CN117925763A - 一种山茶籽多肽的制备方法及其在痤疮中的应用 - Google Patents
一种山茶籽多肽的制备方法及其在痤疮中的应用 Download PDFInfo
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- CN117925763A CN117925763A CN202410228369.1A CN202410228369A CN117925763A CN 117925763 A CN117925763 A CN 117925763A CN 202410228369 A CN202410228369 A CN 202410228369A CN 117925763 A CN117925763 A CN 117925763A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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- A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
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- A—HUMAN NECESSITIES
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
本发明公开了一种山茶籽多肽的制备方法及其在痤疮中的应用。本发明将山茶籽粉加水再调节pH7.5~8.5后40~60℃恒温震荡提取,离心,取上清液过滤然后调节pH3.5~4.5以沉淀蛋白质;然后以山茶籽蛋白为原料加水配制蛋白水溶液,超声处理,然后调pH中性,调温度45~55℃,依次加入木瓜蛋白酶和风味蛋白酶分别进行酶解;离心收集上清液,然后进行透析、冷冻干燥得到山茶籽多肽。将制备的山茶籽多肽用于化妆品中,起到抗炎作用,同时有效抑制痤疮丙酸杆菌和限制性马拉色菌的生长,调节皮肤微生态,进而改善痤疮,有利于皮肤健康。
Description
技术领域
本发明属于化妆品技术领域,尤其涉及一种山茶籽多肽的制备方法及其在痤疮中的应用。
背景技术
山茶为山茶科山茶属,常绿、长寿的油料木本植物,山茶科植物主要集中在中国南方的长江中下游的省区。山茶属植物中富含多种活性成分,也是日化产品的重要功效添加物,具有抗氧化、抗菌、抗过敏、抗炎、促进伤口愈合等多种效果。山茶籽饼粕为山茶籽提取山茶籽油后的副产品,拥有多种有益成分:蛋白15%,各种糖类物质40%,油脂物质5%,各类纤维6%,皂素10-14%,单宁2%等。目前,山茶籽饼粕一般被用于田地增肥或者直接丢弃,造成了极大的资源浪费。山茶籽饼粕中蛋白含量高,可以进行多肽资源开发,具有开发生物功能活性多肽药物资源的潜力。
痤疮是一种累及毛囊皮脂腺单位的慢性炎症性疾病,近年来其发病率不断上升,发病年龄范围也越来越广。痤疮最常累及面部、胸部和背部,中重度的痤疮会出现脓疱、结节、囊肿,甚至瘢痕,可能会破坏患者的外观。炎症和皮肤微生态失衡是诱发痤疮的重要因素。痤疮丙酸杆菌过度增殖通过多种方式促炎导致痤疮发生。痤疮丙酸杆菌代谢过程将皮脂中的甘油三酯分解为游离脂肪酸,可以诱导粉刺的形成。其细胞壁上病原相关分子模式、水解皮脂产生的游离脂肪酸等物质均可通过刺激TLR2等途径启动皮肤固有免疫系统,诱导产生炎症介质,加重炎症反应。马拉色菌与难治性痤疮相关,限制性马拉色菌具有较强的脂酶活性,促进毛囊口角化,并可以诱导角质形成细胞分泌炎症因子,加剧炎症反应。
目前痤疮的外用药包括维A酸、过氧苯甲酰、抗生素、壬二酸类,口服药包括抗生素、异维生A酸、激素类药物等,都不适合长期、大量使用,因此研发一种安全有效的改善痤疮的制剂是很有必要的。
