Glycosides from the leaves of Fraxinus Hubeiensis

Fraxinus hubeiensis is a plant endemic to China and widely used as folk medicine to treat various diseases. However, its chemical constituents have never been reported sufficiently. Thus, the primary objective of this study was to investigate the phytochemical constituents and biological activities of F. hubeiensis leaves. Hence, combined column chromatographic and spectroscopic techniques were used to identify and characterize the secondary metabolites such as a pair of 3-keto-glycoside epimers (1) and (2), along with five known compounds (3 ~ 7). The results of α-glucosidase inhibitory activity exhibited that 1 and 2 had moderate activity with IC50 values of 359.50 and 468.43 µM, respectively, compared to a positive control acarbose with the IC50 value of 164.08 µM. However, Compounds 1–6 were shown to be inactive against the tested microbes. Supplementary Information The online version contains supplementary material available at 10.1186/s13065-023-01070-6.


Introduction
Fraxinus hubeiensis belongs to the family Oleaceae which comprises about 27 genera and more than 400 species in the world [1,2].About 30 species of the Fraxinus genus native to China and some of which were used in traditional Chinese Medicines as Cortex Fraxini [3], commonly known as 'Qin-Pi' in Chinese.Because of its very narrow distribution and relatively long growth cycle, F. hubeiensis, as a rare species, has received little attention [4].In folk applications, it has also been widely used in the chemotaxonomic research on Fraxinus and elucidating its active ingredients, and indicate great potential for the development of novel plant-derived hypoglycemic drugs.
Initially, the mixture (1 and 2) was obtained and exhibited a single spot in TLC and one peak detected over a rountine HPLC equipment.Its 1 H and 13 C NMR spectra showed two sets of resonances respectively, and can be assigned clearly to each compound, which suggested the mixture was a racemic.The above deduction was in accordance with its optical activity which was close to zero.To confirm, subsequent chiral HPLC of the mixture resulted in the purification of two enantiomers in a ratio of 1:2, with opposite optical rotation values.
β-D-ribo-hex-3-ulopyranoside (2), was also obtained as colorless gum.The molecular formula was determined to be C 8 H 14 O 6 based by HR-ESI-MS peak at [M + Na] + peak at m/z 229.0697 (calcd for 229.0688).Interesting that, the 1 H and 13 C-NMR signals were different from those of its enantiomer 1, even when they were mixed together.Meanwhile, the HMBC cross-peaks confirmed the structure of 2 (Fig. 2), which was previously found as a biotransformation product in the Coleus forskohlii root culture induced from Agrobacterium rhizogenes [8].The large coupling constant (J = 7.9 Hz) of the anomeric proton indicated it had β-glycosidic configuration.Therefore, the structure of 2, as a new natural product, was determined as β-D-ribo-hex-3-ulopyranoside.
The α-glucosidase inhibition assay was carried out for a pair of 3-keto-glycoside epimers 1 and 2. Compounds 1 and 2 exhibited moderate activities with their IC 50 values of 359.50 and 468.43µM respectively, compared to acarbose with the IC 50 value of 164.08µM (Table 2).Meanwhile, the antimicrobial activities of compounds 1-6 were tested, while none of them exhibited activities.

General experimental procedures
Optical rotations were measured on an Autopol-III automatic polarimeter (Rudolph Research Analytical, USA).IR spectra were recorded on an Agilent Technologies Cary 630 FTIR spectrophotometer with KBr pellets.NMR spectra were acquired on Bruker AV-600 spectrometer using methanol-d 4 (CD 3 OD) or chloroform-d 1 (CDCl 3 as references, locked to the deuterium signal of the solvent.Chemical shifts were given in parts per million (ppm), and coupling constants in hertz (Hz).HRESIMS were measured on a Waters UHPLC-H-CLASS/XEVO G2-XS Q-TOF mass spectrometer (Waters, USA).Semi-preparative HPLC was performed using an Agilent 1260 Infinity II liquid chromatograph system equipped with UV-VIS detector.And a reversedphase C18 column (Agilent Eclipse XDB, 250 × 9.4 mm., 5 μm) and a chiral-phase column (NQ (2)-RH, 250 × 4.6 mm, 5 μm) were used for purification and enantioselective analysis.Thin-layer chromatography (TLC) was conducted using silica gel GF 254 (Qingdao Haiyang Chemical Co.Ltd.) plates.Open column chromatography was performed using silica gel (Qingdao Haiyang Chemical Co. Ltd., 200-300 mesh) and Sephadex LH-20 (Pharmacia Biotech Ltd.) and reverse-phase C 18 silica gel ODS (Merck & Co., Inc.USA).All the solvents were of analytical grade and were purchased from Beijing Chemical Company Ltd.TLC spots were observed under UV light or by spraying with 5% sulfuric acid vanillin solution.

