Two flavanone compounds from litchi (Litchi chinensis Sonn.) seeds, one previously unreported, and appraisal of their α-glucosidase inhibitory activities

Food Chemistry 127 (2011) 1760–1763

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Two flavanone compounds from litchi (Litchi chinensis Sonn.) seeds, one previously unreported, and appraisal of their a-glucosidase inhibitory activities Shen Ren, Duoduo Xu, Zhi Pan, Yang Gao, Zhenguo Jiang, Qipin Gao ⇑ Research and Development Center, Changchun University of Traditional Chinese Medicine, Changchun 130117, China

a r t i c l e

i n f o

Article history: Received 7 December 2010 Received in revised form 28 January 2011 Accepted 12 February 2011 Available online 17 February 2011 Keywords: Litchi chinensis seeds Flavanone compounds a-Glucosidase inhibitory activity

a b s t r a c t Using a bioactivity-guided method, a new and a previously reported flavanone glycoside were isolated from 50% ethanol extract of litchi (Litchi chinensis Sonn.) seeds. Column chromatography using macroporous resin, silica gel and Sephadex LH-20 was applied during the isolation, and the method of PMP derivatisation was performed to investigate the component sugar of the compounds. The structures of the compounds were elucidated by 1H, 13C, and 2D NMR (HMQC, HMBC) spectroscopy and HR-ESIMS. Compound 1 was identified as (2R)-naringenin-7-O-(3-O-a-L-rhamnopyranosyl-b-D-glucopyranoside), a new, natural product that had been synthesised previously. Compound 2 was (2S)-pinocembrin7-O-(6-O-a-L-rhamnopyranosyl-b-D-glucopyranoside). The two isolated compounds showed activity in the a-glucosidase inhibition assay. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Diabetes mellitus is a chronic disease not only in developed countries but also in developing countries because of changes in human lifestyles and dietary habits (Horton, 1995). a-Glucosidase plays an important role in the control of blood glucose levels in the body and is the key enzyme catalysing the final step in the digestive process of carbohydrates. Inhibitors of a-glucosidase retard the liberation of D-glucose from complex dietary carbohydrates and delay glucose absorption, thus reducing plasma glucose levels and suppressing post-prandial hyperglycaemia (Holman, 1998; Lebovitz, 1997; Murai et al., 2002). Several a-glucosidase inhibitors, such as acarbose (Chiasson et al., 2002), miglitol (Pogano et al., 1995) and voglibose (Murai et al., 2002) are used widely in clinics to regulate blood glucose levels of patients, although they can cause negative effects such as abdominal distension, flatulence, meteorism and possibly diarrhoea (Hollander, 1992). In recent years, many efforts have been made to search for potential and effective a-glucosidase inhibitors from natural sources to develop a physiologically functional food or lead compounds for antidiabetes treatment (Matsui et al., 2001; Mcdougall et al., 2005; Niwa, Doi, & Osawa, 2003; Yoshikawa, Morikawa, Matsuda, Tanabe, & Muraoka, 2002). Litchi (Litchi chinensis Sonn.) is a tropical and subtropical fruit of the Sapindaceae family, originating in south-east Asia. The fruits are favoured by consumers for their delicious taste, attractive

colour and abundant nutrition (Jiang, Duan, Joyce, Zhang, & Li, 2004). Presently, litchi arils are consumed both as fresh and processed fruits, whereas litchi seeds are used as a traditional Chinese medicine. Recent work has shown that litchi seeds could be used as a readily accessible natural antioxidant source, and could potentially be used as a functional food ingredient or natural preservative (Prasad et al., 2009). Furthermore, it was reported that litchi seeds possess anti-diabetic effects (Guo et al., 2003), and that they have a-glucosidase inhibitory (Yang & Liang, 2004), antihyperlipidaemic, antiplatelet, antitumour (Xu, Xie, Hao, Jiang, & Wei, 2010), and antiviral activities (Chen, Wu, Gu, & Chen, 2007). Previous chemical studies revealed that flavones are the main components of litchi seeds. The goal of the present study was to isolate and identify the constituents of flavones from litchi seeds and evaluate their anti-a-glucosidase inhibitory activities. 2. Experimental section 2.1. Plant materials Litchi ( L. chinensis Sonn.) seeds were purchased from a local herbal pharmacy in Changchun. The plant was identified by Professor Minglu Deng, Pharmacy College, Changchun University of Traditional Chinese Medicine. A voucher specimen was deposited in the herbarium of the same university. 2.2. Chemicals and reagents

