Rhizomes of P. polyphylla were collected from Malyadhor, Bageshwar, Uttarakhand, India (30°08’48.8’’ N; 79°58’14.0’’ E; an altitude of 2348 m) in October 2020. Rhizomes were used as an explant to establish the callus culture. The explants were washed in flowing tap water for 20 min before being immersed in Bavistin solution (0.1%; BASF India Ltd.), containing Tween 20 (2% v/v) in distilled water. The explants were rinsed thrice with sterilized distilled water to eliminate any excess Bavistin and detergent. Moreover, explant surfaces were cleaned with mercuric chloride aqueous solution. (HgCl₂; 0.05%, w/v; 2 min) before being repeatedly washed with sterilized distilled water in a laminar airflow (LAF) cabinet (Thermadyne, Faridabad, India). In aseptic conditions, the explants were inoculated on MS medium (Murashige and Skoog, 1962) containing TDZ (0.0 to 2.0 µM), sucrose (3%), and agar (0.8%) maintained at 5.8 pH. The inoculated agar plates were kept at 25 ± 2°C for 16 and 8 hours of light and dark cycles respectively on racks with cool fluorescent tubes (Philips 40 W; 42.0 and 60.0 mol/m2/s irradiance within and outside the culture flasks, respectively). To achieve the optimum growth, sub-culturing was carried out at 6-week intervals. Optimum growth of callus was observed on MS medium containing 0.5 µM TDZ. Further, callus was sub-cultured in ½ MS liquid medium in addition to 0.5 µM TDZ to establish callus suspension culture. All the cultures were kept in the culture room.

To generate suspension culture, 0.50 g of callus was placed into 100 mL of ½ MS liquid medium and 0.5 µM TDZ in Erlenmeyer flasks (Borosil India Ltd., Mumbai). The callus was extracted at an interval of seven days for eight weeks and filtered through Whatman No. 1 papers. The experiment was performed in triplicates. The fresh weight of the callus biomass was determined and its average was calculated. The dry weight (DW) was recorded at a temperature of 50°C in a hot air oven. The suspension of callus culture was kept in a culture chamber on a rotary shaker at 90 revolutions per min in a culture room.

TDZ, MeJA, and YE were used to determine their effect on the development of callus suspension. In the Erlenmeyer flasks, callus (0.5g) was placed in the100 ml of ½ MS liquid medium with increasing doses of TDZ (0.0-2.0 M), MeJA (10-200 µM), or YE (100 to 1000 mg/l). The experiment was performed in triplicates. The average callus biomass weight was evaluated after incubation of 4 weeks. The flasks without TDZ, MeJA, or YE were used as control samples.

The extraction of phenolic compounds from callus suspension culture was carried out to check the effect of TDZ, MeJA, or YE. The callus was thoroughly cleansed with distilled water and then allow to dry in a hot air oven at 50°C for 48 hours. After that, dried callus were mashed with a mortar and pestle and the resulting powder (100mg) was dissolved in 10 ml of 80% (v/v) ethanol for extraction at 50-60 °C for 7-8 hrs, following which the solvent was vaporized to a dry state. The residues were then dissolved in 10 ml of distilled water and used to measure the antioxidant activity, total flavonoid content, total phenolic content, and total tannin contents (Swain and Hillis, 1959; Giri et al., 2012; Raomai et al., 2015; Lepcha et al., 2019). Total phenolic content was calculated as mg Gallic Acid Equivalent (GAE)/g dry weight of sample, while total tannin content was measured as mg Tannic Acid Equivalent (TAE)/g dry weight of the sample, respectively. The estimation of total flavonoids was represented as mg quercetin equivalent/g sample.

The plant samples were analyzed for dioscin and diosgenin (steroidal saponin) content following the method of Raomai et al. (2015). For diosgenin, the plant sample was prepared by refluxing the ground plant material with 50 ml H₂SO₄ for 4 hours and further extracted with 50 ml Ethyl acetate in a separating funnel thrice. The organic layer was collected and passed over anhydrous NaSO₄ and evaporated. The residue was dissolved in ethanol and filtered with 0.45-micron filter paper. This filtrate was further used for the quantification of diosgenin.

For dioscin, ground sample (5 gm) was mixed with ethanol-water mixture (7:3 v/v), and incubated at 50°C for 15 min. The mixture was then sonicated in the ultra-sonicate bath for 15 min, centrifuged and the supernatant was collected and filtered with 0.45 micron filter paper. This filtrate was further used for the quantification of dioscin.

