Elicitor-mediated enhancement of biomass, polyphenols, mangiferin production and antioxidant activities in callus cultures of Salacia chinensis L.

Abstract

The present investigation aimed to improve callus biomass, polyphenolic content, biosynthesis of mangiferin and biological potential following application of different elicitor treatments for medicinally important Salacia chinensis L. The leaf-derived callus cultures were established on Murashige and Skoog’s (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D: 2.0 mg/l) and 6-benzylaminopurine (BAP: 1.5 mg/l). These cultures were treated with different elicitors viz. jasmonic acid (JA), methyl jasmonate (MeJA) and yeast extracts (YE). The highest calli biomass (five-fold increase within 4 weeks) was achieved when callus was treated with JA (75 µM). The callus obtained on MS medium supplemented with 2,4-D (2.0 mg/l), BAP (1.5 mg/l) and treated with JA (75 µM) displayed augmented values for total phenolics, flavonoids and mangiferin contents. Besides, same treatment elicits the calli for antioxidant properties as evaluated by 2,2-diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) and metal chelating assays. This is the first report on the elicitation study in genus Salacia and, therefore, the discoveries suggested that, S. chinensis calli might be a perfect source for large-scale production of industrially important secondary metabolites. Concurrently data provide accumulated information demonstrating its prominent antioxidant effect revealing its potential without disturbing natural resources.

Introduction

Over-developing interest of bioactive compounds from natural resources has enforced the implication of diverse biotechnological tools for sustainable utilization of medicinally important plants (Salma et al. 2018). Plant tissue culture is among the attainable biotechnological strategies which offer enormous scope for production of plantlets, conservation, restoration as well as the use of in vitro cultures for production of bioactive metabolites under controlled conditions, independent of seasonal and topographical conditions (Smetanska 2008; Chavan et al. 2018). One of the important advantage of in vitro cultures is one can achieve the differential accumulation and biosynthesis of natural products using elicitors and precursors. For quite few decades, plant cell cultures were utilized for synthesis of a few industrially significant secondary metabolites (Rao and Ravishankar 2002; Espinosa-Leal et al. 2018). Additionally, exploiting cell culture elicitation offers a striking elective way to reinforce yield, defeating the constrained accessibility and scaling-up of biologically active and medicinally significant metabolites in several in vitro cultures (Zafar et al. 2017; Ahmad et al. 2019; Khan et al. 2019).

Genus Salacia (family—Celastraceae) comprises over 200 woody lianas, shrubs or small trees. The genus is having multi-potent species which are used in food and pharmaceutical industries. Salacia chinensis L. is commonly known as Saptarangi is among the foremost-investigated medicinal plants and convenient source of mangiferin, salacinol and kotalanol, which are known for their strong antidiabetic, anticancerous and anti-HIV properties (Yoshikawa et al. 2001; Thuan 2005; Silpraist et al. 2011; Chavan et al. 2015b). More recently, mangiferin has been effectively used for preventing neurodegeneration in Alzheimer's and Parkinson's disease (Feng et al. 2019). Besides, the plant is chief source of several number of metabolites belonging to diverse groups viz. polyphenols, alkaloids, glycosides, anthocyanidins, quinones, friedo-oleanones, terpenoids, coumarins, steroids, saponins, tannins, gums and mucilage (Majid et al. 2016a; Ghadage et al. 2017). The commercial utilization of Salacia species into Asian and European Countries as packing’s of root concentrates and tablets in the late 1990s provides the stage for its familiarization into the international pharmaceutical market. At present, the global exchange of Salacia species has developed into a multimillion-dollar industry (Dubey et al. 2011). Unavoidably, the developing worldwide interest for Salacia species (including S. chinensis) crude materials came about in over-abuse of its wild populaces in Asian Countries (Chavan et al. 2015a).