⇑ Corresponding author. Tel./fax: +86 431 86172070. E-mail addresses: [email protected] (S. Ren), [email protected] (D. Xu), [email protected] (Z. Pan), [email protected] (Y. Gao), jiangzhenguo_2009 @163.com (Z. Jiang), [email protected] (Q. Gao). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.02.054

a-Glucosidase from baker’s yeast and p-nitrophenyl-a-D-glucopyranoside (PNPG), which was used as a synthetic substrate, were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO).

S. Ren et al. / Food Chemistry 127 (2011) 1760–1763

Acarbose (Glucobay, Bayer, Leverkusen, Germany), which was used as a synthetic inhibitor of a-glucosidase, was purchased from a local pharmacy. All 96-well plates were purchased from Corning Incorporated (Corning, NY). All other chemicals and solvents used for extraction and isolation were of analytical grade and were purchased from Beijing Reagent Company (Beijing, China).

2.3. General apparatus The 1H, 13C and 2D NMR (HMQC, HMBC) spectra were acquired on Bruker AV400 spectrometers using TMS as an internal standard. Chemical shifts are reported in d values. High resolution mass spectrometry experiments were recorded on an IonSpec Ultima 7T FT-ICR-MS (Varian, Palo Alto, CA) with an electrospray source operating in the negative ion mode. CD spectra were measured on a JASCO J-810 circular dichroism spectrometer (Peking University Health Science Center). A Multiscan MK3 microplate reader was purchased from Thermo Electron Corporation (Waltham, MA). Column chromatography was performed on macroporous resin D101 (Cangzhou BonChem Co. Ltd., China), silica gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., Shandong, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). All purifications were monitored by pre-coated thin-layer chromatography (TLC) plates with silica gel G and F254 (Qingdao Haiyang Chemical Co. Ltd., Shandong, China). All TLC spots were visualised under UV light (k = 254 and 365 nm) and were stained with a 10% H2SO4 solution in ethanol followed by heating.

2.4. Extraction and isolation The dried litchi seeds (10 kg) were crushed with a hammer and then extracted three times with 50% aqueous ethanol for 2 h at 70 °C. The concentrated material was suspended in water, reextracted with chloroform and then extracted with n-butanol. Each fraction was evaporated under vacuum to yield chloroform, n-butanol and water fractions. The n-butanol fraction, which displayed the highest activity on a-glucosidase inhibitory assay, was divided into a water-soluble fraction (WS) and a water-insoluble fraction (WI). The WI fraction was applied to a D101 macroporous resin, and elution was initiated with 30% ethanol and increased with a gradient to 90% ethanol. The fractions collected in the 30–60% ethanol range were combined according to activity and eluted on a silica gel column with CHCl3-MeOH mixtures, gradually increasing the amount of MeOH to 100% MeOH. The fractions were concentrated in vacuo, resulting in seven fractions (fractions 1–7) based on their behaviours on the TLC plates. Fraction 6 was chromatographed on a Sephadex LH-20 column with MeOH:CHCl3 (4:1) to obtain a subfraction with two yellow spots on the TLC plate; it was further subjected to a silica gel using CHCl3:MeOH:HCOOH (10:1:0.1) as eluent to yield Compound 1 (4 mg, 0.000025%), and semi-pure Compound 2 (8 mg, 0.00008%), which was further purified by Sephadex LH-20 using MeOH.

