新疆双峰驼乳酪蛋白血管紧张素转化酶抑制肽的酶法制备和抑制特性

对新疆双峰驼乳酪蛋白分别进行胃蛋白酶和胰蛋白酶的单酶和双酶联合水解,用反相高效液相色谱外标法,对产生的马尿酸含量进行检测,测定水解产物血管紧张素转化酶（angiotensin converting enzyme,ACE）抑制活性。通过米式方程对比水解产物与卡托普利之间的竞争关系,并用50、10、3 kDa的超滤膜对水解液进行超滤,测定截留液ACE抑制活性。结果表明：驼乳酪蛋白单酶水解12 h过程中,胰蛋白酶水解4 h,水解度达到3.7%,胃蛋白酶水解4 h,水解度达到16.58%,胰蛋白酶水解2 h,水解     

The development of angiotensin I-converting enzyme inhibitor derived from chicken bone protein

In order to produce angiotensin I‐converting enzyme (ACE) inhibitor for application in functional food, chicken bones were gathered from a meat processing factory and then hydrolyzed with Alcalase, pepsin and trypsin for 12 h. The hydrolysates were lyophilized, stored at ?80°C and tested experimentally every 2 h for pH value, peptide content, degree of hydrolysis (DH), electrophoresis and activity of ACE inhibitor. The hydrolysates of Alcalase had the highest peptide content and DH. The components of more than 66 kDa had disappeared in hydrolysates of Alcalase and trypsin after 2 h of hydrolysis. The hydrolysates of Alcalase were more active in inhibiting ACE, especially when hydrolyzed at 4 and 8 h, and also had low IC50 values of 1.960 and 0.945 mg/mL. According to the results of DH and electrophoresis, the higher activity of ACE inhibitor is assumed to be derived from the low molecular peptides in hydrolysates of Alcalase. Chicken leg bone has a high potential to be utilized to develop ACE inhibitory peptides as a potential ingredient of functional food intended to alleviate hypertension.     

Identification of an antihypertensive peptide derived from chicken bone extract

A novel angiotensin‐converting enzyme (ACE) inhibitory peptide was isolated and purified from chicken bone extract by enzymatic digestion. The peptide was defined as an ACE inhibitor, and it demonstrated antihypertensive activity following oral administration to spontaneously hypertensive rats (SHRs). The results of this study suggest that peptides derived from an extract of chicken bones, administered orally, have the ability to reduce the blood pressure of SHRs significantly over a short period of time (3 h). Moreover, the blood pressure then remains low for 3 h. This peptide derived from chicken bones may therefore have great value as a short‐term remedy for chronic conditions such as high blood pressure. The amino acid sequence of the peptide was YYRA (Tyr‐Tyr‐Arg‐Ala), which was the origin of the Ig heavy chain V region (27–30 position). The IC₅₀ value of its synthetic peptide was 33.9 μg/mL. We suggest that the ACE inhibitory and antihypertensive peptides derived from chicken bone extract may contribute to develop physiologically functional foods or improve food functionality.     

Determination of angiotensin-I converting enzyme inhibitory peptides in chicken leg bone protein hydrolysate with alcalase

This study aims to identify peptides with angiotensin-I converting enzyme (ACE) inhibitory activity in hydrolysate from chicken leg bone protein hydrolyzed with alcalase for 4 h (A4H). The hydrolysate has demonstrated potent in vitro ACE inhibitory activity, and has been shown to attenuate the development of hypertension and cardiovascular hypertrophy in spontaneously hypertensive rats (SHR). A4H is competitive for ACE and was separated using high-performance liquid chromatography (HPLC) with a gel filtration column (Superdex Peptide HR 10/30). The results show that A4H is a mixed non-competitive inhibitor. Eighteen fractions were detected after separation of A4H, and most of them showed ACE inhibitory activity. Five fractions with strong ACE inhibitory activities (above 50%) were labeled from A to E. In addition, there were 10 peptides, consisting of 5–10 amino acid residues that were identified from fraction D that exhibited the strongest ACE inhibitory activity. Three of the identified peptides corresponded to peptides derived from collagen type I and chicken muscular protein. It is revealed that A4H has several peptides that possess ACE inhibitory activities.     

