CN117999080A - 包含复合人参皂苷组合物的抗炎组合物 - Google Patents
包含复合人参皂苷组合物的抗炎组合物 Download PDFInfo
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- CN117999080A CN117999080A CN202180102314.0A CN202180102314A CN117999080A CN 117999080 A CN117999080 A CN 117999080A CN 202180102314 A CN202180102314 A CN 202180102314A CN 117999080 A CN117999080 A CN 117999080A
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- ginsenoside
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- inflammatory
- red ginseng
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Abstract
技术问题:提供一种副作用小或无副作用,且具有抗炎活性功效的天然物质组合物。技术方案:本发明人使用溶剂,从红参中提取出复合人参皂苷,将其吸附在吸附树脂上,利用溶剂解吸对其进行部分纯化后,混合红参提取浓缩液进行抗炎活性试验,结果证实其抗炎活性显著,并利用其制备了抗炎组合物。该组合物可用于食品、皮肤外用制剂、化妆品组合物等。
Description
技术领域
本发明涉及一种包含从红参中提取并加工的复合人参皂苷组合物的抗炎组合物,及包含该组合物的药学组合物、化妆品、食品、牙膏、口腔清洁剂等。
背景技术
炎症是体内对损伤、感染或被免疫系统识别为外来物质的分子做出的反应。炎症的特征表现为组织疼痛、肿疮或功能障碍。炎症性疾病表现为组织或器官的免疫系统被异常激活,这可能导致组织或器官出现功能异常或诱发疾病。
炎症性疾病是全球主要死因之一。炎症性疾病会入侵各个器官及组织,例如:血管、心脏、脑、神经、关节、皮肤、肺、眼、胃肠管、肾脏、甲状腺、副肾、胰脏,肝和肌肉。炎症性疾病的治疗一直是制药公司和研发人员关注的对象。最近该领域出现很多相关方面的研究,目前针对炎症性疾病的疗法包括:使用非特异性药物缓解症状,减少炎症及减缓疾病进展等,但此类疗法存在严重的药物副作用和耐药性问题。
目前广泛使用的抗炎药物为非甾体消炎药,但众所周知非甾体消炎药会出现肾功能障碍、胃肠管障碍,肝障碍等严重的副作用(RainsfordKD.,Subcell biochem.,42:3-27,2007;Guruprasad P.Aithal.,Rheumatology.,7:139-150,2011;Praveen P.N.Rao etal.,Pharmaceuticals.,3:1530-1549,2010)。
因此,相关研究正在如火如荼进行中,旨在从天然物质中寻找具有抗炎活性功效、副作用小或无副作用的新药。
发明内容
要解决的技术问题
本发明的目的在于提供一种副作用小或无副作用,具有抗炎活性功效的天然物质组合物,旨在证明从人参中提取的人参皂苷及包含其的组合物具有抗炎效果,并以此为基础,提供一种强效抗炎物质。
另外,本发明的目的在于提供利用上述抗炎物质的食品、药品、化妆品、口腔清洁剂、牙膏等。
技术方案
本发明人使用溶剂,优选地,使用可食用溶剂,从人参中提取出复合人参皂苷,将其吸附在吸附树脂上,利用溶剂解吸对其进行部分纯化后,混合红参提取浓缩物,制备成复合人参皂苷组合物,然后,对其进行抗炎活性试验,结果证实其抗炎活性显著。
该组合物可用于口腔炎、牙龈炎等口腔炎症的预防或治疗剂、牙膏组合物、皮肤外用制剂、化妆品组合物和食品等。
有益效果
本发明的复合人参皂苷组合物表现为抑制NO生长,抑制促炎细胞因子表达及优异的抗炎活性。
