Identification of a new angiotensin-converting enzyme (ACE) inhibitor from Thai edible plants

Food Chemistry 165 (2014) 92–97

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Identification of a new angiotensin-converting enzyme (ACE) inhibitor from Thai edible plants Arunee Simaratanamongkol a, Kaoru Umehara c, Hiroshi Noguchi c, Pharkphoom Panichayupakaranant a,b,⇑ a

Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand Phytomedicine and Pharmaceutical Biotechnology Excellent Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand c School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan b

a r t i c l e

i n f o

Article history: Received 14 February 2014 Received in revised form 21 April 2014 Accepted 14 May 2014 Available online 22 May 2014 Keywords: Apium graveolens Apiaceae Angiotensin converting enzyme ACE inhibitor Junipediol A 8-O-b-D-glucoside

a b s t r a c t Eight Thai edible plants were tested for their inhibitory activity against an angiotensin-converting enzyme (ACE) using an in vitro assay. The methanol extract of Apium graveolens exhibited significant ACE inhibitory activity with an IC50 value of 1.7 mg/ml, and was then subjected to an isolation procedure that resulted in identification of a pure active constituent, junipediol A 8-O-b-D-glucoside (1-b-D-glucosyloxy-2-(3-methoxy-4-hydroxyphenyl)-propane-1,3-diol) (1), which had good ACE inhibitory activity with an IC50 value of 76 lg/ml. Another eight known compounds, isofraxidin-b-D-glucoside (2), roseoside (3), apigenin-7-O-b-D-glucoside (4), luteolin-7-O-b-D-glucoside (5), icariside D2 (6), apiin (7), chrysoeriol-7-O-b-D-apiosylglucoside (8), and 11,21-dioxo-3 b,15a,24-trihydroxyurs-12-ene-24-Ob-D-glucopyranoside (9) were also identified. Although each of these five constituents (2–6) isolated from the same fraction as 1 showed no activity at concentrations of 500 lM, together, when each was present at 300 lg/ml, they enhanced the inhibitory activity of 500 lM of 1 from 64% to 81%. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Hypertension is the term used to describe high blood pressure, and it is a worldwide chronic disease. Untreated hypertension can lead to stroke, coronary heart disease, kidney dysfunction, disability and death. Hypertension is defined as a systolic blood pressure above 140 mmHg and/or a diastolic blood pressure above 90 mmHg (Chen et al., 2009). The use of angiotensin-converting enzyme (ACE) inhibitors is well established as one of the main therapeutic agents for the treatment of hypertension. ACE is a component of the renin– angiotensin–aldosterone system. It plays a key role in the homoeostatic mechanism of mammals, by contributing to the maintenance of normal blood pressure and to the electrolyte balance, and by being involved in the regulation and control of the arterial pressure. ACE inhibitors are also employed for the prophylactic control of diabetic nephropathy and for the treatment of heart failure. Several ACE inhibitors, such as captopril, enalapril, lisinopril and temocapril, are now in clinical use for the treatment of

⇑ Corresponding author at: Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand. Tel./fax: +66 74 428220. E-mail address: [email protected] (P. Panichayupakaranant). http://dx.doi.org/10.1016/j.foodchem.2014.05.080 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

hypertension. An advantage of the ACE inhibitors over other anti-hypertensive drugs is a reduction of any CNS side effects. However, all the synthetic ACE inhibitor drugs produce some side effects, such as coughing, rashes and taste aversion, thus justifying the search for new natural ACE inhibitors that may be safer and also more economical to use (Chen et al., 2009). A peptide isolated from snake venom is one source of a natural ACE inhibitor that was used as lead compound for the development of synthetic ACE inhibitors, such as captopril (Bhuyan & Mugesh, 2011). Moreover, several plant derived compounds are also a source of natural ACE inhibitors. It has been reported that several plant extracts, such as those from Glycine max (Rho, Lee, Chung, Kim, & Lee, 2009), Cassia tora (Hyun, Lee, Kang, Chung, & Choi, 2009), Rosa damascene (Kwon et al., 2010), Curcuma longa (Bhullar, Jha, Youssef, & Rupasinghe, 2013) and Lactuca sativa (Lagemann, Dunkel, & Hofmann, 2012), contained ACE inhibitors. Although some Thai edible plants, such as A. graveolens, Morinda citrifolia, Moringa oleifera, Solanum torvum, have been traditionally used for their anti-hypertensive effect, reports on their ACE inhibitory activity are rarely found. A study to detect ACE inhibitors from Thai edible plants is therefore of interest, and possibly important for the development of naturally derived anti-hypertensive agents. In this study, the ACE inhibitory activity of extracts from some edible plants commonly consumed in Thailand was investigated.

93

A. Simaratanamongkol et al. / Food Chemistry 165 (2014) 92–97

injection volume was 10 ll, and hippuric acid was detected by monitoring the absorbance of the eluents at 228 nm.