Despite the fact that, Salacia species contains several numbers of biologically active compounds, the utility of plant tissue culture techniques are restricted to the development of micropropagation conventions for their conservation and that excessively confined with S. chinensis, S. reticulata and S. oblonga (Dhanasri et al. 2013; Deepak et al. 2015; Majid et al. 2016b; Laxmi et al. 2018). However, in previous report (Chavan et al. 2015a), we have effectively developed an in vitro propagation system along with elevated accumulation of mangiferin during various regeneration stages of S. chinensis. More recently, Bagnazari et al. (2018) assessed the antioxidant and antidiabetic potential of aerial parts of in vitro regenerated plantlets of S. chinensis. Till date, no report has been published on elicitor-treated increase in biomass, industrially important biomolecules with in vitro pharmacological properties in callus cultures of genus Salacia.

Circumstantial literature motivate us to explore whether elicitor-treated undifferentiated callus cultures of S. chinensis can upgrade the production of biomass, metabolites and biological properties, hence following objectives were focused during the current investigation: (i) optimization of elicitor treatment for extensively growing callus cultures; (ii) appraisal of the total content of phenolics and flavonoids, (iii) evaluation of mangiferin accumulation using RP-HPLC and (iv) characterizing callus extract (CE) for its antioxidant capacity using 2,2-diphenyl-2-picrylhydrazyl, ferric-reducing antioxidant power and metal chelating assay.

Materials and methods

Plant material

Samples of S. chinensis were collected from Amboli locality (15° 58′ 05.6″ N; 73° 59′ 48.7″ E; altitude: 739 m) of the Northern Western Ghats, India. The authentically identified specimen was at Herbarium, Department of Botany, Shivaji University, Kolhapur (Voch. No. JC/SC/04).

Chemicals, reagent and analytical instruments

Phenolic acid (gallic acid), flavonoid (quercetin), 2,2,-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ), mangiferin (99% pure), jasmonic acid (JA) and methyl jasmonate (MeJA) were procured from Sigma-Aldrich (St. Louis, MO, USA). Yeast extract were procured from Merck, India. N,N-Dimethyl formamide (DMF) and methanol (MeOH) were of HPLC grade (Spectrochem, India). The chemicals used for tissue culture experiments were from Himedia, India and Qualigens, India. The hot air oven utilized in this study was from Thermo Scientific, Germany. The optical density measurements for various experiments including polyphenolics and antioxidant capacity were recorded utilizing UV–visible spectrophotometer (Shimadzu UV1800, Japan). Waters chromatographic system (Model no. HPLC-W6590, Waters, Milford, USA) with dual UV absorbance detector (W-2487) was used for quantitation of mangiferin content.

Establishment and maintenance of callus cultures

The callus cultures were initiated by inoculating healthy leaf pieces of S. chinensis as portrayed by Chavan et al. (2015a). The leaves washed under running tap water were kept in labogent solution (0.5%, v/v) for 5 min, treated with HgCl₂ (0.1%) for 3 min and washed thrice with sterile water. The surface disinfected leaf pieces were transferred aseptically on MS medium (Murashige and Skoog 1962) fortified with 2,4-D (2.0 mg/l) and BAP (1.5 mg/l). The media was enriched with sucrose (30 g/l), solidified with 2.0 mg/l ClariGel and autoclaved at 121 °C for 20 min after adjusting the pH to 5.8. The cultures were maintained at 25 ± 5 °C with provision of light irradiance (40 µmol/m/s) for 16 h photoperiod.

Preparation and treatment of elicitors

The stock solutions of elicitor viz. jasmonic acid and methyl jasmonate were prepared in 96% ethanol and filter-sterilized through 0.45 μM Millipore filter (Minisart, Germany). Various concentrations of JA (25–125 µM) and MeJA (50–250 µM) were added to MS medium after sterilization containing a mixture of 2,4-D (2.0 mg/l) and BAP (1.5 mg/l). Yeast extract was added to culture medium with concentration ranging from 100 to 500 mg/l. Range of the elicitor treatments were chosen based on the preliminary experiments.

