酪蛋白抗凝血肽的分离纯化及其抗凝血活性测定

本实验通过比较混合酶解和复合酶解酪蛋白的水解液活性,获得抗凝血活性最高的组合,为“菠萝蛋白酶+胰蛋白酶+中性蛋白酶+木瓜蛋白酶”复合酶解酪蛋白组。然后选择截留分子量为1 kD和3 kD的超滤膜对抗凝血活性最高的组合进行超滤,实验结果表明截留分子量为1 kD的超滤浓缩液抗凝血活性最高。对超滤浓缩液进行抗凝血实验,实验结果表明,兔血︰1 kD浓缩液（V︰V）= 6︰4和兔血︰1kD浓缩液（V︰V）=1:1组合都是48 h后血液没有凝固;体外溶栓实验表明,置于浓缩液中的血栓条24 h后红色褪去,证明1 kD超滤浓缩液具有较好的溶栓和抗凝血作用。对1 kD超滤浓缩液进行脱盐,然后采用凝胶色谱法进行分子量分布测定,由数据分析可知分子量主要集中在500～180、1 000～500、2 000～1 000范围。     

酪蛋白抗凝血肽的分离纯化及其抗凝血活性测定

目的 本论文采用复合酶解法制备酪蛋白抗凝血肽,然后对其进行了分离及抗凝血活性测试。方法 本实验通过比较混合酶解和复合酶解酪蛋白的水解液活性,获得抗凝血活性最高的组合,为“菠萝蛋白酶+胰蛋白酶+中性蛋白酶+木瓜蛋白酶”复合酶解酪蛋白组。然后选择截留分子量为1 kD和3 kD的超滤膜对抗凝血活性最高的组合进行超滤,实验结果表明截留分子量为1 kD的超滤浓缩液抗凝血活性最高。结果 超滤浓缩液进行抗凝血实验,实验结果表明,兔血︰1 kD浓缩液(V︰V)=6︰4和兔血︰1kD浓缩液(V︰V)=1︰1组合都是48 h后血液没有凝固;体外溶栓实验表明,置于浓缩液中的血栓条24 h后红色褪去,证明1 kD超滤浓缩液具有较好的溶栓和抗凝血作用。1 kD超滤浓缩液进行脱盐,然后采用凝胶渗透色谱法进行分子量分布测定,由数据分析可知分子量主要集中在500～180、1 000～500、2 000～1 000范围。结论 按照本实验方法制备的酪蛋白抗凝血肽具有较好的溶栓和抗凝血作用。     

胰酶药物

<正> 胰酶是具有生理活性的复合酶,是国际医药卫生界公认的疗效确切的生化药品之一。主要含有胰蛋白酶、胰糜蛋白酶、胰淀粉酶和胰脂肪酶,还有羧肽酶、核糖核酸酶、弹性蛋白酶及激肽释放酶等。1983年以前,我国生产的胰酶是控制单项     

红非鲫(Oreochromis niloticus)鱼皮胶原蛋白酶解条件的研究

目的研究单酶和复合酶水解红非鲫鱼皮胶原蛋白制备具有抑制血管紧张素转换酶(ACE)生物活性的寡聚肽的工艺条件。方法根据6种单酶的水解产物对ACE抑制作用的测定结果,确定了两种酶即菠萝蛋白酶和alcalase 2.4L碱性蛋白酶组成复合水解酶。采用正交试验确定了这两种酶单独水解鱼皮胶原蛋白的最适酶解条件。结果与结论菠萝蛋白酶和alcalase 2.4L蛋白酶的最适酶解温度,pH,酶/底物,时间,底物浓度分别为45℃,pH4.5,4000U.g-1,4h,6%和55℃,pH7.5,6000U.g-1,2h,4%。在此基础上,进行复合酶水解实验,结果表明先采用菠萝蛋白酶水解,再用alcalase 2.4L蛋白酶水解,效果更佳。采用该双酶复合水解工艺,所得水解产物的水解度为30.43%,体外对ACE的抑制率达68.6%。     

