In vitro α-glucosidase inhibitory activity of phenolic constituents from aerial parts of Polygonum hyrcanicum

Background and the purpose of the study The early stage of diabetes mellitus type 2 is associated with postprandial hyperglycemia. Hyperglycemia is believed to increase the production of free radicals and reactive oxygen species, leading to oxidative tissue damage. In an effort of identifying herbal drugs which may become useful in the prevention or mitigation of diabetes, biochemical activities of Polygonum hyrcanicum and its constituents were studied. Methods Hexane, ethylacetate and methanol extracts of P. hyrcanicum were tested for α-glucosidase inhibitory, antioxidant and radical scavenging properties. Active constituents were isolated and identified from the methanolic extract in an activity guided approach. Results A methanolic extract from flowering aerial parts of the plant showed notable α-glucosidase inhibitory activity (IC50 = 15 μg/ml). Thirteen phenolic compounds involving a cinnamoylphenethyl amide, two flavans, and ten flavonols and flavonol 3-O-glycosides were subsequently isolated from the extract. All constituents showed inhibitory activities while compounds 3, 8 and 11 (IC50 = 0.3, 1.0, and 0.6 μM, respectively) were the most potent ones. The methanol extract also showed antioxidant activities in DPPH (IC50 = 76 μg/ml) and FRAP assays (1.4 mmol ferrous ion equivalent/g extract). A total phenol content of 130 mg/g of the extract was determined by Folin-Ciocalteu reagent. Conclusion This study shows that P. hyrcanicum contains phenolic compounds with in vitro activity that can be useful in the context of preventing or mitigating cellular damages linked to diabetic conditions.


Introduction
The early stage of diabetes mellitus type 2 is associated with postprandial hyperglycemia due to impaired aftermeal acute insulin secretion. Hyperglycemia is believed to increase the production of free radicals and reactive oxygen species, leading to oxidative tissue damage and diabetic complications such as nephropathy, neuropathy, retinopathy, and memory impairment [1]. Glucosidases are a group of digestive enzymes which break down the dietary carbohydrates into simple monosaccharides. Glucosidase inhibitors such as acarbose reduce the rate of carbohydrate digestion and delay the carbohydrate absorption from the digestive tract. Therefore, they have a potential to prevent the development of type 2 diabetes mellitus by lowering the after-meal glucose levels [2].
Polygonum species are valuable medicinal plants which possess interesting biological activities such as antiinflammation [3], cardiovascular protection [4], neuroprotection [5], and mitigation of biochemical processes involved in age-related neurodegenerative disorders such as Alzheimer's [6] and Parkinson's disease [7]. It is believed that these beneficial effects are, at least in part, due to antioxidant and radical scavenging properties of the plant. Moreover, some Polygonum species were reported to possess glucosidase inhibitory properties. Phenylpropanoid glycosides of P. sachalinense [8] and tannins of P. cuspidatum [9] were subsequently identified as active compounds.
Polygonum hyrcanicum is an endemic species that grows widely in northern areas of Iran [10]. In folk medicine of the Turkmen Sahra region (southeast of the Caspian Sea), decoctions made from aerial parts of the plant are used for the treatment of liver problems, anemia, hemorrhoids, and kidney stones [11]. To our knowledge, no biological or phytochemical investigation has been carried out with this species. To explore the plant's properties with respect to potential prevention or mitigation of cellular damages linked to diabetic conditions, different extracts of P. hyrcanicum were tested for α-glucosidase inhibitory, antioxidant and radical scavenging properties. Active constituents were isolated and identified from the methanolic extract.

Plant materials
Aerial parts of Polygonum hyrcanicum Rech. f. at full flowering stage were collected in September 2008 near the village of Veresk (Mazandaran Province) in the north of Iran. The plant material was identified by the forth co-author. A voucher specimen (6729-TEH) has been deposited at the Herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences.

Extraction and isolation
Shade-dried aerial parts of the plant (1200 g) were cut to small pieces and macerated with n-hexane, ethyl acetate, and methanol, successively, at room temperature (3 × 48 hours with each solvent). The extracts were concentrated under reduced pressure, then freeze dried, resulting in dry extracts of hexane (14 g), ethyl acetate (12 g), and methanol (150 g).

Sugar analysis
Sugar moieties of glycoside structures were detected by GC-MS analysis after acid hydrolysis and derivatization with L-cysteine methyl ester and silylation [12].
α-Glucosidase inhibition assay α-Glucosidase inhibitory activities were evaluated according to the chromogenic method described by McCue et al. (2005), with some modifications [13]. The enzyme solution contained 20 μl α-glucosidase (0.5 unit/ml) and 120 μl 0.1 M phosphate buffer (pH 6.9). p-Nitrophenylα-D-glucopyranoside (5 mM) in the same buffer (pH 6.9) was used as a substrate solution. Ten microliters of test samples, dissolved in DMSO at various concentrations, were mixed with enzyme solution in microplate wells and incubated for 15 min at 37°C. Twenty microliters of substrate solution were added and incubated for an additional 15 min. The reaction was terminated by adding 80 μl of 0.2 M sodium carbonate solution. Absorbance of the wells was measured with a microplate reader at 405 nm, while the reaction system without plant extracts was used as control. The system without α-glucosidase was used as blank, and acarbose was used as positive control. Each experiment was conducted in triplicate. The enzyme inhibitory rates of samples were calculated as follows: Inhibition%¼½ðcontrol absorptionÀsample absorptionÞ =controlabsorption Á Â100: The IC 50 values of samples were calculated and reported as the mean ± standard deviation (SD) of three experiments.

