Sodium nitroprusside enhances biomass and gymnemic acids production in cell suspension of Gymnema sylvestre (Retz.) R.Br. ex. Sm.

Gymnemic acids (a group of triterpenoid saponins) found in Gymnema sylvestre (Retz.) R.Br. ex Sm. works as the main hypoglycaemic active components. These can be the potential active pharmaceutical ingredient (API) to be used by pharmaceutical industries in modern medicines against diabetes. The present study aims to investigate the effectiveness of sodium nitroprusside (SNP) treatment for enhancement of cell suspension culture biomass and to quantify the production of deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII contents. Callus was obtained from in vitro derived leaves of G. sylvestre on MS medium fortified with 3.0 mg/L 2, 4-d (2, 4-dichlorophenoxyacetic acid) and 1.0 mg/L Kn (Kinetin), and the same were used further to produce cell suspension cultures. Cell suspensions were exposed to different concentrations of SNP (5, 10, 20 and 40 μM) and data were collected at 20, 30 and 40 days. Out of the tested concentrations, 20 μM SNP had the highest level of cell culture growth (398.94 ± 8.32 g/L Fresh cell weight (FCW) and 40.00 ± 0.75 g/L Dry cell weight (DCW) on 40-day as compared to control (MS with 3.0 mg/L 2, 4-d + 1.0 mg/L Kn). High-performance liquid chromatography analysis showed that maximum accumulation of deacylgymnemic acid (5.51 mg/g DCW), gymnemagenin (2.80 mg/g DCW) and gymnemic acid XVII (2.08 mg/g DCW) in 20 μM SNP treatment which is (13.43, 13.86 and17.33 folds) higher than the respective control at 40 days exposure. This research suggests that G. sylvestre cell suspension culture with optimal SNP elicitation treatment could be used as a good strategy for the large-scale production of these secondary metabolites at the industrial level. This research suggests that Gymnema sylvestre cell suspension culture with SNP elicitation treatment could be used as a good strategy for the large-scale production of these secondary metabolites at the industrial level.


Introduction
Gymnema sylvestre (Retz.) R.Br. ex Sm. belongs to the family Apocynaceae, it is a slow growing perennial woody climber. It contains many phytomolecules among them gymnemic acids (GAs) and gymnemasaponins are major secondary metabolites classified as triterpenoid saponins having oleanane type structural conformations. These are glycosidic compounds having one or more glucose or its derivative unit directly attached to them through ester bond. These compounds are proved to block intestinal site hence decreasing overall glucose adsorption therefore used for clinical applications (Laha and Paul 2019;Khan et al. 2019). The plant takes more than two years to be fully mature and can be harvested only twice a year. The plant leaf is the part which majorly bears GAs that is only ≥ 2% w/w of dry leaves. G. sylvestre has been used in Ayurveda medicine since antiquity to treat diabetes, malaria and snake bite (Anjum and Hasan 2013;Khan et al. 2019). Ayurvedic practitioners prescribe this plant for the treatment of dyspepsia, jaundice, asthma, leucoderma and bronchitis (Anis et al. 2000;Laha and Paul 2019;Khan et al. 2019). Kanetkar et al. (2007) and Singh et al. (2008) found multiple potential applications mentioned in Ayurveda, Siddha and Unani system of medicine in India for treating diverse human complaints, but only a few achieved scientific information on its secondary metabolites production.
Plant cell and tissue cultures ensured that cell suspension cultures have been explored to serve as an alternative source for large scale production of useful secondary metabolites (Thorat et al. 2017). Also, accumulation of secondary metabolite in in vitro cultures outperformed those seen in plants cultivated in the field (Das and Bandyopadhyay 2020). Accumulation of secondary phytoconstituents in cell suspension or callus culture is highly dependent on different types of elicitors which lead to activate biosynthetic pathway route key enzymes (Sharan et al. 2019;Pandey et al. 2020).
The recent studies showed that signalling molecules involved in the regulation of plant metabolite synthesis include reactive oxygen species, nitric oxide (NO), calcium ion, polyamines (PAs) salicylic acid and jasmonic acid (Ma et al. 2013). Sodium nitroprusside (SNP), highly reactive bioactive molecule that plays an important role in plant tissue and organ culture such as growth and development of plant (Kolbert et al. 2008), stimulates callogenesis regeneration response in Albizzia lebbeck (Kalra and Babbar 2010), enhances callus and multiple shoot induction (Sarropoulou and Maloupa 2017;Subiramani et al. 2019;Pandey et al. 2020), promote root formation and callus induction (Hesami et al. 2020) and enhanced shoot regeneration and improved salinity stress in soybean (Karthik et al. 2019). Ötvös et al. (2005) reported that SNP in combination with 2,4-d significantly stimulated cell division and embryogenic cell growth in Medicago sativa. In Hyoscyamus niger, supplementation of SNP at 50 μM increased callus fresh weight (Samsampour et al. 2018). As a result, this led us to study the effect of sodium nitroprusside (SNP) in secondary metabolites production from suspension culture of G. sylvestre for enhancement of deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII metabolites.