发明内容
有鉴于此,本发明提供了一种山茶籽多肽的制备方法及其在痤疮中的应用。本发明采用水提酸沉、酶解获得山茶籽多肽,将制备的山茶籽多肽用于化妆品中,起到抗炎作用,同时有效抑制痤疮丙酸杆菌和限制性马拉色菌的生长,调节皮肤微生态,进而改善痤疮,有利于皮肤健康。
本发明首先提供了一种山茶籽多肽的制备方法,包括以下步骤:
S1:将山茶籽粉加水混合,调pH 7.5~8.5后40~60℃恒温震荡提取,离心,取上清液过滤;
S2:将S1得到的上清液调pH 3.5~4.5以沉淀蛋白,离心收集蛋白沉淀;
S3:以S2得到的山茶籽蛋白为原料加水配制蛋白水溶液,超声处理,然后调pH中性,调温度45~55℃,依次加入木瓜蛋白酶和风味蛋白酶分别进行酶解,每一次酶解完毕后均需要水浴灭酶;
S4:离心收集上清液,然后装入透析袋(截留分子量为3500Da)透析,透析后取保留液,经冷冻干燥得到山茶籽多肽。
进一步的,S1步骤,山茶籽粉与水的比例为1:10~18(w/v),提取时间为2~3h。
进一步的,S3步骤,蛋白水溶液浓度为15~20mg/mL;采用100~200W超声处理15~30min。
进一步的,S3步骤,水浴灭酶温度为80~100℃,时间为10~15min。
进一步的,S5步骤中,透析温度为2~8℃,透析时间为72~96h。
上述方法制备的山茶籽多肽,在制备治疗痤疮的化妆品中的应用。
本发明还公开了所述的山茶籽多肽调节皮肤菌群,当山茶籽多肽浓度为20mg/mL,抑制痤疮丙酸杆菌CCSM0331增殖,抑制率为100%;抑制限制性马拉色菌CICC 33081生长,抑制率80.36%。
本发明还公开了所述的山茶籽多肽的抗炎效果,5mg/mL山茶籽多肽能显著地降低LPS诱导引起的巨噬细胞RAW264.7产生炎症因子IL-6的表达水平,下降率为39.86%,具有良好抗炎功效。
相对于现有技术,本发明具有如下有益效果:本发明提供了一种山茶籽多肽的制备方法,以山茶籽饼粕为原料,采用水提酸沉、酶解获得山茶籽多肽。该方法获得的山茶籽多肽可有效抑制痤疮丙酸杆菌和限制性马拉色菌生长,调节皮肤微生态,同时降低LPS诱导的巨噬细胞RAW264.7产生的炎症因子IL-6的水平,显示出良好的抗炎效果,进而改善痤疮。
附图说明
图1为本发明1.25、2.5、5、10、20mg/mL浓度山茶籽多肽对痤疮丙酸杆菌CCSM0331的抑制率;
图2为本发明1.25、2.5、5、10、20mg/mL浓度山茶籽多肽对限制性马拉色菌CICC33081的抑制率;
图3为本发明0.1、0.5、1、5、10mg/mL浓度山茶籽多肽对巨噬细胞RAW264.7细胞毒性;
图4为本发明5mg/mL浓度山茶籽多肽对LPS引起的巨噬细胞RAW264.7表达IL-6的影响。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。本实施例以本发明技术方案为前提进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
以下各实施例和对比例中,如无特别说明的原料或处理技术,则表明其均为本领域的常规市售原料产品或常规处理技术。
山茶籽饼粕:来源于山茶籽榨油后残渣。
实验菌株:痤疮丙酸杆菌CCSM0331:分离自人体健康脸颊皮肤;限制性马拉色菌CICC 33081:购自中国工业微生物菌种保藏中心。
胰酪大豆胨液体培养基(TSB):胰酪胨17g/L,大豆木瓜蛋白酶水解物3g/L,氯化钠5g/L,磷酸氢二钾2.5g/L,葡萄糖2.5g/L,121℃高压灭菌15min,备用。
改良Dixon培养基(mDixon):麦芽浸粉36g/L,牛胆粉20g/L,蛋白胨6g/L,甘油2g/L,油酸2g/L,121℃高压灭菌15min,备用。
实施例1:山茶籽多肽的制备
(1)山茶籽饼粕粉碎过60目筛,获得山茶籽粉。
