Gymnema sylvestre

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Abstract

Gymnemic acids, a pentacyclic triterpenoid, present in the Gymnema sylvestre R.Br. leaves, are known to be very effective against curbing of diabetes and obesity. The present work reports on the use of statistical methods and precursors (both natural and synthetic) for the enhanced production of gymnemic acids from G. sylvestre solid state callus cultures. The significance of essential medium constituents such as plant growth regulators and vitamins sources were investigated through Plackett-Burman design through three different cell culture media. The next step involved the interaction between the significant factors in the significant cell culture media through response surface methodology (RSM). Gymnemic acid production increased considerably from 291.45 ± 0.05 µg/g dcw to 386.88 ± 0.23 ug/g dcw after RSM. Precursors were added in different concentrations to check the effect they generate on gymnemic acid production; wherein natural precursors included tomato juice, carrot juice, coconut water and coconut milk while squalene and casein hydrolysate acted as synthetic precursors. The maximum enhanced gymnemic acid content was observed to be at 492.37 ± 0.17 mg/g DCW with addition of 700 mg/l of squalene.

Keywords:

Gymnema sylvestre; gymnemic acids; plant cell culture; statistical media optimization; precursors

Abbreviations

BA 6- Benzyl amino purine

2,4-D 2,4 Dichloro phenoxy acetic acid

Kn Kinetin

IAA Indole acetic acid

NAA Naphthalene acetic acid

IBA Indole butyric acid

TH Thiamine hydrochloride

PH Pyridoxine hydrochloride

NA Nicotinic acid

RSM Response surface methodology

CJ Carrot juice

TJ Tomato juice

CW Coconut water

CM Coconut milk

CH Casein hydrolysate

Sq Squalene

DW Dry weight

MS Murashige and Skoog medium (Murashige and Skoog, 1962)

B5 Gamborg's medium (Gamborg and Miller, 1968)

WPM Woody Plant Medium (Lloyd and McCown, 1980)

D/W Distilled water

DCW Dry cell weight

Introduction

Plant tissue culture has long been used to ensure the survival of many medicinal, industrially important and rare plants. With the advent of technology, fundamental principles of tissue culture started getting used for production of industrially important phytoconstituents. The production, extraction and analysis of phytoconstituents from callus cultures have therefore assumed importance (Dicosmo and Misawa, 1995).

Gymnema sylvestre (Asclepiadaceae), a vulnerable species which is slow growing and most importantly, a perennial medicinal woody climber found in central and peninsular India. A potent anti diabetic plant having excellent usage in folk, ayurvedic and homeopathic systems of medicine for treating asthma, inflammations and snakebite. Leaves of this species yield acidic glycosides and anthroquinones, exhibiting anti diabetic, anti sweetener and anti-inflammatory activities (Komalavalli and Rao, 2000). G. sylvestre leaves contain triterpene saponins (both oleanane and dammarene classes); wherein oleanane saponins were identified to be gymnemic acids and gymnemasaponins (Dateo and Long, 1973), whereas the dammarene saponins are gymnemasides (Liu and others 1992). Till date, the chemistry of these acids and related compounds has been extensively studied (Sinsheimer and Manni, 1965; Sinsheimer and others 1970; Yoshikawa and others 1989). Gymnemic acids are known to delay the glucose absorption in the blood system and in the recent past gymnemic acid based formulations are the new obesity-controlling agents (Kanetkar and others 2007).

Optimization of media components for bioprocesses and especially for plant cell culture includes the identification and subsequent concentration ranges of relatively significant media components. These include plant growth regulators, vitamins, minerals, and culture conditions. Traditionally, this aspect had been done by the “single factor” optimization method; which is time consuming and hence, cannot be relied upon, if the bioprocess is to be taken up a larger scale. Hence, in order enhance the system under study; an in-depth understanding of the optimization of media parameters should be sought. This can be achieved by devising statistical methods; which will give the end user the optimum concentration levels of the individual components (Chattopadhyay and others 2002).

To the best of our knowledge, there are no reports documenting the use of statistical approaches and precursors for the enhanced gymnemic acid production in solid state cultures. Previous reports on gymnemic acid production have been restricted to single factor method at both solid state and suspension cultures (Gopi and Vatsala, 2006, Devi and others 2006, Saha and others 2008). In the present study, successful attempts were made to understand the significance and interaction for enhancement of gymnemic acid production through the usage of precursors; both natural and synthetic, in a medium previously optimised by statistical methods.

