天龙粉酶解工艺中最佳用酶的研究

目的探索天龙粉经过4种蛋白酶酶解后对肺腺癌A549细胞株的抑制作用,筛选出最佳用酶。方法天龙粉分别经过胰蛋白酶、胃蛋白酶、胰凝乳蛋白酶和弹性蛋白酶酶解,采用茚三酮比色法测定蛋白质的水解度,同时以天龙粉水提物作为对照组,用甲基噻唑蓝(MTT)法测定4种酶水解物对肺腺癌A549的抑制率。结果对照组不同药物浓度对肺腺癌A549细胞无明显抑制作用;弹性蛋白酶、胰蛋白酶对天龙粉的水解度分别为77.50%、28.02%,给药组不同药物浓度对肺腺癌A549细胞的抑制率明显高于对照组;胰凝乳蛋白酶和胃蛋白酶的水解度分别为34.63%、72.80%,给药组不同药物浓度对肺腺癌A549细胞无明显抑制作用。结论天龙粉酶解工艺的最佳用酶是弹性蛋白酶,其次是胰蛋白酶。     

蜈蚣粉酶解工艺最佳用酶的初步筛选

目的分别利用胃蛋白酶、胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶对蜈蚣粉进行酶解并选取最佳用酶来制备抗肿瘤多肽。方法利用4种酶对蜈蚣粉进行酶解,采用MTT体外抗肿瘤实验方法,以蛋白质的水解度及酶解提取液对乳腺癌MCF-7细胞的抑制率为指标,同时以蜈蚣粉的水溶性蛋白质含量为标准,评价4种酶对蜈蚣粉的酶解效果并筛选出最佳用酶。结果胃蛋白酶对蜈蚣粉的水解度为10.44%;胰蛋白酶对蜈蚣粉的水解度为28.29%,在0.125 mg·m L~(-1)及0.25 mg·m L~(-1)的较小浓度下对乳腺癌MCF-7细胞的抑制率低于对照组,但随浓度的增大抑制率逐渐增大,在0.5~2 mg·m L~(-1)浓度时,胰蛋白酶在4种酶中达到最大抑瘤效果;胰凝乳蛋白酶和弹性蛋白酶的水解度分别为50.45%、44.16%,当浓度>0.5 mg·m L~(-1)时其对乳腺癌MCF-7细胞的抑制率高于对照组,在浓度为0.5mg·m L~(-1)时抑制率分别为2.86%、36.37%,且随浓度增大抑制率增大。结论蜈蚣粉酶解工艺最佳用酶为胰蛋白酶,其次是胰凝乳蛋白酶和弹性蛋白酶,而胃蛋白酶活性较低可以去除。     

鱼腥草注射液的被动皮肤过敏试验及体外过敏试验

目的探索鱼腥草注射液中的过敏原及其致敏机制,并建立药物过敏原筛选的方法。方法分别用鱼腥草注射液、鱼腥草蒸馏液、吐温80、阴性对照物和阳性对照物对BALB/C小鼠、SD大鼠进行异种被动皮肤过敏试验,并对RBL-2H3细胞进行体外致敏试验。检测BALB/C小鼠和BN大鼠抗血清中IgE的含量、被动皮肤过敏反应中大鼠蓝斑直径大小及体外致敏实验中肥大细胞的脱颗粒反应。结果鱼腥草注射液A组、鱼腥草注射液B组、吐温80组、鱼腥草蒸馏液组和阳性对照组的BALB/C小鼠和BN大鼠血清中的IgE水平均显著升高。当BALB/C小鼠抗血清稀释比例为1∶2时,SD大鼠被动皮肤过敏反应均呈阳性;BALB/C小鼠抗血清稀释比例为1∶8时,仅鱼腥草注射液B组和阳性对照组SD大鼠呈阳性;BALB/C小鼠抗血清稀释比例为1∶32时,各组SD大鼠均未见阳性。体外过敏试验结果显示:鱼腥草注射液组、吐温80组、鱼腥草蒸馏液组和阳性对照组的肥大细胞脱颗粒百分率、β-氨基己糖苷酶释放率和上清中组胺释放量,与阴性对照组相比均有显著性增加。结论吐温80为鱼腥草注射液的主要过敏原,其致敏机制是由IgE介导的,经过吐温80的首次致敏和二次激发出现有细胞因子及炎性介质参与的机体异常的免疫调节,引起一系列过敏症状。鱼腥草蒸馏液中含有其他引起过敏反应的过敏原。体外过敏试验结果与动物试验结果一致,体外致敏模型可用于药物过敏原的筛选。     

