CN107875373A - 一种抗真菌人工合成多肽喷雾组合及其制备方法 - Google Patents
一种抗真菌人工合成多肽喷雾组合及其制备方法 Download PDFInfo
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Abstract
一种抗真菌人工合成多肽喷雾组合,包括抗真菌人工合成多肽液和喷雾基液;所述抗真菌人工合成多肽液包括:抗真菌多肽合成液、山梨糖醇、单聚合度完全脱乙酰壳寡糖、去离子水;所述喷雾基液包括:黄秋葵多糖提取物、甘油、抗性糊精、维生素E、乳酸、水杨酸、L‑半胱氨酸、N‑乙酰半胱氨酸、赖氨酸、库拉索芦荟、谷胱甘肽、去离子水。该喷雾组合由抗真菌人工合成多肽液和喷雾基液经真空沸腾混合、低温均质、灌装而成。本发明具有广谱抗真菌效果,可以用于治疗真菌感染和皮肤疾病,具有无毒副作用、无耐药性、价格低廉等优点,具有重要的临床应用价值。
Description
技术领域
本发明涉及生物合成领域,具体说是一种抗菌喷雾组合的制备技术,更具体地说是涉及一种抗真菌人工合成多肽喷雾组合及其制备方法。
背景技术
真菌感染,轻者仅累及皮肤黏膜,重者可致某个器官侵袭性感染,如口腔炎、阴道炎、血液感染和深部组织感染。我国台湾地区的调查报告显示,仅深部念珠菌感染30 天内死亡率为 36.7%。Hoffmann调查研究了巴西130名深部真菌感染患者,侵袭性真菌类感染病的病死率可高达 49.2%,病人的平均住院时间为 53.5 天,给患者带来极大的痛苦和经济负担。浅部皮肤真菌类病的治疗一般以外用药为主,如特比萘芬、克霉唑、萘替芬、布替萘芬、联苯苄唑、咪康唑、酮康唑、制霉菌素、舍他霉素等霜剂和软膏和两性霉素 B 泡腾片等。深部真菌类感染较难治愈,目前用于治疗深部真菌类感染的药物有两性霉素B、伊曲康唑、氟康唑、制霉菌素、卡泊芬净等。然而随着深部真菌类感染的治疗,出现耐药性甚至多重耐药性。世界卫生组织认为耐药菌是威胁人类健康的“三大元首”之一,因此探索新型抗生素迫在眉睫,因此开发一种新型的抗真菌药物具有重要意义。
抗菌肽是一类具有抗菌活性肽的简称,一般含有10 到50个氨基酸残基不等,一般带有2 到7个正电荷,有两亲结构,具有抗细菌、真菌、病毒和抗肿瘤活性。抗菌肽是生物防御系统中产生的活性物质,是组成生物先天免疫的重要部分。1972年,瑞典科学家首次分离得到抗菌肽天蚕素(Cecropin)。40多年以来,人们从细菌、真菌、昆虫、两栖类动物、哺乳动物等不同的生物体内发现大量不同种类的抗菌肽。截至到2017年12月,抗菌肽数据库(APD,http://aps.unmc.edu/AP/main.php)已收录多达2927种抗菌肽。
与传统抗生素相比,该人工合成抗真菌多肽Ala-Arg-Ile-Leu-Ser-Ile-Leu-Arg-His-Gln-Gln具有很多优势,该抗菌肽分子量小,仅由11个氨基酸组成;抗菌谱广,对念珠菌类真菌具有广谱抗菌活性,对细菌和癌细胞都有抑制活性;抑菌作用强,特别是对临床上极为难治的白色念珠菌具有较高的抑菌活性, MIC90=9.5μg/mL,与Antimicrobial PeptideDatabase (APD)中已收录的1047种抗真菌多肽相比,其抑菌活性同比占80%以上,无耐药性产生等,有望取代传统抗生素。