Materials and Methods

Chemicals

α- naphthalene acetic acid (α-NAA), benzyl adenine (BA), kinetin, indole acetic acid, indole butyric acid, agar (extra pure), sucrose, di-sodium EDTA, ready prepared Murashige and Skoog [MS] (Murashige and Skoog, 1962), Gamborg's medium [B5] (Gamborg and Miller, 1968) and Woody Plant Medium [WPM] (Lloyd and McCown, 1980) media were procured from HIMEDIA Lab. Pvt. Ltd. Mumbai. Nicotinic acid, pyridoxine HCl, thiamine HCl, ethanol, casein hydrochloride were procured from S. D. Fine chemicals Ltd., Mumbai. Squalene was procured from Sigma-Aldrich, USA. Tomatoes, carrots, coconut water and freshly cut coconuts were obtained from the local market at Matunga, Mumbai.

Standard gymnemagenin (92% pure) was procured as a gift sample from M/s Laila Impex, Hyderabad, India.

Plant material

Young leaves were taken as explants, collected from an elite vine (5 years old) in the gardens at the Institute of Chemical Technology (ICT) gardens, Mumbai, India. The plant was identified at the Institute of Chemical Technology (ICT), where the voucher specimens have been maintained.

Explant sterilization

The leaves were first washed under tap water (15 min) and then with distilled water (10 min), before transferring them to a beaker containing 500 ml of distilled water with two drops of Teepol® - detergent. Constant stirring for 10 min enhanced the rate of disinfection. They were rinsed with six changes of distilled water. Under aseptic conditions, explants were washed in freshly prepared 70 % alcohol for 30s followed by 4 – 6 changes of sterile glass distilled water (D/W). The final step included 0.1 % mercuric chloride for 1 min concluded by 6-7 changes of sterile distilled water of 1 min duration each. The explants were then placed in between pieces of sterile tissue paper to remove excess moisture. The leaves were cut into small pieces and then inoculated into cell culture media.

All media used were adjusted to pH 5.8 using 0.1 N HCl or 0.1 N NaOH, and sterilized in an autoclave at 121°C for 20 min. All vitamins and growth regulators were filter sterilized using 0.22 m filter discs (Millipore, India) and added to autoclaved and cooled medium.

Induction of callus culture systems and environmental conditions

20 ml portions of media were dispensed into culture tubes (23×150 mm, Borosil, Mumbai, India), and plugged with non-absorbent cotton wrapped in one layer of wire gauze cloth. All cultures were maintained at 25 ± 2 °C and a relative humidity of 55–60 % under cool-white fluorescent light having an intensity of 12 Lux and a 16 h (light)/ 8 h (dark) photoperiod, for a period of 30 days.

Analytical determinations

Estimation of biomass on dry cell weight basis

The callus obtained after the final subculture was dried at 60°C for 24 h. after which the dry weight was recorded and the cells were subjected to conversion from acid to genin for analytical quantification (Kanetkar and others 2006). After the final hydrolysis step, the methanolic extract centrifuged at 10, 000 rpm and the supernatant was taken for HPTLC analysis. The pH of the extract was adjusted in between 7-8 for enabling the stability and subsequent application of the samples for analysis.

Estimation of gymnemagenin

The analysis of gymnemagenin was carried out as previously reported (Kanetkar and others 2006). After the adjusting of pH, 10 ml of the sample was applied on the TLC plate in triplicates for enabling the repeatability of the analysis. In order confirm the peak resolution and separation of the compound, reference standard was also applied on the plate along with the samples. The genin was thus identified by comparing the chromatogram of reference standard gymnemagenin to that obtained in the samples.

Optimization of major nutrients for gymnemic acid production in solid state culture using statistical methods:

Plackett-Burman design for evaluation of significance of medium components

In order to give justice to the screening process via the statistical approach, it was decided to take into consideration media components such as plant growth regulators (callus induction) and vitamins (enhancement of growth) for the Plackett-Burman design (Plackett & Burman, 1946) and then implement the design on 3 cell culture media for understanding their significance on the gymnemic acid production. The aim was to observe the effects of these components in the subsequent media on the cell growth and in turn gymnemic acid production. Plant growth regulators – BA, 2, 4-D, Kn, NAA, IAA and IBA; cell culture vitamins – pyridoxine hydrochloride, thiamine hydrochloride and nicotinic acid and three cell culture media – Murashige and Skoog [MS] (Murashige and Skoog, 1962), Gamborg's medium [B5] (Gamborg and Miller, 1968) and Woody Plant Medium [WPM] (Lloyd and McCown, 1980) were included in this design. This will prove to be a dual approach; cell culture media as well as plant growth regulators get screened for their significance on gymnemic acid production.

