Comparative secondary metabolite expression in callus cultures and mother plant in Barleria prionitis L.

The present study is the first report on the quantitative analysis of secondary metabolites in callus cultures of Barleria prionitis L. and comparison with the mother plant. Callus was obtained from stem internode (on MS medium with 0.5 mg l−1 NAA, 0.5 mg l−1 BAP and 300 mg l−1 ascorbic acid), and leaf (on MS medium with 1.0 mg l−1 2,4-D, 0.5 mg l−1 BAP, 300 mg l−1 ascorbic acid ) explants and, multiplied by subculturing thrice at an interval of 25 days. Calli and mother plant counterparts were extracted in four solvents (methanol, ethanol, acetone, and distilled water) and examined for active compounds and antioxidant potential. Callus cultures not only preserved the mother plant’s metabolite profile but also displayed elevated levels. Leaf-derived callus surpassed stem-derived callus in most of the studied parameters. The highest total phenolic content (21.46 mg GAE g−1 FW) and total flavonoid content (24.58 mg of RE g−1 FW) were observed in methanol extract of leaf-derived callus, representing a 3-fold and 2-fold increase over mother plant leaf, respectively. Antioxidant capacity based on FRAP and DPPH assay was highest in methanol extract of leaf-derived callus (7-fold and 3-fold increase over mother plant, respectively) while ABTS activity was highest (122-fold increase) in acetone extract of leaf-derived callus. HPTLC analysis revealed enhanced concentrations of squalene (10-fold) and SME (2.3-fold) in acetone and methanol extract of leaf-derived callus, respectively, compared to mother explants. The results of RP-HPLC for phenolics showed the highest gallic acid content (99-fold increase) in ethanol extract of stem-derived callus whereas catechol was maximum (37-fold increase) in aqueous extract of leaf-derived callus. These findings suggest that callus cultures of B. prionitis L. can be a potential source of active metabolites. Further, cell suspension cultures can be produced from the callus, which could be an avenue for the large-scale production of bioactive compounds. For the first time, active components of B. prionitis L. were determined quantitatively in callus cultures and compared with the mother plant.


Introduction
The capacity of plant cells, tissue, and organ cultures to produce valuable biochemical compounds has been recognized almost since the inception of plant biotechnology (Chatterjee and Ghosh 2020).Comparative phytochemical evaluation of in vitro cultures with the mother plant is necessary to ensure their authenticity in terms of chemical composition.Furthermore, it serves as a basis for quality assurance (Khan et al. 2021) and the tissue cultures with better quality than the mother plant can thus be recommended for scaling-up production of useful compounds.There are many research reports that focused on such comparative analyses in medicinal plant species and some of them revealed the synthesis of certain phytochemicals at a higher level than in vivo plants (Riahi et al. 2022).
Barleria prionitis L. is a medicinal shrub belonging to the family Acanthaceae.It is used for the treatment of various conditions like toothache, bleeding gums, whooping cough, inflammation, stiffness of limbs, enlargement of the scrotum and sciatica (Gangaram et al. 2022).Its important active principles are shanzhiside methyl ester (SME), barlerin, and squalene apart from phenolics and flavonoids.SME is an iridoid glycoside that has been reported to show various bioactivities, such as anti-inflammatory, anti-depressant, anti-diabetic, antioxidant, and neuroprotective effects (Ghule et al. 2012).Some of the possible mechanisms of action of SME include inhibiting the production of pro-inflammatory cytokines, stimulating the expression of β-endorphin in spinal microglia, modulating the activity of monoamine oxidase and serotonin transporter, enhancing the insulin signaling pathway, scavenging free radicals, and protecting against neuronal damage (Ghule et al. 2020;Sun et al. 2022).
Squalene is a triterpene that is widely distributed in nature and is an intermediate in the cholesterol biosynthesis pathway.It has been reported to exhibit various biological activities, such as antioxidant, anticancer, antidiabetic, antiinflammatory, neuroprotective, skin hydrating and emollient effects (Lou-Bonafonte et al. 2018).It has multiple industrial applications in different fields such as pharmaceuticals, cosmetics, food supplements and biofuels (Lozano-Grande et al. 2018).Both SME (Ghule et al. 2012) and squalene (Nidhi et al. 2013) have been quantified and isolated in B. prionitis using techniques like HPTLC, flash chromatography and GC-MS and the structure of these compounds was elucidated with the aid of extensive NMR spectral studies.
The tremendous medicinal potential of B. prionitis demands its use as a prospective drug resource to treat many ailments (Farrukh 2022) and utilizing its tissue cultures to extract candidate active compounds would be an ideal choice.A literature survey showed that few attempts have been made toward tissue culture of B. prionitis L. (Lone et al. 2011;Kumar and Rani 2016;Singh et al. 2015;Premjet et al. 2010;Shukla et al. 2011;Kumari et al. 2013) but none of these undertook phytochemical characterization of tissue cultures except one report on qualitative analysis of secondary metabolites in callus cultures (Kumari et al. 2013).
Callus culture plays a vital role in the production of highvalue plant-derived products such as pharmaceuticals, nutraceuticals, flavors, fragrances, and natural dyes.It offers a range of benefits, including renewable and sustainable production, manipulation of biosynthetic pathways through the optimization of media and physical conditions, and provide insights into metabolic processes (Chandran et al. 2020).In callus cultures, several investigators have reported the synthesis of industrially important secondary compounds such as resveratrol, artemisin, α-tocopherol, ajmaline, serpentine, reserpine, flavones, paclitaxel, and stilbene (Efferth 2019).Additionally, callus cultures can be used to develop cell suspension cultures which can be cultivated in bioreactors to upscale the production.
To exploit tissue cultures for industrial use, it is imperative to undertake their extensive phytochemical characterization.The literature indicated a gap in phytochemical studies in tissue cultures of B. prionitis L. Therefore, the present investigation aimed to evaluate the antioxidant capacity and contents of phenolics, flavonoids, shanzhiside methyl ester (SME) and squalene in leaf-derived and stem-derived callus cultures in comparison to the mother plant leaf and stem.To the best of our knowledge, this is the first report of its kind in B. prionitis L. tissue cultures.