卡介菌多糖核酸的抗炎和抗过敏作用

目的　研究卡介菌多糖核酸 (BCG PSN)的抗炎、抗过敏作用。方法　观察对磷酸组胺所致豚鼠皮肤瘙痒的影响 ;采用二甲苯所致小鼠耳廓肿胀及角叉菜胶所致大鼠足跖肿胀实验观察抗炎作用 ;以 2 ,4 二硝基氟苯所致小鼠皮肤迟发型变态反应、大鼠同种被动皮肤过敏反应及小鼠异种被动皮肤过敏反应 ,探讨抗过敏作用。结果　豚鼠隔日肌内注射BCG PSN(0 1,0 2 ,0 4mg·kg-1) 3wk ,对磷酸组胺所致皮肤瘙痒无明显影响。小鼠隔日肌内注射BCG PSN(0 15 ,0 30 ,0 6 0mg·kg-1) 3wk ,可剂量依赖性地抑制二甲苯所致耳廓肿胀和耳异种被动皮肤过敏反应 ,高剂量时也可显著抑制 2 ,4 二硝基氟苯诱导的迟发型变态反应。大鼠隔日肌内注射BCG PSN(0 1,0 2 ,0 4mg·kg-1) 3wk ,可剂量依赖性地抑制角叉菜胶所致足跖肿胀和同种被动皮肤过敏反应。收稿日期 :2 0 0 3 -12 -2 4,修回日期 :2 0 0 4-0 2 -12作者简介 :刘桂珍 ( 1965 -) ,女 ,主管实验师 ,研究方向 :心血管药理学。Tel:0 73 1 2 3 5 5 0 77;胡长平 ( 1969-) ,男 ,博士 ,副教授 ,通迅作者 ,研究方向 :心血管药理学 ,Tel:0 73 1 2 3 5 5 0 77,E mail:huchangping2 0 0 1@yahoo .com .cn结论　BCG PSN对急性炎症、速发型变态反应和迟发型变态反应具有抑制作用。     

扇贝边酶解物抗氧化作用研究

目的探讨不同蛋白酶酶解扇贝边所得酶解物对羟自由基(·OH)的清除效果及酶解物的体内抗氧化作用。方法通过胰蛋白酶、胃蛋白酶、木瓜蛋白酶、复合风味酶、枯草杆菌蛋白酶的扇贝边酶解物对·OH的清除活性筛选酶种,并优化其酶解条件。用酶解物灌胃小鼠,测定小鼠血液中的谷胱甘肽过氧化物酶(GSH-PX)活力、超氧化物歧化酶(SOD)活力和肝脏中的丙二醛(MDA)含量。结果枯草杆菌蛋白酶和木瓜蛋白酶酶解扇贝边所得酶解物具有较好的·OH清除效果,其清除率分别为84 .37%和79.93% ,这两种酶解物均可提高小鼠血液中GSH-PX活力和SOD活力,并降低小鼠肝脏中的MDA含量。结论扇贝边酶解物具有不同程度的体内抗氧化活性作用     

酶解条件控制对透明质酸工业化提取的影响

本研究采用粗胰酶水解鸡冠从中提取透明质酸.结果显示,分批次加入酶、控制酶解温度45℃、pH 8～9,酶解效果较好.透明质酸收率0.4%～0.6%.平均分子量4.0×10~5～1.0×10~6,蛋白质含量5%～15%.     

