Phenolics from Bidens bipinnata and their amylase inhibitory properties

Fitoterapia 83 (2012) 1169–1175

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Phenolics from Bidens bipinnata and their amylase inhibitory properties Xiao-Wei Yang a, 1, Min-Zhu Huang a, b, 1, Yong-Sheng Jin a,⁎, Lian-Na Sun a, Yan Song a, Hai-Sheng Chen a,⁎ a b

School of Pharmacy, Second Military Medical University, Shanghai, 200433, China No. 117th Hospital of PLA, Hangzhou, 310012, China

a r t i c l e

i n f o

Article history: Received 6 December 2011 Accepted in revised form 6 July 2012 Available online 17 July 2012 Keywords: Bidens bipinnata Flavonoids Biscoumaric acid derivative α-Amylase inhibitory activity

a b s t r a c t A new chlorinated flavonoid, 3, 6, 8-trichloro-5, 7, 3′, 4′-tetrahydroxyflavone (1), a new biscoumaric acid derivative, 4-O-(2″, 3″-O-diacetyl-6″-O-p-coumaroyl-β-D-glucopyranosyl)-pcoumaric acid (2), and 8, 3′, 4′-trihydroxyflavone-7-O-β-D-glucopyranoside (3) together with twenty-four known compounds (4–27) were isolated from the whole plant of Bidens bipinnata. All chemical structures were established on the basis of UV-, MS- and NMR (1H, 13C, 1H–1H COSY, HMQC and HMBC) spectroscopic data. Some of the isolated compounds were tested for the inhibition of α-amylase. The result showed that isookanin (6) was a potent inhibitor of α-amylase (IC50 =0.447 mg/ml). © 2012 Elsevier B.V. All rights reserved.

1. Introduction Bidens bipinnata belongs to the Asteraceae family, and is widely distributed in tropical, subtropical and temperate regions [1]. The whole herb has been used as folk medicine against various diseases, such as sore throat, chronic diarrhea, malaria, dysentery, acute nephritis, etc. [2]. Pharmacological studies showed that the whole plant of B. bipinnata also has antihypertensive, anti-liver fibrosis and antidiabetic activities [3,4]. In Southwest of China, B. bipinnata has been used to treat diabetes with a long history. Anecdotal information also received from Traditional Healers on B. bipinnata used to treat diabetic disease. Biological effects have been evidenced [5,6] that its ethyl acetate extract has potent anti-diabetic activities in diabetic rats. But none of researches has revealed the most potent components of B. bipinnata which is responsible for the anti-diabetic activity. α-Amylase belongs to the family of glycoside hydrolase enzymes that break down starch into glucose molecules by acting on α-1,4-glycosidic bonds. It is now believed that inhibition of α-amylase can significantly decrease the ⁎ Corresponding authors. Tel./fax: +86 21 81871250. E-mail addresses: [email protected] (Y.-S. Jin), [email protected] (H.-S. Chen). 1 These two authors shared the first authorship. 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.07.005

postprandial increase of blood glucose level after a mixed carbohydrate diet and therefore can be an important strategy in the management of hyperglycemia linked to type 2 diabetes [7]. During our search for biologically active compounds for the treatment of diabetes, an ethyl acetate and a n-BuOH extracts derived from B. bipinnata were investigated and led to the isolation of three new compounds along with twenty-four known compounds. This paper describes the structural characterization of these new compounds and reports on the αamylase inhibitory activity of some isolated compounds. 2. Experimental section 2.1. General methods Optical rotations were measured on a Perkin-Elmer 341 polarimeter. IR and UV spectra were recorded on Bruker Vector-22 and Shimadzu UV-2550 UV-visible spectrophotometers, respectively. NMR spectra were obtained on a Bruker Avance 600 MHz or Avance 300 MHz NMR spectrometer in MeOD or DMSO-d6 with TMS as an internal standard. ESI-MS and HRESI-MS were acquired on a LC-MS (Thermo scientific, Finnigan LCQ Deca XP MAX) and a Q-TOF micro mass spectrometer (Waters, Milford, MA), respectively.

