CN113975263B - 一种抑制真菌的组合物及其制备方法和应用 - Google Patents
一种抑制真菌的组合物及其制备方法和应用 Download PDFInfo
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- CN113975263B CN113975263B CN202111222255.9A CN202111222255A CN113975263B CN 113975263 B CN113975263 B CN 113975263B CN 202111222255 A CN202111222255 A CN 202111222255A CN 113975263 B CN113975263 B CN 113975263B
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- selenomethylselenocysteine
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- candida albicans
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Abstract
本发明公开了一种抑制真菌的组合物及其制备方法,属于药剂学领域,具体涉及含L‑硒甲基硒代半胱氨酸的组合物水凝胶制剂的制备方法和应用。L‑硒甲基硒代半胱氨酸对真菌,如白色念珠菌的酵母态及菌丝态都有明显的抑制作用,能够有效针对真菌感染的疾病进行干预和治疗。本发明的L‑硒甲基硒代半胱氨酸可以与常用抗真菌药物以及肾上腺皮质激素类药物联合用药起到更佳的治疗效果。水凝胶是优良的药物载体,并且有良好的生物相容性和滞留性能,能够有效延长药物作用时间,增强药效。本发明的L‑硒甲基硒代半胱氨酸首次被公开为抑制真菌的天然生物活性氨基酸,有望为临床真菌感染疾病及复发性真菌疾病提供新的治疗方案。
Description
技术领域
本发明属于药剂学领域,具体涉及一种含L-硒甲基硒代半胱氨酸的组合物的制备及应用。
背景技术
真菌是一种具真核的、产孢的、无叶绿体的真核生物,包含霉菌、酵母菌、蕈菌以及其他人类所熟知的菌菇类。临床上常见的致病性真菌包括糠秕马拉色菌、毛癣菌、孢子丝菌、白色念珠菌等。糠秕马拉色菌是一种表面感染真菌,多寄居于机体富有皮脂腺的部位,在高温、潮湿、多汗等情况下易侵犯人体皮肤浅表的角质层和毛干,引起慢性、轻微症状或无症状的感染。毛癣菌是一种皮肤癣菌,易侵犯皮肤表面角质化的组织,引起皮肤癣症。孢子丝菌可引起皮下组织感染,经外伤感染侵入皮下,可也经口进入消化道或经呼吸道感染进而引起其他脏器或全身性感染。白色念珠菌是临床上最为常见、致病力最强的一种条件致病性真菌,通常存在于人的口腔、阴道等腔体中及皮肤表面,可通过侵犯这些部位引起皮肤念珠菌病、黏膜念珠菌病以及内脏中枢神经念珠菌病等。当机体免疫力下降或者正常菌群相互制约作用失调时,或者基体因手术或者其他外力受到创伤时,白色念珠菌可以繁殖并引发疾病。另外,由于一些具有免疫抑制作用或激素类药物的使用,真菌由共生菌转化为致病菌的可能性大大增加。目前,复发性念珠菌性疾病日益增加,临床已出现对唑类耐药的念珠菌,治疗难度加大。因此,寻找预防和治疗真菌感染的新治疗策略极为重要。