Resource of plant material and culture condition
In this study, Gymnema sylvestre immature follicles were obtained from the field of CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow. The immature follicles were sterilized according to our previous experiment (Mahendran et al. 2021). The immature embryo was inoculated on MS (Murashige and Skoog 1962) basal medium with sucrose (3.0%) and agar (0.8%) to initiate aseptic seedlings. The seedlings were maintained in cooling white fluorescent light (60 μmol m -2 s -1 ) with light/dark (16/8 h) of photoperiod at 25 ± 2 °C. In vitro derived young leaves were utilized as explants for the establishment of callus and cell suspension culture of G. sylvestre. Murashige and Skoog (1962) medium, 2,4-Dichlorophenoxyacetic, (2,4-d), Kinetin (Kn), and Sodium nitroprusside (SNP) were purchased plant tissue culture grade from Himedia (Mumbai, India). HPLC grade methanol and acetonitrile obtained from Merck (Mumbai, India) and ultra-pure water was prepared using a Milli-Q (Millipore, France) was used. Deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII standards were supplied by Sigma-Aldrich (Münich, Germany).

Cell suspension culture conditions
In the present study, cell suspension cultures initiated from 30 days old whitish friable callus (1 g) that was inoculated into 50 mL of MS liquid medium supplemented with 2,4-d (3 mg/L) and Kn (1.0 mg/L) combination served as a control. The cultures were incubated on a shaker (100 rpm) at 25 ± 2 °C for 16/8 h (light/dark).

Sodium nitroprusside (SNP) elicitation
In the present investigation, SNP was dissolved in distilled sterile water and sterilized through syringe filters then used at different concentrations (5, 10, 20 and 40 μM). SNP supplemented on the same day of cell suspension initiated and the cells were harvested at 20, 30 and 40 days. Cell suspension culture was collected from the medium at 20, 30 and 40 days for determination of cell growth biomass accumulation (fresh and dry cell weight) and yield of GAs contents.
At the end of each culture intervals, the cell suspension was harvested from the culture medium for fresh cell weight and dry cell weight measurement. Harvested cells passed through a two-layer muslin cloth to remove the water from the cell and measured the fresh cell biomass weight. Further, cells biomass was placed on a Petri dish and dried at 50 °C in a hot air oven for 24 h and cell dry biomass measured.

Morphology of suspension cells
100 mg cells from suspension cell cultures were checked in 1 mL 0.005% (w/v) neutral red and acridine orange solution to analysis the morphological variations during growth phases. 30 μL of stained cells were transferred to the slide and observed under the light microscope (Olympus, India).

Sample and standards stock preparation
Exactly weighed 1 g of dried biomass obtained from cell culture and wild plant leaves were grounded into a relatively homogenous powder using a grinder. Powder was then defatted using 10 mL of petroleum ether at 50 °C for 6 h and dried. Sample was then extracted with a mixture of 70:30 (Water: Methanol) at 50 °C for 6 h for complete extraction of gymnemic acids. The extract was dried using rotatory evaporator and 10 mg of each of prepared extract was weighed accurately and dissolved in methanol (1 mL) for making final concentration of 10 mg/mL and kept 15 min for sonication and filtered on a 4 mm membrane filter and kept at 4 °C until further use.
Deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII standards were supplied by Sigma-Aldrich (Münich, Germany). Standard stocks were prepared by diluting each of them separately at a concentration of 1 mg/mL in methanol. 20 μL of injection volume of sample was injected into the HPLC system.

Quantification of deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII
Our previously reported gradient method was used for the quantification of GAs in samples Mahendran et al. (2021). The chromatographic conditions were Waters HPLC system built with a binary pump, photodiode array detector (PDA) and Rheodyne injector. SunFire C18 column (5 µm, 250 × 4.6 mm) and Empower Pro software (Waters, USA) was used for analysis. The detection was achieved at 205 nm, column temperature kept at 30 °C and the flow rate was 1 mL/min. The deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII contents were quantified using peak area obtained through HPLC.