2.5. Characteristic data of compounds (2R)-Naringenin-7-O-(3-O-a-L-rhamnopyranosyl-b-D-glucopyranoside) (1): pale yellow solid, HR-ESIMS: m/z 579.17313 [MH], calculated for C27H32O14; UV kMeOH nm: 283, 327. CD: max [h]330  3375, [h]288 + 350. 1H, 13C NMR and HMBC: See Table 1. (2S)-Pinocembrin-7-O-(6-O-a-L-rhamnopyranosyl-b-D-glucopyranoside) (2): pale yellow solid, HR-ESIMS: m/z 563.17530 [M-H], calculated for C27H32O13; UV kMeOH max nm: 284, 327. CD: [h]330 + 2562, [h]282  27905, [h]218 + 26436. 1H, 13C NMR: See Table 1.

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2.6. a-Glucosidase inhibitory assay The a-glucosidase inhibitory experiment was performed according to the chromogenic method described by Kim, Wang, and Rhee (2004), with some modifications, using a-glucosidase from baker’s yeast. A substrate solution of p-nitrophenyla-D-glucopyranoside was prepared in a phosphate buffer and adjusted to pH 6.8 to simulate intestinal fluid. A 40 lL solution (0.04 units/ml, dissolved in phosphate buffer, pH 6.8) of a-glucosidase was preincubated at 37 °C for 5 min with 40 lL of the respective test solution (dissolved in phosphate buffer, pH 6.8, solutions of acarbose and samples at various concentrations). The blank solution had phosphate buffer in place of the sample solution. The enzymatic reaction was initiated by adding 20 lL of PNPG (0.5 mM), and the mixture was incubated for another 30 min at 37 °C. The reaction was terminated by adding 100 lL of sodium carbonate solution (0.1 M, pH 9.8). Inhibition of a-glucosidase was determined by measuring the optical density (OD) of the p-nitrophenol released from PNPG at 405 nm using a microplate reader. The a-glucosidase inhibitory activity was calculated using following formula:

Inhibition ratioð%Þ ¼ 100  ðODblank  ODsample Þ=ODblank 2.7. PMP derivatisation Fraction 6 (10 mg) was hydrolysed with 2 M trifluoroacetic acid (10 ml) for 8 h at 100 °C in a sealed glass tube. The excess acid was removed completely at 70 °C by nitrogen flow, and the hydrolysed products were extracted with CHCl3 and H2O (v/v 1:1) twice. The H2O layer was evaporated to dryness to prepare for derivatisation. The reaction was carried out (Yang et al., 2005) by mixing the H2O fraction of hydrolysed products and 5 ml of 0.3 M NaOH. Then, 6 ml of a 0.5 M PMP-methanol solution were added followed by incubation for 30 min in a 70 °C water bath. The sample was subsequently cooled to room temperature, and 0.3 M HCl was added to neutralise the solution to pH 7. Finally, the solution was extracted with CHCl3 three times to remove any of the remaining PMP reagent. The H2O extract was collected, and the derivatives of the saccharide were tested on an HPLC. Derivatisation of the standard reference material of monosaccharide used the same method. HPLC was performed on a Zorbax Eclipse XDB-C18 analytical column (Agilent 1200, Agilent, Santa Clara, CA) (i.d. 4.6 mm  150 mm) at 40 °C. The absorbance was measured at 250 nm. Gradient system A: Phosphate buffer (KH2PO4–NaOH, pH = 6.8): acetonitrile (85:15, V/V). Gradient system B: Phosphate buffer (KH2PO4– NaOH, pH = 6.8): acetonitrile (60:40, V/V), flow rate: 0.9 ml/min. The elution was performed with linear gradient profiles (Table 4).

3. Results and discussion 3.1. Bioassay-guided extraction The 50% ethanol extracts of the litchi seeds showed strong inhibitory activity toward a-glucosidase. The crude extract was partitioned by CHCl3, n-butanol and H2O. The n-butanol fraction demonstrated a higher enzyme inhibitory activity than the other two fractions. Then the n-butanol fraction was divided into a water-soluble (WS) fraction and a water-insoluble (WI) fraction. The enzyme assay showed that the inhibitory activity of the WI was higher than that for WS. Therefore, the WI was applied to a D101 macroporous resin and eluted with 30% to 90% ethanol in 10% ethanol increments. The fractions of 30%, 40%, 50% and 60% ethanol displayed significant inhibition to a-glucosidase, whereas the 70–90% ethanol fractions showed lower inhibition (Table 2).