酪蛋白和鱼精蛋白对胰岛素酶降解和口服降血糖作用的影响

目的研究酪蛋白和鱼精蛋白对酶降解胰岛素(INS)的保护作用及对大鼠灌胃给予INS溶液和微球后降血糖作用的影响。方法采用HPLC法测定α-糜蛋白酶和胰蛋白酶溶液中INS的残余量,考察酪蛋白和鱼精蛋白对体外酶降解INS的保护作用；制备INS溶液及肠溶微球,在促吸剂n-［8-(2-羟基苯甲酰基)氨基］辛酸钠(SNAC)存在下,大鼠灌胃给药研究酪蛋白和鱼精蛋白单独及合并使用对INS溶液和微球降血糖作用的影响。结果酪蛋白对α-糜蛋白酶体外降解INS有较好的抑制作用,鱼精蛋白不能抑制α-糜蛋白酶对INS的降解,但对胰蛋白酶降解INS的抑制作用优于酪蛋白。鱼精蛋白和酪蛋白均可增加INS溶液和肠溶微球的降血糖作用,两者合用效果更佳。在SNAC、鱼精蛋白和酪蛋白存在下,INS肠溶微球比INS溶液有更强而持久的降血糖作用。结论酪蛋白和鱼精蛋白可通过竞争性与酶结合机制增加INS在肠道消化酶中的稳定性,对INS口服吸收有利。     

2种地龙饮片不同提取法体外抗凝活性对比研究

目的 研究酒制对地龙体外抗凝活性的影响，为地龙临床应用提供科学依据。方法 分别采用传统水提法和仿生酶解提取法对生品地龙、酒制地龙进行提取，以活化部分凝血活酶时间（activated partial thromboplastin time，APTT）、凝血酶原时间（prothrombin time，PT）、凝血酶时间（thrombin time，TT）和抗凝血酶活力为指标进行抗凝活性测定，并采用BCA蛋白测定法测定可溶性蛋白、多肽含量。结果 采用水提取法时，PT和抗凝血酶滴定法均显示酒地龙与生地龙抗凝血活性相当，TT测定结果显示酒地龙抗凝血活性强于生地龙，APTT测定结果则显示地龙具有促凝血活性，且在一定范围内，浓度越高，促凝效果越强，蛋白、多肽含量为酒地龙大于生地龙；采用仿生酶解提取法时，APTT和PT测定结果显示酒地龙的抗凝血活性强于生地龙，并且蛋白、多肽含量测定结果显示酒地龙要多于生地龙，但TT和抗凝血酶滴定法并未比较出生品与酒制品之间的差异。结论 与传统水提法相比，仿生酶解提取法能更多地提取出蛋白多肽类成分，仿生酶解提取液具有更强的抗凝血活性，2种提取法均显示地龙酒制有利于可溶性蛋白、多肽的溶出，能增强体外抗凝血活性。     

从生产胰岛素的胰渣中制备胰酶、胰蛋白酶及糜蛋白酶

目的 从中性乙醇提取胰岛素后的残渣中提取制备高活性的胰酶,并从中分离纯化出胰蛋白酶(T)、糜蛋白酶(CT).方法 在胰渣中加入活化剂活化后,用PEG-6000沉淀、丙酮脱脂干燥得到胰酶;胰酶经CM-纤维素纯化得到胰糜蛋白酶混合制剂,再经CM-sepharose FF层析分离得胰蛋白酶和糜蛋白酶.结果 胰酶回收率为12.1％,胰蛋白酶、胰脂肪酶、胰淀粉酶的活性分别为5.40、39.72、76.72 U·mg-1,3种酶的活性比例达1∶7.4∶14.2.胰蛋白酶、糜蛋白酶的效价分别为2505、1003 U·mg-1;相对于胰酶,二者的活性回收分别为48.93％、61.73％.结论 从胰渣中制得的胰酶活性较高(约9倍于《中国药典》2010年版中的标准);制得的T及CT的活力也达到《中国药典》2010年版中的标准.     

