Acylated Flavonoid from Vaccinium Corymbosum ( Ericaceae ) Flowers with Yeast α-Glucosidase Inhibitory Activity

Purpose: To isolate and identify chemical constituents with yeast α-glucosidase inhibitory activity from the methanol extract of V. myrtillus (blueberry) flowers. Methods: The active fraction (ethyl acetate extract) were chromatographed on C18 MPLC column, Sephadex LH-20 column and semi-preparative HPLC column. The isolated compounds were identified by the extensive H-nuclear magnetic resonance spectroscopy (NMR), C-NMR, 2D-NMR and high resolution mass spectral (HR-MS) analyses. Results: Two phenolic compounds, an acylated flavonoid and a coumaric acid derivative, were isolated and identified as isorhamnetin-3-O-(6''-O-coumaroyl)-β-D-glucoside (1) and cis-cinnamic acid methyl (2). Compound 1 showed powerful α-glucosidase inhibitory activity and in this regard, was superior to the positive drug, acarbose. Conclusion: Compounds 1 and 2 were isolated for the first time from this species and the genus of Vaccinium. This is the first report on characterization of these phenolic compounds and the possible utilization of blueberry flowers for nutraceutical and functional food applications.


INTRODUCTION
Postprandial hyperglycemia is recognized as characteristic of type 2 diabetes mellitus and plays an important role in the development of some chronic complications including circulatory disease, stroke, hypertension, blindness, kidney failure, uremia and gangrene of the lower limbs [1].It is well known that inhibition of carbohydrate hydrolysis enzymes linked to diabetes is considered an effective approach to treat or prevent type 2 diabetes mellitus.Therefore, αglucosidase and α-amylase are frequently used to screen therapeutic agents derived from the natural plants and isolated compounds for control of postprandial hyperglycemia [2].Vaccinium corymbosum, also called the northern highbush blueberry, is a species of blueberry native to eastern North America.It is a deciduous shrub growing to 6 -12 feet tall and wide.
There are a great number of interests worldwide in blueberry because of its high content of beneficial constituents (such as anthocyanins, flavonols, tannins, stilbenoids and phenolic acids) and potential health benefits.Among those constituents, anthocyanins are considered to be one of the dominant blueberry polyphenols.It has been reported that blueberry extracts and its phytochemicals exhibit antioxidant [3], αamylase and α-glucosidase inhibitory activity [4], anti-proliferation [5], lipid-lowering activity [6] and cardioprotective effect [7].
Recently blueberry fruits extracts have been shown to exhibit carbohydrate enzyme inhibitory activities but the active compounds were not identified [4].The purpose of this study was to isolate and investigate the hypoglycemic activity compounds against inhibition of yeast αglucosidase in vitro.

EXPERIMENTAL Plant material
The flowers of high bush blueberry were collected from Morgan Farms (North Kingstown, RI, USA) in May 2009 and authenticated by Mr J Peter Morgan (a senior gardener of University of Rhode Island).A voucher specimen (16JPM51-VCJ51309FL) was deposited in the Heber-Youngken Garden and Greenhouse at the College of Pharmacy, University of Rhode Island (Kingston, RI, USA).

Extraction and chromatography
The flowers (1.3 kg, fresh weight) were extracted exhaustively with MeOH (3 × 4.3 L) at room temperature to yield a dried MeOH extract (138 g).A portion of the extract (133 g) was resuspended in H 2 O (750 mL) and partitioned with EtOAc (3 × 750 mL) and n-butanol to yield a dried EtOAc extract (33 g) and n-butanol soluble fractions, respectively.

Yeast α-Glucosidase inhibitory assay
Yeast α-Glucosidase inhibitory activity was determined as described in the literature [10].Briefly, a mixture of 50 μL of different concentrations of each of the samples (ethyl acetate fraction, n-buthanol fraction or two pure compounds) and 100 μL of 0.1 M phosphate buffer (pH 6.9) containing yeast α-glucosidase solution (1.0 U/ml) was incubated in 96 well plates at 25 °C for 10 min.After this preincubation period, 50 μL of 5 mM pNPG solution in 0.1 M phosphate buffer (pH 6.9) was added to each well at predetermined intervals.The reaction mixtures were incubated at 25 °C for 5 min.Absorbance was recorded at 405 nm before and after incubation with a micro-plate reader (SpectraMax M2) and compared to that of the control which had 50 μL buffer solutions instead of test samples.α-Glucosidase inhibitory activity, expressed as inhibition (%),was calculated as as in Eq 1.
where Ac is the difference between the absorbance values of the control at 5 znd 0 min, and As is the difference between the absorbance values of the sample at 0 and 5 min.

α-Glucosidase inhibitory activity
Table 1 shows the α-glucosidase inhibitory activity of the crude extracts and two isolated compounds from blueberry flowers.The ethyl acetate and n-buthanol soluble fractions showed promising α-glucosidase inhibitory activity compared with the positive control drug, acarbose.The IC 50 values of ethyl acetate and nbuthanol fractions, and acarbose were 132.7 μg/mL, 32.3 μg/mL and 129.6 μg/mL (or 200.7 μM), respectively.Although the n-buthanol fraction showed better activity than ethyl acetate fraction, HPLC chromatography (data not shown) showed that the ethyl acetate extract exhibited more detectable peaks.Therefore, further isolation was conducted on the ethyl acetate fraction.
The α-glucosidase inhibitory activity of the two isolated compounds was tested at the original concentration of 2 mg/mL.Compound 1 showed > 50 % inhibition activity (p  0.05), while compound 2 showed < 50 % ( p > 0.05).Hence, compound 1 was further tested and the IC 50 was calculated (Table 1).

DISCUSSION
Compound 1 showed better yeast α-glucosidase inhibitory activity than the positive drug acarbose (Table 1), which was in agreement with previous reports that many flavonoids from plants have been reported as α-glucosidase inhibitors [11].The present study also suggests that coumaroylated moiety could increase the activity of the flavonoid glycosides, which was in agreement with those concluded from the studies of acylated anthocyanins and flavonol monorhamnosides against α-glucosidase [12,13].

Figure 1 :
Figure 1: Chemical structures of the compounds (1 and 2) isolated from blueberry flowers.

1 H-NMR and 13 C
-NMR spectrum of compound 2 exhibited similar signals to the coumaroyl moiety mentioned above.The coupling constant of J = 12.8 Hz indicated a cis-configuration.The structure of compound 2 was confirmed as ciscinnamic acid methyl by comparison with published NMR data[9].

Isorhamnetin- 3 -
O-(6''-O-coumaroyl)-β-D-glucoside (1) is a promising α-glucosidase inhibitor.Therefore, blueberry flower is potentially a good source of α-glucosidase inhibitors for hyperglycemic therapy.Further studies are needed to continue to isolate other the chemical constituents of the active fractions and to determine the α-glucosidase inhibitory mechanism of the active compound (Compound 1).

Table 1 :
Yeast α-glucosidase inhibitory activity (mean ± SD, n = 3) of crude extracts and isolated compounds of Vaccinium corymbosum IC50 values of isolated pure compounds expressed as μM; data shown as mean ± SD (n = 3); different letters within the same column indicate significant difference at p  0.05 by Duncan's test. c