硒作为生物的必需元素及含硒酶的活性中心,对于机体的体液免疫功能和细胞免疫功能有显著的增强作用。因此,补充适量的硒有助于增强基体的免疫功能。硒能够明显提高吞噬细胞的细胞存活率以及吞噬能力,而缺乏硒时,中性粒细胞的游走能力明显降低。此外,硒元素对于淋巴细胞的增殖、分化具有促进效果,相同的对于T淋巴细胞的细胞毒效应也具有促进效果。硒元素在调节人体免疫方面具有一定的作用,在局部补充时,能够调节预防或治疗部位免疫,对于预防和治疗真菌的感染有重要作用。
本发明首次研究发现,L-硒甲基硒代半胱氨酸在≥36.2 μg/mL时几乎可完全抑制酵母态白色念珠菌的生长,并且对菌丝态白色念珠菌也有较好的抑制作用。目前L-硒甲基硒代半胱氨酸抑制白色念珠菌的机制尚未明确,有待进一步研究。
L-硒甲基硒代半胱氨酸作为可有效抑制真菌、调节免疫功能的化合物,将其制备为预防或治疗真菌感染的制剂或医疗器械非常有临床价值。凝胶剂大多拥有良好的生物相容性、优良的成形性、一定的粘附性,能够较好的与感染部位贴合,包裹感染部位,对于皮肤、口腔、阴道、手术植入物的真菌感染的预防或治疗优势明显。乳膏剂、擦剂、喷雾剂有良好的延展性,使用方便,多应用于皮肤表面疾病,其适用于皮肤表面创伤的白色念珠菌感染。栓剂是阴道直肠给药的首选剂型,而阴道感染真菌的可能性也较大。因此,制备含L-硒甲基硒代半胱氨酸的上述剂型组合物对于临床预防、治疗真菌的感染是非常有意义的。
发明内容
为解决临床现有唑类耐药菌治疗难度大的问题,本发明提供了一种抑制真菌的组合物及其制备方法和应用,为预防和治疗阴道、口腔、皮肤创口、植入医疗器械引起的真菌感染以及复发性真菌疾病提供了新的治疗策略。
优选的,本发明所述的含L-硒甲基硒代半胱氨酸的组合物除了L-硒甲基硒代半胱氨酸为主要起效物质外还可选择性联合其他抗真菌药物使用,包括:抑菌药物硝酸咪康唑、益康唑、酮康唑、氟康唑、伊曲康唑、伏立康唑、咪康唑、泊沙康唑、米卡芬净、卡泊芬净、两性霉素B、特比萘芬中的一种或几种;肾上腺皮质激素类药物地塞米松、氯倍他索、可的松、氢化可的松、氟轻松中的一种或几种。
优选的,本发明所述的含L-硒甲基硒代半胱氨酸的组合物还包括药剂学上可接受的药用辅料,包括凝胶基质、黏度调节剂、防腐剂、表面活性剂、助悬剂、抗氧剂、稳定剂、渗透压调节剂和保湿剂中的一种或几种。
优选的,本发明所述的含L-硒甲基硒代半胱氨酸的组合物中的凝胶基质为泊洛沙姆407和泊洛沙姆188,其中泊洛沙姆407含量为5-45%(W/W),泊洛沙姆188含量为0-20%(W/W)。
优选的,本发明所述的含L-硒甲基硒代半胱氨酸的组合物中的渗透压调节剂和保湿剂为甘油,其含量为0-350 g/L。
含L-硒甲基硒代半胱氨酸的组合物的制备方法包括以下步骤: L-硒甲基硒代半胱氨酸加入纯化水中,搅拌均匀,加入甘油至溶液中,搅拌均匀,将泊洛沙姆407和泊洛沙姆188加入溶液中,在4-20℃下搅拌6-24 h使其充分溶胀,辐照灭菌。
另外,本发明公开了上述的抑制真菌的组合物用于预防或治疗皮肤创口、阴道、口腔的真菌感染以及复发性真菌疾病中的应用。
本发明还公开了上述抑制真菌的组合物为凝胶剂,但不局限于凝胶剂,还包括栓剂、乳膏剂、洗剂、擦剂、涂膜剂、喷雾剂等剂型。
本发明产生的有益效果是:本发明的抑制真菌的组合物对于酵母态白色念珠菌具有较强的抑制作用, 24 h时L-硒甲基硒代半胱氨酸(≥36.2 μg/mL)对白色念珠菌的抑菌率能够达到90%以上。
本发明的抑制真菌的组合物对于菌丝态白色念珠菌具有较强的抑制作用,镜检表明无论在液体培养基或是固体培养基,L-硒甲基硒代半胱氨酸都能够抑制白色念珠菌菌丝的生长,主要体现为 L-硒甲基硒代半胱氨酸处理的白色念珠菌菌丝数量大幅减少,菌丝长度明显缩短。