Plant material and callus culture
The wild plants of B. prionitis L. were collected from Thane in Maharashtra, India and maintained in the greenhouse at the School of Biotechnology and Bioinformatics, D. Y. Patil Deemed to be University, CBD Belapur.Plants were authenticated at the Botanical Survey of India, Pune (voucher specimen No. DYP01TH).Based on the preliminary phytochemical screening, a well-grown plant with a promising metabolite profile was selected as an explant donor (mother plant).
Callus was established from the soft stem and young leaf explants as per the protocol described by Ranade (2022).Internodal segments from stems were washed under running tap water and surface sterilized by pre-treatment with sodium hypochlorite (1% active chlorine; v/v) for 7 min, 0.1% mercuric chloride (w/v) for 10 min followed by thorough washing with sterile distilled water and inoculated on callus induction medium i.e.MS medium (Murashige and Skoog 1962) containing 0.5 mg l −1 naphthalene acetic acid (NAA), 0.5 mg l −1 6-benzylaminopurine (BAP) and 300 mg l −1 ascorbic acid.Callus obtained was multiplied by repeated subculture (four cycles) at an interval of 25 days on the callus induction medium containing 200 mg l −1 ascorbic acid, 100 mg l −1 polyvinylpyrrolidone (PVP) and 20 mg l −1 citric acid (Ranade 2022).
Leaf explants were surface sterilized by pre-treatment with sodium hypochlorite (1% active chlorine; v/v) for 7 min, 0.1% mercuric chloride (w/v) for 8 min followed by thorough washing with sterile distilled water and inoculated on MS medium supplemented with 1.0 mg l −1 2,4-dichlorophenoxy acetic acid (2,4-D), 0.5 mg l −1 BAP, 300 mg l −1 ascorbic acid (callus induction medium).Callus obtained was multiplied by repeated subculture (three cycles) at an interval of 25 days on the callus induction medium containing 200 mg l −1 ascorbic acid, 100 mg l −1 PVP and 20 mg l −1 citric acid (Ranade 2022).The multiplication phase (after the last subculture) callus was used as the experimental material.

