LWT - Food Science and Technology 43 (2010) 655–659
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The antioxidant, angiotensin converting enzyme inhibition activity, and phenolic compounds of bamboo shoot extracts Eun-Jin Park 1, Deok-Young Jhon* Department of Food Science and Human Nutrition, Chonnam National University, Gwangju, 500-757 Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2008 Received in revised form 14 September 2009 Accepted 6 November 2009
This study was undertaken to evaluate the functional properties of two of the most popular species of edible bamboo shoots in Korea (Phyllostachys pubescens and Phyllostachys nigra). Powdered bamboo shoots were extracted with methanol and an aqueous suspension of the obtained methanol extract was partitioned successively with chloroform, ethyl acetate, and butanol, leaving a residual water extract. All obtained extracts were evaluated for their antioxidant capacity and antimicrobial activity, angiotensin converting enzyme (ACE) inhibition activity, and ascorbic acid and phenolic compound content. Methanol and water fractions showed a particularly high ascorbic acid contents. The ethyl acetate fraction contained a high concentration of phenolic compounds. Among all extracts, the ethyl acetate and butanol fractions showed particularly high antioxidant activity. Methanol extract had a significantly higher ACE inhibitory activity than other extracts. None of the extracts inhibited the tested bacteria. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Bamboo shoots Antioxidant Antimicrobial ACE inhibition Phenolic compound
1. Introduction Bamboo is a group of genera of evergreen plants belonging to the Poaceae or grass family. Bamboo shoots are the immature and edible culms arising from the rhizomes. Shoots emerge in early spring, can grow quickly at over 1 m per day, and usually become lignified (woody) in 2–3 days (Luo, Xi, Fu, & Lu, 2002; Wang, 2002; Zhang, Yang, Han, & Dong, 2000). For these reasons, bamboo shoots have limitations for consumption and storage. Among the edible 60–90 genera of bamboo shoots, Phyllostachys pubescens and Phyllostachys nigra are the major species cultivated in Korea (statistical data of Korea Forest Research Institute, 2005). Bamboo leaves (Phyllostachys Sieb. et Zucc.) have antioxidant capacity due to high polyphenol content (Lu, Wu, Shi, Dong, & Zhang, 2006). Park, Lim, Kim, Choi, and Lee (2007) reported that a butanol extract of bamboo leaves (Sasa borealis) exhibited significant antioxidant capacity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Ethanol extracts of bamboo (Phyllostachys bambusoides) have a nitrite scavenging ability (Lim, Na, & Baik, 2004). Kim, Cho, Lee, Ryu, and Shim (2001) reported that extracts of bamboo leaves and stems (Phyllostachys spp.) showed strong antibacterial activities. Most research studies
* Corresponding author. Department of Food Nutrition, Chonnam National University, 300 YongBong-Dong, Gwangju, 500-757 Republic of Korea. Tel.: þ82 62 530 1335; fax: þ82 62 530 1339. E-mail address:
[email protected] (D.-Y. Jhon). 1 Present address: Department of Biology, Kyung Hee University HoeGi-Dong 1, DongDaeMun-Gu, Seoul 130-701, Republic of Korea. 0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2009.11.005
have investigated the functional activities of bamboo leaves and stems. However, only a few studies have reported on the functional properties of bamboo shoots. Wang and Ng (2003) reported on the antifungal protein (dendrocin) isolated from bamboo shoots (Dendrocalamus latiflora Munro). Thus, further studies of the functional and bioactive properties of bamboo shoots are needed. Solvent extraction is frequently used for isolation of antioxidant, of which extraction yield is dependent on the solvent and method of extraction. Several extraction techniques have been reported for extraction using solvents with different polarities, such as methanol, ethanol, chloroform, ethyl acetate, acetone, petroleum ether, butanol, and water (Cheung, Cheung, & Ooi, 2003; Singh, Murthy, & Jayaprakasha, 2002). Extracted fractions have been assayed for their functional properties, such as antioxidant capacity, angiotensin converting enzyme inhibition activity, antimicrobial activity, nitrite scavenging ability, and the presence of phenolic compounds. Plant phenols are bioactive compounds of interest because they are an important group of strong natural antioxidants, and some of them are potent antimicrobial compounds. Free radicals produced by radiation, chemical reactions and several reactions with various compounds may contribute to oxidative damage of lipids, protein, and nucleic acids in living cells (Morrissey & O’Brien, 1998). The mechanism of antioxidant capacity may involve the scavenging of free radicals (Dini, Tenore, & Dini, 2006). Plant foods are potential sources of natural antioxidants, such as vitamin C, tocopherol, carotenoids, flavonoid, and phenolic compounds which prevent free radical damage (Diplock et al., 1998; Paganga, Miller, & Rice-Evan, 1999; Vinson, Hao, Su, & Subik, 1998).
