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α-Glucosidase and α-amylase inhibitory activities of guava leaves
Food Chemistry 123 (2010) 6–13
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a-Glucosidase and a-amylase inhibitory activities of guava leaves Hui Wang a, Yang-Ji Du b, Hua-Can Song a,* a b
School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China School of Chemistry and Environment, South China Normal University, Guangzhou 510006, China
a r t i c l e
i n f o
Article history: Received 18 November 2008 Received in revised form 31 January 2010 Accepted 22 March 2010
Keywords: Guava leaves a-Glucosidase and a-amylase inhibitor Flavonoid compounds Structure-activity relationship Diabetes
a b s t r a c t The 75% ethanol extract from guava (Psidium guajava Linn.) leaves was extracted further, in turn, with CH2Cl2, EtOAc and n-BuOH to afford four fractions, CH2Cl2-soluble, EtOAc-soluble, n-BuOH-soluble and residual extract fractions. Both the n-BuOH-soluble and EtOAc-soluble fractions showed high inhibitory activity against a-glucosidase and a-amylase. Seven pure flavonoid compounds, quercetin (1), kaempferol (2), guaijaverin (3), avicularin (4), myricetin (5), hyperin (6) and apigenin (7), were isolated (using enzyme assay-guide fractionation method) from the n-BuOH-soluble and EtOAc-soluble fractions. The structures of these pure compounds were determined on the basis of MS and NMR data and the activities of these compounds were evaluated. Compounds 1, 2 and 5 showed high inhibitory activities, with IC50 values of 3.5 mM, 5.2 mM and 3.0 mM against sucrase, with IC50 values of 4.8 mM, 5.6 mM and 4.1 mM against maltase and with IC50 values of 4.8 mM, 5.3 mM and 4.3 mM against a-amylase, respectively. We found that myricetin showed the most powerful activity among these compounds with a 70% inhibition against sucrase at a concentration of 1.5 mg/ml. The hydroxyl group at the 3-position on the A-ring and a number of hydroxyl groups attached to the C-ring played important roles in the inhibition activity. There was an obvious synergistic effect (the mixing action of two compounds) against a-glucosidase, but against a-amylase this was not found. This is the first study of the active compositions of guava leaves and the biological activity of the active compositions against a-glucosidase and a-amylase. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a metabolic disease caused by deficiency in insulin secretion. The disease is developing, along with an increase in both obesity and ageing, in the general population. It is a great challenge now, because about 5% of the global population is affected by DM (WHO, 2002). Insulin secretion deficiency results in an increase of blood glucose level and causes serious damage to body systems, such as blood vessels and nerves (Matsui et al., 2007). One of the therapeutic approaches is to decrease the postprandial hyperglycaemia by retarding absorption of glucose by inhibition of carbohydrate-hydrolysing enzymes, such as aamylase and a-glucosidase (Bhandari, Nilubon, Gao, & Kawabata, 2008; Lebovitz, 1997; Rhabasa-Lhoret & Chiasson, 2004). From this point of view, many efforts have been made to search for more effective and safe inhibitors of a-glucosidase and a-amylase from natural materials to develop physiological functional food to treat diabetes (Bhandari et al., 2008; Kim, Wang, & Rhee, 2004; Matsuura, Asakawa, Kurimoto, & Mizutani, 2002; Matsuura et al., 2004; Nishioka, Kawabata, & Aoyama, 1998; Toda, Kawabata, & Kasai, 2000).
As a folk medicine, guava leaves were used to prevent and treat diabetes for many years in China (Wang & Liu, 2005). It is reported that the extract from guava leaves possesses inhibition activity against a-glucosidase (Maruyama et al., 1985; Mukhtar, Ansari, Ali, Naved, & Bhat, 2004; Mukhtar, Ansari, Bhat, Naved, & Singh, 2006; Oh et al., 2005) and could improve the hypercoagulable state in diabetes (Hsieh, Lin, Yen, & Chen, 2007). However, the studies on anti-diabetic effects of guava leaves were mainly focused on the activity of the extract; the active components of the extract were not ascertained, and polyphenols, flavonoids, pentacyclic triterpenoids and other compounds in the guava leaves were only speculated to account for the observed hypoglycaemic effects of the extract (Maruyama et al., 1985; Ojewole, 2005; Wang & Liu, 2005; Takashi et al., 2006; Wang, Liu, & Ju, 2005). We decided to study guava leaves by isolating and identifying the active compositions of guava leaves by enzyme assay-guided fractionation and further to understand how the extract of guava leaves acts against a-glucosidase and a-amylase. 2. Materials and methods 2.1. Materials
* Corresponding author. Tel.: +86 020 84110918; fax: +86 020 84112245. E-mail address: [email protected] (H.-C. Song). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.03.088
Guava leaves were purchased from a local herbal shop in Guangzhou, China. The rat intestinal acetone powder, the porcine
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pancreatic a-amylase and azure starch were supplied by Sigma Aldrich Co. (USA). ICN Alumina B, Akt.-I was purchased from Eschwege (Germany). Acarbose, used as a standard, was produced by Hangzhou zhongmei huadong pharmaceutical Co. ltd. All the chemicals used were of analytical grade and were purchased from Guangzhou Chemical Reagent Company (Guangzhou, China), unless otherwise stated. 2.2. Preparation of extracts and determination of the content of the active compositions Dried guava leaves (6 kg) were cut into small pieces and twice extracted with 75% ethanol under reflux to give the 75% ethanol extract. The extract was suspended in water and extracted, respectively, with CH2Cl2, EtOAc and n-BuOH, to offer four extracts, CH2Cl2-soluble, EtOAc-soluble, n-BuOH-soluble and residual extract fractions, and then each extract was evaporated to dryness under reduced pressure, while the residual extract fraction was frozen to dryness. So, in total, five extracts were obtained. A small amount of each fraction was redissolved in 50% dimethyl sulfoxide (DMSO) and these mixture solutions were subjected to sucrase, maltase and porcine pancreatic a-amylase inhibitory activity assays. The relative amounts of individual component of each extract fraction were determined by HPLC, using a UV detector and using a Phenomenix Luna (II) column (250 4.6 mm ID, 5 lm). The HPLC data were obtained on a LC-10A VP liquid chromatograph (SHIMADZU). The mobile phase was a mixture of aqueous solutions of 0.1% H3PO4 and CH3CN, and the polarity was changed by changes of the ratio of CH3CN. Injection volume was 20 lm and the flow rate was 1 ml/min. Detection was set at 360 nm. 2.3. Isolation of active compounds from EtOAc-soluble and n-BuOH soluble fractions The EtOAc-soluble fraction (98 g) was subjected to a silica gel chromatography column, using a petroleum-ether/EtOAc system as eluent, and the eluent polarity was increased by increasing the ratio of EtOAc during the process. The separation was monitored by TLC and four fractions were obtained. As fraction 3 [petroleum-ether:EtOAc = 3:7 (v:v)] and fraction 4 [petroleum-ether: EtOAc = 1:9 (v:v)] showed strong inhibitory activities against aglucosidase and a-amylase, then were further separated by a silica gel chromatography column with a petroleum-ether/EtOAc system. Then a further separation was completed by a combination of Sephadex LH-20 column chromatography, using MeOH as eluent, and using reversed-phase TLC to monitoring the isolation. The n-BuOH-soluble fraction (110 g) was chromatographied over the highly porous synthetic resin, Diaion HP-20 (Mitsubishi kasei, Tokyo) (ø 10 cm 40 cm h) using H2O–CH3OH as eluent, followed by an increase of the amount of MeOH during the process; eight fractions were obtained. The enzyme inhibitory activity of each fraction was determined and fraction 5 [H2O:CH3OH = 1:1 (v:v)], fraction 6 [H2O:CH3OH = 3:7 (v:v)] and fraction 7 [100% CH3OH] showed stronger activity, compared with the other fractions. These three active fractions were subjected to a RP-18 chromatography column, eluting with H2O–CH3OH, followed by an increase of the ratio of CH3OH; sub-fractions were obtained and were further purified by Sephadex LH-20 column chromatography using a reverse-phase TLC as a monitor.
