CN115006472B - 一种茶油烫伤膏及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种茶油烫伤膏及其制备方法和应用。其制备方法为:油茶籽通过固定化蛋白酶缓冲液提取后滤过液过大孔树脂,经过液经超滤膜过滤和冷冻干燥获得油茶多肽,截留液经固定化多糖水解酶解和过滤液冷冻干燥获得油茶低聚糖,吸附液经乙醇水溶液洗脱后,真空干燥得油茶皂苷,茶油中加入脂肪酶水解反应后,再加入油茶低聚糖,反应得到茶油低聚糖结合物,再将其与油茶多肽和油茶皂苷分别配成缓冲液,高速搅拌并过微孔滤膜制得茶油低聚糖结合物包被油茶多肽的纳米乳液,冷冻干燥即可。该产品可以在防止感染的同时,调节皮下组织脂肪、糖和蛋白代谢平衡,从而在促进伤口愈合同时,减少疤痕的形成。
Description
技术领域
本发明属于医药领域,具体涉及一种茶油烫伤膏及其制备方法和应用。
背景技术
烧烫伤是由于火焰、高温蒸汽或液体等导致的组织损伤,主要是皮肤或粘膜,严重可伤及皮下组织。可引起局部疼痛、皮肤红肿、水疱、破损等。创面处理和及时施药可有效减轻继续损伤,并加速恢复,但用药不当,不仅延缓恢复,而且会产生疤痕。疤痕是由于伤口在愈合过程中,局部微环境的变化,使胶原蛋白的合成与分解功能平衡紊乱,导致疤痕胶原纤维的堆积和排列异常形成的。常用的药物包括防感染药和去腐生肌药等,如抗生素、一些活血化瘀和滋补中药等,并不能改变胶原蛋白的代谢平衡,所以即使控制炎症和促进增生,但不一定能消除疤痕。
油茶在我国有大量种植,油茶籽产量大、价格低,且为食品级原料,适合长期使用。茶油和茶皂素具有治疗烫伤的作用,CN106913745A、CN106913746A公开了一种含有茶油的烫伤膏及其制备方法,CN109394680A公开了一种含茶皂素的烧烫伤软膏及其制备方法和应用。茶油具有隔离空气防止伤口感染作用,茶皂素具有抗炎镇痛作用,然而它们只能改善红肿症状,不能从皮下组织营养代谢方面改善受伤组织的微环境。
将油茶籽中成分重新装配,以制备改善烧烫伤组织代谢微环境,以加速修复的药膏,具有良好的临床应用价值。
发明内容
为了克服现有技术的缺点与不足,本发明的首要目的在于提供一种茶油烫伤膏。
本发明的另一目的在于提供所述茶油烫伤膏的制备方法。
本发明的再一目的在于提供所述茶油烫伤膏在制备治疗皮肤创伤药物中的应用。
一种茶油烫伤膏的制备方法,包括如下步骤:
(1)脱脂茶粕中加入其质量1%~10%固定化蛋白酶和其质量8~30倍的50~70℃pH5~10缓冲液提取1~3h后,过滤,滤液用大孔树脂吸附8~24h;
(2)将步骤(1)所得未被吸附液用截留分子量5~20kDa的膜过滤,滤液冷冻干燥24~48h,得油茶多肽;截留液加入其质量0.5%~5%的固定化多糖水解酶,50~70℃反应1~3h后过滤,滤液冷冻干燥24~48h,得油茶低聚糖;将吸附后的大孔树脂用1~3倍大孔树脂质量的体积分数为50%~70%的乙醇水溶液洗脱,洗脱液经50~70℃、0.01~0.1MPa干燥3~8h,得油茶皂苷;
(3)茶油加入其质量5%~10%脂肪酶和8~30倍的50~70℃pH5~10缓冲液反应1~3h后,加入茶油质量5~10倍的有机溶剂和0.5~1.5倍的步骤(2)所得油茶低聚糖,继续反应1~3h后,静置8~24h,上层液经50~70℃,0.01~0.1MPa浓缩干燥3~8h,得茶油低聚糖结合物;
