Two new glycosides from Stachys geobombycis C.Y.Wu

Abstract Two new compounds geobomlin A (1) and geobomlin B (2) were isolated from the roots of Stachys geobombycis C. Y. Wu. Structural determinations were established principally by two-dimensional NMR and MS data analyses. Geobomlin B showed moderate inhibitory activity against α-glucosidase with IC50 = 248.77 μM. We have also determined the mechanism by which geobomlin B elicit its inhibitory effect on α-glucosidase, for which we have established a competitive inhibition mode. Docking studies confirmed our results on geobomlin B α-glucosidase inhibitory properties. Graphical Abstract


Introduction
Stachys geobombycis C. Y. Wu (¼ S. geobombycis var.geobombycis), belonging to the family Lamiaceae, which is widely distributed in southern China (National Compilation group of Chinese Herbal Medicine 1996; Zhang et al. 2008).The roots of this plant have long been used in folk medicine to treat phthisis, cough, sucking blood, continuous sweating, asthma, weak, blood deficiency, infantile malnutrition, scald and so on (Zhou et al. 2019).The extracts of this plant were reported to exhibit anti-oxidant and anti-tumor activity (Zhang et al. 2004;Jin et al. 2010).Previous chemical studies on the plants of S. geobombycis have led to the isolation and identification of some compounds, such as terpene, flavone and volatile oil (Tang et al. 2002;Jin 2007).And a derivative of syringic acid and an iridoid glycoside were found form this plant in our previous phytochemical research (Zhou et al. 2019).In order to find more interesting bioactive compounds from this plant, the chemical compositions were restudied by us.As a result, two new glycosides were isolated from the plant for this time.In addition, since we have been engaged in the screening of hypoglycemic active compounds, and some glycosides have been found to have a-glucosidase inhibitory (Yan et al. 2021;Zhou et al. 2021), this time we also evaluated the inhibitory activity of a-glucosidase of these two glycosides.Herein, we report the isolation, structure elucidation and biological activities of these compounds, geobomlin A (1) and geobomlin B (2), respectively.
The compounds 1 and 2 were evaluated for their against a-glucosidase inhibitory activity.Acarbose is an effective a-glucosidase inhibitor, and it was used as the positive control in this study.The results showed that compound 2 displayed moderate inhibitory activity against a-glucosidase with IC 50 value at 248.77 lM (Supplementary material, Table S1).In addition, According to Figure S2, the inhibition of compound 2 on the enzyme showed a competitive inhibition (Ali et al. 2021).
To gain insights into the inhibition mechanisms of compound 2 towards a-glucosidase, docking simulation was performed by the AutoDock Vina suite.Docking simulations indicated that multiple hydrogen bonding interactions were observed between the hydroxy groups of acarbose and polar residues such as Asp-307, Asp-242, His-280, Tyr-158 and Thr-310 (Figure S22A) which fits well with the competitive model of acarbose.Interestingly, compound 2 did not detect complete occupation the position for substrate binding of the a-glucosidase.Instead, clear hydrogen bond networks were found in residues Arg-315, Gln-279, His-280, Ser-241 and Ser-441 (Figure S22B).Furthermore, calculation of the affinity of the above inhibitors showed acarbose with a binding energy of À9.0 kcal/mol and compound 2 with À8.6 kcal/mol, which showed that they can stably bind to a-glucosidase.

Plant material
The roots of S. geobombycis was collected in September 2019 from GuiGang, a city of the Guangxi Zhuang Autonomous Region, China, and was authenticated by associate professor Deqing Huang (Guilin Medical University, College of Pharmacy).A voucher specimen has been deposited in the Natural Products Chemistry Laboratory, College of Pharmacy, Guilin Medical University (Gulin, China), registration No. 201909001.

Extraction and isolation
The power-dried roots of S. geobombycis (25 kg) were heated for extractions with H 2 O three times, each time for two hours.The aqueous phase was applied to a D101 macroporous adsorbent resin column eluting with H 2 O, 50% EtOH and 95% EtOH successively.The 50% EtOH exaction (672.0 g) was applied to silica gel (100-200 mesh) column chromatography, eluting with a CHCl 3 -MeOH gradient system (v/v, 9:1, 8:2, 2:1, 1:1, 0:1), to yiled five fractions (Fr.A $ Fr.E) based on the TLC analysis.The Fraction D (369.0 g) was chromatographed on a MCI column, eluting with a MeOH-H 2 O system 0% $ 100%, yielded fraction D1 $ D26 based on the TLC analysis.The Fr.D7 was separated by Agilent LC1260 HPLC (flow rate 3.0 mL/min, 17% MeOH-H 2 O) to afford compound 1 (16.8 mg).Then the S. geobombycis extracted by pure water are dried in the sun.The dried roots were heated for reflux extractions with 95% EtOH three times, each time for two hours.The extract was concentrated in vacuo to afford a brown residue.Then it was suspended in H 2 O, and partitioned with EtOAc and n-BuOH, successively.The n-BuOH extraction (39.0 g) was subjected to a Sephadex LH-20 column and eluted with 100% MeOH to afford fifteen subfractions Fr.A 0 $ Fr.O 0 .The Fr.E 0 was separated by Agilent LC1260 HPLC (flow rate 3.0 mL/min, 5% MeOH-H 2 O) to afford compound 2 (13.5 mg).

Bioactivity assay
The assay was performed as reported method (Tao et al. 2013;S ¸€ ohreto glu et al. 2017) with the positive control of acarbose.The compounds were prepared into 1% DMSO reserve solution, and the compounds and acarbose were diluted to different concentrations in a buffer solution (phosphate buffer, pH ¼ 6.8) for experiments.Then, added 50 mL of the compounds (1, 15, 75, 150, 300, and 700 lM) to the 96-well plate.The concentration setting of acarbose was the same as the compounds.Then, incubated at 37 C for 15 min, added 5 mM 4-PNPG solution 50 mL, and interacted at 37 C.The absorbance value was measured at 405 nm.a-glucosidase inhibition (%) ¼ [(A control -A sample )/A control ] Â 100%.Where A control is the activity of enzyme without compound/positive and A sample is the activity of enzyme with compound/positive at different concentrations.The inhibitory mechanism assay was applied with varying the concentration of the enzyme in the reaction mixture, and the inhibition type was then assayed by the Lineweaver-Burk plot.

Molecular docking
Molecular docking is a common method used to study the interaction between ligand and protein.The crystal structure of a-glycosidase (PDB ID: 3A4A) was downloaded from the RCSB Protein Data Bank.Blind docking was carried out on Autodack program.Before docking, all water molecules were removed, and hydrogen atoms were added to generate the PDBQT file.The ligands structure were transformed into MOL2 file by Chem3D, followed by energy minimization.With regard to the docking result, the docking model with the lowest binding energy was selected as the best result.The docking results were analyzed using Pymol software.

Conclusion
In this study, we reported the isolation, structure elucidation and against a-glucosidase inhibitory activity of two new compounds, named geobomlin A (1) and geobomlin B (2). Com-paring with the positive control acarbose, we found that the compound 2 possessed moderate a-glucosidase inhibitory activity with an IC 50 value of 248.77 lM.This compound inhibited the enzyme in a competitive manner.In addition, molecular docking results also showed that compound 2 could inhibit the activitly of a-glucosidase.Therefore, the studies of the new compounds found in this work can provide experimental basis for the development and utilization of this plant.