Growth kinetics and biomass production

Callus from exponential developmental stage (20th day) was harvested and inoculated individually either on control medium [MS + 2,4D (2.0 mg/l) + BAP (1.5 mg/l)] or alongside elicitor treatments. The cultures were incubated at 25 ± 5 °C with provision of light irradiance (40 µmol/m/s) for 16 h photoperiod. Increase in callus biomass in response to varied elicitor treatments were measured as fresh (FW) and dry (DW) weights after 30 days of incubation. Calli were carefully taken out from culture vessels, washed gently with sterile water, pressed softly on paper to the excess water and weighed for fresh weight. The calli harvested were oven dried at 45 °C for 24 h to determine dry weight (DW). However, callus proliferation frequency (CPF) was calculated using following formula,

$$\text{CPF}\left(\%\right)=\frac{\text{No}.\text{of}\text{culture}\text{vessels}\text{with}\text{increased}\text{values}\text{for}\text{calli}\text{biomass}}{\text{Total}\text{no}.\text{of}\text{culture}\text{vessels}\text{per}\text{elicitor}\text{treatment}}\times 100.$$

Extract preparation for polyphenols and antioxidant assays

Extract preparation was carried out using the previously described method for S. chinensis by Chavan et al. (2013) with little change. The calli harvested from 30 days of elicitor treatments were oven dried at 45 °C for 24 h were crushed to fine powder. One gram of dried callus powder was extracted with 100 ml of MeOH for 10 min. CE was prepared using steam bath-assisted extraction (SBAE) on stirred thermal water bath (Equitron, India) with consistent temperature (70 °C). The extracts were filtered through Whatman No. 1 filter paper and adjusted to 100 ml and were used for further analysis.

Determination of polyphenol content

Total phenolic content (TPC) was estimated spectrophotometrically using Folin–Ciocalteu method (Singleton and Rossi 1965). A mixture of CE (0.125 ml) and 1.8 ml of ten-fold diluted Folin–Ciocalteu reagent were permitted to react at 25 °C for 6 min. After addition of 15% Na₂CO₃ (1.2 ml), the reaction mixture was kept at room temperature for 90 min and the readings were recorded at 765 nm.

Total flavonoid content (TFC) was determined using aluminium chloride calorimetric method described by Chang et al. (2002) with little alteration. CE (0.5 ml), 10% AlCl₃ (0.1 ml), MeOH (1.5 ml), 1 M potassium acetate (0.1 ml) and distilled water (2.8 ml) were vortex for 7 min. After 30 min incubation at room temperature, the absorbance of the mixture was measured at 416 nm. The results were compared with standard curves of gallic acid and quercetin for TPC and TFC, respectively, and were expressed as milligram equivalent per gram dry weight.

RP-HPLC analysis of mangiferin from elicitated callus cultures

Extraction and quantitative determination of mangiferin was carried out by following the previously described method for S. chinensis (Chavan et al. 2015b). One gram of callus powder was added to 100 ml of N,N-dimethyl formamide (DMF, 30%); suspension was mixed well and heated for 10 min of exposure period (SBAE). After cooling, final volume was adjusted to 100 ml with DMF. Extracts filtered through 0.45 mm nylon filter (Axiva) were transferred into an Agilent amber vial and stored at 4 °C until chromatographic analysis. 10 mg of standard mangiferin was dissolved in 50 ml of DMF (30%), warmed on steam bath for 10 min and cooled at room temperature. Final volume was adjusted to 100 ml with DMF (30%). The chromatographic system and conditions were maintained as per the previous report described for S. chinensis (Chavan et al. 2015b). The separation was completed on Waters C18 column (Princeton SPHER, 5 μ, 250 × 4.6 mm). Mobile phase comprising of “A” (0.2% triethylamine pH 4.0 with orthophosphoric acid) and “B” (acetonitrile) was used for separation with 89% “A” as to 11% “B” in an isocratic mode. The injection volume was 20 μl and the flow rate was maintained at 1 ml/min with detection wavelength of dual λ absorbance detector at 250 and 260 nm. The analysis was done for 10 min for standard as well as samples. The content of mangiferin was recorded as mg/g DW.

