HRCs system is one of the best functional criteria for the development of this crop and sustainable supply of plant materials with quality assurance to the market 37,40. Despite the economically important, promising features and advancements, the production of P. sidoides-derived pharmaceuticals by HRs has not yet been commercially exploited. In the present study, three elite HRLs of P. sidoides, were evaluated during 35 days of suspension culture for their antioxidant potential to support the modern claims of their medicinal uses. The HRs were tested for preliminary phytochemical screening, enzymatic-antioxidants resposes and antibacterial activities.
In this investigation, suspension culture treatment resulted in enhanced PAL activities, antioxidant enzyme accumulation, and stress levels (H2O2) in HRLs of P. sidoides derived by genetic transformation (Agrobacterium tumefaciens C58C1 (pRiA4)), particularly at the highest time of harvesting. PAL activity had an increased activity on the 21th day, stronger at (0.96 ± 0.019 µg CA/s/mg pro; HRL15) than at other harvesting times. This work showed that both the PAL activity and phenolics accumulations were enhanced dramatically during the final culture period treatment, indicating that PAL is the critical enzyme in regulating the phenolic acid accumulations in the P. sidoides HRCs. The CAT activity in treated HRLs reached the maximum on the 7th and 19th days, as well as SOD activity, reached to its peak on the 19th day. Similarly, Karatoprak et al. 41 displayed higher levels of SOD activity (5.50 U/ml and 5.67 U/ml) compared to the control roots (4.48 U/ml). In this regard, Shi et al. 42 showed that SOD and CAT activities in sorbitol-treated HRs of Salvia miltiorrhiza reached the maximum in the first days of treatment, and there was no correlation between tanshinone accumulation and enzyme activity. Chayjarung et al. 43 demonstrated that HRCs of peanut in an early stage of elicitor exposure (at 24 h), had significant increase in the antioxidant enzymes (CuZn-SOD, APX, and GPX).
This research also revealed the accumulation of PPO, GPX, and APX levels in three HRLs of P. sidoides, declined significantly with increasing exposure times. Whereas, different times of harvesting, especially at the 6th and 9th cycles cause particular enhanced H2O2 accumulation in HRLs by decreasing the activity of H2O2-metabolizing enzymes. A search of the literature did not reveal any information about the hydrogen peroxide scavenging capacity of HRLs of P. sidoides. According to previous results, Izuegbuna et al. 44 showed the scavenging ability of P. inquinans leaves was high and dose-dependent, which could be a result of high levels of phenolic compounds and flavonoids with high amounts of polyphenol, and antioxidant activities 45. The overall results showed that, HRL10 cultured on MS medium exhibited higher means of enzymatic activity (PAL, PPO and CAT contents) than the other HRLs. On the other hand, a comparison with the means of H2O2 activity showed that low concentration in HRL10 at the same tested times (35 days, Fig. 3). These changes could be attributed to its phenolic compounds which act as synthetic antioxidants to prevent lipid oxidation and decomposition of hydroperoxides in living tissues 46.
Pelargonium contains a rich spectrum of tannin, flavonoid, phenolic acids and coumarin derivatives 47,48, whose accumulation is influenced by the activities of enzymes related to their synthesis and oxidation. Strong correlations (r = 0.43**) between the PAL activity, and PPO, demonstrated on a similar physiological basis metabolism for these two enzymes as phenylperpanoids pathway. These results are in a good line with previous reports that showed a positive relationship between PAL and PPO enzymes activity in Calotropis procera HRs after addition of magnetic nanoparticles (MNPs) and salicylic acid (SA) 49. Positive and significant correlation between H2O2 with PPO (r = 0.32**), PAL (r = 0.25*), SOD (r = 0.28**) and CAT (r = 0.29**), indicating at high antioxidant capacity of these enzymes to scavenging peroxyl radicals in HRCs of P. sidoides. The lack of correlation between the antioxidant enzymes (APX, and GPX) with PAL, as a marker of phenolics metabolism, suggests that the determination of these enzymes is not an indicator for evaluating the changes in phytochemical marker compounds of HRCs in P. sidoides.
Here, the HRs and control samples (untransformed root and shoot plants) express ten compounds, including distinct phenolic acids (GA, CGA, CAF, and TA), hydroxycinnamic acid derivatives (FA, and p-COA), coumarins (Um), and flavonoids (QE, LU, and RH) variations. An analytical HPLC method for the detection of TA, CGA, LU, and FA in ethanolic HR extracts has not been published previously.
The phytochemical screening of HRCs of P. sidoides resulted in an increased production of Um, CGA, RH, TA, FA, LU, and p-CoA contents in the HRs extracts compared with untransformed roots, control shoots and roots. Results obtained revealed that, the content of detected phenolic acids has been different in variants harvest times with different sources of HRs. Generally, all the HRCs of the leaves sources have been shown high production of phenolic acids compared to the control. The alterations occurring in SMs production of transformed plants may be due to the content of polyamine metabolism, irritation to auxins, agropine synthesis (ags) and co-expression of (rol) genes, rolA, rolB, rolC, and rolD 50. As previously reported, molecular analysis of these RLs confirmed the presence of TL and TR T-DNA parts, represented by rolB, rolC, aux and ags respectively 26. This suggests that biosynthetic capacity for accumulation of different classes of SMs can be due to the rolC gene-induced from the A. rhizogenes plasmid 51. But the real mechanism of advancement is still not well-founded and needs more systematic studies of expression of critical enzymes involved in superior metabolic pathways.