Ethanolic extract was employed in an HPLC analytical system (Merck Hitachi, Japan). The samples were separated in an isocratic manner using an isocratic mobile phase comprising acetonitrile: ethanol in a ratio of 80:20 v/v. The flow rate was kept at 1 ml/min in the C-18 column (Purosphere, Merck). The volume of the sample used was 20 µl. The retention duration of comparable external standards was used to identify phenolic chemicals. The compounds were identified with the help of retention time of reference standard. Diosgenin or dioscin were quantified with help of calibration curve of peak areas of external standards recorded at 196 nm. The standard curve of peak area for each compound was prepared at five different concentrations ranging from 05 to 20 µg/ml and linear regression curve between peak area and concentration was used for quantification ( Supplementary File S1 ). The results were expressed in mg/100 g or µg/g.

Three invitro methods were followed for the assessment of the antioxidant activity of the samples. viz ABTS assay, DPPH assay and FRAP assay.

Complete experimental design is presented in Figure 1 . The experiments were conducted twice in a randomized block design, with triplicates for each treatment. Mean values from various treatments were assessed using SPSS version 7.5. The difference in the mean values was determined at significance (at p 0.05) using Duncan’s Multiple Range Test (DMRT).

Within 6 weeks of inoculation, rhizomes transplanted to MS medium containing 0.5 µM TDZ displayed brittle and light-yellow colored calli. These rhizomes derived callus were further transferred in liquid MS media supplemented with 0.5 µM TDZ. The outcomes show that P. polyphylla rhizome-derived callus grows at its best on MS medium containing 0.5 µM TDZ. Similar outcomes have been seen with callus induction in seeds of several medicinal plants that needed plant growth regulators (PGRs; Hong et al., 2008; Giri et al., 2012). These rhizomes derived callus were subsequently used to plot a growth curve and carry out additional experiments.

Callus development on the MS medium containing 0.5 µM TDZ for 8 weeks of culture exhibited a 15-fold increase in biomass (fresh weight). The study revealed 7 days of a lag phase, a log phase extending from 7 to 30 days and lastly, a stationary phase was present ( Figure 2 ). The late log phase callus (after 32 days), was employed for further examinations.

The influence of varying concentrations of TDZ (0.0-2.0 µM), MeJA (10-200 µM), or YE (100 to 1000 mg/l) on callus biomass were examined ( Table 1 ). Higher callus biomass resulted from the addition of TDZ. The best TDZ concentration for biomass production was 0.5 µM, however, higher TDZ concentrations (2.0 µM) harm biomass output.

An analysis of MeJA’s impact on callus biomass was also conducted. MeJA was observed to harm biomass production at higher concentrations (200 µM), and it was not found to be effective in increasing biomass. Elicitors like MeJA have been demonstrated to regulate the mitotic cycle in plant cells, restricting cell division and biomass increase by preventing cell division in the G1 phase before entering the S phase as signalling molecules in the plant defence system. As a result, under high MeJA concentrations, biomass production declines or ceases entirely (Kang et al., 2004; Ho et al., 2018; Mendoza et al., 2018). YE displayed a similar pattern; at a dosage of 250 mg/l, it promoted callus growth and boosted biomass production, however at 1000 mg/l, the biomass was reduced to 1.32 g FW at 2.5 mg/l ( Table 1 ).

All three components i.e., total phenol, flavonoids, and tannin showed increased amounts with increasing TDZ concentration ( Table 1 ). While the highest quantity of total phenol (49.66 ± 4.43 GAE/g DW), flavonoids (34.67 ± 3.22 QE/g DW), and tannin (57.66 ± 4.22 TAE/g DW) all had been recorded at 50 µM MeJA, the maximum amount of biomass production (2.98 ± 0.08), flavonoids (34.67 ± 3.22 QE/g DW), and tannin were all observed at 0.5 µM TDZ.

According to studies by Jalalpour et al. (2014) and Ayoola-Oresanya et al. (2021), MeJA was responsible for increasing phenolic and flavonoid accumulation in Taxus baccata cell culture. Earlier, MeJA greatly increased the amount of alkaloid in O. liukiuensis hairy roots produced when they were elicited in a liquid medium (Asano et al., 2004). However, a further rise in MeJA concentration (200 µM) harms the production of biomass, phenol, flavonoids, and tannin ( Table 1 ). It should be noted that the use of PGRs and elicitors to examine callus biomass production and the build-up of secondary metabolites in a variety of species led to equivalent results, (Sudha and Ravishankar, 2003; Liu et al., 2007; Yesil-Celiktas et al., 2007; Giri et al., 2012).