黄芩苷的致过敏作用机制

目的：黄芩苷是双黄连粉针剂的主要有效成分,有报道称双黄连注射液过敏反应与黄芩苷有关,本研究旨在阐明黄芩苷的致过敏作用机制,探索一种能够避免多种中药制剂引起过敏的方法。方法：本文采用活性酯法制备黄芩苷全抗原,建立黄芩苷致豚鼠过敏性变态反应的动物模型;通过被动皮肤过敏反应试验（PCA）、豚鼠肥大细胞脱颗粒、离体回肠收缩试验分析黄芩苷致过敏作用的变应原性;采用酶联免疫吸附试验和放射过敏原吸附试验测定血清中黄芩苷特异性抗体。结果：黄芩苷与牛血清蛋白（BSA）、人血清白蛋白（HSA）耦联成功。致敏豚鼠激发后,迅速出现呼吸困难、小便失禁直到呼吸停止死亡等症状,组织HE染色显示豚鼠出现了典型的速发型过敏反应特征。被动皮肤过敏反应试验表明致敏豚鼠体内同时存在黄芩苷特异性IgE和IgG型抗体,IgE和IgG两抗体之间无相关性。19只致敏豚鼠特异性IgG抗体阳性率为84％,特异性IgG水平与特异性IgG抗体效价之间有相关性（P〈0．05）。     

牛樟芝水提取物抗过敏作用及其机制研究

目的 研究牛樟芝水提物的抗过敏作用并初步探讨其作用机制。方法 采用大鼠被动皮肤过敏模型、小鼠迟发型超敏模型、小鼠全身皮肤瘙痒模型、小鼠腹腔毛细血管通透性模型,观察牛樟芝水提物在体抗过敏作用。采用体外培养的RBL-2H3肥大细胞,观察牛樟芝水提物对细胞增殖及凋亡的影响,初步探讨其抗过敏反应的作用机制。结果 体内试验中,牛樟芝水提物显著降低大鼠被动皮肤过敏反应,明显降低小鼠耳肿胀度、胸腺指数及脾指数,提高右旋糖酐所致皮肤瘙痒阈值,减少小鼠瘙痒次数,抑制组胺所致血管通透性上升;体外试验中,其剂量相关性的抑制RBL-2H3细胞增殖,促进细胞凋亡。结论 牛樟芝水提取物有一定的抗过敏作用,其机制与促进RBL-2H3细胞凋亡有关。     

卡介菌多糖-核酸抗Ⅰ型过敏反应作用及机制探讨

目的研究卡介菌多糖-核酸(BCG-PSN)的抗I型过敏反应作用,并探讨其可能的作用机制。方法建立卵白蛋白致敏大鼠血清引起的大鼠同种被动皮肤过敏反应(PCA)模型、磷酸组胺诱导的大鼠皮肤血管通透性升高模型、低分子右旋糖酐诱导的小鼠全身皮肤瘙痒模型,探讨BCG-PSN在体抗I型过敏反应作用;卵白蛋白致敏大鼠腹腔肥大细胞主动脱颗粒试验法探讨BCG-PSN离体抗I型过敏反应作用;ELISA法检测外周血中IL-4、IFN-γ水平和IgE抗体滴度,探讨BCG-PSN抗I型过敏反应可能的作用机制。结果大鼠隔日肌内注射BCG-PSN(0.026,0.077,0.232mg·kg-1)3wk、小鼠隔日肌内注射BCG-PSN(0.025,0.075,0.225mg·kg-1)3wk,均可剂量依赖性抑制卵白蛋白诱导的大鼠PCA,缓解低分子右旋糖酐诱导的小鼠全身皮肤瘙痒,对磷酸组胺诱导的大鼠皮肤血管通透性增高无影响;BCG-PSN(10-6mg·mL-1～10-3mg·mL-1)可浓度依赖性抑制卵白蛋白诱导的肥大细胞脱颗粒反应,最大抑制率为69.12%,抑制肥大细胞脱颗粒反应的半数抑制浓度(IC50)为7×10-5mg·mL-1;与生理盐水对照组比较,BCG-PSN三个剂量组1∶2、1∶8、1∶32三个血清稀释度蓝斑平均OD值均明显降低(P<0.05),外周血中IL-4水平明显降低(P<0.05),IFN-γ水平明显升高(P<0.05)。结论BCG-PSN可剂量依赖性抑制I型过敏反应作用,其抗I型过敏反应的可能机制与抑制血清IgE的生成,减少外周血中IL-4水平,增加外周血中IFN-γ水平有关。     