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Table 1 1 H, 13C and HMBC (400/100 MHz) data for compounds 1 and 2 in CD3OD. 1

2

1

Position 2 3a 3b 4 5 6 7 8 9 10 10 20 , 60 30 , 50 40 Glu-10 0 20 0 30 0 40 0 50 0 60 0 Rha-10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 60 0 0

H (d, J = Hz) 5.43 (d, J = 14.4, 2.8) 3.19 (d, J = 16.8, 14.4) 2.78 (d, J = 16.8, 2.8)

6.19 (s) 6.17 (s)

7.32 (d, J = 7.8) 6.82 (d, J = 7.8) 5.12 (d, J = 7.5)

5.26 (br s)

1.18 (d, J = 6.0)

13

C 81.0 44.3

HMBC 20 , 60

198.8 165.3 98.2 167.0 97.1 165.0 105.3 131.1 129.4 116.7 159.5 100.0 78.6 81.9 70.8 78.5 62.6 102.6 71.2 72.0 74.0 68.4 17.1

3 6, 8 8 G-1 6

1

6.31 (s)

7.60 7.48 7.48 5.04

G-3 R-3

3.2.1. Isolation of Compound 1 and 2 The 30–60% ethanol fractions of the D101 macroporous resin were combined and were subjected to a silica gel column, eluting with CHCl3:MeOH mixtures. Fraction 6 (CHCl3:MeOH 10:3) showed activity in the a-glucosidase assay. This fraction was further fractionated by Sephadex LH-20 and silica gel column to yield Compounds 1 and 2. 3.2.2. PMP derivatisation The results of the PMP derivatisation showed that glucose and rhamnose were both in fraction 6, consistent with the NMR data. 3.2.3. Spectral analysis Compound 1: The HR-ESIMS of Compound 1 showed an [M-H] ion at m/z 579.17313, indicating a molecular formula of C27H32O14. Analysis of the 1D- and 2D-NMR spectra with heteronuclear direct or long-range correlations allowed assignment of 1H and 13C NMR signals, which are tabulated in Table 1. The 1H-NMR spectra of compound 1 showed AB and ABX type aromatic proton signals at d 6.17 (1H, s), 6.19 (1H, s), 6.82 (2H, d,

(d, J = 7.2) (m) (m) (d, J = 7.2)

4.781 (br s)

R-6 R-1 R-4, R-5

3.2. Isolation and identification of the compounds

198.4 165.3 98.4 167.2 97.4 164.6 105.3 140.5 127.8 130.1 130.1 101.5 75.0 78.2 71.6 77.5 67.7 102.5 72.4 72.7 74.4 70.1 18.2

6.31 (s)

6, 8 2, 20 , 60 , 30 , 50 20 , 60 30 , 50 & 20 , 60 G-3 G-3, G-5 G-2, G5,6 G-2,G-3,G-5,G-6 G-2, G-3

Using a bioassay-guided separation method, the constituents responsible for the effect on a-glucosidase were isolated.

13 C 80.9 44.5

H (d, J = Hz) 5.62 (dd, J = 12.4, 2.8) 3.25 (dd, J = 17.2, 12.4) 2.93 (dd, J = 17.2, 2.8)

1.27 (d, J = 6.0)