2. Materials and methods 2.1. Plant materials Eight plants (Table 1) were collected from Songkhla Province, Thailand during 2010. Voucher specimens were deposited in the herbarium of the Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand. The plant material was dried at 50 °C for 24 h in a hot air oven, then reduced to powder using a grinder, and the powder was passed through a sieve (No. 45). 2.2. Reagents Hippuric acid (HA), N-hippuryl-L-histydyl-L-leu-cine (HHL), and angiotensin I-converting enzyme (ACE) from rabbit lung were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). 2.3. Preparations of plant extracts for the screening of ACE inhibitory activity The dried plant powders (20 g) were extracted twice with ethyl acetate (100 ml  2) under reflux conditions for 1 h. The extracts were combined and concentrated under reduced pressure to produce the ethyl acetate extracts. The marcs were subsequently extracted with methanol (100 ml  2) and concentrated under reduced pressure to produce the methanol extracts. 2.4. ACE inhibitory assay The activity of ACE was determined using HHL as the substrate with some modifications (Wanasundara et al., 2002). The assay was conducted in 100 mM sodium borate buffer (pH 8.3) containing 300 mM NaCl. The buffer was used to dilute samples, enzyme, and substrate. Each sample solution (50 ll) with 50 ll of 2.5 mM HHL was pre-incubated at 37 °C for 5 min. The reaction was initiated by addition of 50 ll of ACE solution (50 mU/ml), and the mixture was incubated at 37 °C for 60 min. The reaction was stopped by heating at 100 °C for 4 min. The reaction mixture was assayed for its hippuric acid content using HPLC. The IC50 value was defined as the concentration of inhibitor required to inhibit 50% of the ACE activity under the assay conditions and was determined by regression analysis of the ACE inhibition (%) versus the log of the inhibitor concentration. 2.5. Quantitative HPLC determination of hippuric acid HPLC analysis was carried out using the HPLC Jasco AS-2055 Plus Intelligent Sampler, PU-2085 Plus Semi-micro HPLC Pump, and MD-2010 Plus Multiwavelength Detector. Data analysis was performed using software Chrom NAV. A column TOSOH TSK gel ODS 100 V 4.6  250 mm, and isocratic solvent system (17.5% v/v acetonitrile in 0.1% formic acid water) were employed. The

2.6. Extraction and isolation of 1–9 from A. graveolens The dried whole plant material from A. graveolens (2.7 kg) was successively extracted with ethyl acetate (6 L  3), and methanol (6 L  3) by maceration for 3 days. The extracts were concentrated under reduced pressure at 40 °C to yield the ethyl acetate (30 g) and methanol (375 g) dry extracts. The methanol extract (250 g) was further suspended in water (1.4 L) and partitioned with ethyl acetate (8  0.4 L) to yield the ethyl acetate- (86 g) and water-soluble (150 g) fractions. The water-soluble fraction was subjected to Diaion HP-20 column chromatography using water, 50% v/v methanol, 75% v/v methanol, methanol, and ethyl acetate as eluents (5, 20, 16, 10, and 2 L, respectively) to yield the following fractions: water (109 g), 50% v/v methanol (23 g), 75% v/v methanol (3.4 g), methanol (1.8 g), and ethyl acetate (1 g). The 50% v/v methanol-soluble fraction was chromatographed on a silica gel column (6  50 cm) and fractionated (500 ml for each fraction) using a chloroform–methanol gradient solvent system (9:1 and 8:2 (v/v); of 11 and 15 L, respectively). Fractions were collected and pooled by TLC analysis to afford 7 combined fractions. Fraction 4 [0.6 g, eluted with chloroform–methanol (9:1)] was subjected to a semi-preparative HPLC [Inert Sustain ODS column with methanol–water (20:80)] to yield 2 (2 mg; tR 35 min). Fraction 5 [2.5 g, eluted with chloroform–methanol (9:1)] was subjected to a semi-preparative HPLC (Inertsil Ph-3 column) to yield 3 [3 mg; tR 38 min with acetonitrile–water (12.5:87.5)], 5 [1 mg; tR 41 min with methanol–water (35:65)], and 4 [1 mg; tR 63 min with methanol–water (35:65)]. Fraction 6 [1.4 g, eluted with chloroform–methanol (8:2)] was subjected to the semi-preparative HPLC [Inertsil Ph-3 column with methanol– water (7.5:92.5)] to yield 1 (15 mg; tR 25 min) and 6 (3 mg; tR 54 min) (Fig. 1). The 75% v/v methanol-soluble fraction was subjected to the semi-preparative HPLC (Inertsil Ph-3 column) to yield 7 [260 mg; tR 36 min with methanol–water (40:60)], 8 [300 mg; tR 63 min with methanol–water (35:65)], and 9 [11 mg; tR 42 min with methanol–water (50:50)] (Fig. 1). 2.7. Identification of 1–9 Junipediol A 8-O-b-D-glucoside (1): Colourless amorphous powder; 1H NMR (400 MHz, DMSO-d6): d 6.83 (d, J = 2 Hz, H-2), 6.67 (d, J = 8 Hz, H-5), 6.65 (dd, J = 8, 2 Hz, H-6), 4.14 (d, J = 8 Hz, H-10 ), 3.93 (dd, J = 9.5, 6.5 Hz, H-8), 3.74 (s, OCH3), 3.68 (dd, J = 11.5, 6 Hz, H-9), 3.65 (dd, J = 9.5, 7 Hz, H-8), 3.57 (dd, J = 11.5, 6 Hz, H-9), 2.85 (m, H-7); 13C NMR (100 MHz, DMSO-d6): d 147.1 (C-3), 144.8 (C-4), 132.2 (C-1), 120.2 (C-6), 115.0 (C-5), 112.6 (C-2), 103.2 (C-10 ), 76.8 (C-30 ), 76.7 (C-50 ), 73.4 (C-20 ), 70.6 (C-40 ), 70.1 (C-8), 63.1 (C-9), 61.1 (C-60 ), 55.5 (OCH3), 47.4 (C-7). Based on these 1H NMR and