美洲大蠊抗肝纤维化活性提取物的酶解制备工艺研究

目的 优选中性蛋白酶酶解美洲大蠊脱脂膏制备抗肝纤维化活性提取物的工艺。方法 以美洲大蠊脱脂膏的水解度、活性提取物的得率和体外活性为评价指标,在单因素考察（酶用量、酶解时间、pH值、酶解温度、底物浓度）的基础上,采用L₉（3⁴）正交试验优化中性蛋白酶酶解制备抗肝纤维化活性提取物的工艺。结果 最佳酶解工艺为酶用量0.3%,酶解时间2 h,pH值8.5,酶解温度45℃,底物密度1.07 g·mL^-1。工艺验证试验结果表明,美洲大蠊脱脂膏的水解度为15.8%,活性提取物的得率从0.54%提高到0.72%,其24,48,72 h的IC₅₀值分别为（118.37±3.12）,（115.16±2.48）,（105.00±6.35）μg·mL^-1。结论 研究得到美洲大蠊抗肝纤维化活性提取物的酶解制备工艺,该工艺得率较高,简单可行,为美洲大蠊抗肝纤维化药物的研发奠定基础。     

口服用糜胰蛋白酶及其片剂的工艺

<正> 注射用结晶糜胰蛋白酶为我国首次从猪胰中提取制得的蛋白水解酶,1976年由苏州地区科技组,卫生局组织鉴定。该药具有糜蛋白酶与胰蛋白酶二种酶协同水解蛋白质肽链的作用它能液化脓液和坏死组织,从而净化创面,有利于新生肉芽的生长,促进治愈。同从牛胰中提取的α——糜蛋白酶或胰蛋白酶比较,它具有疗效高、适应症广、付作用小、使用安全等优点。对內牛胰中提取的糜蛋白酶或胰蛋白酶有     

用丙酮法制备胰酶工艺的探索

<正> 胰酶系胰脏提取的酶的混合物,含有胰蛋白酶,糜蛋白酶,多种肽酶,脂肪酶及淀粉酶等,体内蛋白质的消化主要靠胰蛋白酶。现在国内基本上采用乙醇低浓度激活,高浓度沉淀的制备工艺,高浓度的乙醇会使脂肪酶失去活性,为了保证胰酶中三种主要酶的天然活性,我们对胰酶生产工艺进行了改革,其结果如下:一、工艺路线     

Deamination of beta-phenylethylamine by monoamine oxidase--inhibition by imipramine

The oxidative deamination of tyramine (Tyr), 5-hydroxytryptamine (5-HT), and β-phenylethylamine (PEA) by mitochondrial preparations of rabbit lung and brain was inhibited by imipramine. This tricyclic iminodibenzyl antidepressant drug was most effective in decreasing the deamination of PEA: at 1 × 10^?4M imipramine, deamination of PEA, Tyr and 5-HT was inhibited by approximately 70, 45 and 45 per cent, respectively, when either lung or brain mitochondrial monoamine oxidase (MAO) preparations were used. Imipramine-induced inhibition of MAO was shown to be of a mixed type based on Lineweaver-Burk plots, but was found to be completely reversible. The desmcthyl and didesmethyl derivatives of imipramine were equally as effective as the parent drug in inhibiting the deamination of PEA, whereas the N-oxide analog of imipramine was less effective as an inhibitor of this reaction. These results support the premise that the action of imipramine as a clinically effective antidepressive agent may be related to its inhibitory effect on the specific form of MAO which deaminates PEA.     

Augmentation by serrapeptase of tissue permeation by cefotiam

Cefotiam (CTM) is a new cephalosporin with a broad spectrum of activity against both Gram-positive and Gram-negative microorganisms. Cephalosporins are widely used for prophylaxis of infections in patients undergoing thoracotomy. Augmentation by serrapeptase on tissue permeation of CTM was examined in 35 thoracotomy patients with lung cancer. The subjects were divided into two groups according to the method of the administration of CTM. Group I consisted of 17 subjects, each of whom received a single dose of 2 g of CTM alone by an instillation for 30 minutes. Group II consisted of 18 subjects, each of whom received a combination of CTM and serrapeptase; serrapeptase was given 2 tablets (10 mg) each time for three times/day until the day before surgery, and then CTM was administered by the same procedure. The following results were obtained: Individual difference was observed for the permeation of CTM into tissues. Pathologic differences also affected the permeation. Nevertheless, the CTM levels in pulmonary tissues reached about a half of those in the blood in both the single dose group and the combination group, hence sufficient concentrations exceeding MIC80 for main microorganisms that caused infections in the lung were obtained. The concentrations of CTM in inflammatory tissues have showed lower levels than those of normal tissues in both CTM single dose and the combination groups. Decrease of blood flow volume may have contributed to the reduction in levels of CTM in the inflammatory tissues. The ratio of the concentration of the drug in pulmonary tissues to that in the blood was 29.1 +/- 2.5% in the single dose group, and 44.2 +/- 6.0% in the combination group, the latter showing quite a significant increase (P less than 0.05). Combined administrations of CTM and serrapeptase deserves more trials in the case when surgical treatments of the lung are performed. An antiinflammatory effect of serrapeptase in the respiratory system is expected, and in addition, the combined use of CTM and serrapeptase should stimulate permeation of the antibiotic into tissues.     

