Botanical Studies (2012) 53: 207-214.
BIOCHEMISTRY
Antioxidant activities of different wild bitter gourd
(Momordica charantia L. var. abbreviata Seringe) cultivars
Yeh-Lin LU1,8, Yuh-Hwa LIU2,3,8, Jong-Ho CHYUAN4, Kur-Ta CHENG5, Wen-Li LIANG6 * and
Wen-Chi HOU6,7 *
1School of Pharmacy, Taipei Medical University, Taipei, Taiwan
2Division of Gastroenterology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
3Department of Primary Care Medicine, Taipei Medical University, Taipei, Taiwan
4Hualien District Agricultural Research and Extension Station, Hualien, Taiwan
5Department of Biochemistry, School of Medicine, Taipei Medical University, Taipei, Taiwan
6Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan
7Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, Taipei, Taiwan
(Received April 6, 2011; Accepted June 16, 2011)
ABSTRACT. Antioxidant activity assays were conducted using water (H) and methanolic (M) extracts of sixteen cultivars from indigenous wild bitter gourd (Momordica charantia L. var. abbreviata Seringe, MCA) in Taiwan. The scavenging activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals were different among MCA cultivars and the concentrations of 50% scavenging activity (IC50) for the effective cultivar was 181 (ig/mL (H) and 246 (ig/mL (M) in the former extract and 148 (ig/mL (H) and 37 (ig/mL (M) in the latter. For inhibitory activities against Cu2+-induced low-density-lipoprotein peroxidation, most MCA cultivars at 4000 (ig/mL (especially for M extracts) showed protective activities and were equivalent to 0.8 mM Trolox by thiobarbituric acid reactive substance assays. These useful data may help promote the use and further research of MCA as an antioxidant in the health food industry
Keywords: Momordica charantia L. var. abbreviata Seringe (MCA); Antioxidant activity; 2,2-diphenyl-1-picrylhydrazyl (DPPH); Low-density-lipoprotein peroxidation.
Abbreviations: DMPO, 5,5-dimethyl-1-pyrroline-Ar-oxide; DPPH, 2,2-diphenyl-1-picrylhydrazyl; H extracts, water extracts; LDL, low density lipoprotein; M extracts, methanolic extracts; MC, Momordica charantia L.; MCA, Momordica charantia L. var. abbreviata; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reacti­ve substance.
INTRODUCTION
ties of natural compounds in fruits, vegetables, and herbal medicines, such as echinacoside in Echinaceae root (Hu and Kitts, 2000), anthocyanin (Espin et al., 2000), various phenolic compounds (Rice-Evans et al., 1997), and a hy-drolysable tannin, geraniin, from Phyllanthus urinaria (Lin et al., 2008).
Active oxygen species and free radical-mediated reac­tions are involved in degenerative and pathological pro­cesses such as aging (Ames et al., 1993; Harman, 1995), cancer, coronary heart disease, and Alzheimer's disease (Ames, 1983; Gey, 1990; Smith et al.; 1996; Diaz et al., 1997). Many epidemiological results indicate an associa­tion between people who have a diet rich in fresh fruits and vegetables and a decrease in the risk of cardiovascular diseases and certain forms of cancer (Salah et al., 1995). Several reports have focused on the antioxidant activi-
The wild bitter gourd (Momordica charantia L. var. ab-breviata Seringe, MCA), normally smaller than domesti­cated bitter gourd (Momordica charantia L., MC), belongs to the family Cucurbitaceae. The fresh fruits of MC and MCA are frequently used as vegetables in Taiwan, MC also being used as a traditional medicine and listed in the Chinese pharmacopoeia Ben Cao Kong Mu. MC extract partitions reportedly show many pharmacological activi­ties (Grover and Yadav, 2004), including hypoglycemic (Miura et al., 2001; Rathi et al., 2002; Kar et al., 2003), anti-bacterial (Omoregbe et al., 1996), anti-viral (Lee-Huang et al., 1990), cytotoxic (Lee-Huang et al., 1995),
8These two authors contributed equally to this work. *Corresponding authors: E-mail: wchou@tmu.edu.tw; Fax: 886-2-2378-0134 (Wen-Chi HOU); E-mail: wenlee@tmu.edu.tw.
(Wen-Li LIANG).
