Sodium Nitroprusside Enhances Biomass And Gymnemic Acids Production In Cell Suspension of&nbsp;Gymnema Sylvestre (Retz.) R.Br. Ex. Sm.

doi:10.21203/rs.3.rs-172209/v1

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Original Article

Sodium Nitroprusside Enhances Biomass And Gymnemic Acids Production In Cell Suspension of Gymnema Sylvestre (Retz.) R.Br. Ex. Sm.

https://doi.org/10.21203/rs.3.rs-172209/v1

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published 28 Mar, 2021

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Gymnema sylvestre (Retz.) R.Br. ex Sm. is widely used as an efficient Ayurvedic traditional medicine for the treatment of diabetes. Phytochemical investigations of this plant showed gymnemic acids (a group of triterpenoid saponins) as the main active components. The present study aims to investigate the effectiveness of sodium nitroprusside (SNP) treatment for enhancement of cell suspension culture biomass and to evaluate their deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII contents of G. sylvestre. 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 2.0 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 FCW and 40.00 ± 0.75 g/L DCW) on 40-day as compared to control. 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.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Suspension culture

Elicitation

Medicinal plant

Secondary metabolites

Metabolic disorders have emerged as a major health problem around the world, as reported by the WHO. Diabetes and obesity are categories of metabolic disorders that are characterized by high blood sugar levels associated with carbohydrate and fat metabolism. Diabetes and obesity are a leading cause of mortality worldwide, with an estimated 422 million people worldwide and estimated to be seventh leading cause of death by 2030 (Hossain et al., 2020). WHO has estimated that diabetes deaths will rise by two-thirds between 2008 and 2030. Clinically, diabetes is related to the inability of beta cells to produce enough pancreatic hormone insulin. Research into effective antidiabetic agents to prevent this disease has fuelled extensive research worldwide on new therapeutic drug discovery strategies targeting notorious metabolic characteristics antidiabetic products (Pappahan et al., 2019; Rasouli et al., 2020). On this basis, many plant secondary metabolites and their semi-synthetic analogues have found significant roles for the management of diabetics because of their structural diversity and their ability to overcome multiple metabolic disorders (Tabatabaei-Malazy et al., 2015). In this post-genomic period, the use of plant-based natural active phytochemicals in the production of antidiabetic drug development phase have become more refined through a better understanding of their structure-based high-throughput metabolic targets (Vieira et al., 2019; Pappachan et al., 2019; Jugran et al., 2020; Artasensi et al., 2020).

The most practised antidiabetic drug development process chiefly remained engrossed in searching for novel drugs focus on glucose-lowering level by defining their mechanisms of action and site-specific binding behaviour amongst phytochemicals (Belete, 2020). Diverging research efforts around the world have led to the successful launch of the hypoglycaemic agent such as insulin secretagogues, insulin sensitization and inhibitors of glucose-absorbing drugs in the gastrointestinal tract as potential antidiabetic drugs for the direct treatment of different types of diabetics (Pappachan et al., 2019; Belete, 2020). However, over the years, the accuracy of these anti-hyperglycemic drugs or metabolites in managing or preventing diabetes has remained difficult to achieve because of the trend in glucose metabolism (Belete, 2020). Whereas managing with these difficulties in treatment of diabetes, the restorative benefits of gymnemic acids, an antisweet and most promising molecule of naturally occurring triterpenoid saponin glycosides, have picked up noticeable quality through the disclosure of their inalienable gut glucosidase inhibition capacity connected to its generally remarkable blood-glucose-lowering effect and initiate insulin release from β-cells (Alkefai et al., 2019; Saha and Pal, 2020).

Gymnema sylvestre (Retz.) R.Br. ex Sm. belongs to the family Asclepiadaceae and has been used in Ayurveda medicine since antiquity and used to treat diabetes, malaria and snake bite (Anjum and Hasan, 2013; Khan et al., 2019). In Ayurveda, Ayurvedic practitioner prescribed 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. G. sylvestre contains many phytomolecules among them GAs and gymnemasaponins are major secondary metabolites and are classified as oleanane saponins (triterpenoid saponins) for clinical application (Laha and Paul, 2019; Khan et al., 2019).