Plant materials
The dried leaves of F. hubeiensis were collected in the Bonsai garden of Jingzhou in China, in June 2017.Plant materials were identified by one of the authors (Prof.Qing-Lai Wu).A voucher specimen (No.DJBL201706) was deposited at TCM and Ethnomedicine Innovation & Development International Laboratory in Hunan University of Chinese Medicine.

Extraction and isolation
The dried leaves of F. hubeiensis (5.0 kg) were pulverized and immersed in 40 L 95% aqueous EtOH for three times at room temperature, each time 10 days.The solvent was evaporated under vacuum to obtain a crude extract (1.01 g), and the extract was suspended in 2 L H 2 O and partitioned successively with petroleum ether (PE, 60 ~ 90 °C) and ethyl acetate (EtOAc), to yield PE (311.0 g), EtOAc (189.0 g), and water (420.1 g) fractions.

α-glucosidase inhibitory assay
The α-glucosidase inhibition assay was carried out on the basis of the method reported with minor modifications [14,15].Briefly, 2µL of test sample (0.1, 0.25, 0.5, 1.0 mmol/L) dissolved in 98µL of PBS buffer (0.1 mol/L, pH 6.8) and 25µL α-glucosidase (0.2 U/mL) solution were mixed and pre-incubated at 37 °C for 20 min in a 96-well microplate.Then, 25µL p-nitrophenyl-α-Dglucopyranoside (pNPG, 4 mmol/L) solution was added to initiate the reaction.The 96-well microplate was incubated at 37 °C for an additional 15 min before being stopped with 50µL Na 2 CO 3 (0.2 mol/L).The absorbance of each well was measured at a wavelength of 405 nm, and the data were recorded and measured in parallel for three times.In this experiment, acarbose was used as the positive control, PBS buffer was used as the blank group, and DMSO solution was used as the negative control group.Other reagents were consistent with the sample experimental group.The absorbance at 405 nm was measured by microplate reader, and the inhibition rate was calculated.
The α-glucosidase inhibitory activity was expressed as percent inhibition and was calculated as follows:

Antimicrobial activity
Compounds 1-6 were screened for antimicrobial activities against the tested microbes (S. aureus, B. subtilis, E. coli, P. aeruginosa and C. albicans) by the previously reported method [16,17].Regrettably, compounds 1-6 exhibited no activities.Perhaps for these isolates, the rang of strains should be expanded to test for the activity.However, because of no sufficient of these compounds isolated from this plant, it was not able to screen them more broadly for antimicrobial activities.

Conclusion
In summary, seven compounds were isolated from the leaves of F. hubeiensis including two ketoglycosides, one methylinsitol, three coumarins and salicylic acid.All compounds except 5 were isolated from F. hubeiensis for the first time.This study is the first reported of successful isolation of a pair of 3-ketoglycoside isomers by chiralphase HPLC, which are undescribed compounds from nature.The results of α-glucosidase inhibitory indicated that 1 and 2 possessed moderate activities.It was worth mentioning that the activity of 1 with α-configuration was better than that of 2 with β-configuration, so the subsequent work on structural modification based on the ketose-type skeleton and their structure-activity relationship deserves further study.According to traditional uses of the plant, compounds 1-6 were evaluated for antimicrobial effect.Unfortunately, these isolates had no activity.Therefore, more in-depth phytochemistry and pharmacological research is worth continuing in the future.
a Acarbose was used as a positive control