(4)将步骤(2)所得油茶多肽和其质量分数10%~50%的油茶皂苷与其质量5~20倍的步骤(3)所得茶油低聚糖结合物,分别用pH5~10缓冲液配制成5%~10%的分散液,在5000~20000转/min搅拌下,先将油茶皂苷分散液加入到茶油低聚糖结合物分散液中,搅拌10~60min后,再加入油茶多肽分散液,继续搅拌10~60min后,过0.1~0.5μm微孔滤膜,冷冻干燥,即获得所述的茶油烫伤膏。
优选的,步骤(1)中所述的脱脂茶粕通过如下方法制备得到:将茶粕粉碎,过20~30目筛,加入正已烷,搅拌,转速3000~4000转/min离心20~30min,所得固体物即为所述的脱脂茶粕。
优选的,步骤(1)中所述的固定化蛋白酶为吸附或交联在水不溶性载体上的中性蛋白酶、碱性蛋白酶、木瓜蛋白酶中的一种或多种组合。
优选的,所述的水不溶性载体为介孔二氧化硅。
优选的,所述的固定化蛋白酶通过如下方法制备得到:在1%酶缓冲液中,加入蛋白酶质量10倍的介孔二氧化硅,混合均匀后再加入3%戊二醛,60℃反应12h,过滤。
优选的,步骤(1)中所述的所述大孔树脂为弱极性大孔树脂,包括但不限于AB-8、HPD100、HPD400、D101、YKDH-2,其用量为脱脂茶粕质量的1~3倍。
优选的,步骤(2)中所述的固定化多糖水解酶为吸附或交联在水不溶性载体上的果糖酶、乳糖酶和葡聚糖酶的一种或多种组合。
优选的,所述的水不溶性载体为介孔二氧化硅。
更优选的,所述的固定化蛋白酶通过如下方法制备得到:在1%酶缓冲液中,加入多糖水解酶质量10倍的介孔二氧化硅,混合均匀后再加入3%戊二醛,60℃反应12h,过滤。
优选的,步骤(3)中所述的有机溶剂为乙酸乙酯、石油醚和环已烷中的一种或多种组合。
优选的,步骤(1)、(3)和(4)中所述的缓冲液为醋酸缓冲液、磷酸缓冲液、Tris-HCl缓冲液中的任意一种。
一种茶油烫伤膏,通过上述制备方法制备得到。该茶油烫伤膏是以茶油低聚糖结合物为壳,油茶多肽为核,油茶皂苷为乳化剂组成的纳米乳膏。
上述茶油烫伤膏在制备治疗皮肤创伤药物中的应用。
所述茶油烫伤膏可以在防止感染的同时,调节皮下组织脂肪、糖和蛋白代谢平衡,从而在促进伤口愈合同时,减少疤痕的形成。
本发明的原理:脱脂茶粕经蛋白酶缓冲液提取为油茶多糖、多肽和皂苷的混合液,经大孔树脂吸附分离得到油茶皂苷,穿过液再经超滤膜分离得到油茶多糖和多肽,油茶多糖进一步在多糖水解酶作用下得到低聚糖。茶油在脂肪酶作用下水解为脂肪酸,并与低聚糖形成结合物。茶油低聚糖结合物与油茶多肽在油茶皂苷作用下乳化成纳米颗粒,由于茶油低聚糖结合物的疏水部分与油茶多肽的疏水基团相连接,形成茶油低聚糖结合物为壳和油茶多肽为核的纳米结构。该结构使之易进入组织细胞,并在细胞内酶作用下释放多肽、低聚糖和脂肪酸,从而可调节细胞内的多肽、糖和脂肪水平,以避免胶原蛋白的过度合成和疤痕的形成。
本发明相对于现有技术具有如下的优点及效果:
(1)本发明制备了一种茶油低聚糖结合物包载油茶多肽的纳米制剂,有利于皮肤的渗透和细胞的吸收。
(2)本发明克服了油脂、多糖、皂苷只作用的单一性,且不能调节代谢的缺点,可改善烧烫伤组织代谢微环境,抑制胶原蛋白的过度增生,减少疤痕的产生。
(3)本发明提供的方法反应条件温和,易于工业化生产。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。
