Stimulation of phenolic compounds accumulation and antioxidant activity in in vitro culture of Salvia tebesana Bunge in response to nano-TiO2 and methyl jasmonate elicitors

In the present study, we first attempted to achieve an efficient procedure for optimizing callogenesis from apical meristem and leaf explants of Salvia tebesana on MS media containing different concentrations of BAP alone and in combination with 2,4-D. Then, the inducing effect of nano-TiO2 (10, 60, and 120 mg L−1) and methyl jasmonate (50, 100, and 200 µM), as abiotic elicitors were studied on the enhancement of phenolic compounds, rosmarinic acid, and some individual flavonoids as well as antioxidant capacity of callus. According to the results, the highest callogenesis rate (100 and 93.33, respectively) and DW (0.55 ± 0.03 and 0.36 ± 0.02 g, respectively) per responsive explant were achieved from apical meristem on MS media containing "BAP1 + 2,4-D1" mg L−1 and from leaf explant on the medium supplemented with "BAP0.5 + 2,4-D1" mg L−1. The elicitation with 10 and 60 mg L−1 nano-TiO2 (respectively for apical meristem and leaf), and 50 µM MeJa could significantly promote the production of predominant phenolic derivatives in S. tebesana calli, where the highest content of total phenolics, O-diphenols, phenolic acid, flavonoid, flavone and flavonol, proanthocyanidin was recorded. As well as results declared that the increasing of rosmarinic acid was more correlated with nano-TiO2 treatment than MeJa elicitation. Moreover, the highest content of apigenin (0.33 ± 0.02 μg g−1 DW) was detected after MeJa-elicitation (50 µM), while the maximum level of quercetin (2.62 ± 0.09 μg g−1 DW) and rutin (13.79 ± 0.87 μg g−1 DW) were obtained after exposure to 60 mg L−1 nano-TiO2, both from leaf-derived calli. While a significant positive correlation was recorded between antioxidant assays (DPPH and FRAP) and phenolic derivatives of treated calli; a very strong correlation occurred between the content of rosmarinic acid of apical meristem-derived calli and DPPH and FRAP values (r2 = − 0.921 and r2 = − 0.913, P < 0.01 respectively). Our results showed that the combination of in vitro culture and elicitation would be a good technique to successfully produce and enhance the content of pharmacologically valuable metabolites in S. tebesana. Nano-TiO2 and methyl jasmonate elicitors in a concentration-dependent manner increased the phenolic compounds, specific flavonoids, and antioxidant properties in the callus culture of Salvia tebesana Bunge.


Introduction
Salvia tebesana Bunge is a small shrub plant, which its geographical distribution restricts to Iran, Afghanistan, and Pakistan. In Iran, it has only been reported in a limited area of South Khorasan province (Khatamzaz 2002). This endemic plant belongs to the genus Salvia genus (family Lamiaceae), with over 900 species that have been identified around the world, and about 17 species are endemic to Iran (Walker et al. 2004). Salvia L. contains a large assortment of secondary metabolites, including essential oils, phenolic acids, flavonoids, and terpenoids (Russo et al. 2013). Studies have also shown that phenolic compounds can exhibit a broad range of physiological-therapeutic properties, such as anti-inflammatory, anti-allergic, antioxidant, antimicrobial, anti-proliferative activities, antithrombotic, and cardiovascular protective effects. Because of their high antioxidant activities, phenolic compounds function as the main bioactive compounds to determine the antioxidant power in foods (Ulubelen and Topcu 1992;Taârit et al. 2012;Aleebrahim-Dehkordy et al. 2016). In addition to high levels of flavonoids (Đorđević et al. 2000;Kamatou et al. 2008), it has been proved that one of the most abundant phenolic acids present in Salvia species is rosmarinic acid (RA) (Bandoniene et al. 2005;Koşar et al. 2008;Jadeja and Devkar 2014). This compound has also been reported to have antibacterial, antiviral, antioxidant, and anti-inflammatory effects (Petersen and Simmonds 2003).
In vitro production of callus-derived secondary metabolites is a suitable technique for continuous and mass production of these valuable compounds with high efficiency for a short time (Ourmazdi and Chalabian 2006). Callus induction from plant tissues is influenced by several factors, such as genotype, explant type, growth regulators, culture medium, type of nutrients, environmental conditions as well as elicitors (Hasanloo et al. 2009). Moreover, elicitors' roles in culture conditions to boost the production of a diversity of metabolites have been confirmed (Zhao et al. 2005). Recent studies have shown that these inductions highly depend on the alteration of physiological responses and the accumulation of phytoalexins under stress conditions (Baskaran et al. 2012). Generally, the application of various elicitors, like nano-titanium dioxide (nano-TiO 2 ) and methyl jasmonate (MeJa) with a concentration-dependent manner, is remarkably advisable to promote the generation and accumulation of phenolic compounds during the culture process (Ge and Wu 2005;Bahreini et al. 2015). Results of the current literature have indicated that the nano-TiO 2 effect on the induction and accumulation of phenolic compounds was studied in different plant species, such as Salvia officinalis (Ghorbanpour 2015), Papaver somniferum , Aloe vera (Raei et al. 2014), and Foeniculum vulgare (Bahreini et al. 2015). It has also been proved that the production of phenolic compounds, especially rosmarinic acid, can be promoted after MeJa elicitation in Salvia virgata (Dowom et al. 2017), Salvia officinalis (Grzegorczyk and Wysokińska 2009), Salvia miltiorrhiza (Ge and Wu 2005) and Exacum affine Balf. f. ex Regel (Skrzypczak-Pietraszek et al. 2014).
Up to the present time, very little information is available about secondary metabolites of S. tebesana as a traditional medicinal plant (Goldansaz et al. 2017;Eghbaliferiz et al. 2018;Fotovvat et al. 2018). Although Hemmati et al. (2020) has studied the effect of some growth hormones (BAP, 2,4-D, and NAA) on tissue culture and phenolic compounds accumulation of S. tebesana in vitro, there still is no report available regarding the effect of elicitors on secondary metabolites of S. tebesana. This study was done for the first time (a) to create an efficient system to optimize the culture condition and hormonal composition for the large-scale production of callus, (b) to find the optimal concentration of nano-TiO 2 and MeJa elicitors to enhance the level of phenolic secondary metabolites and antioxidant properties, and (c) to evaluate the content changes of rosmarinic acid and some individual flavonoids (apigenin, quercetin, and rutin) in elicitor-treated calli.