RP-HPLC法分析重组人胰高血糖素类多肽-1（7-36）肽图谱

目的:建立重组人胰高血糖素类多肽-1(7-36)[rhGLP-1(7-36)]胰蛋白酶或V_8蛋白酶肽图分析方法。方法:酶解缓冲液分别为1%碳酸氢铵缓冲液(pH7.8)和磷酸盐缓冲液(pH7.8),酶与底物的比例为1∶10,酶解时间分别为3h和6h。采用ODS柱(250mm×4.6 mm,5μm),以三氟醋酸-水(0.1∶100)为流动相A,三氟醋酸-水-乙腈(0.1∶5∶95)为流动相B。胰蛋白酶洗脱梯度:0-30min,流动相A与B的比例为80:20→55:45。V_8蛋白酶洗脱梯度:0-8min,流动相A与B的比例为99:1→95:5;8-55 min,比例为95:5→48:52。流速:1.0mL·min~(-1),检测波长214nm,柱温:37℃。结果:无论用胰蛋白酶或V_8蛋白酶酶解rhGLP-1(7-36),控制好酶与底物的比例及酶解的时间,都可以得到重现性好的稳定的肽图。结论:本方法可用于重组人胰高血糖素类多肽-1(7-36)胰蛋白酶和V_8蛋白酶肽图分析,简便,可行。     

Participation of histamine H1 and H2 receptors in passive cutaneous anaphylaxis-induced scratching behavior in ICR mice

Scratching behavior associated with passive cutaneous anaphylaxis was examined and compared to that induced by compound 48/80 or histamine in ICR mice. Elicitation of passive cutaneous anaphylaxis, and intradermal injections of compound 48/80, histamine or serotonin induced both scratching behavior and vascular permeability increase in ICR mice. In mast cell-deficient WBB6F1-W/Wv mice, although histamine induced scratching behavior and vascular permeability increase, passive cutaneous anaphylaxis was not observed. Cetirizine and terfenadine significantly inhibited the scratching behavior and vascular permeability increase caused by passive cutaneous anaphylaxis, compound 48/80 and histamine. The histamine H1 receptor antagonists inhibited the vascular permeability increase almost completely, whereas they failed to abolish the scratching behavior. Famotidine and ranitidine significantly inhibited the scratching behavior caused by histamine. The histamine H2 receptor antagonists did not affect the vascular permeability increase caused by histamine. The combination of cetirizine and ranitidine abolished the histamine-induced scratching behavior. The combination, however, failed to potentiate the inhibition of passive cutaneous anaphylaxis-induced scratching behavior significantly. The results indicated that histamine induces scratching behavior in ICR mice through both histamine H1 and H2 receptors, and that histamine plays a major role in passive cutaneous anaphylaxis-induced scratching behavior. Histamine might also play an important role in compound 48/80-induced scratching behavior.     

Topical application of luteolin inhibits scratching behavior associated with allergic cutaneous reaction in mice

The present study is aimed at evaluating the effects of luteolin on the scratching behavior associated with an allergic cutaneous reaction in mice. Elicitation of passive cutaneous anaphylaxis, and intradermal injections of compound 48/80, histamine or serotonin induced scratching behavior in ICR mice. Models of irritant contact dermatitis and allergic contact dermatitis were prepared by the topical application of 2,4-dinitrochlorobenzene (DNCB) on the ears of mice. Topical application of luteolin at concentrations of 20 and 100 mug/site significantly inhibited the number of scratching incidents associated with passive cutaneous anaphylaxis, and a similar tendency was also observed in histamine-, serotonin- and compound 48/80-evoked cutaneous reactions. The vascular permeability increase induced by passive cutaneous anaphylaxis or histamine injection was also significantly reduced by luteolin. Luteolin showed a potent inhibition on the ear thickness increase in models of irritant contact dermatitis and allergic contact dermatitis. In conclusion, luteolin significantly inhibited the scratching behavior associated with allergic cutaneous anaphylaxis. Its effects against pruritus are mainly attributed to its inhibition of mediator release from activated mast cells and direct antagonist effects on the released mediators which may act as local pruritogens.     