本发明的抑制真菌的组合物的凝胶剂能够对白色念珠菌性阴道炎起到较好的干预作用,动物试验结果表明L-硒甲基硒代半胱氨酸凝胶剂能够有效减少白色念珠菌并大幅度减轻大鼠阴道粘膜损伤并显著抑制阴道上皮TNF-α、IL-1α、IL-1β等炎症因子的水平。
附图说明
图1. L-硒甲基硒代半胱氨酸对酵母态白色念珠菌的抑制作用(A:与L-SeMC孵育24 h后白色念珠菌的细胞活力;(B)不同浓度L-SeMC孵育下白色念珠菌的生长曲线;(C)CFU微量稀释法测定白色念珠菌的存活率)。
图2. L-硒甲基硒代半胱氨酸对假菌丝态白色念珠菌的抑制作用(A:L-SeMC在液体培养基中孵育24小时后白色念珠菌的生长情况;B:L-SeMC在液体培养基中孵育24小时后白色念珠菌的显微形态观察;C:L-SeMC在固体培养基中孵育不同时间后白色念珠菌的显微形态观察。箭头表示白色念珠菌出芽)。
图3. 与L-硒甲基硒代半胱氨酸孵育24 h后白色念珠菌菌丝的生长情况(A:生长对照组;B:36.2 μg/mL组; C:72.4 μg/mL组; D:144.8 μg/mL组) 。
图4. L-硒甲基硒代半胱氨酸和L-硒甲基硒代半胱氨酸水凝胶的阴道安全性评价。
图5. SD雌性大鼠各个生理时期阴道分泌物涂片(a:发情前期未角质化的上皮细胞较多,呈现圆或椭圆状有核细胞;b:发情期角质化的上皮细胞较多,呈不规则状无核细胞;c:发情后期角质化的上皮细胞、未角质化的上皮细胞、白细胞各占约1/3;d:发情间期绝大多数为白细胞,呈圆形较小)。
图6. L-硒甲基硒代半胱氨酸水凝胶对大鼠阴道分泌物数量的影响 (A:固体培养基;B:液体培养基) 。
图7. L-硒甲基硒代半胱氨酸水凝胶对大鼠阴道切片的组织病理学分析;
图8. L-硒甲基硒代半胱氨酸水凝胶对白色念珠菌性阴道炎的抑制效果的免疫组化分析;
图9. L-硒甲基硒代半胱氨酸水凝胶抑制骨科植入物白色念珠菌感染的扫描电镜分析(A:假体组;B:造模组;C:干预组)。
图10. L-硒甲基硒代半胱氨酸水凝胶抑制骨科植入物白色念珠菌感染的病理切片分析 (A:正常组;B:假体组;C:造模组;D:干预组)。
具体实施方式
以下结合实施例进一步描述本发明,但不限制本发明。
实施例1
1. L-硒甲基硒代半胱氨酸对酵母态白色念珠菌的抑制作用
菌株的选择:本实验的菌株为白色念珠菌SC5314,使用的白色念珠菌均为3-5代。
白色念珠菌的培养:试验器具灭菌消毒,超净工作台提前紫外灭菌30 min。取5mLSDB培养基于一次性无菌摇菌管中,加入5 μL链霉素,用一次性无菌接种环从SDA培养皿勾取一个白色念珠菌菌落,伸入摇菌管培养基中,摇晃3-5次。将摇菌管置于恒温振荡培养箱中,30℃,200 rpm培养过夜。
L-硒甲基硒代半胱氨酸溶液的配置:称量L-硒甲基硒代半胱氨酸,用超纯水配置成14.48 mg/mL的母液,使用0.22μm微孔滤膜过滤除菌,将母液用SDB培养基稀释为289.6、144.8、72.4、36.2、18.1、9.05、4.52、2.26 μg/mL,备用。
L-硒甲基硒代半胱氨酸抑制酵母态白色念珠菌试验:
试验器具灭菌消毒,超净工作台提前紫外灭菌30 min。取培养的白色念珠菌1 mL于试管中,逐渐加入生理盐水稀释,并于0.5号麦氏比浊管比浊,得到白色念珠菌浓度。将白色念珠菌用SDB培养基稀释至约2*105 cfu/mL,备用。取96孔板6块,于第1列、第12列、第1行、第8行加入200 μL无菌水,第2-10列加入100 μL稀释好的白色念珠菌菌液,第10行加入100μL SDB培养基,第11列加入200 μL SDB培养基,第2-9列分别加入289.6、144.8、72.4、36.2、18.1、9.05、4.525、2.2625 μg/mL的L-硒甲基硒代半胱氨酸 SDB溶液。其中,第2-9列为干预组、第10列为对照组、第11列祛除培养基干扰。
将96孔板置于37℃培养约24 h,然后使用排枪于给药、生长对照、空白对照各孔加入MTT溶液50μL,37℃孵育4 h,取出,吸弃上层废液,加入150 mL DMSO,吹打均匀。使用酶标仪测定490 nm波长处的吸光度,并计算L-硒甲基硒代半胱氨酸的抑菌率。
结果:如图1所示,当L-硒甲基硒代半胱氨酸浓度大于等于36.2 μg/mL时可有效抑制白色念珠菌的生长。
2. L-硒甲基硒代半胱氨酸对菌丝态白色念珠菌的抑制作用
白色念珠菌的培养:试验器具灭菌消毒,超净工作台提前紫外灭菌30 min。取5 mLspider液体培养基于一次性无菌摇菌管中,加入5 μL链霉素,用一次性无菌接种环从SDA培养皿勾取一个白色念珠菌菌落,伸入摇菌管培养基中,摇晃3-5次。将摇菌管置于恒温振荡培养箱中,37℃,200 rpm培养过夜。
L-硒甲基硒代半胱氨酸溶液的配置:称量L-硒甲基硒代半胱氨酸用超纯水配置成14.48 mg/mL的母液,使用0.22μm微孔滤膜过滤除菌,备用。
(1)L-硒甲基硒代半胱氨酸抑制菌丝态白色念珠菌试验(spider液体培养基):
试验器具灭菌消毒,超净工作台提前紫外灭菌30 min。称量酵母浸膏2.4 g、甘露醇0.12 g、磷酸氢二钾0.24 g、琼脂3.6 g加入纯化水240 mL,121℃高压蒸汽灭菌15 min,分装到3个灭菌过的玻璃瓶中,约60℃时,分别加入L-硒甲基硒代半胱氨酸母液,使L-硒甲基硒代半胱氨酸含量为144.8、72.4、36.2 μg/mL,倒入培养皿中,待其凝固备用。取1 mL白色念珠菌于试管中,加入生理盐水稀释,并于0.5号麦氏比浊管比浊,得到白色念珠菌浓度。将白色念珠菌用spider培养基稀释至约1*105 cfu/mL,备用。
取玻璃试管12支,分为4组,每组3支。其中,一组加入4 mL稀释的白色念珠菌;一组加入3960 μL稀释的培养基和40 μL L-硒甲基硒代半胱氨酸母液,使其L-硒甲基硒代半胱氨酸含量为144.8 μg/mL;一组加入3980 μL稀释的培养基和20 μL L-硒甲基硒代半胱氨酸母液,使其L-硒甲基硒代半胱氨酸含量为72.4 μg/mL;一组加入3990 μL稀释的培养基和10μL L-硒甲基硒代半胱氨酸母液,使其L-硒甲基硒代半胱氨酸含量为36.2 μg/mL。37℃,200rpm培养约48 h。显微镜下观察。
结果如图2所示,随着L-硒甲基硒代半胱氨酸浓度升高,白色念珠菌假菌丝长度越短,L-硒甲基硒代半胱氨酸有良好的抑制白色念珠菌菌丝生长的作用。
(2)L-硒甲基硒代半胱氨酸抑制菌丝态白色念珠菌试验(spider固体培养基):
试验器具灭菌消毒,超净工作台提前紫外灭菌30 min。取培养的白色念珠菌1mL于试管中,逐渐加入生理盐水稀释,并于0.5号麦氏比浊管比浊,得到白色念珠菌浓度。将白色念珠菌用spider培养基稀释至约1*107、1*106、1*105、1*104 cfu/mL,备用。
