α-Amylase and α-Glucosidase Inhibitory Saponins from Polyscias fruticosa Leaves

Three bisdesmosidic saponins 3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucuronopyranosyl] oleanolic acid 28-O-β-D-glucopyranosyl ester (1), polyscioside D (2), and 3-O-{β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-glucuronopyranosyl} oleanolic acid 28-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranosyl ester (3) were isolated from a methanol extract of Polyscias fruticosa (L.) Harms leaves. Compound 1 was obtained as a main constituent and compound 3 was reported for the first time and named as polyscioside I. Saponin 1 inhibited porcine pancreas α-amylase and yeast α-glucosidase activities while 2 and 3 were inactive. Synergistic inhibitory effect on α-amylase was observed from the combination of low concentrations of 1 and acarbose. The findings suggest the use of P. fruticosa and itsmajor saponin 1 for the prevention and treatment of diabetes and its complications.


Introduction
Polyscias fruticosa (L.) Harms (synonyms Nothopanax fruticosus (L.) Miq. and Panax fruticosus L.), belonging to the family Araliaceae, is widely used in Vietnam as a tonic agent for the treatment of ischemia and inflammation and to increase blood flow in the brain. The leaves of this plant are also eaten as a salad [1]. Previous studies showed that P. fruticosa leaf extracts exhibited antipyretic, antiinflammatory, analgesic, and molluscicidal properties [2,3] and antidiabetic activity [4]. A few reports on the chemical composition of this plant indicated the presence of polyacetylene [5] and saponins [6]. Diabetes is a group of metabolic diseases characterized by chronic hyperglycemia resulting from deficiency in insulin secretion or action. Recent reports revealed that a high postprandial plasma glucose level is more harmful than fasting blood glucose. Therefore, it is important to control the postprandial blood glucose level to reduce complications and mortality. One therapeutic approach for treating diabetes is to decrease postprandial glycemia by inhibiting enzymes responsible for carbohydrate hydrolysis, such as -glucosidase and -amylase [7].
Our search for antidiabetic agents of natural origin identified a methanol extract of P. fruticosa leaves that showed significant inhibitory activity against -glucosidase and -amylase. A phytochemical investigation of the methanol extract of P. fruticosa leaves led to the isolation of three bisdesmosidic saponins (   Tokyo, Japan). NMR experiments were carried out on a Bruker AM500 FT-NMR spectrometer (Bruker, Rheinstetten, Germany) using tetramethylsilane (TMS) as internal standard. The HR-ESI-MS was recorded on API Q-STAR PULSAR I of Applied Biosystem. The bioassay absorbance was read by xMark Microplate Spectrophotometer reader (Bio-Rad Laboratories, USA).

Plant
Material. The leaves of Polyscias fruticosa were collected in Tien Hai, Thai Binh, in July 2011 and identified by Professor Tran Huy Thai, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. The voucher specimens were deposited at the herbarium of the Institute of Ecology and Biological Resources.

Extraction and Isolation.
The air-dried and powdered P. fruticosa leaves (2.0 kg) were extracted with methanol (5 L × 3 times) at room temperature. The combined extracts were concentrated to give 300 g of crude extract, which was then resuspended in water (1.5 L) and successively partitioned with hexane and ethyl acetate (each 0.5 L × 3 times) to obtain 75 and 92 g of hexane and ethyl acetate residues, respectively. The water residue was filtered through a Diaion HP-20 column eluted by 0%, 25%, and 100% methanol in water. The 100% methanol-eluted fraction was diluted with water (1 : 1 v/v) and kept overnight at 4 ∘ C. Compound 1 as white crystals (500 mg) was obtained by filtering. The filtrate was concentrated and chromatographed on a silica gel column eluted with a gradient of 0%, 50%, and 100% methanol in ethyl acetate to afford three fractions, F1-F3. Fraction F2 was fractionated on a silica gel column eluted with chloroformmethanol-water (50 : 10 : 1 v/v) to afford compound 2 (10 mg). Compound 3 (20.5 mg) was purified from F3 by a RP-18 column (methanol-water 1 : 1 v/v).  Acid Hydrolysis of 3. Compound 3 (10 mg) was heated in 2 N HCl (5 mL) at 80 ∘ C for 2 h, and then the solution was extracted with ethyl acetate (2 mL × 3). The sugar products in the aqueous layer were identified as glucose (Rf 0.65), galactose (Rf 0.60), and glucuronic acid (Rf 0.12) in comparison with standards by silica gel thin layer chromatography (developed with acetone-methanol-water 10 : 9 : 1 and sprayed by 10% sulfuric acid solution containing 2% vanillin). After preparative thin layer chromatography, the optical rotation of each sugar fraction was checked. The positive optical rotation values led to the assignment of D-glucuronic acid, D-glucose, and D-galactose [9].