Sample and preparation of extract
Four samples were studied: Mother plant leaf, leaf-derived callus, mother plant stem and stem.-derivedcallus.Fresh material of each sample was ground in liquid nitrogen followed by extraction in four different solvents i.e. ethanol, methanol, acetone and distilled water (aqueous).The sample-to-solvent ratio was 1:10 w/v (5 g of crushed sample in 50 ml of solvent).The resulting mixture was kept on a shaker overnight (100 rpm) for 10 h to facilitate better extraction.The insoluble material was removed by centrifugation at 4000×g for 15 min (Superspin R-V/Fa, Plasto crafts, India) and the extracts were concentrated by evaporation using rotavapor (Rotary Vacuuma 'Digital Bath', Superfit Continental Pvt. Ltd.India).
The extracts were prepared in triplicate, each time using callus from separate cultures as well as leaf and stem from different regions of the mother plant.

Total phenolic content
The total phenolic content (TPC) was estimated by the Folin Ciocalteu (FC) method (Supritha and Radha 2018).The reaction mixture consisted of 0.5 ml of sample extract (1 g l −1 ), 2.5 ml 10-fold diluted FC reagent and 2 ml 7.5% sodium carbonate (Na 2 CO 3 ).It was incubated in dark at room temperature for 30 min followed by a record of absorbance at 760 nm against a blank using a UV visible spectrophotometer (UV-1700 Pharma Spec, Shimadzu).The total phenolic content was calculated from the standard curve of gallic acid and expressed as mg gallic acid equivalent (GAE) g −1 FW.

Total flavonoid content
The total flavonoid content (TFC) was measured by aluminium chloride colorimetric assay (Akkol et al. 2008). 1 ml of extract (1 g l −1 ) or standard (rutin) was added to 1 ml of 2%aluminum chloride (AlCl 3 ) and 3 ml (5%) sodium acetate.The mixture was incubated at 20 °C for 2.5 h and the absorbance was recorded against the blank at 440 nm using a UV visible spectrophotometer.The total flavonoid content was calculated from the standard curve of rutin and expressed as mg rutin equivalents (RE) g −1 FW.
For the DPPH-FRSA assay, ascorbic acid was used as standard.The ability of extracts (0.01to 0.1 mg ml −1 ) to scavenge DPPH radical was assessed and % DPPH inhibition was calculated using the formula: where Abs control is the absorbance of the control (standard) and Abs sample is the absorbance of the test sample (extract).
The scavenging activities of all concentrations of a sample were plotted on the graph and regression analysis was done.Antioxidant activity was expressed as IC50 value (mg ml −1 ) calculated from the graph.
For the ABTS assay, ascorbic acid was used as standard.The ability of extracts (0.01 to 0.1 mg ml −1 ) to scavenge ABTS radical was assessed and % ABTS inhibition was calculated using the formula: where Abs control is the absorbance of the control (standard) and Abs sample is the absorbance of the test sample (extract).
The scavenging activities of all concentrations of a sample were plotted on the graph and regression analysis was done.Antioxidant activity was expressed as IC50 value (µg ml −1 ) calculated from the graph.
For the FRAP assay, antioxidant activity based on ferric ion-reducing ability was calculated using a standard curve of ascorbic acid at 593 nm.The FRAP result was expressed as mg of ascorbic acid equivalent antioxidant capacity g −1 FW (mg AEAC.g −1 FW).

Quantification of phenolics-HPLC analysis
RP-HPLC (Waters Model 2487 with UV detector) was performed for the quantification of phenolic compounds in callus and mother plant samples.The stationary phase was C18 column (5 µM).The standards of gallic acid, catechol, caffeic acid, ferulic acid and p-coumarin were prepared in methanol (1000 ppm) and diluted to 50 ppm.The elution system was composed of 20% methanol, 1% acetic acid and 80% water with a linear gradient scheme and the detector was adjusted at 280 nm (Ranade 2022).Identification of phenolic compounds was based on the comparison of their retention time with the standard chromatogram of a mixture of pure phenolic compounds.The concentration of phenolic compounds was calculated from the peak area measurement and was expressed as mg g −1 .

Quantification of shanzhiside methyl ester (SME)
A validated HPTLC method for the determination of SME in B. prionitis was employed (Ghule et al. 2012).HPTLC system CAMAG LINOMAT 5, including Linomat V sample applicator, UV detector, a CAMAG twin-trough TLC chamber, CAMAG TLC Scanner 3, and software WinCATS-[Linomat 5_130806] were used.Aluminum-backed HPTLC plates (10 cm × 20 cm) with 200-nm thickness of silica gel 60 F254 (Merck, Darmstadt, Germany), prewashed with methanol, were used.SME as a marker (200.0 µg ml −1 ) in methanol was spotted on the TLC plate at concentrations of 400, 600, 800, 1000 and 1200 ng for preparation of the standard curve.For every TLC plate, at least one spot of SME (600 ng) was applied along with the spots of samples.The mobile phase used was chloroform and methanol in the ratio of (8:2).The compound SME was detected at 254 nm and HPTLC chromatograms along with peak data were obtained.The quantity of SME was calculated from the standard curve and expressed as µg g −1 FW.

