu et al. 1992; Rana and Avadhoot 1992; Masayuki et al. 1997).
The total saponin fraction in leaves of G. sylvestre, commonly known as gymnemic acid (GA), belongs to a group of oleanane-type triterpene glycosides (Maeda et al. 1989; Sahu et al. 1996). However, GA is not one single compound but in fact consists of several related compounds (Kiuchi et al. 1992; Kurihara 1992). A number of triterpene saponins along with oleanane-type saponins (Ye et al. 2000, 2001) as well as dammarane-type saponins (Yoshikowa et al. 1992) have been reported from this plant time-to-time by different group of workers (Hooper 1889; Stöcklin 1967; Rao and Sinsheimer 1968, 1971; Sinsheimer and Rao 1970; Sinsheimer et al. 1970; Dateo and Long 1973;Chakravarti and Debnath 1981; Liu et al. 1992; Yoshikawa et al. 1993a). G. sylvestre leaf contains more than 20 saponin glycosides. The major saponin fraction comprises of gymnemic acid (the anti-sweet principle) which is a complex mixture of at least nine closely related acidic glycosides (Yoshikawa et al. 1991), also 23-hydroxylongispinogenin, gymneastrogenin (pentahydroxyoleane-12-ene) and a few dammarane derivatives as aglycones, that makes the purification and characterization of gymnemic acid a difficult task. Neverthless, a quantitative analysis of gymnemic acid by HPLC analysis of gymnemagenin, the common aglycone obtained on hydrolysis has been reported by Toshihiro et al. (1994). Growing commercial interest in the production of GA for the development of pharmacological agents posed the interest to investigate the in vitro production of active principles in G. sylvestre cultures.
Jasmonic acid (JA) and its methyl ester methyl jasmonate (MJ), have been proposed to be key signaling compounds in the process of elicitation leading to the accumulation of various secondary metabolites. The jasmonates have been previously applied in order to overproduce triterpene saponins in many plants, ginsenosides from Panax ginseng (Lu et al. 2001), saikosaponin from Bupleurum falcatum roots fragments (Aoyagi et al. 2001), and soyasaponin in cultures of Glycyrrhiza glabra (Hayashi et al. 2003). Jasmonates have also been reported to play an important role in signal transduction processes that regulate defense genes in plants (Farmer and Ryan 1990; Walling 2000). Triterpenoids were induced by the treatment of yeast elicitor preparation in Scutellaria baicalensis cell suspension cultures (Yoon et al. 2000), yeast extract also stimulated ginsenosides in Panax ginseng cell suspension cultures (Lu et al. 2001).
The presence of active principles gymnemic acid and gymnemagenin was successfully investigated in the cultured undifferentiated cells of G. sylvestre (Gopi and Vatsala 2006) and in cell suspension cultures of G. sylvestre (Subathra Devi et al. 2006). Abiotic stressers like blue light and fluorescence light have been shown to significantly influence the higher rate of gymnemic acid production in callus cultures (Ali Ahmed et al. 2009). The effect of plant growth regulators in combination with salt stress was reported to influence the gymnemic acid accumulation in callus cultures (Kumar et al. 2010). But till-date no attempts have been reported to study the elicitor-enhanced production of secondary metabolites in G. sylvestre.
This interest in gymnemic acid motivated us to investigate the production by in vitro cultures of G. sylvestre using elicitors. In this study, the influence of various elicitors on secondary metabolite production in cell suspension cultures of Gymnema sylvestre was investigated.
Materials and methods
Cell suspension cultures were established from in vitro grown leaf callus. Callus was induced in in vitro leaf tissue explants cultured on MS-agar solidified medium (Murashige and Skoog 1962) supplemented with 0.5 mg l−l 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l−l naphthaleneacetic acid (NAA), 2 mg l−l 6-benzyladenine (BA), 0.1 mg l−l picloram. Callus (about 200 mg) was inoculated to 25 ml MS liquid medium supplemented with same PGRs except 2, 4-D in a 100 ml conical flask for 20-day period and cultured at 110 rpm on rotatory shaker-incubator at 25°C in dark. Elicitors were added from the day of inoculation and cells were` removed at regular time periods each at 5, 10, 15 and 20 days, cells were collected by filtration for the gymnemic acid analysis.