Table 1 Plant materials used in this study. Family

Botanical name

Voucher number

Part used

Anacardiaceae Apiaceae Dioscoreaceae Moraceae Moringaceae Rubiaceae Saururaceae Solanaceae

Anacardium occidentale Linn. A. graveolens L. Tacca chantrieri Andre Ficus racemosa Linn. M. oleifera Lam. M. citrifolia L. Houttuynia cordata Thumb. S. torvum Sw.

SKP SKP SKP SKP SKP SKP SKP SKP

Leaves Whole plants Leaves Fruits Leaves Leaves Aerial parts Fruits

009 012 062 117 118 165 173 180

01 01 20 06 13 13 08 19

15 01 07 01 03 01 18 01 15 01 03 01 03 01 20 01

94

A. Simaratanamongkol et al. / Food Chemistry 165 (2014) 92–97

MeOH extract of A. graveolens (250 g) EtOAc/ H2O

Aqueous fr (150 g)

EtOAc fr (86 g)

Diaion HP-20 column

H2O fr (109 g)

50% MeOH fr (23 g)

75% MeOH fr (3.4 g)

100% MeOH fr (1.8 g)

EtOAc fr (1 g)

Si gel Column CHCl3-MeOH (9:1 and 8:2) system 9:1 I (0.1 g)

8:2 II (0.6 g)

III (0.8 g)

HPLC, ODS column 20% MeOH 2 (2 mg)

IV (0.6 g)

V (2.6 g)

VI (6.3 g)

VII (2.6 g) HPLC, Ph-3 column 7.5% MeOH

HPLC, Ph-3 column 12.5% MeCN 35% MeOH

3 (3 mg)

5 (1 mg)

4 (1 mg)

1 (15 mg)

6 (3 mg)

75% MeOH fr (3.4 g)

HPLC, Ph-3 column 40% MeOH 7 (260 mg)

HPLC, Ph-3 column 35% MeOH

8 (300 mg)

HPLC, Ph-3 column 50% MeOH

9 (11 mg)

Fig. 1. Purification process of compounds 1–9.

13

C NMR data, 1 was identified as junipediol A 8-O-b-D-glucoside by comparison with the data reported previously (Comte, Allais, Chulia, Vercauteren, & Pinaud, 1997). Isofraxidin-b-D-glucoside (2): White amorphous powder; 1H NMR (400 MHz, CD3OD): d 7.86 (d, J = 9.5 Hz, H-4), 7.00 (s, H-5), 6.34 (d, J = 9.5 Hz, H-3), 5.33 (d, J = 7.6 Hz, H-10 ), 4.02 (s, –OCH3), 3.89 (s, –OCH3); 13C NMR (100 MHz, CD3OD): d 162.6 (C-2), 151.5 (C-6), 145.8 (C-4), 144.1 (C-8), 143.5 (C-7), 142.4 (C-9), 116.7 (C-10), 115.8 (C-3), 104.3 (C-10 ), 62.5 (–OCH3), 57.2 (–OCH3). Based on these 1H NMR and 13C NMR data, 2 was identified as isofraxidinb-D-glucoside by comparison with the data reported previously (Tsukamoto, Hisada, & Nishibe, 1985). Roseoside (3): White amorphous powder; 1H NMR (400 MHz, CD3OD): d 5.88 (s, H-8), 5.85 (s, H-4), 5.83 (s, H-7), 4.41 (qui, H-9), 4.32 (d, J = 8 Hz, H-10 ), 3.85 (dd, J = 12, 2.5 Hz, H-60 ), 3.62 (dd, J = 12, 6 Hz, H-60 ) 3.34 (t, J = 8 Hz, H-30 ), 3.24 (m, H-50 ), 3.23 (t, J = 8 Hz, H-40 ), 3.16 (t, J = 8 Hz, H-20 ), 2.52 (d, J = 17 Hz, H-2), 2.16 (d, J = 17 Hz, H-2), 1.91 (d, J = 1.5 Hz, H-13), 1.28 (d, J = 6.5 Hz, H-10), 1.03 (s, H-11), 1.02 (s, H-12); 13C NMR (100 MHz, CD3OD): d 201.2 (C-3), 167.2 (C-5), 135.3 (C-8), 131.6 (C-7), 127.2 (C-4), 102.8 (C-10 ), 80.0 (C-6), 78.2 (C-30 ), 78.0 (C-50 ), 77.3 (C-9), 75.3 (C-20 ), 71.7 (C-40 ), 62.9 (C-60 ), 50.7 (C-2), 42.4 (C-1), 24.7 (C-12), 23.4 (C-11), 21.2 (C-10), 19.5 (C-13). Based on