Plant material
Plant collecting and in vitro growth condition was performed according to our previous research (Hemmati et al. 2020). The plant grown in Hoagland nutrient solution (Hoagland and Arnon 1950) was used as a source of apical meristem and leaf explants. Both explants rinsed 3 times with sterile distilled water, followed by immersing in 70% (v/v) methanol (for 30 s and once). Leaf explants were also plunged in 5% (v/v) sodium hypochlorite (3 min and once). The final wash of both explants was done 3 times with sterile distilled water.

Culture condition and callus induction
Because one of the aims of the present study was to assess the effect of elicitors on the best callus-inducing hormonal medium, some hormonal conditions related to Hemmati et al. study (2020) were nominated to evaluate the best conditions. Apical meristem and leaf explants were cultured on MS medium with different concentrations of BAP (0, 0.5, and 1 mg L −1 ) and 2,4-D (0, 0.5, 1, and 1.5 mg L −1 ). Three replications were considered for each treatment, and 1 3 five explants were cultured in each jar. After 3 weeks of dark treatment, the samples were exposed to 16 h of light and 8 h of darkness at 25 ± 2 °C for 4 weeks. Subculturing was performed in the 7 th week with the same hormone concentrations. 90-day-old calli were harvested, and some morphological characteristics, including the percentage of callus induction and fresh and dry weight (FW and DW), were recorded. The percentage of callogenesis was calculated based on the following equation: The data obtained from the best growth rates of callus (the highest percentage of callogenesis and callus DW) was used to determine the best culture media.
The culture of selected media was performed in a like manner to the first experiment till the 7th week. At the end of the 7th week and during the subculturing, elicitor solutions (10, 60, and 120 mg L −1 nano-TiO 2 ; 50, 100, and 200 μM MeJa) were added to basal culture media. The calli were harvested after 10 days of elicitors' treatment, and the callus biomass (FW and DW) was estimated.

Extraction for phytochemical analysis
The 60-day-old calli were dried in an oven at 40 °C for 48 h, and then, 80% (v/v) methanol was added to the callus powder in a ratio of 1 to 100 (w/v). The obtained extracts were placed in an ultrasonic device (Parsonic 2600 s, Japan) for 30 min at a temperature of 30 °C and a frequency of 40 kHz. Then the extracts were filtered with Whatman paper (No. 1), and the solvent was evaporated under air condition. To prepare the final extract, the powder (0.0015 g) was dissolved in 5 mL of 80% (v/v) methanol (Annegowda et al. 2012). This solution was used as a "methanolic extract" for all the phytochemical assays.