Nevertheless, elicitation by yeast extract had a suppressive effect on the synthesis of total phenol, flavonoids, and tannin. Several workers have also noted comparable outcomes (Asano et al., 2004; Xu et al., 2016; Pisitpaibool et al., 2021). Table 1 compares the effectiveness of each treatment with the control.

All the phenolic substances discovered in this investigation have the potential to act as antioxidants. The biological effects of phenolic compounds, alkaloids, flavonoids, and tannins found in plants include anti-cancerous, anti-inflammatory, and antioxidant properties, among others (Miller, 1996; Nithya et al., 2016; Muniyandi et al., 2019). Three separate in vitro experiments used to determine the antioxidant activity of all phenolics revealed diverse results ( Table 1 ). With treatment with 50 µM MeJA, the highest level of antioxidant activity as measured by DPPH, ABTS, and FRAP was observed. The maximal antioxidant activity was estimated to be 6.89 mM Ascorbic acid Equivalent/100 g dry weight of the sample as depicted by ABTS assay, compared to 2.21 mM Ascorbic Acid Equivalent/100 g of dry weight for the DPPH assay. For the FRAP experiment, 50 µM MeJA was reported to demonstrate the highest antioxidant activity (2.88 mM Ascorbic Acid Equivalent/100 g of dry weight). All three assays followed depicted the highest antioxidant activity at 50 µM MeJA, however, these assays resulted in different ranges of antioxidant potential for each experiment. The variations observed in antioxidant activity as observed by multiple tests are most likely connected to the respective modes of action of three assays. The reduction of a ferric analog is the foundation of the FRAP assay. In the presence of any antioxidant, the tripyridyl-triazine Fe (TPTZ)+++ Fe+++ complex is transformed into a vivid blue color Fe (TPTZ)++ at low pH (Wong et al., 2006). Differences in antioxidant activity have been found by other researchers as a result of the different techniques used (Surveswaran et al., 2007; Rawat et al., 2011; Giri et al., 2012).

In the control, reduced antioxidant activity was determined by all three of the assays used in the study. Minimum antioxidant activity was recorded with DPPH assay (1.09 mM Ascorbic Acid Equivalent/100 g of dry weight) and maximum was recorded for ABTS assay (6.89 mM Ascorbic Acid Equivalent/100 g of dry weight). Similar results have been reported in recent studies (Jalalpour et al., 2014; Makowczyńska et al., 2015; Ayoola-Oresanya et al., 2021).

The antioxidant potential and total phenolic contents of the in vitro generated callus (best cultures of TDZ, MeJA, and YE) and control were considerably different. Total phenol concentration in invitro callus suspension culture was more than twice higher than in control ( Table 1 ). In addition, compared to the control, the flavonoids and tannin levels dramatically increased. TDZ and MeJA grown callus were shown to have significantly (p 0.05) higher antioxidant activity in comparison with control (reported as mM Ascorbic Acid Equivalent/100 g of dry weight). However, using the DPPH assay, no appreciable difference in antioxidant activity was found. Many studies have shown how different PGRs and elicitors can be used to produce or augment secondary metabolites and other advantageous bioactive compounds (Giri et al., 2012; Jalalpour et al., 2014; Makowczyńska et al., 2015; Ayoola-Oresanya et al., 2021). The elicitor MeJA (50 µM concentration) was found to be quite successful at increasing the total phenol, flavonoids, and tannin content in P. polyphylla, whereas TDZ was useful in improving callus biomass.

The production of steroidal saponin (dioscin and diosgenin) from wild rhizomes and in vitro callus (the best cultures of TDZ, MeJA, and YE) were compared. The concentration of dioscin in callus cultures treated with 1.0 µM TDZ and 50 µM MeJA was reported to be more than two times greater.

MeJA is efficient in increasing the production of secondary metabolites in cell cultures and is essential for signal transduction processes that control plant defence responses (Zhao et al., 2005; Singh and Dwivedi, 2018). Although YE indicated an increase in dioscin production, it was not substantial. Similar findings were observed for the generation of diosgenin content, which was considerably higher than that of control ( Figure 3 ). Chromatogram of steroidal saponin diosgenin and dioscin is also presented in Figure 4 . According to the culture method, Wang et al. (2022) recently observed that the MeJA-treated Chinese chives had substantial variations in primary and secondary metabolites. Similar outcomes were also reported by other researchers, who claimed that using MeJA as an elicitor in the culture increased the synthesis of the secondary metabolites (Rodríguez-Sánchez et al., 2020; Deveci et al., 2022; Wang et al., 2022).