下述实施例中所用脱酯茶粕为将茶粕粉碎,过20目筛,向其中加入正已烷试剂,搅拌,转速3000转/min离心20min分离所得;所用固定化酶是在1%酶缓冲液中,加入酶质量10倍的介孔二氧化硅,混合均匀后再加入3%戊二醛,60℃反应12h,过滤所得。
实施例1
(1)1kg脱脂茶粕中加入10g固定化中性蛋白酶和8kg的50℃pH5醋酸缓冲液提取1h后,过滤,滤液用1kg AB-8大孔树脂吸附8h;
(2)将上述未被吸附液用截留分子量5kDa的膜过滤,滤液冷冻干燥24h,得油茶多肽96g;截留液1kg加入5g的固定化果糖酶,50℃反应1h后过滤,滤液冷冻干燥24h,得油茶低聚糖16g;将吸附后的大孔树脂用1kg体积分数为50%的乙醇水溶液洗脱,洗脱液经50℃,0.01MPa干燥3h,得油茶皂苷85g;
(3)茶油1kg加入50g脂肪酶和8kg 50℃pH5醋酸缓冲液反应1h后,加入5kg乙酸乙酯和0.5kg油茶低聚糖,继续反应1h后,静置8h,上层液经50℃、0.01MPa浓缩干燥3h,得茶油低聚糖结合物0.8kg;
(4)将油茶多肽10g与50g的茶油低聚糖结合物和1g油茶皂苷,分别用pH5醋酸缓冲液配制成5%的分散液,在5000转/min搅拌下,先将油茶皂苷分散液加入到茶油低聚糖结合物分散液中,搅拌10min后,再加入油茶多肽分散液,继续搅拌10min后,过0.1μm微孔滤膜,冷冻干燥即得茶油烫伤膏。
实施例2
(1)1kg脱脂茶粕中加入100g固定化碱性蛋白酶和30kg的70℃pH10磷酸缓冲液提取3h后,过滤,滤液用3kgHPD100大孔树脂吸附24h;
(2)将上述未被吸附液用截留分子量20kDa的膜过滤,滤液冷冻干燥48h,得油茶多肽135g;截留液1kg加入50g的固定化乳糖酶,70℃反应3h后过滤,滤液冷冻干燥48h,得油茶低聚糖7g;将吸附后的大孔树脂用9kg体积分数为70%的乙醇水溶液洗脱,洗脱液经70℃,0.1MPa干燥8h,得油茶皂苷130g;
(3)茶油1kg加入100g脂肪酶和30kg 70℃pH10磷酸缓冲液反应1-3h后,加入10kg石油醚和1.5kg油茶低聚糖,继续反应3h后,静置24h,上层液经70℃、0.1MPa浓缩干燥8h,得茶油低聚糖结合物1.1kg;
(4)将油茶多肽10g与200g的茶油低聚糖结合物和5g油茶皂苷,分别用pH10磷酸缓冲液配制成10%的分散液,在20000转/min搅拌下,先将油茶皂苷分散液加入到茶油低聚糖结合物分散液中,搅拌60min后,再加入油茶多肽分散液,继续搅拌60min后,过0.5μm微孔滤膜,冷冻干燥即得茶油烫伤膏。
实施例3
(1)1kg脱脂茶粕中加入50g固定化木瓜蛋白酶和15kg的60℃pH6Tris-HCl缓冲液提取2h后,过滤,滤液用2kg HPD400大孔树脂吸附12h;
(2)将上述未被吸附液用截留分子量10kDa的膜过滤,滤液冷冻干燥36h,得油茶多肽112g;截留液1kg加入25g的固定化葡聚糖酶,60℃反应2h后过滤,滤液冷冻干燥36h,得油茶低聚糖11g;将吸附后的大孔树脂用4kg体积分数为60%的乙醇水溶液洗脱,洗脱液经60℃,0.05MPa干燥5h,得油茶皂苷122g;
(3)茶油1kg加入70g脂肪酶和15kg 60℃pH6Tris-HCl缓冲液反应2h后,加入7kg环已烷和1kg油茶低聚糖,继续反应2h后,静置16h,上层液经60℃、0.05MPa浓缩干燥5h,得茶油低聚糖结合物1.1kg;