Determination of total phenolic compounds
The content of phenolics in elicitor-treated calli was assessed by the Folin-Ciocalteu method (Marinova et al. 2005). For this purpose, 2.5 mL of 10% (v/v) folin was added to 100 μL of "methanolic extract". After 5 min in Rate of callogenesis = number of explants with callus∕ total number of explants used × 100 the dark condition and room temperature, 2 mL of 7.5% (w/v) sodium carbonate was added, and after 1.5 h, the absorption was read by a spectrophotometer (Jasco7800, Germany) at 765 nm. The content of phenolic compounds of any sample was calculated based on the standard curve of gallic acid equivalent (GAE) (y = 0.0032x + 0.0313, r 2 = 0.987), and the result was presented in terms of mg GAE per 100 g DW of callus sample.

Determination of O-diphenols content
The reaction mixture was prepared by adding 2 mL of 50% (v/v) aqueous methanol and 0.5 mL of 5% (w/v) sodium molybdate dihydrate to 100 μL of "methanolic extract". The samples were shaken for 15 min and exposed to darkness at room temperature. Finally, the light absorption of the samples was recorded at 370 nm (Carrasco-Pancorbo et al. 2005). The basis for calculating of total O-diphenols content was the gallic acid graph (y = 0.0042x + 0.0674, r 2 = 0.998), which was expressed as mg of GAE per 100 g DW of callus.

Determination of phenolic acids content
The content of phenolic acids was determined based on the Matkowski method (Matkowski et al. 2008). Distilled water (1.25 mL), hydrogen chloride (250 μL), Arnow's reagent [10% (w/v) aqueous sodium molybdate and 10% (w/v) aqueous sodium nitrite] (250 μL) were added, respectively, to 250 μL of "methanolic extract". After being kept in total darkness for 30 min (25 ± 2 °C), 250 μL of sodium hydroxide with 250 μL of distilled water were added to the sample, and the absorbance was read at 490 nm. The phenolic acid content was calculated based on the standard caffeic acid equivalent (CAE) graph (y = 0.0041x + 0.0258, r 2 = 0.983), and the result was presented in terms of mg of CAE per 100 g DW of callus.

Determination of flavonoids content
A valoum of 300 μL of "methanolic extract" was mixed to 3.4 mL of 30% (v/v) methanol. Then 150 μL of 0.5 M sodium nitrite and 150 μL of 0.3 M aluminum chloride were added, respectively. After keeping the samples in the dark at room temperature for 5 min, 1 mL of 1 M sodium hydroxide was added, and the absorbance was recorded at 510 nm (Matkowski et al. 2008). Catechin equivalent (CATE) was used to prepare the standard curve (y = 0.0018x + 0.0106, r 2 = 0.989), and the result was calculated in mg of CATE per 100 g DW of callus sample.

Determination of flavone and flavonol content
Quercetin equivalent (QE) was used to make the calibration curve for this assay (y = 0.0022x − 0.007, r 2 = 0.982). It was added 250 μL of "methanolic extract" to 50 μL of 10% (w/v) aluminum chloride and 50 μL of 1 M potassium acetate. Then, 750 μL of 80% methanol (v/v) and 1.4 mL of distilled water were added till the final volume reached 2.5 mL. After 30 min at room temperature, the absorbance was read at 415 nm (Kosalec et al. 2004). Using the standard curve line equation obtained from quercetin, the amount of flavone and flavonol in the extract was calculated and expressed as mg QE per 100 g DW of callus sample.

Determination of proanthocyanidins content
In this step, 250 μL of the diluted extract was added to pure methanol (1 mL) and then, to 3 mL of 1% (w/v) vanillin. After vortex (WiseMix, Korea) for 30 s, 2.5 mL of 9 N methanolic sulfuric acid was added. The samples were placed in Ben-Marie (Kavoosh Mega, Iran) for 15 min at 38 °C (in the dark). Finally, the optical absorption of the reaction mixture was recorded at 500 nm (Sun et al. 1998). Total proanthocyanidin content was defined with the standard curve of catechin solution (y = 0.001x + 0.0653, r 2 = 0.939) and expressed as mg of CATE per 100 g DW of callus sample.

Determination of rosmarinic acid content
According to the method described by Öztürk et al. (2010), the concentration of rosmarinic acid in the samples was calculated based on the rosmarinic acid complex with zirconium ions (Zr 4+ ) by spectrophotometer assay. 920 μL of pure ethanol was added to 40 μL of the extract, and the final volume was increased to 1 mL by adding 40 μL of 0.5 M zirconium chloride. About 5 min following the addition of the zirconium salt solution, the absorbance of the sample was measured at 362 nm. Using the obtained equation (y = 0.014x + 0.024, r 2 = 0.991), the amount of rosmarinic acid was calculated as mg of RA per 100 g of callus DW.