these 1H NMR and 13C NMR data, 3 was identified as vomifoliol 30 -O-b-D-glucopyranoside or roseoside by comparison with the data reported previously (Andersson & Lundgren, 1988). Apigenin-7-O-b-D-glucoside (4): Yellow amorphous powder; 1H NMR (400 MHz, DMSO): d 12.95 (s, OH-5), 7.94 (d, J = 8 Hz, H-20 ,60 ), 6.93 (d, J = 8 Hz, H-30 ,50 ), 6.84 (s, H-3), 6.82 (d, J = 2 Hz, H-8), 6.44 (d, J = 2 Hz, H-6), 5.05 (d, J = 7 Hz, H-100 ); 13C NMR (100 MHz, DMSO): d 181.8 (C-4), 164.2 (C-2), 162.8 (C-7), 161.3 (C-40 ), 161.0 (C-5), 156.8 (C-9), 128.5 (C-20 ,60 ), 120.9 (C-10 ), 115.9 (C-30 ,50 ), 105.3 (C-10), 103.0 (C-3), 99.9 (C-100 ), 99.5 (C-6), 94.8 (C-8), 77.1 (C-500 ), 76.4 (C-300 ), 73.0 (C-200 ), 69.5 (C-400 ), 60.5 (C-600 ). Based on these 1H NMR and 13C NMR data, 4 was identified as apigenin-7-O-b-D-glucoside by comparison with the data reported previously (Gohari et al., 2011). Luteolin-7-O-b-D-glucoside (5): Yellow amorphous powder; 1H NMR (400 MHz, DMSO): d 7.43 (dd, J = 8, 2 Hz, H-60 ), 7.40 (d, J = 2 Hz, H-20 ), 6.88 (d, J = 8 Hz, H-50 ), 6.77 (d, J = 2 Hz, H-8), 6.72 (s, H-3), 6.44 (d, J = 2 Hz, H-6), 5.06 (d, J = 7.5 Hz, H-100 ); 13C NMR (100 MHz, DMSO): d 181.7 (C-4), 164.4 (C-2), 162.9 (C-7), 161.0 (C-5), 156.8 (C-9), 150.1 (C-40 ), 145.8 (C-30 ), 121.1 (C-10 ), 119.1 (C-60 ), 115.9 (C-50 ), 113.4 (C-20 ), 105.2 (C-10), 103.0 (C-3), 99.9 (C-100 ), 99.5 (C-6), 94.6 (C-8), 77.1 (C-500 ), 76.3 (C-300 ), 73.1 (C-200 ), 69.5 (C-400 ), 60.6 (C-600 ). Based on these 1H NMR and 13C NMR data,

