IJFS#1090_bozza Ital. J. Food Sci., vol. 30, 2018 - 792 PAPER ANGIOTENSIN I CONVERTING ENZYME INHIBITORY PEPTIDES FROM SWORD BEAN Y. ZHANG, P. CHEN, L. LIU, K. LI, H. WANG and L. WANG* School of Life Science, Beijing University of Chinese Medicine, Fangshan District, Beijing 102488, China *E-mail address: wanglz@bucm.edu.cn ABSTRACT Sword bean is a healthy food and herbal medicine in China. In this study, the main components of sword bean were determined. Albumin, globulin, prolamin and glutelin were hydrolyzed by pepsin and then the angiotensin I converting enzyme (ACE) inhibitory activity was evaluated. Our results showed that glutelin peptides manifested the highest ACE inhibitory activity with inhibitory ratio of 22.10±1.57% followed by prolamin peptides and albumin peptides of 16.77±0.76% and 16.40±0.42%, respectively, at the final concentration of 0.01 mg/mL. Our results strongly suggest that sword bean at some extant have potential to lower blood pressure. Keywords: sword bean, main component, angiotensin I converting enzyme, peptides Ital. J. Food Sci., vol. 30, 2018 - 793 1. INTRODUCTION Hypertension, a major risk factor for cardiovascular and renal diseases, has become the most common serious chronic health problem. The rennin-angiotensin system (RAS) is critically involved in the physiological regulation of blood pressure and pathogenesis of hypertension (CAT and TOUYZ, 2011). ACE, as an essential member of RAS, can catalyzes the conversion of angiotensin (ANG) I to ANG II by removing a carboxyterminal dipeptide (WYSOCKI et al., 2006). Meanwhile, ACE metabolizes bradykinin (BK), a vasodilator, to inactive BK-(1-7). Therefore, ACE inhibitors are effective first-line treatment against essential hypertension (THOMAS et al., 2004), such as captopril, enalapril and lisinopril. However, these synthetic drugs may also cause obvious side effects including cough, loss to taste, renal impairment, and angioneurotic oedema (ANTONIOS et al., 1995). Thus, peptides with potent ACE inhibitory activity derived from natural food provide an effectively alternative treatment (YU et al., 2006). In recent years, ACE inhibitory peptides from natural protein have been successfully isolated, such as corn (YANG et al., 2007), soybean (MALLIKARJUN et al., 2006) and Coix seed (YUAN et al., 2014). Recently, the antihypertensive peptides from traditional Chinese medicine proteins has drawn considerable attention. Sword bean, the seed of the leguminous plant Canavalia gladiate, also has been treated as traditional medicine for containing canavanine, hemagglutinin, and concanavalin A (EKANAYAKE et al., 2006). It has been reported that sword bean may exhibit antioxidant activity of eliminating free radicals and against oxidative stress. In addition, it also has strong anti-inflammatory and anticarcinogenic effects. It is reported that soybean paste containing sword bean exhibits higher ACE inhibitory effects than other soybean pastes (HAN et al., 2015). In this study, this medicinal food was chose to prepare the ACE inhibitory peptides because of its ACE inhibitory activity and rich protein. The aims of this study are: (1) to determine the main components and protein content of sword bean. (2) to obtain peptides with low molecular weight (≤ 3 KD) by hydrolyzing protein with pepsin, and estimate their ACE inhibitory activity. (3) to provide some reference for the clinical drug use of sword bean in traditional Chinese medicine. 2. MATERIALS AND METHODS 2.1. Material Sword bean was purchased from Tongrentang (Beijing, China). The voucher specimen (No. 131121003) was deposited at -20°C. Pepsin, ACE and hippuryl-L-histidyl-L-leucine (HHL) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trifluoroacetic acid (TFA, MS grade) and Acetonitrile (HPLC grade) were purchased from Merck KGaA (Darmstadt, Germany) and Fisher Scientific (Pittsburgh, PA, USA) respectively. All other chemicals and reagents were analytical grade. 