Compounds from Rehmannia glutinosa and the activity to suppress α-glucosidase

Abstract Rehmannia glutinosa was extensively used to control blood sugar in diabetes treatment in tradition Chinese medicine. In the present study, three new compounds, including an iridoid rehmannia A (1) and two ionone rehmannias B-C (7-8), together with fourteen known compounds (2-6 and 9-17), were isolated from the roots of R. glutinosa. The structures of these compounds were determined by physicochemical constants and spectral analysis (1D, 2D-NMR and MS). The effect of 1-17 on α-glucosidase activity was tested in vitro. Compounds 9, 10, and 11 (IC50: 5.0, 3.1, and 6.3 mM) showed moderate activity to suppress α-glucosidase relative to acarbose (IC50 = 3.0 mM). The findings provided some new insights to understand the hypoglycemic effect of R. glutinosa and the development towards the α-glucosidase inhibitor drugs. Graphical Abstract


Introduction
Rehmannia glutinosa Libosch, belonging to the plant of Scrophulariaceae family, is chiefly spread in Henan, Shanxi, Shaanxi, Inner Mongolia and Jiangsu in China.There are six species of Rehmannia recorded in Flora of China, including Rehmannia elata N. E. Brown, Rehmannia henryi N. E. Brown, Rehmannia piasezkii Maxim, Rehmannia chingii Li, Rehmannia solanifolia Tsoong et Chin and Rehmannia glutinosa Libosch (Guo and Xin, 2015).
The phytochemical research on Rehmannia plants revealed that R. glutinosa mainly contained iridoids, ionones, phenylethanoid glycosides, and sugars.Catalpol (Zhou et al. 2015), an iridoid glycoside, may be one of the main active ingredients for the hypoglycemic effect.Catalpol has a comprehensive effect on the metabolism of glucose by activating endogenous opioid pathway, inhibiting carbohydrate hydrolase activity, and improving insulin resistance.As a part of our research to identify anti-diabetes lead compounds, we have firstly conducted a phytochemical study on the roots of R. glutinosa.As a result, three undescribed compounds rehmannias A-C (1, 7, and 8) and fourteen known compounds (2-6 and 9-17) were isolated from the samples.Then, the effect of 1-17 on α-glucosidase activity was further screened.Therefore, we here reported the isolation, structural elucidation, and activity to suppress α-glucosidase of compounds 1-17 (Figure 1).
The 1D NMR data of 1 is very similar to those of rehmaglutin A (Kitagawa et al. 1991), except for the replacement of two sp 3 methylenes in rehmaglutin A by two sp 2 methines in 1.The above differentiation suggested that one pair of additional double bond was formed in 1 as compared with that in rehmaglutin A, which was assigned between C-3 and C-4 due to the HMBC correlations of H-3/C-1, H-3/C-5, H-4/C-3, H-4/C-5, and 1 H-1 H CoSY of H-3/H-4/H-5 (Figure S1).The relative configuration of 1 was determined by the analysis of RoESY spectrum and comparison of chemical shifts with rehmaglutin A. The signals at δ H 2.33 (H-5), 2.33 (H-9), δ C 32.3 (C-5), and 43.1 (C-9) in 1 were very close to those of in rehmaglutin A, suggesting that H-5 and H-9 are both α-oriented (Kitagawa et al. 1991).The RoESY correlations of H-1/H-9, H-6/H-5, H-7/H-5, and H-6/H-7 indicated that H-1, H-6, and H-7 were all in an α-orientation (Figure S2).Therefore, the structure of 1 was fully elucidated and named as rehmannia A.
The 1D NMR spectra of 7 were extremely similar to those of frehmaglutoside H (Feng et al. 2015), except the minor difference in chemical shifts of H-7 and H-8 and coupling constants of H-10.The chemical shifts of H-7 and H-8 in 7 were 6.29 and 6.70 ppm, respectively, which were shifted to 6.07 and 6.19 ppm in frehmaglutoside H.The coupling constant of H-10 in frehmaglutoside H were 5.5 Hz, which were 6.9 Hz in 7. The above minor difference was supposed due to the differentiation of Δ 7,8 and Δ 9,10 configurations between the two compounds.Further 1 H-NMR inspection of 7 showed that the coupling constant of H-7 and H-8 was 16.0 Hz in 7, which was the same as those of H-7 and H-8 in frehmaglutoside H, suggesting that both of them have an E-configuration Δ 7,8 double bond.Then, 7 was supposed to have an Z configuration Δ 9,10 double bond, which was different from the E configuration Δ 9,10 in frehmaglutoside H. Detailed analysis of 2D NMR data confirmed that 7 and frehmaglutoside H possessed the same planar structure.The relative configuration of 7 was judged by the analysis of the RoESY and chemical shifts.The RoESY correlation of H-2/H-14 indicated that H-2 was in β-orientation.H-15 was inferred to be an α-orientation due to the RoESY correlation of H-13/H-15 (Figure S2).The chemical shift of C-6 in 7 and frehmaglutoside H was both 80.9 ppm, indicating that 6-oH was in an β-orientation (Feng et al. 2015).Therefore, the structure of 7 was fully elucidated and named rehmannia B.
The NMR spectra of 8 were comparable to those of rehmaionoside B (Kitagawa et al. 1995), except the replacement of one methyl in rehmaionoside B by one oxymethylene in 8.The above differences suggested that one of the methyls (C-10, C-13, C-14, and C-15) in rehmaionoside B was oxygenated in 8.The HMBC (Figure S1) correlations of H-1′/C-13, H-13/C-1, and H-13/C-6 testified that the glycoside is attached to the C-13 in 8 rather than C-5 in rehmaionoside B. The relative configuration of 8 was judged by the analysis of the RoESY and chemical shifts.The RoESY correlation of H-14/H-15 suggested that H-15 have an β-orientation (Figure S2).6-oH was suggested to be an β configuration due to the similarity of C-6 chemical shift in 8 and trihydroxy-β-ionone monoacetate (obtained from CD data and X-ray crystallographic analysis) (Kitagawa et al. 1991).In addition, H-9 was established to be an α-configuration because the chemical shift of H-9 in 8 was closely similar to that in rehmaionoside B (Kitagawa et al. 1995).Thus, the structure of 8 was fully elucidated and named rehmannia C.