It was found that 18 types of gymnemic acids (GAs) isolated and confirmed from G. sylvester leaves block the receptor site and prevent absorption of glucose into the intestine, thus reducing blood glucose levels (Khan et al., 2019; Laha and Paul, 2019; Tran et al., 2020). Published reports indicated that oral administration of GA-IV to 13.4 mg/kg body weight decreased blood sugar by 60.0% (Sugihara et al., 2000). GA-IV (150 mg/kg) was administered to high fat and fructose diabetic rats to restore antioxidant levels in the liver and kidneys (Karthikeyan et al., 2020). Recently, oral administration of GAs to diabetic rats at 5 mg/kg body weight has shown a significant decrease in blood sugar and increased levels of insulin in plasma and haemoglobin (Mahadeva Rao et al., 2020). Oral administration of 200 mg/kg deacyl gymnemic acid significantly improved the fast plasma glucose level and insulin resistance. Arylated gymnemic acids and olean-15-ene type gymnemic acids showed α-glucosidase inhibition (Alkefai et al., 2018 and 2019). GAs improve insulin release and lower blood lipid concentrations (Barbara et al., 2015). Literature suggests that GA XVII at 0.5 M led to block the sweet taste induced by 0.4 M sucrose and GA IV at A 1.0 mM conquest of sweetness induced by 0.4 mM sucrose (Kazujo et al., 1989; Yoshikawa et al., 1992). GA IV showed inhibitory activity against glucose uptake (Masuyuki et al., 1997).

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 by 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.

Chemicals

Murashige and Skoog (1962) medium, 2,4-Dichlorophenoxyacetic, (2,4-D), Kinetin (Kn), and Sodium nitroprusside (SNP) were purchased plant tissue culture grade from Hi-media (Mumbai, India). Deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII standards were supplied by Sigma-Aldrich (Münich, Germany). HPLC grade methanol and acetonitrile obtained from Merck (Mumbai, India) and ultra-pure water was prepared using a Milli-Q (Millipore, France) was used.

Cell suspension cultures

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 (2.0 mg /L) combination. The cultures were incubated on a shaker (100 rpm) at 25 ± 2 °C for 16/8 h (light/dark).

Treatment of culture with Sodium nitroprusside

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.

Analysis of cell suspension biomass

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

The dried biomass obtained from cell culture and wild plant leaves were grounded into powder. Then, 1 g of sample was extracted in 10 mL of petroleum ether for defatting at 50ºC for 6 h and dried at room temperature. The defatted sample was then extracted using 70:30 (Water: Methanol) at 50ºC for 6 h for complete extraction of gymnemic acids. The extract was dried using rotatory evaporator and kept at 4ºC until further use.

Deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII standard stock solutions were diluted separately at a concentration of 1 mg/mL in methanol. 10 mg of each of the above 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. 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

A newly modified gradient method was developed for the quantification of Bio-markers present in the sample. 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 30ºC and the flow rate was 1 mL/min. The deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII contents were quantified by according to Mahendran et al. (2021).

Statistical analysis

Each experiment was repeated thrice 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.

Initiation and growth of cell suspension cultures

Nitric oxide (NO) is a bioactive molecule which can be generated through the breakdown of NO donor sodium nitroprusside molecules (SNP). Recently, SNP has been reported as an elicitor involved in regulating several signalling responses (Siddiqui et al., 2017; Khezerluo et al., 2018; Hao et al., 2020; Kara et al., 2020). In previous studies, many researchers have reported that SNP treatments are very effective in shoot regeneration of Chrysanthemum cultivars (Arun et al., 2017), Canscora diffusa (Subiramani et al., 2019), Antirrhinum majus (Rezaei Zafarghandi and Rahmati Joneidabad 2020) Valeriana jatamansi (Pandey et al., 2020) and callus induction in Dioscorea opposita (Xu et al., 2009), Hyoscyamus niger (Samsampour et al., 2018) Ficus religiosa (Hesami et al., 2019) Canscora diffusa (Subiramani et al., 2019) and Valeriana jatamansi (Pandey et al., 2020), Furthermore, SNP play important roles in increasing metabolites accumulation (Khezerluo et al., 2018; Pradhan et al., 2020; Tsolmon et al., 2020).