DPPH scavenging activity assay
As described by de Torre et al. (2019), 100 μL of "methanolic extract" and 100 μL of methanolic DPPH solution (0.2 mM) were poured into each well of the 96-well plate, respectively. The plate was incubated in the dark at 25 ± 2 °C for half an hour, and then, its absorbance was read at 490 nm by the Elisa Reader (Gentaur ST2100, America). The antioxidant capacity was calculated in terms of inhibitory concentration (IC 50 ; effective concentration of sample with 50% DPPH inhibitory capacity), and in comparison with the antioxidant capacity of ascorbic acid (as positive control) at the same concentrations of the sample. To calculate IC, the following equation was used: In the above equation, AS represents the adsorption of the extracted sample, AB represents the adsorption of the blank sample (100 μL of extract and 100 μL of pure methanol), and AC represents the adsorption of the control sample (100 μL of pure methanol and 100 μL of DPPH solution).

Ferric reducing antioxidant power (FRAP) assay
The reduction ability of callus extract was analyzed using FRAP assay according to Li et al. (2017) with a minor modification. For this purpose, FRAP reagent was prepared by adding acetate buffer (300 mM; pH 3.6), 10 mM 2,4,6-tri (2-pyridyl)-1,3,5-triazine (TPTZ) solution in 40 mM HCL, and 20 Mm FeCl 3 solution in a volume ratio of 10:1:1. The "methanolic extract" of callus (20 µL) was mixed with 130 µL of FRAP solution and incubated in the dark for 30 min. The absorbance of reaction mixture was assayed through the ELISA system at 630 nm. Ferrous sulfate II (FeSO 4 ) was applied to produce a standard curve (y = 0.0165x + 1.323, r 2 = 0.908). The results were expressed in mg of Fe +2 per 100 g DW of callus sample.

Callus extraction
For LC-MC analysis, both explant-derived calli with the highest content of flavonoids, flavone and flavonols, were selected. 0.25 gr of dry callus powder was dissolved in 2.5 mL of HPLC grade methanol. After 2 h using an ultrasonic bath at a temperature of 30 °C and a frequency of 40 kHz, the solution was filtered through a 45 μm filter, and the solvent was evaporated under air condition (Skerget et al. 2005). For flavonoid extraction of each dry sample, the extractive solution (75% methanol/0.1% formic acid) and continuing process were done according to the method described by Gómez et al. (2018).

LC-MS/MS analysis of specific individual flavonoids
The separation was carried out using the mobile phase containing solvent A and B in gradient. Mobile phase A consisted of 0.1% formic acid in acetonitrile (v/v), and mobile phase B consisted of 0.1% formic acid in H 2 O (v/v). The volume of injection was 5 μL in all samples. Chromatographic separation was performed on an Atlantis T3-C18 3µ, 2.1 × 150 mm column at a flow rate of 0.15 mL min −1 . The column oven temperature was set at 40 °C. Mass spectra were acquired in the positive ion mode (ESI+). The following parameters were used in all experiments: cone voltage, 30 V; capillary voltage, 4 kV; extractor, 2 V; RF lens, 0.2 V; collision energy, 30 eV; gas nebulizer, N2 (grade 5); flow gas, 200 L h −1 ; source temperature, 120 °C; desolation temperature, 300 °C. A Waters Alliance 2695 HPLC-Micromass Quattro micro API Mass Spectrometer was used for the analysis. The reference substances of flavone (apigenin) and flavonols (quercetin and rutin) were obtained from Sigma-Aldrich (St. Luis, MO, USA).

Statistical analysis
Statistical analysis of data was performed using SPSS software (version 25). Using Duncan's multiple range test, the mean values were compared with the probabilities of P ≤ 0.01 and P ≤ 0.05. Pearson's correlation coefficient analysis, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to assess the associations between different phenolic derivatives contents, antioxidant capacity and different samples of apical meristem-derived and leaf-derived calli. The graphs were drawn by EXCEL software (Office 2016).

Callus induction of apical meristem and leaf explants
The results showed that internal hormones significantly affected the callus induction at P ≤ 0.01 (Duncan's multiple range test). We observed that the individually adding of BAP and 2,4-D did not induce to callogenesis. Results for 2,4-D application alone were zero, or near-zero for callus formation of both explants, while apical meristem in the media supplemented with BAP alone was more desired to branch. However, the simultaneous use of these hormones and their interaction significantly increased the callus induction (Table 1). For apical meristem explant, the best callus induction rate with the highest fresh and dry weight was obtained from the media augmented with "BAP 0.5 + 2,4-D 0.5 " and "BAP 1 + 2,4-D 1 " mg L −1 , while in the case of the leaf explant, the highest callogenesis rate, fresh and dry weight was recorded for the media supplemented with "BAP 0.5 + 2.4-D 1 ", "BAP 0.5 + 2.4-D 1.5 " and "BAP 1 + 2.4-D 0.5 " mg L −1 . In terms of appearance, we observed that all the calli showed a friable form with yellow, light, or dark green colors.