因此,本发明的复合人参皂苷组合物可应用于抗炎药学组合物、口腔炎症预防或治疗用牙膏组合物、包括口腔清洁剂在内的口腔用组合物、皮肤炎症治疗用皮肤外用制剂组合物、化妆品组合物和食品组合物等。
附图说明
图1示出的是对不同浓度的BTEX-K样本进行处理后,用LPS诱发炎症48小时后的NO生成结果(**p<0.01,***p<0.001是对比对照组)。
图2示出的是对不同浓度的BTEX-K样本进行处理后,用LPS诱发炎症48小时后的促炎细胞因子的生成结果(*p<0.05,**p<0.01是对比对照组)。
最佳实施方式
本发明涉及一种抗炎组合物,包含复合人参皂苷组合物作为有效成分,其通过以下步骤获得:
步骤1,对红参进行提取、浓缩,获得红参浓缩物;
步骤2,溶解部分红参浓缩物后,使其穿过吸附树脂柱完成吸附,再用乙醇解析,获得包含人参皂苷Rb1、Rb2、Rc和Rd的复合人参皂苷;
步骤3,用α-半乳糖苷酶处理复合人参皂苷,获得发酵复合人参皂苷;及
步骤4,在通过上述步骤1获得的红参浓缩物中,添加通过步骤3获得的发酵复合人参皂苷,
其中,以100重量份的人参皂苷化合物K为准,分别包含7重量份以上的人参皂苷Rg3(R+S)、人参皂苷F2,人参皂苷Rh2和人参皂苷Rb1,优选地,包含7重量份以上,30重量份以下,更优选地,包含10重量份以上,30重量份以下。
在上述步骤4中,红参浓缩物:发酵复合人参皂苷=1~9:9~1(v/v),优选地,以4~6:6~4的比例混合,更优选地,以100重量份的人参皂苷化合物K为准,分别包含7重量份以上的人参皂苷Rg3(R+S)、人参皂苷F2,人参皂苷Rh2和人参皂苷Rb1,优选地,包含7重量份以上,30重量份以下,更优选地,包含10重量份以上,30重量份以下。
另外,本发明涉及一种抗炎组合物,其特征在于,上述复合人参皂苷组合物以100重量份的人参皂苷化合物K为准,包含20重量份以上的人参皂苷Rg3(R+S)、8重量份以上的人参皂苷F2,12重量份以上的人参皂苷Rh2及8重量份以上的人参皂苷Rb1。
另外,本发明涉及一种抗炎组合物,其特征在于,上述复合人参皂苷组合物包含100mg/g以上的人参皂苷化合物K,且分别包含8mg/g以上的人参皂苷Rg3(R+S),人参皂苷F2,人参皂苷Rh2及人参皂苷Rb,优选地,包含8mg/g以上、30mg/g以下,更优选地,包含10mg/g以上,30mg/g以下。
上述吸附树脂柱并无特别限制,其特征在于,优选地,为多孔性吸附树脂柱,更优选地,为HP2MG或HP-20中的一个或多个。
另外,本发明涉及一种抗炎组合物,其特征在于,上述步骤1、2和4中的红参浓缩物为粉末状态或液态。
另外,本发明涉及一种皮肤炎症治疗用皮肤外用制剂组合物,其包含上述抗炎组合物。
另外,本发明涉及一种炎症预防或治疗用药学组合物,其包含上述抗炎组合物。在一个实现例中,上述药学组合物可以是选自由口服剂型、外用制剂、栓剂、无菌注射溶液和喷雾剂组成的组中的一种或多种剂型。
另外,本发明涉及一种口腔炎症预防或改善用牙膏组合物,其包含上述抗炎组合物。
另外,本发明涉及一种口腔炎症预防或改善用口腔清洁剂组合物,其包含上述抗炎组合物。
另外,本发明涉及一种炎症预防或改善用食品组合物,其包含上述抗炎组合物。
本发明中使用的术语“食品”是指含有一种或多种营养素的天然物或加工品,优选地,是指经过一定程度的加工工艺后达到可直接食用的状态,通常,其可包含所有食品、食品添加剂、功能性食品、健康功能食品、健康辅助食品及饮料等。本发明的食品组合物可以制成各种饮料、口香糖、茶、饼干、复合维生素、健康保健食品等形态,但不限于此。本发明的食品组合物的优选摄取量,会随摄取者的状态、体重、症状程度、食品形态和摄取时间而不同,可适当选择。为获得最佳效果,优选地,本发明的食品组合物有效成分的1日摄取量应达到0.2mg/kg至200mg/kg。
