Multidirectional insights into the phytochemical, biological, and multivariate analysis of extracts from the aerial part of Swertia perennis Linnaeus

Abstract Swertia perennis Linnaeus (SP) has been utilised to treat gastritis. We report the qualitative and quantitative phytochemical analysis, antioxidant and enzyme inhibitory activities of SP. The correlation between the biological activities and total bioactive contents of the extracts was also studied via multivariate analysis. Methanol extract contained many active compounds and exhibited good antioxidant activity. Therefore, this was selected for further phytochemical profiling and stability studies. Fourteen compounds were identified by ultra-performance liquid chromatography-electrospray ionisation-orbitrap-mass spectrometry for the first time from this plant. Iridoids, xanthones, and flavonoids were the main components. Methanol extract exhibited good stability and antioxidant capacity in stability studies, with low toxicity, and showed a protective effect on the oxidation of olive and sunflower oils. SP has the potential to be developed and used as an antioxidant, or as urease and XO inhibitors, and its methanol extract could be used as a natural oil stabiliser. Graphical Abstract


Introduction
Swertia perennis Linnaeus (SP) belongs to the genus Swertia and is a perennial herb.SP has been used as medicine to treat dyspepsia and gastritis (Yu et al. 2016).To date, gentiopicrin, thirteen xanthones, and four flavonoid C-glycosides (Hostettmann and Jacot-Guillarmod 1976;Hostettmann and Miura 1977) have been isolated from SP.Therefore, the purpose of this study was to investigate the chemical constituents and biological activities of SP extracts by multivariate analysis.These results will provide the basis for further research and development.