Anti-inflammatory drug actions on allergic responses in guinea-pig skin.

Five non-steroidal anti-inflammatory drugs (indomethacin, naproxen, meclofenamic acid, feprazone and phenylbutazone: NSAIDs) and three glucocorticosteroids (dexamethasone, hydrocortisone and prednisolone) have been tested as local inhibitors of increased vascular permeability in guinea-pig skin. Lesions were induced by histamine or by antigen to evoke type I (passive cutaneous anaphylaxis), type III (reverse passive Arthus) and type IV (delayed hypersensitivity) allergic reactions. NSAIDs and glucocorticosteroids caused either weak, inconsistent inhibition or slight, high-dose inhibition of the response to histamine. None of the drugs tested showed significant inhibition of the type IV response. The NSAIDs caused dose-related inhibition of both type I and type III responses whereas glucocorticosteroids were ineffective. Maximum inhibition with the NSAIDs was never greater than 50--60% Feprazone, meclofenamic acid and indomethacin were the most potent inhibitors of histamine, PCA and Arthus responses respectively. The possible significance of the effects of these anti-inflammatory agents on vascular permeability is discussed.     

Anti-inflammatory activity of the dry distillation tar of delipidated soybean (Glyteer) (3). Effects on type I-type IV allergic reaction

Effects of Glyteer (GL, 5%) on Type I-Type IV allergic reactions were investigated by its topical application to rats and mice, and the effects were compared with those of betamethasone 17-valerate (BV, 0.12%), indomethacin (ID, 1%), and bufexamac (BM, 5%), which were all prepared with the same ointment base. Type I: GL showed inhibitory effects on the 48 hr homologous passive cutaneous anaphylaxis (PCA) in rats. The inhibitory activity of GL on the PCA had the same potency as that of BV (0.12%). GL also inhibited the degranulation of mast cells induced by PCA. Type II: GL did not exert an inhibitory effect on the reversed cutaneous anaphylaxis (RCA) in rats, but BM, ID and BV had an inhibitory activity on the RCA. Type III: BV markedly inhibited the direct passive Arthus reaction in rats. On the other hand, GL, BM and ID had not an inhibitory activity on it. Type IV: GL (0.2, 1 and 5%) showed a concentration-dependent inhibition on the delayed-type hypersensitivity response induced by oxazolone in mice, and the activity was stronger than those of ID and BM. From these results, it is suggested that GL applied externally possesses a potent effect as an anti-allergic drug on Type I and Type IV allergic reactions.     

Antigenicity and cutaneous toxicity of N-(4′-(9-acridinylamino)-3-methoxyphenyl)-methanesulfonamide (AMSA; NSC-249992) in guinea pigs and rabbits

Dermal rashes appearing in personnel involved in the bulk formulation of the antineoplastic agent AMSA prompted this investigation of the allergenicity and cutaneous irritation potential of the drug in rabbits and guinea pigs. Maximization testing of AMSA in guinea pigs resulted in a 100% skin sensitization rate. A 14-day cutaneous irritation study using a modified Draize test did not show any potential of AMSA to produce skin irritation on intact or abraded skin. AMSA did not induce antibodies in rabbits which could be detected by passive hemagglutination, cutaneous anaphylaxis, Arthus reactivity, or delayed hypersensitivity in rabbits. AMSA also failed to sensitize guinea pigs for systemic anaphylaxis.     

On the nature of the chemical mediators involved in anaphylactic reactions in mice

The effects of mepyramine, promethazine, chlorpromazine and lysergic acid diethylamide have been compared on the capillary permeability changes of the skin, produced by histamine, by 5-hydroxytryptamine and by passive cutaneous anaphylaxis in mice. Promethazine, mepyramine and chlorpromazine can inhibit, in decreasing order of activity, the effect of histamine, whilst lysergic acid diethylamide is inactive. Lysergic acid diethylamide and chlorpromazine are equally potent inhibitors of the action of 5-hydroxytryptamine on the peripheral vascular bed, whilst mepyramine is inactive. Promethazine has intermediate activity. Passive cutaneous anaphylaxis is strongly inhibited by chlorpromazine and by promethazine. Mepyramine and lysergic acid diethylamide, each injected alone, affect only weakly the anaphylactic reaction. However, passive cutaneous anaphylaxis is almost completely abolished by the simultaneous injection of the two last antagonists. It is suggested that the anaphylactic reaction in mice is the result of simultaneous release of both mediators, histamine and 5-hydroxytryptamine, each of them strengthening the effect of the other.     

