Phytochemical analysis, antioxidant and hypoglycemic activities of a methanol extract from Stachys brachyclada de Noé ex Coss. leaves

Abstract Stachys brachyclada de Noé ex Coss. (Lamiaceae) is a quite rare medicinal plant endemic to the Mediterranean basin. In this study, seven secondary metabolites from a methanol extract of its leaves have been isolated and identified by a combination of chromatographic and spectroscopic methods (1D and 2D NMR experiments and ESIMS analysis). They include one ethyl 4-hydroxybenzoate (1), three acylated flavone glycosides (2–4), one diapigenin derivative (5) and two flavone aglycones (6–7). Stachysetin (5) was found the major compound of the extract (74.0 mg/g of dry matter). Moreover, the produced extract showed the ability in inhibiting the α-glucosidase enzyme (IC50 = 13.7 µg/mL), in quenching the radical 1,1-diphenyl-2-picrylhydrazyl (EC50 = 74.6 µg/mL), and in reducing the intracellular oxidative stress level in Human Dermal Fibroblast (64% inhibition at 50 µg/mL). Graphical Abstract


Introduction
The genus Stachys L., Lamiaceae family (Lamioideae: Stachydeae), includes about 450 species of herbs and small shrubs worldwide, mostly distributed in the temperate region of the Southwestern Asia and Mediterranean basin (Venditti et al. 2014).Plants of this genus have been used for centuries in various traditional medicines to treat several ailments such as skin inflammations, genital tumors, rheumatic and stomach disorders, sclerosis of the spleen, and ulcers (Venditti, Frezza, Bianco, et al. 2017;Pritsas et al. 2021).The aerial parts of some of them, known as Mountain tea, are consumed in infusion or decoctions due to their sedative, antispasmodic, diuretic, and emmenagogue effects (Venditti, Frezza, Celona, et al. 2017;Tomou et al. 2020).Several Stachys herbs are also used as aromatic plants in foods and to improve the taste of yogurt and jelly (Šliumpaitė et al. 2013).Their edible tubers, rich in carbohydrates and micro-nutrients, are added to the bread or eaten raw (Venditti, Frezza, Bianco, et al. 2017).In the last decades, many biological effects have been reported for Stachys species that might justify the potential benefit on human health associated with their intake.They include antimicrobial, antitoxic, neuroprotective, hepatoprotective, anti-inflammatory and anticancer activities (Bahadori et al. 2019;Tomou et al. 2020).Moreover, the phytochemical investigation has highlighted the presence of different classes of bioactive metabolites including polyphenols, terpenes, phenolic acids, and essential oil that provide functional properties of the genus (Tundis et al. 2014;Bahadori et al. 2019).Considering the great consumption worldwide and the therapeutic value of the Stachys species, this study was focused on the chemical profile and the biological activities of a quite rare one, S. brachyclada de Noé ex Coss.(trivial name: Epiaire à rameaux courts) endemic in Algeria, south of France and Spain, and Sardinia (Italy).It is a small annual herb (10-40 cm) with weak and flexuous stems branched from the base.The leaves, opposite, ovate cordiform, and rounded at the base, are silky-hairy on the margin; the vortex inflorescence, with flowers white or pink, is closed in the apical part.
According to the ethnobotanical investigation, S. brachyclada leaves are used in herbal preparation for the treatment of cutaneous inflammations and infections, and infusion for gastrointestinal disorders.Moreover, the essential oil from the aerial parts is applied to relieve muscle pain or myalgia.However, its medical application and secondary metabolites have not been so far documented.Thus, a methanol extract from S. brachyclada leaves (SB) was produced for chemical investigation using chromatographic and spectroscopic methods.The antioxidant and hypoglycemic activities of SB and its major components were evaluated too.
The ability of dietary polyphenols to improve human well-being reducing the risk of chronic diseases such as diabetes, cardiovascular disease, and neurological impairment are extensively investigated (Del Bo' et al. 2019;Kerbab et al. 2019, Salehi et al. 2019).Based on the polyphenols composition of SB extract, the α-glucosidase inhibition, the free-radical scavenging activity against the DPPH • radical, and the invitro antioxidant capacity in a cell system were assayed.
SB extract inhibited the α-glucosidase enzyme with a potency about 2-fold less than acarbose, an antidiabetic drug used as a positive control (IC 50 = 13.7 and 7.5 µg/ mL, respectively).In the same assay, all isolated compounds inhibited the carbohydrate enzyme with a potency lower than that of the parental extract (Figure S2a) suggesting the synergic action of all components.The α-glucosidase is a hydrolase enzyme involved in the digestion of dietary complex carbohydrate and its inhibition retard the absorption of glucose in the intestinal lumen and decrease the blood glucose level.The control of postprandial hyperglycemia is a strategy used to treat diabetes mellitus type 2 and its complications (Alaribe et al. 2021).The DPPH assay exhibited a significant and dose-dependent antioxidant activity for SB (EC 50 = 74.6 µg/ mL) which should be correlated with its high Total Polyphenols Content (180.0 ± 4.2 mg A /g of dried extract).Among the tested compounds, 6 and 2, possessed a strong ability in quenching DPPH • radical (EC 50 = 18.8 and 20.2 µM, respectively) while the other pure compunds (1, 3-5 and 7) had EC 50 values ranging from 74.5 to 400.1 µM (Figure S2b).In fact, the adjacent hydroxyl groups in the aromatic ring were important for high free scavenging activity (compounds 2 and 6), their methylation (compound 3) or absence (compounds 1, 4-5 and 7) reduced drastically the activity.In agreement with already reported studies, the number and position of hydroxyl groups in the polyphenols structures play a pivotal role in antioxidant activity (Sarian et al. 2017).Finally, the effect of SB and stachysetin (5), its main compound, against intracellular oxidative stress was evaluated (at not cytotoxic concentration, see supplementary section 3.6.2) by the hydrogen peroxide (H 2 O 2 )-induced ROS (reactive oxygen species) release in Human Dermal Fibroblasts (HDF) using the redox probe DCFH 2 (dihydro dichlorofluorescein) (Armentano et al. 2015).Figure S4 displayed that SB reduced the ROS levels in the H 2 O 2 -treated cell line from 50 µg/mL (64% inhibition, p < 0.001) while compound 5 was significantly active (p < 0.01) from 100 µg/mL (ROS inhibition= 31.1%).
The results were in line with other Stachys species (Tomou et al. 2020) such as Stachys cretica L. subsp.mersinaea that possessed strong antidiabetic activity against carbohydrate enzymes (Bahadori et al. 2019) or S. rizeensis R. Bhattacharjee and S. mialhesi de Noé that exerted strong antioxidant effects against different radicals (Tomou et al. 2020;Gerçek et al. 2022).Furthermore, this was the first study on the inhibition of α-glucosidase enzyme and the antioxidant ability of Stachysetin, a rather rare secondary metabolite.

Experimental
See Supplementary Material for the experimental section and figures related to this paper.

Conclusion
This article investigated a methanol extract from S. brachyclada, a not previously studied Stachys species.Seven secondary metabolites belonging to polyphenols classes have been isolated including a phenolic acid ester never reported in Stachys genus.The produced extract exhibited good antioxidant and antidiabetic activities ascribable to its polyphenolic components.The preliminary biological results support the traditional use of S. brachyclada and suggest that this latter might be an attractive ingredient for pharmaceutical and nutraceutical proposals.