2.2. Determination of the proximal compositions Protein, fat, moisture and ash content of sword bean were determined according to the Chinese pharmacopoeia (Commission, 2015). The content of starch was determined by Ji (JI et al., 2016). Ital. J. Food Sci., vol. 30, 2018 - 794 2.3. Sequential extraction of seed protein The seeds were ground into powder by a universal high-speed smashing machine, and then defatted with cooled petroleum ether and dried at 40°C overnight. Albumin, globulin, prolamin and glutelin were then sequentially extracted with deionized water, 0.5 M NaCl, 70% ethanol (containing 0.5% NaAC, 5% β-mercaptoethanol) and 0.0125 M sodium borate buffer (containing 1% SDS, 2% β-mercaptoethanol), respectively. All of the protein solutions were dialysised against deionized water at 4°C for 24 h and then freeze- dried. The samples were stored at -80°C for further analysis. 2.4. Determination of protein molecular weight distribution SDS-PAGE was conducted according to the method of Krizkova (KRIZKOVA et al., 2015) with some modifications to determine the molecular weight distribution of all the protein fractions. All the samples were run for approximately 100 min in 3% stacking gel with a electric current of 10 mA and then for another 100 min in 15% separating gel with 30 mA. After that, the gel was dyed with Coomassie brilliant blue overnight and then decolored with bleaching liquid until the strips were seen clearly. 2.5. Determination of the Amino acid content For determination of amino acid composition, 100 mg samples were subjected to acid hydrolysis with 20 mL of 6 M HCl at 110°C for 24 h. Then the lyophilized hydrosate was dissolved in 0.02 M HCl and analyzed by a amino acid analyzer (L-8900; Hitachi, Tokyo, Japan) (WANG et al., 2008). 2.6. Preparation of enzymatic hydrolysates To produce bioactive peptides, enzymatic hydrolysis method was applied. The protein (2%, w/v) was dissolved in 0.01 M HCl, and pepsin was added with enzyme/substrate ratio of 1/10 (w/w). The mixture was incubated at the temperature of 37°C for 48 h. To terminate the reaction, the mixture was heated 95°C for 5 min. The hydrolysates supernatant was collected after the centrifugation (at 10,000 rpm, 10 min, 4°C). 2.7. Ultrafiltration (UF) of protein hydrolysates To produce low molecular weight peptides, the hydrolysates were passed through ultrafiltration membrane(MWCO, 3 KD). The peptide concentration of each collected fractions was estimated by the Lowry method (SAPAN, and LUNDBLAD, 2015). 2.8. The assay of ACE inhibitory activity The ACE inhibitory activity was determined according to the method reported by Yuan (YUAN et al., 2014). Briefly, the reaction system contained 10 μL sample, 20 μL ACE (2 mU) and 20 μL HHL (2 mM). Sample and ACE were incubated at 37°C for 10 min prior to adding substrate HHL, and then for an additional incubation for 80 min at the same temperature. To terminate the reaction, 100 μL acetonitrile was added. Captopril and borate buffer solution was used as positive and blank control, respectively. ACE inhibitory activity was confirmed by monitoring the formation of HA which was generated by HHL under enzymatic hydrolysis. HA was detected by RP-HPLC on a C18 column (250 × 4.6 mm, 5 μm, Tianhe). The column was eluted by a mobile phase of acetonitrile/water (0.05% Ital. J. Food Sci., vol. 30, 2018 - 795 TFA) at a volume ratio of 25 : 75 with the flow rate of 1 mL/min. The elution was monitored at 228 nm. The ACE inhibitory ratio of each sample was calculated as follows: Inhibitory activity (%) = [ (A-B) ⁄ A] × 100% where A was the HA peak area of blank control; B was the HA peak area in the presence of the sample. 2.9. Statistical analysis All data were conducted in triplicate and expressed as the mean±SD. The SAS 9.3 program was used for Multiple comparison, and P < 0.05 were considered to be significant. 