Statistical analysis
Each experiment was repeated thrice containing 5 replicates each and data expressed in terms of mean ± standard deviation (SD). The significant difference between control and SNP treatment was analysed by Tukey's HSD (honestly significant difference) test using one-way ANOVA with IBM SPSS statistics (version 20.0, USA). Significant was considered when P < 0.05.
A perusal of literature revealed that cell suspension culture has to be the most capable method used to increase the cell growth, biomass and secondary metabolite production in vitro cultures (Mahendran et al. 2018;Hu et al. 2019;Açıkgöz, 2020). In this study, white friable callus obtained from 3.0 mg/L (2, 4-d) + 1.0 mg/L (Kn) (Fig. 1A, B), and same combination was used to establish a culture of cell suspension in which there were no aggregations or clumps of cells noted. In the present study, suspension cell culture growth kinetic was monitored in terms of fresh cell weight (FCW) and dry cell weight (DCW) and results are shown in Fig. 2. Cell suspension growth was shown to be increased greatly with the increasing concentration of SNP. Among the different concentrations of SNP tested, the highest accumulation of FCW and DCW were 71.21 ± 1.56 and 7.05 ± 0.04 g/L observed on 20th day with 20 μM SNP that were 1.43 and 1.41 times higher compared with control. The biomass accumulation in cell culture increased with increasing concentration of SNP up to an optimal level of 20 μM (Fig. 1C). SNP has increased the growth of cell culture biomass at 5-20 μM concentrations tested and the entire exposure period of the study compared with the control. The maximum amount of biomass accumulation was achieved at 40th day with 20 μM SNP (398.94 ± 8.32 g/L FCW and 40.00 ± 0.75 g/L DCW) and was nearly 1.17 folds higher than control. Earlier studies demonstrated that elicitors are very effective for the enhancement of cell growth and increase metabolites accumulation. Furthermore, the type of elicitors, concentration and exposure period favours the biomass accumulation in cell suspension culture (Salma et al. 2018;Zare-Hassani et al. 2019;Açıkgöz, 2020).
Our study confirms the efficiency of SNP treatments, promoted cell growth and biomass accumulation. Similar effectiveness of SNP on cell growth has been reported in Hypericum perforatum . Furthermore, SNP in combination with 2,4-d significantly promoted embryogenic cell in Medicago sativa (Ötvös et al. 2005). Some studies have shown that SNP either alone or in combination treatments are very effective for callus induction (Samsampour et al. 2018;Subiramani et al. 2019;Pandey et al. 2020). Results of the present investigation in G. sylvestre cell suspension cultures suggested that SNP at 40 μM drastically diminished cell growth and biomass accumulation (225.39 ± 5.13 g/L FCW) compared with control (339.08 ± 1.76 g/L FCW) at 40 days (Fig. 2). Similar inhibitory effects have been reported at higher concentrations of elicitors in Psoralea corylifolia (Gajula et al. 2018), Leucas aspera (Vijendra et al. 2020) and Ocimum basilicum (Açıkgöz, 2020).

Morphology of G. sylvestre cell suspension
The morphological differences at different growth times in the suspension cultures are shown in Fig. 1C, F. The cells were rapidly growing and individually or in cell masses of small groups of cells aggregated and settled at the bottom of the flask were detected in the suspension culture (Fig. 1C,  D). The shape of the cells varied in size with round, oval shape or elongated shaped and health of cell visible clearly nuclei and cell components (Fig. 1G, H). In contrast, death cells showed cytoplasm and cell wall shrinkage (Fig. 1E, F, I).

HPLC analysis of gymnemic acids
To enhance the deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII content in suspension culture of G. sylvestre, different concentrations of SNP (5, 10, 20 and 40 μM) and days (20, 30 and 40) were investigated. The results displayed that SNP had significantly boosted the production of deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII at all tested concentration over control. However, SNP at 20 μM dose produced the highest accumulation of deacylgymnemic acid (1.80 mg/g DCW), gymnemagenin (1.91 mg/g DCW), gymnemic acid IV (0.74 mg/g DCW) and gymnemic acid XVII (1.14 mg/g DCW) contents for 20 days of exposure in cell suspension culture, which was represented 6.09, 17.36, 1.01 and 9.5 folds higher compared with the control cultures ( Fig. 3A-D). Similarly,  demonstrated that cell cultures of Catharanthus roseus treated with 10 and 20 mM SNP enhanced formation of catharanthine, ajmalicine and total alkaloids. Khezerluo et al. (2018) found that maximum accumulation of hyoscyamine and scopolamine production (1.2-fold and 1.5-fold) in hairy root culture of Hyoscyamus reticulatus was detected at 50 and 100 μM SNP at 48 and 24 h.
Interestingly, higher content of gymnemagenin (2.86 mg/g DCW) and gymnemic acid IV (1.24 mg/g DCW) was also observed at 5 μM of SNP treated suspension cell culture at 30 and 40 days of culture respectively (Figs. 4B and 5C). Likewise, the maximum (3.25 mg/g) gymnemic acid XVII production was observed at 10 μM SNP at 40 days (Fig. 5D). Similarly, Wang et al. (2009) reported that SNP (10 μM) and cerebroside (30 μg/mL) combination in hairy root callus of Artemisia annua exhibited more effective for improving artemisinin production up to 2.3-fold over the control. In another study, researchers reported that lower concentrations of SNP (15 μM) have been more effective in enhancing catharanthine production in suspension cells culture of C. roseus . Likewise, the maximum 3.2-fold hypericin production in suspension  .

Conclusion
In conclusion, this work suggested that the effects of SNP treatment approach improved cell culture growth (biomass) and yield of gymnemic acids (triterpenoid saponins) content in cell suspension cultures of G. sylvestre. 20 μM SNP treatment and exposure time of 40 days showed the highest rate of cell culture biomass accumulation (398.94 ± 8.32 g/L FCW and 40.00 ± 0.75 g/L DCW) and the maximum production of deacylgymnemic acid (5.51 mg/g DCW), gymnemagenin (2.80 mg/g DCW) and gymnemic acid XVII (2.08 mg/g DCW) compared with the control culture. The present study has demonstrated that establishment of efficient cell suspension cultures of G. sylvestre for biomass and enhanced production of triterpenoid saponins (gymnemic acids) at industrial scale.