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Angiotensin converting enzyme (ACE; peptidyldipeptide hydrolase EC 3,4,15,1) plays an important physiological role in the control of blood pressure. Since ACE activity raises blood pressure, it is recommended to inhibit ACE activity in patients suffering from hypertension (Hernaandex-Ledesma, Martian-Aalvarez, & Pueyo, 2003). Therefore, inhibition of ACE results in an overall antihypertensive effect. Several studies have been conducted using synthetic drugs, which can cause undesirable side effects. Edible plants are natural and abundant resources of bioactive chemicals. Since it is recommended to use several natural foods to inhibit ACE activity, many studies have been investigated ACE inhibitory compounds from foods. Up until now, many different protein hydrolysates, such as soy sauce, tuna, bonito, and soybean, have been reported up to now show ACE inhibition activity (Kinoshita, Yamakoshi, & Ikeuchi, 1993; Kohama et al., 1988; Matsumura, Fujii, Takeda, Sugita, & Shimuzu, 1993; Okamoto, Hanagata, Kawamura, & Yanagida, 1995). The objective of the present study was to analyze the functional properties, such as antioxidant capacity, antimicrobial potential, ACE inhibitory activity, ascorbic acid contents, and phenolic compounds, of solvent extracts of bamboo shoots, and to compare the effect of solvent extraction (methanol, chloroform, ethyl acetate, butanol, and water) on those functional properties. 2. Materials and methods 2.1. Bamboo shoots Two kinds of bamboo shoots (P. pubescens, PP and P. nigra, PN) were used in this experiment. Bamboo shoots were grown and harvested in the spring of 2006 in Dam-Yang, Korea. Harvested bamboo shoots were trimmed immediately, lyophilized, and pulverized using 0.254 mm sieves (FM-681C, Hanil Co., Korea). 2.2. Extraction Each sample of bamboo shoots powder (10 g) was extracted by mixing with a magnetic stirrer with 1500 mL of 99.9 mL/mL methanol at room temperature (20–25 C) for 24 h in 3–5 replicates. The total extract was filtered through Whatman No. 6 filter paper and evaporated in a rotary vacuum evaporator (Vacuum rotary evaporator, Dai-Han Inc., Korea) at 35 C. Methanol (MeOH) extract was partitioned successively with chloroform (CHCl3), ethyl acetate (EtOAc), and butanol (BuOH), leaving residual water fractions (H2O). All obtained extracts, including the residual water fractions, were evaporated in a rotary vacuum evaporator at 35 C in water bath. Each extract was dissolved in 99.9 mL/mL methanol, in concentrations of 1–10 mg/mL, centrifuged at 14,000 g for 20 min, and the supernatant was stored at refrigerator temperature (4 C) before subsequent experiments. 2.3. HPLC analysis of ascorbic acid and phenolic compound For analysis of ascorbic acid and phenolic compounds, each extract was mixed with 99.9 mL/mL methanol and vortexed for 30 min at the highest setting. Mixed samples were centrifuged at 14,000 g for 20 min and the supernatant was filtered through a 0.45 mm syringe filter. Final samples were analyzed by HPLC (LC-900, Jasco International Co., Ltd, Japan). The operating conditions of HPLC for analysis of ascorbic acid and phenolic compounds in bamboo shoots were as follows: Instrument, LC-900 (Jasco International, Co., Ltd, Japan); Column, mBondapakTM C18 (i.d. 3.9 300 mm); Detector, UV-975 (Jasco International, Japan); Detection wavelength, 254 nm; Mobile phase,13 mL/ mL MeOH and 1 mL/mL HAc (gradient elution); Flow rate,1.0 mL/min; Chart speed, 2 mm/min.