Effect of elicitors on polyphenols content of apical meristem-derived callus
The content of different phenolic compounds in apical meristem-derived calli was significantly affected (P ≤ 0.01) by elicitor type and hormonal treatment. As a general result, it can be concluded that highest polyphenolic compounds were observed in the samples on basal media with "BAP 0.5 + 2,4-D 0.5 " mg L −1 . The most content of total phenols (1074.07 ± 47.59 mg 100 g −1 DW) and rosmarinic acid (87.22 ± 0.61 mg 100 g −1 DW) was found in calli treated with 10 mg L −1 nano-TiO 2 and "BAP 0.5 + 2,4-D 0.5 " mg L −1 .
The phenol content of callus in this medium was more than twice that of the control in the same hormonal treatment.

Effect of elicitors on polyphenols content of leaf derived callus
Taking into account the changes of all analyzed compounds in leaf-derived calli, the highest amount of analyzed compounds was observed in media supplemented with MS salts and "BAP 0.5 + 2,4-D 1 " mg L −1 . Total phenolic compound in calli was found to range between 1392.14 ± 37.77 and 292.56 ± 9.42 mg100 g −1 DW. The highest concentration of phenolics was measured in the callus treated with 60 mg L −1 nano-TiO 2 on the medium containing "BAP 0.5 + 2,4-D 1 " mg L −1 hormonal treatment, which was about three times more than the amount (504.53 ± 42.47 mg 100 g −1 DW) recorded in the control sample. Exposure to 60 mg L −1 nano-TiO 2 in the medium supplemented with "BAP 0.5 + 2,4-D 1 " mg L −1 induced the most increment in total O-diphenols, phenolic acids, proanthocyanidins, and rosmarinic acid contents, which were 236.74 ± 9.58, 341.74 ± 9.71, 244.50 ± 4.35, and 113.39 ± 2.08 mg.100 g −1 DW, respectively. However, almost double the content of flavonoids than the control was observed when the lowest concentration of MeJa treatment, 50 µM, was applied to the medium augmented with "BAP 0.5 + 2,4-D 1 " mg L −1 . For all media, effective concentrations of elicitor treatments to increase the amounts of flavone and flavonol were 60 mg L −1 nano-TiO 2 and 50 µM MeJa (Table 3).

Assessment of antioxidant activity
Following the determination of the biophenolic contents, the antioxidant activities of all calli derived from apical meristem and leaf were evaluated by DPPH and FRAP radical scavenging assays. As shown in Figs. 1a and 1b, DPPH (P ≤ 0.01) and FRAP (P ≤ 0.05) results revealed that the highest antioxidant activity of apical meristem-derived callus (IC 50 23.33 ± 2.27 µg mL −1 and 475.77 ± 22.39 mg Fe +2 100 g −1 DW, respectively) was found after treatment with 10 mg L −1 nano-TiO 2 on the media containing "BAP 0.5 + 2,4-D 0.5 " mg L −1 . Moreover, the IC 50 values of elicitor-treated extracts were near to ascorbic acid (IC 50 12.52 ± 0.97 µg mL −1 ). The DPPH antioxidant activity in leaf-derived calli varied from IC 50 88.31 ± 5.14 µg mL −1 to IC 50 25.71 ± 6.91 µg mL −1 . The low-IC 50 value (as the highest antioxidant capacity) was observed in the presence of "BAP 0.5 + 2,4-D 1.5 " mg L −1 and 60 mg L −1 nano-TiO 2 (Fig. 2a) followed by methanol extract of callus on media with 50 μM MeJa and "BAP 0.5 + 2,4-D 1 " mg L −1 . The results from FRAP antioxidant assay revealed that all the extracts showed considerable antioxidant potency when compared to the controls and the application of 60 mg L −1 nano-TiO 2 and 50 µM MeJa resulted in the highest amount of antioxidant activities (Fig. 2b).