(2)将步骤(1)得到的山茶籽粉与水按1:15(w/v)混合,加入0.1M NaOH溶液调pH至8.5后,50℃恒温震荡提取1.5h,离心收集上清。
(3)将步骤(2)得到的上清,0.1M盐酸调pH至4.0以沉淀蛋白,离心收集沉淀,获得山茶籽蛋白。
(4)以实施例1制备的山茶籽蛋白为原料,将其配制成15mg/mL蛋白水溶液,150W超声处理15min,用0.1M NaOH溶液调pH至7.0,温度50℃,加入木瓜蛋白酶5000U/mL,充分混匀,酶解4h,酶解完毕后,沸水浴灭酶15min。
(5)0.1M NaOH溶液调pH至7.0,控制温度50℃,在步骤(4)得到的酶解液加入风味蛋白酶5000U/mL,充分混匀,酶解3h,酶解完毕后,沸水浴灭酶15min。
(6)离心收集上清液,在4℃透析,装入透析袋(截留分子量为3500Da)透析,透析后取保留液经冷冻干燥得到山茶籽多肽。
实施例2:山茶籽多肽的制备
(1)山茶籽饼粕粉碎过60目筛,获得山茶籽粉。
(2)将步骤(1)得到的山茶籽粉与水按1:18(w/v)混合,加入0.1M NaOH溶液调pH至8.0后,50℃恒温震荡提取2h,离心收集上清。
(3)将步骤(2)得到的上清,0.1M盐酸调pH至4.5以沉淀蛋白,离心收集沉淀,获得山茶籽蛋白。
(4)以实施例2制备的山茶籽蛋白为原料,将其配制成20mg/mL蛋白水溶液,200W超声处理15min,用0.1M NaOH溶液调pH至7.0,温度55℃,加入木瓜蛋白酶5000U/mL,充分混匀,酶解2.5h,酶解完毕后,沸水浴灭酶15min。
(5)0.1M NaOH溶液调pH至7.0,控制温度55℃,在步骤(4)得到的酶解液加入风味蛋白酶5000U/mL,充分混匀,酶解2.5h,酶解完毕后,沸水浴灭酶15min。
(6)离心收集上清液,在4℃透析,装入透析袋(截留分子量为3500Da)透析,透析后取保留液经冷冻干燥得到山茶籽多肽。
实施例3:山茶籽多肽的制备
(1)山茶籽饼粕粉碎过60目筛,获得山茶籽粉。
(2)将步骤(1)得到的山茶籽粉与水按1:10(w/v)混合,加入0.1M NaOH溶液调pH至7.5后,60℃恒温震荡提取2h,离心收集上清。
(3)将步骤(2)得到的上清,0.1M盐酸调pH至3.5以沉淀蛋白,离心收集沉淀,获得山茶籽蛋白。
(4)以实施例3制备的山茶籽蛋白为原料,将其配制成15mg/mL蛋白水溶液,150W超声处理15min,用0.1M NaOH溶液调pH至7.0,温度55℃,加入木瓜蛋白酶5000U/mL,充分混匀,酶解4h,酶解完毕后,沸水浴灭酶15min。
(5)0.1M NaOH溶液调pH至7.0,控制温度45℃,在步骤(4)得到的酶解液加入风味蛋白酶5000U/mL,充分混匀,酶解4h,酶解完毕后,沸水浴灭酶15min。
(6)离心收集上清液,在4℃透析,装入透析袋(截留分子量为3500Da)透析,透析后取保留液经冷冻干燥得到山茶籽多肽。
实施例4:山茶籽多肽中蛋白含量测定
(1)采用Folin-酚法测定,配制标准牛蛋白血清溶液,称取0.0253g牛血清蛋白,使用蒸馏水定容至100mL,则配成253μg/ml的标准牛血清蛋白溶液。配制Folin-酚试剂A:①4%碳酸钠溶液;②0.2mol/L氢氧化钠溶液;③1%硫酸铜溶液;④2%酒石酸钾钠溶液。将①与②等体积混合配成碳酸钠-氢氧化钠溶液,③与④等体积混合配成硫酸铜-酒石酸钾钠溶液。将两种溶液按50:1的比例混合,即为Folin-酚试剂A。Folin-酚试剂B为1mol/L的Folin-酚试剂。