MJ was dissolved in sterile distilled water, pH was adjusted to 5.8 and solution was filter-sterilized before use in concentrations ranging from 50, 100, 150 and 200 μM. Yeast extract (0.5, 1, 1.5 and 2 g l−l) and pectin (0.2, 0.4, 0.6 and 0.8%) were dissolved in distilled water and pH was adjusted to 5.8 before adding to the medium. Chitin (50, 100, 150 and 200 mg l−l) was dissolved in 50% H2SO4, diluted with distilled water and pH was adjusted to 5.8. Filter-sterilized elicitor solutions were added to the autoclaved culture medium individually. For the control set of cultures distilled water substituted the elicitors.
Analysis of cell growth
The growth was measured in terms of dry weights (DW) of cell mass. At the end of treatment period (5, 10, 15 and 20 days after inoculation) the suspension cultures were filtered off the culture medium and biomass was subsequently recovered from the filter paper (Whatman No.1) after washing 2–3 times with distilled water. The cell mass was placed between the folds of blotting paper to remove excess of water, dry weight was measured after drying the fresh cell mass in an oven at 45 ± 2°C for 48–72 h till it attains constant weight. To minimize the differences in growth (biomass increase) caused by the variation in inoculum size, growth index (fold) = Wtfinal/Wtinitial was used, where Wtinitial is the weight of inoculum at 0 day of inoculation and Wtfinal is the weight of the cell at harvest. Alternatively cell growth was also measured by sedimented cell volume (SCV). Cell viability was determined by vital staining with methylene blue stain also.
Extraction and analysis of secondary compounds
The major bioactive constituents of G. sylvestre are a group of oleanane-type triterpenoid saponins, known as “gymnemic acid”. Non-availability of the different reference standards makes the analysis job more difficult, therefore the estimation of the gymnemic cid was performed by hydrolyzing the extract first with alkali and then with acid. The gymnemagenin thus obtained was estimated by HPLC using commercially available gymnemagenin (Natural Remedies, Bangalore, India) as standard and the total gymnemic acid content was calculated by applying molecular weight corrections.
For the quantitative analysis of gymnemic acid in callus samples, finely ground dried tissue powder was first mixed with 50% ethanol allowed to dissolve completely for 1 h with frequent shaking. Later subjected to alkaline hydrolysis and subsequently acid hydrolysis in a water bath for 1 h each, finally pH was adjusted to 7.5–8.5 and filtered. For HPLC analysis the extract was filtered through a 0.2 μm micro filter (Millipore). HPLC analysis using Waters, model 2,487, pump 1,515 with 2,487 dual absorbance UV detection and 2414 RI detectors, manual sample injector was conducted. Separation was done using Waters C18 column (3.5 μm Ø 4.6 × 75 mm), the eluents being acetonitrile and water (1:3). The flow rate was 1.5 ml/min and the detector was set at 210 nm. The identity was confirmed by comparison with retention time of standard gymnemagenin (Natural Remedies, Bangalore, India).
The experiments were independently repeated thrice under the same conditions and all analyses were performed in duplicate. Data were analyzed by Microsoft Excel and presented as Mean ± SE.
In MJ treated cell suspension cultures, 15 days of elicitor (50 μM MJ) treatment resulted in highest gymnemic acid content (70.43 ± 0.26% DW) (Fig. 3a). This was followed by 100 μM MJ for 15 days (65.57 ± 0.33% DW), 150 μM for 10 days (51.62 ± 0.28% DW) and 200 μM for 10 days (43.37 ± 0.32% DW) culture period. Prolonged treatment at a higher concentration (200 μM MJ for 20 days of treatment) found to influence negatively on gymnemic acid content. The highest gymnemic acid production 34.38 ± 0.26 mg l−l DW was obtained after 15 days of treatment at 50 μM MJ treated culture, this was 2.8-folds higher compared to respective control (Fig. 3a). Cell growth was also influenced by the addition of MJ resulting in decreased growth fold compared to the respective control (Fig. 4a).