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Column chromatography was performed by using silica gel (200–300 mesh and 10–40 μm; Jiangyou Silica Gel Development Co. Ltd., Yantai, China), Sephadex LH-20 (40–70 μm; Amersham Pharmacia Biotech AB, Uppsala, Sweden) and macroporous adsorptive resin (AB-8; The Chemical Plant of Nankai University, Tianjin, China). Zones were visualized under UV light (254 nm) or by spraying with 10% H2SO4 followed by heating. Alphaamylase from human saliva (Wako Pure Chemicals Ind., Ltd., Osaka, Japan) was used for inhibition of α-amylase assay. 2.2. Plant material The whole plant of B. bipinnata was collected from Kunming, Yunnan province, Southwest China, during August 2007. The plant was identified by Prof. Han-ming Zhang, Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai, China. The voucher specimen has been deposited in the herbarium of the School of Pharmacy, Second Military Medical University (herbarium No. 060916). 2.3. Extraction and isolation Air-dried and powdered whole plant (15 kg) of B. bipinnata was extracted with 80% ethanol (3×80 L) at room temperature 3 times for 24 h. After removal of the solvent from the combined extracts under reduced pressure, a crude extract was obtained. The crude extract was suspended in water and extracted with petroleum ether, ethyl acetate and n-butanol. The ethyl acetate extract (100 g) was subjected to silica gel column chromatography, eluting with a step gradient from 0 to 100% MeOH in CHCl3 to afford six fractions (Fr. 1–6). Fraction 3 was chromatographed on a silica gel column with increasing polarities of CHCl3–MeOH (0–80% MeOH in CHCl3) to give 6 subfractions (Fr. I–VI). Fraction III (1.7 g) was subjected to Sephadex LH-20 with MeOH–H2O (70%) as eluent to afford 1 (8 mg), 2 (13 mg), 4 (28 mg), 5 (13 mg), 6 (28 mg) and 23 (22 mg). Fraction IV (4.5 g) was achieved by reversed phase silica gel (RP-18) and over Sephadex LH-20 repeatedly to yield eight compounds: 3 (26 mg), 7 (32 mg), 8 (38 mg), 9 (45 mg), 10 (25 mg), 11 (35 mg), 12 (30 mg) and 13 (32 mg). The n-BuOH extract (70 g) was subjected to macroporous adsorptive resin (AB-8) with H2O, 30%, 60%, and 90% EtOH as eluent respectively to afford 4 fractions (Fr. A–D). Fr. B (20 g) was chromatographed on silica gel column and eluted with increasing polarities of CHCl3–MeOH to give 4 subfractions (Fr. I–IV). Fr. III was fractioned by column chromatography over Sephadex LH-20 using MeOH as eluent to yield 14 (13 mg), 15 (60 mg) and 16 (11 mg). Fr. IV was chromatographed over Sephadex LH-20 with 70% MeOH–H2O as eluent to give 17 (15 mg), 18 (10 mg), 19 (27 mg), 20 (350 mg), 21 (25 mg), 22 (20 mg), 24 (31 mg), 25 (18 mg), 26 (25 mg) and 27 (28 mg). Compound 1: UV λMeOH max nm: 366.0 and 283.5; +NaOAc: 365.0 and 293.5; +NaOAc + H3BO3: 284.0 and 390.5; +AlCl3 + HCl: 401.0 and 283.5; IR νmax (KBr): 3390 (OH), 2945, 1710 (C_O), 1655, 1590, 1490 cm−1; 1H and 13C NMR spectroscopic data, see Table 2; negative ion ESI-MS m/z 386.93 [M− H]−; HRESI-MS m/z 386.9233 [M− H]− (calcd for C15H7Cl3O6 − H, 386.9229). 20 ¼ −45:3 (c 0.18, MeOH); UV λMeOH Compound 2: ½α  D max nm: 217.5, 223.0, 284.0, 294.0, 303.5; IR νKBr max cm−1:

3419 (OH), 1739 (C_O), 1707 (C_O), 1633 (>C_C), 1279, 1227, 1163 (C\O) and 823;1H and 13C NMR spectroscopic data, see Table 3; negative ion ESI-MS m/z 555.38 [M−H]−, 1110.71 [2M−H]−; HRESI-MS m/z 579.1497 [M+Na]+ (calcd for C28H28O12 +Na, 579.1479). 20 Compound 3: ½α  ¼ þ26:2 (MeOH) UV λMeOH max D nm: 320.5, 258.0; C21H20O11 by ESI-MS at m/z 447 [M−H]− and HRESI-MS at m/z 447.0930 [M−H]− (calcd for C21H20O11 − H, 447.0927), 1H and 13C NMR spectroscopic data see Table 4. 2.4. Inhibition of α-amylase assay The Caraway iodine/potassium iodide (IKI) method [8] was applied with slight modifications to determine the amount of starch hydrolyzed (absorbance at 620 nm). And the widely described drug acarbose was used as positive control. In brief, 100 μl test solution of certain concentration or distilled water (negative control) and 900 μl of 0.01 M sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride) containing α-amylase solution (0.5 mg/ml) or distilled water (blank) were incubated at 37 °C for 10 min. After preincubation, 500 μl of a 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride) was added to each tube. The reaction mixture was then incubated at 37 °C for 10 min. The reaction was stopped with 1.0 ml 10% HCl. This was followed by addition of about 300 μl of the working indicator (iodine-potassium iodide solution). Finally, the reaction mixture was then diluted after adding 10 ml distilled water and absorbance was measured at 620 nm. In the presence of an α-amylase inhibitors less starch would be hydrolyzed into maltose or glucose, and the absorbance value would be decreased. The inhibition of α-amylase activity was calculated as follows:   ðAbss −Absb Þ Inhibitionð% Þ ¼ 1−  100; ðAbscþ −Absc− Þ where Absc+, Absc−, Abss and Absb are defined as absorbance of 100% enzyme activity (only solvent with the enzyme), 0% enzyme activity (only solvent without the enzyme), a test sample (with the enzyme) and a blank (a test sample without the enzyme), respectively. 3. Results and discussion Compound 1 (Fig. 1) was isolated as light yellow powder. ESI-mass spectrum of 1 gave [M+ H]+ at m/z 388.94. The isotopes M + 2 peak and M + 4 peak were observed and the ratio of the M + 4 peak to M + 2 peak to the molecular ion peak was 1:3:3, which suggested the existence of three chlorine atoms in the compound. HRMS of 1 gave [M− H]− at m/z 386.9233 (calcd for C15H7Cl3O6 − H, 386.9229) and established the molecular formula of 1 as C15H7Cl3O6 from the experimentally measured masses of 35Cl and 37Cl isotopes which were 388.9217 (calcd for C15H6O635Cl237Cl) and 390.9180 (calcd for C15H6O635Cl 37Cl2), respectively. As mentioned above, the mass spectrum clearly showed the characteristic isotope molecular ions for three chlorine atoms. UV absorption maxima at 366

X.-W. Yang et al. / Fitoterapia 83 (2012) 1169–1175

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Fig. 1. Chemical structures of compounds 1, 2 and 3.

and 283.5 nm were indicative of the presence of a flavone. The 1 H-NMR spectrum of 1 showed signals for three protons in the aromatic region at δ 7.51 (1H, d, J = 2.0 Hz), δ 7.44 (1H, dd, J = 2.0, 9.0 Hz) and δ 6.96 (1H, d, J = 9.0 Hz), and their corresponding carbon signals appear at δ 116.5, 122.0 and 115.5, respectively. These three protons were readily assigned as aromatic protons H-2′, H-6′ and H-5′ of a flavone ring B. And compared with the 13C-NMR spectrum of luteolin (27), the data of B ring were very similar (Table 2). A (D2O) exchangeable signal at δ 13.04 (1H, s) not linked to any carbon resonance in the HMQC spectrum, correlated with C-6 (δ 104.5) and C-10 (δ 103.3) in the HMBC spectrum was assigned to a chelated hydroxyl group at C-5. In its 13C-NMR spectrum, the lack of signals at δC 130–140 (the signals of C-3 of a flavonol), and observation of signal at 113.0 ppm suggested that a chlorine atom linked at the C-3 position, because the electronegativity of oxygen atom is greater than chlorine atom, which caused downfield shift of the C-3. For further determination of the position of hydroxyl group for compound 1, we conducted UV experiments with addition of UV diagnostic reagent as a tool. The result was shown in Table 1. The 10 nm red shift of band II in UV spectra with addition of NaOAc (unmelted) indicated a hydroxyl group at position 7; the 35 nm red shift of band I in UV spectra with addition of AlCl3/HCl indicated a hydroxyl group at position 5; the 24.5 nm red shift of band I in UV spectra with addition of NaOAc/H3BO3 and a 40 nm blue shift of band I in UV spectra with addition of AlCl3/HCl indicated there was an ortho-phenolic hydroxyl group in ring B, and a 35 nm red shift of band I in UV spectra with addition of AlCl3/ HCl indicated there was an hydroxyl group at position 5. Thus, the rest to chlorine atoms were placed at C-6 and C-8. In comparison with isolated known compound luteolin (27), the downfield shift of the C-3 (Δδ=10.2 ppm), C-6 (Δδ=5.7 ppm), and C-8 (Δδ=5.3 ppm) signals and upfield shift in C-2 (Δδ= 3.1 ppm), C-4 (Δδ=5.7 ppm), C-5 (Δδ=7.2 ppm), C-7 (Δδ= 14.6 ppm) and C-9 (Δδ=7.0 ppm) further agreed with the presence of chlorine atoms at C-3, 6, 8 (Table 1). Thus the structure of compound 1 was unambiguously elucidated as 3, 6, 8-trichloro-5, 7, 3′, 4′-tetrahydroxyflavone (see Fig. 1). The 1H NMR and 13C NMR spectrum data were shown in Table 1. The chlorinated flavone compound 1 is a very interesting discovery, because chlorinated flavonoid is very rare, especially

