Alfa glucosidase inhibitory, anti inflammatory activities and a new furanocoumarin derivative of Ruellia tuberosa

Abstract Rullia tuberosa L. is used for treatment of diabetes mellitus, anti-inflammation, etc. However, its hypoglycaemic and anti inflammatory activities has not been investigated so far. In the present study, the α-glucosidase inhibitory, anti inflammatory activities of the extract of this plant were investigated. Our results showed that the crude extract as well as ethyl acetate and methanol fractions showed α-glucosidase inhibitory activity with IC50 of 15.84, 4.73 and 8.27 µg/ml, respectively. In addition, the hexane and ethyl acetate fractions are capable of inhibiting LPS-induced NO production with IC50 of 17.41 and 23.95 µg/mL, respectively. From the ethyl acetate and methanol fractions, eight compounds, including isobargaptol 5-O-β-D-glucopyranoside (1), syringaresinol (2), catechin (3), pulmatin (4), stigmast-4-en-3-on (4), verbascoside (5), hydroxymethylfurfural (6), rutin (7), and homoplantaginin (8) were extracted and isolated. Their chemical structures were elucidated by spectroscopic method including MS, 1 D and 2 D- NMR and comparison with the literature values. Graphical Abstract


Introduction
Diabetes mellitus is a chronic disease which relates to hyperglycaemia as a result of defects in insulin secretion, insulin action, or both. The goal of treatment of diabetes is to control and keep blood glucose levels at normal or near-normal levels, of which limiting increases postprandial blood glucose (Unwin and Alberti 2006). To control blood glucose, in addition to losing weight, exercise and eating healthily, people still use drugs, including a-glucosidase enzyme inhibitors. The a-glucosidase inhibitors prevent the digestion of carbohydrates, leading to reduce postprandial absorption of glucose, thereby delaying the increase in blood glucose (Haque et al. 2011).
Current literature shows an association of diabetes mellitus and secondary complications with chronic inflammation. Apart from anti-inflammatory drugs, various hypoglycaemic agents have also been found to reduce inflammation that could contribute to improved outcomes (Vishal et al. 2016). Inflammation has been implicated as an important aetiological factor in the development of both type1 and type 2 diabetes mellitus (Montane et al. 2014). Morever, hyperglycaemia promotes auto-oxidation of glucose to form free radicals. This is the main factor account for the development of diabetes (Tripathi and Chandra 2009). Therefore, preventing the progression of diabetes by adding the hypoglycaemic, anti-inflammatiory and antioxidant compounds is a meaningful study. The plant Ruellia tuberosa L. is a Vietnamese folk remedy in the treatment of diabetes mellitus, anti-inflammation and diuretic agent etc. (Bich et al. 2004). This plant possesses significant blood glucose lowering effect in alloxan-induced diabetic rat and rabbit (Shahwara et al. 2011;Rajan et al. 2012), and the leaves extract inhibited lipoxygenase enzyme (Somia et al. 2005). From the ethyl acetate fraction of R. tuberosa L., five flavonoids as cirsimaritin, cirsimarin, cirsiliol 4-glucoside, sorbifolin, pedalitin together with betulin, vanillic acid, and indole-3-carboxaldehyde were extracted and isolated (Lin et al. 2006). Flavonoid compounds included apigenin, luteolin, 3,5diglucoside, apigenin-7-O-glucuronide, apigenin glucoside, apigenin rutinoside, luteolin glucoside, and flavone glycoside were also reported in R. tuberosa L. (Wagner et al. 1971;Nair and Subramanian 1974). Continuing our study on chemical and bioactivities of the plant (Trinh et al. 2018), the aim of this present work is to evaluate the inhibition of a-glucosidase enzyme and anti-inflammation activity as well as isolate phytochemicals from active fraction of R. tuberosa collected in Tiengiang province, Vietnam.