抗炎组合物包含上述复合人参皂苷组合物作为有效成分,其可与药剂学领域的常用载体调配,按照常规方法配制成注射剂、口服剂、外敷剂等剂型。例如:可配制成液剂、糖浆剂、胶囊剂、颗粒剂、粉末、软膏、乳液、凝胶、乳霜等制剂,并通过多种途径用药。在各类剂型中,例如,注射用组合物优选等张性水溶液或悬浮液,提及的组合物经灭菌或含有助剂(防腐剂、稳定剂、润湿剂或乳化剂溶液促进剂、调节渗透压的盐/或缓冲剂)。另外,其还可以含有其他对治疗有用的物质。药剂学上的可用载体包括:乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、黑洋槐、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石粉、硬脂酸镁及矿物油等。另外,上述组合物还可以包括:润滑剂、润湿剂、调味剂、乳化剂和防腐剂。本发明的组合物可配制成颗粒剂、散剂、液剂、片剂、胶囊剂或干糖浆剂等口服制剂和注射剂等非口服制剂,但不限于此。
以此种方式制备的药剂学制剂可根据目的用于皮下给药、肌肉给药或口服给药、静脉给药、及以涂抹皮肤、涂抹口腔、涂抹牙龈等方式的给药。用量方面,每日给药量为0.001μg/㎏~100mg/kg,可分一次或数次给药。针对特定患者的给药用量水平,会根据患者的体重、年龄、性别、健康状态、使用时间、使用方法,疾病严重程度而有所变化。
另外,抗炎组合物包含上述复合人参皂苷组合物作为有效成分,其特征在于,是一种皮肤炎症治疗用皮肤外用制剂。外敷剂包含本发明的抗炎组合物作为有效成分,其可以按照常规制备方法制备成任一种形态。作为一例,在制备乳霜型外敷剂时,使本发明的抗炎组合物包含在普通的水包油(O/W)或油包水(W/O)乳霜基质中,并根据需要使用香料、螯合剂、色素、抗氧化剂、防腐剂等,同时,还可以以改善物性为目的合成蛋白质、矿物质、维生素等或同时使用天然材料。
另外,本发明涉及一种化妆品组合物,其包含上述复合人参皂苷组合物作为有效成分。其可根据需要添加pH调节剂、香料、乳化剂、防腐剂等,并按照常规化妆品制备方法配制成化妆水、凝胶、水溶性粉、脂溶性粉、水溶性液体、乳霜或精华液等剂型。
另外,本发明涉及一种口腔炎症预防或改善用牙膏组合物,其包含上述复合人参皂苷组合物作为有效成分。其可根据需要添加乳香精油、蜂胶提取物、焦磷酸钠、氟化钠、醋酸生育酚、羟基磷灰石、甜味剂、增稠剂、研磨剂、润湿剂或表面活性剂等,并按照常规牙膏组合物制备方法配制成剂型。
另外,本发明涉及一种口腔炎症预防或改善用口腔用组合物,其包含上述复合人参皂苷组合物作为有效成分。其可根据需要添加增溶剂、润湿剂、表面活性剂、润滑剂、香料、甜味剂、防腐剂或药效剂等,并按照常规口腔用组合物制备方法配制成剂型。其可制备成选自由口香糖(gum)、口腔清洗剂、口腔清洁剂、口腔用喷雾剂、口腔用软膏剂、口腔用贴剂或其组合组成的组中的剂型。
具体实施方式
以下,结合下述实施例对本发明的具体方法作更详细的说明。但本发明的范围并不限于这些实施例的记述,其对于本领域技术人员来说是显而易见的。
实施例1:制备复合人参皂苷
购买韩国产干红参(4年根)作为实验原料(忠清南道锦山、德源人参)。从干红参中提取复合人参皂苷。在60℃条件下,12小时内,用50%乙醇对10kg的干红参重复提取、浓缩3次,获得红参浓缩液。
红参浓缩液的总量为5.6kg(63brix),用水溶解其中的5.5kg后,使其穿过填充有合成吸附树脂(HP2MG,三菱化学或HP-20,Diaion)的吸附柱完成吸附,再用95%乙醇进行解析,获得460g包含人参皂苷Rb1、Rb2、Rc及Rd等的复合人参皂苷。
实施例2:制备红参浓缩液粉末
从通过实施例1制备的5.6kg红参浓缩液中另取100g,减压浓缩后,获得71g的红参浓缩液粉末。
实施例3:制备发酵复合人参皂苷
以通过上述实施例1获得的复合人参皂苷100g作为基质,在50mM乙酸钠缓冲溶液(pH4.5)中,与曲霉来源的α-半乳糖苷酶(0.75g)在60℃恒温水槽中反应84小时以上,制备了发酵复合人参皂苷(41g)。
通过上述方法制备的发酵复合人参皂苷中包含人参皂苷化合物K、人参皂苷F2、人参皂苷Rh2等。
实施例4:制备复合人参皂苷组合物(以下与“BTEX-K”互用)
在通过实施例3制备的100g发酵复合人参皂苷中,混合通过实施例2制备的40g红参浓缩液粉末,制备了复合人参皂苷组合物BTEX-K。
复合人参皂苷组合物BTEX-K中除人参皂苷化合物K=100mg/g之外,还包含人参皂苷F2、人参皂苷Rh2等多种人参皂苷。