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triglyceride-lowering (Senanayake et al., 2004), and anti-inflammatory activities (Kobori et al., 2008). The MCA extracts activated peroxisome proliferator-activated recep­tor a (Chao and Huang, 2003), and had anti-inflammatory (Lii et al., 2009), and antioxidant activities (Wu and Ng, 2008). Wu and Ng (2008) used one MCA cultivar to pre-pare separate hot-water and ethanolic extracts to investi-gate antioxidant and anti-radical activities. In this report, water (H) and methanolic (M) extracts of sixteen cultivars from indigenous wild bitter gourd (Momordica charantia L. var. abbreviata Seringe, MCA) in Taiwan were used for antioxidant activity assays. We found that scavenging ac-tivities against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals were different among MCA cultivars. These useful data might promote its use as an antioxidant worthy of further research in the health food industry.
zerland) and expressed as AA517. We measured means of triplicates and performed Trolox in parallel. We used deionized water or methanol as a blank experiment and calculated the scavenging activity of DPPH radicals (%) by the equation (AA517blank - AA517s—e) + AA517blank x 100%. IC50 stands for the concentration of 50% scavenging activity. We presented the quantitative data as the mean 士 SD of three independent experiments.
Scavenging activities of MCA extracts against hydroxyl radicals as detected by electron spin resonance (ESR) spectrometry
The hydroxyl radical was generated by the Fenton reac­tion (Kohno et al., 1991; Liu et al., 2010). A total mixture of 500 μL included a fixed concentration of H extracts or M extracts at 500 μg/mL, 5 mM of DMPO, and 0.05 mM ferrous sulfate. The potent H extracts from cultivars D, J, and P or the M extract from cultivar F were then used to perform dose-dependent scavenging activity assays. After mixing, the solution was transferred to an ESR quartz cell, placed in the cavity of an ESR spectrometer, and hydrogen peroxide added to a final concentration of 0.25 mM. De-ionized water or methanol was used instead of the sample solution for the control experiments. After 40 seconds, the relative intensity of the DMPO-OH spin-adduct signal was measured. All ESR spectra were recorded at ambient tem­perature (298 K) on a Bruker EMX-6/1 ESR spectrometer equipped with WIN-ESR SimFonia software version 1.2. The conditions of ESR spectrometry were as follows: cen­ter field, 345.4 士 5.0 mT; microwave power, 8 mW (9.416 GHz); modulation amplitude, 5 G; modulation frequency, 100 kHz; time constant, 0.6 seconds and scan time, 1.5 min. The quantitative data were presented as the mean 士 SD of three independent experiments
MATERIALS AND METHODS
Materials
5,5-dimethyl-1-pyrroline-N-oxide (DMPO), DPPH, ferrous sulfate, and low density lipoprotein (LDL, 500 μg protein/mL) were purchased from Sigma Chemical Co. (St. Louis, MO). Hydrogen peroxide (33%) was from Wako Pure Chemical Industries (Osaka, Japan). Trolox and thio-barbituric acid (TBA) were purchased from E. Merck Inc. (Darmstadt, Germany). The other chemicals and reagents were from Sigma Chemical Co. (St. Louis, MO, USA).
Preparations of MCA extracts
Sixteen fresh MCA cultivars were obtained fpr experi­ments from Jong-Ho Chyuan (Hualien District Agricul­tural Research and Extension Station, Hualien, Taiwan) and were assigned the letters A-P. Cultivars A and D contained the mature (AG and DG) and ripened (AY and DY) samples. After being washed, each de-seeded MCA (200 g) was cut into strips and blended with 200 mL de-ionized water. After 30 min centrifugation at 12500 xg, the supernatants were saved as crude water extracts which were lyophilized as H extracts. Each de-seeded MCA (200 g) was cut into strips and blended with 200 mL methanol, and then left for 3-days. After filtering, the residue was extracted with methanol under the same procedure twice. The filtrates were collected, concentrated by rotary vacu­um evaporator and then lyophilized as M extracts.
Protecting Cu2+ induced low density lipoprotein (LDL) peroxidation with MCA extracts
The capacity of H extracts or M extracts of MCA at a fixed concentration of 4000 fig/mL against Cu2+-induced human LDL oxidation in a total 100 (L sample volume was measured by thiobarbituric acid reactive substances (TBARS) assay at wavelength 532 nm (Yan et al., 1995). The LDL (500 μg protein/mL) was incubated at 37°C un-der air in a 10 mM phosphate buffer (pH 7.4) containing 10 μM CuSO4 for 24 h with or without MCA extracts. The reaction was stopped by adding EDTA with a final con-centration of 100 μM. For TBARS determination, 100 (L of TBA solution (0.4 mg TBA in 80 mL of 10% trichlo-roacetic acid) was added to 20 μL of reaction mixture, mixed and heated at 90°C for 40 min. After centrifugation at 12500 xg for 10 min, the 100 μL of supernatants were positioned at 96-wells and the absorbance at 532 nm was measured using ELISA reader. A student's t-test was used for comparison between two treatments. A difference be-tween the blank and each treatment (*p < 0.05; **p < 0.01) or the blank and the control (##p < 0.01) was considered statistically significant.