Determination of antioxidant potential of callus extracts

The antioxidant capacity of CE extract was evaluated using three different assays viz. 2,2-diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) and metal chelating antioxidant power. The antioxidant capacity using DPPH assay of CE was measured using method developed by Brand-Williams et al. (1995). The stock solution of reagent prepared (24 mg of DPPH in 100 ml of MeOH) was stored at − 20 °C. 10 ml of reagent added to 45 ml of MeOH to obtain absorbance value of 1.1 ± 0.02 at 517 nm on UV–Vis Spectrophotometer. CE (100 µl) was mixed with DPPH solution (2.9 ml) and allowed to react for 30 min in dark at room temperature. The absorbance of resulting solution was recorded at 517 nm and the results were expressed as antioxidant capacity (%) using following formula,

$$\text{Antioxidant}\text{capacity}\left(\%\right)=\frac{A\text{control}-A\text{sample}}{A\text{control}}\times 100,$$

where A is the absorbance.

The FRAP assay was performed according to the procedure described by Benzie and Strain (1996). The working FRAP reagent was formulated with a combination of 20 mM FeCl₃·6H₂O, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl and 300 mM acetate buffer (pH 3.6), in 1:1:10 ratio and mixture was heated in hot water bath for 10 min at 37 °C. CE (100 µl) was allowed to react with 2.9 ml of FRAP reagent in the 3.0 ml of reaction mixture. After 30 min incubation in dark, the absorbance of ferrous–tripyridyltriazine complex was measured at 593 nm and the FRAP values were determined as optical density readings.

Metal chelating antioxidant power assay was performed according to Dinis et al. (1994) with little modification. A reaction mixture was prepared with 2 mM FeCl₃ (50 µl), deionized water (2750 µl) and CE (100 µl) and the reaction was initiated after addition of 5 mM ferrozine solution (100 µl). A mixture was allowed to stand for 20 min at room temperature, inhibition of ferrozine to Fe₂⁺ complex was recorded at 562 nm and antioxidant potential was expressed using the formula,

$$\text{Inhibition}\left(\%\right)=\frac{A\text{control}-A\text{sample}}{A\text{control}}\times 100,$$

where A is the absorbance.

Experimental design and data analysis

The callus culture experiments were conducted three times with 20 replicates per treatment and data were analyzed using one-way ANOVA. The significant differences among the means were assessed by Dunnett multiple comparison test. All phytochemical and biological potential determination experiments were repeated thrice. The data shown represents the mean ± standard error (SE) for three independent experiments. All statistical analysis was performed using GraphPad Instat (GraphPad Soft-ware, Inc, USA) and Microsoft Excel (Microsoft Co. ltd.)