(4)将油茶多肽10g与100g的茶油低聚糖结合物和3g油茶皂苷,分别用pH6Tris-HCl缓冲液配制成7%的分散液,在10000转/min搅拌下,先将油茶皂苷分散液加入到茶油低聚糖结合物分散液中,搅拌30min后,再加入油茶多肽分散液,继续搅拌30min后,过0.3μm微孔滤膜,冷冻干燥即得茶油烫伤膏。
实施例4
(1)1kg脱脂茶粕中加入20g固定化中性蛋白酶和10kg的55℃pH6醋酸缓冲液提取1.5h后,过滤,滤液用1.5kg D101大孔树脂吸附12h;
(2)将上述未被吸附液用截留分子量10kDa的膜过滤,滤液冷冻干30h,得油茶多肽109g;截留液1kg加入10g的固定化果糖酶,55℃反应1.5h后过滤,滤液冷冻干燥30h,得油茶低聚糖9g;将吸附后的大孔树脂用5kg体积分数为55%的乙醇水溶液洗脱,洗脱液经55℃、0.02MPa干燥4h,得油茶皂苷90g;
(3)茶油1kg加入60g脂肪酶和10kg 55℃pH6醋酸缓冲液反应1.5h后,加入6kg乙酸乙酯和0.8kg油茶低聚糖,继续反应1.5h后,静置12h,上层液经55℃、0.05MPa浓缩干燥5h,得茶油低聚糖结合物0.7kg;
(4)将油茶多肽10g与80g的茶油低聚糖结合物和2g油茶皂苷,分别用pH5.5醋酸缓冲液配制成7%的分散液,在8000转/min搅拌下,先将油茶皂苷分散液加入到茶油低聚糖结合物分散液中,搅拌20min后,再加入油茶多肽分散液,继续搅拌20min后,过0.3μm微孔滤膜,冷冻干燥即得茶油烫伤膏。
实施例5
(1)1kg脱脂茶粕中加入80g固定化碱性蛋白酶和20kg的65℃pH8磷酸缓冲液提取2.5h后,过滤,滤液用2.5kg YKDH-2大孔树脂吸附20h;
(2)将上述未被吸附液用截留分子量15kDa的膜过滤,滤液冷冻干燥40h,得油茶多肽127g;截留液1kg加入40g的固定化葡聚糖酶,65℃反应2.5h后过滤,滤液冷冻干燥40h,得油茶低聚糖8g;将吸附后的大孔树脂用7kg体积分数为65%的乙醇水溶液洗脱,洗脱液经65℃,0.06MPa干燥6h,得油茶皂苷116g;
(3)茶油1kg加入80g脂肪酶和20kg 65℃pH8磷酸缓冲液反应2.5h后,加入8kg环已烷和1.2kg油茶低聚糖,继续反应2.5h后,静置20h,上层液经65℃、0.06MPa浓缩干燥6h,得茶油低聚糖结合物1.2kg;
(4)将油茶多肽10g与180g的茶油低聚糖结合物和4g油茶皂苷,分别用pH8磷酸缓冲液配制成8%的分散液,在15000转/min搅拌下,先将油茶皂苷分散液加入到茶油低聚糖结合物分散液中,搅拌40min后,再加入油茶多肽分散液,继续搅拌40min后,过0.4μm微孔滤膜,冷冻干燥即得茶油烫伤膏。
对比例1
按实施例1制备方法,但在步骤(3)中不加入油茶低聚糖,所制得的产品为对照1。
对比例2
按实施例1制备方法,但在步骤(4)中将茶油低聚糖结合物替换为卵磷脂,所制得的产品为对照2。
测试1:
实施例1-5制得的茶油低聚糖结合物的组成分析
方法:将实施例1-5制得的茶油低聚糖结合物,取适量按照中华人民共和国农业行业标准-食用菌中粗多糖含量的测定(NY/T1676-2008)测定样品中的糖含量,按照食品安全国家标准-食品中脂肪酸的测定(GB5009.168-2016)第一法测定样品中的脂肪酸含量。
结果:实施例1-5制得的茶油低聚糖结合物中的糖和脂肪酸含量见表1,每100g产品中含糖21.2-31.5%和脂肪酸63.8-73.4%,说明该结合物中同时含有糖和脂肪酸。
表1 茶油低聚糖结合物的含量组成