A. Simaratanamongkol et al. / Food Chemistry 165 (2014) 92–97

5 was identified as luteolin-7-O-b-D-glucoside by comparison with the data reported previously (Gohari et al., 2011). Icariside D2 (6): Colourless amorphous powder; 1H NMR (400 MHz, CD3OD): d 7.03 (d, J = 8.6 Hz, H-2,6), 6.93 (d, J = 8.8 Hz, H-3,5), 4.75 (d, J = 7.6 Hz, H-10 ), 3.66 (t, J = 7.1 Hz, H-8), 2.66 (t, J = 7.1 Hz, H-7); 13C NMR (100 MHz, CD3OD): d 157.6 (C-4), 134.3 (C-1), 130.8 (C-2,6), 117.8 (C-3,5), 102.6 (C-10 ), 78.1 (C-30 ), 78.0 (C-50 ), 74.9 (C-20 ), 71.4 (C-40 ), 64.3 (C-8), 62.5 (C-60 ), 39.4 (C-7). Based on these 1H NMR and 13C NMR data, 6 was identified as 4-(2-hydroxyethyl)phenyl-b-D-glucopyranoside or icariside D2 by comparison with the data reported previously (Miyase, Ueno, Takizawa, Kobayashi, & Oguchi, 1989). Apiin (7): Yellow amorphous powder; 1H NMR (400 MHz, DMSO): d 7.95 (d, J = 8.8 Hz, H-20 ,60 ), 6.94 (d, J = 8.8 Hz, H-30 ,50 ), 6.85 (s, H-3), 6.80 (d, J = 2 Hz, H-8), 6.43 (d, J = 2 Hz, H-6), 5.35 (d, J = 1 Hz, H-1000 ), 5.16 (d, J = 7.1 Hz, H-100 ), 3.92 (m, H-4000 ,5000 ), 3.71 (m, H-300 ), 3.66 (dd, J = 10,2 Hz, H-600 ), 3.52 (m, H-200 ), 3.49 (m, H-2000 ), 3.48 (m, H-600 ), 3.47 (m, H-500 ), 3.20 (m, H-400 ); 13C NMR (100 MHz, DMSO): d 181.9 (C-4), 164.2 (C-2), 162.6 (C-7), 161.3 (C-5), 161.07 (C-40 ), 156.8 (C-9), 128.5 (C-20 , 60 ), 120.9 (C-10 ), 115.9 (C-30 , 50 ), 108.7 (C-1000 ), 105.3 (C-10), 103.0 (C-3), 99.3 (C-100 ), 98.1 (C-6), 94.7 (C-8), 79.2 (C-3000 ), 76.9 (C-500 ), 76.7 (C-200 ), 76.0 (C-300 ), 75.8 (C-2000 ), 73.9 (C-4000 ), 69.8 (C-4000 ), 64.1 (C-5000 ), 60.5 (C-600 ). Based on these 1H NMR and 13C NMR data, 7 was identified as apigenin-7-O-b-D-apiosylglucoside or apiin by comparison with the data reported previously (Yoshikawa et al., 2000). Chrysoeriol-7-O-b-D-apiosylglucoside (8): Yellow amorphous powder; 1H NMR (400 MHz, DMSO): d 7.59 (dd, J = 8,2 Hz, H-60 ), 7.58 (d, J = 2 Hz, H-20 ), 6.96 (s, H-3), 6.94 (d, J = 8 Hz, H-50 ), 6.84 (d, J = 2 Hz, H-8), 6.43 (d, J = 2 Hz, H-6), 5.35 (d, J = 1 Hz, H-1000 ), 5.15 (d, J = 7.5 Hz, H-100 ), 3.92 (m, H-4000 ,5000 ), 3.90 (s, –OCH3), 3.71 (m, H-300 ), 3.66 (dd, J = 10,2 Hz, H-600 ), 3.52 (m, H-200 ), 3.49 (m, H-2000 ), 3.48 (m, H-600 ), 3.47 (m, H-500 ), 3.20 (m, H-400 ); 13C NMR (100 MHz, DMSO): d 181.9 (C-4), 164.1 (C-2), 162.6 (C-7), 161.0 (C-5), 156.9 (C-9), 150.9 (C-40 ), 148.0 (C-30 ), 121.3 (C-10 ), 120.5 (C-60 ), 115.8 (C-50 ), 110.4 (C-20 ), 108.7 (C-1000 ), 105.3 (C-10), 103.4 (C-3), 99.3 (C-200 ), 98.2 (C-6), 94.9 (C-8), 79.2 (C-3000 ), 77.0 (C-500 ), 76.7 (C-2000 ), 76.1 (C-300 ), 75.8 (C-200 ), 73.9 (C-4000 ), 69.8 (C-400 ), 64.1 (C-5000 ), 60.5 (C-600 ), 55.9 (–OCH3). Based on these 1H NMR and 13C NMR data, 8 was identified as chrysoeriol-7-O-b-D-apiosylglucoside by comparison with the data reported previously (Bucar et al., 1998). 11,21-Dioxo-3b,15a,24-trihydroxyurs-12-ene-24-O-b-D-glucopyranoside (9): White amorphous powder; 1H NMR (400 MHz, CD3OD): d 5.68 (s, H-12), 4.24 (d, J = 7.8 Hz, H-10 ), 4.22 (d, J = 10 Hz, H-15), 4.02 (d, J = 10 Hz, H-24), 3.85 (dd, J = 12, 2 Hz, H-60 b), 3.77 (d, J = 10 Hz, H-24), 3.66 (m, H-60 a), 3.30 (m, H-3), 3.2–3.3 (m, H-30 - 50 ), 3.15 (d, J = 7.8 Hz, H-20 ), 2.71 (m, H-1 b), 2.46 (d, J = 13.2 Hz, H-22 b), 2.43 (s, H-9), 2.22 (m, H-20), 2.16 (m, H-18), 2.12 (d, J = 13.2 Hz, H-22a), 1.86 (m, H-2 b), 1.85 (m, H-7 b), 1.75 (m, H-6a), 1.74 (m, H-7a), 1.65 (m, H-16a), 1.60 (m, H-2a), 1.55 (m, H-6 b), 1.48 (m, H-1a), 1.42 (m, H-16 b), 1.30 (s, H-23), 1.20 (s, H-25–27), 1.05 (m, H-19), 1.02 (d, J = 6.3 Hz, H-30), 1.01 (d, J = 6.3 Hz, H-29), 1.01 (s, H-28), 0.85 (m, H-5); 13C NMR (100 MHz, CD3OD): d 213.8 (C-21), 201.6 (C-11), 165.7 (C-13), 132.4 (C-12), 105.3 (C-10 ), 80.4 (C-50 ), 78.3 (C-3), 78.0 (C-30 ), 75.2 (C-20 ), 73.2 (C-24), 71.8 (C-40 ), 68.1 (C-15), 62.9 (C-60 ), 62.9 (C-9), 59.6 (C-18), 56.8 (C-5), 55.5 (C-22), 51.3 (C-20), 50.4 (C-14), 47.7 (C-8), 43.9 (C-4), 42.6 (C-19), 40.6 (C-16), 40.5 (C-17), 40.3 (C-1), 38.2 (C-10), 37.2 (C-7), 28.7 (C-2), 28.3 (C-28), 23.3 (C-23), 19.5 (C-26), 19.4 (C-6), 18.6 (C-29), 17.4 (C-25), 15.6 (C-27), 12.7 (C-30). Based on these 13C NMR data, 9 was identified as 11,21-dioxo-3b,15a,24-trihydroxyurs-12-ene-24-O-b-D-glucopyranoside by comparison with the data reported previously (Zhou et al., 2009).