DMSO-d6 or CD3OD as solvents. ESI-MS data were obtained on a THEROMO MAX spectrometer (USA). 2.5. Determination of intestinal a-glucosidase inhibitory activity The rat intestinal sucrase and maltase inhibitory activities of prepared samples were determined using a literature method (Nishioka et al., 1998; Bhandari et al., 2008; Gao et al., 2008) with a slight modification. The substrate (sucrose: 56 mM, 0.2 ml; maltose: 3.5 mM, 0.35 ml) in 0.1 M potassium phosphate buffer (pH 7.0, 0.2 ml) was mixed with 0.1 ml of the plant extracts in 50% aqueous DMSO. After pre-incubation at 37 °C for 5 min, 0.4 ml of sucrase solution was added to the mixture. The mixture containing maltase was made by the same method, whereas 0.1 ml of DMSO was used in place of the plant extract for the blank sample. After being thoroughly mixed, both sample and blank test tubes were incubated at 37 °C for 15 min and then the reaction was stopped by adding 1.5 ml of 2 M Tris-HCl buffer (pH 6.9). The reaction mixture was passed through a basic alumina column (ø 6 mm 35 mm h) to eliminate phenolic or acidic compounds. The amount of liberated glucose was determined by the glucose oxidase method, using a commercial test kit (Glucose-B TestKit). The mixture of filtrate and glucose kit solution was incubated in a 96-well microplate at 37 °C for 30 min. The optical densities (OD) of the wells were measured at 490 nm and the inhibitory activity was calculated using the following formula:
Inhibitory activity ð%Þ ¼ ðODcontrol ODtest sample Þ=ODcontrol 100 For the extracts, inhibitory activities are shown as inhibiting percentages at the concentration of 1.5 mg/ml (Table 1); for the pure isolated compounds 1–7, inhibitory activities are shown as inhibiting percentages at the concentration of 1.5 mg/ml and IC50 values (Table 2) at the same time. 2.6. Determination of porcine pancreatic a-amylase inhibitory activity Porcine pancreatic a-amylase inhibitory activity of samples was determined using a literature method (Hansawasdi, Kawabata, & Kasai, 2000; Bhandari et al., 2008; Gao et al., 2008.) with a slight modification. Briefly, Starch azure (8 mg, used as substrate) was suspended in 0.5 M Tris-HCl buffer (pH 6.9) containing 0.01 M CaCl2 and soaked in boiling water for 5 min. Then, the starch azure solution was preincubated at 37 °C for 5 min. The test samples (0.2 ml) in 50% DMSO and 0.2 ml of PPA solution (A-3176; 6.25 U/ml) were added to each assay sample, whereas 0.1 ml 0.5 M Tris-HCl buffer was used in place of the plant extract for the blank sample. After being thoroughly mixed, both the sample and the blank test tubes were incubated at 37 °C for 10 min and the reaction was stopped by adding 0.1 ml of 50% acetic acid. The reaction mixture was then centrifuged (3000 rpm, 4 °C) for
Table 1 a-Glucosidasese and a-amylase inhibitory effects of the extracts of guava leaves. Plant extract
a-Glucosidase
Ethanol extract CH2Cl2-soluble EtOAc-soluble n-Butanol-soluble Water-soluble Acarbose
2.4. Determination of the structures of main active compositions of extracts 1
H NMR and 13C NMR spectra of the isolated pure compounds were recorded with a Varian INONA400 instrument, using
Inhibition (%)a,b
a b
a-Amylase
Sucrase
Maltase
38.3 ± 4.2 18.3 ± 5.3 46.3 ± 8.3 63.5 ± 5.6 34.5 ± 4.9 48.4 ± 1.3
33.4 ± 6.3 15.2 ± 7.2 40.6 ± 4.5 47.7 ± 3.7 27.3 ± 2.0 79.8 ± 0.5
31.7 ± 3.1 9.3 ± 2.1 43.9 ± 8.7 54.4 ± 6.6 29.3 ± 7.9 52.1 ± 0.8
Values represent the means ± standard deviation (SD) of n = 3 duplicate assays. The concentration of all test samples was 1.5 mg/ml.
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Table 2 Inhibitory activities of isolated flavonoid compounds against a-glucosidase and a-amylase. Flavonoid Myricetin Quercetin Avicularin Guaijaverin Hyperin Kaempferol Apigenin Acarbose a b
Rat intestinal sucrase Inhibition (%)a,b IC50 (mM)a
Rat intestinal maltase Inhibition (%)a,b IC50 (mM)a
a-amylase
70.2 ± 4.5 64.5 ± 3.1 39.0 ± 2.6 36.4 ± 2.5 34.3 ± 3.8 50.6 ± 2.7 23.1 ± 2.4 48.4 ± 1.3
53.7 ± 2.3 51.1 ± 4.7 31.7 ± 3.5 33.8 ± 4.2 39.7 ± 3.7 45.0 ± 0.4 27.6 ± 3.9 79.8 ± 0.5
53.9 ± 5.6 52.1 ± 7.3 43.3 ± 6.9 44.3 ± 5.8 40.4 ± 5.7 50.7 ± 8.3 31.4 ± 3.1 52.1 ± 0.8
3.0 ± 0.1 3.5 ± 0.3 6.5 ± 0.7 6.2 ± 0.6 7.5 ± 0.8 5.2 ± 0.4 >30 2.5 ± 0.1
Inhibition(%)a,b IC50 (mM)a
4.1 ± 0.2 4.8 ± 0.4 7.6 ± 0.2 6.6 ± 0.5 7.8 ± 0.6 5.6 ± 0.1 >30 0.9 ± 0.1
4.3 ± 0.3 4.8 ± 0.1 5.9 ± 0.5 5.6 ± 0.2 6.1 ± 0.3 5.3 ± 0.2 >30 2.3 ± 0.1
Values represent the means ± standard deviation (SD) of n = 3 duplicate assays. The concentration of all test samples was 1.5 mg/ml.
Table 3 The possible synergistic activities of the isolated flavonoid compounds against a-glucosidase and a-amylase. Flavonoid
a
Sucrase
a-Amylase
Maltase
IC50 (mM)a
Interaction
IC50 (mM)a
Interaction
IC50 (mM)a
Interaction
Quercetin Myricetin Quercetin + myricetin
3.5 ± 0.3 3.0 ± 0.1 2.0 ± 0.2
Synergistic
4.8 ± 0.4 4.1 ± 0.2 3.2 ± 0.4
Synergistic
4.8 ± 0.1 4.3 ± 0.3 4.5 ± 0.3
None
Hyperin Avicularin Hyperin + avicularin
7.5 ± 0.8 6.5 ± 0.7 4.5 ± 0.5
Synergistic
7.8 ± 0.6 7.6 ± 0.2 5.0 ± 0.3
Synergistic
6.1 ± 0.3 5.9 ± 0.5 6.0 ± 0.2
None
Kaempferol Quercetin Kaempferol + quercetin
5.2 ± 0.4 3.5 ± 0.3 2.6 ± 0.6
Synergistic
5.6 ± 0.1 4.1 ± 0.2 4.2 ± 0.1
Synergistic
5.3 ± 0.2 4.3 ± 0.3 5.1 ± 0.4
None
Values represent the means ± standard deviation (SD) of n = 3 duplicate assays.
5 min. The absorbance of the supernatant was measured at 595 nm and the inhibitory activity was calculated using the following formula:
Inhibitory activity ð%Þ ¼ ðODcontrol ODtest sample Þ=ODcontrol 100 The Inhibitory activities of the samples against a-amylase are shown as inhibiting percentages and IC50 values (Table 2). 2.7. Evaluation of synergy of isolated compounds against aglucosidase and a-amylase In order to determine the synergy of the isolated compounds against a-glucosidase and a-amylase, five pure isolated compounds were selected to form three samples by mixing the solutions of two different compounds with the same concentration (1.5 mg/ml) and the same volume (1.5 ml). These three samples are quercetin–myricetin, hyperin–avicularin, quercetin–kaempferol, their activities against a-glucosidase and a-amylase were determined as IC50 values by the method mentioned above and their IC50 values are listed in Table 3. 3. Results and discussion 3.1. Isolation of active compounds and structural determination Seven pure compounds were separated from the EtOAc-soluble fraction by the method mentioned above; they were identified as quercetin (1, 98.3 mg), kaempferol (2, 15.6 mg), guaijaverin (3, 19.9 mg), avicularin (4, 23.5 mg), myricetin (5, 7.5 mg), hyperin (6, 9.6 mg) and apigenin (7, 11.4 mg), respectively (their structures are shown in Fig. 1), by comparing the obtained MS and NMR data with those reported. Five pure compounds were obtained
from n-BuOH-soluble fraction and they were identified as 1 (203.6 mg), 2 (15.6 mg), 3 (50.3 mg), 4 (31.4 mg) and 6 (23.6 mg). In addition, the purity of each individual compound isolated was also determined by HPLC with values more than 99%. Compound 1, yellow powder. 1H NMR (DMSO-d6, 400 MHz): d 6.13 (1H, d, J = 2.0 Hz, H-6), 6.35 (1H, d, J = 2.0 Hz, H-8), 6.84 (1H, d, J = 8.0 Hz, H-50 ), 7.52 (1H, dd, J = 2.0, 8.4 Hz, H-60 ), 7.65 (1H, d, J = 2.0 Hz, H-20 ). 13C NMR (DMSO-d6, 100 Hz): d 93.7 (C-8), 98.5 (C-6), 103.3 (C-10), 115.4 (C-20 ), 115.9 (C-50 ), 120.8 (C-60 ), 122.3 (C-10 ), 136.1 (C-3), 145.4 (C-30 ), 147.1 (C-2), 148.0 (C-40 ), 156.5 (C-5), 161.1 (C-9), 164.2 (C-7), 176.2 (C-4, C@O). Negative ESI-MS m/z 301 [MH]. The MS data and the NMR data are consistent with those of quercetin reported by Guvenalp and Demirezer (2005) and Zheng, Cheng, Chao, Wu, and Wang (2008). Compound 1 was identified as quercetin. Compound 2, yellow powder. 1H NMR (DMSO-d6, 400 MHz): d 6.15 (1H, d, J = 2.0 Hz, H-6), 6.39 (1H, d, J = 2.0 Hz, H-8), 6.88 (2H, d, J = 8.8 Hz, H-30 , H-50 ), 8.00 (2H, d, J = 8.8 Hz, H-20 , H-60 ). 13C NMR (DMSO-d6, 100 MHz): d 93.8 (C-8), 98.6 (C-6), 103.4 (C-10), 115.8 (C-30 , C-50 ), 122.0 (C-10 ), 129.9 (C-20 , C-60 ), 136.0 (C-3), 147.2 (C-2), 156.5 (C-5), 159.5 (C-40 ), 161.1 (C-9), 164.2 (C-7), 176.3 (C-4, C@O). Negative ESI-MS m/z 285 [MH]. The NMR data are consistent with those reported by Pelter, Ward, and Gray (1976). Compound 2 was identified as kaempferol. Compound 3, yellow needles. 1H NMR (CD3OD, 400 MHz): d 3.43 (1H, dd, J = 3.2, 13.6 Hz, H-200 ), 3.63 (1H, dd, J = 3.2, 8.4 Hz, H-300 ), 3.81 (2H, dd, J = 3.6, 9.6 Hz, H-500 ), 3.88 (1H, dt, J = 1.6, 8.4 Hz, H-400 ), 5.14 (1H, d, J = 6.4 Hz, H-100 ), 6.17 (1H, d, J = 2.0 Hz, H-6), 6.36 (1H, d, J = 2.0 Hz, H-8), 6.85 (1H, d, J = 8.8 Hz, H-50 ), 7.55 (1H, dd, J = 2.4, 8.8 Hz, H-60 ), 7.73 (1H, dd, J = 2.4 Hz, H-20 ). 13 C NMR (CD3OD, 100 MHz): d 65.5 (C-500 ), 71.4 (C-300 ), 72.7 (C200 ), 67.7 (C-400 ), 93.2 (C-8), 98.4 (C-6), 103.2 (C-100 ), 104.1 (C-10), 114.7 (C-20 ), 116.0 (C-50 ), 121.4 (C-60 ), 121.6 (C-10 ), 134.2 (C-3),
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OH O
HO
HO
Quercetin (1)
HO
OH HO
OH O
OH O
Myricetin (5)
OH
HO
OH
Avicularin (4)
OH
OH O
OH
O O
OH
OH O
O
OH
OH OH
OH OH
Guaijaverin (3)
Kaempferol (2)
O
O
O OH O
O
OH
O
OH OH O
OH O
HO
O
HO
O
OH
OH
OH
OH
HO
HO
HO
HO
HO
O
OH OH
Hyperin (6)
OH O
Apigenin (7)
Fig. 1. Structures of isolated flavonoid compounds from guava leaves.