Correlation, principal component analysis and hierarchical cluster analysis
The results of Pearson's correlation showed a significant and positive correlation (P < 0.01) between estimated polyphenol derivatives and assayed antioxidant capacities (Fig. 3). A very strong correlation was recorded between the content of rosmarinic acid of apical meristem-derived calli and DPPH and FRAP values (r 2 = − 0.921 and r 2 = − 0.913, P < 0.01 respectively). While there were highly statistically significant correlations between total phenolic content of all extracts and two radicals scavenging indexes, correlations of these assays with phenolic acids, flavonoids, flavones, and proanthocyanidins were somewhat weaker The weakest correlation occurred between the O-diphenols content of leaf-derived calli and DPPH scavenging activity and FRAP assay (r 2 = 0.501 and r 2 = 0.491, respectively).
Subsequently, the quantitation data of phenolic derivatives and antioxidant potentials were subjected to PCA, to explore the differences in secondary metabolites among callus extracts (9 variables × 35 cases). The results showed that there were two eigenvalues higher than 1.00 and the two principal components explain about 80.45% of the total variance (Table 4). The first principal component, PC1, has high loadings with total contents of phenols, O-diphenols, flavonoid, flavone and flavonol, DPPH, and FRAP with 44.79% of the total variance; confirming more the data variation for the metabolites that PC2. The second component, accounting for 35.66% of the total variance, is highly positively correlated with total contents of proanthocyanidin, phenolic acid, and rosmarinic acid. The relations among different compounds based on the two PCs are illustrated in Fig. 4.
To gain insights into the relationships among different samples of apical meristem-derived calli and leaf-derived calli studied, HCA by applying the Ward's method, was performed based on the data of nine analyzed variables. Based on this analysis, various objects of apical meristem-derived calli (Fig. 5a) and leaf-derived calli (Fig. 5b) were clustered into two groups. In first group of both, the first accession of apical meristem-derived calli dendrogram consisted of callus treated with 10 mg L −1 nano-TiO 2 on the medium augmented with "BAP 0.5 + 2,4-D 1 " mg L −1 while the first subgroup of leaf-derived calli dendrogram consisted of sample treated with 60 mg L −1 nano-TiO 2 on the medium fortified with "BAP 0.5 + 2,4-D 1 " mg L −1 . The hierarchical clustering results provided more insight into the distribution of studied parameters among different samples; confirming our results about the best media with the highest phenolic derivatives and antioxidant properties.