Scavenging activities of MCA extracts against DPPH radicals as detected by spectrophotom-etry
The 60 μl of MCA extracts (H extracts or M extracts, 25, 50, 100, and 200 μg in the final amounts were added to 20 μl of 1 M Tris-HCl buffer (pH 7.9), and then mixed with 120 μl of 100 μM DPPH in methanol to the final con-centrations of 60 μM for 20 min under dark at room tem-perature (Hou et al., 2002; Lin et al., 2008). The decrease in absorbance at 517 nm was measured using ELISA read-er (TECAN Sunrise microplate reader, Mannedorf, Swit-
LU et al. — Antioxidant activities of wild bitter gourd
209
RESULTS AND DISCUSSION
de-seeded MCA. The recovery rate was around 2%, and the M extracts had higher recovery rate than did H ex­tracts, except for cultivars F, H, I, J, and N.
Cultivars A and D contained the mature (green flesh, AG and DG) and ripe (yellow flesh, AY and DY) samples. Table 1 shows the recovery rate of H and M extracts from
Active oxygen species and free radical-mediated reactions are involved in degenerative and pathological processes such as aging (Ames et al., 1993; Harman, 1995), cancer, coronary heart disease, and Alzheimer's disease (Ames, 1983; Gey, 1990; Smith et al., 1996; Diaz et al., 1997). Several in vitro assay systems are used to evaluate the antioxidant potential in foods. DPPH radicals are widely used in model systems, to investigate the scavenging activities of several natural compounds. The color of DPPH radicals changed from purple to yellow, and the absorbance at a wavelength of 517 nm was decreased. Table 2 shows the results of IC50 of H and M extracts against DPPH radicals. We found that the M extracts generally showed better DPPH scavenging activities than H extracts did, and that extracts from mature flesh had better DPPH scavenging activities than extracts from ripe flesh. Among 16 MCA cultivars, the H extracts (IC50, 181 ug/mL) or M extracts (IC50, 246 ug/mL) of cultivar N exhibited the best DPPH scavenging activities, equivalent to that of 25.5 ug Trolox/g of H extracts and 16.88 ug Trolox/g of M extracts. Wu and Ng (2008) reported that hot water extracts and ethanolic extracts from one MCA showed DPPH scavenging activities (IC50) of 129.94 and 156.89 ug/mL, respectively. These differences
Table 1. The recovery rate of water (H) and methanolic (M) extracts of sixteen indigenous wild bitter gourd (Momordica charantia L. var. abbreviata Seringe, MCA) cultivars from Tai­wan.
No.
Extracts
Flesh weight (g)
Extract weight Recovery
(g) (%)
AG (matured)
M
203.6
4.0340
1.98
H
208.0
4.1111
1.98
AY (ripen)
M
219.3
4.2451
1.94
H
197.8
3.2808
1.66
B
M
196.4
3.3131
1.69
H
207.4
2.9549
1.42
C
M
200.2
3.3338
1.67
H
236.9
2.5125
1.06
DG (matured)
M
208.5
5.3540
2.57
H
205.5
3.4029
1.66
DY (ripen)
M
208.2
4.4179
2.12
H
216.7
2.5205
1.16
E
M
108.6
2.4727
2.28
H
207.1
4.1341
2.00
F
M
53.7
1.1295
2.10
H
52.5
1.5951
3.04
G
M
165.5
3.4774
2.10
H
214.0
4.2893
2.00
H
M
210.3
4.1944
1.99
H
250.1
5.4477
2.18
I
M
200.0
3.6930
1.85
H
200.0
4.1726
2.09
J
M
145.2
2.7742
1.91
H
202.5
5.4629
2.70
K
M
236.5
6.8875
2.91
H
131.2
3.5497
2.71
L
M
201.4
4.6538
2.31
H
162.8
1.2791
0.79
M
M
204.2
4.9257
2.41
H
221.0
1.2620
0.57
N
M
223.9
4.8464
2.16
H
212.8
4.8912
2.30
O
M
178.8
3.1145
1.74
H
183.2
3.0729
1.68
P
M
180.5
3.9271
2.18
 
H
193.7
3.4371
1.77
Table 2. The concentration for 50% inhibition (IC50) against DPPH radicals from water (H) and methanolic (M) extracts of sixteen wild bitter gourd cultivars.