In yeast extract treated cultures, at 0.5 g l−l concentration for 20 days of treatment elicited the highest gymnemic acid (5.32 mg/500 mg DW) production. While at other concentrations also elicited higher production of gymnemic acid, with 1.5 g l−l concentration for 15 days (2.24 mg/500 mg DW), 2 g−l concentration for 20 days (1.95 mg/500 mg DW) of treatment and 1 g−l concentration for 10 days (1.67 mg/500 mg DW) compared to respective controls. The highest yield of gymnemagenin elicited with 0.5 g−l of yeast extract, 20 days is sixfolds higher than the respective control (Fig. 3b). Yeast extract also affected cell growth as indicated by the decrease in dry weight (Fig. 4b).
In Pectin treated cultures, at 0.6% pectin concentration for 20 days of treatment elicited highest gymnemic acid (64.19 ± 0.19%DW) production. In 0.2% pectin treated cultures there was a gradual accumulation of gymnemic acid content till the 15th day (46.7 ± 0.31% DW), while in 0.4% treated cultures there was an initial increase in gymnemic acid content (49.07 ± 0.17% DW) till 5th day, that decreased thereafter. A gradual increase in gymnemic acid content was observed in 0.6% pectin treated cultures, that reached highest value of 64.19 ± 0.19% DW at the end of 20 days of culture period. While, in cultures treated with 0.8% pectin similar trend was observed but with low levels of gymnemic acid accumulation (36.12 ± 0.25% DW). Of the pectin concentrations tested for elicitation, 0.6% showed better elicitation (64.19 ± 0.19%DW) over other concentrations, resulting in highest gymnemic acid production (23.7 ± 0.19 mg l−1DW) at the end of culture period, that was 2.65-fold greater than the respective control (Fig. 3c). Pectin also affected the cell growth in a concentration dependent manner (Fig. 4c).
In chitin treated cultures, at both 50 and 100 mg l−1 there was a gradual accumulation of gymnemic acid content up to 15th day, with slight decrease thereafter till 20th day, with 100 mg l−1 chitin (62.72 ± 0.25% DW) resulted accumulation higher gymnemic acid (50.76 ± 0.22% DW) than 50 mg l−1. The other two chitin concentrations 150 mg l−1 (42.26 ± 0.16% DW) and 200 mg l−1 41.83 ± 0.18% DW) also showed elicitation after 5 days of treatment. Of four concentrations of chitin, 100 mg l−1 elicited highest accumulation of gymnemic acid (23.46 ± 0.25 mg l−1 DW) that is more than 2.62-folds increase over the respective controls (Fig. 3d). Chitin also affected the cell growth in a concentration dependent manner (Fig. 4d).
It has been previously reported that elicitor treatment could be effective for enhancing secondary metabolite synthesis in cell suspension cultures (Bohlmann et al. 1995; Yazaki et al. 1997; Delauny et al. 2007; Sharan et al. 1998). The present study was initiated to study the influence of four different elicitors MJ, yeast extract, pectin and chitin on production of gymnemic acid in cell suspension cultures. Many reports emphasizes the importance of MJ in eliciting secondary metabolite production in cell and tissue cultures (Kittipongpatana et al. 2002; Choi et al. 2005; Gandzovska et al. 2007; Lystvan et al. 2009; Ana Coste et al. 2011; Junge Qu et al. 2011). MJ treatment has enhanced the accumulation of triterpenoid saponins in cell and tissue cultures of various plants, ginsenoside content in cultured cells (Lu et al. 2001) and hairy roots (Yu et al. 2002; Ali et al. 2006; Kim et al. 2009a, 2009b) of P. ginseng, saikosaponin in roots fragments of Bupleurum falcatum (Aoyagi et al. 2001; Kim et al. 2011), soyasaponin biosynthesis in cultured cells of Glycyrrhiza glabra (Hayashi et al. 2003; Lee et al. 2004; Shabani et al. 2009), asiaticoside production in whole plants (Mangas et al. 2006) and hairy roots (Kim et al. 2007) of Centella asiatica. Researchers have widely used MJ at a concentration up to 100 μM to increase the secondary metabolite production in in vitro cultures (Hayashi et al. 2003; Bae et al. 2006; Kim et al. 2010; Korsangruang et al. 2010) with plant cells or organs coming into direct