95

3. Results and discussion Prior to determination of the ACE inhibitory activity of these plant extracts, the responsiveness of the assay system was calibrated with captopril, a positive control, and it showed ACE inhibitory activity with an IC50 value of 1.56 nM, which was in good agreement with a previous report (Nunes-Mamede, De Mello, & Martins, 1990). Among the sixteen extracts from eight Thai edible plants that were investigated for their ACE inhibitory activity, the methanol extract of A. graveolens gave the highest percentage of ACE inhibitory activity (82.3%, at a concentration of 5 mg/ml), and exhibited a significant IC50 value of 1.7 mg/ml, followed by the methanol extract of S. torvum (76.2%, at a concentration of 5 mg/ml) and the ethyl acetate extract of A. occidentale (64.2%, at a concentration of 5 mg/ml). The other plant extracts showed only a weak inhibitory activity with a percentage of ACE inhibitory activity lower than 60% each tested at a concentration of 5 mg/ml) (data not shown). Thus, these three edible plants may be able to contribute to hypotensive effects by being inhibitors of angiotensin converting enzyme in the renin–angiotensin system. The methanol extract of A. graveolens was the one selected and subjected to isolation of the ACE inhibitor. The methanol extract of A. graveolens (250 g) was partitioned between ethyl acetate and water. The water soluble fraction was subjected to Diaion HP-20 column chromatography and eluted with water, 50% v/v methanol, 75% v/v methanol, and methanol, successively. These fractions were then used to determine their ACE inhibitory activity. The most active fraction (50% v/v methanol fraction) that gave more than 60% inhibitory activity, at 1.5 mg/ml was further subjected to silica gel column chromatography and eluted with chloroform–methanol–water as a gradient solvent system to afford 7 combined fractions. Further purification of the active fractions was carried out using preparative HPLC to give 6 pure compounds. The isolated compounds were identified by comparisons of their spectroscopic data and optical rotations with the values reported in the literature (Andersson & Lundgren, 1988; Comte et al., 1997; Gohari et al., 2011; Miyase et al., 1989; Tsukamoto et al., 1985) as junipediol A-8-O-b-D-glucoside (1), isofraxidin-b-D-glucoside (2), roseoside (3), apigenin-7-O-b-D-glucoside (4), luteolin-7-O-b-D-glucoside (5), icariside D2 (6) (Fig. 2). The active 75% v/v methanol fraction was also fractionated by using semi preparative HPLC to yield 3 pure compounds. These isolates were identified as apiin (7), chrysoeriol-7-O-b-D-apiosylglucoside (8), and 11,21-dioxo-3 b,15a,24-trihydroxyurs-12-ene24-O-b-D-glucopyranoside (9) (Fig. 2) by comparisons of their spectroscopic data (Bucar et al., 1998; Yoshikawa et al., 2000; Zhou et al., 2009). The ACE inhibitory activity of the isolated compounds was tested by assessing the hippuric acid content produced from HHL using HPLC monitoring the absorbance at 228 nm and evaluated for their IC50 values. Compound 1 showed good ACE inhibitory activity, with an IC50 of 75.6 lg/ml (210 lM). Other isolates scarcely showed activity even at a concentration of 500 lM (data not shown). However, the inhibitory activity of 1 was enhanced to 66% when 250 lM of 1 was mixed with 300 lg/ml of the other five compounds (2–6) present in the same fraction together. Moreover, these five compounds (2–6) increased the activity to 81% in the case when 500 lM of 1 was employed (Table 2). The five compounds may enhance the activity by interacting with ACE at different region from 1. Some plant derived phenolic compounds have been evaluated for their ACE inhibitory activity, and their QSAR analysis revealed that the number of hydroxyl groups played an important role in their activity. Compound 1 exhibited higher activity than those phenolic compounds with a better IC50 value, however, its SAR study may help us to develop more powerful ACE inhibitors, as