No.
H extracts
M extracts
IC50 (Hg/mL)
IC50 (Hg/mL)
AG
521 士6
562 士19
AY
726 士26
> 1000
B
570 士34
> 1000
C
519 士23
519 士5
DG
> 1000
405 士14
DY
> 1000
672 士20
E
> 1000
384 士7
F
528 士34
398 士6
G
> 1000
> 1000
H
446 士14
364 士4
I
> 1000
902 士28
J
677 士30
734 士7
K
> 1000
259 士8
L
> 1000
> 1000
M
> 1000
> 1000
N
181 士6
246 士8
O
> 1000
766 士24
P
720 士19
460 士15
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Botanical Studies, Vol. 53, 2012
might be due to a different cultivar or the 25 μM DPPH concentrations Wu and Ng (2008) used compared to our presently reported 60 μM.
The hydroxyl radical was generated by the Fenton reaction, and was trapped by DMPO to form DMPO-OH
adducts. We used the intensities of the DMPO-OH spin signals on ESR spectrometry to evaluate the scavenging activities of water (Figure 1A) and methanolic (Figure 1B) extracts from 16 MCA cultivars at a fixed concentration of 500 μg/mL. Based on the results of DMPO-OH intensities
Figure 1. The intensity changes of (A) water and (B) methanolic extracts (500 μg/mL) of sixteen wild bitter gourd cultivars against DMPO-OH adducts by electron spin resonance. Sixteen MCA cultivars (A to P) were used in experiments with cultivars A and D containing the mature (AG and DG) and ripe (AY and DY) samples. Deionized water was used instead of sample (water extracts, H; methanolic extracts, M) for the control experiments.
LU et al. — Antioxidant activities of wild bitter gourd
211
(Figure 1), the scavenging activities against hydroxyl radicals among MCA cultivars were very different, as shown in Table 3. H extracts generally showed better hydroxyl radical scavenging activities than M extracts did, and extracts from mature flesh had better results than those from ripe flesh. The H extracts from mature D, J, and P cultivars had hydroxyl radical scavenging activities over 90% at 500 μg/mL, and M extracts from cultivar F had the highest, at 500 μg/mL (Table 3). Therefore, the different concentrations of the above-mentioned extracts were used to calculate the concentration of 50% hydroxyl radical scavenging activity. The IC50 was 151, 190, and 148 (g/mL, respectively, for H extracts of mature D, J, and P cultivars and 37 μg/mL for M extracts of cultivar F.
Free radicals can damage macromolecules in cells, such as DNA, proteins and lipids in membranes (Halliwell, 1999). Lipid peroxidation products (such as malondialdehyde) can damage proteins and DNA (Ester-bauer et al., 1991). LDL peroxidation contributes to the development of atherosclerosis (Steinbrecher, 1987) and its delay or prevention is an important antioxidant function. The TBARS assay reveals the degrees of Cu2+-induced human LDL peroxidation. Figure 2 shows the protective effect of water (A) and methanolic (B) extracts from 16 MCA cultivars at concentrations of 4 mg/mL against Cu2+-induced human LDL peroxidation, using 0.8 mM Trolox (T) as comparison. Either H or M extracts
Table 3. The inhibition (%) against hydroxyl radicals from water (H) and methanolic (M) extracts (500 (g/mL) of sixteen wild bitter gourd cultivars.
Figure 2. Effects of (A) water and (B) methanolic extracts (4000 (g/mL) of sixteen wild bitter gourd cultivars against Cu2+-in-duced LDL peroxidation determined by thiobarbituric acid meth­ods. Sixteen MCA cultivars (A to P) were used in experiments with cultivars A and D containing the mature (AG and DG) and ripe (AY and DY) samples. Lipid peroxidation was determined as TBARS (A532 nm). The C (control), B (blank) and T (0.8 mM Trolox) were added for comparison. The quantitative data were presented as mean 士 SD of two independent experiments. A stu­dent's t-test was used for comparison between two treatments. A difference between the blank and each treatment (*p < 0.05; **p < 0.01) or the blank and the control (##p < 0.01) was considered statistically significant.
H extracts
M extracts
No.