contact with elicitor. Sensitivity of cells to elicitor concentration differs with plants (Mangas et al. 2006). In Panax ginseng cell suspension culture highest saponin production was reported at 500 μM MJ concentration (Lu et al. 2001), in our study also MJ was effective up to 200 μM concentration and the results revealed that the inhibitory effect of MJ on cell growth was not significant at its lower concentration especially at 50 μM, this was evident that at this concentration there was biomass increase in the initial 10 days of treatment period and this could be correlated with the elevated increase in gymnemic acid accumulation (70.43 ± 0.27% DW) in the next 5 days of treatment period. Even at 100 μM MJ concentration gymnemic acid accumulation continued to be higher (65.57 ± 0.33% DW) than respective controls, and it was only at 200 μM concentration for longer duration, MJ showed negative influence on both biomass and gymnemic acid accumulation. The negative effect of MJ at higher concentrations (0.2 mM) on cell growth was also reported in buckwheat (Fagopyrum esculentum) suspension cultures (Hu et al. 2011), hairy root cultures of ginseng (Kim et al. 2009a, b). Of the different concentrations of MJ tested, at 50 μM resulted in highest accumulation of gymnemic acid content (2.8-folds) followed by 100 μM MJ concentration (2.61-folds), compared to respective control and 15 days of treatment found to be optimum for elicitation. Our results are in agreement with the previous findings, that the longer the elicitor was in contact with the cells, the greater the increase in the secondary metabolites accumulation (Walker et al. 2002; Ali et al. 2006) and also suggest that G. sylvestre cells are not much sensitive to inhibitory effect of MJ at below 200 μM concentrations. Similar findings on the dose-dependency of MJ on centellosides and phytosterol production were reported in Centella asiatica suspension cultures (Bonfill et al. 2011). The effect of MJ or of any other elicitor is dependent on a number of factors which may interact, these include the elicitor’s specificity, concentration, the duration of treatment and the growth stage of the culture (Holden et al. 1988).
Besides the MJ, other elicitors are also found to stimulate biosynthesis of secondary metabolites (Ren and West 1992; Chen and Chen 2000; Lu et al. 2001; Flores-Sánchez et al. 2002). In yeast extract treated cultures, 0.5 g l−1 yeast extract was found to be optimum for elicitation and required duration is 20 days. It was therefore quite evident from the study that yeast extract (5.25-folds) and MJ (2.8-folds) markedly promoted the gymnemic acid production. There are many reports on yeast elicitor induced production of triterpenoids and other secondary metabolites in cell and tissue cultures of many plants, such as Scutellaria baicalensis (Yoon et al. 2000), Panax ginseng (Lu et al. 2001), Centella asiatica (Kim et al. 2004), Angelica gigas (Rhee et al. 2010) and Pueraria candollei (Korsangruang et al. 2010). In our study, yeast extract was found to be more effective in inducing gymnemic acid production than MJ and other two elicitors used. In the present study pectin at 0.6% 20 days of culture period has enhanced gymnemic acid production by 2.65-folds, while chitin at 100 mg l−1 increased the gymnemic acid production by 2.62-folds after 15 days of treatment. Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (Flores-Sánchez et al. 2002).
In conclusion addition of four different elicitors to cell suspension cultures of G. sylvestre increased the gymnemic acid content. Of the four elicitors tested yeast extract was evaluated to be superior over the other three elicitors tested with MJ standing second. All the elicitors affected the growth of the cells on a concentration and time duration dependent manner.
We thank Dr. Manjappa and Dr. Suresh, Department of Chemistry, Bapuji Institute of Engineering and Technology, Davangere, for the help in HPLC analysis.
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