144.5 (C-30 ), 148.5 (C-40 ), 156.9 (C-2), 157.2 (C-8), 161.5 (C-5), 164.5 (C-7), 178.0 (C-4, C@O). Negative ESI-MS m/z 433 [MH]. The MS data and the NMR data are identical with those in the literature (Arima & Danno, 2002). Compound 3 was identified as guaijaverin. Compound 4, yellow crystals. 1H NMR (400 MHz, CD3OD): d 3.49 (m, sugar-2H, H-500 ), 3.86 (1H, dd, J = 1.6, 4.0 Hz, H-300 ), 3.90 (1H, dd, J = 3.2, 5.2 Hz, H-200 ), 4.32 (1H, dt, J = 1.6, 2.8 Hz, H-400 ), 5.45 (1H, d, J = 6.8, H-100 ), 6.18 (1H, d, J = 2.0 Hz, H-6), 6.36 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.4 Hz, H-50 ), 7.47 (1H, dd, J = 2.0, 8.4 Hz, H-60 ), 7.51 (1H, d, J = 1.6 Hz, H-20 ). 13C NMR (CD3OD, 100 MHz): d 61.1 (C-500 ), 77.2 (C-300 ), 81.8 (C-200 ), 87.5 (C-400 ), 93.3 (C-8), 98.4 (C-6), 104.1 (C-10), 108.0 (C-100 ), 114.9 (C-50 ), 115.4 (C-20 ), 121.5 (C-60 ), 121.6 (C-10 ), 133.4 (C-3), 144.9 (C-30 ), 148.4 (C-40 ), 157.1 (C-9), 157.9 (C-2), 161.6 (C-5), 164.5 (C-7), 178.5 (C-4, C@O). Negative ESI-MS m/z 433 [MH]. The MS data and the NMR data are identical with those in the literature (Zhang et al., 2005). Compound 4 was identified as avicularin. Compound 5, yellow brown powder. 1H NMR (DMSO-d6, 400 MHz): d 6.14 (1H, s, H-6), 6.33 (1H, s, H-8), 7.20 (2H, d, J = 1.6 Hz, H-20 , H-60 ). 13C NMR (DMSO-d6, 100 MHz): d 93.6 (C-8), 98.5 (C-6), 103.3 (C-10), 107.5 (C-20 , C-60 ), 121.1 (C-10 ), 136.2 (C-40 ), 136.2 (C-3), 146.1 (C-30 , C-50 ), 147.2 (C-2), 156.4 (C-9), 161.1 (C-5), 164.2 (C-7), 176.1 (C-4, C@O). Negative ESI-MS m/z 317 [MH]. The NMR data are consistent with those in the literature (Pelter et al., 1976); compound 5 was identified as myricetin. Compound 6, yellow powder. 1H NMR (CD3OD, 400 MHz): d 3.44–3.82 (m, sugar-H), 5.14 (1H, d, J = 7.6 Hz, H-100 ), 6.17 (1H, s, H-6), 6.37 (1H, s, H-8), 6.83 (1H, d, J = 8.0 Hz, H-50 ), 7.56 (1H, d, J = 8.4 Hz, H-60 ), 7.81 (1H, s, H-20 ). 13C NMR (CD3OD, 100 MHz): d 60.4 (C-600 ), 68.5 (C-400 ), 71.7 (C-200 ), 73.6 (C-300 ), 75.7 (C-500 ), 93.2 (C-8), 98.4 (C-6), 103.8 (C-100 ), 103.9 (C-10), 114.6 (C-20 ), 116.3 (C-50 ), 121.4 (C-10 ), 121.4 (C-60 ), 134.4 (C-3), 144.3 (C-30 ), 148.4 (C-40 ), 156.3 (C-2), 157.3 (C-9), 161.5 (C-5), 164.6 (C-7), 178.0 (C-4, C@O). Negative ESI-MS m/z 463 [MH]. The MS data and the NMR data are consistent with those in the literature (Zhang et al., 2005); compound 6 was identified as hyperin. Compound 7, pale yellow powder. 1H NMR (DMSO-d6, 400 MHz): d 7.87 (2H, dd, J = 2.4, 8.8 Hz, H-20 , H-60 ), 6.87 (2H, dd, J = 2.4, 8.8 Hz, H-30 , H-50 ), 6.72 (1H, d, J = 2.4 Hz, H-3), 6.43 (1H, d, J = 2.0 Hz, H-8), 6.14 (1H, d, J = 2.0 Hz, H-6). 13C NMR (DMSOd6, 100 MHz): d 94.3 (C-8), 99.2 (C-6), 103.2 (C-3), 104.0 (C-10),
121.5 (C-10 ), 116.3 (C-30 , C-50 ), 128.8 (C-20 , C-60 ), 157.7 (C-9), 161.5 (C-5), 161.8 (C-40 ), 164.1 (C-7), 164.5 (C-2), 182.1 (C-4, C@O). Negative ESI-MS m/z 269 [MH]. The NMR data are consistent with those in the literature (Ren & Yang, 2001); compound 7 was identified as apigenin. 3.2. Activities of samples against a-glucosidase and a-amylase 3.2.1. Activities of extracts The inhibitory activities of crude 75% ethanol extract of guava leaves were determined at the concentration of 1.5 mg/ml against a-glucosidase and a-amylase (Table 1). The inhibiting percentages of 75% ethanol extract against a-glucosidase were 38.3 ± 4.2% (sucrase) and 33.4 ± 6.3% (maltase), respectively; against a-amylase, inhibition was 31.7 ± 3.1%. After the 75% ethanol extract was extracted respectively with CH2Cl2, EtOAc and n-BuOH, four extracts (CH2Cl2-soluble, EtOAcsoluble, n-BuOH-soluble and residual fraction) were obtained and the inhibitory activities of these four extracts were determined at the concentration of 1.5 mg/ml against a-glucosidase and a-amylase (Table 1). The inhibiting percentages of the EtOAc-soluble extract against a-glucosidase were 46.3 ± 8.3% (sucrase) and 40.6 ± 4.5% (maltase), respectively; against a-amylase, inhibition was 43.9 ± 8.7%. The inhibiting percentages of the n-BuOH extract against a-glucosidase were 63.5 ± 5.6% (sucrase) and 47.7 ± 3.7% (maltase), respectively; against a-amylase inhibition was 54.4 ± 6.6%. Table 1 shows that, at the concentration of 1.5 mg/ml the sequences of inhibitory effects against a-glucosidase and against aamylase had the same order as follows:
n-BuOH-soluble > EtOAc-soluble > 75% Ethanol-extract > residual fraction > CH2 Cl2 -soluble 3.2.2. Activities of isolated compounds and relationships of structure and activity As the EtOAc-soluble and n-BuOH-soluble extracts showed obviously higher inhibitory activities than did the 75% ethanol extract against a-glucosidase and a-amylase, we supposed that the main active compositions of the 75% ethanol extract of guava leaves were basically in the n-BuOH-soluble fraction and EtOAcsoluble fraction, and both of the extracts were further investigated
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by isolating the active components and by evaluating the activity of the isolated compounds against a-glucosidase and a-amylase. Their IC50 values and inhibiting percentages, at the concentration of 1.5 mg/ml of the seven isolated compounds, are shown in Table 2. Although the inhibitory activities of the seven isolated compounds were still lower than that of the therapeutic drug, acarbose (IC50 = 2.5 ± 0.1 mM for rat intestinal sucrase, 0.9 ± 0.1 mM for rat intestinal maltase and 2.3 ± 0.1 for porcine pancreatic a-amylase), the data clearly indicate the potential of these compounds as inhibitors of a-glucosidase and a-amylase. On the basis of the IC50 values in Table 2, it was found that the sequence of inhibitory activities of these seven isolated compounds against sucrase, maltase and a-amylase are as follows:
Myricetin > quercetin > kaempferol > guaijaverin > avicularin > hyperin > apigenin Among the seven isolated compounds, myricetin, quercetin and kaempferol exhibited significant inhibitory activities against aglucosidase and a-amylase. The IC50 values were lower than 8 mM, especially for myricetin and quercetin, and the IC50 values were lower than 5 mM. Apigenin only showed a weak inhibitory activity with more than 30 mM for the IC50 value. Quercetin, kaempferol, myricetin and apigenin are all derivatives of 5,7-dihydroxy-2-phenylchromen-4-one; guaijaverin, avicularin and hyperin are glycosides of quercetin with different sugars. So the structures of all seven compounds can be shown as the 2-substituted-phenyl-chromen-4-one in Fig. 2. Fig. 2 shows that all the atoms forming the A-ring, B-ring and Cring, would take a sp2 hybrid form, which allows these atoms to form a ‘‘large conjugated-system”. We deduce that the ‘‘large conjugated-system” skeleton could be necessary for these compounds to act upon a-glucosidase and a-amylase. We could draw three conclusions from the above. (1) The hydroxyl on the 3-position of the flavonoid plays an important rule in the inhibitory activity against a-glucosidase and a-amylase. This conclusion could be elucidated by the following. Apigenin, lacking the 3-OH, possesses a weaker inhibitory percentage (about 20– 30%) and a higher IC50 value (more than 30 mM), but quercetin, kaempferol and myricetin, possessing the 3-OH, show obviously higher inhibitory activities than apigenin could do. In addition, the glycosylation of the 3-OH of the flavonoid was also unfavourable to the inhibitory activity (the inhibitory percentages of glycoside compounds of quercetin were lower than that of quercetin itself). Comparing the inhibitory percentages of these compounds, the order of inhibitory ability was obtained:
3-Hydroxylated flavonoid > flavonoid glycoside > 3-unhydroxylated flavonoid Similar results are also observed for the IC50 values. So the free 3-position hydroxyl group was a key functional group for inhibiting a-glucosidase and a-amylase.(2) The number of hydroxyl substitution on the C-ring would affect the inhibitory activity. Comparing the inhibitory activity of 40 -hydroxylated, 30 ,40 -dihy-
B
O 2 A 4
C
3
O Fig. 2. Structure of 2-substitutedphenyl-chromen-4-one.
droxylated and 30 ,40 ,50 -trihydroxylated flavonoids (corresponding to kaempferol, quercetin and myricetin, respectively) at the concentration of 1.5 mg/ml, we found that the inhibitory activity increases considerably with the increase of the number of hydroxyl groups on the C-ring.(3) A sugar group attached to the flavonoid nuclei at the 3-position does not notably affect its inhibitory activity. The decreasing order of the inhibitory activity against rat intestinal a-glucosidase and porcine pancreatic a-amylase was:
Guaijaverin > avicularin > hyperin So, the five-carbon glycoside could exhibit a higher activity than the six-carbon glucoside. But the inhibition percentage order of these three glucosides did not accord with that of the IC50; for example, hyperin possessed a maximum inhibition percentage (39.7 ± 3.7%) against rat intestinal maltase and showed the strongest inhibitory activity. The effect of the glycosyl structure on their activities was unclear. The conclusions above were also supported by the work of Tadera, Minami, Takamatsu, and Matsuoka (2006). 3.2.3. Synergistic action of isolated flavonoid compounds against aglucosidase and a-amylase Comparing the activities of isolated compounds with those of the extracts, it appears that the inhibiting percentages of isolated compounds are not obviously higher than that of n-BuOH-soluble extract or the EtOAc-soluble extract at the same concentration, even myricetin and quercetin, which displayed the most powerful activities among these seven isolated compounds, only had inhibitory percentage close to the n-BuOH-soluble extract or the EtOAcsoluble extract. On the other hand, Peng et al. (2003) and Cushnie and Lamb (2005), respectively, reported that flavonoid compounds could show a synergistic effect on antimicrobial activity and antioxidation. Both of the reasons noted above induced us to investigate the operation of a synergistic effect that contributes to the enhancement of inhibitory activity of a flavonoid in the presence of other flavonoids, in order to collectively account for the anti-diabetic activity and understand the mechanism of action of flavonoid compounds existing in guava leaves. Table 3 shows that inhibitory activities of three two-mixed samples, quercetin–myricetin, hyperin–avicularin and quercetin– kaempferol, display a significant improvement and would exhibit an obvious synergistic action against a-glucosidases, including sucrase and maltase, by comparing the IC50 value of the two-mixed samples with that of the original single composition. For example, the IC50 value of myricetin–quercetin (2.0 mM) was lower than that of the individual myricetin (3.0 mM) or quercetin (3.5 mM). However, we did not found these three two-mixed samples performing the synergistic action against a-amylase. 3.2.4. Analysis of HPLC data In order to investigate the relationship of activity and composition of extracts, the content of the active components of these extracts was determined by HPLC, using pure quercetin (1), kaempferol (2), guaijaverin (3), avicularin (4), myricetin (5), hyperin (6) and apigenin (7) as references. The HPLC chromatograms of the mixture of seven pure compounds, 75% ethanol extract, CH2Cl2-solube extract, EtOAc-soluble extract, n-BuOH-soluble extract and residual extract are shown in Figs. 3–8, respectively. Fig. 3 is the HPLC chromatogram of the mixture of seven pure compounds, this Figure is used as a standard to determine the active compound in the other Figures. HPLC showed that the total relative amounts of three components, namely hyperin, guaijaverin and avicularin in the 75% EtOH, CH2Cl2-soluble fraction, EtOAc-soluble fraction, n-BuOH-soluble fraction and residual extract were 84.5%, 61.2%, 82.6%, 87.1% and 67.5%, respectively (Table 4), which is in agreement with inhibitory activity of each extract. This result confirmed that glucosides
H. Wang et al. / Food Chemistry 123 (2010) 6–13
Fig. 3. HPLC chromatograms of the mixture of seven pure compounds.
Fig. 4. HPLC chromatograms of 75% ethanol extract.
Fig. 5. HPLC chromatograms of CH2Cl2-soluble extract.
Fig. 6. HPLC chromatograms of EtOAc-soluble extract.
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H. Wang et al. / Food Chemistry 123 (2010) 6–13
Fig. 7. HPLC chromatograms of n-C4H9OH-soluble extract.
Fig. 8. HPLC chromatograms of residual extract.
Table 4 The relative amounts of individual component in extract fractions. Flavonoid
Hyperin Guaijaverin Avicularin Myricetin Quercetin Kaempferol Apigenin
75%EtOH
CH2Cl2
EtOAc
n-C4H9OH
Residual extract
Time (min)
Content (%)
Time (min)
Content (%)
Time (min)
Content (%)
Time (min)
Content (%)
Time (min)
Content (%)
17.342 21.951 23.321 28.142 37.453 43.752 45.577
18.0 35.6 30.9 0.58 7.44 5.50 1.97
17.199 21.806 23.156 28.272 37.265 43.638 45.387
11.4 26.0 23.8 1.10 34.9 0.43 2.22
17.430 21.992 23.318 28.508 37.365 43.627 45.398
13.1 34.7 34.8 1.93 12.9 2.24 0.29
17.177 21.904 23.278 28.314 37.533 43.790 45.607
29.1 32.2 25.9 1.42 7.13 2.90 1.43
17.726 22.251 23.570 28.427 37.587 43.947 45.826
25.3 27.2 15.0 4.34 9.80 4.75 13.6
should be the main active components in these extracts to inhibit a-glucosidase and a-amylase. Quercetin was the main component among the individual compounds isolated from EtOAc-soluble or n-C4H9OH-soluble extracts, but we found from the HPLC chromatograms of the EtOAc-soluble or n-C4H9OH-soluble extracts that quercetin was not the main component. The reason for this phenomenon may be that the hyperin, guaijaverin and avicularin were hydrolysed into quercetin during the process of column chromatographic isolation by the acidity of the resin material.
nents could confer an obvious synergistic effect to inhibitory activity against a-glucosidase, but not against a-amylase, which is found for the first time in this study on anti-diabetic activity of isolated compounds from guava leaves. The results give scientific support for the proper use of guava leaves in folk medicine for the treatment of diabetes and this work could help to develop medicinal preparations or nutraceutical and functional foods for diabetes and related conditions.
4. Conclusion
This research was financially supported by the Guangdong Food Industry Institute.