in higher plants [9]; and this is the first chlorinated flavonoids found in B. bipinnata. The structure of luteolin (27) was very similar to compound 1 and the only difference between them was the existence of 3, 6, 8-trichloro substituents. In the process of extraction no chlorinated solvents were used and the only chlorinated solvent CHCl3 was used in the process of silica gel column chromatography, so the possibility that compound 1 was artificial was very low. To further prove that compound 1 is a real natural product, luteolin and mixed luteolin with other isolated flavones were treated with methanol–chloroform (0–80% MeOH in CHCl3) to imitate the isolation conditions (different temperatures, and various lengths of time). After those compounds were evaluated by LC-MS, no artificial chlorinated flavones were found. To date, more than 4500 halogenated natural products have been discovered [10], but which commonly appear in the metabolites by bacteria, fungi or marine organisms. The

Table 1 1 H NMR and

13

C NMR data of compound 1 and luteolin (27) in DMSO-d6.

Compound 1 1

H NMR

2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ \OH

a

7.51 (1H, d, J = 2.0)

6.96 (1H, d, J = 9.0) 7.44 (1H, dd, J = 2.0, J = 9.0) 13.04 (1H, s) 9.98 (1H, s) 9.55 (1H, s)

Luteolin 13

C NMRa 160.8 qC 113.0 qC 175.9 qC 150.0 qC 104.5 qC 149.6 qC 99.2 qC 154.4 qC 103.3 qC 120.7 qC 116.5 CH 145.5 qC 156.8 qC 115.5 CH 122.0 CH

1

7.40 (1H, d, J = 2.4)

13 C NMRa 163.9 qC 102.8 CH 181.6 qC 157.2 qC 98.8 CH 164.2 qC 93.8 CH 161.4 qC 103.6 qC 118.9 qC 113.4 CH

6.88 (1H, d, J = 9.0)

145.7 qC 149.7 qC 116.0 CH

H NMR

6.65 (1H, s, H-3)

6.18 (1H,d, J = 1.8) 6.44 (1H, d, J = 1.8)

7.41 (1H, dd, J = 2.4, J = 9.0) 12.95 (1H, s)

121.5 CH

Carbon multiplicities were determined by DEPT experiments.

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Fig. 2. Chemical structures of compounds 4–27.