绵羊乳酪蛋白血管紧张素转换酶抑制肽的制备与鉴定

以绵羊乳酪蛋白为原料,采用中性蛋白酶水解制备生物活性肽,研究酶解时间对酶解产物水解度和血管紧张素转换酶（angiotensin converting enzyme,ACE）抑制率的影响,利用液相色谱-质谱联用技术对水解产物进行分析和鉴定,筛选具有潜在ACE抑制作用的生物活性肽。结果表明：酶解时间达到300 min时,水解度最高值达9.65%,ACE抑制率为84.55%;当ACE抑制率达到50%时,所需肽段YYQQRP浓度为5～10 μmol/L;利用超高效液相色谱飞行时间质谱仪对酶解后的多肽进行序列分析,     

A study of in vivo antihypertensive properties of enzymatic hydrolysate from chicken leg bone protein

Numerous attempts have been made to develop angiotensin I converting enzyme (ACE) inhibitors from various sources of food protein. Generally chicken leg bones are discarded after industrial chicken meat processing without any substantial benefit. In previous studies, chicken leg bone proteins were hydrolyzed by various enzymes and the results demonstrated that Alcalase hydrolysates have considerable ACE inhibiting activities. In this study, the best ACE inhibitory hydrolysate (A4) (which was derived from chicken leg bone protein by Alcalase after 4 h incubation) was orally administrated (50 mg/kg bw) in spontaneously hypertensive rats (SHRs) to investigate its antihypertensive effects. After oral administration of A4, a maximal reduction activity of about 26 mmHg was found at 4 h and maintained to 8 h. Moreover, SHRs treated with A4 (50 mg/kg bw/day) for eight weeks exhibited a reduction in systolic blood pressure, which is as significant as the effects of Captopril ( P  < 0.05). These results suggested that chicken leg bones have a high potential for utilization to develop ACE inhibitors as potential food ingredients intended to alleviate hypertension.     

Angiotensin Converting Enzyme in Bovine Ovarian Follicular Fluid and its Relationship with Oestradiol and Progesterone

The purpose of the present study was to identify angiotensin converting enzyme (ACE) in bovine ovarian follicular fluid and to relate the ACE activity to the phase of the oestrous cycle, pregnancy, and the follicular fluid concentrations of oestradiol and progesterone. The ACE activity was similar to that found in bovine serum and was completely inhibited by the specific ACE inhibitor captopril. The 50% inhibitory concentration (IC₅₀) was 1.4 × 10^?8 mol/l (range 0.8 × 10^?8 to 5.0 × 10^?8 mol/l; n=6), which is similar to that found in bovine and human serum. The ACE activity did not differ in the pre‐ovulatory and luteal phase, pregnancy or cystic follicles. It correlated with the follicular fluid concentration of progesterone in cycling cows (ρ=0.476; p < 0.005; n=36), but did not correlate with the diameter of the follicles, the follicular fluid concentration of oestradiol or the ratio between the oestradiol and progesterone concentrations. The demonstration of ACE in bovine ovarian follicular fluid provides further evidence for the presence of a local renin–angiotensin system in the bovine ovary.     

In vitro effects of Yunnan Baiyao on canine hemangiosarcoma cell lines

Yunnan Baiyao is a Chinese herbal medicine that has been utilized for its anti‐inflammatory, haemostatic, wound healing and pain relieving properties in people. It has been utilized in the veterinary profession to control bleeding in dogs with hemangiosarcoma (HSA) and has been anecdotally reported to prolong survival times in dogs with this neoplasm. This study evaluated the in vitro activity of Yunnan Baiyao against three canine HSA cell lines after treatment with increasing concentrations of Yunnan Baiyao (50, 100, 200, 400, 600 and 800 µg mL^?1) at 24, 48 and 72 h. Mean half maximum inhibitory concentration (IC₅₀) at 72 h for DEN, Fitz, SB was 369.9, 275.9 and 325.3 µg mL^?1, respectively. Caspase‐3/7 activity increased in correlation with the IC₅₀ in each cell line which was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, APO‐BRDU Kit; BD Biosciences, San Jose, CA, USA) assay. VEGF in cell supernatant was also quantified. Overall, the study found that Yunnan Baiyao causes dose and time dependent HSA cell death through initiation of caspase‐mediated apoptosis, which supports future studies involving Yunnan Baiyao.     