Seven major active components, quercetin, myricetin, avicularin, guaijaverin, hyperin and kaempferol, were isolated from extracts of guava leaves. We found that (1) the major active components of these extracts were glycosides of quercetin and there is an obvious relationship between the structure and activity in these isolated active compounds; (2) a mixture of two compo-
Acknowledgements
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
a-Glucosidase and a-amylase inhibitory activities of guava leaves Hui Wang a, Yang-Ji Du b, Hua-Can Song a,* a b
School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China School of Chemistry and Environment, South China Normal University, Guangzhou 510006, China
a r t i c l e
i n f o
Article history: Received 18 November 2008 Received in revised form 31 January 2010 Accepted 22 March 2010
Keywords: Guava leaves a-Glucosidase and a-amylase inhibitor Flavonoid compounds Structure-activity relationship Diabetes
a b s t r a c t The 75% ethanol extract from guava (Psidium guajava Linn.) leaves was extracted further, in turn, with CH2Cl2, EtOAc and n-BuOH to afford four fractions, CH2Cl2-soluble, EtOAc-soluble, n-BuOH-soluble and residual extract fractions. Both the n-BuOH-soluble and EtOAc-soluble fractions showed high inhibitory activity against a-glucosidase and a-amylase. Seven pure flavonoid compounds, quercetin (1), kaempferol (2), guaijaverin (3), avicularin (4), myricetin (5), hyperin (6) and apigenin (7), were isolated (using enzyme assay-guide fractionation method) from the n-BuOH-soluble and EtOAc-soluble fractions. The structures of these pure compounds were determined on the basis of MS and NMR data and the activities of these compounds were evaluated. Compounds 1, 2 and 5 showed high inhibitory activities, with IC50 values of 3.5 mM, 5.2 mM and 3.0 mM against sucrase, with IC50 values of 4.8 mM, 5.6 mM and 4.1 mM against maltase and with IC50 values of 4.8 mM, 5.3 mM and 4.3 mM against a-amylase, respectively. We found that myricetin showed the most powerful activity among these compounds with a 70% inhibition against sucrase at a concentration of 1.5 mg/ml. The hydroxyl group at the 3-position on the A-ring and a number of hydroxyl groups attached to the C-ring played important roles in the inhibition activity. There was an obvious synergistic effect (the mixing action of two compounds) against a-glucosidase, but against a-amylase this was not found. This is the first study of the active compositions of guava leaves and the biological activity of the active compositions against a-glucosidase and a-amylase. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a metabolic disease caused by deficiency in insulin secretion. The disease is developing, along with an increase in both obesity and ageing, in the general population. It is a great challenge now, because about 5% of the global population is affected by DM (WHO, 2002). Insulin secretion deficiency results in an increase of blood glucose level and causes serious damage to body systems, such as blood vessels and nerves (Matsui et al., 2007). One of the therapeutic approaches is to decrease the postprandial hyperglycaemia by retarding absorption of glucose by inhibition of carbohydrate-hydrolysing enzymes, such as aamylase and a-glucosidase (Bhandari, Nilubon, Gao, & Kawabata, 2008; Lebovitz, 1997; Rhabasa-Lhoret & Chiasson, 2004). From this point of view, many efforts have been made to search for more effective and safe inhibitors of a-glucosidase and a-amylase from natural materials to develop physiological functional food to treat diabetes (Bhandari et al., 2008; Kim, Wang, & Rhee, 2004; Matsuura, Asakawa, Kurimoto, & Mizutani, 2002; Matsuura et al., 2004; Nishioka, Kawabata, & Aoyama, 1998; Toda, Kawabata, & Kasai, 2000).
As a folk medicine, guava leaves were used to prevent and treat diabetes for many years in China (Wang & Liu, 2005). It is reported that the extract from guava leaves possesses inhibition activity against a-glucosidase (Maruyama et al., 1985; Mukhtar, Ansari, Ali, Naved, & Bhat, 2004; Mukhtar, Ansari, Bhat, Naved, & Singh, 2006; Oh et al., 2005) and could improve the hypercoagulable state in diabetes (Hsieh, Lin, Yen, & Chen, 2007). However, the studies on anti-diabetic effects of guava leaves were mainly focused on the activity of the extract; the active components of the extract were not ascertained, and polyphenols, flavonoids, pentacyclic triterpenoids and other compounds in the guava leaves were only speculated to account for the observed hypoglycaemic effects of the extract (Maruyama et al., 1985; Ojewole, 2005; Wang & Liu, 2005; Takashi et al., 2006; Wang, Liu, & Ju, 2005). We decided to study guava leaves by isolating and identifying the active compositions of guava leaves by enzyme assay-guided fractionation and further to understand how the extract of guava leaves acts against a-glucosidase and a-amylase. 2. Materials and methods 2.1. Materials
* Corresponding author. Tel.: +86 020 84110918; fax: +86 020 84112245. E-mail address: [email protected] (H.-C. Song). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.03.088
Guava leaves were purchased from a local herbal shop in Guangzhou, China. The rat intestinal acetone powder, the porcine
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pancreatic a-amylase and azure starch were supplied by Sigma Aldrich Co. (USA). ICN Alumina B, Akt.-I was purchased from Eschwege (Germany). Acarbose, used as a standard, was produced by Hangzhou zhongmei huadong pharmaceutical Co. ltd. All the chemicals used were of analytical grade and were purchased from Guangzhou Chemical Reagent Company (Guangzhou, China), unless otherwise stated. 2.2. Preparation of extracts and determination of the content of the active compositions Dried guava leaves (6 kg) were cut into small pieces and twice extracted with 75% ethanol under reflux to give the 75% ethanol extract. The extract was suspended in water and extracted, respectively, with CH2Cl2, EtOAc and n-BuOH, to offer four extracts, CH2Cl2-soluble, EtOAc-soluble, n-BuOH-soluble and residual extract fractions, and then each extract was evaporated to dryness under reduced pressure, while the residual extract fraction was frozen to dryness. So, in total, five extracts were obtained. A small amount of each fraction was redissolved in 50% dimethyl sulfoxide (DMSO) and these mixture solutions were subjected to sucrase, maltase and porcine pancreatic a-amylase inhibitory activity assays. The relative amounts of individual component of each extract fraction were determined by HPLC, using a UV detector and using a Phenomenix Luna (II) column (250 4.6 mm ID, 5 lm). The HPLC data were obtained on a LC-10A VP liquid chromatograph (SHIMADZU). The mobile phase was a mixture of aqueous solutions of 0.1% H3PO4 and CH3CN, and the polarity was changed by changes of the ratio of CH3CN. Injection volume was 20 lm and the flow rate was 1 ml/min. Detection was set at 360 nm. 2.3. Isolation of active compounds from EtOAc-soluble and n-BuOH soluble fractions The EtOAc-soluble fraction (98 g) was subjected to a silica gel chromatography column, using a petroleum-ether/EtOAc system as eluent, and the eluent polarity was increased by increasing the ratio of EtOAc during the process. The separation was monitored by TLC and four fractions were obtained. As fraction 3 [petroleum-ether:EtOAc = 3:7 (v:v)] and fraction 4 [petroleum-ether: EtOAc = 1:9 (v:v)] showed strong inhibitory activities against aglucosidase and a-amylase, then were further separated by a silica gel chromatography column with a petroleum-ether/EtOAc system. Then a further separation was completed by a combination of Sephadex LH-20 column chromatography, using MeOH as eluent, and using reversed-phase TLC to monitoring the isolation. The n-BuOH-soluble fraction (110 g) was chromatographied over the highly porous synthetic resin, Diaion HP-20 (Mitsubishi kasei, Tokyo) (ø 10 cm 40 cm h) using H2O–CH3OH as eluent, followed by an increase of the amount of MeOH during the process; eight fractions were obtained. The enzyme inhibitory activity of each fraction was determined and fraction 5 [H2O:CH3OH = 1:1 (v:v)], fraction 6 [H2O:CH3OH = 3:7 (v:v)] and fraction 7 [100% CH3OH] showed stronger activity, compared with the other fractions. These three active fractions were subjected to a RP-18 chromatography column, eluting with H2O–CH3OH, followed by an increase of the ratio of CH3OH; sub-fractions were obtained and were further purified by Sephadex LH-20 column chromatography using a reverse-phase TLC as a monitor.