2.4. DPPH radical scavenging activity Radical scavenging activity was determined using a 2,2diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay with modification (Joyeux, Mortier, & Fleurentin, 1995; Robards, Sanchez-Moreno, Larrauri, & Sauracalixto, 1998). Eight hundred ml of each extracts was mixed with 400 mL of 0.4 mmol/L methanolic DPPH. Mixtures were vortexed for 30 sec at the highest setting and left for 30 min in the dark. After that, absorbance was measured at 517 nm using MeOH as blank. The scavenging activity of the DPPH radical was calculated using the following equation: Scavenging activity (%) ¼ 100 (A0 A1)/A0, where A0 is the absorbance of the methanol control, and A1 is the absorbance in the presence of bamboo shoots extracts. The inhibition concentration (IC50) was defined as the amount of extract required for 50% reduction of free scavenging activity. The IC50 values were obtained from the resulting inhibition curves. Results were compared with the activity of butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), ascorbic acid, and catechin (Sigma, St. Louis, MO, USA) as control antioxidants. 2.5. ACE inhibition activity The ACE (angiotensin-converting enzyme) inhibition activity was measured spectrophotometrically using Hip-His-Leu (N-hippuryl-1-histidyl-leucine tetrahydrate, Sigma, USA) as a substrate. Ten mL of bamboo shoot extracts were mixed with 100 mL of substrate and 40 mL of angiotensin converting enzyme (peptidyldipeptidase A, dissolved in 0.01 mol/L potassium phosphatase monobasic and 0.5 mol/L sodium chloride, amended to pH 7.0, Sigma, USA). Mixtures were incubated at 37 C for 60 min and 125 mL of 1 mol/L HCl was added to stop the reaction. After that, samples were mixed with 850 mL of ethyl acetate, vortexed for 1 min at the highest setting, and centrifuged at 14,000 g for 20 min. Five hundred mL of the supernatant was dried at 100 C. Dried samples were mixed with 1 mL of deionized water, vortexed for 1 min at the highest setting, and absorbance was measured at 230 nm using deionized water as a blank. ACE inhibition activity was calculated using the following equation: ACE inhibition activity (%) ¼ [(C0 – Cb) – (S0 – Sb)/(C0 – Cb)] 100, where C0 is the absorbance of the deionized water control, Cb is the absorbance of the deionized water sample, S0 is the absorbance of the tested sample, and Sb is the absorbance without sample. 2.6. Antimicrobial activity In order to determine the antimicrobial activity of extracts, the paper disc method was used. The following microorganisms were tested: Escherichia coli (ATCC 35210), Enterococcus faecium (ATCC 19434), Ent. faecalis (ATCC 29212), and Streptococcus mutans (ATCC 27352). Each strains of E. coli, Enterococcus spp., and Strep. mutans was enumerated on LB agar (0.1 g/100 mL tryptone; 0.5 g/100 mL yeast extract; 1 g/100 mL sodium chloride; and 1.5 g/100 mL agar), MRS agar (Difco, Detroit, MI, USA), and m-BHI agar [modified Brain Heart Infusion agar: BHI broth (Difco) supplemented with 0.5 g/100 mL yeast extract; 2 g/100 mL glucose; and 1.5 g/100 mL agar], respectively. Three strains, including E. coli, Ent. faecium, and Ent. faecalis, were incubated at 37 C for 24 h, and Strep. mutans was incubated at 37 C for 72 h in an anaerobic jar (BBL, USA). Each microorganism culture (107–8 cfu/mL) at 2 mg/100 mL (v/v) total weight was mixed with cooled agar medium, poured onto the surface of agar, and cooled to be solidified. Five hundred mL of extracts, which were filtered using 0.45 mm syringe filter, were loaded on 8 mm sterilized paper discs (Toyo Roshi Kaisha Ltd., Japan) and allowed to dry. The loaded discs were placed on the agar surface,
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3.2. Analysis of ascorbic acid and phenolic compounds
Table 1 The extraction yields of powdered bamboo shoots by various solvents. Solvent
Yield (g/100 g, w/w)a P. pubescens (PP)
Methanol extract Chloroform fraction Ethyl acetate fraction Butanol fraction Water fraction
30.3 4.2 0.7 8.4 15.4
2.1b 0.3 0.0 1.1 1.0
P. nigra (PN) 24.3 5.6 1.0 5.5 12.1
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1.8 0.2 0.1 0.8 0.9
a Yield ratios (g/100 g, w/w) ¼ [solid in extract or fraction (g)/raw material (100 g, dry weight)]. b All experiments were replicated three times and results are presented as mean SE.
rehydrated with 0.1 mL of deionized water, and the compounds allowed to diffuse in the hood for 1 h. The plates were incubated at 37 C for 24–72 h. After incubation, inhibition zones formed around the disc were measured with a transparent ruler in millimeters.
