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 rst attempted to achieve an ecient procedure for optimizing callogenesis from apical meristem and leaf explants of S. tebesana on MS media containing different concentrations of BAP alone and in combination with 2,4-D. Then, the inducing effect of nano-TiO 2 (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 avonoids as well as antioxidant capacity of callus extracts. 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 "BAP 1 + 2,4-D 1 " mg L − 1 and from leaf explant on the medium supplemented with "BAP 0.5 + 2,4-D 1 " mg L − 1 . The elicitation with 10 and 60 mg L − 1 nano-TiO 2 (respectively for apical meristem and leaf), and 50µM MeJa could signicantly promote the production of predominant phenolic derivatives in S. tebesana calli, where the highest content of total phenolics, O-diphenols, phenolic acid, avonoid, avone and avonol, proanthocyanidin was recorded. Additionally, in increasing the amount of rosmarinic acid of callus, nano-TiO 2 treatment was more effective than the elicitation with MeJa. Also, the highest content of Apigenin (0.33 ± 0.02 µg g − 1 DW) was detected after MeJa-elicitation (50µM), while the maximum level of Quercetin (2.61 ± 0.09 µg g − 1 DW) and Rutin (13.79 ± 08 µg g − 1 DW) were obtained after exposure to 60 mg L − 1 nano-TiO 2 , both from leaf-derived calli. While a signicant 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 (r 2 = -0.921 and r 2 = -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. Multiple Range Test. BAP: 6-benzylaminopurine, 2,4-D: 2,4-dichlorophenoxyacetic acid. results of the present study showed that regardless of the type of elicitor used, the calluses 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 compared with other combination ratios of 2,4-D and BAP. Similarly, the research of Hemmati et al. (2020) on Salvia tebesana showed that the low combinations of 2,4-D (0.5 mg L -1 ) and BAP and 1 mg L -1 phenolic callus obtained from shoot apical meristem and leaf explants, but Chaâbani et al. MS medium L 2,4-D L BAP suitable medium (1 mg L − 1 ). Also, the present study showed that elicitation with nano-TiO 2 and MeJa at low concentrations, signicantly promote the production of predominant phenolic derivatives in S. tebesana calli. Additionally, in increasing of rosmarinic acid, Quercetin, and Rutin, nano-TiO 2 treatment was more effective than MeJa elicitation. Our results showed that the combination of in vitro culture and


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 identi ed 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, avonoids, and terpenoids (Russo et al. 2013). Studies have also shown that phenolic compounds can exhibit a broad range of physiologicaltherapeutic properties, such as anti-in ammatory, anti-allergic, antioxidant, antimicrobial, anti-proliferative activities, anti-thrombotic, and cardiovascular protective effects. Because of high antioxidant activities, phenolic compounds function as the main bioactive compounds to determine the antioxidant power in foods (Ulubelen and Topcu, 1992 This compound has also been reported with antibacterial, antiviral, antioxidant, and anti-in ammatory 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 e ciency for a short time (Ourmazdi and Chalabian 2006). Callus induction from plant tissues is in uenced 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 for boost the production of a diversity of metabolites have been con rmed (Zhao et al. 2005). Recent studies have shown that these inductions are 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 ( . 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 o cinalis (Grzegorczyk and Wysokińska 2009), Salvia miltiorrhiza (Ge and Wu 2005) and Exacum a ne Balf. f. ex (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 cell and tissue culture or the effect of elicitors on secondary metabolites of S. tebesana. This study was done for the rst time (a) to create an e cient system to optimize the culture condition and hormonal composition for the large-scale production of callus, (b) to nd 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 RA and some individual avonoids (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. For sterilization of explants, the samples rinsed 3 times with sterile distilled water, followed by immersing in 70% (v/v) methanol (for 30 sec and once).
Leaf explants were also plunged in 5% (v/v) sodium hypochlorite (3 min and once). The nal wash of both explants was done 3 times with sterile distilled water.

Culture conditions 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's study were nominated to evaluate the best conditions (Hemmati et al. 2020). 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 ve 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 7th 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: Rate of callogenesis = number of explants with callus / total number of explants used × 100 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.

Preparation of elicitors
Two elicitors, including nano-TiO 2 (10, 60, and 120 mg L -1 ) and MeJa (50, 100, and 200 μM), were used. The stock of nano-TiO 2 (stock #: US3490, US Research Nanomaterials, Inc.) was prepared by dissolving the speci ed amounts (1.2 mg) of nano-TiO 2 powder in 10 mL of sterile distilled water and dispersed using ultrasonic vibration by providing 40 kHz wave signals at the temperature of 40 °C for 25 min (Ghorbanpour 2015). To prepare MeJa stock, a certain amount of it was dissolved in 96% ethanol (Wang et al. 2015). Both elicitors were ltered and sterilized using 0.22 μm lters.