Results and discussion

Callus induction and biomass accumulation

The well-growing callus cultures were achieved from young and healthy leaf explants (sterilized with 0.1% HgCl₂ for 3 min) of S. chinensis on MS medium with 2,4-D (2.0 mg/l) and BAP (1.5 mg/l) as described by Chavan et al. (2015a). Noticeably, the calli showed three distinctive coloring patterns (pale-yellow–brown–black) all through the 30 days’ incubation time (Fig. 1A–C). As per the previous reports from same laboratory, 30 days’ incubation period was found optimal for obtaining higher values for calli biomass in S. chinensis (Chavan et al. 2015a). The blackening of calli might be due to the accumulation of polyphenols. However, blackening does not affect the callus proliferation rate. In contrast, Gao et al. (2020) reported heavy browning phenomenon hinders proliferation rate in callus cultures of Paeonia suffruticosa. However, they have identified browning linked genes through transcriptome sequencing which will help to reduce this phenomenon. In the present study, the calli harvested from 20th day of culture were transferred to different elicitor treatments by keeping steady hormonal supplementation. The biomass of callus was recorded as fresh and dry weight after a period of 30 days of cultures under the influence of various elicitor treatments (Table 1). The rate of callus growth and morphology was altered by the elicitor treatment. The calli pieces transferred on MS medium in presence of lower concentrations of JA was produced dark brown to blackish and friable calli; however, MeJA treatments yielded brown, compact and hard callus (Table 1). All calli lines were undifferentiated. The fresh and dry weight was significantly higher when calli were treated with JA followed by MeJA and YE. Among the treatments, the weights of the calli ranged from 1.92 ± 0.1 g to 8.10 ± 0.6 g FW and 0.13 ± 0.03 to 0.81 ± 0.03 g DW, respectively. This demonstrated the differential responses of calli of S. chinensis to various elicitor treatments. JA treatment (75 µM) heightened biomass accumulation (8.10 ± 0.6 g FW) in 95% of cultures with growing callus (Table 1, Fig. 2). The findings affirmed that, 4.97 and 4.7-fold enhancement in fresh and dry callus biomass, respectively, when calli were treated with JA (75 µM). Notably, comparable calli biomass production (3.95 ± 1.1 g FW) was observed when cultures were treated with MeJA (100 µM). The present outcomes confirmed the arousing proficiency of elicitors on triggering of cells to achieve higher growth than control cultures. Likewise, elicitor-dependent in vitro growth and development has been reported for medicinally important plants viz. Rosa hybrid (Ram et al. 2013) and Rauvolfia serpentine (Zafar et al. 2017).

Fig. 1.

Establishment of callus cultures of S. chinensis on MS medium fortified with 2,4,D (2.0 mg/l) and BAP (1.5 mg/l). A Pale-yellow callus, B brownish callus and C black-colored callus

Table 1.

Callus proliferation frequency (CPF) and biomass profile in response with elicitor treatments

S. no.	Elicitor	Conc.	CPF (%)	Biomass (g)		Remark
				FW	DW
1	Elicitor free		90	1.63 ± 0.3	0.17 ± 0.01	Light Brown
2	Jasmonic acid (μM)	25	90	3.30 ± 0.7*	0.29 ± 0.03*	Dark brown, friable
3		50	90	4.33 ± 1.0**	0.38 ± 0.04**	Black, friable
4		75	95	8.10 ± 0.6**	0.81 ± 0.03**	Black, semi-hard
5		100	90	5.03 ± 0.8**	0.45 ± 0.07**	Black, semi-hard
6		125	85	2.81 ± 0.4*	0.31 ± 0.03**	Black, compact
7	Methyl jasmonate (μM)	50	70	2.12 ± 0.8 ns	0.28 ± 0.02*	Brown, compact
8		100	70	3.95 ± 1.1**	0.38 ± 0.03**	Brown, compact
9		150	75	3.29 ± 0.4*	0.33 ± 0.02**	Brown, semi-hard
10		200	70	3.25 ± 0.4*	0.21 ± 0.06ns	Brown, semi-hard
11		250	65	2.55 ± 0.3*	0.13 ± 0.03ns	Brown, hard
12	Yeast extract (mg/l)	100	75	1.92 ± 0.1 ns	0.24 ± 0.02ns	Yellowish, compact
13		200	75	1.98 ± 0.9ns	0.24 ± 0.05ns	Yellowish, watery
14		300	60	2.76 ± 0.5*	0.29 ± 0.01*	Semi-green
15		400	65	2.06 ± 0.5ns	0.25 ± 0.03*	Greenish, friable
16		500	65	2.30 ± 0.8*	0.19 ± 0.07ns	Greenish, friable

Values are significantly different at ns—non significant, *P < 0.05 and **P < 0.01 level as compared by Dunnett multiple comparisons test

Fig. 2.