目前被广泛接受的观点是抗菌肽通过下述几种不同的作用机制来发挥抗菌作用。破坏细胞膜:破坏细胞膜的完整性,导致细胞内溶物大量外渗,最终导致微生物死亡,从而发挥抗菌活性。对细胞核染色体的影响:部分抗菌肽可与微生物核酸结合,抑制DNA复制,影响细胞核染色体的正常功能。抑制细胞壁的合成。
该人工合成抗真菌多肽有着新颖的作用机制,该人工合成抗真菌多肽是一种非破膜的小肽,虽然其影响细胞膜的稳定性,但对细胞膜结构完整性没有影响。该人工合成抗真菌多肽通过非跨膜转用机制进入真菌细胞内,直接作用于细胞内部结构引起一系列信号通路转导。例如:该人工合成抗真菌多肽引起线粒体膜电位降低,从而使线粒体膜通透性增加,致使线粒体膜内溶物从线粒体内渗出到胞质中,电子传递受损。其次,该人工合成抗真菌多肽打破了细胞内Ga2+离子平衡,甚至可引真菌发生凋亡现象。
该抗菌肽因为作用机理特殊,微生物对其不易产生耐药性,抗真菌具有广谱性,合成价格低廉。为此,我们提出一种抗真菌人工合成多肽喷雾组合及其制备方法。
发明内容
本发明的目的在于提供一种抗真菌人工合成多肽喷雾组合及其制备方法。具体方案为:一种抗真菌人工合成多肽喷雾组合,包括抗真菌人工合成多肽液和喷雾基液;其特征在于抗真菌人工合成多肽液成分按重量比为:抗真菌多肽合成液0.2~0.4份、山梨糖醇20~40份、单聚合度完全脱乙酰壳寡糖50~80份、去离子水400份;所述喷雾基液成分按质量比:黄秋葵多糖提取物1~3份、甘油5~10份、抗性糊精1~10份、维生素E 2~10份、乳酸0.1~1份、水杨酸0.1~1份、L-半胱氨酸1~3份、N-乙酰半胱氨酸1~3份、赖氨酸0.5~1份、库拉索芦荟0.5~1份、谷胱甘肽0.5~1份、去离子水450~480份;真菌人工合成多肽液和喷雾基液的配比为质量比500:500。
一种抗真菌人工合成多肽喷雾组合,其特征在于:所述的抗真菌人工合成多肽的序列为Ala-Arg-Ile-Leu-Ser-Ile-Leu-Arg-His-Gln-Gln。
一种抗真菌人工合成多肽喷雾组合,其特征在于:所述的单聚合度完全脱乙酰壳寡糖为壳二糖、壳四糖、壳六糖的任意一种。
一种抗真菌人工合成多肽喷雾组合,其特征在于:所述的抗性糊精仅限于土豆抗性糊精或小麦抗性糊精中的任意一种。
本发明还公开一种抗真菌人工合成多肽喷雾组合的制备方法,其特征是以抗真菌人工合成多肽为功能原料,以山梨糖醇、单聚合度完全脱乙酰壳寡糖、黄秋葵多糖提取物、甘油、抗性糊精、维生素E、乳酸、水杨酸、L-半胱氨酸、N-乙酰半胱氨酸、赖氨酸、库拉索芦荟、谷胱甘肽、去离子水为辅助原料,原料中各组分均以重量计,包括以下工艺步骤:
(1)将抗真菌多肽合成液0.2~0.4份、山梨糖醇20~40份、单聚合度完全脱乙酰壳寡糖50~80份溶于400份去离子水中,置于真空釜中,真空度为300 Pa,温度控制在42-54℃,沸腾混合30-60s,控制水分流失,制成抗真菌人工合成多肽液12-16℃保存备用;
(2)将黄秋葵多糖提取物1~3份、甘油5~10份、抗性糊精1~10份、维生素E 2~10份、乳酸0.1~1份、水杨酸0.1~1份、L-半胱氨酸1~3份、N-乙酰半胱氨酸1~3份、赖氨酸0.5~1份、库拉索芦荟0.5~1份、谷胱甘肽0.5~1份溶于450~480份去离子水中,置于真空釜中,真空度为300 Pa,温度控制在42-54℃,沸腾混合30-60s,控制水分流失,制成喷雾基液12-16℃保存备用;