A total of 11 (n) variables (nine media components and two dummy factors) were studied in twelve (n + 1) experiments. Factors evaluated were BA (0.2, 0.6 mg/l), 2,4-D (0.2, 0.6 mg/l), IBA (0.2, 0.4 mg/l), IAA (0.2, 0.4 mg/l), NAA (0.1, 0.4 mg/l), Kn (0.1, 0.4 mg/l), thiamine hydrochloride (1, 10 mg/l), pyridoxine hydrochloride (1, 10 mg/l) and nicotinic acid (1, 10 mg/l) were taken from preliminary studies (results not shown) to study their significance on gymnemic acid production. The s in the parenthesis indicate the high and low values of the respective variables. The experimental design protocol was developed with the help of Design-Expert version 6.0.10 software (Stat-Ease) and the response observed for the three media is represented in Table 1.

The explants used for the experiments were taken from a seven year old vine and then inoculated in test tubes containing the combination of the medium as per the design. The experiments were done in triplicates. The cultures were incubated as per the conditions mentioned previously. The cells (callus) obtained were allowed to acclimatize after their induction was done; for a period of 2 weeks before they could be harvested. The dry cell weight and gymnemic acid content through gymnemagenin were analyzed for each set.

Concentration optimization of significant components

Experimental design – Box Behnken

Optimization refers to improving the performance of a system, a process, or a product in order to obtain the maximum benefit from it. Response surface methodology (RSM) is one such optimization tool; an empirical statistical modeling technique employed for multiple regression analysis using quantitative data obtained from properly designed experiments to solve multivariate equations simultaneously (Kalil and others, 2000). RSM is used to determine the optimum nutrient concentrations, for the production of gymnemic acids.

Once the significant variables were obtained from Plackett–Burman design, RSM was performed to optimize the medium components for enhanced gymnemic acid production for three independent variables. The Box Behnken (Box and Behnken, 1960) design was chosen in order to enhance the interaction between the variables for generation of a maximum response and also for the fact that it allows the efficient estimation of the first- and second-order coefficients of the mathematical model. (Bezerraa and others 2008). These designs are not only efficient but also economical when compared to their 3k designs; otherwise used for a larger number of variables. The design generated included a total of 17 experimental runs in order to optimize the levels of the independent variables for the gymnemic acid production in solid state culture. Regression analysis was performed on the data obtained from the design experiments. Coding of the variables was done according to the Eqn 1.

i =1, 2, 3, . . . , k (1)

where: xi is the coded value of an independent variable; Xi is the actual value of an independent variable; X0 is the actual value of an independent variable at centre point; and ∆X is the step change value of an independent variable. The experiments were carried out at in triplicates, which was necessary to estimate the variability of measurements. The relationship of the independent variables and the response was calculated by the second order polynomial Eqn 2.

k k

Y = β0 +Σ βi Xi +Σ βii Xi Xj +Σi Σj βij Xi Xj (2)

i =1 i =1 i < j

Y is the predicted response; β0 a constant; βi the linear coefficient; βii the squared coefficient; and βij the cross-product coefficient, k is number of factors.

The second order polynomial coefficients were calculated and the subsequent response plots were obtained using the software package Design Expert Version 6.0.10 to estimate the responses of the dependent variable.

Use of precursors (natural and synthetic) for enhanced production of gymnemic acid production

A study was initiated for investigating the effects of precursors; both natural and synthetic, on gymnemic acid production in solid state callus culture. Various concentrations of carrot juice, tomato juice, coconut water, coconut milk, casein hydrolysate and squalene were added in the culture medium. For the preparation of the juices, thoroughly washed carrots were crushed in a juicer to produce quantity sufficient juice using double distilled water, and this was considered as 100% carrot juice (e.g.100 ml of juice was prepared from 100 g of carrot). This juice was filtered twice through muslin cloth. The juice so obtained was boiled for 10 min and filtered again through muslin cloth to remove all agglomerates. Both tomato and carrot juices were prepared using the same procedure (Bule and Singhal, 2009). Fresh coconut water was filtered sterilized and used in various concentrations.

Coconut water is the liquid part of the coconut (Cocos nucifera) endosperm and is a byproduct of copra and desiccated coconut industry. The application of coconut water as a precursor for growth can be justified by its chemical composition (Survase and others 2005). Coconut water also contains high concentration of amino acids viz.: alanine, arginine, aspartic acid, cystine, glutamic acid and so on; which makes it a good growth enhancing factor on a total basis. Hence, it was considered for the present study.