The In Vitro Secretion of Acetylcholinesterase by Adult Stages of Heligmosomoides polygyrus: the Effects of Broad‐Spectrum Anthelmintics

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 ± 0.07 µmol min^?1 l^?1 mg^?1) than for female (0.56 ± 0.04 µmol min^?1 mg^?1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug‐free medium (RPMI medium supplemented with 0.5 % BSA) for 24 h or continuously exposed to the drugs in supplement‐free medium (24 h), the concentration‐ and time‐dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50 % (IC₅₀) relative to drug‐free controls were estimated. The IC₅₀ values ranged from 0.012 µM (IVM) to 2.96 µM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad‐spectrum anti‐nematodal activity is discussed.     

The influence of fig proteases on the inhibition of angiotensin I‐converting and GABA formation in meat

The purposes of this research were to use fig protease for texture tenderizing, and to inhibit angiotensin I‐converting enzyme (ACE) action and γ‐aminobutyric acid (GABA) formation of meat. Liberated peptides by the enzymatic action of fig protease in processing meat and free amino acids were determined and ACE inhibitory activity was assayed. Meat treated with fig protease became tender as indicated by shear force value (SFV) which was half of those of non‐fig treated meat during storage even at 5°C. Liberated peptides, free amino acids and GABA increased while extremely low levels of Glu were detected after storage. The optimal temperature of fig protease against meat was 80°C. However, the activity of fig protease decreased after pre‐heating more than 40°C. High ACE inhibitory activity of a mixture of fig and meat was found around 80°C, and the value corresponded to the amount of liberated peptide. A lot of liberated peptides were found at 60–80°C and pasterization of meat product becomes convenient to produce peptides. Production of ACE inhibitory peptides and GABA can be expected as the healthy functional meat product such as antihypertensive activity and improve brain function.     

Survival of dietary antigens in the digestive tract of calves intolerant to soybean products

Preruminant calves were given a series of feeds containing heated soybean flour. Digesta collected from the small intestine were analysed by haemagglutination inhibition assay for soluble soybean antigens. The presence of undenatured glycinin and beta-conglycinin in ileal contents was associated with digestive disturbances. In vitro experiments showed that, under optimal conditions of pH for protease activity, beta-conglycinin but not glycinin, was unaffected by pepsin and both antigens were resistant to rennin and trypsin. The solubility of glycinin and beta-conglycinin in saline extracts remained high over pH ranges of 2 to 3 and 6 to 10 but under conditions of intermediate pH, about 4.5, solubility was minimal. It was concluded that preruminant calves suffer from gastrointestinal hypersensitive responses to certain soybean products because major proteases of the digestive tract fail to denature soluble antigenic constituents of the soybean protein.     

Digestive enzymes along digestive tract of a carnivorous fish Glyptosternum maculatum (Sisoridae,Siluriformes)

This study attempted to determine the presence, distribution and levels of digestive proteases, pepsin, amylase and lipase in the homogenates of digestive tract of a freshwater, cold‐adapted, carnivorous teleost of Tibet, Glyptosternum maculatum (n = 10, mean weight: 129.6 ± 28.9 g, mean length: 195.1 ± 16.6 mm, approximately 6 years old), at different temperatures and pH levels. The descending order of protease activity was as follows: stomach (pepsin, 16.16 ± 0.96 U/mg protein), anterior intestine (3.18 ± 0.25 U/mg protein), posterior intestine (1.76 ± 0.21 U/mg protein) and middle intestine (1.52 ± 0.23 U/mg protein). Amylase activity levels in descending order were as follows: anterior intestine (0.0062 ± 0.0007 U/mg protein), stomach (0.0032 ± 0.0009 U/mg protein), middle intestine (0.0023 ± 0.0005 U/mg protein) and posterior intestine (0.0023 ± 0.0004 U/mg protein). The highest lipase activity was found in the anterior intestine (0.83 ± 0.25 U/mg protein), followed by posterior intestine (0.51 ± 0.19 U/mg protein), middle intestine (0.48 ± 0.09 U/mg protein) and stomach (0.39 ± 0.10 U/mg protein). The optimal temperature and pH level for protease were 50 °C and 9.0–10.0, respectively, along the intestine. The pepsin optima in the stomach were 30 °C and 2.0 respectively. The optimal temperature for amylase was found to be 30 °C along the digestive tract and the optimal pH was 7.0 in the intestine and 6.0 in the stomach. The optimal temperature and pH levels of lipase were 30 °C, pH 6.0 for the stomach and 40–50 °C, pH 8.0 for the intestine, respectively. Hepato‐somatic index was 1.35 ± 0.2% and relative intestine length was 0.90 ± 0.19. The weight ratio of the stomach and intestine to body weight was 1.63 ± 0.43% and 1.57 ± 0.24% respectively.     