3. RESULTS 3.1. Proximal compositions of sword bean The proximal compositions of sword bean were presented in Table 1. The starch content of sword bean ranked first (36.59±2.93%). As the member of Leguminosae, the protein content of sword bean accounted for 31.95±0.24%. The moisture and ash contents of this medicinal food all conformed to the requirements of the Chinese pharmacopoeia. Table 1. Proximal compositions (%) of sword bean. Protein Crude fat Moisture Ash Starch Sword bean 31.95 (±0.24) 0.71 (±0.04) 8.33 (±0.01) 3.16 (±0.04) 36.59 (±2.93) Results were expressed as the mean±SD (n = 3). 3.2. Protein fractions distribution of sword bean Protein patterns of sword bean were shown in Table 2. Considerable variability among albumin, globulin, prolamin and glutelin was observed. Albumin had the highest percentage of 70.93±0.25% followed by globulin of 16.75±0.51%. The prolamin and glutelin contents of this leguminous seeds were rather low. Insoluble protein in the residue only accounted for 5.83±0.04% indicating effective extraction of protein. Table 2. Protein pattern of sword bean (% of total protein). Albumin Globulin prolamin Glutelin Residual Sword bean 70.93a (±0.25) 16.75b (±0.51) 1.48e (±0.02) 7.02c (±0.26) 5.83d (±0.04) Results were expressed as the mean±SD (n = 3). Different letters of indicated having significantly different (P < 0.05). Ital. J. Food Sci., vol. 30, 2018 - 796 3.3. SDS-PAGE pattern of protein fractions of sword bean The molecular weight (MW) distributions of different protein fractions for sword bean were detected by SDS-PAGE, which was shown in Fig. 1. Albumin and globulin resolved into similar subunits ranging from 97 KD to 19 KD, with the major subunit of 50 KD. The bands of prolamin and glutelin were heterogeneous ranging from 57 to 14.4 KD and 97 to 19 KD, respectively. Figure 1. The molecular weight distribution of proteins extracted from sword bean. Lane 1, marker; Lane 2, albumin; Lane 3, globulin; Lane 4, prolamin; Lane 5, glutelin. 3.4. Amino acid composition of seeds flour For sword bean, a total of 13 kinds of amino acid were detected. Including almost all the essential amino acids and semi-essential amino acids for human beings. From Table 3, it could be seen that Phe had the highest percentage of 4.55±0.11 mg/100 mg, while His recorded the lowest (0.50±0.01 mg/100 mg) in sword bean. 3.5. ACE inhibitory activity assay The RP-HPLC method was utilized to estimate ACE inhibitory activity of peptide mixtures (≤ 3 KD). The blank control displayed a strong peak area of HA (Fig. 2a), while the positive control (captopril, final concentration of 2×10-9 mol/L) manifested a strong ACE inhibition ratio of 91.64±0.07% (Fig. 2b). The glutelin peptides (≤ 3 KD) revealed the highest ACE inhibitory activity at the final concentration of 0.01 mg/mL with 22.10±1.57 (Fig. 2c). All results were showed in Table 4. Ital. J. Food Sci., vol. 30, 2018 - 797 Table 3. The amino acid content of sword bean (mg/100 mg). Amino acid Sword bean (mg/100 mg) Asp 2.84±0.03 Thra 1.32±0.03 Ser 1.49±0.02 Glu 3.26±0.07 Gly 0.88±0.02 Ala 0.68±0.01 Cys - Vala 2.74±0.06 Meta - Ilea 1.07±0.02 Leua 1.69±0.05 Tyr - Phea 4.55±0.11 Lysa 1.47±0.02 Hisa 0.50±0.01 Trpa Arga - 1.56±0.02 Pro - a(semi-) essential amino acid for human, - not detected. The data was expressed as the mean±SD (n = 3). Elution time (min) Elution time (min) HA HHL b HA HHL a Ital. J. Food Sci., vol. 30, 2018 - 798 Elution time (min) Figure 2. RP-HPLC chromatograms of (a) blank control, (b) positive control (captopril, final concentration of 2×10-9 mol/L), (c) glutelin peptides (≤ 3 KD) of 0.01 mg/mL. The mobile phase consisted of 25% acetonitrile (containing 0.05% TFA), eluting at a flow rate of 1.0 mL/min and the absorbance of eluent was detected at 228 nm. Table 4. ACE inhibition rate (%) of peptides (≤ 3 KD) from different protein fractions. Peptide ACE inhibition rate (%) Albumin peptides 16.40±0.42b Globulin peptides 12.72±0.29c Prolamin peptides 16.77±0.76b Glutelin peptides 22.10±1.57a Results were expressed as the mean±SD (n = 3). The samples were measured at the final concentration of 0.01 mg/mL. Different letters of indicated having significantly different (P < 0.05). 