Material
The root tuber of R. glutinosa were purchased from Chinese traditional medicine market in Luosiwan, Kunming city, Yunnan province, on oct.2021, and authenticated by Prof. Xuanqin Chen (Kunming university of Science and Technology).The sample specimens (KMuST20211017) were stored in the Key Laboratory of Phytochemistry, Kunming university of Science and Technology

Determination of α-glucosidase inhibitory activity
The experiments were carried out in 96-well plates.The sample solution (dissolved in DMSo and diluted to 2 mg/mL) was mixed with 0.3 u/mL α-glucosidase (10 µL) (SIGMA) in 2.0 mM phosphate buffer (pH = 6.8).After 10 min pre-incubation, 2.0 mM p-nitrophenyl-α-D-glucopyranoside (pNPG, 20 µL) (TCI) was added and the solution was incubated at 37 °C for 20 min.Then, 0.2 mo1/L Na 2 Co 3 solution (150 µL) was added to stop the reaction in the end.The absorbance of the released p-nitrophenol (pNP) was measured at 405 nm using a microplate reader.Acarbose (SIGMA) was used as a positive control.The inhibition rate of α-glucosidase and IC 50 value of each sample can be calculated according to the formula

Acid hydrolysis of 7 and 8
Compounds 7 and 8 (both 2.0 mg) were dissolved in 1.2 N HCl-MeoH (1:2, 4 ml) and heated at 75 °C for 12 h.After cooled to room temperature, the mixture was extracted with CHCl 3 .Then, the water layer was neutralized with 5% NaoH and evaporated to afford the sugar fraction.The residue was dissolved in water, and analyzed by HPLC coupled to a Jasco oR-2090 chiral detector (Jasco Electric Co. Ltd., Tokyo, Japan) and VertisepTM sugar LMP column (7.8 × 300 mm i.d., mobile phase water, flow rate 0.4 ml/ min, temperature 80 °C, Vertical Chromatography Co. Ltd., Nonthaburi, Thailand).Then, the retention time and optical rotation of the sugar produced in the acid hydrolysis were compared with those of standard glucose substance.

Conclusions
In summary, chemical investigation led to the isolation of seventeen compounds from R. glutinosa, including three new ones.Bioassay showed that compounds 9, 10, and 11 (IC 50 : 5.0, 3.1, and 6.3 mM) showed moderate activity to suppress α-glucosidase activity.The findings provided some new insights to understand the hypoglycemic effect of R. glutinosa and the development towards the α-glucosidase inhibitor drugs.
control group absorbance, A B : blank group absorbance, A S : sample group absorbance, A SB : sample blank group absorbance