X.-W. Yang et al. / Fitoterapia 83 (2012) 1169–1175

larger representation of chlorinated and brominated metabolites probably reflects the abundance of chloride and bromide ions in microenvironments of terrestrial and marine producer organisms [11]. Chlorinated flavonoids are extremely rare natural products, so the biogenesis of compound 1 was properly related to the microenvironments. Compound 2 (Fig. 1) was obtained as white powder that analyzed for the molecular formula C28H28O12 by HRESI-MS at m/z 579.1497 [M+ Na]+(calcd for C28H28O12 Na 579.1479). The 1H NMR and 13C NMR data of compound 2 could be readily attributed to phenylpropanoid glycosides. Eight aryl proton signals (2H, δ 6.79, d, J = 7.8 Hz, H-3′, H-5′; 2H, δ 7.01, d, J = 7.8 Hz, H-3, H-5; 2H, δ 7.55, d, J = 7.8 Hz, H-2′, H-6′; 2H, δ 7.61, d, J = 7.8 Hz, H-2, H-6) were attributed to two 1,4-disubstituted (A2B2 system) moieties. Another four signals at δH 6.40 (1H, d, J = 16.0 Hz, H-8), δH 6.42 (1H, d, J =16.0 Hz, H-8′), δH 7.49 (1H, d, J =16.0 Hz, H-7), δH 7.52 (1H, d, J = 16.0 Hz, H-7′) were attributed to two sets of trans-olefinic protons. From this combined information it was possible to deduce the presence of two trans-p-coumaroyl groups. The 1H NMR spectrum also revealed the presence of two acetyl groups [δ 2.01 (3H, s) and 1.98 (3H, s)] and two (D2O) exchangeable signals at δ 10.02 (1H, s) and 12.26 (1H, s, \COOH). Additionally, a series of signals between δ 3.63 and 5.56 indicated a sugar moiety (anomeric proton H-1″ [δ 5.56 (1H, d, J =7.8 Hz)]), and six signals between δC 60 and 100 in the 13C NMR spectrum indicated a glucoside. Moreover, the large coupling constant (d, J = 7.8 Hz) of the characteristic anomeric proton (δ 5.56, H-1″) indicated that the sugar was in the β-configuration. The HMBC spectrum showed correlations from H-1″ (δ 5.56) to C-4 (δ 157.81), from H-6″ (δ 4.43) to C-9′ (δ 166.34), from H-2″ (δ 5.15) to one acetyl carbonyl (δ 169.21), and from H-3″ (δ 4.96) to the other acetyl carbonyl (δ 169.68), thus indicating that the two acetyl groups are placed at C-2″ and C-3″, respectively, while the two p-coumaroyl groups were linked at C-1″ and C-6″ (Fig. 3). This assumption was subsequently confirmed by comprehensive analysis of 1H–1H COSY, HMQC, and HMBC data, which allowed the complete assignment of all 1H and 13C NMR signals (Table 2), confirming the structure assignment as shown in Fig. 1. Compound 3 (Fig. 1) was obtained as pale yellow powder, analyzed for the molecular formula C21H20O11 by ESI-MS at m/z 447 [M−H]− and HRESI-MS at m/z 447.0930 [M−H]− (calcd for C21H20O11 −H, 447.0927), which was further confirmed by 13 C NMR and DEPT spectra. Analysis of the 1H-NMR and HMQC spectra assigned three broad 1H singlets at δ 8.5–10.0 as phenolic protons. Three aromatic protons signals (δ 7.46, d, J=2.0 Hz; δ 7.35, dd, J=2.0 Hz, 8.5 Hz; δ 6.87, d, J=8.5 Hz) indicated an ABX system on ring B, and these proton signals were assigned to H-2′, H-6′ and H-5′ by HMQC analysis. Likewise, another three aromatic protons signals (δ 7.21, d, J=8.0 Hz; δ 7.07, d, J= 8.0 Hz; δ 6.71, s) were readily assigned to H-5 and H-6 of ring A and H-3 of ring C. Moreover, the large coupling constant (d, J= 8.0 Hz) of the characteristic anomeric proton (δ 4.94) indicated that the sugar was in the β-configuration. Comparing the 1H and 13 C NMR data with known compound 22 [12], the moiety was identified as β-D-glucopyranoside. In HMBC spectrum (Fig. 4), the correlations from H-5 (δ 7.21) to C-4 (δ 182.3), C-5 (δ 124.8) and the signal at δ 152.2, H-6 (δ 7.07) to the signal at δ 152.2 and C-6 (δ 112.8), C-5 (δ 124.8), indicated the signal at δ 152.2 was C-7. And H-3 (δ 6.71) to C-4 (δ 182.3) and C-1′ (δ 123.3), and

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Fig. 3. The key HMBC of compound 2.