4. DISCUSSION Food-derived ACE inhibitory peptides can provide an effectively alternative treatment for hypertension. There are different methods to produce ACE inhitory peptides from precursor proteins, such as enzymatic hydrolysis (CHEN et al., 2007), microbial fermentation (YAMAMOTO et al., 1994) and chemical synthesis. Among these methods, enzymatic hydrolysis is the most commonly used method (YUAN et al., 2014). There are a great number of studies have proved that food-derived protein hydrolysates and peptides possess ACE inhibitory activity (BALTI et al., 2010; LASSISSI et al., 2014; LEE et al., 2010). Soybean paste containing sword beans exhibits higher ACE inhibitory effects (HAN et al., 2015). Research has shown that the presence of hydrophobic amino acids can increase ACE inhibitory activity (LI et al., 2004). Our study showed that sword bean included all the essential amino acids (except Met) and semi-essential amino acids for human beings. Moreover, sword bean protein may become effective sources for preparation of ACE inhibitory peptides because of its high proportion of hydrophobic amino acid and proline, with 44.58% of total amino acid. Albumin, globulin, prolamin and glutelin were sequentially extracted, which is of benefit to study different proteins activity. The high levels of protein and starch make sword bean good sources of these nutrients. 5. CONCLUSION Our study mainly focused on the ACE inhibitory activity of protein hydrolysates. The result showed that glutelin peptides manifested the highest ACE inhibitory activity with HA HHL c Ital. J. Food Sci., vol. 30, 2018 - 799 inhibitory ratio of 22.10±1.57% followed by prolamin peptides and albumin peptides of 16.77±0.76% and 16.40±0.42%, respectively, at the final concentration of 0.01 mg/mL. After further separation, purification and structural identification of hydrolysates (≤ 3 KD), bioactive peptides with better antihypertensive activity might be obtained. Our data might contribute to further research into food derived antihypertensive compounds, meanwhile it also provides some reference for the clinical drug use of sword bean in traditional Chinese medicine. ACKNOWLEDGEMENTS This study has received financial support from the Natural Science Foundation of China (NO. 81872972) and Basic Scientific Research Funding Project of Beijing University of Chinese Medicine (NO. 2015-JYB-JSM S026). Thanks to all the people who joined and helped in this study. REFERENCES Antonios T.F. and MacGregor G.A. 1995. Angiotensin converting enzyme inhibitors in hypertension: potential problems. Journal of Hypertension 13:S11-S16. DOI: doi.org/10.1097/00004872-199509003-199500003. Balti R., Nedjar-Arroume N., Bougatef A., Guillochon D. and Nasri M. 2010. Three novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttlefish (Sepia officinalis) using digestive proteases. Food Research International 43(4):1136-1143. DOI: doi.org/1110.1016/j.foodres.2010.1102.1013. Cat A.N.D. and Touyz R.M. 2011. A new look at the renin-angiotensin system-Focusing on the vascular system. Peptides 32(10):2141-2150. DOI: doi.org/2110.1016/j.peptides.2011.2109.2010. Chen Q., Xuan G.-D., Fu M.-L., He G.-Q., Wang W., Zhang H.