DPPH free radical scavenging activity
The antioxidant activities of the extracts were determined with the DPPH assay according to an established protocol [14]. Extract solutions of 500, 250, 100, and 50 μg/ml were prepared in methanol. Each test tube contained 1 ml of the samples and 2 ml freshly prepared DPPH solution of 40 μg/ml in methanol. Negative control tubes were the same as the test tubes, except that they did not include DPPH. Absorbance of the mixtures were recorded at 517 nm after 30 minutes, against the blank covets of DPPH solution. Vitamin E was used as a positive control. All samples were assayed in triplicate and IC 50 values were calculated.

FRAP assay
Antioxidant activities of plant extracts were evaluated by monitoring their ferric-reducing abilities [15]. Freshly prepared FRAP reagent contained 5 ml FeCl3 (20 mM) plus 5 ml of a 10 mM TPTZ solution in 40 mM HCl and 50 ml of 300 mM acetate buffer (pH = 3.6). One hundred microliters of the samples, dissolved in methanol at various concentrations, were mixed with 3 ml FRAP reagent and incubated at 37°C for 10 minutes; the absorptions at 593 nm were recorded. A calibration curve was generated in the range of 125-750 μM ferrous sulfate (FeSO 4 .7H2O). Vitamin E was used as a positive control and the results were expressed as mmol ferrous ion equivalent per gram of extracts.

Determination of total phenolic content
Total phenolic contents of extracts were assessed using Folin-Ciocalteu reagent [16]. The reagent was diluted 10-fold with distilled water. Two hundred microliters of appropriate dilutions of extracts were added to 1.5 ml reagent and allowed to stand at room temperature for 5 minutes. Sodium bicarbonate solution (1.5 mL, 60 g/L) was added to the mixture and stored at room temperature for an additional 90 minutes; absorptions at 725 nm were recorded. Known concentrations of gallic acid (0-100 μg/ml in methanol) were applied as standard samples and a calibration curve was created. Total phenolic contents were expressed as mg of gallic acid equivalents (GAE) per gram of dry extracts.

Result and discussion
Sequential extraction of the aerial parts of P. hyrcanicum yielded 1.0% of hexane, 0.8% of ethyl acetate, and 12.5% of methanol extracts, respectively. The methanol extract of P.
Subsequently, α-glucosidase inhibitory activities of phenolic compounds 1-13, isolated from the methanolic extract, were evaluated. The results are reported in Table 2. All constituents showed interesting inhibitory activities while compounds 3, 8 and 10 (IC50 = 0.3, 1.0, and 0.6 μM, respectively) were the most potent ones. The α-glucosidase inhibitory activities of compounds 4, 5, 6, and 12 have not been reported in the literature previously. Comparing the IC 50 values of tested flavonoids shows that hydroxyl substitution affects the inhibitory activity so that increasing number of free phenolic groups results in higher activity.
Since oxidative stress is considered as a key factor in the pathogenesis of diabetic complications, antioxidant properties of the extracts were also studied. DPPH radical scavenging activity and ferric reducing power of P. hyrcanicum extracts are summarized in Table 1. The methanolic extract showed noticeable antioxidant activities in both DPPH (IC 50 = 76.0 μg/ml) and FRAP (1.4 mmol ferrous ion equivalent/g) assays compared to vitamin E (IC 50 = 14.1 μg/ml, FRAP value of 2.4 mmol ferrous ion equivalent/g) as the positive control. The ethyl acetate extract was a moderate antioxidant, while the hexane extract was not active at the concentrations tested. The antioxidant properties were in accord with the total phenol content of the extracts (Table 1). Methanol and ethyl acetate extracts contained 135.0 mg and 20.3 mg gallic acid equivalent/g, respectively, and the hexane extract was free of phenolic constituents.

Conclusion
In the perspective of identifying traditional herbal drugs which might be useful in preventing or mitigating cellular damages related to diabetes, we carried out the first study of the Persian edible plant, P. hyrcanicum. The methanolic extract, in particular, showed promising α-glucosidase, antioxidant, and radical scavenging activities and thirteen phenolic compounds were purified in an activity-guided approach. All the isolated compounds (IC 50 = 0.3-7.6 μM) were more potent than the positive control acarbose (IC 50 = 13.5 μM). This study suggests that P. hyrcanicum is a promising source of active compounds that can prevent the development of diabetes mellitus type 2 and its complications. While these in vitro results are of a preliminary nature, further investigation of P. hyrcanicum, in particular, in vivo pharmacological testing of the methanolic extract is warranted. These studies will provide a more in depth picture on the potential of this interesting traditional Persian plant.