Growth and proliferation of callus on jasmonic acid (75 µM) treated cultures of S. chinensis

The callus cultured in presence of higher concentrations of MeJA (250 µM) demonstrated the reduced biomass productivity (FW: 2.55 ± 0.3 g, DW: 0.13 ± 0.03 g) in relation to the lower MeJA concentration. So also, among all the yeast extract treatments, no demonstrable decrease or increase in biomass has been observed as compared to control cultures (Table 1). Similarly, lower concentrations showed negligible response during callus induction and accumulation of secondary metabolites in Dregea volubilis (Yogananth et al. 2019). In contrast, Vijayalakshmi and Shourie (2019) recently reported the yeast extract triggered biomass accumulation in callus cultures of licorice. More recently, phytohormone regulated calli biomass production has been demonstrated for S. chinensis and S. macrosperma (Chavan et al. 2015a; Mahendra et al. 2020). However, the current investigation on elicitor treatment is crucial to meet fairly higher biomass production and therefore increment in accumulation of metabolites of interest in callus cultures of S. chinensis.

Polyphenolic profile

In the present study, the content for total phenolics and flavonoids was explored from dried callus samples treated with various elicitors for S. chinensis. The content of total phenolics and flavonoids ranged between 44.00 ± 1.34 to 68.49 ± 0.90 mg GAE/g DW and 8.89 ± 0.33 to 26.18 ± 0.35 mg QE/g DW, respectively (Table 2). The calli treated with JA (75 µM) demonstrated the most noteworthy content of phenolics (68.49 ± 0.90 mg GAE/g DW), which was 29% higher than the control (Table 2) and higher flavonoids content (26.18 ± 0.35 mg QE/g DW) in S. chinensis. Also, Astello-Garciaet et al. (2013) recommended the addition of jasmonic acid treatment for improved production of polyphenolic contents in callus cultures of Opuntia robusta. On contrary, yeast extract demonstrated the least impact on polyphenol accumulation in callus cultures of S. chinensis. The total contents of phenolics (44.00 ± 1.34 mg GAE/g DW) and flavonoids (8.89 ± 0.33 mg QE/g DW) were found minimal in calli treated with yeast extract (500 mg/l), which was beneath the levels recorded in control cultures (Table 2). The significant decrease in the flavonoid content was observed at the different concentrations of methyl jasmonate and yeast extract-treated callus culture extract. Similarly, the declined levels of flavonoid content in response to elicitor treatment were reported in cell suspension cultures of Phoenix dactylifera (Al-Khayri and Naik 2020). Literature survey suggests that there are various reports on elicitated production of polyphenolic compounds in callus cultures of medicinal plants (Khan et al. 2019); however, the present work is the first attempt on standardization of elicitor treatments for optimal metabolites production in callus cultures for genus Salacia.

Table 2.

Efficiency of elicitors on total phenolics and flavonoids content in callus cultures of S. chinensis

S. no.	Elicitor	Conc.	TPC (mg GAE/g DW)	TFC (mg QE/g DW)
1	Elicitor free		48.63 ± 1.80	20.18 ± 0.91
2	Jasmonic acid (μM)	25	55.34 ± 0.72ns	24.10 ± 1.00*
3		50	62.00 ± 2.21*	19.23 ± 0.30ns
4		75	68.49 ± 0.90**	26.18 ± 0.35**
5		100	66.22 ± 1.11**	20.13 ± 0.40ns
6		125	61.30 ± 0.90*	16.76 ± 0.12ns
7	Methyl jasmonate (μM)	50	51.10 ± 0.94ns	20.23 ± 1.20ns
8		100	61.54 ± 0.34*	19.68 ± 0.24ns
9		150	52.80 ± 0.12ns	11.28 ± 1.10ns
10		200	52.11 ± 0.70ns	18.36 ± 0.88ns
11		250	49.89 ± 1.00ns	16.61 ± 0.60ns
12	Yeast extract (mg/l)	100	50.40 ± 1.88 ns	12.35 ± 1.00ns
13		200	51.96 ± 2.20ns	18.90 ± 0.98ns
14		300	50.78 ± 2.00ns	10.28 ± 0.44ns
15		400	44.23 ± 1.20ns	12.30 ± 0.39ns
16		500	44.00 ± 1.34ns	08.89 ± 0.33ns