取各组别spider固体培养基各3皿。将每个培养皿分为3排4列共12个方形区域,每列设3个区域为三次重复,1~4列方形区域中央滴加的菌液分别滴加1*107、1*106、1*105、1*104 cfu/mL白色念珠菌菌液2 μL。待菌液被充分吸收后,于恒温振荡培养箱中37℃倒置培养48 h。
结果如图3所示,随着L-硒甲基硒代半胱氨酸浓度升高,白色念珠菌菌丝长度越短,白色念珠菌菌落越小,L-硒甲基硒代半胱氨酸有良好的抑制白色念珠菌菌丝生长的作用。
3. L-硒甲基硒代半胱氨酸及其水凝胶对SD雌性大鼠阴道组织的安全性评价
动物分组:将24只SD雌性大鼠(雌性,6-8周)随机分为4组(n = 6/组),即对照组、空白泊洛沙姆凝胶组、L-SeMC溶液组、L-SeMC凝胶组。L-SeMC溶液组和L-SeMC凝胶组中的L-SeMC浓度均为5 mg/mL。未进行任何处理的健康大鼠作为对照。
实验步骤:将大鼠头朝下放置,缓慢注入100 μL凝胶于大鼠阴道内,保持头朝下约1 min,进行凝胶化。连续7天,每2天给予大鼠游离L-SeMC或凝胶。然后对小鼠实施安乐死,收集阴道,用生理盐水冲洗后用4%多聚甲醛溶液固定。然后将样品脱水并包埋在石蜡中。取5 μm厚度的组织切片,用苏木精伊红(H&E)染色,然后用SOPTOP显微镜(Sunny Opticaltechnology co., LTD, China)成像。
结果分析:分析各组处理后的阴道组织切片,评价其阴道内给药后的阴道安全性。如图4所示,空白泊洛沙姆凝胶、L-SeMC溶液、L-SeMC凝胶处理后的阴道组织与健康阴道非常相似,未见病理改变。
4. 含L-硒甲基硒代半胱氨酸的泊洛沙姆凝胶对白色念珠菌引起的阴道炎的抑制作用
泊洛沙姆凝胶的制备:将L-硒甲基硒代半胱氨酸(300 μg/mL)加入纯化水中,搅拌均匀,加入甘油5%,加入泊洛沙姆407 17.6%(W/W)泊洛沙姆188 1.15%(W/W),放入-4℃冷藏12 h,使其溶胀均匀,搅拌均匀。
动物:使用SD雌性大鼠建立白色念珠菌感染的阴道炎模型。
造模及干预:将SD雌性大鼠(约200 g)适应性喂养7天。于第一天和第三天皮下注射50 mg/kg(5 mg/100g)环磷酰胺后,然后于第二天始每两天皮下注射0.1 mL苯甲酸雌二醇溶液一次,直至实验结束。在注射戊酸雌二醇期间,每天用移液器吸取生理盐水20 μL注入大鼠阴道,冲洗两次,吸出,置于载玻片上,盖上载玻片,光学显微镜观察,判断各大鼠的生理周期,各时期状态如图5。大鼠处于发情期可进行下一步操作。其中40只大鼠随机分成四组,正常组取无菌生理盐水溶液100 μL,注入大鼠阴道中,连续接种三天;造模组、空白凝胶组、L-硒甲基硒代半胱氨酸溶液组、L-硒甲基硒代半胱氨酸水凝胶组取100 μL含白色念珠菌的生理盐水溶液(108 cfu/mL),注入大鼠阴道中,连续接种三天。然后于第四天向阴道注入100 μL无菌生理盐水,稀释100倍后取50 μL迅速滴加于SDA平板培养基上,涂布器涂布均匀,置于30℃培养24 h,观察白色念珠菌生长情况,若培养的白色念珠菌大于106 cfu/mL,造模成功。
微生物培养:干预结束后,用100 μL无菌生理盐水冲洗大鼠阴道后迅速将冲洗液滴加于SDA平板培养基上,使用涂布器涂布均匀,置于30℃培养24 h,观察各组的白色念珠菌生长情况。
确定造模成功后,正常组和造模组连续7天向大鼠阴道注入100 μL无菌生理盐水,干预组连续7天向大鼠阴道注射100 μL L-硒甲基硒代半胱氨酸水凝胶。