Quantification of squalene
Squalene was quantified using the HPTLC system described in the previous point.Squalene (10 µg ml −1 in acetone) was spotted on the TLC plate at concentrations of 50, 100, 150, 200 and 250 ng for preparation of the standard curve.For every TLC plate with sample spots, at least one spot of SQ (150 ng) was applied.The mobile phase used was n-hexane.The TLC plates were air-dried and sprayed with copper sulphate reagent.The TLC plates were then baked in the oven at 120 ℃ for 10 min for the development of spots.The separation of compounds was visualized under CAMAG UV detector at 550 nm.The standard curve of peak area versus concentration was plotted and linear regression analysis was carried out.The quantity of SQ was calculated from the standard curve and expressed as µg g −1 FW.

Statistical analysis
Experiments were conducted in a randomized design.Data were expressed as means ± standard deviation (SD) of three biological replicates (extracts).Assays for TPC, TFC and antioxidant activity were run in triplicates while HPLC and HPTLC were run once, for each of the triplicate extracts.Data were analyzed by the one-way analysis of variance (ANOVA), followed by post-hoc Fisher's LSD test (p < 0.05), using GraphPad Prism 5.0.Pearson's correlation coefficients (r) between TPC, TFC and antioxidant activity assays were determined using Microsoft Excel 2007.

Results and discussion
The current study compared the biosynthetic capacity of callus cultures of B. prionitis L. to that of the mother plant.As previously described by Kumari et al. (2013) in B. prionitis L., we observed the accumulation of parental active chemicals in the callus.Similarly, various researchers have shown a high degree of resemblance between the phytochemical makeup of wild plants' aerial organs and undifferentiated cells (Rameshkumar et al. 2018).In our investigation, a quantitative comparison of metabolites and antioxidant efficacy between the callus and the mother plant indicated significantly (p < 0.05) larger amounts in the former.

Total phenolic content (TPC) and total flavonoid content (TFC)
In the present study, callus derived from both leaf and stem explants showed higher TPC (2.5-3-fold) and TFC (2-fold) compared to the mother part equivalent.The highest phenolic and flavonoid content (21.46 mg GAE g −1 and 24.58 mg RE g −1 , respectively) was noted in the methanol extract of leaf-derived callus (Table 1).Like our observations, higher TPC in leaf-derived callus over the mother leaf has been reported in L. umbellata (Govindaraju et al. 2019).It is important to consider that callus cultures are complex systems and the interplay of genetic, epigenetic, hormonal, and environmental factors in the tissue culture process can lead to variations in metabolite production.In vitro conditions are experienced as stress by the cultured tissues and to combat stress the tissues accumulate antioxidants (Najhah et al. 2021).The high levels of active compounds in hazelnut callus were attributed to the presence of plant growth regulators and nutrients in the culture media (Hazrati et al. 2022).Different reports are found from the literature, whereby some authors observed lower flavonoid contents in stemderived callus compared to the mother plant in S. kakudensis (Manivannan et al. 2015) while others noticed higher TFC in leaf-derived callus than the mother leaf in L. pumila var.alata (Najhah et al. 2021).These observations indicate that the concentrations vary depending on plant species and plant part.
We discovered that phenolic (1.5 to 2-fold) and flavonoid (1.3-fold) concentrations were greater in leaf-derived callus over stem-derived callus, which was in line with the trend observed in mother counterparts.Our results are consistent with previous reports in Maytenus emarginata (Moteriya et al. 2014a) and Gloriosa superba L. (Moteriya et al. 2014b), where leaf-derived callus was richer in flavonoids than stem callus.According to Pérez-Alonso and Jiménez (2011), the chemical composition of callus varies according to the origin of the explant as the inherent differences in the biosynthetic capabilities of the explants may be carried over to the cultures derived from these explants.Leaf and stem tissues within a plant encounter environmental stressors differently resulting in distinct metabolic profiles, especially regarding defensive polyphenol compounds (Pérez-Alonso and Jiménez 2011).Like our findings, prior studies in B. prionitis revealed that the leaf was a better source of phenolic compounds than the stem (Jaiswal et al. 2010;Sharma et al. 2014).
In the present study, the capacity of different solvents to extract TPC in leaf-derived callus was almost similar while Govindaraju et al. (2019) reported the highest phenolics in methanol extract of L. umbellate callus.We found maximum TFC in methanol extracts of both calli, contrary to which Moteriya et al. (2014a) identified the best extraction of flavonoids in acetone.The effect of solvent on metabolite extraction is relatively straightforward and proven.