(3)将制成抗真菌人工合成多肽液和喷雾基液按质量比500:500加入到均质机中,补充氮气流速为330L/h,均质转速400~600r/min,均质时间4-6min,同时控制均质体系温度12-16℃;
(4)将均质后的抗真菌人工合成多肽喷雾组合灌装至喷雾瓶中,充氮气30s,流速300L/h,除去喷雾瓶中空气,12-16℃保存备用,即可得成品。
本发明的特点:(1)本发明所使用的抗真菌多肽,为人工合成多肽,价格低廉、无污染、无致病源、无过敏源、抗菌广谱,无耐药性等优点。另外该抗真菌多肽抗菌机制清晰,婴幼儿或孕妇使用安全可靠。
(2)本发明在科学理论和实践指导下精选配方,科学配伍抗真菌人工合成多肽和多种有益成分,组分经辨证配伍,作用上相互佐助,提出的抗真菌人工合成多肽喷雾组合组成较为简单,功能成分明晰,作用机制明晰。抗真菌人工合成多肽作为抗真菌主要功能成分对皮下真菌感染如足菌肿、孢子丝菌病、着色芽生菌病和系统性真菌感染如念珠菌病、曲霉病、隐球菌病、接合菌病和马内菲青霉病具有根治效果;添加维生素E、乳酸、水杨酸、L-半胱氨酸、N-乙酰半胱氨酸、赖氨酸、甘油等辅助功能成分有效防止病患部位瘙痒、起皮,能够加强抗真菌人工合成多肽的治疗效果;添加单聚合度完全脱乙酰壳寡糖、黄秋葵多糖提取物、甘油、抗性糊精、库拉索芦荟、谷胱甘肽等对病患部位具有修复作用,恢复患部皮肤水嫩光滑,提高病患部位的免疫力,防止反复感染。
(3) 在在抗真菌人工合成多肽喷雾组合的制备过程中采用“真空沸腾混合+氮气低温均质+氮气灌装”的工艺,避免空气及氧气对肽的氧化,充分糅合抗真菌人工合成多肽和辅助功能成分的功能活性成分,最大程度的保持抗真菌肽的生物活性,方法简单,操作方便,成本低,而且经济有效,使用方便,具有较好的经济效益和社会效益。
具体实施方式
实施例1
一种抗真菌人工合成多肽喷雾组合,包括抗真菌人工合成多肽液和喷雾基液;其特征在于抗真菌人工合成多肽液成分按重量比为:抗真菌多肽合成液0.2份、山梨糖醇20份、单聚合度完全脱乙酰壳寡糖50份、去离子水450份;所述喷雾基液成分按质量比:黄秋葵多糖提取物1份、甘油5份、抗性糊精1份、维生素E 2份、乳酸0.1份、水杨酸0.1份、L-半胱氨酸1份、N-乙酰半胱氨酸1份、赖氨酸0.5份、库拉索芦荟0.5份、谷胱甘肽0.5份、去离子水450份;真菌人工合成多肽液和喷雾基液的配比为质量比500:500。
所述的抗真菌人工合成多肽的序列为Ala-Arg-Ile-Leu-Ser-Ile-Leu-Arg-His-Gln-Gln。
所述的单聚合度完全脱乙酰壳寡糖为壳二糖。
所述的抗性糊精仅为土豆抗性糊精。
一种抗真菌人工合成多肽喷雾组合的制备方法,原料中各组分均以重量计,包括以下工艺步骤:
(1)将抗真菌多肽合成液0.2份、山梨糖醇20份、单聚合度完全脱乙酰壳寡糖50份溶于450份去离子水中,置于真空釜中,真空度为300 Pa,温度控制在42℃,沸腾混合30s,控制水分流失,制成抗真菌人工合成多肽液12℃保存备用;
(2)将黄秋葵多糖提取物1份、甘油5份、抗性糊精1份、维生素E 2份、乳酸0.1份、水杨酸0.1份、L-半胱氨酸1份、N-乙酰半胱氨酸1份、赖氨酸0.5份、库拉索芦荟0.5份、谷胱甘肽0.5份溶于450份去离子水中,置于真空釜中,真空度为300 Pa,温度控制在42℃,沸腾混合30s,控制水分流失,制成喷雾基液12℃保存备用;