96

A. Simaratanamongkol et al. / Food Chemistry 165 (2014) 92–97

OH O

HO HO

O

CH

OH

OH

MeO

OH O

HO HO

OMe

O

O

OMe

OH

O

OH

2

1 OH

OH O

HO HO

O

HO HO

HO

OH

OH O

O

O

OH

O

OH O

4

3 OH HO HO

CH2OH CH2

OH O

O

O

OH

OH OH O

OH

5

O O

HO HO

OH

OH

OH O

HO HO HO

6

O

OH

O

OH

7

OH O O

O

O

OCH3

O

O

O H

OH O OH

O

OH O

OH HO HO HO

O

OH

OH HO HO

8

O

HO O CH2 H

H

OH

OH

9 Fig. 2. Structure of compounds 1–9.

Table 2 Synergistic ACE inhibitory activity of the isolates from A. graveolens. Samples 1 1 1 1 1

(500 lM) + 2–6 (300 lg/ml) (250 lM) + 2–6 (300 lg/ml) (500 lM) (250 lM) (125 lM)

ACE inhibitory activity (%) 80.7 ± 1.8 65.9 ± 2.5 64.0 ± 4.2 52.8 ± 3.6 41.6 ± 2.3

n = 3.

members of the catechol group were reported to contribute the greatest activity (Al Shukor et al., 2013). ACE inhibitors are in general the first line of therapy for hypertension, heart failure, myocardial infarction and diabetic nephropathy. It is critical for the metabolism of two key vasomotor peptides, such as the vasoconstrictor angiotensin II and the vasodilator bradykinin. The latter acts with nitric oxide (NO) synthesis by stimulating NO synthase (NOS). A. graveolens is a popular and common vegetable throughout the world (Lin, Lu, & Harnly,

2007) and well-known for being a rich source of flavonoids. Some groups have quantified the flavonoid contents of apigenin, luteolin, and chrysoeriol derivatives by using HPLC. Apigenin was reported to have a vascular protective effect mediated by attenuating NO reduction (Jin et al., 2009), and luteolin was shown to increase the eNOS promoter activity and eNOS mRNA expression (Li, Xia, Brausch, Yao, & Forstermann, 2004). Although some of these glycosides were inactive in our ACE inhibitory assay, A. graveolens does contain chemical ingredients with a potential for hypertension therapy since these aglycones are possibly produced when they are taken orally. 4. Conclusions An ACE inhibitor, junipediol A-8-O-b-D-glucoside was purified from the crude methanol extract of A. graveolens, together with eight known compounds. This is the first report of junipediol A 8-O-b-D-glucoside in A. graveolens. Then junipediol A 8-O-b-D-glucoside might be a lead compound to develop ACE inhibitor from

A. Simaratanamongkol et al. / Food Chemistry 165 (2014) 92–97

a natural source, and a standard marker for quality control of A. graveolens extracts used in nutraceutical applications and even for controlling hypertension.

Acknowledgements The authors wish to thank the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Programme (Grant No. PHD/0120/ 2552) for financial support in this research. Also thanks to Dr. Brian Hodgson for assistance with the English.