样品来源 | 糖含量% | 脂肪酸含量% |
实施例1 | 26.4 | 71.6 |
实施例2 | 29.5 | 67.5 |
实施例3 | 21.2 | 73.4 |
实施例4 | 31.5 | 63.8 |
实施例5 | 27.3 | 69.4 |
测试2:
实施例1-5制得的茶油烫伤膏纳米粒径和油茶多肽载药量的测定
方法:将实施例1-5制得的茶油烫伤膏、对照1和对照2,采用马尔文纳米粒度仪测定其粒径,并取适量通过微柱离心法除去游离多肽,按照中华人民共和国出入境检验检疫行业标准-出品乳、蛋、豆类食品中蛋白质含量的测定考马斯亮蓝法(SN/T3926-2006)测定样品中的蛋白含量,计算油茶多肽载药量。
结果:实施例1-5制得的茶油烫伤膏的粒径和油茶多肽含量见表2,其粒径为214-273nm,载药量为4.6-15.4%,说明该产品为负载了油茶多肽的纳米制剂。对照1无载药,说明茶油脂肪酸不具备包载能力;对照2有少量载药,说明脂质体可包载油茶多肽,但载药量低。
表2 茶油烫伤的粒径和油茶多肽载药量
样品来源 | 粒径nm | 油茶多肽载药量% |
实施例1 | 273 | 15.4 |
实施例2 | 214 | 4.6 |
实施例3 | 236 | 8.7 |
实施例4 | 255 | 11.2 |
实施例5 | 227 | 5.3 |
对照1 | 158 | 0 |
对照2 | 243 | 1.7 |
测试3:
实施例1制得的茶油烫伤膏的应用效果
方法:64只大鼠,雄性,体重280±20g,饲养1周后,剃毛器脱毛后,用体积分数70%的酒精消毒,然后腹腔注射水合氯醛(体积分数10%,350mg/kg)麻醉,将直径3cm铁棒置入沸水中15min,取出后迅速用纱布擦干,一端紧贴大鼠背部皮肤,持续7s,形成深Ⅱ度烫伤模型。烫伤后0.5h将实验大鼠随机分为8组,分别为阴性对照组(生理盐水组),阳性对照组(磺胺嘧啶银软膏组),给药组(实施例1茶油组、油茶皂苷组、油茶多肽组、油茶低聚糖组、茶油烫伤膏)和对照2组,每组8只,分笼饲养。每天1次,每次每只大鼠给药约20mg。于第2天、第7天、第14天早上8时测量烫伤创面面积,观察疤痕产生情况。按下列公式计算创面愈合率:创面愈合率=(原始创面面积-未愈合创面面积)/原始创面面积×100%。
结果:结果见表3,相比于阴性对照组,对照2、茶油、油茶皂苷、油茶多肽和油茶低聚糖均有一定的促进创面愈合的作用,但效果比阳性对照弱,而茶油烫伤膏创面愈合率显著优于其他组。14天后,茶油烫伤膏组创面无疤痕,而其他组均有疤痕,说明茶油烫伤膏发挥了各成分的协同作用,具有更强促进创面愈合和减少疤痕产生的作用效果。
表3 大鼠创面愈合率
组别 | 数量 | 第2天 | 第7天 | 第14天 |
阴性对照组 | 8 | 1.4±2.1 | 6.7±4.2 | 16.3±6.2 |
阳性对照组 | 8 | 5.2±2.7 | 28.5±13.6 | 37.6±14.3 |
茶油组 | 8 | 3.1±3.3 | 11.4±5.2 | 21.2±6.4 |
油茶皂苷组 | 8 | 3.4±4.1 | 17.3±7.6 | 28.4±11.7 |
油茶多肽组 | 8 | 2.6±2.4 | 7.8±4.9 | 19.3±7.2 |
油茶低聚糖组 | 8 | 3.1±2.9 | 8.7±6.3 | 18.4±5.8 |
茶油烫伤膏组 | 8 | 6.7±4.5 | 35.4±16.7 | 56.8±18.2 |