(3)将制成抗真菌人工合成多肽液和喷雾基液按质量比500:500加入到均质机中,补充氮气流速为330L/h,均质转速400r/min,均质时间4min,同时控制均质体系温度12℃;
(4)将均质后的抗真菌人工合成多肽喷雾组合灌装至喷雾瓶中,充氮气30s,流速300L/h,除去喷雾瓶中空气,12℃保存备用,即可得成品。
实施例2
一种抗真菌人工合成多肽喷雾组合,包括抗真菌人工合成多肽液和喷雾基液;其特征在于抗真菌人工合成多肽液成分按重量比为:抗真菌多肽合成液0.3份、山梨糖醇30份、单聚合度完全脱乙酰壳寡糖65份、去离子水465份;所述喷雾基液成分按质量比:黄秋葵多糖提取物2份、甘油8份、抗性糊精5份、维生素E 6份、乳酸0.5份、水杨酸0.5份、L-半胱氨酸2份、N-乙酰半胱氨酸2份、赖氨酸0.75份、库拉索芦荟0.75份、谷胱甘肽0.75份、去离子水465份;真菌人工合成多肽液和喷雾基液的配比为质量比500:500。
所述的抗真菌人工合成多肽的序列为Ala-Arg-Ile-Leu-Ser-Ile-Leu-Arg-His-Gln-Gln;
所述的单聚合度完全脱乙酰壳寡糖为壳四糖。
所述的抗性糊精为小麦抗性糊精。
本发明还公开一种抗真菌人工合成多肽喷雾组合的制备方法,原料中各组分均以重量计,包括以下工艺步骤:
(1)将抗真菌多肽合成液0.3份、山梨糖醇30份、单聚合度完全脱乙酰壳寡糖65份溶于465份去离子水中,置于真空釜中,真空度为300 Pa,温度控制在48℃,沸腾混合45s,控制水分流失,制成抗真菌人工合成多肽液14℃保存备用;
(2)将黄秋葵多糖提取物2份、甘油8份、抗性糊精5份、维生素E 6份、乳酸0.5份、水杨酸0.5份、L-半胱氨酸2份、N-乙酰半胱氨酸2份、赖氨酸0.75份、库拉索芦荟0.75份、谷胱甘肽0.75份溶于465份去离子水中,置于真空釜中,真空度为300 Pa,温度控制在48℃,沸腾混合45s,控制水分流失,制成喷雾基液14℃保存备用;
(3)将制成抗真菌人工合成多肽液和喷雾基液按质量比500:500加入到均质机中,补充氮气流速为330L/h,均质转速500r/min,均质时间5min,同时控制均质体系温度14℃;
(4)将均质后的抗真菌人工合成多肽喷雾组合灌装至喷雾瓶中,充氮气30s,流速300L/h,除去喷雾瓶中空气,14℃保存备用,即可得成品。
实施例3
一种抗真菌人工合成多肽喷雾组合,包括抗真菌人工合成多肽液和喷雾基液;其特征在于抗真菌人工合成多肽液成分按重量比为:抗真菌多肽合成液0.4份、山梨糖醇40份、单聚合度完全脱乙酰壳寡糖80份、去离子水480份;所述喷雾基液成分按质量比:黄秋葵多糖提取物3份、甘油10份、抗性糊精10份、维生素E 10份、乳酸1份、水杨酸1份、L-半胱氨酸3份、N-乙酰半胱氨酸3份、赖氨酸1份、库拉索芦荟1份、谷胱甘肽1份、去离子水480份;真菌人工合成多肽液和喷雾基液的配比为质量比500:500。
所述的抗真菌人工合成多肽的序列为Ala-Arg-Ile-Leu-Ser-Ile-Leu-Arg-His-Gln-Gln。
所述的单聚合度完全脱乙酰壳寡糖为壳六糖。
所述的抗性糊精为土豆抗性糊精。