Values are significantly different at ns—non significant, *P < 0.05 and **P < 0.01 level as compared by Dunnett multiple comparisons test

Mangiferin accumulation

Mangiferin (a xanthone glucoside) is an active principal compound predominantly present in different organs of S. chinensis and known for broad spectrum of pharmacological properties including antidiabetic, anticancer and anti-HIV. In the current study, the quantification and accumulation of the mangiferin in the elicitor-treated callus extract is determined using RP-HPLC (Fig. 3A, B). Based on the RP-HPLC analysis, all samples were found to contain mangiferin; however, the change in the contents was observed according to the elicitor treatment (Fig. 4). The results of current study revealed that, JA (75 µM) supported the maximum calli biomass production along with elevated content of mangiferin. Similarly, Chavan et al. (2015a) reported the media composition which supports the higher biomass production also supported the higher accumulation of mangiferin in S. chinensis. MeJA also supported the mangiferin accumulation while the yeast extract was not effective in terms of increased mangiferin accumulation in callus cultures of S. chinensis. Eliciting the cultures with JA (75 µM) and MeJA (100 µM) triggered the mangiferin production with values of 8.493 ± 0.193 mg/g DW (Fig. 3B) and 8.439 ± 0.645 mg/g DW, respectively, which was in excess of about 1.5-fold than the un-elicited cultures (control). So also, various studies recommended the essentiality of adding elicitors in the callus cultures for the production of bioactive drugs (Sailo et al. 2018; Sarmadi et al. 2018; Ahmad et al. 2019; Rajan et al. 2020). The outcomes of current study showed that, the rise in biomass resulted in enhancing the biosynthesis and accumulation of mangiferin in callus cultures of S. chinensis. These results are in accordance with the biosynthesis of anticancer alkaloids (vincristine and vinblastine) in callus cultures of periwinkle (Mekky et al. 2018). The calli treated with yeast extract was able to produce minimal contents of mangiferin. Incidentally, these values were less than the amounts of mangiferin accumulated in un-elicited cultures (Fig. 4).

Fig. 3.

RP-HPLC chromatogram of mangiferin. A Standard mangiferin, B callus treated with JA (75 µM)

Fig. 4.

Mangiferin content estimated by RP-HPLC in jasmonic acid (75 µM) elicitated callus cultures of S. chinensis. Values are significantly different at ns—non significant, *P < 0.05 and **P < 0.01 level as compared by Dunnett multiple comparisons test

Antioxidant properties

In the current study, the existence of noteworthy amount of polyphenols and mangiferin recommend their conceivable contribution to antioxidant activity. However, it is complicated to accurately assess the antioxidant capacity of a system having various components using single test. As, each method changes as far as principle and environmental conditions; therefore, diverse reaction mechanisms are needed to authenticate antioxidant potential of the plant extracts (Jauhari et al. 2019). Hence, 2,2-diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) and metal chelating mechanisms were used to assess the antioxidant potential of elicitor-treated calli extracts of S. chinensis. Antioxidant potential of all the elicitor-treated callus samples are given in Fig. 5A–C.

Fig. 5.