(2)分别准确吸取牛血清白蛋白标准储备液0.0、0.1、0.2、0.4、0.6、0.8和1.0mL,置于具塞试管中,各管加水至1mL,再分别加Folin-酚试剂A 5.0mL,在30℃水浴中保温10min,再分别加入Folin-酚试剂B 0.5mL,立刻振荡混匀,在30℃水浴中保温30min,冷却后,在660nm波长处测定吸光度,蛋白含量以mg/mL表示,绘制标准曲线y=0.0009x+0.0079(R2=0.9994);
(3)取1g实施例1所述的山茶籽多肽溶于9mL蒸馏水中,梯度稀释后,取1mL稀释至一定倍数的山茶籽多肽水溶液置于具塞试管中,加Folin-酚试剂A 5.0mL,在30℃水浴中保温10min,再加入Folin-酚试剂B 0.5mL,立刻振荡混匀,在30℃水浴中保温30min,冷却后,在660nm波长处测定吸光度。代入标准曲线计算本发明山茶多肽的蛋白含量为303.18mg/g。
以实施例1制备的山茶籽多肽进行后续的试验。
实施例5:山茶籽多肽对痤疮丙酸杆菌增殖的影响
(1)配置山茶籽多肽水溶液,浓度为80、40、20、10、5mg/mL,过0.22μm滤膜除菌后,与无菌双倍浓度TSB培养基以1:1比例混合得到浓度为40、20、10、5、2.5mg/mL的山茶籽多肽溶液;
(2)制备痤疮丙酸杆菌CCSM0331菌悬液,将活化了的痤疮丙酸杆菌CCSM0331挑取菌落接种于装有7mL的TSB液体培养基的试管中,置于37℃厌氧培养60h,将其OD600调至0.3000~0.4000,继续稀释1000倍备用;
(3)实验组的96孔板单孔加100μL菌液和100μL不同浓度的样品,实验空白组添加100μLTSB和100μL不同浓度的样品,无菌控制孔加200μLTSB,生长控制孔加100μLTSB和100μL菌液,每组做4个复孔;
(4)将96孔板在37℃厌氧培养72h,使用酶标仪测量OD600值,计算抑制率,结果如图1所示。
抑制率=1-(实验组OD值-实验空白组OD值)/(生长控制孔OD值-无菌控制孔OD值)×100%
由图1可知,山茶籽多肽可以有效抑制痤疮丙酸杆菌CCSM0331的增殖,即使在1.25mg/mL浓度下对痤疮丙酸杆菌CCSM0331的抑制率也能达到38.15%,在≥10mg/mL浓度下可以完全抑制痤疮丙酸杆菌CCSM0331的增殖。
实施例6:山茶籽多肽对限制性马拉色菌增殖的影响
(1)配置山茶籽多肽水溶液,浓度为80、40、20、10、5mg/mL,过0.22μm滤膜除菌后,与无菌双倍浓度mDixon培养基以1:1比例混合得到浓度为40、20、10、5、2.5mg/mL的山茶籽多肽溶液;
(2)制备马拉色菌的菌悬液:将活化了两代的马拉色菌挑取单菌落接种于装有7mL的mDixon液体培养基的试管中,置于32℃摇床中,以150r/min的速度振荡培养72h,调整菌体OD600在0.7-0.8范围,继续稀释100倍备用;
(3)实验组的96孔板单孔加100μL菌液和100μL不同浓度的样品,实验空白组添加100μL mDixon培养基和100μL不同浓度的样品,无菌控制孔加200μL mDixon培养基,生长控制孔加100μL mDixon培养基和100μL菌液,每组做4个复孔;
(4)将96孔板在32℃培养72h,使用酶标仪测量OD600值,计算抑制率,结果如图2所示。
抑制率=1-(实验组OD值-实验空白组OD值)/(生长控制孔OD值-无菌控制孔OD值)×100%
由图2可知,山茶籽多肽在各个浓度下对限制性马拉色菌CICC 33081均有50%以上的抑制率,并且随浓度变化不明显。在20mg/mL浓度下对限制性马拉色菌CICC 33081抑制率最高,达80.36%,即使在1.25mg/mL浓度下对限制性马拉色菌CICC 33081也有59.33%的抑制率。
实施例7:山茶籽多肽降低LPS诱导巨噬细胞RAW264.7产生细胞因子IL-6的测试
(1)RAW264.7细胞活力实验