Antioxidant activity
Plant antioxidant activity is mostly due to polyphenols, which function via multiple mechanisms and may thus be evaluated using a variety of assays.In the present study, all three assays i.e., DPPH, FRAP and ABTS revealed higher antioxidant activity in the callus compared to the mother plant, with the increase being more pronounced in leafderived callus.As shown in Table 2, the leaf-derived callus had the highest FRAP value in methanol extract (253.77mg AAE g −1 ), representing an increase of 7-fold over the mother plant leaf.At the same time, based on DPPH and ABTS assays, leaf callus showed the highest activity, as reflected by the lowest IC50 of 16.55 mg ml −1 (3-fold increased activity in methanol extract) and 3.08 µg ml −1 (122-fold increased activity in acetone extract), respectively.Our results agree with the previous reports of higher FRAP and DPPH activity in leaf callus over mother leaf in C. fistula (Bahorun et al. 2002) and pumila (Najhah et al. 2021), respectively.Contrastingly, field-grown leaves showed three times more ABTS activity than leaf-derived calli in blueberry and blackberry (Kolarević et al. 2021).
With a few exceptions, better antioxidant activity was often seen in leaf and leaf callus over stem and stem callus in the current study.This could be associated with higher levels of phenolics and flavonoids found in leaf tissues over the stem.We recorded higher DPPH radical scavenging activity in the acetone extract of the leaf (IC50: 26.98 mg ml −1 ) Correlation studies were performed to gain insight into the relationship between polyphenolic compounds and the antioxidant potential of B. prionitis L. Our results revealed mostly a significant (p < 0.05) positive correlation between TPC, TFC, FRAP, DPPH and ABTS assays, although in variable degrees (Table 3).The negative correlation values seen for DPPH and ABTS assays in fact mean a positive correlation because DPPH and ABTS radical scavenging potential were expressed as IC50 values, which implies the lower the value, the higher the antioxidant capacity (Terpinc et al. 2012).Both TPC and TFC had a strong correlation with FRAP (r > 0.75 in leaf and leaf callus and r > 0.9 in all except the mother stem, respectively).On the other hand, the correlation of TPC and TFC with DPPH (r = − 0.68 to − 0.98 and r = − 0.58 to − 0.98, respectively) and ABTS (r = − 0.821 to − 0.918 and r > − 0.9, respectively) was generally high to moderate.These disparities in the correlation of TPC and TFC with the three assays could be related to the variations in their composition and component concentrations as well as in their interaction with each other and with ABTS, DPPH radicals, and ferric ions (Terpinc et al. 2012).
Different antioxidant activity assays work by different mechanisms, consequently, they may have low correlations with each other.However, in the present study, an overall good correlation was seen between DPPH and ABTS activity (r > 0.889; p < 0.05) and FRAP with DPPH (r = − 0.834 to − 0.949; p < 0.05) and ABTS activity (r = − 0.729 to − 0.940; p < 0.05) (Table 3), that indicated

Quantification of phenolics
According to the RP-HPLC data, only two phenolic compounds (gallic acid and catechol) were detected in some of the samples (Table 4).The mother plant contained gallic acid in both leaf and stem while catechol was in the stem in the present study.Such variation in the distribution pattern of phenolics has also been noticed by Singh et al. (2023) in B. prionitis L. and the authors reported the presence of vanillin and caffeic acid in addition to gallic acid in both leaf and stem.In the present study, callus expressed the parental phenolics in most of the solvent extracts.Gallic acid was detected in all the extracts of stem-derived callus with the highest content of 99.08 µg g −1 FW found in ethanol extract contrary to its absence in the mother counterpart (Fig. 1) while leaf-derived callus exhibited gallic acid only in aqueous extract (11.16 µg g −1 FW) and this amount was 10-fold higher than the mother plant leaf.Catechol was detected only in the aqueous extracts of the mother plant stem (5.22 µg g −1 FW) and leaf-derived callus (37.43 µg g −1 FW).While increased accumulation or exclusive presence of phenolics in the callus suggests that their expression was influenced by culture conditions, their absence in the samples may indicate that their synthesis was beyond the limits of detection.