2.7. Statistical analysis Three replicate trials for each experiment were performed. Analysis of variance was performed and means separated using the ANOVA procedure of SAS (SAS Institute, Cary, NC, USA). Mean values were reported for all analyses and separated using Duncan’s multiple range tests. Significant differences between values were presented at a significance level of P ¼ 0.05.
3. Results and discussion 3.1. Extraction yield Efficiency of extraction is an important factor for the comparison of functional activities. Several researchers reported that relatively higher antioxidant capacity and phenolic acid contents were observed from methanolic extracts (Sosulski, Krygier, & Hogge, 1982; Zielinski & Kozlowska, 2000) compared to other solvents. Therefore, crude methanolic extracts was selected as a basis for further partitioning by several solvents, such as chloroform, ethyl acetate, butanol, and water. The extraction yields (expressed as w/w percentages) of the bamboo shoots extracts is shown in Table 1. The extraction yield of methanol extracts were 30.3 g/100 mL (PP) and 24.3 g/100 mL (PN), respectively. The relative extraction yield of other solvents decreased in the following order (PP and PN): water fractions (15.4 and 12.1 g/100 mL) > butanol fractions (8.4 and 5.5 g/100 mL) > chloroform fractions (4.2 and 5.6 g/100 mL) > ethyl acetate fractions (0.7 and 1.0 g/100 mL). This implies that most of the soluble components in bamboo shoots were high polarity.
Oxidative stress is defined in general as an excessive amount of free radicals, such as superoxide (O 2 ) and hydroxyl radical (OH ), as well as hydrogen peroxide (H2O2) (Cerutti, 1991). ROS are strongly associated with aging, carcinogenesis, and cardiovascular disease (Moskovitz, Yim, & Choke, 2002). Cells have several antioxidant mechanisms to prevent the effects of ROS. Antioxidative enzymes, such as superoxide dismutase and calatase, ascorbic acid (vitamin A), and tocopherol (vitamin E) have effective ROS scavenging properties (Fridovich, 1999). Phenolic compounds, also, are antioxidants and have been isolated from fruits, vegetables, grains, medicinal plants, nuts, herbs and edible oils (Goli, Barzegar, & Sahari, 2005; Hayouni, Abedrabba, Bouix, & Hamdi, 2007; Kuti & Konuru, 2004; Li, Wong, Cheng, & Chen, 2008; Liyana-Pathirana & Shahidi, 2006; Yanishlieva & Marinova, 2001). Therefore, the determination of phenolic acid content is necessary before measuring antioxidant capacity. In this investigation, the five bamboo shoots extracts were evaluated for content of ascorbic acid and phenolic compounds using HPLC analysis. Table 2 and 3 shows ascorbic acid content and phenolic compound compositions of individual extracts. The ascorbic acid content, on a dry weight basis, were as follows (PP and PN): methanol, 154.7 and 195.3 mg; chloroform, 1.0 and 9.3 mg; ethyl acetate, 51.0 and 162.5 mg; butanol, 50.9 and 114.3 mg; and water, 136.3 and 231.8 mg per 100 g of bamboo shoots. Methanol extracts and water fractions showed particularly high ascorbic acid content. In bamboo shoots, eight phenolic acids (protocatechuic acid, p-hydroxybenzoic acid, catechin, caffeic acid, chlorogenic acid, syringic acid, p-coumaric acid, and ferulic acid) were identified and quantified by HPLC. The most abundant compounds were protocatechuic acid, p-hydroxybenzoic acid, and syringic acid. Syringic acid was a most abundant acid of the butanol fraction of PP. In ethyl acetate fractions, six and eight compounds were detected in PP and PN, respectively. Ethyl acetate fractions of PP contained protocatechuic acid, 0.5 mg; p-hydroxybenzoic acid, 2.9 mg; caffeic acid, 0.3 mg; syringic acid, 1.3 mg; p-coumaric acid, 1.3 mg; and ferulic acid, 0.5 mg