7天后将大鼠脱颈处死,将阴道部位取出,浸泡于4%多聚甲醛固定。一部分标本用于免疫组化分析,一部分标本经脱钙、脱水、石蜡包埋后,进行HE染色。
各组大鼠阴道分泌物中的白色念珠菌定量分析(图6)显示:在第7天时,正常组和L-硒甲基硒代半胱氨酸水凝胶组几乎没有白色念珠菌生长,而空白凝胶组和L-SeMC溶液组的白色念珠菌数量大于2000 cfu/mL,因此,含L-硒甲基硒代半胱氨酸的水凝胶对白色念珠菌有非常好的抑制效果。
各组大鼠阴道切片HE染色结果(图7)显示:正常组和L-硒甲基硒代半胱氨酸水凝胶组的HE切片相似,几乎没有炎性损伤,而空白凝胶组和L-SeMC组存在明显的炎性损伤。因此,含L-硒甲基硒代半胱氨酸的水凝胶可显著抑制白色念珠菌引起的阴道炎。
各组大鼠阴道切片的免疫组化结果(图8)显示:正常阴道中TNF-α、IL-1α和IL-1β含量极低,而白色念珠菌感染可显著上调阴道TNF-α、IL-1α和IL-1β的表达。空白泊洛沙姆凝胶和L-SeMC溶液 (5 mg/mL)均不能缓解白色念珠菌感染引起的TNF-α、IL-1α和IL-1β的过表达。而L-SeMC水凝胶(5 mg/mL)可显著抑制这些促炎细胞因子的上调。这说明含L-硒甲基硒代半胱氨酸的水凝胶可能是通过调节相关的促炎细胞因子来抑制白色念珠菌引起的阴道炎。
5. L-硒甲基硒代半胱氨酸水凝胶对假体周围白色念珠菌感染的预防作用
水凝胶的制备:将L-硒甲基硒代半胱氨酸(300 μg/mL)加入纯化水中,搅拌均匀,加入5%甘油,搅拌均匀,加入泊洛沙姆407 17.6%(W/W)泊洛沙姆188 1.15%(W/W),放入-4℃冷藏12 h,使其溶胀均匀,搅拌均匀。
动物:将C57小鼠(15-20g)适应性喂养7天后随机分成4组,每组10只,正常组、假体组、造模组、干预组。正常组不进行任何手术干预,假体组只植入镍钛合金丝不接种白色念珠菌菌液,造模组植入镍钛合金丝后接种菌液,干预组植入镍钛合金丝后接种菌液并加入L-硒甲基硒代半胱氨酸凝胶。具体操作为:无菌条件下,在每只小鼠的右膝上开一个切口,用无菌的1 mL注射器针头手工扩髓。股骨远端逆行置入镍钛合金丝(直径~0.5 mm)。用移液器吸取2 μL白念珠菌的生理盐水培养液培养液 (约1×106 cfu/mL)至合金丝结合处。干预组另外注射2 μL含L-硒甲基硒代半胱氨酸的泊洛沙姆凝胶至白念珠菌接种后的合金丝结合处。手术部位用可吸收缝线缝合。两周后,处死小鼠,取各组标本,采用下列方法评价抗感染效果。
扫描电镜分析:每组取植入关节标本,进行SEM分析。
病理切片分析:取各组小鼠关节标本,小心取出移植物后用10%福尔马林固定过夜后,脱钙、脱水、石蜡包埋、切片后进行HE染色。
扫描电镜分析(图9)显示,造模组在镍钛合金丝表面形成成熟而厚的生物膜,而假体组没有附着白色念珠菌,说明在实验条件下成功建立了假体周围感染模型,干预组镍钛合金丝表面无明显的白色念珠菌附着,说明L-硒甲基硒代半胱氨酸的水凝胶能够预防植入假体周围感染白色念珠菌。
各组病理切片(图10)表明:与正常组相比,假体组、干预组的HE切片无明显变化,而造模组骨小梁数量减少,中性粒细胞带形态比例明显增加,说明组织病理学改变是由于白念珠菌定植所致,而非假体植入所致,干预组的HE切片无明显变化,因此证明,含L-硒甲基硒代半胱氨酸的水凝胶能够预防植入假体周围感染白色念珠菌。
实施例2
一种含L-硒甲基硒代半胱氨酸的乳膏:
1.L-硒甲基硒代半胱氨酸的乳膏的处方:
油相:凡士林8份;液体石蜡3份;羊毛脂1份;十八醇3份;单双硬脂酸甘油酯2.5份;S-40酯4.5份;
水相:EDTA二钠0.05份;甘油5份;三乙醇胺1份;卡波姆-980 0.2份;冰片1份;氯倍他索0.05份;L-SeMC 0.01份;超纯水加至100份。
2.L-硒甲基硒代半胱氨酸的乳膏的制备工艺:
1)将卡波姆-980加入纯化水中,于600-800 rpm搅拌,室温溶胀; 2.将油相所有成分加热至70-90℃熔化,于75℃保温;3. 将水相剩余成分(除冰片)加入溶胀好的卡波姆-980中,于600-800 rpm搅拌,60-80℃加封口膜保温;2)待油相完全熔化后,于高速剪切均质机搅拌时将水相倒入油相中,温度维持在70-90℃,高速剪切均质10-30 min,200-30000rpm;5. 边搅拌边降温,转速维持在200-30000 rpm,待降温至40-50℃时,加入冰片,继续搅拌10-30 min;6. 用搅拌器搅拌乳膏,转速200-400 rpm,直至乳膏降至室温。
Claims (10)
1.L-硒甲基硒代半胱氨酸在制备预防或治疗皮肤创口、阴道、口腔的真菌感染及复发性真菌疾病的药物中的应用,所述真菌为白色念珠菌。
2.如权利要求1所述的应用,其特征在于:所述预防或治疗皮肤创口、阴道、口腔的真菌感染及复发性真菌疾病的药物为抑制真菌的组合物,由主药和药用辅料组成,所述主药为L-硒甲基硒代半胱氨酸单独构成,或联合抑菌药物及肾上腺皮质激素类药物构成。
3.如权利要求2所述的应用,其特征在于:所述L-硒甲基硒代半胱氨酸在组合物中含量为0.05-10mg/mL。
4.如权利要求2所述的应用,其特征在于:所述抑菌药物包括硝酸咪康唑、益康唑、酮康唑、氟康唑、伊曲康唑、伏立康唑、咪康唑、泊沙康唑、米卡芬净、卡泊芬净、两性霉素B、特比萘芬中的任一种或几种;所述肾上腺皮质激素类药物包括地塞米松、氯倍他索、可的松、氢化可的松、氟轻松中的任一种或几种。
5.如权利要求2所述的应用,其特征在于:所述药用辅料为药剂学上可接受的药用辅料,包括凝胶基质、黏度调节剂、pH调节剂、防腐剂、表面活性剂、助悬剂、抗氧剂、稳定剂、渗透压调节剂和保湿剂中的任一种或几种。
6.如权利要求5所述的应用,其特征在于:渗透压调节剂和保湿剂为甘油,其在抑制真菌的组合物中的含量为0-350g/L;所述凝胶基质为泊洛沙姆、卡波姆、纤维素类、明胶、海藻酸钠、壳聚糖中的任一种或几种。
7.如权利要求6所述的应用,其特征在于:凝胶基质为泊洛沙姆407和泊洛沙姆188中的一种或两种,其中泊洛沙姆407在抑制真菌的组合物中的含量为5-45wt%,泊洛沙姆188在抑制真菌的组合物中的含量为0-20wt%。
8.如权利要求7所述的应用,其特征在于配方如下:泊洛沙姆407 5-45wt%,泊洛沙姆1880-20wt%,L-硒甲基硒代半胱氨酸0.05-10mg/mL,甘油0-350g/L,余量为水。
9.如权利要求8所述的应用,其特征在于所述抑制真菌的组合物采用如下方法制备得到:L-硒甲基硒代半胱氨酸加入水中,搅拌均匀,加入甘油至溶液中,搅拌均匀,将泊洛沙姆407和泊洛沙姆188加入溶液中,在4-20℃下搅拌6-24h使其充分溶胀,辐照灭菌。
10.如权利要求2所述的应用,其特征在于:所述抑制真菌的组合物为凝胶剂、栓剂、乳膏剂、洗剂、擦剂、涂膜剂或喷雾剂。
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