Discussion
The combination of plant hormones in in vitro culture has been reported necessary to stimulate a high frequency of callus induction (Hong et al. 2008;Giri et al. 2012). In this work, the simultaneous use of BAP and 2,4-D significantly increased callus induction rate. We observed that highest callogenesis rate was for apical meristem explant on the Fig. 1 Antioxidant activity was assessed using DPPH (a) and FRAP (b) assays in apical meristem-derived callus. IC 50 represents the levels of biophenols required to scavenge 50% of DPPH radicals. All data are expressed as the mean ± SE. AA, Ascorbic acid 1 3 medium augmented with" BAP 1 + 2,4-D 1 " mg L −1 and for leaf explant on the medium containing "BAP 0.5 + 2.4-D 1 " mg L −1 . Hemmati et al. (2020) also reported similar results concerning the promotive effect of BAP and 2,4-D in callus production of different explants for S. tebesana. Hussein et al. (2014) showed the enhancing effects of BAP and 2,4-D on the callus induction in S. officinalis hypocotyl. The positive influence of simultaneous use of BAP and 2,4-D for callogenesis was also reported previously for several Salvia species (Lemraski et al. 2014;Modarres et al. 2018) and other plant species (Wang and Bao 2007;El-shafey et al. 2019). A remarkable point is a balance between auxin and cytokinin in the culture medium as a key regulator of in vitro callogenesis, leading to increased cell division as an important process for callus induction (Johri and Mitra 2001;Roy and Banerjee 2003).
The results of the present study showed that regardless of the type of elicitor used, the calli of both apical meristem and leaf explants on MS medium supplemented with the lower concentrations of auxin (2,4-D) and cytokinin (BAP) can produce more phenolic compounds when compared to the other combination ratios of 2,4-D and BAP. Similarly, the research of Hemmati et al. (2020) on S. tebesana showed that the low combinations of 2,4-D (0.5 mg L −1 ) and BAP Fig. 2 Antioxidant activity was assessed using DPPH (a) and FRAP (b) assays in leaf-derived callus. IC 50 represents the levels of biophenols required to scavenge 50% of DPPH radicals. All data are expressed as the mean ± SE. AA, Ascorbic acid (0.5 and 1 mg L −1 ) caused the maximum phenolic accumulation in calli obtained from shoot apical meristem and leaf explants, but Chaâbani et al. (2015) confirmed MS medium augmented with 2.0 mg L −1 2,4-D and 1.0 mg L −1 BAP seemed to be the most suitable medium for the highest production of total phenols in leaf-derived callus of Crataegus azarolus L. var. aronia in comparison to lower 2,4-D/BAP ratio. In agreement with our results, Maharik et al. (2009) and El-Baz et al. (2010) suggested that the addition of a lower level of auxin (NAA) to the culture medium than BAP yielded the most phenolic derivatives contents in in vitro cultures. The type and concentration of plant hormones in the culture medium influence the pathways involved producing secondary metabolites (Adie et al. 2007). The biosynthesis of most phenolics dominantly takes place through the shikimate/arogenate pathway producing three amino acids of tryptophan, tyrosine, and phenylalanine. These amino acids are also involved in the synthesis of some hormones, in particular auxins (Ruiz and Romero 2001). Phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) are the key enzymes of this pathway (Acamovic and Brooker 2005). Due to the confirmed effect of auxin (IAA and 2,4-D) on gene expression of corresponding enzymes, as well the activation or the inhibition of their activities, the variation in the biosynthesis rate of phenolic compounds is normally happening (Tanaka and Uritani 1979;Chaâbani et al. 2015).
Based on the obtained results, it can be concluded that both nano-TiO 2 and MeJa stimulate the production of polyphenols when compared to controls. In treatment with nanoparticles, the application of 10 mg L −1 nano-TiO 2 and 60 mg L −1 nano-TiO 2 led to the most increase of phenolic derivatives in calli derived from apical meristem and leaf, respectively; which were about 1.5 times higher than the amounts observed in other nano-TiO 2 treated samples. The promotive effect of nano-TiO 2 elicitor on phenolic compounds accumulation has also been recorded in seedling of S. officinalis (Ghorbanpour 2015), seedling of S. officinalis (Mazarie et al. 2019), cell culture of Aloe vera (Raei et al. 2014), and in vitro cultures of other species such as Papaver somniferum  and Lallemantia iberica (Sattari et al. 2020). It has been   suggested that the use of nanoparticles in plant cell culture is highly important to produce secondary metabolites (Asl et al. 2019). Due to the oxidative stress caused by different kinds of elicitor treatments in target cells (Aghdam et al. 2016;Mohammadi et al. 2014), non-bio-elicitors including nanoparticles stimulate the plants to increase immune responses and increase PAL gene expression, leading to an increase in secondary metabolites synthesis, especially phenolic compounds, which are a part of the systemic acquired resistance process (Rajendran et al. 1994;Feizi et al. 2012;Singh and Dwivedi. 2018). Studies have also shown that elicitors, including nano elicitors, have a positive effect on increasing the absorption of some nutrients, especially nitrates, leading to increased expression of genes involved in the synthesis of phenolic compounds (Bais et al. 2004;Lemraski et al. 2014;Baenas et al. 2014;Kavianifar et al. 2018;Rivero-Montejo et al. 2021). However, studies have shown that the effects of TiO 2 nanoparticles are highly concentration-dependent.
That is to say, if the concentration exceeded the particular threshold, the adverse and cytotoxic effects will appear in plants (Ghosh et al. 2010;Rafique et al. 2014;Mohammadi et al. 2016). Similar results regarding a decreased promotive effect of nano-TiO 2 at higher concentrations were reported by Raei et al. (2014), who observed that the treatment with 120 mg L −1 nano-TiO 2 reduced the amount of Aloin in the tissue culture of Aloe vera. Treatment with non-optional concentrations of nano elicitors probably can cause the overproduction amount of oxidative materials and disturbance in the cellular redox balance leading to many severe damages to plant cell structures, proteins, and DNA (Afaq et al. 1998;Raei et al. 2014; Rafique et al.