J = 7.8) and 7.32 (2H, d, J = 7.8) ppm, and the signals at d 2.78 (1H, dd, J = 16.8, 2.8), 3.19 (1H, dd, J = 16.8, 14.4), and 5.43 ppm (1H, d, J = 14.4, 2.8), which could be assigned to H-3 and H-2, respectively. Finally, two anomeric protons d 5.12 (d, J = 7.5) and 5.26 (br s) ppm and one tertiary methyl group at d 1.18 (d, J = 6.0) ppm were indicative of glucose and rhamnose as sugar moieties. In the 13C-NMR spectra, two anomeric carbons were observed at d 100.0, 102.6 ppm, and the signal due to C-7 (d 167.0 ppm) was shifted upfield and the signal due to C-300 (d 81.9 ppm) of glucose was shifted downfield. Moreover, the HMBC spectrum showed two correlation peaks of C-7 and C-100 of the glucose, and the C-300 of the glucose and C-100 0 of the rhamnose, the linkage between glucose and C-7 and C-300 of the inner glucose and terminal rhamnose were determined (Table 1). The 1H and 13C chemical shift assignments as well as the linkage between the anomeric carbon and protons of sugar were determined from HMQC and HMBC spectra. In addition, C-2 of the flavanone was a chiral carbon, by comparison with the published CD spectrum; it was determined to be in the R configuration (Prescott, Stamford, Wheeler, Firmin, 2002). According to these data, the structure of Compound 1 was established as (2R)-naringenin7-O-(3-O-a-L-rhamnopyranosyl- b-D-glucopyranoside). Compound 2: The HR-ESIMS of Compound 2 showed an [M-H] ion at m/z 563.17530, indicating a molecular formula of C27H32O13.

Table 2 Inhibition of a-glucosidase by litchi seeds extracts. Inhibition (%) mg/ml

Control 50% ethanol extract

n-butanol extract

CHCl3 extract

H2O extract

Water-insoluble fraction

Water-soluble fraction

30–60% ethanol fraction of macroporous resin

70–90% ethanol fraction of macroporous resin

1 0.1 0.01

89.1 72.3 63.7

99.0 79.6 59.5

64.2 43.8 15.9

92.6 89.7 52.1

98.9 97.9 58.1

93.5 81.2 55.0

95.4 89.0 50.4

83.0 63.9 57.1

99.7 98.7 61.5

S. Ren et al. / Food Chemistry 127 (2011) 1760–1763 Table 3 Inhibition of a-glucosidase by isolated compounds.

References

Inhibition (%) mg/ml 1 0.1 0.01

Control 91.3 75.5 62.3

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1 56.8 40.2 33.3

2 58.8 43.6 22.5

Table 4 HPLC gradient elution of the PMP derivatisation. Time (min)

Gradient A (%)

Gradient B (%)

0–10 10–30 30–40

100 ? 92 92 ? 80 80

0?8 8 ? 20 20

Comparison with the reported data in the literature suggested that Compound 2 was (2S)-pinocembrin-7-O-(6-O-a-L-rhamnopyranosyl-b-D-glucopyranoside) (Hanawa, Yamada, Nakashima, 2001; Li et al., 2009). (NMR data see Table 1.) 3.3. Biological activity of isolated compounds The isolated compounds were further examined for the inhibition of a-glucosidase. Table 3 shows the inhibitory activity of the isolated compounds. Although the potency of inhibition was still lower than the acarbose control, which could be caused by linkage of the saccharides, the observed data indicated the potency of the flavones as inhibitors to a-glucosidase. In general, the two compounds exhibited dose-dependent inhibitory activities. 4. Conclusions Although litchi seeds have been used to treat diabetes in traditional Chinese medicine, there are few reports regarding the chemical components or active compounds in its extract. In this study, we focused on the chemical components and inhibitory effect on a-glucosidase of litchi seeds. Two flavanone glycosides were obtained. The results from this study provide scientific support to the utilisation of litchi seeds for the treatment of diabetes, and the results demonstrate the potency of litchi seeds for medicinal preparations or functional foods. Acknowledgements We are grateful to Mr. Yang Xiu-wei, College of Pharmacy, Peking University, Mrs. Liu Shu-ying, Jilin Ginseng Academy and Mr. Liu Zhi-qiang, Changchun Institute of Applied Chemistry Chinese Academy of Sciences. This research was supported financially by the Technology Development Program of Jilin Province (No.20080719).

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