Previous documents show that increased photosynthetic capacity due to Mo supply contributes to the accumulation of carbohydrates, such as fructose and glucose.We observed that lipids, fibers, and saponins were significantly increased in CA1. Flavonoids were increased reasonably in CA3.Different impacts of Mo were observed in different species/cultivars, which might be due to different genetic backgrounds and the variable uptake of Mo.Phenolics play essential roles in plant development; these aromatic benzene ring compounds with one or more hydroxyl groups are produced by plants mainly for protection against stress .
In our study, the Mo priming of Canavalia seeds significantly increased the phenolic content of CA3 sprouts; a non-significant increase was found in CA2 and, surprisingly, a slight decrease was noticed in CA1. This might be due to differences in endogenous molecular pathways of different species. This link to the inherent levels of mechanisms may also lead to differences in the enhancing patterns of the individual phenolic compounds after Mo treatment, which we observed in our study. A similar trend was observed in DAHPS, the first enzyme of the shikimate pathway, which converts PEP and E4-P into 3-dehydroquaianate. Further, to assess the impact of Mo seed priming and enhanced phenolic production on the pharmacological properties of plants, we performed antioxidant and antidiabetic assays. FRAP, ABTS, and DPPH assays were used to evaluate the antioxidant capacity of species samples spectrophotometrically.
An increase in antioxidant potential was observed after Mo treatment in all species. In the FRAP assay, the increase was significant in both CA1 and CA3. CA3 also expressed a significant changein antioxidant activity, measured via ABTS assay. This correlates to the above findings that more impacts of Mo on synthetic pathways were observed in CA3. Antioxidant activity has recently become a target for product development in the pharmaceutical and cosmetics industries .Canavalia species are of medicinal importance due to their potential antioxidant properties. In a study, Canavalia gladiata extract, at the concentration of 2 mg/mL, showed an antioxidant effect comparable to that of ascorbic acid of the control group . Plant growth and antioxidant capacity are greatly dependent upon N availability. Higher N improves the stress tolerance of plants via enhancement of the antioxidant ability and inhibition of lipid peroxidation. Mo primarily improves the nitrogen fixation to the plant and increases its antioxidant potential, which we observed in our study. This also might be the reason for the enhanced antidiabetic potential of Canavalia species assessed by GI, α-amylase inhibition activity, and α-glucosidase inhibition activity.
Terpenoids and flavonoids of Canavalia gladiata are reported to play a role in lowering glucose levels and possessing antioxidant potential.The Mo-mediated enhanced concentrations of terpenoids and flavonoids might have led to the increased antioxidant and antidiabetic activities of Canavalia sprouts in our study. For sample preparation, we used an ETA 0067 grinder with grinding stones, VIPO mini grinder, followed by homogenization by Vibrom S2 , and a cryogenic grinder accompanied by liquid nitrogen. Supercritical fluid extraction using SE-1 extractor and a steam distillatory apparatus according to CSN 58 0110 and CSN 6571 were successively applied for extraction and subsequent determination of the total content of Canavalia oils. Approximately 500 mg of each sample was transferred into an extraction column for SFE extraction. The extraction was performed at 40 MPa for 60 min, and extractor and restrictor temperatures were 80 and 120 ◦C, respectively. The extract was further trapped into a hexane layer inside a trapping vessel.The determination of phenolics and their precursor metabolites was carried out using an ultra-performance liquid chromatography system coupled with a quadrupole mass spectrometer provided with an ESI source according to Wang et al.’s method.
Phenolic and flavonoid levels were identified by comparing the standard mixture of different phenols and flavonoids to the relative retention time. The concentration of each compound was calculated using the peak area of the corresponding standard. In addition, deoxy-d-arabino 201 heptulosonate-7-phosphate synthase activity was analyzed according to Wang et al., . This enzyme catalyzes the reactions in cinnamic and shikimic acid pathways that are involved in phenylpropanoid biogenesis and therefore in the biosynthesis of coumarins. Samples were first homogenized in 3 mL 50 mM Tris-HCL buffer . The assay mixture contained 0.1 mM erythrose-4-phosphate, 0.2 mM phosphoenolpyruvate, and 0.1 mM MnSO4/0.1 mM CoCl2. In total, the reaction was initiated by enzyme addition and terminated by 25% trichloroacetic acid addition.