per 100 g of bamboo shoots. Catechin and chlorogenic acid were detected only in the PN extracts. The PN extracts contained protocatechuid acid, 1.5 mg; p-hydroxybenzoic acid, 8.1 mg; catechin, 10.6 mg; caffeic acid, 1.5 mg; chlorogenic acid, 4.1 mg; syringic acid, 2.5 mg; p-coumaric acid, 3.5 mg; and ferulic acid, 1.85 mg per 100 g of bamboo shoots. There were no phenolic acids in water fractions of the tested extracts. However, ethyl acetate fractions contained a high phenolic acid concentration, followed by butanol fractions. It has been reported that the correlation between antioxidant capacity of plant materials and their phenolic compound content is statistically significant (Velioglu, Mazza, Gao, & Oomah, 1998). Although the concentrations and varieties of phenolic compounds were highest in the ethyl acetate fractions, the
Table 2 Ascorbic acid and phenolic compounds in bamboo shoot (Phyllostachys pubescens, PP) extracts by various solvents. Compound
Contents (mg/100 g, dry weight basis) MeOH
CHCl3
EtOAc
BuOH
H2O
Ascorbic acid Protocatechuic acid p-Hydroxybenzoic acid Catechin Caffeic acid Chlorogenic acid Syringic acid p-Coumaric acid Ferulic acid
154.7 8.5a 2.8 0.2 1.7 0.0 0 0 0 0 0 0
1.0 0.0 0.1 0.0 0.4 0.0 0 0 0 0 0 0
51.0 3.2 0.5 0.0 2.9 0.0 0 0.3 0.0 0 1.3 0.0 1.3 0.0 0.5 0.0
50.9 1.7 1.3 0.0 0.6 0.0 0 0 0 6.7 0.2 0 0
136.3 4.2 0 0 0 0 0 0 0 0
a
All experiments were replicated three times and results are presented as mean SE.
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Table 3 Ascorbic acid and phenolic compounds in bamboo shoot (Phyllostachys nigra, PN) extracts by various solvents. Compound
Ascorbic acid Protocatechuic acid p-Hydroxybenzoic acid Catechin Caffeic acid Chlorogenic acid Syringic acid p-Coumaric acid Ferulic acid a
Contents (mg/100 g, dry weight basis) MeOH
CHCl3
EtOAc
195.3 3.4a 0 5.4 0.2 0 0 0 0 0 0
9.3 0.8 0 0.6 0.0 0 0 0 1.8 0.0 0 0
162.5 1.5 8.1 10.6 1.5 4.1 2.5 3.5 1.8
5.7 0.0 1.2 1.9 0.0 0.3 0.0 0.1 0.0
BuOH
H2O
114.3 3.2 0 1.4 0.0 0 0 3.5 0.5 2.1 0.1 0 0
231.8 10.1 0 0 0 0 0 0 0 0
All experiments were replicated three times and results are presented as mean SE.
extraction yields of those were extremely low and hence their contribution was insignificant. 3.3. Scavenging activity on DPPH radicals DPPH radical scavenging activity, which is a mechanism of measuring the decrease in DPPH radical absorption after exposure to radical scavengers, frequently can be used to rapidly determine antioxidant capacity. Comparison of antioxidant capacities between extracts of bamboo shoots is shown in Table 4 and radical scavenging activity expressed as IC50. A wide range of antioxidant capacity among the tested fractions was observed. The IC50 of each fraction increased in the following order (PP and PN): ethyl acetate fractions (0.8 and 0.4 mg/mL) z butanol fractions (0.7 and 0.8 mg/mL) > chloroform fractions (4.0 and 2.3 mg/mL) z methanol extracts (3.6 and 3.4 mg/mL) > water fractions (4.7 and 5.3 mg/mL). Strong DPPH radical scavenging activity was also found in the ethyl acetate and butanol fractions possessing high phenolic contents (Tables 2 and 3). However, those activities were significantly lower than those of the control antioxidants, such as BHT, BHA, ascorbic acid, and catechin. From the earlier result, it was found that the water and chloroform fractions had relatively lower antioxidant capacities due to their lower phenolic compounds contents. Overall, the antioxidant capacities of the fractions were highly correlated with their total phenolic contents and these results are also similar to those of previous findings (Beta, Nam, Dexter, & Sapirstein, 2005; Bouaziz, Chamkha, & Sayadi, 2004; Kuti & Konuru, 2004; Liyana-Pathirana & Shahidi, 2006).