Antioxidant capacity of elicitor-treated callus cultures of S. chinensis. A DPPH assay, B FRAP assay and C metal chelating assay. Values are significantly different at ns—non significant, *P < 0.05 and **P < 0.01 level as compared by Dunnett multiple comparisons test

Among the elicitor treatments, JA (75 µM) exhibited the most elevated antioxidant capacity (88.3 ± 4.3%) in DPPH assay (Fig. 5A). The alterations in ability of extracts to reduce Fe₃⁺ to Fe₂⁺ radicals were seen in FRAP assay and a similar propensity was seen in the metal chelating assay supported the formation of ferrozine to Fe₂⁺ complex. The maximum antioxidant capacity in FRAP (2.350 ± 0.08 OD) and metal chelating assays (77.4 ± 2.3%) recorded in calli treated with JA (75 µM) and YE (400 mg/l), respectively (Fig. 5B, C). Considering all three assays (DPPH, FRAP and metal chelating), JA-treated calli showed greater antioxidant potential when compared to other elicitor treatments and control. The least antioxidant activity values such as 64 ± 1.3% (in DPPH assay), 1.499 ± 0.2 OD (in FRAP assay) and 58.5 ± 1.5% (in metal chelating assay) were exhibited by the calli extract obtained from YE treatment (500 mg/l in DPPH and FRAP) and MeJA (250 µM), respectively (Fig. 5A–C). The findings of the present study revealed the chemical elicitation remarkably improved the antioxidant potential of callus cultures of S. chinensis. Recently, few researchers have assessed the impact of plant growth regulators on antioxidant capability of callus and aerial parts of micropropagated Salacia spp. (Chavan et al. 2015a; Bagnazari et al. 2018; Mahendra et al. 2020); however, the present study is the first and sole report describing elicitor-mediated increase in antioxidant potential of callus cultures of S. chinensis.

Conclusion

Our investigation demonstrated the heightened results when diverse elicitor treatments were applied for callogenesis, biomass accumulation, and production of active phytochemicals coupled with antioxidant potential in S. chinensis. This is the first report on elicitation studies in callus cultures for S. chinensis (perhaps for genus Salacia). Among various elicitors, JA (75 µM) enhanced the callus proliferation frequency with higher calli biomass accumulation. Moreover, same elicitor treatment resulted in increased polyphenol, mangiferin accumulation as well as antioxidant properties. Our findings provide evidence that, the use of elicitor treatment is promising approach for improved calli biomass production, secondary metabolites and antioxidant properties in S. chinensis. Various bioactive and industrially important compounds are found in S. chinensis; therefore, further study is necessary for optimization of efficiency of diverse class of elicitors, precursors, genetic engineering approach, and triggering the cultures within bioreactors would be advantageous for exploiting the full potential of this taxa.

Abbreviations

2,4-D

2,4-Dichlorophenoxyacetic acid

BAP

6-Benzylaminopurine

CE

Callus extract

CPF

Callus proliferation frequency

DMF

N,N-Dimethyl formamide

DPPH

2,2-Diphenyl-2-picrylhydrazyl

DW

Dry weight

FRAP

Ferric-reducing antioxidant power

FW

Fresh weight

GAE

Gallic acid equivalent

JA

Jasmonic acid

MeJA

Methyl jasmonate

MeOH

Methanol

MS

Murashige and Skoog’s medium

QE

Quercetin equivalent

RP-HPLC

Reversed-phase high-performance liquid chromatography

SBAE

Steam bath-assisted extraction

SE

Standard error

TFC

Total flavonoid content

TPC

Total phenolic content

TPTZ

2,4,6-Tripyridyl-s-triazine

YE

Yeast extract

Author contributions

JJC conceived, designed and performed the experiments, wrote the draft of manuscript. PRK, SGJ, VMN, STG performed the experiments, contributed to review and edit the manuscript. SRP contributed to resources and analyzed the data.

Funding

The present work was financially supported by Science and Engineering Research Board (SERB) of Department of Science and Technology (DST), Govt. of India, New Delhi (No. SB/FT/LS-259/2012) through Fast-Track Scheme for Young Scientist.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.