DMSO-d6 or CD3OD as solvents. ESI-MS data were obtained on a THEROMO MAX spectrometer (USA). 2.5. Determination of intestinal a-glucosidase inhibitory activity The rat intestinal sucrase and maltase inhibitory activities of prepared samples were determined using a literature method (Nishioka et al., 1998; Bhandari et al., 2008; Gao et al., 2008) with a slight modification. The substrate (sucrose: 56 mM, 0.2 ml; maltose: 3.5 mM, 0.35 ml) in 0.1 M potassium phosphate buffer (pH 7.0, 0.2 ml) was mixed with 0.1 ml of the plant extracts in 50% aqueous DMSO. After pre-incubation at 37 °C for 5 min, 0.4 ml of sucrase solution was added to the mixture. The mixture containing maltase was made by the same method, whereas 0.1 ml of DMSO was used in place of the plant extract for the blank sample. After being thoroughly mixed, both sample and blank test tubes were incubated at 37 °C for 15 min and then the reaction was stopped by adding 1.5 ml of 2 M Tris-HCl buffer (pH 6.9). The reaction mixture was passed through a basic alumina column (ø 6 mm 35 mm h) to eliminate phenolic or acidic compounds. The amount of liberated glucose was determined by the glucose oxidase method, using a commercial test kit (Glucose-B TestKit). The mixture of filtrate and glucose kit solution was incubated in a 96-well microplate at 37 °C for 30 min. The optical densities (OD) of the wells were measured at 490 nm and the inhibitory activity was calculated using the following formula:
Inhibitory activity ð%Þ ¼ ðODcontrol ODtest sample Þ=ODcontrol 100 For the extracts, inhibitory activities are shown as inhibiting percentages at the concentration of 1.5 mg/ml (Table 1); for the pure isolated compounds 1–7, inhibitory activities are shown as inhibiting percentages at the concentration of 1.5 mg/ml and IC50 values (Table 2) at the same time. 2.6. Determination of porcine pancreatic a-amylase inhibitory activity Porcine pancreatic a-amylase inhibitory activity of samples was determined using a literature method (Hansawasdi, Kawabata, & Kasai, 2000; Bhandari et al., 2008; Gao et al., 2008.) with a slight modification. Briefly, Starch azure (8 mg, used as substrate) was suspended in 0.5 M Tris-HCl buffer (pH 6.9) containing 0.01 M CaCl2 and soaked in boiling water for 5 min. Then, the starch azure solution was preincubated at 37 °C for 5 min. The test samples (0.2 ml) in 50% DMSO and 0.2 ml of PPA solution (A-3176; 6.25 U/ml) were added to each assay sample, whereas 0.1 ml 0.5 M Tris-HCl buffer was used in place of the plant extract for the blank sample. After being thoroughly mixed, both the sample and the blank test tubes were incubated at 37 °C for 10 min and the reaction was stopped by adding 0.1 ml of 50% acetic acid. The reaction mixture was then centrifuged (3000 rpm, 4 °C) for
Table 1 a-Glucosidasese and a-amylase inhibitory effects of the extracts of guava leaves. Plant extract
a-Glucosidase
Ethanol extract CH2Cl2-soluble EtOAc-soluble n-Butanol-soluble Water-soluble Acarbose
2.4. Determination of the structures of main active compositions of extracts 1
H NMR and 13C NMR spectra of the isolated pure compounds were recorded with a Varian INONA400 instrument, using
Inhibition (%)a,b
a b
a-Amylase
Sucrase
Maltase
38.3 ± 4.2 18.3 ± 5.3 46.3 ± 8.3 63.5 ± 5.6 34.5 ± 4.9 48.4 ± 1.3
33.4 ± 6.3 15.2 ± 7.2 40.6 ± 4.5 47.7 ± 3.7 27.3 ± 2.0 79.8 ± 0.5
31.7 ± 3.1 9.3 ± 2.1 43.9 ± 8.7 54.4 ± 6.6 29.3 ± 7.9 52.1 ± 0.8
Values represent the means ± standard deviation (SD) of n = 3 duplicate assays. The concentration of all test samples was 1.5 mg/ml.
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Table 2 Inhibitory activities of isolated flavonoid compounds against a-glucosidase and a-amylase. Flavonoid Myricetin Quercetin Avicularin Guaijaverin Hyperin Kaempferol Apigenin Acarbose a b
Rat intestinal sucrase Inhibition (%)a,b IC50 (mM)a
Rat intestinal maltase Inhibition (%)a,b IC50 (mM)a
a-amylase
70.2 ± 4.5 64.5 ± 3.1 39.0 ± 2.6 36.4 ± 2.5 34.3 ± 3.8 50.6 ± 2.7 23.1 ± 2.4 48.4 ± 1.3
53.7 ± 2.3 51.1 ± 4.7 31.7 ± 3.5 33.8 ± 4.2 39.7 ± 3.7 45.0 ± 0.4 27.6 ± 3.9 79.8 ± 0.5
53.9 ± 5.6 52.1 ± 7.3 43.3 ± 6.9 44.3 ± 5.8 40.4 ± 5.7 50.7 ± 8.3 31.4 ± 3.1 52.1 ± 0.8
3.0 ± 0.1 3.5 ± 0.3 6.5 ± 0.7 6.2 ± 0.6 7.5 ± 0.8 5.2 ± 0.4 >30 2.5 ± 0.1
Inhibition(%)a,b IC50 (mM)a
4.1 ± 0.2 4.8 ± 0.4 7.6 ± 0.2 6.6 ± 0.5 7.8 ± 0.6 5.6 ± 0.1 >30 0.9 ± 0.1
4.3 ± 0.3 4.8 ± 0.1 5.9 ± 0.5 5.6 ± 0.2 6.1 ± 0.3 5.3 ± 0.2 >30 2.3 ± 0.1
Values represent the means ± standard deviation (SD) of n = 3 duplicate assays. The concentration of all test samples was 1.5 mg/ml.
Table 3 The possible synergistic activities of the isolated flavonoid compounds against a-glucosidase and a-amylase. Flavonoid
a
Sucrase
a-Amylase
Maltase
IC50 (mM)a
Interaction
IC50 (mM)a
Interaction
IC50 (mM)a
Interaction
Quercetin Myricetin Quercetin + myricetin
3.5 ± 0.3 3.0 ± 0.1 2.0 ± 0.2
Synergistic
4.8 ± 0.4 4.1 ± 0.2 3.2 ± 0.4
Synergistic
4.8 ± 0.1 4.3 ± 0.3 4.5 ± 0.3
None
Hyperin Avicularin Hyperin + avicularin
7.5 ± 0.8 6.5 ± 0.7 4.5 ± 0.5
Synergistic
7.8 ± 0.6 7.6 ± 0.2 5.0 ± 0.3
Synergistic
6.1 ± 0.3 5.9 ± 0.5 6.0 ± 0.2
None
Kaempferol Quercetin Kaempferol + quercetin
5.2 ± 0.4 3.5 ± 0.3 2.6 ± 0.6
Synergistic
5.6 ± 0.1 4.1 ± 0.2 4.2 ± 0.1
Synergistic
5.3 ± 0.2 4.3 ± 0.3 5.1 ± 0.4
None
Values represent the means ± standard deviation (SD) of n = 3 duplicate assays.
5 min. The absorbance of the supernatant was measured at 595 nm and the inhibitory activity was calculated using the following formula:
Inhibitory activity ð%Þ ¼ ðODcontrol ODtest sample Þ=ODcontrol 100 The Inhibitory activities of the samples against a-amylase are shown as inhibiting percentages and IC50 values (Table 2). 2.7. Evaluation of synergy of isolated compounds against aglucosidase and a-amylase In order to determine the synergy of the isolated compounds against a-glucosidase and a-amylase, five pure isolated compounds were selected to form three samples by mixing the solutions of two different compounds with the same concentration (1.5 mg/ml) and the same volume (1.5 ml). These three samples are quercetin–myricetin, hyperin–avicularin, quercetin–kaempferol, their activities against a-glucosidase and a-amylase were determined as IC50 values by the method mentioned above and their IC50 values are listed in Table 3. 3. Results and discussion 3.1. Isolation of active compounds and structural determination Seven pure compounds were separated from the EtOAc-soluble fraction by the method mentioned above; they were identified as quercetin (1, 98.3 mg), kaempferol (2, 15.6 mg), guaijaverin (3, 19.9 mg), avicularin (4, 23.5 mg), myricetin (5, 7.5 mg), hyperin (6, 9.6 mg) and apigenin (7, 11.4 mg), respectively (their structures are shown in Fig. 1), by comparing the obtained MS and NMR data with those reported. Five pure compounds were obtained
from n-BuOH-soluble fraction and they were identified as 1 (203.6 mg), 2 (15.6 mg), 3 (50.3 mg), 4 (31.4 mg) and 6 (23.6 mg). In addition, the purity of each individual compound isolated was also determined by HPLC with values more than 99%. Compound 1, yellow powder. 1H NMR (DMSO-d6, 400 MHz): d 6.13 (1H, d, J = 2.0 Hz, H-6), 6.35 (1H, d, J = 2.0 Hz, H-8), 6.84 (1H, d, J = 8.0 Hz, H-50 ), 7.52 (1H, dd, J = 2.0, 8.4 Hz, H-60 ), 7.65 (1H, d, J = 2.0 Hz, H-20 ). 13C NMR (DMSO-d6, 100 Hz): d 93.7 (C-8), 98.5 (C-6), 103.3 (C-10), 115.4 (C-20 ), 115.9 (C-50 ), 120.8 (C-60 ), 122.3 (C-10 ), 136.1 (C-3), 145.4 (C-30 ), 147.1 (C-2), 148.0 (C-40 ), 156.5 (C-5), 161.1 (C-9), 164.2 (C-7), 176.2 (C-4, C@O). Negative ESI-MS m/z 301 [MH]. The MS data and the NMR data are consistent with those of quercetin reported by Guvenalp and Demirezer (2005) and Zheng, Cheng, Chao, Wu, and Wang (2008). Compound 1 was identified as quercetin. Compound 2, yellow powder. 1H NMR (DMSO-d6, 400 MHz): d 6.15 (1H, d, J = 2.0 Hz, H-6), 6.39 (1H, d, J = 2.0 Hz, H-8), 6.88 (2H, d, J = 8.8 Hz, H-30 , H-50 ), 8.00 (2H, d, J = 8.8 Hz, H-20 , H-60 ). 13C NMR (DMSO-d6, 100 MHz): d 93.8 (C-8), 98.6 (C-6), 103.4 (C-10), 115.8 (C-30 , C-50 ), 122.0 (C-10 ), 129.9 (C-20 , C-60 ), 136.0 (C-3), 147.2 (C-2), 156.5 (C-5), 159.5 (C-40 ), 161.1 (C-9), 164.2 (C-7), 176.3 (C-4, C@O). Negative ESI-MS m/z 285 [MH]. The NMR data are consistent with those reported by Pelter, Ward, and Gray (1976). Compound 2 was identified as kaempferol. Compound 3, yellow needles. 1H NMR (CD3OD, 400 MHz): d 3.43 (1H, dd, J = 3.2, 13.6 Hz, H-200 ), 3.63 (1H, dd, J = 3.2, 8.4 Hz, H-300 ), 3.81 (2H, dd, J = 3.6, 9.6 Hz, H-500 ), 3.88 (1H, dt, J = 1.6, 8.4 Hz, H-400 ), 5.14 (1H, d, J = 6.4 Hz, H-100 ), 6.17 (1H, d, J = 2.0 Hz, H-6), 6.36 (1H, d, J = 2.0 Hz, H-8), 6.85 (1H, d, J = 8.8 Hz, H-50 ), 7.55 (1H, dd, J = 2.4, 8.8 Hz, H-60 ), 7.73 (1H, dd, J = 2.4 Hz, H-20 ). 13 C NMR (CD3OD, 100 MHz): d 65.5 (C-500 ), 71.4 (C-300 ), 72.7 (C200 ), 67.7 (C-400 ), 93.2 (C-8), 98.4 (C-6), 103.2 (C-100 ), 104.1 (C-10), 114.7 (C-20 ), 116.0 (C-50 ), 121.4 (C-60 ), 121.6 (C-10 ), 134.2 (C-3),
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H. Wang et al. / Food Chemistry 123 (2010) 6–13
OH O
HO
HO
Quercetin (1)
HO
OH HO
OH O
OH O
Myricetin (5)
OH
HO
OH
Avicularin (4)
OH
OH O
OH
O O
OH
OH O
O
OH
OH OH
OH OH
Guaijaverin (3)
Kaempferol (2)
O
O
O OH O
O
OH
O
OH OH O
OH O
HO
O
HO
O
OH
OH
OH
OH
HO
HO
HO
HO
HO
O
OH OH
Hyperin (6)
OH O
Apigenin (7)
Fig. 1. Structures of isolated flavonoid compounds from guava leaves.