References Al Shukor, N., Van Camp, J., Gonzales, G. B., Staljanssens, D., Struijs, K., Zotti, M. J., et al. (2013). Angiotensin-converting enzyme inhibitory effects by plant phenolic compounds: A study of structure activity relationships. Journal of Agricultural and Food Chemistry, 61, 11832–11839. Andersson, R., & Lundgren, L. N. (1988). Monoaryl and cyclohexenone glycosides from needles of Pinus sylvestris. Phytochemistry, 27, 559–562. Bhullar, K., Jha, A., Youssef, D., & Rupasinghe, H. (2013). Curcumin and its carbocyclic analogs: Structure–activity in relation to antioxidant and selected biological properties. Molecules, 18, 5389–5404. Bhuyan, B. J., & Mugesh, G. (2011). Angiotensin converting enzyme inhibitors in the treatment of hypertension. Current Science, 101, 21–27. Bucar, F., Ninov, S., Ionkova, I., Kartnig, T., Schubert-Zsilavecz, M., Asenov, I., et al. (1998). Flavonoids from Phlomis nissolii. Phytochemistry, 48, 573–575. Chen, Z. Y., Peng, C., Jiao, R., Wong, Y. M., Yang, N., & Huang, Y. (2009). Antihypertensive nutraceuticals and functional foods. Journal of Agricultural and Food Chemistry, 57, 4485–4499. Comte, G., Allais, D. P., Chulia, A. J., Vercauteren, J., & Pinaud, N. (1997). Three phenylpropanoids from Juniperus phoenicea. Phytochemistry, 44, 1169–1173. Gohari, A. R., Ebrahimi, H., Saeidnia, S., Foruzani, M., Ebrahimi, P., & Ajani, Y. (2011). Flavones and flavone glycosides from Salvia macrosiphon Boiss. Iranian Journal of Pharmaceutical Research, 10, 247–251. Hyun, S. K., Lee, H., Kang, S. S., Chung, H. Y., & Choi, J. S. (2009). Inhibitory activities of Cassia tora and its anthraquinone constituents on angiotensin-converting enzyme. Phytotherapy Research, 23, 178–184.

97

Jin, B. H., Qian, L. B., Chen, S., Li, J., Wang, H. P., Bruce, I. C., et al. (2009). Apigenin protects endothelium-dependent relaxation of rat aorta against oxidative stress. European Journal of Pharmacology, 616, 200–205. Kwon, E. K., Lee, D. Y., Hyungjae, L., Kim, D. O. K., Baek, N. I. N., Kim, Y. E., et al. (2010). Flavonoids from the buds of Rosa damascena inhibit the activity of 3hydroxy-3-methylglutaryl-coenzyme a reductase and angiotensin I-converting enzyme. Journal of Agricultural and Food Chemistry, 58, 882–886. Lagemann, A., Dunkel, A., & Hofmann, T. (2012). Activity-guided discovery of (S)malic acid 10 -O-b-gentiobioside as an angiotensin I-converting enzyme inhibitor in lettuce (Lactuca sativa). Journal of Agricultural and Food Chemistry, 60, 7211–7217. Li, H., Xia, N., Brausch, I., Yao, Y., & Forstermann, U. (2004). Flavonoids from artichoke (Cynara scolymus L.) up-regulate endothelial-type nitric-oxide synthase gene expression in human endothelial cells. Journal of Pharmacology and Experimental Therapeutics, 310, 926–932. Lin, L., Lu, S., & Harnly, J. (2007). Detection and quantification of glycosylated flavonoid malonates in celery, Chinese celery, and celery seed by LC–DAD–ESI/ MS. Journal of Agricultural and Food Chemistry, 55, 1321–1326. Miyase, T., Ueno, A., Takizawa, N., Kobayashi, H., & Oguchi, H. (1989). Ionone and lignan glycosides from Epimedium diphyllum. Phytochemistry, 28, 3483–3485. Nunes-Mamede, M. L., De Mello, F. G., & Martins, A. R. (1990). Effect of pmercuribenzoate on the subestimation of angiotensin-converting enzyme measurement during chick retina development. Journal of Neuroscience Methods, 31, 7–11. Rho, S. J., Lee, J.-S., Chung, Y. I., Kim, Y.-W., & Lee, H. G. (2009). Purification and identification of an angiotensin I-converting enzyme inhibitory peptide from fermented soybean extract. Process Biochemistry, 44, 490–493. Tsukamoto, H., Hisada, S., & Nishibe, S. (1985). Coumarins from bark of Fraxinus japonica and F. mandshurica var. japonica. Chemical and Pharmaceutical Bulletin, 33, 4069–4073. Wanasundara, P. K. J. P. D., Ross, A. R. S., Amarowicz, R., Ambrose, S. J., Pegg, R. B., & Shand, P. J. (2002). Peptides with angiotensin I-converting enzyme (ACE) inhibitory activity from defibrinated, hydrolyzed bovine plasma. Journal of Agricultural and Food Chemistry, 50, 6981–6988. Yoshikawa, M., Uemura, T., Shimoda, H., Kishi, A., Kawahara, Y., & Matsuda, H. (2000). Medicinal foodstuffs. XVIII. Phytoestrogens from the aerial part of Petroselinum crispum MILL. (PARSLEY) and structure of 600 -acetylapiin and a new monoterpene glycoside, petroside. Chemical and Pharmaceutical Bulletin, 48, 1039–1044. Zhou, K., Zhao, F., Liu, Z., Zhuang, Y., Chen, L., & Qiu, F. (2009). Triterpenoids and flavonoids from celery (Apium graveolens). Journal of Natural Products, 72, 1563–1567.