Hippuryl l histidyl l leucine hhl

Green soybeans were obtained from a local farm (Harbin, China). Salt, paprika, and ginger powder were purchased from a supermarket (Beijing, China). B. velezensis CAU263 (CGMCC NO: 20318) isolated from Chinese traditional douchi was used in this study. Fibrinogen from bovine plasma, thrombin, DPPH, ABTS, angiotensin I‐converting enzyme (ACE), 2,4,6‐tris (2‐pyridyl)‐s‐triazine (TPTZ), (±)‐6‐hydroxy‐2,5,7,8‐tetramethyl‐chromane‐2‐carboxylic acid (trolox), 3‐tert‐butyl‐4‐hydroxyanisole (BHA), and hippuryl‐L‐histidyl‐L‐leucine (HHL) were obtained from Sigma‐Aldrich Co. Ltd. Peptone and yeast extract were obtained from Oxoid Co. Ltd. Rutin was gained from Shanghai Yuanye Biotechnology Co. Ltd. Gallic acid was obtained from Shanghai Macklin Biochemical Co. Ltd. L (+)‐ascorbic acid (VC) was acquired from Fujifilm Wako Pure Chemical Corporation. Other chemical reagents were obtained from Beijing Chemical Works.

Muscovy ducks (Cairina moschata) blood was obtained from Anqing Yongqiang Agricultural Science and Technology Co., Ltd. (Anqing, China). Alkaline protease (S10154, 200 U/mg) was purchased from Yuanye Biotechnology (Shanghai, China). Bicinchoninic acid (BCA) protein assay kit (Biosharp, Shanghai, China) was employed for protein content determination. ACE from rabbit lung (0.1 U/mL) and hippuryl–L–histidyl–L–leucine (HHL) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, formic acid and triethylamine were HPLC grade, while other reagents were of analytical grade.

Mature artichoke flowers (C. scolymus L.) were collected from the Region of Murcia (Spain). Vacuum-packed A. diaperinus L. larvae were provided by Proti-Farma (Holland). ACE from rabbit lung, the ACE synthetic substrate, hippuryl-L-histidyl-L-leucine (HHL), and 1,1-diphenil-2-picrylhydrazyl) (DPPH) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA).

Cyclina sinensis was purchased at the Dinghai Nanzhen fish market in Zhoushan City, China. After being positively identified by Professor Zhao Shenglong of Zhejiang Ocean University, the samples were shipped in ice to our biology laboratory within 30 minutes of purchase. Cyclina sinensis was cleaned, dehulled, washed with pure water, and then homogenized with a grinder (5000 rpm, 5 min). The tissue homogenate was incubated 3 times with 0.1 M NaOH, and each incubation lasted 2 h, then filtered with a gauze. The filter was then washed with water until the pH reached neutrality, and the homogenate was dehydrated and stored in sealed bags at −20 °C until the next separation and purification steps.
Trypsin (from bovine pancreas, 200 U/mg) and pepsin (from porcine gastric mucosa, 300 U/mg) were purchased from YTHX Biotechnology Co., Ltd. (Beijing, China). ACE (from rabbit lung), and the ACE synthetic substrate hippuryl-l-histidyl-l-leucine (HHL) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Other chemicals and reagents used were of analytical grade, except acetonitrile and trifluoroacetic acid (TFA), which were HPLC grade (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China).

All chemicals used were of analytical grade and purchased from Merck KGaA (Darmstadt, Germany) except for 95% aqueous ethanol which was purchased from R & M Marketing (Essex, UK). Phenylephrine (PE) hydrochloride, acetylcholine (ACh) chloride, Ang I, Ang II, BK, ACE, hippuryl-L-histidyl-L-leucine (HHL), and Tween® 80 were from Sigma-Aldrich Co. (St. Louis, MO, USA). Antagonists of angiotensin II type 2 receptor (AT₂R), PD 123319, and Mas receptor, A-779, were from Bachem (Bubendorf, Switzerland) and Tocris Bioscience (Bristol, UK), respectively.

Inhibitory activity of aqueous extracts was analyzed following a method by Hayakari et al. [22 (link)] https://dx.doi.org/10.17504/protocols.io.tb4eiqw. Hippuryl-L-histidyl-L-leucine (HHL) (Sigma) was hydrolyzed by ACE to yield hippuric acid and histidyl-leucine. This method relies on the colorimetric reaction of hippuric acid with 2, 4, 6-trichloro-s-triazine (TT) (Sigma). The tests were performed in triplicate and antihypertensive activity was quantified by a regression analysis of ACE inhibitory activity (%) versus phenolic compounds concentration in aqueous extract and defined as an IC₅₀ value, that is, the concentration of phenolic compounds required to produce 50% ACE inhibition under the conditions described. For angiotensin-converting enzyme inhibitory assay, lisinopril (antihypertensive drug) was used as control.

The reagents o-phthalaldehyde (OPA), chlorohydric acid (37 %), phenol (>99 %), 29-fluorenylmethyl chloroformate (FMOC), mobile phase (methanol, acetonitrile), hippuryl-L-histidyl-L-leucine (HHL), and ACE (ACE 3.4.15.1, 5.1 U/mg) were obtained from Sigma-Aldrich (Oakville, Ontario, Canada) and carbamoylcholine chloride (212385-M), phenylephrine hydrochloride (Pg126), N-nitro-L-arginine methyl ester hydrochloride (N5751, L-NAME) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Alcalase® 2.4 L (commercial protease obtained from fermentation of Bacillus licheniformis, non-specific serine endopeptidase) was supplied by Novozymes (Bagsværd, Denmark). All reagents implemented in this study were analytical grade.