胃蛋白酶和胰蛋白酶对大豆凝集素的稳定性研究

本研究以纯化的大豆凝集素(SBA)为研究对象,体外研究了动物体内两大主要蛋白消化酶即胃蛋白酶和胰蛋白酶对其酶解作用。试验通过SDS-PAGE方法分析了SBA经蛋白酶酶解后的降解程度;利用间接竞争ELISA方法测定其在酶解过程中的活性变化。结果表明:SBA可缓慢地被胃蛋白酶降解,SBA浓度越高,完全降解需要的时间越长;随着SBA的降解,SBA的活性也逐渐减小,随着酶解时间的延长活性消失速度也逐渐变慢,并且在本研究选取的酶浓度作用下,SBA浓度越高,活性减小的速度越快,但活性完全消失的时间延长。然而,SBA不能被胰蛋白酶降解,并且酶解过程中SBA的活性也保持完好,说明SBA对胰蛋白酶具有很好的稳定性。因本研究结果将为进一步揭示大豆凝集素在动物体内的消化动力学及其对机体的抗营养作用机理研究奠定理论基础。     

The pharmacokinetics and in vitro cyclooxygenase selectivity of deracoxib in horses

Davis, J. L., Marshall, J. F., Papich, M. G., Blikslager, A. T., Campbell, N. B. The pharmacokinetics and in vitro cyclooxygenase selectivity of deracoxib in horses. J. vet. Pharmacol. Therap. 34 , 12–16. The purpose of this study was to determine the pharmacokinetics of deracoxib following oral administration to horses. In addition, in vitro equine whole blood cyclooxygenase (COX) selectivity assays were performed. Six healthy adult horses were administered deracoxib (2 mg/kg) orally. Plasma samples were collected prior to drug administration (time 0), and 10, 20, 40 min and 1, 1.5, 2, 4, 6, 8, 12, 24, and 48 h after administration for analysis with high pressure liquid chromatography using ultraviolet detection. Following PO administration, deracoxib had a long elimination half‐life (t_1/2k₁₀) of 12.49 ± 1.84 h. The average maximum plasma concentration (C_max) was 0.54 μg/mL, and was reached at 6.33 ± 3.44 h. Bioavailability was not determined because of the lack of an IV formulation. Results of in vitro COX selectivity assays showed that deracoxib was selective for COX‐2 with a COX‐1/COX‐2 ratio of 25.67 and 22.06 for the IC₅₀ and IC₈₀, respectively. Dosing simulations showed that concentrations above the IC₈₀ for COX‐2 would be maintained following 2 mg/kg PO q12h, and above the IC₅₀ following 2 mg/kg PO q24h. This study showed that deracoxib is absorbed in the horse after oral administration, and may offer a useful alternative for anti‐inflammatory treatment of various conditions in the horse.     

Pharmacokinetics and distribution of minocycline in mature horses after oral administration of multiple doses and comparison with minimum inhibitory concentrations

Reasons for performing study: Minocycline holds great potential for use in horses not only for its antimicrobial effects but also for its anti‐inflammatory and neuroprotective properties. However, there are no pharmacokinetic or safety data available regarding the use of oral minocycline in horses. Objectives: To determine pharmacokinetics, safety and penetration into plasma, synovial fluid, aqueous humour (AH) and cerebral spinal fluid (CSF) of minocycline after oral administration of multiple doses in horses and to determine the minimum inhibitory concentrations (MIC) of minocycline for equine pathogenic bacteria. Methods: Six horses received minocycline (4 mg/kg bwt q. 12 h for 5 doses). Thirty‐three blood and 9 synovial fluid samples were collected over 96 h. Aqueous humour and CSF samples were collected 1 h after the final dose. Minocycline concentrations were measured using high pressure liquid chromatography. The MIC values of minocycline for equine bacterial isolates were determined. Results: At steady state, the mean ± s.d. peak concentration of minocycline in the plasma was 0.67 ± 0.26 µg/ml and the mean half‐life was 11.48 ± 3.23 h. The highest trough synovial fluid minocycline concentration was 0.33 ± 0.12 µg/ml. The AH concentration of minocycline was 0.09 ± 0.03 µg/ml in normal eyes and 0.11 ± 0.04 µg/ml in blood aqueous barrier‐disrupted eyes. The mean CSF concentration of minocycline was 0.38 ± 0.09 µg/ml. The MIC values were determined for 301 isolates. Minocycline concentrations were above the MIC₅₀ and MIC₉₀ for many gram‐positive equine pathogens. Potential relevance: This study supports the use of orally administered minocycline at a dose of 4 mg/kg bwt every 12 h for the treatment of nonocular infections caused by susceptible (MIC≤0.25 µg/ml) organisms in horses. Further studies are required to determine the dose that would be effective for the treatment of ocular infections.     