Coconut water was taken from local coconut vendor. It was filtered twice through muslin cloth and then filter sterilized before addition to the RSM optimised medium in various concentrations. For coconut milk preparation, the milk from coconuts (minimum 3) was drained and deproteinized by boiling for 10 min. It was autoclaved separately in volumes less than 25 ml; after filtration through Whatman No. 1 filter paper. It was stored at -70°C and thawed before addition to the RSM optimized culture medium in variable concentrations (Purohit, 2004). A working stock of 1 mg/ml was prepared from the original stock solution of squalene of purity 99% (Sigma-Aldrich, USA)

Tomato and carrot juices (5 – 20%; v/v), coconut water (10 – 20%; v/v), coconut milk (5 – 20%; v/v), squalene (100 – 1000 mg/l) and casein hydrolysate (0.2 – 1 g/l) were added to the RSM optimized medium components and their responses are recorded in Table 4.

Results and Discussion

Evaluation of significant media parameters for gymnemic acid production in solid state culture using Plackett-Burman design

Plackett–Burman experiments highlighted the importance of optimising culture variables in attaining higher gymnemic acid production. The results of data analysis for the effect of eleven selected parameters on the growth of G. sylvestre and gymnemic acid production are shown in Table 1. Among the eleven variables which were likely to play a significant role in enhancing gymnemic acid production, three factors, viz.: BA, NAA and Kn were found to be most significant and the medium which was the most significant was observed to be Gamborg's medium (B5).

Plant growth regulators have always played a major role in induction of callus from the explants; hence, here too a similar response has been observed. BA, NAA and Kn are observed to be most influential not only for the growth but also for the production of gymnemic acids. The dummy variable incorporated in the design did not exhibit any impact on gymnemic acid production. This system was further subjected to response surface methodology.

Concentration optimization of screened components using Box Behnken design

To examine the combined effect of three different plant growth regulators (independent variables) varied over three levels including three replicates at the centre point on gymnemic acid production; an experimental design with 17 runs was formed. The three significant factors that were identified by Plackett–Burman design i.e. BA, NAA and Kn were further optimized using RSM in Gamborg's medium (B5); the significant medium.

Second order polynomial equation was used to correlate the independent process variables, Xi, with gymnemic acid production. The second order polynomial coefficient for each term of the equation determined through multiple regression analysis using the Design Expert. Table 3 shows the response from the Box Behnken matrix where the maximum gymnemic acid production to be at 385.36 ± 0.23 mg/g dcw.

The analysis of variance (ANNOVA) as reported in Table 4 showed that this regression model is highly significant. The Model F-value of 10.905 implies that the model is significant. Model F-value is calculated as ratio of mean square regression and mean square residual. Model P-value (Prob > F) is very low (0.0024). This resignifies the significance of the model. The P values were used as a tool to check the significance of each of the coefficients, which, in turn, are necessary to understand the pattern of the mutual interactions between the test variables. The smaller the magnitude of the P, the more significant is the corresponding coefficient. Values of P less than 0.05 indicate model terms are significant. Furthermore, the experimental and predicted values (Table 3) shows the agreement between the yield predicted by the model and the experimental data is very strong, with a difference less than 1.37% of extraction rate.

The P-values were used as a tool to check the significance of each coefficient, which also indicates the interaction effects between each independent variable. The model indicates that among the test variables used in the study, C (Kn), AC (BAxKn) and BC (NAAxKn) are significant model terms. Kinetin (P = 0.0034) in individual capacity had the largest effect on gymnemic acid production. The mutual interaction between BA – Kn (P= 0.0059) and NAA – Kn (P= 0.0030) were also found to be important.

The corresponding second-order response model (see Eqn. 2) that was found after analysis for the regression was:

Yield (mg/g) = 213.50083 + 243.00000 * BA + 326.27500 * NAA + 333.18333 * Kn - 281.77778 * BA2 - 368.50000 * NAA2 - 410.44444 * Kn2 - 203.00000 * BA * NAA - 228.44444 * BA * Kn - 541.00000 * NAA * Kn

The fit of the model was also expressed by the coefficient of determination (R2), which was found to be 0.8623, indicating that 86.23 % of the variability in the response could be explained by the model. This is also evident from the fact that the plot of predicted versus experimental Fig 1. Almost all points are lying in close proximity of zero error line showing that the prediction of experimental data is quite satisfactory. Accordingly, three-dimensional graphs were generated for the pair-wise combination of the three factors. Graphs are given here to highlight the roles played by various factors (Fig 2). From the central point of the contour plot or from the bump of the 3D plot the optimal composition of medium components was identified.