对照2组 | 8 | 2.3±2.1 | 7.1±3.8 | 17.7±6.2 |
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受所述的实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (6)
1.一种茶油烫伤膏在制备治疗皮肤创伤药物中的应用,其特征在于:所述的茶油烫伤膏通过包括如下步骤的方法制备得到:
(1)脱脂茶粕中加入其质量1%~10%固定化蛋白酶和其质量8~30倍的50~70℃ pH5~10缓冲液提取1~3h后,过滤,滤液用大孔树脂吸附8~24h;
(2)将步骤(1)所得未被吸附液用截留分子量5~20kDa的膜过滤,滤液冷冻干燥24~48h,得油茶多肽;截留液加入其质量0.5%~5%的固定化多糖水解酶,50~70℃反应1~3h后过滤,滤液冷冻干燥24~48h,得油茶低聚糖;将吸附后的大孔树脂用1~3倍大孔树脂质量的体积分数为50%~70%的乙醇水溶液洗脱,洗脱液经50~70℃、0.01~0.1MPa干燥3~8h,得油茶皂苷;
(3)茶油加入其质量5%~10%脂肪酶和8~30倍的50~70℃ pH5~10缓冲液反应1~3h后,加入茶油质量5~10倍的有机溶剂和0.5~1.5倍的步骤(2)所得油茶低聚糖,继续反应1~3h后,静置8~24h,上层液经50~70℃,0.01~0.1MPa浓缩干燥3~8h,得茶油低聚糖结合物;
(4)将步骤(2)所得油茶多肽和其质量分数10%~50%的油茶皂苷与其质量5~20倍的步骤(3)所得茶油低聚糖结合物,分别用pH5~10缓冲液配制成5%~10%的分散液,在5000~20000转/min搅拌下,先将油茶皂苷分散液加入到茶油低聚糖结合物分散液中,搅拌10~60min后,再加入油茶多肽分散液,继续搅拌10~60min后,过0.1~0.5μm微孔滤膜,冷冻干燥,即获得所述的茶油烫伤膏;
步骤(1)中所述的固定化蛋白酶为吸附或交联在水不溶性载体上的中性蛋白酶、碱性蛋白酶、木瓜蛋白酶中的一种或多种组合;
步骤(2)中所述的固定化多糖水解酶为吸附或交联在水不溶性载体上的果糖酶、乳糖酶和葡聚糖酶的一种或多种组;
所述的水不溶性载体为介孔二氧化硅。
2.根据权利要求1所述的应用,其特征在于:
所述的固定化蛋白酶或固定化多糖水解酶通过如下方法制备得到:在1%酶缓冲液中,加入酶质量10倍的介孔二氧化硅,混合均匀后再加入3%戊二醛,60℃反应12h,过滤。
3.根据权利要求1所述的应用,其特征在于:
步骤(1)中所述的所述大孔树脂为AB-8、HPD100、HPD400、D101或YKDH-2,其用量为脱脂茶粕质量的1~3倍。
4.根据权利要求1所述的应用,其特征在于:
步骤(3)中所述的有机溶剂为乙酸乙酯、石油醚和环已烷中的一种或多种组合。
5.根据权利要求1所述的应用,其特征在于:
步骤(1)、(3)和(4)中所述的缓冲液为醋酸缓冲液、磷酸缓冲液、Tris-HCl缓冲液中的任意一种。
6.根据权利要求1所述的应用,其特征在于:
步骤(1)中所述的脱脂茶粕通过如下方法制备得到:将茶粕粉碎,过20~30目筛,加入正已烷,搅拌,转速3000~4000转/min离心20~30min,所得固体物即为所述的脱脂茶粕。
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