indicative of an overall anti-hypertensive effect. Figs.1 and 2 show the dose-response curve for the ACE inhibition activities of bamboo shoots extracts at 1, 5, and 10 mg/mL concentrations. The inhibitory activities increased proportional to extract concentration. The results showed that methanol extracts had significantly higher ACE inhibitory activity than other extracts with increasing concentrations. Butanol and ethyl acetate fractions showed higher ACE inhibition activities than did chloroform and water fractions. Synthetic drugs, such as captopril and benazepril, were used for comparison of ACE inhibitory activity with the tested compounds (Vermeirssen, Camp, & Verstraete, 2002). To obtain approximately 50% ACE inhibitory activity, 3.6 ng/mL of captopril were needed (Chen, Chang, Chung, & Chou, 2007). In our study, 3.5 (PP) and 6.0 (PN) mg/ml of methanol extracts, the ones showing a highest ACE activity, were needed to produce activity similar to captopril. Although the ACE inhibition activities of bamboo shoots extracts were significantly less than those of synthetic drugs, it is useful to know that bamboo shoot extracts did have potent ACE inhibitory compounds. 3.5. Antimicrobial activity tests None of the fractions extracted from either species bamboo shoots inhibited any of the tested bacteria, such as E. coli, Ent. faecium, Ent. faecalis, and Strep. mutans, at 1, 5, and 10 mg/mL concentrations (data not shown). Zhu, Zhang, Lo, and Lu (2005) reported that the minimum inhibitory concentrations (MICs) used for fungi were at or below 2.5 mg/mL and for bacteria were at or above 2.5 mg/mL. The concentrations tested in our experiment did
3.4. Effect of extracts on the activity of ACE 80
Table 4 Antioxidant capacity of solvent fractions from the powdered bamboo shoots and control antioxidant by DPPH radical scavenging method. Fraction
Antioxidant capacitya (IC50: mg/mL) P. pubescens (PP)
Methanol extract Chloroform fraction Ethyl acetate fraction Butanol fraction Water fraction BHT BHA Ascorbic acid Catechin
3.6 4.0 0.8 0.7 4.7
0.2b 0.0 0.0 0.0 0.3
0.006 0.008 0.009 0.023
0.0 0.0 0.0 0.0
P. nigra (PN) 3.4 2.3 0.4 0.8 5.3
0.3 0.0 0.0 0.0 0.8
0.006 0.008 0.009 0.023
0.0 0.0 0.0 0.0
a Amount of bamboo shoots extract required for 50% reduction of free radical scavenging activity. b All experiments were replicated three times and results are presented as mean SE.
70
ACE Inhibition activity (%)
In the ACE inhibition tests, ACE catalyses the degradation of the substrate, Hip-His-Leu, so that ACE activity might be derived from the decrease in absorbance after reaction. Therefore, inhibition of ACE is
60 50 40 30 20 10 0 0
2
4
6
8
10
12
Concentration (mg/ml) Fig. 1. Angiotensin converting enzyme (ACE) inhibition activity by solvent fractions from powdered bamboo shoots (Phyllostachys pubescens, PP). C, methanol fraction; B chloroform fraction; : ethyl acetate fraction; 6 butanol fraction; - water fraction.
E.-J. Park, D.-Y. Jhon / LWT - Food Science and Technology 43 (2010) 655–659
80
ACE Inhibition activity (%)
70 60 50 40 30 20 10 0 0
2
4
6
8
10
12
Concentration (mg/ml) Fig. 2. Angiotensin converting enzyme (ACE) inhibition activity by solvent fractions from powdered bamboo shoots (Phyllostachys nigra, PN). C methanol fraction; B chloroform fraction; : ethyl acetate fraction; 6 butanol fraction; - water fraction.
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