The optimal concentrations for the three components as obtained from the maximum point of the model were calculated to be as 0.28 mg/l, 0.24 mg/l and 0.17 mg/l for BA, NAA and Kn respectively. By substituting levels of the factors into the regression equation, the maximum predictable response for gymnemic acid production was calculated and was experimentally verified. The maximum production of gymnemic acid obtained experimentally using the optimized medium was 385.36 ± 0.23 mg/g dcw, which is in correlation with the predicted value of 386.18 mg/g dcw by the RSM regression study.

Use of precursors for enhancement of gymnemic acid production

In order to observe the effects of precursors on the gymnemic acid production callus was induced in the RSM optimized medium with the addition of precursors. Carrot and tomato juice were used as natural precursors since these plants accumulate carotenoids and contain active precursors of polyprenyl pyrophosphate, the pathway leading to the formation of squalene like compounds and in turn oleanane saponins i.e. gymnemic acids. Coconut water and coconut milk are reported to exhibit a cytokine like response for the enhancement of growth of callus and were hence used for the present studies.

Tomato and carrot juices when added to the RSM optimized medium in varying concentrations ranging from 5 – 20 %; v/v. gave the maximum gymnemic acid content at (399.48 ± 0.18 mg/g dcw) 15% tomato juice and (402. 47 ± 0.08 mg/g dcw) 20 % carrot juice. 18% coconut water and 10% coconut milk gave a maximum response at 392.49 ± 0.61 mg/g dcw and 386.02 ± 0.23 mg/g dcw, respectively. Coconut water contains sucrose, glucose and fructose and other constituents as proteins, citric acid and mineral salts (De Sousa and others, 2005). These compounds play an important role in both the growth of cells as well as gymnemic acid production. Casein hydrolysate; also known as edamin, is the milk protein digest composed of amino acids and other substances. This complex yet undefined product is used as an additive in cell culture media and is the non-specific source of organic nitrogen for boosting the growth of cells. 0.6 g/l casein hydrolysate gave a maximum response of 396.19 ± 0.15 mg/g dcw.

The main pathway leading to the formation of gymnemic acids is the acetate – melavonate (Ac–MVA) pathway (Agarwal and others 2000) and the cyclization of 2, 3-oxidosqualene is the first committed step in triterpenoid biosynthesis (Dubey and others 2003). 2, 3-oxidosqualene; obtained from squalene, is considered to be the starting material for production of oleanane saponins i.e. gymnemic acids, as they are formed in the same biochemical pathway; hence forming the basis of the logic behind its addition in the tissue culture medium (Haralampidis and others 2002). The logic behind its addition was that to induce a supplementary trigger to the effect already induced by the media components previously optimized through RSM for the enhanced gymnemic acid production. This hypothesis worked in our favor as the over all maximum production was at 700 mg/l concentration of squalene (492.37 ± 0.18 mg/g DCW). This response was the maximum response observed on an overall basis for the precursors. An interesting phenomenon was observed in the responses generated during the squalene addition. At 900 and 1000 mg/l concentration, there is no induction of callus; which indicates that squalene in higher concentrations is toxic for the explant. 2 shows the various callus developed during the work; wherein (a) shows the callus developed through Plackett-Burman, (b) shows the callus developed through RSM and (c) shows the callus developed by addition of squalene (700 mg/l) to the RSM optimized medium.

Table 6 gives the overall summary of the production of gymnemic acids through the strategies discussed in this report. Plackett-Burman design not only gave the significant parameters (media components) but also the significant medium. Gamborg's medium recorded the maximum gymnemic acid production 291.45 ± 0.05 µg/g dcw. RSM optimized medium (B5 medium) with 0.28 mg/l (BA), 0.24 mg/l (NAA) and 0.17 mg/l (Kn) gave a maximum gymnemic acid production at 386.88 ± 0.23 ug/g dcw. The addition of precursors turned out to be a good exercise; wherein squalene (700 mg/l) triggered an additional effect on the production and it was recorded to be 492.37 ± 0.17 mg/g dcw.

Conclusion

Gymnema sylvestre R. Br. is a plant system that has been understudied till date especially at callus culture level. Here, we present the successful attempt for studying the production parameters and their effect on gymnemic acid production. A combination of statistical design and precursor addition was the approach successfully implemented and such combinations seem to be the need of the hour for enhancing production of essential metabolites. Using the statistical methods, the yield of gymnemic acids was significantly increased (291.45 ± 0.05 µg/g after Plackett-Burman to 386.88 ± 0.23 ug/g dry cell weight after RSM) with minimum number of experiments. RSM was fairly accurate in predictive modeling and media optimization, which suggests that the relation between the concentration of plant growth regulators and yield of gymnemic acids can be reasonably approximated by quadratic non-linearity. Precursor study also gave foundation to the hypothesis of addition of starting compound (squalene) to the RSM optimized medium at very low concentrations.

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