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 g/L and 7.05 ± 0.04g/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. 1). 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 (Fig. 1C-F). 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 (Xu et al., 2005). 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. 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-F). 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 & J). In contrast, death cells showed cytoplasm and cell wall shrinkage (Fig. 1H, I and L).

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, Xu et al. (2005) 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 24h.

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 (Fig. 4B and 5C). Likewise, the maximum 3.25 mg/g gymnemic acid XVII production was observed at 10 µM SNP at 40 days (Fig. D). 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 (Xu and Dong, 2005). Likewise, the maximum 3.2-fold hypericin production in suspension culture was observed at 15.0 mmol/L SNP at 14 days (Xu et al., 2005).

In the present study, SNP at 20 µM treatment at 30 days resulted in the highest deacylgymnemic acid (2.32 mg/g DCW) Fig. 4A, whereas 40 µM SNP treatment at 30 days cell suspension culture period produced maximum of gymnemagenin (2.53 mg/g DCW), gymnemic acid IV (1.24 mg/g DCW) and gymnemic acid XVII (1.90 mg/g DCW) contents over control and 20 µM SNP (Fig. 4. B, C & D). Nevertheless 20 µM SNP treatment yielded higher 13.43 (5.51 mg/g DCW) fold deacylgymnemic acid (Fig. 5A), 13.86 fold (2.80 mg/g DCW) gymnemagenin (Fig. 5B) and 17.33 fold (2.08 mg/g DCW (Fig. 5D, Fig. 6A-C) gymnemic acid XVII production in G. sylvestre cell suspension cultures compared with respective control, however, this deacylgymnemic acid, gymnemagenin and gymnemic acid XVII yield was found to be 3.11, 1.78 and 1.18 fold lesser at SNP (40 µM) treated cultures compared with respective SNP 20 µM. In contrast to this study, Tsolmon et al. (2020) in their investigation of Sophora alopecuroides cell suspension culture, reported that accumulation of oxymatrine content was 18.6 µg/g higher in suspension culture treated at higher 200 µM Jasmonic acid (JA) and 50 µM SNP combination. The ginsenoside accumulation was recorded highest at 200 µM of SNP treatment (Rahimi et al., 2016).

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 ± 0.75g /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.

Acknowledgements

First author gratefully acknowledges the financial support by SERB, New Delhi, India for awarding DST-SERB-National Post-Doctoral Fellowship (PDF/2017/000368). The authors are also thankful to the Director, CSIR-CIMAP for providing necessary facilities.

Author contributions: GM: designed the experiment, performed tissue culture experiments, contributed to writing and corrected manuscript. DK: designed the experiment, performed HPLC experiments and analysed data. SKV: performed tissue culture experiments, analysed data, reviewed and edited manuscript. AC, ZIW and ZH: performed tissue culture experiments. PKR: conceptualization, methodology, formal analysis and editing, LR: conceptualization, supervision, funding acquisition, review, editing and improved the manuscript.

Compliance with ethical standards

Conflict of interest: All authors declare that there is no conflict of interest.

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published 28 Mar, 2021

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31 Jan, 2021
First submitted to journal
24 Jan, 2021

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Sodium Nitroprusside Enhances Biomass And Gymnemic Acids Production In Cell Suspension of Gymnema Sylvestre (Retz.) R.Br. Ex. Sm.

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Journal Publication

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Abstract

Figures

Introduction

Material And Methods

Resource of plant material and culture condition

Chemicals

Cell suspension cultures

Treatment of culture with Sodium nitroprusside

Analysis of cell suspension biomass

Morphology of suspension cells

Sample and Standards Stock preparation

Quantification of deacylgymnemic acid, gymnemagenin, gymnemic acid IV and gymnemic acid XVII

Statistical analysis

Results And Discussion

Initiation and growth of cell suspension cultures

Hplc Analysis Of Gymnemic Acids

Conclusion

Declarations

References

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Journal Publication

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