Antagonism of morphine by beta-melanocyte-stimulating hormone and by tetracosactin

Using the decerebrate—spinal Lloyd preparation morphine depressed evoked mono- and polysynaptic reflex activity, β-melanocyte-stimulating hormone enhanced monosynaptic reflex activity, and tetracosactin had no effect. When morphine injection was preceded either by β-melanocyte-stimulating hormone or by tetracosactin a statistically significant depression was not observed. The stimulant actions of β-melanocyte-stimulating hormone did not appear to account for its capacity to antagonize morphine. The fall of blood pressure which follows the administration of morphine in this preparation was not antagonized by the prior administration of either polypeptide.     

Analgesia induced by brief footshock is inhibited by 5-hydroxytryptamine but unaffected by antagonists of 5-hydroxytryptamine or by naloxone

Exposure to footshock (1 mA) for 30 sec induced a marked analgesia that was enhanced by pretreatment with the 5HT synthesis inhibitor, p-chlorophenylalanine, and attenuated by the 5HT releasing drugs p-chloroamphetamine and fenfluramine, by the 5HT re-uptake inhibitor, fluoxetine and by the 5HT agonists, 5-methoxy-N,N-dimethyltryptamine and MK212. However, agonists, quipazine and trifluoromethylphenylpiperazine, with greated reported affinities for 5HT binding sites on rat brain membranes than MK212 were without effect as were the antagonists metergoline, methysergide, cyproheptadine, mianserine and methiothepin. The specific opioid antagonist naloxone was also without effect. The results in general indicate that analgesia induced by brief footshock (1 mA, 30 sec) is inversely related to 5HT availability but thereis little evidence of involvement of known 5HT receptors.     

Synergism by caffeine and by cocaine of the motor control deficit produced by midazolam

To evaluate the effects of caffeine and cocaine on the impairment of discriminative motor control produced by midazolam, rats were trained to hold a force transducer operated with a paw so that it remained between upper and lower limits of a force band for a continuous 1.5-s period to deliver each food pellet. Acute doses of 3 mg/kg midazolam SC impaired motor performance. Except for one animal, caffeine (10-40 mg/kg IP) had little or no effect on performance, while cocaine (3.75-22.5 mg/kg IP) produced dose-related impairment. When each dose of caffeine was combined with 3 mg/kg midazolam, a marked synergism in motor performance impairment occurred. Cocaine plus midazolam produced mainly an additive synergism. The conspicuous synergistic action of caffeine on the motor control deficit produced by midazolam contrasts with the typical antagonism found between the benzodiazepines and methylxanthines when performance is evaluated by psychomotor tests not requiring fine motor control.     

The inhibition of cyclo-oxygenase of rabbit platelets by aspirin is prevented by salicylic acid and by phenanthrolines.

Salicylic acid, 1,10- and 1,7-phenanthroline prevented inhibition by aspirin of platelet aggregation and of generation of thromboxane A2 due to arachidonic acid, to the ionophore A21387, to thrombin and to collagen. Dithiothreitol, another drug which prevents aggregation and formation of thromboxane A2, but only reversibly, failed to interfere with the inhibition by aspirin. Irreversible inhibition by indomethacin and by the substrate analogue 5,8,11,14-tetraynoic acid was also unaffected by salicylic acid or by 1,10-phenanthroline, which thus probably exert a specific interaction with the aspirin-binding site. Inactivation of platelet cyclo-oxygenase with arachidonic acid led to inhibition of the formation of thromboxane A2 and of aggregation due to arachidonic acid itself and to collagen, but barely affected aggregation by thrombin, even though generation of thromboxane A2 was blocked. Use of salicylic acid and of reversible inhibitors of cyclo-oxygenase may help to unravel the mechanism of inhibition due to other agents.     

