In vitro elicitation and detection of apigenin, catalpol and gallic acid in hairy root culture of Plantago major L. and assessment of cytotoxicity and anti-bacterial activity of its methanolic extract

Abstract The aim of this study was to establish the hairy root (HR) culture of Plantago major to evaluate the accumulation of apigenin, catalpol and gallic acid after elicitation and investigate the biological activity of its methanolic extraction. The highest transformation frequency was obtained by Agrobacterium rhizogenes strain A4, 0.5 mg/L 6-Benzylaminopurine in pre-cultivation medium, 150 µM acetosyringone in co-cultivation medium (1/2 MS), and immersion method for inoculation of leaf explants. The production of apigenin, catalpol and gallic acid compounds were significantly affected by treatment of 1.18 mM AgNO3 at 24 h which yielded 4.30, 8.24 and 2.89-fold increase, respectively. The assessment of anti-bacterial activity showed that the methanolic extracts of the HRs elicited with 1.18 mM AgNO3 were significantly active against Proteus vulgaris (PTCC 1182) (MIC = 25 mg/mL and MBC = 25 mg/mL). Furthermore, the MTT assay revealed that the methanolic extracts of the HRs were cytotoxic on the SW-480 cell (IC50=337.56 ± 1.82 µg/mL). Graphical Abstract


Introduction
Plantago major, commonly known as great plantain, is an important medicinal plant that belongs to family of Plantaginaceae and grows wild as a native plant in Asia, Europe, and America. 275 Plantago species have been identified around the world (Wang et al. 2015). P. major contains a number of active compounds including terpenoids, alkaloids, iridoid glycosides, flavonoids, phenolic acid derivatives, polysaccharides, vitamins and fatty acids in its seeds, leaves, flowers and roots. This perennial medicinal plant possesses several biological properties such as anti-inflammatory, antiviral, anti-bacterial, anti-oxidant, anti-cancer and wound-healing activities (Zubair et al. 2016;Adom et al. 2017). P. major also showed a great potential for in vitro culture, in vitro shoot organogenesis and in vitro plant regeneration (Rahamooz-Haghighi et al. 2018). Hairy root (HRs) of Plantago lanceolata were studied in order to investigate two caffeic acid glycoside esters,Verbascoside and Plantamoside by Fons et al. 1999. In our previous study, HR induction in P. lanceolata was also optimized and then contents of secondary metabolites were affected by different elicitors (Rahamouz Haghighi et al. 2021). According to the previous studies, valuable medicinal compounds exist in P. major such as apigenin, catalpol and gallic acid. These compounds are known to possess, anti-inflammatory, anti-oxidant and cytotoxic properties (Morais et al. 2010;Elhassan et al. 2019;Salehi et al. 2019). To the best of our knowledge there is no report in application of elicitors for induction of secondary metabolites, analysis of compounds, and biological activity of P. major HRs .

Hairy root induction
At preparation of explants and after the infection by A. rhizogenes especially in injection method, the wound sites of the explants that were not cultured in the medium containing 6-Benzylaminopurine (BAP), released black secretions containing polyphenols which can affect the HR induction rate ( Figure S1A and B). Duncan's multiplerange test showed that the treatment of explants with BAP had a significant difference on the induction of HRs ( Figure S1C and D). The time of inoculation did not show a positive effect in HR induction rate, since the period of inoculation increased from 5 to 10 and 15 min, the root induction rate decreased in both types of explants ( Figure  S2). Prolonged exposure (15 min) resulted in necrosis of explants, especially in the injection method. HR induction of leaf, petiole and stem explants of P. major by A. rhizogenes carried out by immersion and injection methods ( Figures S3 and S4). Both methods were similar on HR induction of leaf explants except the transformation with ATCC15834 bacterial strain ( Figure S5A). The injection method was able to increase the induction of HR in stem explants ( Figure S5B). The addition of 150 mM acetosyringone to the inoculation medium was able to increase the frequency of transformation from 16% and 6% to 83.33% and 75% in leaf and stem explants by A4 bacterial strain (Table S1). Data analysis by Duncan test indicates that the induction of HRs in co-cultivation with 1 = 2 -Murashige and Skoog medium (MS) salt was more effective than full MS salt ( Figure S6). The successful transformation of abundant species takes place by applying 1 = 2 salts-strength MS as co-cultivation medium (Machado et al. 1997). Infected explants by A. rhizogenes transferred to media after co-cultivation in 1 = 2 MS or 1 = 2 -strength Gamborg's (B 5 ) basal medium. The results showed that the HRs obtained on the 1 = 2 B 5 medium had a higher growth rate ( Figure S7). The HRs on the MS media were more delicate. The calli formed on HRs after one month. It has been suggested that the standard B 5 medium is suitable for the optimal growth of the HRs (Nguyen et al. 1992). The inoculated explants by A. rhizogenes were transferred to 1 = 2 B 5 medium with different concentrations of sucrose for 10 days. HR growth was reduced in the media containing more and less than standard concentration (30 g/L) of sucrose ( Figure S8). In fact, high concentrations of sugar in the culture medium can lead to osmotic stress (Rao and Ravishankar 2002). In the optimum condition, HR induction was observed with different frequency using A4, A13, ATCC15834 and MSU440 bacterial strains, respectively ( Figure S9). PCR analysis confirmed the integration of the rolB gene into the genome of P. major HRs ( Figure S10). The sequence of the eluted PCR product was sequenced to confirm the presence of the rolB gene (Table S11) (Valimehr et al. 2014).