anomeric proton (δ 4.94) to C-7 (δ 152.2) confirmed the structure as 8,3′,4′-trihydroxyflavone-7-O-β-D-glucopyranoside, which was further confirmed by comparing the 1H and 13C NMR data with known compound 22 (Table 4). The 1H NMR and 13C NMR spectrum were shown in Table 3. By comparing their physical and spectroscopic data with those reported in the literature or analyzing their 2D NMR spectral data, the known compounds were identified as okanin 4′-O-β-D-(2″,4″,6″-triacetyl)-glucopyranoside (4) [13], 4-O-(6″-O-p-coumaroyl-β-D-glucopyranosyl)-pcoumaric acid (5) [2], isookanin (6) [14], 3, 5-di-Ocaffeoylquinic acid (7) [15], 3,5-di-O-caffeoylquinic acid methyl ester (8) [15], 6,7,3′,4′-tetrahydroxy aurone (9) [16], 7,8-dihydroxycoumarin (10) [17], 4,5-di-O-caffeoylquinic acid (11) [18], hyperoside (12) [12], 5,8,4′-trihydroxyflavone‐ 7-O-β-D-glucopyranoside (13) [19], 5,3′-dihydroxy‐3,4′-dimethoxyflavone-7-O-β-D-glucopyranoside (14) [20], chlorogenic acid (15) [21], methyl chlorogenate (16) [22], okanin 4-methyl ether-3′-O-β-D-glucoside (17) [23], okanin 4′-O-β-D(6″-trans-p-coumaroyl)-glucopyranoside (18) [13], okanin Table 2 1 H NMR and

13

C NMR data of compound 2 in DMSO-d6. 1

P-coumaroylmoiety 1 2,6 7.61 3,5 7.01 4 7 7.49 8 6.40 9 1′ 2′,6′ 7.55 3′,5′ 6.79 4′ 7′ 7.52 8′ 6.42 9′ Glucosemoiety 1″ 2″ 3″ 4″ 5″ 6″

13

C NMRa

H NMR

5.56 4.96 5.15 3.63 4.01 4.25 4.43

(2H, d, J = 7.8) (2H, d, J = 7.8) (1H, d, J = 16.0) (1H, d, J = 16.0)

(2H, d, J = 7.8) (2H, d, J = 7.8) (1H, d, J = 16.0) (1H, d, J = 16.0)

(1H, (1H, (1H, (1H, (1H, (1H, (1H,

d, J = 7.8) t, J = 8.4) t, J = 8.4) m, overlap) m, overlap) d, J = 9.6, J = 10.8) d, J = 6.0, J = 10.8)

OAc 2.01 (3H, s) 1.98 (3H, s) a

125.0 129.8 115.8 157.8 145.0 113.8 167.6 128.7 130.4 116.6 159.9 143.2 117.6 166.3

96.8 71.2 73.5 67.6 74.6 62.6

qC CH CH qC CH CH qC qC CH CH qC CH CH qC

CH CH CH CH CH CH2

169.7 qC 169.2 qC 20.7 CH3 20.4 CH3

Carbon multiplicities were determined by DEPT experiments.

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X.-W. Yang et al. / Fitoterapia 83 (2012) 1169–1175 Table 4 The inhibition of α-amylase activities by the selected compounds.

Fig. 4. The key HMBC of compound 3.

(19) [12], centaurein (20) [24], quercitin-3,4′-dimethyl ether-7-O-rutinoside (21) [17], isookanin-7-O-β-Dglucopyranoside (22) [12], 7,3′,4′-trihydroxyflavone (23) [25], 3,5,7-trihydroxy-3′,4′-dimethoxyflavone (24) [26], 3,5dihydroxy-3′,5′-dimethoxyflavone-7-O-β-D-glucopyranoside (lagotiside) (25) [27], 3,4,2′,3′-tetrahydroxychalcone-4′-Oβ-D-glucopyranoside (Okanin-4′-glc) (26) [28] and luteolin (27) [29] (see Fig. 2). The α-amylase inhibitory activities of some of the compounds isolated from the ethyl acetate fraction of B. bipinnata were determined. The results were shown in Table 4. Compound 6, isookanin, showed potent inhibitory activities (Table 5) with IC50 =0.447 mg/ml. The sensitivity of glycosidases to various inhibitors depends critically on their origin, so the α-amylase from human saliva was used for our preliminary screening in order to improve screening efficiency. Our previous study showed that the ethyl acetate extract of B. bipinnata has potent anti-diabetic activities in diabetic rats. But screened isolated