Results and discussion
The crude extract of Ruellia tuberosa was fractionated over flash column chromatography eluting successively with n-hexane, ethyl acetate, and methanol, respectively, to yield n-hexane (80 g), ethyl acetate (95 g), and methanol fractions (148 g). In a-glucosidase inhibitory assay, crude ethanol extract, ethyl acetate and methanol fractions showed activity with IC 50 of 15.84, 4.73 and 8.27 mg/mL (Supplementary material Table  S1), respectively. In addition, the hexane along with ethyl acetate fractions showed anti-inflammatory activity through supression of nitric oxide production with IC 50 of 17.41 and 23.95 mg/mL (Supplementary material Table S1), respectively. The ethyl acetate and methanol fractions were chromatographed on silica gel, sephadex LH-20 and octadecyl silane columns to give eight compounds 1-8 ( Figure 1).
The HMBC spectrum displayed the correlations between H-3 and H-4 with carboxyl carbon signal at d C 160.1. Morever, proton H-4 correlated with two oxygenated olefinic carbon resonances at d C 152.0 and 146.7 which were attributed to C-5 and C-8a in coumarin skeleton, respectively. Carbon C-4a was determined through HMBC correlation between H-3 with signal at d C 107.1. In addition, the cross-peak between anomeric proton d H 5.02 with C-5 in HMBC spectrum proving that glucose residue attached to C-5 in coumarin framework. The singet signal at d H 7.41 was asigned to H-6 due to HMBC correlations between H-6 with C-5, C-4a, C-7 and C-8. Therefore, furan unit attached to coumarin framework at C-7 and C-8. In HMBC spectrum, two olefinic proton signals at [d H 8.06 (1H, d, 2.0 Hz) and 7.19 (1H, d, 2.0 Hz)] correlated to each other and shared cross peak with C-7, C-8. Futhermore, only signal at d H 7.19 gave a strong correlation with C-8a indicating that these two proton were H-2' and H-3', respecively. These data strongly confirmed that compound 1 was an angular 5-oxygenated furanocoumarin derivative (Dharmatti et al. 1962). Compared with isobargapten, all nmr data were matched, except for the presence of glucose moiety at C-5 in compound 1 instead of methyl group (Taryn et al. 2013). Based on the above spectroscopic data, compound 1 was established as isobargaptol 5-O-b-D-glucopyranoside.
Compounds 1, 2, 3 and 5 were evaluated for their a-glucosidase inhibitory and anti inflammatory activities. The results indicated that compound 2 showed appreciable inhibitory effect against LPS-induced NO production with IC 50 values of 14.37 mM, while other compounds exhibited moderate acitivity with IC 50 value were in range 75.32 to 312.04 mM (Supplementary material Table S2). In a-glucosidase inhibitory activity, compound 2 and 5 indicated moderate a-glucosidase inhibitory activity which were comparable with that of possitive sample acarbose. The IC 50 values of remain compounds were weaker than acarbose (Supplementary material Table S2).

Plant material
The whole plant of Ruellia tuberosa L. were collected in Tiengiang province, Vietnam in July, 2017. A voucher specimen (D072017) has been deposited in Institute of Applied Materials Science (VAST, HoChiMinh city, Vietnam) by Dr. Luu Hong Truong.

General experimental procedures
Melting points were determined on an Electrothermal 9100 melting point apparatus. Specific rotation was measured on digital polarimeter (Kruss, Hamburg, German). NMR spectra were recorded on a Bruker AM500 FTNMR spectrometer (Bruker, Karlsruhe, German) using TMS as an internal standard, Institute of Chemistry (Vietnam Academy of Science and Technology, Hanoi, Vietnam). HR-ESI-MS were performed on a MicroOTOF-Q mass spectrometer (Bruker, Karlsruhe, German), University of Science (National University, HoChiMinh city, Vietnam). TLC was performed on precoated silica gel 60 F254 (1.05554.0001, Merck, Darmstadt, German) and RP-18 F254S plates (1.15685.0001, Merck, Darmstadt, German). The detection of TLC zones were done by exposure to UV light at 254 or 365 nm or a solution of FeCl 3 /EtOH or H 2 SO 4 /EtOH. Column chromatography was performed on silica gel (240-430 mesh, Merck, Darmstadt, German), ODS Merck,Darmstadt,German) or Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

Extraction and isolation
The dried powder of whole plant (5.0 kg) was extracted with 96% ethanol at room temperature for four times (4 Â 30 L) and filtered. The filtrate was then concentrated on a rotary evaporator to obtain crude ethanol extract (350 g). The extract was fractionated on a flash silica gel column, eluted successively with n-hexane, ethyl acetate, and methanol, respectively to yield n-hexane (80 g), ethyl acetate (95 g), and methanol fractions (148 g).

a-Glucosidase inhibitory activity assay
A reaction mixture containing 500 ll of 67 mM phosphate buffer (pH 6.8), 20 ll of 0.3 U/ml a-glucosidase in cold deionised water and 20 ll of sample was pre-incubated in thermoregulator for 5 min at 37 C, and then 50 ll of 5 mM PNP-G solution was added to the mixture. After further incubation at 37 C for 30 min, the reaction was stopped by adding 2440 ll of 100 mM Na 2 CO 3 (pH 9.6). The released PNP was monitored spectrophotometrically by measuring UV absorbance at 400 nm. Acarbose was used as positive control. The percentage of a-glucosidase inhibition by the sample was calculated by the following formula: where A C is the absorbance of the control and A S is the absorbance of the tested sample.

Nitrite determination
RAW 264.7 cells (5 Â 10 4 cells/well in 96-well plates) were examined with extracts or isolated compounds for 1 h prior to LPS (1 lg/mL) stimulation for 24 h. Nitrite in culture medium was measured by using Griess reagent. Absorbance was subsequently measured at 540 nm, using a microplate reader. The amount of nitrite was obtained by an extrapolation from a standard curve with sodium nitrite.  Table S3.

Conclusions
Crude ethanol extract as well as ethyl acetate and methanol fractions of Ruellia tuberosa exhibited a-glucosidase inhibitory activity. In addition, the hexane and ethyl acetate fractions inhibited LPS-induced NO production in LPS-stimulated RAW 264.7 macrophages. From the ethyl acetate and methanol fractions, eight compounds (1-8) were extracted and isolated. Among them, 1 is a new compound. Compound 2 showed appreciable inhibitory effect on NO production. In a-glucosidase inhibitory assay, compound 2 and 5 indicated good a-glucosidase inhibitory activity.