Results and discussion
Phytochemical studies of SP indicated the presence of 11 types of phytochemicals (Table S1, Figures S1-S41).Several reports indicate that these constituents act as antioxidants and enzyme inhibitors (Xu et al. 2021;Chen et al. 2022).Fourteen chromatographic peaks were identified from the UPLC-MS data of methanol extract (Table S2).The detailed compound identification is described in the supplementary material.Methanol extract contained seven iridoids, three xanthones, two flavonoids, one phenolic, and gentiobiose (Figure S42).The UPLC results are shown in Figure S43 and the MS and MS/MS results are shown in Figures S44-S75.
The yield of SP extraction in various solvents was investigated, the levels of eight active components were measured (Tables S3-S4): total phenolic content (TP he C), total flavonoid content (TFC), total carbohydrate content (TCC), total protein content (TP ro C), total triterpenoid content (TT ri C), total phenolic acid content (TPAC), condensed tannin content (CTC), and gallotannin content (GC).Methanol extract contained mainly iridoid glycosides, flavonoid glycosides, and phenolic glycosides (Table S2).This is in line with Swertia chirayita's findings (Singh and Chauhan 2012).Methanol extract was found to have the highest TP ro C and TP he C, due to the high levels of antioxidants in extract (Chen et al).Aqueous methanol is suitable solvent for extracting TFC from plants of the genus Swertia (Kshirsagar et al. 2020).If our findings are verified, then using methanol directly will lead to the isolation of more flavonoid compounds.
The results of the antioxidant activity are shown in Tables S5-S6, and Figures S76-S77.Methanol extract had a good ability to scavenge DPPH and ABTS, which could be attributed to its high mangiferin, swertianolin, isoorientin and isovitexin content (Singh and Chauhan 2012;Yang et al. 2015).Aqueous extract exhibited the highest metal chelating ability, this may be due to the fact that it contains xanthone glycosides.The results of the enzyme-inhibitory activity are shown in Table S7.All tested extracts showed effective urease inhibitory activity.This may explain why SP has been used to treat gastritis.Methanol extract showed good XO inhibitory activity because it contained the XO inhibitors mangiferin, isoorientin, and isovitexin (An et al. 2021).
Except for TT ri C, all four polar solvent extracts (water, methanol, ethanol, and acetone) showed high active ingredient content, which explains why they performed well in antioxidant activity assays.Despite the fact that four non-polar solvent extracts (ethyl acetate, ethyl ether, dichloromethane, and hexane) showed low active ingredient content, they were chosen because they made iridoids extraction easier.The iridoids have a good enzyme-inhibitory action (Kırmızıbekmez et al. 2021), and our results corroborate this.
The content of active ingredients in methanol extract was high, and the antioxidant activity was good.Therefore, it was selected for further stability studies.The results of the stability studies of methanol extract are shown in Figures S78-S80.Previous studies have shown the effect of acidic conditions on the hydrolysis of compounds (Chen et al. 2022), such as the cleavage of sugar moieties connected to phenolic compounds, which may explain why methanol extract had high TP he C values and high antioxidant activity at pH 1 and 3.The in vitro digestive stability results indicated that the high TP he C values under stomach conditions were because the acidic environment is conducive to the dissolution of phenolic components.
The results of the oxidative stability studies of oils are shown in Figures S81-S83.Combining the results, we found that adding a small dose of methanol extract of SP to extra virgin olive oil (EVOO) had a good antioxidant effect, and a greater effect was observed with EVOO-1000 than with EVOO-250.However, the opposite result was found for cold-pressed sunflower oil (CPSO).EVOO is rich in the monounsaturated fatty acid, oleic acid, which is stable and not easily oxidised.EVOO also contains antioxidants, such as hydroxytyrosol, which is a powerful antioxidant (Zang et al. 2020).CPSO is rich in the polyunsaturated fatty acid, linoleic acid, which is unstable and easily oxidised.
A cell viability assay showed that methanol extract is relatively safe (Table S8, Figure S84).Pearson's correlation between the total bioactive compounds and the biological activities is shown in Figure S85.Pearson's correlation between the biological activities is shown in Figure S86.
This study used a univariate statistical method, and there was a statistically significant difference between the samples; p < 0.05 were considered to be significant for all evaluated content determination and biological activities assessed separately.However, multiple comparison analysis using Least Significant Difference post hoc analysis revealed that some samples exhibited equal content and activities against some content determination and biological activities.Therefore, samples were classified on the basis of their content determination and biological activity similarities, and the experimental dataset was submitted to principal component analysis (PCA).The experimental results are shown in Figure S87.Three principal components (PCs) explaining 80.4% of the data variance were gained (Figure S87D).The contributions of content determination and biological activities to the three PCs are shown in Figure S87E.PC1 summarises 44.6% of the data variance and differentiates samples based on content determination and antioxidant capacity.The values for PC2 and PC3, representing enzyme inhibitory activity and metal chelating ability, were 26.7% and 9.1%, respectively.By analyzing the factorial maps (Figure S87A-C), it was observed that the extracts of methanol, ethanol, and acetone were distinct from the other extracts.Taking into account all active compounds and the biological activity results, methanol extract was determined as the primary study object.
Furthermore, the separation between other samples was poor, making it difficult to interpret the experimental results.Therefore, the PCA results were used for hierarchical clustering analysis.Analysis of the first three PCs from PCA revealed five clusters (Figure S88).The first cluster, ethyl acetate extract, displayed the highest superoxide radical, a-amylase, and BchE inhibition.The second cluster, composed of methanol, ethanol, and acetone extracts, displayed high TCC, TP he C, TFC, TPAC, CTC, and GC and high antioxidant activity and XO inhibition.The third cluster, aqueous extract, displayed the highest TP ro C value and the highest metal chelating activity and urease inhibition.The fourth cluster, ethyl ether extract, displayed the highest a-glucosidase, AchE, and tyrosinase inhibition.The fifth cluster, which contained dichloromethane and hexane extracts, displayed the highest TT ri C value and effectively prevented b-carotene bleaching.

Conclusion
The current study compares different solvent extracts from SP in terms of their bioactive compounds and biological activities.Methanol extract revealed excellent antioxidant activity with a high content of active ingredients.To provide more detailed information on chemical profile, UPLC-MS analysis of methanol extract was performed and iridoids, xanthones, and flavonoids were found to be the main components.Methanol extract exhibited good stability and antioxidant capacity in stability studies, with low toxicity, and also showed a protective effect on the oxidation of oils.Regarding inhibitory effects against key enzymes of clinical relevance, all tested extracts showed different activities against these enzymes.According to our findings,