RAW 264.7细胞在5% CO2、37℃的培养条件下,利用含10%胎牛血清的高糖DMEM培养基正常培养。当细胞生长至80-90%汇合时,用胰酶将其消化并按照8×105cells/mL的密度接种于96孔板。细胞贴壁24h后,加入不同浓度的待测样品(0.1、0.5、1、5、10mg/mL浓度的山茶籽多肽水溶液)分别处理细胞24h,每个实验组设置3个平行孔,用DPBS缓冲液清洗1次孔中细胞后,采用CCK-8试剂盒于450nm处检测细胞活力。其中,以水作为溶剂对照组(NT)。
相对细胞活性%=(样品组OD值/溶剂对照组OD值)×100%
结果如图3所示,从图3可以看出,与对照组相比,山茶籽多肽的添加量在0.1~10mg/mL时,未显示细胞毒性,而且能显著促进巨噬细胞RAW264.7细胞的生长。
(2)LPS诱导的RAW264.7细胞IL-6含量测定实验
根据细胞活力的结果,选定山茶籽多肽的5mg/mL浓度,LPS(1μg/mL)诱导细胞炎症实验,同时设立地塞米松(DEX,80μM)作为抑制IL-6的阳性对照组,以水作为溶剂对照组(NT)。
RAW264.7细胞株在5%CO2、37℃的培养条件下,利用含10%胎牛血清的高糖DMEM培养基正常培养。当细胞生长至80-90%汇合时,用胰酶将其消化并按照8×105cells/ml的密度接种于96孔板。细胞贴壁24h后,加样处理细胞,每组设3个平行孔。处理细胞24h后取上清液,用IL-6ELISA试剂盒进行IL-6炎症因子的检测。
IL-6相对生成量%=(样品组IL-6含量/溶剂对照组IL-6含量)×100%
实验结果表明(图4),正常细胞产生少量的IL-6细胞因子,而LPS处理后,细胞分泌大量的炎症因子IL-6,加入5mg/mL山茶籽多肽后,可显著降低炎症细胞因子IL-6的量,山茶籽多肽对炎症因子IL-6抑制率达39.86%。
上述实施例仅为本发明的优选实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (10)
1.一种山茶籽多肽的制备方法,其特征是,包括以下步骤:
S1:将山茶籽粉加水混合,调pH 7.5~8.5后40~60℃恒温震荡提取,离心,取上清液过滤;
S2:将S1得到的上清液调pH 3.5~4.5以沉淀蛋白,离心收集蛋白沉淀;
S3:以S2得到的山茶籽蛋白为原料加水配制蛋白水溶液,超声处理,然后调pH中性,调温度45~55℃,依次加入木瓜蛋白酶和风味蛋白酶分别进行酶解;
S4:离心取上清液,装入透析袋进行透析,透析袋截留分子量为3500Da,透析后取保留液冷冻干燥得到山茶籽多肽。
2.如权利要求1所述的一种山茶籽多肽的制备方法,其特征是,所述S1步骤,山茶籽粉与水的质量体积比为1:10~18,提取时间为2~3h。
3.如权利要求1所述的一种山茶籽多肽的制备方法,其特征是,所述S3步骤,蛋白水溶液浓度为15~20mg/mL;采用100~200W超声处理15~30min。
4.如权利要求1所述的一种山茶籽多肽的制备方法,其特征是,所述S3步骤,每一次酶解完毕后均需要水浴灭酶。
5.如权利要求4所述的一种山茶籽多肽的制备方法,其特征是,所述S3步骤,水浴灭酶温度为80~100℃,时间为10~15min。
6.如权利要求1所述的一种山茶籽多肽的制备方法,其特征是,所述S5步骤中,透析温度为2~8℃,透析时间为72h~96h。
7.权利要求1-6中任一项所述方法制备的山茶籽多肽。
8.权利要求7所述的山茶籽多肽,在制备治疗痤疮的化妆品中的应用。
9.如权利要求8所述的应用,其特征是,所述的山茶籽多肽调节皮肤菌群,抑制痤疮丙酸杆菌和限制性马拉色菌增殖。
10.如权利要求8所述的应用,其特征是,所述山茶籽多肽降低LPS诱导引起的巨噬细胞RAW264.7产生炎症因子IL-6的表达水平,具有抗炎功效。
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