Quantification of shanzhiside methyl ester (SME) and squalene (SQ) by HPTLC method
In the present study, SME was detected in both leaf (methanol, acetone and aqueous extract) and stem (methanol and acetone extract) of the mother plant in concentrations ranging from 49.69 to 182.49 µg g −1 FW.In previous studies in B. prionitis, SME amounts were reported to be 4.91% w/w in whole aerial parts (Ghule et al. 2012), 2.62% w/w in leaf (Kaur et al. 2014) and 52% w/w in stem followed by leaf and root (Singh et al. 2023).We found that the callus derived from both the explants also showed the presence of SME, in concentrations significantly higher than the mother plant counterparts (Table 5) and the highest content of 423.82 µg g −1 FW (2.3-fold increase) was recorded in methanol extract of leaf-derived callus (Fig. 2).Contrasting to our results, Premjet et al. (2010) reported the absence of SME in leaf callus of B. prionitis L. based on the results of TLC and HPLC.In our study, methanol was the best solvent for the extraction of SME in all samples and this agrees with the observations reported by Ghule et al. (2012) and Kaur et al. (2014).
According to GC-MS analysis, squalene was detected in the roots of B. prionitis L. (Nidhi et al. 2013).In the current investigation, HPTLC data revealed the presence   of squalene in all samples of the mother plant and callus except methanol extracts of the mother plant leaf.Out of the two calli, only leaf-derived callus showed more squalene than the mother counterpart in all the extracts, and the highest content of 14.23 µg g −1 FW (10-fold increase) was obtained in acetone extract (Table 5; Fig. 2).Popa et al. (2015) also suggested that solvents like acetone are preferable for extraction of squalene due to its low polarity.Like our results, a 2.3-fold increased accumulation of squalene was observed in leaf-derived callus over leaves of field-grown plants of Nilgirianthus ciliates and this rise was attributed to the presence of PGRs in the callus culture medium (Rameshkumar et al. 2018).In the current study, the antioxidant activity of leaf-derived callus corresponded with the squalene concentration in all the extracts indicating the contribution of squalene as an antioxidant, as previously described (Lou-Bonafonte et al. 2018).We observed that the leaf-derived callus had higher (12 to 17-fold) squalene content over the stemderived callus (0.30 to1.15 µg g −1 FW), despite a reverse pattern noticed in the mother part equivalents (Table 5).This unexpected paradox could possibly be due to a shift in the metabolic activity of leaf and stem during the dedifferentiation process (Awada et al. 2019).

Conclusion
Our investigations confirmed the presence of bioactive compounds of B. prionitis L. in its callus cultures.When compared to the mother plant, callus cultures exhibited higher antioxidant activity and accumulation of phenolics, flavonoids, squalene and shanzhiside methyl ester.Leaf-derived callus outperformed stem-derived callus in most of the studied parameters.Methanol extracts of leafderived callus had the highest TPC, TFC, and SME levels, as well as a 10-fold increase in squalene content.The best antioxidant activity based on DPPH and FRAP assays was found in methanol extract of leaf-derived callus while its acetone extract had the highest ABTS radical scavenging activity.The highest concentrations of gallic acid and catechol were found in the ethanol extract of stem callus and aqueous extract of leaf callus, respectively.These findings imply the significance of leaf-derived callus cultures for the synthesis of active principles of B. prionitis L. To realize its commercial potential, bioactive compounds must be produced in suspension culture in a bioreactor, coupled with the implementation of yield improvement strategies such as optimization of media and culture conditions, precursor feeding and elicitation.

Fig. 1
Fig. 1 RP-HPLC chromatogram of phenolics-ethanol extract of mother plant stem (A) and stem-derived callus (B) showing the absence and presence of gallic acid (GA), respectively; aqueous

Table 1
Total phenolic and flavonoid contents in callus and mother plant of B. prionitis

Table 5 HPTLC
-based quantification of shanzhiside methyl ester (SME) and squalene in callus culture and mother plant of B. prionitis