Compd.a

Abss (x  s)

Absb (x  s)

Inhibition (%)

2 3 6 15 17 19 20 21 26 27 acarbose

0.314 ± 0.005 0.520 ± 0.013 0.418 ± 0.007 0.240 ± 0.004 0.248 ± 0.003 0.365 ± 0.008 0.341 ± 0.024 0.220 ± 0.003 0.510 ± 0.004 0.127 ± 0.004 0.452 ± 0.002

0.543 ± 0.007 0.760 ± 0.007 0.556 ± 0.021 0.505 ± 0.004 0.510 ± 0.008 0.525 ± 0.004 0.602 ± 0.027 0.480 ± 0.004 0.693 ±0.058 0.548 ± 0.020 0.459 ± 0.010

25 22 55 14 15 48 15 15 41 −37 97

a Concentration = 0.556 mg/ml, Absc+ and Absc− of negative control were 0.153 ± 0.001 and 0.460 ± 0.012, respectively.

Table 5 The inhibition of α-amylase activities of compound 6. Concentration (mg/ml)

Abss (x  s)

Absb (x  s)

Inhibition (%)

IC50 (mg/ml)

0.556 0.185 0.062 0.015 0.556 (acarbose)

0.446 ± 0.004 0.408 ± 0.009 0.378 ± 0.017 0.369 ± 0.008 0.466 ± 0.010

0.484 ± 0.014 0.478 ± 0.029 0.488 ± 0.037 0.489 ± 0.012 0.484 ± 0.010

65 39 −2 −11 95

0.447

Abs + and Abs − of negative control were 0.132 ± 0.004 and 0.489 ± 0.010, respectively. Table 3 1 H NMR and

13

C NMR data of compounds 3 and 22 in DMSO-d6.

Compound 3 1

H NMR

2 3

4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″

Compound 22 13

C NMR

154.0 6.71(1H, s)

7.21 (1H, d, J = 8.0 Hz) 7.07 (1H, d, J = 8.0 Hz)

7.46 (1H, d, J = 2.0 Hz)

6.87 (1H, d, J = 8.0 Hz) 7.35 (1H, dd, J = 2.0, 8.0 Hz) 4.94 (1H, d, J = 8.0 Hz) 3.31(1H, m) 3.36 (1H, m) 3.20 (1H, m) 3.47 (1H, m) 3.19 (1H, m), 3.72 (1H, m)

112.0

182.3 124.8 112.8 152.2 132.4 148.2 117.1 123.3 114.3 145.5 145.4 116.0 118.3

1

H NMR

5.41 (1H, dd, J = 12.0, 3.0 Hz) 2.70 (1H, dd, J = 12.0, 3.0 Hz) 3.10 (1H, dd, J = 12.0, 3.0 Hz) 7.23 (1H, d, J = 9.0 Hz) 6.86 (1H, d, J = 9.0 Hz)

6.89 (1H, d, J = 2.0 Hz)

6.75 (1H, d, J = 8.0 Hz) 6.78 (1H, dd, J = 2.0, 8.0 Hz)

13

C NMR

79.1 43.4

191.0 117.9

compounds did not show potent inhibitory activities except isookanin. So further studies on the constituents of B. bipinnata and their effects on the target enzymes of treatment of diabetes are needed. Therefore, our study revealed the anti-diabetic potential of isookanin isolated from the ethyl acetate extract of B. bipinnata for the first time and could be helpful in developing medicinal preparations or nutraceutical and functional foods for diabetes and related symptoms from B. bipinnata. Acknowledgments

108.9 150.7 135.1 150.7 116.5 130.1 114.2

This research is the result of financial support from the Shanghai Leading Academic Discipline Project (No. B906). We thank the Analytical and Testing Center of Second Military Medical University for the NMR and IR experiments and Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for the optical rotation determination.

145.1 145.5 115.2

Appendix A. Supplementary data

117.9

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2012.07.005.

101.5

101.5

73.2 75.8 69.6 77.3 60.6

73.7 75.8 69.7 77.0 60.6

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