Recently, Panara et al. 12 identified chemical characterization of P. sidoides root originated from several classes via UPLC-QToF-MS. Studies have shown that the concentration of Um of field-selected plants is regulated by the annual rainfall of the collection sites 52. Due to difficulties in synthesizing of Um, there is a need to identify alternative methods for the production of the compound. Thus, in vitro cultures of P. sidoides have been studied as viable sources of Um. The analysis of HRs extracts from the three HRLs revealed that increasing root Um concentrations were strongly related to both increasing exposure times of culture, especially in the 3th week of growing in the suspension culture. Furthermore, the HRs from the 3th week of culture is potentially the most economical for harvesting owing to their high Um yield. Additionally, average HRs Um concentration varied significantly different from the Um concentrations of shoot and root plant samples of the tissue culture. This suggests that it may be possible to use HRCs of the P. sidoides to identify best lines with high root Um concentrations and hence bioactivity. This would be valuable for sourcing the best biotechnological propagation material for cultivation studies and commercial operations. The suspension culture application of the P. sidoides has been demonstrated to increase coumarin production in HRCs previously by (Yousefian et al. 26), showing that eliciting treatments can be used to manipulate plant bioactive contents. However, in this study, applications of HR suspension culture had a significantly effect in the content of the root coumarins in particular Um. According to Colling et al. 37, the concentrations of main phytochemical compounds such as (QE, GA, CAF, and p-COA) in transgenic roots of P. sidoides were found absent or present at deficient levels compared to our results. Our results confirmed various earlier studies, which expounded that the HRs declared higher polyphenolic compounds, e.g., in Hypericum perforatum 53, and Ligularia fischer 54. The authors showed that both the level of CGA, 3-feruloylquinic acid, and the total amount of identified phenolic acids in the transformed shoots were 1.3 times higher than in control shoots of Hypericum perforatum 55. Furthermore, in the other species, such as Bitter melon (Momordica charantia L.), (Cucurbitaceae) the concentration of CGA, GA, FA, CAF, p-COA, and t-cinnamic acid was significantly increased in jasmonic acid (JA) and salicylic acid (SA) elicited HRs in compared to non-elicited HRs 56.
Although the mechanism of HRC on Um content needs to be further clarified. These results suggested that the metabolic response of P. sidoides HRs to different harvest intervals may be associated with allocation of the carbon flow between the various conflicting pathways 57.
The accumulation of various compounds in HRs of P. sidoides is influenced by the activation of enzymes related to their synthesis and oxidation 39,40. Correspondingly, Hayashi et al. 58 showed that in cultured cells of Glycyrrhiza glabra regulation of various SMs are at least partially due to activation of key enzymes, which catalyze their biosynthesis. Similar results have also been reported in the establishment and maintenance of long-term cell cultures of Thymus persicus, which the highest and the lowest content of terpenoids, were obtained during different times of culture 59.
In this study, the HRCs exhibit higher antibacterial activities. These results support the in vitro cultivation of HRs to supply as a natural product to prevent inflammation, oxidative stress and microbial related disorders, similar to the previous reports 2,14,60. Antimycobacterial and immunomodulatory efficacy of P. sidoides could be attributed to the presence of highly oxygenated coumarins (7-hydroxy-5, 6-di-methoxycoumarin; 6,8-dihydroxy-5,7-dimethoxycoumarin) gallic acid-derivatives, flavonoids, phenolic and hydroxycinnamic acid-derivatives were tested for this particular biological activity 2,9. Here we confirmed the presence of some of these compounds in the transgenic lines. Bacterial inhibition by this HR clone extract is likely in most of the HR clones analyzed by Colling et al. 37 which exhibited moderate to high activity against the two Gram-positive (Bacillus subtilis and S. aureus) and two Gram-negative bacteria (E. coli and Klebsiella pneumoniae). Similarly, HRs of Brassica rapa ssp. rapa 61, Momordica charantia 62, Fagopyrum tataricum Gaertn 63 and Raphanus raphanistrum 64 have also been contained high amounts of phenolic and flavonoid compounds which exhibited high antioxidant activities.
In the present study, there are differences in the antimicrobial effects of various extracts of HRLs and control samples in P. sidoides. Similarly, the study of Mativandlela et al. 14,16, reported higher antimicrobial and especially antitubercular activities of P. sidoides root extracts against some human and plant pathogens, with ethanol and acetone root extracts rather than chloroform extracts. Lewu et al. 65 reported that there was a broad-spectrum inhibitory effect of acetone and methanol extracts of the shoot and root of the P. sidoides herb on the Gram-negative and Gram-positive bacterial and fungal species. The Pelargonium methanolic extracts of HRs showed a more selective antibacterial action against the tested Gram-positive micro-organisms S. aureus and Gram-negative micro-organisms E. coli. The HRLs displayed reduced antimicrobial activity compared to the methanolic control extracts. The antibacterial activity of HRL4 against S. aureus and E. coli suggested that HRL4 may delay (lower) microbial contamination. P. sidoides extract, with its toxicological properties, could be employed as an indigenous medical system for the successful phytopharmaceutical. It also has the potential as a treatment for acute bronchitis and symptoms of the common cold 66.
Furthermore, flavonoid derivatives may contribute to the anti-oxidant activity of the Pelargonium extracts. Therefore, in vitro-regulation of phenolic compounds biosynthesis in P. sidoides HR may offer additional advantages of quantitative and qualitative improvements of these medicinally important metabolites.