Assay for -Glucosidase Inhibition.
The yeast -glucosidase (G0660, Sigma-Aldrich) enzyme inhibition assay was performed by the digestion of p-nitrophenyl--Dglucopyranoside [10]. The sample solution (2 L dissolved in DMSO) and 0.5 U/mL -glucosidase (40 L) were mixed in 120 L of 0.1 M phosphate buffer (pH 7.0). After 5 min preincubation, 5 mM p-nitrophenyl--D-glucopyranoside solution (40 L) was added and the solution was incubated at 37 ∘ C for 30 min. The absorbance of released 4-nitrophenol was measured at 405 nm by using a microplate reader (Molecular Devices, CA). Acarbose was used as positive control (IC 50 650.4 g/mL).

Assay for -Amylase Inhibition.
The porcine pancreasamylase (A3176, Sigma-Aldrich) enzyme inhibitory activity was measured using the method reported by Ashok Kumar et al. [11] with slight modifications. Substrate was prepared by dissolving 2-chloro-4-nitrophenyl--D-maltotrio side (93834, Sigma-Aldrich) in phosphate buffer (pH 7.0). The sample (2 L dissolved in DMSO) and 0.5 unit/mL -amylase (50 L) were mixed in 100 L of 0.1 M phosphate buffer (pH 7.0). After 5 min preincubation, substrate solution (50 L) was added and the solution was incubated at 37 ∘ C for 15 min. The absorbances were measured at 405 nm. Acarbose was used as positive control (IC 50 57.6 g/mL).

Results and Discussion
Compound 3 was obtained as a white crystal and its high-resolution electrospray ionization mass spectrometry spectrum revealed an ion peak at  seven CH 3 , ten CH 2 , five CH, and 8 C signals ( Table 1). The aglycone was identified as oleanolic acid, a common triterpene of saponins in the genus Polyscias [6]. The C values of C-3 at C 88.3 and C-28 at C 175.2 suggested that compound 3 was a bisdesmosidic saponin with sugar units attached to these positions. In addition, five sugar moieties were recognized by five anomeric methine groups at C 103.6 (C-1 ), 103. The inhibitory effects of the isolated compounds against porcine pancreas -amylase and yeast -glucosidase were evaluated in comparison with the antidiabetic acarbose ( Table 2). Compound 1 potently inhibited -amylase andglucosidase with IC 50 values of 27.1 and 440.5 g/mL, respectively. In contrast, compounds 2 and 3 did not show any effect on both enzymes. Dou et al. reported that the glucuronic acid unit at C-3 and glucosidic moiety at C-28 of the aglycone were the functional groups essential for -amylase andglucosidase inhibition; the addition or replacement of glucose by another sugar unit might thus abolish the inhibitory activity [12]. In oral sucrose tolerance tests, several oleanolic acid glycosides showed significant antihyperglycemic activity in the oral sucrose tolerance in rats [13,14]. However, the 2 -O--D-glucopyranosyl unit attached to the glucuronic acid moiety was assumed to markedly reduce the hypoglycemic effect [15]. Consistent with this, in our study, compounds 2 and 3 possessing a 2 -O--D-glucopyranosyl unit were inactive regarding -amylase and -glucosidase inhibition. Previous studies showed that P. fruticosa leaf extracts strongly inhibited rat intestinal -glucosidase [16] and exhibited hypoglycemic and antihyperglycemic activity in normal and streptozotocin-diabetic albino rats [4]. Our results suggest that the observed hypoglycemic activity might be related to the inhibition of -amylase and -glucosidase enzymes.
It was of interest to establish whether compound 1 and acarbose interact synergistically or additively in the inhibition of -glucosidase and -amylase. Therefore, the assay was performed in solutions containing these agents alone or in a mixture at different concentrations. The results showed that the combination of compound 1 and acarbose produced a synergistic inhibitory effect on porcine pancreas -amylase ( Figure 2) but had no effect on yeast -glucosidase (data not shown). Interestingly, higher synergistic inhibition was found from the combination of low concentrations of individual agents. At 1.0 g/mL, compound 1 and acarbose alone exhibited only 1.5% and 4.2% inhibition, respectively. However, their combination increased the inhibition up to 32.8%. At a higher concentration of 10 g/mL, the percentage of -amylase inhibition of the mixture was equal to the sum of compound 1 and acarbose, suggesting that they produced additive inhibition. A slight increase in inhibition was obtained from the mixture in comparison with that achieved by the individual agents at 100 g/mL. These findings indicate that saponin 1 produced synergistic effects on porcine pancreas -amylase inhibition when combined with a low concentration of acarbose. Acarbose is a potent -glucosidase inhibitor and has been used in the treatment of type 2 diabetes. It can also be used in combination with other agents, such as sulfonylurea and metformin, in patients with diabetes. However, the long-term use and high-dose administration of this drug cause severe side effects, such as diarrhea, flatulence, and abdominal or stomach pain [17][18][19]. Our study showed that the combination of P. fruticosa saponin 1 with acarbose at a low concentration produced more synergistic inhibition than either drug alone, suggesting that they might provide a significant clinical benefit in delaying postprandial hyperglycemia and diminishing the adverse effects of acarbose. , were isolated from the leaves of P. fruticosa. Compound 1 showed inhibitory effects against porcine pancreas -amylase and yeast -glucosidase activities and produced a synergistic effect with acarbose in -amylase inhibition. Further study is needed to clarify the mechanism of enzyme inhibition as well as the hypoglycemic properties of saponin 1.