Elicitation
The culture of selected media was performed in a like manner to the rst experiment till the 7th week. At the end of the 7th week and during the subculturing, elicitor solutions were added to basal culture media. The calluses were harvested after 10 days of elicitors' treatment, and the callus biomass (FW and DW) was estimated.

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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 2600s, Japan) for 30 min at a temperature of 30 °C and a frequency of 40 kHz. Then the extracts were ltered with Whatman paper (No. 1), and the solvent was evaporated under air condition. To prepare the nal extract, the extract 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 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 the callus sample.

Determination of total 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 Odiphenols 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 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 avonoids 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 (Zhishen et al. 1999). Catechin (CAT) equivalent 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 avone and avonol 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 nal 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 avone and avonol in the extract was calculated and expressed as mg QE per 100 g DW of callus sample.

Determination of total 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 sec, 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 de ned 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 nal 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) from the standard curve of RA, the amount of rosmarinic acid was calculated as mg of RA per 100 g of DW of callus sample.
Antioxidant activity DPPH scavenging activity assay 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 coe cient analysis was used to assess the associations between different parameters. The graphs were drawn by EXCEL software (O ce 2016).

Callus induction of apical meristem and leaf explants
The results showed that internal hormones signi cantly affected the callus induction at p ≤ 0.01 (Duncan's multiple range test). The individually adding of BAP and 2,4-D are shown that they were not successful for callogenesis. Results obtained for 2,4-D 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 signi cantly 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 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 (Fig. 1).
In terms of appearance, all the calli were also observed in friable form with yellow, light, or dark green colors.  Concerning the content of phenolic acids and proanthocyanidins, the result showed that 10 mg L − 1 nano-TiO 2 and 50 µM MeJa treatments on calli generated on "BAP 0.5 + 2,4-D 0.5 " mg L − 1 caused the highest content of these compounds (Table 2).

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 these compounds was observed in media with MS salts and "BAP 0.5 + 2,4-D 1 " mg L − 1 hormones. Total phenolic compound in calluses 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 with "BAP 0.5 + 2,4-D 1 " mg L − 1 hormonal treatment, which was about 3 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 callus, respectively.
However, almost double the content of avonoids than the control was observed when the lowest concentration of MeJa treatment, 50 µM, was applied to the medium with "BAP 0.5 + 2,4-D 1 " mg L − 1 . For all media, effective concentrations of treatments to increase the amounts of avone and avonol in calli were 60 mg L − 1 nano-TiO 2 and 50 µM MeJa (Table 3). 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 results of Pearson's correlation showed a signi cant and positive correlation (P ≤ 0.01) between estimated polyphenol derivatives and assayed antioxidant capacities. A very strong correlation was recorded between the content of rosmarinic acid of apical meristemderived calli and DPPH and FRAP values (r 2 = -0.921 and r 2 = -0.913, P < 0.01 respectively). While there were highly statistically signi cant correlations between total phenolic content of all extracts and two radicals scavenging assays, correlations of these assays with phenolic acids, avonoids, avones, and proanthocyanidins were somewhat weaker (Fig. 4). The weakest correlation occurred between the O-diphenols content of leaf-derived calli and DPPH scavenging activity and FRAP assay (r² = 0.501 and r² = 0.491, respectively).

Discussion
The combination of plant hormones in the medium has been reported necessary to stimulate a high frequency of callus induction

Conclusion
In conclusion, it was successfully determined 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, signi cantly promote the production of predominant phenolic derivatives in S. tebesana calli. Additionally, in increasing of rosmarinic acid, Quercetin, and Rutin, nano-TiO 2 treatment was more effective than MeJa elicitation. 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.

Declarations
The authors declare that they have no relevant con ict of interest.

Figure 2
Antioxidant activity was assessed using DPPH (a) and FRAP (b) assays in apical meristem-derived callus. IC50 represents the levels of biophenols required to scavenge 50% of DPPH radicals. All data are expressed as the mean ± SE. AA, Ascorbic acid.

Figure 3
Antioxidant activity was assessed using DPPH (a) and FRAP (b) assays in leaf-derived callus. IC50 represents the levels of biophenols required to scavenge 50% of DPPH radicals. All data are expressed as the mean ± SE. AA, Ascorbic acid.