-B. and Ruan H. 2007. Effect of angiotensin І-converting enzyme inhibitory peptide from rice dregs protein on antihypertensive activity in spontaneously hypertensive rats. Asia Pacific journal of clinical nutrition 16(S1):281-285. Commission C.P. 2015. Pharmacopeia of People's Republic of China (vol. 3). China Medical Science Press (In Chinese). ISBN:978-977-5067-7337-5066. Ekanayake S., Nair B.M., Asp N.G. and Jansz E.R. 2006. Effect of processing of sword beans (Canavalia gladiata) on physicochemical properties of starch. Starch‐Stärke 58(5):215-222. DOI: doi.org/210.1002/star.200500449. Han S. S., Hur S.J. and Lee S.K. 2015. A comparison of antioxidative and anti-inflammatory activities of sword beans and soybeans fermented with Bacillus subtilis. Food & Function 6(8):2736-2748. DOI: doi.org/2710.1039/C2735FO00290G. Ji H.F., Liu L., K.L., Zhang Y.Y. and Wang L.Z. 2016. Protein composition analysis and cytotoxicity of gulbulin hydrolysates of Brucea javanica seeds. China Journal of Chinese Materiamedica 41:4210-4215 (In Chinese). doi:4210.4268/cjcmm20162221. Krizkova S., Dostalova S., Michalek P., Nejdl L., Kominkova M., Milosavljevic V., Moulick A., Vaculovicova M., Kopel P. and Adam V. 2015. SDS-PAGE as a tool for hydrodynamic diameter-dependent separation of quantum dots. Chromatographia 78(11-12):785-793. DOI: doi.org/710.1007/s10337-10015-12893-z. Lassissi T.A., Hettiarachchy N.S., Rayaprolu S.J., Kannan A. and Davis M. 2014. Functional properties and Angiotensin-I converting enzyme inhibitory activity of soy–whey proteins and fractions. Food Research International 64:598-602. doi:510.1016/j.foodres.2014.1007.1015. Lee S.-H., Qian Z.-J. and Kim S.-K. 2010. A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats. Food Chemistry 118(1):96-102. DOI: doi.org/110.1016/j.foodchem.2009.1004.1086. Li G.-H., Le G.-W., Shi Y.-H. and Shrestha S. 2004. Angiotensin I–converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutrition research 24(7):469-486. DOI: doi.org/410.1016/j.nutres.2003.1010.1014. Ital. J. Food Sci., vol. 30, 2018 - 800 Mallikarjun Gouda K., Gowda L.R., Rao A.A. and Prakash V. 2006. Angiotensin I-converting enzyme inhibitory peptide derived from glycinin, the 11S globulin of soybean (Glycine max). Journal of Agricultural and Food Chemistry 54(13):4568-4573. DOI: doi.org/4510.1021/jf060264q. Sapan C.V. and Lundblad R.L. 2015. Review of methods for determination of total protein and peptide concentration in biological samples. PROTEOMICS-Clinical Applications 9(3-4):268-276. DOI: doi.org/210.1002/prca.201400088. Thomas D.A., Grant P.J., Turner A.J. and Hooper N.M. 2004. Evaluation of angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin in angiotensin peptide metabolism. Biochemical Journal 383(1):45-51. DOI: doi.org/10.1042/BJ20040634. Wang L., Xu C., Qu M. and Zhang J. 2008. Kernel amino acid composition and protein content of introgression lines from Zea mays ssp. mexicana into cultivated maize. Journal of Cereal Science 48(2):387-393. DOI: doi.org/310.1016/j.jcs.2007.1009.1014. Wysocki J., Ye M., Soler M.J., Gurley S.B., Xiao H.D., Bernstein K.E., Coffman T.M., Chen S. and Batlle D. 2006. ACE and ACE2 activity in diabetic mice. Diabetes 55(7):2132-2139. DOI: doi.org/2110.2337/db2106-0033. Yamamoto N., Akino A. and Takano T. 1994. Antihypertensive effects of different kinds of fermented milk in spontaneously hypertensive rats. Bioscience, biotechnology, and biochemistry 58(4):776-778. DOI: doi.org/710.1271/bbb.1258.1776. Yang Y., Tao G., Liu P. and Liu J. 2007. Peptide with angiotensin I-converting enzyme inhibitory activity from hydrolyzed corn gluten meal. Journal of Agricultural and Food Chemistry 55(19):7891-7895. DOI: doi.org/7810.1021/jf0705670. Yu Y., Hu J., Miyaguchi Y., Bai X., Du Y. and Lin B. 2006. Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin. Peptides 27(11):2950-2956. DOI: doi.org/2910.1061/j.peptides.2006.2905.2025. Yuan J., Liang Y., Cui S., Zhang X., Wang L. and Qiao Y. 2014. Angiotensin I converting enzyme inhibitory and antioxidant activity of adlay (Coix lacryma-jobi l. Var. Ma-yuen stapf) glutelin hydrolysate. Italian Journal of Food Science 26(3):282-288. Paper Received November 8, 2018 Accepted June 25, 2018