Fig. 6
Choromatogram of standard samples of (1) quercetin, (2) rutin, and (3) apigenin (a); Choromatogram of leaf-derived callus extract on MS medium supplemented with "BAP 0.5 + 2,4-D 1 " mg L −1 and 60 mg L −1 TiO 2 (b); LC/MS analysis of apigenin, quercetin and rutin content (μg g −1 DW) in treated and non-treated samples of api-cal meristem-derived callus on the medium containing "BAP 0.5 + 2,4-D 0.5 " mg L −1 (c, d, e); and leaf-derived callus on the medium containing "BAP 0.5 + 2,4-D 1 " mg L −1 (f, g, h). Each value represents mean ± SE 1 3 2014). The destructive effect on DNA and negative consequence on gene expression eventually lead to a decrease in the production of active substances (Lok et al. 2006;Raei et al. 2016). In this work, the pattern of phenolic derivatives accumulations in MeJa-elicited calli varied in an elicitor concentration-dependent manner. In a way that 50 μM MeJa had a dramatically stimulating effect on the synthesis of analyzed compounds, and with increasing the levels of MeJa to 200 μM, the concentration of all phenolics decreased by almost half. Remarkably, the MeJa at 50 μM was also more effective at enhancing the total flavonoids and its individuals' content in calli obtained from both explants in comparison with the best nano-TiO 2 treatments (10 and 60 mg L −1 ). This result is in agreement with the findings of Hashemyan et al. (2020) for Teucrium polium, with emphasis on the lower MeJa-elicitation (50 µM) for the highest contents of polyphenols. More promotive effects of the lower dose of exogenously applied MeJa on phenolic, flavonoid and/or phenolic acid contents were informed in shoot culture of S. virgata (11.2 ppm) (Dowom et al. 2017 (Ho et al. 2018). Induced production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been suggested as one of the potent signaling processes in the MeJa-treated elicitation leading to the overproduction of secondary metabolites, including polyphenols (Walker et al. 2002;Kim et al. 2005). Over-activation or activation of the phenylpropanoid pathway genes, PAL (Wang and Wu 2005;Sun et al. 2013), CHS (Suzuki et al. 2005;Sánchez-Sampedro et al. 2005), C4H (Wang et al. 2014;Yousefian et al. 2020), and 4CL (Yu et al. 2019;Yousefian et al. 2020) under conditions of MeJa treatment would be the reasons for these influence. Taking into account the results of many researches, the burst of oxidative stress and subsequent toxicity damage have been observed in plant samples exposed to a high level of MeJa (Scandalios 1993;Di-Qiu et al. 1999;Mir et al. 2019;Ho et al. 2020). So it is important to optimize the MeJa concentration for improving secondary metabolite synthesis in in vitro culture system (Ho et al. 2020).
However and according to our unpublished data on S. tebesana, total phenolic content and total flavonoid level in hydro-methanolic leaf and stem extracts of wild plant were 1923.1 ± 35.1 mg, 487.6 ± 21.9, 725.7 ± 22.1 and 356.2 ± 16.4 mg 100 g −1 DW, respectively that was found to be higher than the corresponding compounds in callus extracts. Similarly, the comparative studies of Mathew and Sankar (2012) showed a much significantly greater phenol content in the leaves of three Ocimum species than in callus culture. The higher content of some individual phenolics (rutin and caffeic acid) in the aerial part of Fabiana imbricata plant (the maxima between 0.99 to 3.35% and 0.22 to 1.15% w/w, respectively) than ones for callus samples has also been determined by Schmeda-Hirschmann et al. (2004).
Phenolic and flavonoid compounds can protect cells from oxidation by electrolyzing and purifying the ROS produced by the electron transfer system in optical synthesis (Shen et al. 2009). The significant positive influence of increasing phenolic derivatives contents on antioxidant activities, due to the elicitation of nanoparticles and methyl jasmonate (summarized in Fig. 3), shows that the callus of S. tebesana is a potent scavenger of free radicals. The present data were in accordance with the results of experiments on S. verticillata (Tepe et al. 2007), S. officinalis (Grzegorczyk and WysokIńska 2009;Ghorbanpour 2015;Vosoughi et al. 2018), S. miltiorrhiza (Dong et al. 2010), S. tebesana (Hemmati et al. 2020), S. nemorosa (Heydari et al. 2020), S. multicaulis (Salehi et al. 2018), Nepeta binaloudensis (Sagharyan et al. 2019), Graecoanatolica dinarica (Krstić-Milošević et al. 2017), Orthosiphon stamineus (Lim et al. 2013), indicating the relationship between the concentration of phenolic compounds and antioxidant properties. Meanwhile, some differences in the order of the samples between the DPPH and FRAP assays of "methanolic extracts" of both explantsderived callus may be ascribed to the different solvents used in each assay (Kouka et al. 2017).

Conclusion
Our results revealed that S. tebesana has good callogenesis potential, with the highest callogenesis rate and callus dry weight for explants on MS media supplemented with the combined combination of BAP (0.5 and 1 mg L −1 ) and 2,4-D (1 mg L −1 ). Also, the present study showed that elicitation with nano-TiO 2 and MeJa at low concentrations, significantly promote the production of predominant phenolic derivatives in S. tebesana calli. As well as results declared that the increasing of rosmarinic acid, quercetin, and rutin was more correlated with nano-TiO 2 treatment than MeJa elicitation. It can be concluded that the combination of in vitro culture and elicitation would be a good technique to successfully produce and enhance the content of pharmacologically valuable metabolites in S. tebesana.