本发明还公开一种抗真菌人工合成多肽喷雾组合的制备方法,原料中各组分均以重量计,包括以下工艺步骤:
(1)将抗真菌多肽合成液0.4份、山梨糖醇40份、单聚合度完全脱乙酰壳寡糖80份溶于480份去离子水中,置于真空釜中,真空度为300 Pa,温度控制在54℃,沸腾混合60s,控制水分流失,制成抗真菌人工合成多肽液16℃保存备用;
(2)将黄秋葵多糖提取物3份、甘油10份、抗性糊精10份、维生素E 10份、乳酸1份、水杨酸1份、L-半胱氨酸3份、N-乙酰半胱氨酸3份、赖氨酸1份、库拉索芦荟1份、谷胱甘肽1份溶于480份去离子水中,置于真空釜中,真空度为300 Pa,温度控制在54℃,沸腾混合60s,控制水分流失,制成喷雾基液16℃保存备用;
(3)将制成抗真菌人工合成多肽液和喷雾基液按质量比500:500加入到均质机中,补充氮气流速为330L/h,均质转速600r/min,均质时间6min,同时控制均质体系温度16℃;
(4)将均质后的抗真菌人工合成多肽喷雾组合灌装至喷雾瓶中,充氮气30s,流速300L/h,除去喷雾瓶中空气,16℃保存备用,即可得成品。
验证实例:
抗真菌人工合成多肽喷雾组合抗真菌使用效果对比,患者使用实验,观察抗真菌人工合成多肽喷雾组合对真菌感染患者的治疗效果。
测试人数600名,年龄23-50岁,性别各半,患者病程0.5-3年,平均病程1.8年,其中股癣76例、体癣103例、花斑癣45例、甲癣84例、孢子子丝菌病41例、霉菌性阴道炎73例、霉菌性包皮龟头炎80例、霉菌性外阴炎98例。随机分成5组,每组120人,各组两组患者在年龄、病程、症状等方面无明显的差异,无统计学意义(P>0.05),具有可比性。
空白对照组:阳性对照组:对患者外喷生理盐水1.5 ml,平均每天喷雾2次(早晚各一次)。其中,股癣、体癣以及花斑癣的患者用药疗程是2周;手癣、足癣患者用药疗程是4周;孢子丝菌病患者的用药疗程是12周以上;霉菌性阴道炎、霉菌性包皮龟头炎和霉菌性外阴炎患者的治疗疗程是1周;而甲癣患者的用药疗程是8周以上。
阳性对照组:对患者外喷盐酸特比萘芬片1.5 ml,平均每天喷雾2次(早晚各一次)。其中,股癣、体癣以及花斑癣的患者用药疗程是2周;手癣、足癣患者用药疗程是4周;孢子丝菌病患者的用药疗程是12周以上;霉菌性阴道炎、霉菌性包皮龟头炎和霉菌性外阴炎患者的治疗疗程是1周;而甲癣患者的用药疗程是8周以上。
实验组为3组,分别对患者外喷实例一、二、三的抗真菌人工合成多肽喷雾组合1.5ml,平均每天喷雾2次(早晚各一次)。其中,股癣、体癣以及花斑癣的患者用药疗程是2周;手癣、足癣患者用药疗程是4周;孢子丝菌病患者的用药疗程是12周以上;霉菌性阴道炎、霉菌性包皮龟头炎和霉菌性外阴炎患者的治疗疗程是1周;而甲癣患者的用药疗程是8周以上。
试验期间,试验部位停用其他任何药物,受试者分别将受试样品敷于患处,每天两次(早晚各一次),对受试者受试的患处,编号,肉眼观察,按照规定的评估标准分别记录结果。患者治疗的总体有效率包括显效和痊愈。痊愈:患者的全部临床症状都消失,真菌学的检查显示结果为阴性;显效:患者的全部症状都减轻,真菌学的检查显示结果为阴性;有效:患者的全部症状都减轻,真菌学的检查显示结果为阳性;无效:患者的全部症状没有改善,真菌学的检查显示结果为阳性。
表1 各组患者治疗效果的疗效比