Local injuries by accidental ingestion of corrosive substances by children

Own data and analysis of previous publications show that situations where accidental ingestion of corrosive substances by children may have happened are frequent, but severe corrosive esophagitis leading to perforation or stricture formation is very rare. In case of suspected esophageal injury, esophagoscopy and glucocorticoid treatment become necessary. The evaluation of the initial symptoms in patients from our own material and from the literature indicates that all children with serious esophageal burns had one or more of the following symptoms: visible burns in the oral cavity, hypersalivation, retching, vomiting, retrosternal or epigastric pain, cardiovascular collaps, airway stenosis. Hence, children with an uncertain history of ingestion and without any of these symptoms need not be treated. After ingestion of liquid substances, but never of dry or granular products, lesions in the esophagus without accompanying burns in the oral cavity were observed. The evaluation of 1158 cases of accidental ingestions of several types of household products and a collection of data from the literature on the causticity of these substances shows that cleaners containing mainly detergents and phosphates (with pH values generally between 9 and 11), and household bleaches on sodium hypochlorite basis are relatively harmless. Drain cleaners (NaOH), decalcifiers (formic acid) and detergents for automatic dish washing machines (metasilicates) are very caustic and are responsible for the majority of serious accidents in children.

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Zusammenfassung Eigenes Zahlenmaterial und eine Durchsicht früherer Veröffentlichungen ergeben, daß Situationen, in denen Kinder versehentlich ätzende Haushaltsmittel zu sich genommen haben (oder haben könnten), häufig sind, daß aber schwere Ösophagusverätzungen mit Perforationen oder Strikturbildung im Kindesalter sehr selten sind. Beim Verdacht auf eine erheblichere Ösophagusverätzung werden Ösophagoskopie und Glukokortikosteroidbehandlung erforderlich. Alle Kinder mit erheblichen Ösophagusverätzungen, über deren Aufnahmestatus in unseren Unterlagen oder in Publikationen Angaben gemacht worden sind, hatten mindestens eines der folgenden Symptome: sichtbare Ätzspuren in Mund und Rachen, Hypersalivation, Würgen, Erbrechen, Schmerzen hinter dem Brustbein oder im Oberbauch, Kreislaufkollaps, Luftwegsstenose. Demnach brauchen Kinder, deren Anamnese nur fraglich ist, und die keines der genannten Symptome haben, nicht behandelt zu werden. Bei trockenen Substanzen waren stets Ätzspuren in der Mundhöhle sichtbar, wenn es zu Ösophagusverätzungen gekommen war; bei Ösophagusschäden durch Flüssigkeiten fehlen gelegentlich Nekrosen in der Mundhöhle. Die Auswertung von 1158 Verätzungsunfällen bei Kindern mit verschiedenen Haushaltsprodukten, und eine Zusammenstellung bereits bekannter, relevanter Daten zeigt, daß Allzweckreiniger auf Detergentien- und Polyphosphat-Basis (in der Regel mit pH-Werten zwischen 9 und 11) und Haushaltsbleichen (Natriumhypochlorit) verhältnismäßig harmlos sind. Reiniger für maschinelles Geschirrspülen (Metasilikate), Abflußreiniger (Natronlauge) und Entkalker (Ameisensäure) sind stark ätzend und verursachen die Mehrzahl der schweren Verätzungsunfälle bei Kindern.

     

Dose-dependent reductions by naloxone of analgesia induced by cold-water stress

Animals exposed to cold-water swims, rotation, or inescapable shocks, display analgesia comparable to that of 10 mg/kg of morphine. The present study investigated whether a narcotic antagonist would eliminate analgesia induced by cold-water swims. In one group of 12 rats, naloxone at 0, 1, 5, 10 and 20 mg/kg was administered at weekly intervals immediately preceding forced cold-water swims (2°C for 3.5 min) and alterations in flinch-jump thresholds were determined 30 min thereafter. In a second group of six rats, the effects of the same dose range of naloxone were determined upon normal flinch-jump thresholds. Naloxone dose-dependently attenuated the cold-water swim-induced analgesia up to a maximal reduction of 50% at 20 mg/kg. In contrast, all doses of naloxone had no effects upon normal flinch-jump thresholds. Since low doses of naloxone completely abolish morphine-induced analgesia, the present data suggest that the analgesia induced by stress is not identical to that of opiates.