Induction of IgE antibody responses by protein allergens: inter-laboratory comparisons.

There is a growing interest in the development of methods for the evaluation of the allergenic potential of novel proteins. One approach is the measurement of specific IgE antibody production stimulated by systemic (intraperitoneal; i.p.) exposure of BALB/c strain mice. In the current investigations, inter-laboratory comparisons have been performed of IgE antibody production induced in mice by food proteins of differing sensitizing potential. Female BALB/c strain mice (n=5) were exposed to 0.1% peanut agglutinin, an allergenic constituent of peanuts, to 2% ovalbumin (OVA), a major allergenic constituent of hens' egg, or to a protein considered to lack significant allergenicity, potato agglutinin (5%). Specific IgE antibody was measured by homologous passive cutaneous anaphylaxis assay and IgG and IgG1 antibody production was analysed by enzyme-linked immunosorbent assay (ELISA). Two independent experiments were conducted in each laboratory, but with all serological analyses conducted in one of the laboratories. Each of the proteins induced vigorous IgG and IgG1 antibody responses, with no statistically significant differences in titres recorded between laboratories. Furthermore, OVA and potato agglutinin induced responses of equivalent immunogenicity with respect to both IgG and IgG1 antibody titres. Administration of peanut agglutinin and OVA each stimulated marked IgE antibody responses in every experiment. In the two laboratories, titres ranged from 1:32 and 1:64 for peanut agglutinin, and from 1:8 and 1:32 for OVA. In contrast, exposure to potato agglutinin failed to induce vigorous IgE production, with no detectable IgE (negative with neat serum), or titres of 1 (positive with neat serum only) recorded. These data demonstrate that the induction of IgE antibody by food proteins of differing allergenic potential is a relatively robust phenomenon and transferable between laboratories. Furthermore, these results provide additional evidence that the measurement of antibody (IgE) responses in BALB/c mice may allow discrimination between allergens and those materials that apparently lack allergenicity.     

杨梅素对小鼠被动皮肤过敏反应的影响

目的研究杨梅素的抗过敏作用。方法采用小鼠同种、异种被动皮肤过敏反应(PCA)、右旋糖酐致小鼠全身瘙痒反应和2,4-二硝基氯苯(DNCB)所致小鼠耳廓皮肤迟发型超敏反应(DTH)实验,探讨杨梅素的抗过敏作用。结果杨梅素显著抑制小鼠同种、异种被动皮肤过敏反应和右旋糖酐引起的小鼠瘙痒反应,并能抑制DNCB诱导的DTH。结论杨梅素具有抗过敏作用,机制可能与抑制Ⅰ、Ⅳ型变态反应有关。     

Anti-inflammatory properties of zinc protoporphyrin disodium (Zn-PP-2Na).

Anti-inflammatory properties of zinc protoporphyrin disodium (Zn-PP-2Na) were studied. Zn-PP-2Na exhibits anti-allergic action against type III and IV reactions (passive Arthus reaction in rats and tuberculin-induced footpad reaction in mice), but does not affect type I and II reactions (homologous passive cutaneous anaphylaxis in mice and reversed cutaneous anaphylaxis in rats). Zn-PP-2Na also clearly inhibits type II collagen-induced arthritis in mice. The agent inhibits general arthritis symptoms, anti-type-II collagen antibody production and type II collagen-induced delayed type hypersensitivity (DTH) in arthritic mice. Zn-PP-2Na, however, did not affect carrageenin-induced paw edema and histamine- and serotonin-induced skin reactions in rats. Zn-PP-2Na inhibits IL-1-induced mouse lymphocyte proliferation, but does not affect PMA-induced O2- generation from guinea-pig neutrophil. These results indicate that Zn-PP-2Na inhibits type II collagen-induced arthritis in mice due to the antagonism of IL-1 activity and the inhibition of DTH against type II collagen.