144.5 (C-30 ), 148.5 (C-40 ), 156.9 (C-2), 157.2 (C-8), 161.5 (C-5), 164.5 (C-7), 178.0 (C-4, C@O). Negative ESI-MS m/z 433 [MH]. The MS data and the NMR data are identical with those in the literature (Arima & Danno, 2002). Compound 3 was identified as guaijaverin. Compound 4, yellow crystals. 1H NMR (400 MHz, CD3OD): d 3.49 (m, sugar-2H, H-500 ), 3.86 (1H, dd, J = 1.6, 4.0 Hz, H-300 ), 3.90 (1H, dd, J = 3.2, 5.2 Hz, H-200 ), 4.32 (1H, dt, J = 1.6, 2.8 Hz, H-400 ), 5.45 (1H, d, J = 6.8, H-100 ), 6.18 (1H, d, J = 2.0 Hz, H-6), 6.36 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.4 Hz, H-50 ), 7.47 (1H, dd, J = 2.0, 8.4 Hz, H-60 ), 7.51 (1H, d, J = 1.6 Hz, H-20 ). 13C NMR (CD3OD, 100 MHz): d 61.1 (C-500 ), 77.2 (C-300 ), 81.8 (C-200 ), 87.5 (C-400 ), 93.3 (C-8), 98.4 (C-6), 104.1 (C-10), 108.0 (C-100 ), 114.9 (C-50 ), 115.4 (C-20 ), 121.5 (C-60 ), 121.6 (C-10 ), 133.4 (C-3), 144.9 (C-30 ), 148.4 (C-40 ), 157.1 (C-9), 157.9 (C-2), 161.6 (C-5), 164.5 (C-7), 178.5 (C-4, C@O). Negative ESI-MS m/z 433 [MH]. The MS data and the NMR data are identical with those in the literature (Zhang et al., 2005). Compound 4 was identified as avicularin. Compound 5, yellow brown powder. 1H NMR (DMSO-d6, 400 MHz): d 6.14 (1H, s, H-6), 6.33 (1H, s, H-8), 7.20 (2H, d, J = 1.6 Hz, H-20 , H-60 ). 13C NMR (DMSO-d6, 100 MHz): d 93.6 (C-8), 98.5 (C-6), 103.3 (C-10), 107.5 (C-20 , C-60 ), 121.1 (C-10 ), 136.2 (C-40 ), 136.2 (C-3), 146.1 (C-30 , C-50 ), 147.2 (C-2), 156.4 (C-9), 161.1 (C-5), 164.2 (C-7), 176.1 (C-4, C@O). Negative ESI-MS m/z 317 [MH]. The NMR data are consistent with those in the literature (Pelter et al., 1976); compound 5 was identified as myricetin. Compound 6, yellow powder. 1H NMR (CD3OD, 400 MHz): d 3.44–3.82 (m, sugar-H), 5.14 (1H, d, J = 7.6 Hz, H-100 ), 6.17 (1H, s, H-6), 6.37 (1H, s, H-8), 6.83 (1H, d, J = 8.0 Hz, H-50 ), 7.56 (1H, d, J = 8.4 Hz, H-60 ), 7.81 (1H, s, H-20 ). 13C NMR (CD3OD, 100 MHz): d 60.4 (C-600 ), 68.5 (C-400 ), 71.7 (C-200 ), 73.6 (C-300 ), 75.7 (C-500 ), 93.2 (C-8), 98.4 (C-6), 103.8 (C-100 ), 103.9 (C-10), 114.6 (C-20 ), 116.3 (C-50 ), 121.4 (C-10 ), 121.4 (C-60 ), 134.4 (C-3), 144.3 (C-30 ), 148.4 (C-40 ), 156.3 (C-2), 157.3 (C-9), 161.5 (C-5), 164.6 (C-7), 178.0 (C-4, C@O). Negative ESI-MS m/z 463 [MH]. The MS data and the NMR data are consistent with those in the literature (Zhang et al., 2005); compound 6 was identified as hyperin. Compound 7, pale yellow powder. 1H NMR (DMSO-d6, 400 MHz): d 7.87 (2H, dd, J = 2.4, 8.8 Hz, H-20 , H-60 ), 6.87 (2H, dd, J = 2.4, 8.8 Hz, H-30 , H-50 ), 6.72 (1H, d, J = 2.4 Hz, H-3), 6.43 (1H, d, J = 2.0 Hz, H-8), 6.14 (1H, d, J = 2.0 Hz, H-6). 13C NMR (DMSOd6, 100 MHz): d 94.3 (C-8), 99.2 (C-6), 103.2 (C-3), 104.0 (C-10),
121.5 (C-10 ), 116.3 (C-30 , C-50 ), 128.8 (C-20 , C-60 ), 157.7 (C-9), 161.5 (C-5), 161.8 (C-40 ), 164.1 (C-7), 164.5 (C-2), 182.1 (C-4, C@O). Negative ESI-MS m/z 269 [MH]. The NMR data are consistent with those in the literature (Ren & Yang, 2001); compound 7 was identified as apigenin. 3.2. Activities of samples against a-glucosidase and a-amylase 3.2.1. Activities of extracts The inhibitory activities of crude 75% ethanol extract of guava leaves were determined at the concentration of 1.5 mg/ml against a-glucosidase and a-amylase (Table 1). The inhibiting percentages of 75% ethanol extract against a-glucosidase were 38.3 ± 4.2% (sucrase) and 33.4 ± 6.3% (maltase), respectively; against a-amylase, inhibition was 31.7 ± 3.1%. After the 75% ethanol extract was extracted respectively with CH2Cl2, EtOAc and n-BuOH, four extracts (CH2Cl2-soluble, EtOAcsoluble, n-BuOH-soluble and residual fraction) were obtained and the inhibitory activities of these four extracts were determined at the concentration of 1.5 mg/ml against a-glucosidase and a-amylase (Table 1). The inhibiting percentages of the EtOAc-soluble extract against a-glucosidase were 46.3 ± 8.3% (sucrase) and 40.6 ± 4.5% (maltase), respectively; against a-amylase, inhibition was 43.9 ± 8.7%. The inhibiting percentages of the n-BuOH extract against a-glucosidase were 63.5 ± 5.6% (sucrase) and 47.7 ± 3.7% (maltase), respectively; against a-amylase inhibition was 54.4 ± 6.6%. Table 1 shows that, at the concentration of 1.5 mg/ml the sequences of inhibitory effects against a-glucosidase and against aamylase had the same order as follows:
n-BuOH-soluble > EtOAc-soluble > 75% Ethanol-extract > residual fraction > CH2 Cl2 -soluble 3.2.2. Activities of isolated compounds and relationships of structure and activity As the EtOAc-soluble and n-BuOH-soluble extracts showed obviously higher inhibitory activities than did the 75% ethanol extract against a-glucosidase and a-amylase, we supposed that the main active compositions of the 75% ethanol extract of guava leaves were basically in the n-BuOH-soluble fraction and EtOAcsoluble fraction, and both of the extracts were further investigated
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H. Wang et al. / Food Chemistry 123 (2010) 6–13
by isolating the active components and by evaluating the activity of the isolated compounds against a-glucosidase and a-amylase. Their IC50 values and inhibiting percentages, at the concentration of 1.5 mg/ml of the seven isolated compounds, are shown in Table 2. Although the inhibitory activities of the seven isolated compounds were still lower than that of the therapeutic drug, acarbose (IC50 = 2.5 ± 0.1 mM for rat intestinal sucrase, 0.9 ± 0.1 mM for rat intestinal maltase and 2.3 ± 0.1 for porcine pancreatic a-amylase), the data clearly indicate the potential of these compounds as inhibitors of a-glucosidase and a-amylase. On the basis of the IC50 values in Table 2, it was found that the sequence of inhibitory activities of these seven isolated compounds against sucrase, maltase and a-amylase are as follows:
Myricetin > quercetin > kaempferol > guaijaverin > avicularin > hyperin > apigenin Among the seven isolated compounds, myricetin, quercetin and kaempferol exhibited significant inhibitory activities against aglucosidase and a-amylase. The IC50 values were lower than 8 mM, especially for myricetin and quercetin, and the IC50 values were lower than 5 mM. Apigenin only showed a weak inhibitory activity with more than 30 mM for the IC50 value. Quercetin, kaempferol, myricetin and apigenin are all derivatives of 5,7-dihydroxy-2-phenylchromen-4-one; guaijaverin, avicularin and hyperin are glycosides of quercetin with different sugars. So the structures of all seven compounds can be shown as the 2-substituted-phenyl-chromen-4-one in Fig. 2. Fig. 2 shows that all the atoms forming the A-ring, B-ring and Cring, would take a sp2 hybrid form, which allows these atoms to form a ‘‘large conjugated-system”. We deduce that the ‘‘large conjugated-system” skeleton could be necessary for these compounds to act upon a-glucosidase and a-amylase. We could draw three conclusions from the above. (1) The hydroxyl on the 3-position of the flavonoid plays an important rule in the inhibitory activity against a-glucosidase and a-amylase. This conclusion could be elucidated by the following. Apigenin, lacking the 3-OH, possesses a weaker inhibitory percentage (about 20– 30%) and a higher IC50 value (more than 30 mM), but quercetin, kaempferol and myricetin, possessing the 3-OH, show obviously higher inhibitory activities than apigenin could do. In addition, the glycosylation of the 3-OH of the flavonoid was also unfavourable to the inhibitory activity (the inhibitory percentages of glycoside compounds of quercetin were lower than that of quercetin itself). Comparing the inhibitory percentages of these compounds, the order of inhibitory ability was obtained:
3-Hydroxylated flavonoid > flavonoid glycoside > 3-unhydroxylated flavonoid Similar results are also observed for the IC50 values. So the free 3-position hydroxyl group was a key functional group for inhibiting a-glucosidase and a-amylase.(2) The number of hydroxyl substitution on the C-ring would affect the inhibitory activity. Comparing the inhibitory activity of 40 -hydroxylated, 30 ,40 -dihy-
B
O 2 A 4
C
3
O Fig. 2. Structure of 2-substitutedphenyl-chromen-4-one.
droxylated and 30 ,40 ,50 -trihydroxylated flavonoids (corresponding to kaempferol, quercetin and myricetin, respectively) at the concentration of 1.5 mg/ml, we found that the inhibitory activity increases considerably with the increase of the number of hydroxyl groups on the C-ring.(3) A sugar group attached to the flavonoid nuclei at the 3-position does not notably affect its inhibitory activity. The decreasing order of the inhibitory activity against rat intestinal a-glucosidase and porcine pancreatic a-amylase was:
Guaijaverin > avicularin > hyperin So, the five-carbon glycoside could exhibit a higher activity than the six-carbon glucoside. But the inhibition percentage order of these three glucosides did not accord with that of the IC50; for example, hyperin possessed a maximum inhibition percentage (39.7 ± 3.7%) against rat intestinal maltase and showed the strongest inhibitory activity. The effect of the glycosyl structure on their activities was unclear. The conclusions above were also supported by the work of Tadera, Minami, Takamatsu, and Matsuoka (2006). 3.2.3. Synergistic action of isolated flavonoid compounds against aglucosidase and a-amylase Comparing the activities of isolated compounds with those of the extracts, it appears that the inhibiting percentages of isolated compounds are not obviously higher than that of n-BuOH-soluble extract or the EtOAc-soluble extract at the same concentration, even myricetin and quercetin, which displayed the most powerful activities among these seven isolated compounds, only had inhibitory percentage close to the n-BuOH-soluble extract or the EtOAcsoluble extract. On the other hand, Peng et al. (2003) and Cushnie and Lamb (2005), respectively, reported that flavonoid compounds could show a synergistic effect on antimicrobial activity and antioxidation. Both of the reasons noted above induced us to investigate the operation of a synergistic effect that contributes to the enhancement of inhibitory activity of a flavonoid in the presence of other flavonoids, in order to collectively account for the anti-diabetic activity and understand the mechanism of action of flavonoid compounds existing in guava leaves. Table 3 shows that inhibitory activities of three two-mixed samples, quercetin–myricetin, hyperin–avicularin and quercetin– kaempferol, display a significant improvement and would exhibit an obvious synergistic action against a-glucosidases, including sucrase and maltase, by comparing the IC50 value of the two-mixed samples with that of the original single composition. For example, the IC50 value of myricetin–quercetin (2.0 mM) was lower than that of the individual myricetin (3.0 mM) or quercetin (3.5 mM). However, we did not found these three two-mixed samples performing the synergistic action against a-amylase. 3.2.4. Analysis of HPLC data In order to investigate the relationship of activity and composition of extracts, the content of the active components of these extracts was determined by HPLC, using pure quercetin (1), kaempferol (2), guaijaverin (3), avicularin (4), myricetin (5), hyperin (6) and apigenin (7) as references. The HPLC chromatograms of the mixture of seven pure compounds, 75% ethanol extract, CH2Cl2-solube extract, EtOAc-soluble extract, n-BuOH-soluble extract and residual extract are shown in Figs. 3–8, respectively. Fig. 3 is the HPLC chromatogram of the mixture of seven pure compounds, this Figure is used as a standard to determine the active compound in the other Figures. HPLC showed that the total relative amounts of three components, namely hyperin, guaijaverin and avicularin in the 75% EtOH, CH2Cl2-soluble fraction, EtOAc-soluble fraction, n-BuOH-soluble fraction and residual extract were 84.5%, 61.2%, 82.6%, 87.1% and 67.5%, respectively (Table 4), which is in agreement with inhibitory activity of each extract. This result confirmed that glucosides
H. Wang et al. / Food Chemistry 123 (2010) 6–13
Fig. 3. HPLC chromatograms of the mixture of seven pure compounds.
Fig. 4. HPLC chromatograms of 75% ethanol extract.
Fig. 5. HPLC chromatograms of CH2Cl2-soluble extract.
Fig. 6. HPLC chromatograms of EtOAc-soluble extract.
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H. Wang et al. / Food Chemistry 123 (2010) 6–13
Fig. 7. HPLC chromatograms of n-C4H9OH-soluble extract.
Fig. 8. HPLC chromatograms of residual extract.
Table 4 The relative amounts of individual component in extract fractions. Flavonoid
Hyperin Guaijaverin Avicularin Myricetin Quercetin Kaempferol Apigenin
75%EtOH
CH2Cl2
EtOAc
n-C4H9OH
Residual extract
Time (min)
Content (%)
Time (min)
Content (%)
Time (min)
Content (%)
Time (min)
Content (%)
Time (min)
Content (%)
17.342 21.951 23.321 28.142 37.453 43.752 45.577
18.0 35.6 30.9 0.58 7.44 5.50 1.97
17.199 21.806 23.156 28.272 37.265 43.638 45.387
11.4 26.0 23.8 1.10 34.9 0.43 2.22
17.430 21.992 23.318 28.508 37.365 43.627 45.398
13.1 34.7 34.8 1.93 12.9 2.24 0.29
17.177 21.904 23.278 28.314 37.533 43.790 45.607
29.1 32.2 25.9 1.42 7.13 2.90 1.43
17.726 22.251 23.570 28.427 37.587 43.947 45.826
25.3 27.2 15.0 4.34 9.80 4.75 13.6
should be the main active components in these extracts to inhibit a-glucosidase and a-amylase. Quercetin was the main component among the individual compounds isolated from EtOAc-soluble or n-C4H9OH-soluble extracts, but we found from the HPLC chromatograms of the EtOAc-soluble or n-C4H9OH-soluble extracts that quercetin was not the main component. The reason for this phenomenon may be that the hyperin, guaijaverin and avicularin were hydrolysed into quercetin during the process of column chromatographic isolation by the acidity of the resin material.
nents could confer an obvious synergistic effect to inhibitory activity against a-glucosidase, but not against a-amylase, which is found for the first time in this study on anti-diabetic activity of isolated compounds from guava leaves. The results give scientific support for the proper use of guava leaves in folk medicine for the treatment of diabetes and this work could help to develop medicinal preparations or nutraceutical and functional foods for diabetes and related conditions.
4. Conclusion
This research was financially supported by the Guangdong Food Industry Institute.
Seven major active components, quercetin, myricetin, avicularin, guaijaverin, hyperin and kaempferol, were isolated from extracts of guava leaves. We found that (1) the major active components of these extracts were glycosides of quercetin and there is an obvious relationship between the structure and activity in these isolated active compounds; (2) a mixture of two compo-
Acknowledgements
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