(500 μg/mL)
(500 μg/mL)
Inhibition (%)
Inhibition (%)
AG
86.75 土 1.90
64.38 土 1.72
AY
66.07 ± 3.37
63.34 ± 0.44
B
86.37 ± 2.04
69.97 ± 0.95
C
74.21 ± 3.90
70.77 ± 1.45
DG
93.63 ± 2.39
82.56 ± 0.30
DY
87.47 ± 3.75
66.24 ± 1.81
E
87.60 ± 2.38
71.99 ± 0.86
F
70.39 ± 5.22
89.55 ± 1.32
G
62.03 ± 1.56
60.24 ± 0.84
H
68.51 ± 5.49
79.34 ± 1.41
I
72.15 土 1.99
67.43 ± 0.61
J
94.83 ± 1.01
78.08 ± 8.12
K
83.00 ± 1.51
68.46 ± 4.65
L
83.01 ± 3.78
62.93 ± 8.72
M
60.51 ± 1.36
70.50 ± 6.88
N
82.44 ± 3.49
68.48 ± 3.83
O
76.12 土 1.26
67.79 ± 6.97
P
92.17 土 0.77
66.86 ± 6.00
exhibited protective effects against LDL peroxidation with significant differences to the blank (*p < 0.05; **p < 0.01, Figure 2). M extracts (Figure 2B) generally showed better protective effects against LDL peroxidation than H extracts did (Figure 2A). Most M extracts (4000 (g/mL) from the 16 MCA cultivars showed equally protective effects as 0.8 mM Trolox and almost retarded LDL peroxidations compared to the control. Wu and Ng (2008) used a single MCA cultivar to prepare hot-water and ethanolic extracts to investigate antioxidant and anti-radical activities. They found that hot-water extracts contained higher total flavonoids (62 mg/g) than that of ethanolic extracts (44.0 mg/g), however, the former contained lower phenolic
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contents (51.6 mg/g) than that of the latter (68.8 mg/g). Our report made clear that the anti-radical and antioxidant activities are very different among cultivars. Kubola and Siriamornpun (2008) reported that gallic acid, tannic acid, (+)-catechin, caffeic acid, p-coumaric acid, ferulic acid, benzoic acid, and other unidentified compounds may contribute to the total antioxidant activities in different parts of MC fractions.
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In conclusion, active oxygen species and free radical-mediated reactions are involved in degenerative and patho­logical processes. There has been a recent emphasis on the problems associated with aging-related diseases, including neurodegenerative diseases (e.g., Alzheimer's, Parkinsons, and Huntington's disease). The intricate causes of the ag­ing process are still a matter of extensive speculation, and give rise to many theories. In particular, the role of reac­tive oxygen species is a prerequisite nowadays for under­standing this process (Abrass, 1990; Bickford et al., 2000; Schulz et al., 2000). Our study shows that sixteen different indigenous wild bitter gourd cultivars in Taiwan exhibited different antioxidant and anti-radical activities. The poten­tial antioxidant activities of these compounds should be further investigated for possible use in producing health foods.
Acknowledgments. We want to express thanks for finan­cial support (SKH-TMU-100-07) from the Shin Kong Wu Ho-Su Memorial Hospital.
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Botanical Studies, Vol. 53, 2012
不同品種山苦瓜抗氧化活性研究
呂岳寐1 劉玉華2,3 全中和4 鄭可大5 梁文俐6 侯文琪6,7
1臺北醫學大學藥學系
2新光醫院肝膽腸胃科
3臺北醫學大學一般醫學科
4花蓮區農業改良場
5臺北醫學大學醫學系生化學科
6臺北醫學大學生藥學研究所
7臺北醫學大學附設醫院中草藥研究中心
本實驗以花蓮區農業改良場提供之臺灣原生16種不同品種山苦瓜(Momordica charantia L. var.
abbreviata Seringe, MCA)，分別以冷水（水抽物，H)與甲醇（甲醇抽取物，M)抽取，進行抗氧化活
性分析。結果發現品種之間清除DPPH自由基與氫氧自由基的活性都不相同，最有效品種之50%有效
濃度清除DPPH自由基為181 ig/mL (H)及246 ig/mL (M)；最有效品種之50%有效濃度清除氫氧自由
基為148 ig/mL (H)及37 ig/mL (M)。在抑制銅離子誘導低密度脂蛋白過氧化活性表現，特別是甲醇抽
取物（M)在4000 ig/mL濃度下，表現出保護效果（以TBARS表示）幾乎與0.8 mM Trolox效果相
當。以上的結果顯示，山苦瓜抽取物未來也許可以開發為天然抗氧化保健食品。
關鍵詞：山苦瓜；抗氧化活性；DPPH自由基；低密度脂蛋白過氧化。