Elicitation of hairy roots
Line 1 was selected based on rapid growth compared to other lines ( Figure S12). The pH in all media was adjusted between 5.3 À 5.6. Dry mass of samples elicited by 1.18 mM silver nitrate (AgNO 3 ), 0.58 mM AgNO 3 , 100 mg/L chitosan, 5 mg/L yeast extract and 2.5 mg/L yeast extracts (147.85, 145.12, 142.57, 139.54 and 135.66 mg, respectively) were higher than non-elicited HRs samples after 24 h (128.189 mg).

HPLC
HPLC chromatograms of apigenin, catalpol and gallic acid standards are depicted in Figure S13. In the HRs treated by 100 mg/L chitosan, 50 mg/L chitosan and 2.5 mg/L yeast extract, the level of apigenin, catalpol and gallic acid was significantly increased after 24 h. In 48 h after the treatment of elicitors, the biomass and contents of these compounds in the HRs decreased which may be related to the metabolite's secretion into the culture medium. The results showed that the yields of apigenin, catalpol and gallic acid in HRs induced by 1.18 mM AgNO 3 was 4.30, 8.24 and 2.89-fold increase in comparison to the control sample after 24 h of treatment, respectively ( Figure S14A-C). . The content of apigenin, catalpol and gallic acid in the wild P. major roots (1.97, 9.57 and 1.44-fold increase, respectively) and aerial part (1.95, 5.49 and 3.12-fold increase, respectively) is more than non-elicited HRs (control) ( Figure S14). It is revealed that AgNO 3 elicits phytoalexin production and consequently increases the yield of tropane alkaloids in Datura metel HRs (Angelova et al. 2006).

Antibacterial potential of Plantago major
The striking result to emerge from the data is that the HR treated with 1.18 mM AgNO 3 has a significant effect against P. vulgaris (PTCC 1182) (20 ± 0.5 mm) and showed a significant difference with non-treated HRs extracts (P-value < 0.05 ( Figure  S15). On the other hand, low inhibitory effects of all extracts were found on E. coli (ATCC 10536). The in vitro anti-bacterial potential of all extracts was less than that of Kanamycin. The remarkable result to emerge from the data is that after 24 h, the growth of P. vulgaris (The most sensitive bacteria to plant extracts) with no treatment showed 8.39-fold increase, while the ratio of the viability of P. vulgaris treated with the extracts of HR elicited by 1.18 mM AgNO 3 , non-treated HRs, root and aerial parts were 0.42, 1.12, 0.86 and 0.56, respectively. Based on these results it can be presumed that the growth of bacteria is inhibited. These results indicate a statistically significant anti-bacterial activity associated with these extracts after 24 h ( Figure S16). MBC values for all bacteria are listed in Table S2. Holetz et al. showed that P. major has some degree of anti-bacterial activity (Holetz et al. 2002). Table S3 presents that the methanolic extracts of aerial, root and HR exhibited significant cytotoxic activity against SW-480 cancer cell in a dose-dependent manner. However, none of the methanolic extracts showed significant cytotoxic activity against HEK-293 normal cell. Moreover, the HR extracts of P. major also demonstrated the minimum cytotoxic effect on HEK-293 (IC 50 :6142.5 mg/mL). Researchers reported that P. major extract can also inhibit the carcinogenesis of breast (MCF-7), melanoma (UACC-62) (G alvez et al. 2003), human transformed cells HCT-15, SQC-UISO, OVCAR and KB (Velasco-Lezama et al. 2006).

Conclusions
In this study, we introduced a reliable protocol to establish P. major HRs. It investigated the effect of some elicitors on apigenin, catalpol and gallic acid content of HRs. Consequently, based on these findings it could be concluded that these extracts possess multi-biological activity including potential anti-bacterial and anti-cancer activities.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran (Grant number: A-12-848-35).