Alliuocide G, a new flavonoid with potent α-amylase inhibitory activity from Allium cepa L.

Chemical investigation of the ethyl acetate fraction of the dried outer scales of Allium cepa L. afforded one new flavonoid named alliuocide G, together with four known compounds. Their structures were unambiguously established on the bases of NMR spectroscopy ( 1 H, 13 C, DEPT, and HMBC) and mass spectrometry. The isolated compounds showed potent α-amylase inhibitory activities, in addition the new compound had antioxidant activity.


Introduction
High dietary intakes of fruits and vegetables have been reported to correlate with a low risk of degenerative diseases from epidemiological evidence.The protective effects have been attributed partly to the various antioxidative compounds present in fruits and vegetables.The most abundant types of antioxidative compounds in human diet are flavonoids 1 .Previous phytochemical studies of the A. Cepa L. (family: Liliaceae) (white and red onion) have resulted the isolation of flavonoids [2][3][4][5][6] , especially high levels of quercetin and its derivatives 2 , anthocyanins 7,8 , thiosulfinates, sulfides, sulfoxides 3 and peptides 9 .This study undertook the isolation and structure elucidation of one new flavonoid named alliuocide G, together with four known compounds.The isolated compounds were tested for their α-amylase inhibitory activities, in addition the new compound was evaluated for its antioxidant activity using DPPH assay.

Results and Discussion
The methanolic extract of the air-dried brownish scales of onions was concentrated to dryness and successively partitioned between n-hexane, ethyl acetate, n-butanol and water.The ethyl acetate fraction afforded one new flavonoid named alliuocide G (2) and four known compounds (1, 3-5).

Extraction and isolation
White onions were purchased at the Faculty of Agriculture, Al-Azhar University, Assiut, Egypt.The air-dried brownish scales of onions (400 g) was extracted several times with methanol (2 L  5) and evaporated to yield 36 g.The latter was suspended in distilled water (100 ml) then partitioned between n-hexane (500 mL × 4), ethyl acetate (500 mL × 4) and n-butanol (500 mL × 3), successively.Each fraction was concentrated under reduced pressure to give n-hexane fraction (4.6 g), ethyl acetate fraction (8.6 g), n-butanol fraction (10.3 g) and aqueous (12.2 g) residue.The ethyl acetate fraction (8.6 g) was subjected to vacuum liquid chromatography (VLC) using CHCl3-MeOH gradients to afford 5 fractions.Fraction II (0.96 g) was chromatographed over silica gel column using CHCl3-MeOH gradients to get compound 1 (32 mg).Fraction III (2.3 g) was chromatographed on Sephadex LH-20 eluted with MeOH to yield subfractions A-C.Subfraction A (0.38 g) was subjected to silica gel column using CHCl3-MeOH gradients to afford compound 3 (26 mg).Subfraction C (0.71 g) was subjected to RP-18 column using MeOH-H2O gradient to give compound 2 (18 mg).Fraction V (3.7 g) was chromatographed over silica gel column using CHCl3-MeOH gradients to obtain compound 4 (13 mg), and 5 (44 mg).Further purification of compounds 4 and 5 were accomplished by RP-18 column eluted with MeOH-H2O gradients.α-Amylase inhibitory activity 10,12 The method is based on assay of α-amylase by EnzCheck ® Amylase Assay Kit (E-11954) was purchased from Molecular Probes (GmbH, Germany).The provided stock solution of DQ starch and α-amylase enzyme were diluted with the reaction buffer (pH 6.9) according to the reported protocol 12 .To the microplate wells, 50 µg/10 µL of the tested compound in DMSO, 50 µL of the diluted enzyme and 40 µL of the reaction buffer were added and allowed to stand for 5 min at room temperature then 100 L of DQ starch was added.The fluorescence intensity of the digestion products from the DQ starch (with or without compounds) was measured using a kinetic assay program in the Tecan Genios microplate reader at max 48510 nm starting from zero min to 60 min at 10 min intervals.All determinations were performed in triplicate.The αamylase inhibitory activity of each tested compound was measured in relation to acarbose (Ac) (Kohlpharmam GmbH, Germany) set as 100% α-amylase inhibitory activity.The percentage of α-amylase activity and α-amylase inhibition was calculated using the following equations:  All isolated compounds were evaluated for α-amylase inhibitory activity as shown in table 2, new compound 2 showed potent activity compared with acarbose.Apparently, α-amylase inhibitory activity increased with the presence of hydroxyl groups at 3`, and 4`-position of the Bring 13 .
Compound 2 was dissolved in HPLC MeOH to obtain a concentration of 20 m /mL.Then was mixed with DPPH (118  10 -5 ) and allowed to stand for half an hour for any reaction to occur.The UV absorbance was recorded at 517 nm.The experiment was performed in triplicate and the average absorption was noted.The antioxidant activity was calculated using the following equation; Compound 2 have 66.4 % antioxidant activity according to published structure activity relationship of flavonoids [15][16][17][18] compared with propyl gallate (a known synthetic antioxidant) set as 100 % antioxidant activity.
with the sample at 60 min, 0 s F : Fluorescence with the sample at 0 min, 60 c F : Fluorescence of the control at 60 min, 0 c F : Fluorescence of the control at 0 min.% α-amylase inhibition  100  % α-amylase activity.

Table 2 .
α-Amylase inhibitory activity of the isolated compounds