通过表1可以看出,抗真菌人工合成多肽喷雾组合实验组的总体有效率为98.33%,阳性对照组的有效率为80%,空白对照组的总有效率为0%,抗真菌人工合成多肽喷雾组合抗真菌的治疗效果优于其他组,比较差异具有统计学意义(P<0.05)。
对本领域的技术人员来说,可根据以上描述的技术方案以及构思,做出其它各种相应的改变,而所有的这些改变都应该属于本发明 权利要求的保护范围之内。
Claims (5)
1.一种抗真菌人工合成多肽喷雾组合,包括抗真菌人工合成多肽液和喷雾基液;其特征在于抗真菌人工合成多肽液成分按重量比为:抗真菌多肽合成液0.2~0.4份、山梨糖醇20~40份、单聚合度完全脱乙酰壳寡糖50~80份、去离子水400份;所述喷雾基液成分按质量比:黄秋葵多糖提取物1~3份、甘油5~10份、抗性糊精1~10份、维生素E 2~10份、乳酸0.1~1份、水杨酸0.1~1份、L-半胱氨酸1~3份、N-乙酰半胱氨酸1~3份、赖氨酸0.5~1份、库拉索芦荟0.5~1份、谷胱甘肽0.5~1份、去离子水450~480份;真菌人工合成多肽液和喷雾基液的配比为质量比500:500。
2.根据权利要求1所述的抗真菌人工合成多肽喷雾组合,其特征在于:所述的抗真菌人工合成多肽的序列为Ala-Arg-Ile-Leu-Ser-Ile-Leu-Arg-His-Gln-Gln。
3.根据权利要求1所述的抗真菌人工合成多肽喷雾组合,其特征在于:所述的单聚合度完全脱乙酰壳寡糖为壳二糖、壳四糖、壳六糖的任意一种。
4.根据权利要求1所述的抗真菌人工合成多肽喷雾组合,其特征在于:所述的抗性糊精仅限于土豆抗性糊精或小麦抗性糊精中的任意一种。
5.一种抗真菌人工合成多肽喷雾组合,其特征是以抗真菌人工合成多肽为功能原料,以山梨糖醇、单聚合度完全脱乙酰壳寡糖、黄秋葵多糖提取物、甘油、抗性糊精、维生素E、乳酸、水杨酸、L-半胱氨酸、N-乙酰半胱氨酸、赖氨酸、库拉索芦荟、谷胱甘肽、去离子水为辅助原料,原料中各组分均以重量计,包括以下工艺步骤:
(1)将抗真菌多肽合成液0.2~0.4份、山梨糖醇20~40份、单聚合度完全脱乙酰壳寡糖50~80份溶于400份去离子水中,置于真空釜中,真空度为300Pa,温度控制在42-54℃,沸腾混合30-60s,控制水分流失,制成抗真菌人工合成多肽液12-16℃保存备用;
(2)将黄秋葵多糖1~3份、甘油5~10份、抗性糊精1~10份、维生素E 2~10份、乳酸0.1~1份、水杨酸0.1~1份、L-半胱氨酸1~3份、N-乙酰半胱氨酸1~3份、赖氨酸0.5~1份、库拉索芦荟0.5~1份、谷胱甘肽0.5~1份溶于450~480份去离子水中,置于真空釜中,真空度为300 Pa,温度控制在42-54℃,沸腾混合30-60s,控制水分流失,制成喷雾基液12-16℃保存备用;
(3)将制成的抗真菌人工合成多肽液和喷雾基液按质量比500:500加入到均质机中,补充氮气流速为330L/h,均质转速400~600r/min,均质时间4-6min,同时控制均质体系温度12-16℃;
(4)将均质后的抗真菌人工合成多肽喷雾组合灌装至喷雾瓶中,充氮气30s,流速300L/h,除去喷雾瓶中空气,12-16℃保存备用,即可得成品。
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