补料日龄对哺乳期仔猪消化酶活性的影响研究

为研究补料日龄对哺乳期仔猪消化道主要消化酶活性的影响,试验采用单因素完全随机试验设计,选取母猪分娩时间相差在48h之内、泌乳性能良好、产仔数接近的12窝仔猪,分4个处理组,分别于仔猪出生后7、10、15日龄和21日龄开始补料。结果表明:各试验组仔猪胃凝乳酶活性呈先升高后降低趋势,15日龄时仔猪胃凝乳酶活性达到最高(P<0.05);10日龄补料组到28日龄断奶时胃蛋白酶活性与胰蛋白酶活性最高(P<0.05);7日龄与10日龄补料组仔猪28日龄断奶时脂肪酶与淀粉酶活性最高(P<0.05)。本试验证明,仔猪出生后10日龄补料对28日龄断奶仔猪消化酶活性影响最小。     

In-vitro antiviral efficacy of ribavirin and interferon-alpha against canine distemper virus

Canine distemper is a highly contagious disease with high incidence and lethality in the canine population. The objective of this study was to evaluate the efficacy of antiviral action with ribavirin (RBV), interferon-alpha (IFNα), and combinations of RBV and IFNα against canine distemper virus (CDV). Vero cells inoculated with CDV were treated with RBV, IFNα, and combinations of these drugs. The efficacy to inhibit viral replication was evaluated by adding the compounds at different times to determine which step of the viral replicative process was affected. Both drugs were effective against CDV in vitro. The IFNα was the most active compound, with an average IC₅₀ (50% inhibitory concentration) value lower than the IC₅₀ of the RBV. Ribavirin (RBV) was more selective than IFNα, however, and neither drug showed extracellular antiviral activity. The combination of RBV and IFNα exhibited antiviral activity for the intra- and extracellular stages of the replicative cycle of CDV, although the intracellular viral inhibition was higher. Both RBV and IFNα showed high antiviral efficacy against CDV, and furthermore, RBV + IFNα combinations have shown greater interference range in viral infectivity. These compounds could potentially be used to treat clinical disease associated with CDV infection.     

牛乳酪蛋白初级抗菌肽粉乳基料的制备

为制备抗菌活性较好的肽粉乳基料,以新鲜牛乳为原料提取酪蛋白,以酪蛋白酶解物对大肠杆菌、沙门氏菌和金黄色葡萄球菌的抑菌圈直径为指标,从木瓜蛋白酶、碱性蛋白酶、中性蛋白酶和胰蛋白酶中筛选出一种蛋白酶水解牛乳酪蛋白,并通过单因素试验和正交试验确定该酶水解牛乳酪蛋白优化水解条件。结果表明：木瓜蛋白酶水解牛乳酪蛋白,其酶解物对3种菌均有较好的抑制作用;优化水解工艺条件为：酶解温度50℃、酶解时间4.5h、加酶量4500U/g、pH5.5、底物质量浓度6g/100mL;牛乳酪蛋白酶解物及初级肽粉乳基料稳定性均较好。     

In vitro anti‐tubulin effects of mebendazole and fenbendazole on canine glioma cells

Benzimidazole anthelmintics have reported anti‐neoplastic effects both in vitro and in vivo. The purpose of this study was to evaluate the in vitro chemosensitivity of three canine glioma cell lines to mebendazole and fenbendazole. The mean inhibitory concentration (IC₅₀) (±SD) obtained from performing the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay after treating J3T, G06‐A, and SDT‐3G cells for 72 h with mebendazole were 0.030 ± 0.003, 0.080 ± 0.015 and 0.030 ± 0.006 μM respectively, while those for fenbendazole were 0.550 ± 0.015, 1.530 ± 0.159 and 0.690 ± 0.095 μM; treatment of primary canine fibroblasts for 72 h at IC₅₀ showed no significant effect. Immunofluorescence studies showed disruption of tubulin after treatment. Mebendazole and fenbendazole are cytotoxic in canine glioma cell lines in vitro and may be good candidates for treatment of canine gliomas. Further in vivo studies are required.