【表1】
实验例1:细胞活力分析(MTTassay)
1)将细胞(Raw264.7)接种到96孔板中,每孔8×103个,然后,在CO2培养基中培养24小时。
2)去除培养基,更换为按不同浓度添加使用乙醇的样品提取物的100ul新培养基后,培养24小时。
【表2】
号码 | 样品 | 浓度 |
1 | 人参皂苷化合物K | 1,5,10,20μg/ml |
2 | BTEX-K | 5,10,20,30,40,50,100μg/ml |
3)加入MTT溶液后,反应4小时。
4)使用分光光度计在450nm(420~480nm)波长处测量吸光度(参考值为600nm)。
实验例2:NO生成测定
1)将细胞(Raw 264.7)悬浮于含有10%胎牛血清的DMEM培养基中。
2)将悬浮的细胞接种到孔板中,每孔8×103个,然后在CO2培养基中培养24小时。
3)培养24小时后,用PBS洗涤贴壁细胞两次,对稀释至下表所示浓度的样品,预处理24小时后,进行100ng/ml LPS(Escherichia coli 0111;B4)处理,然后培养48小时。
【表3】
号码 | 样品 | 浓度 |
1 | 人参皂苷化合物K | 1,5,10μg/ml |
2 | BTEX-K | 5,10,20μg/ml |
4)48小时后,进行离心分离,取上清液,用格里斯试剂(Griessreagent)(0.2%N-(1-萘基)乙二胺溶液:0.2%磺胺溶液=1:1)在540nm处测量吸光度。
5)利用亚硝酸钠溶液,制作校准曲线,并通过吸光度计算亚硝酸盐浓度。
6)通过对比仅进行LPS处理的组,评估活性。
实验例3:细胞因子测定(ELISA)
1)将细胞(Raw 264.7)悬浮于含有10%胎牛血清的DMEM培养基中。
2)将悬浮的细胞接种到96孔板中,每孔8×103个,然后在CO2培养基中培养24小时。
3)24小时后,用PBS洗涤贴壁细胞两次,将稀释至表3所示浓度的取证样品放入新培养基中预处理24小时后,进行100ng/ml LPS(Escherichia coli 0111;B4)处理,然后培养48小时。
【表4】
4)48小时后,进行离心分离,取上清液,使用小鼠ELISA试剂盒(R&D Systems,Inc.,USA)对IL-1β、IL-6进行定量。
结果1:细胞活力
由于溶解样品的溶剂是乙醇,因此,对Raw264.7细胞在乙醇中的细胞活力进行了分析。将不同浓度的乙醇添加到Raw264.7细胞培养基中,培养24小时,然后,通过MTT分析证实,乙醇浓度达到2%时,Raw264.7细胞死亡率开始超过40%。因此,将样品溶剂的乙醇浓度固定在1%以下。
另外,还确认了在阳性对照组(即不同浓度的人参皂苷化合物K)中的Raw264.7细胞活力。结果证实,人参皂苷化合物K浓度达到20ug/ml时,细胞活力开始下降。因此,将人参皂苷化合物K的样品浓度固定在1、5和10ug/ml后,进行了以下实验。
另外,以不同浓度给药样品BTEX-K,以确认Raw264.7细胞活力。结果证实,BTEX-K浓度达到30ug/ml时,Raw264.7的细胞活力开始下降。因此,将BTEX-K样品浓度固定在5、10和20ug/ml后,进行了以下实验。
在Raw264.7细胞细胞中,进行不同浓度的炎症诱导试剂LPS处理,并测试了细胞活力和NO生成。结果证实,细胞活力在100ng/ml和200ng/ml的LPS浓度下增加,NO生成仅在100ng/ml的LPS浓度下增加。因此,将LPS的浓度定为100ng/ml后,进行了实验。
结果2:一氧化氮生成
如图1所示,在Raw264.7细胞中对不同浓度的BTEX-K进行处理后,培养24小时,然后,添加100ng/ml的LPS,48小时后对NO浓度进行确认,结果证实,在20ug/ml的BTEX-K浓度下显著降低(**p<0.01)。
结果3:促炎细胞因子测定
在Raw264.7细胞中对不同浓度的BTEX-K进行处理后,培养24小时,再添加100ng/ml的LPS,48小时后对细胞因子的生成情况进行确认,结果证实,在20ug/ml的BTEX-K浓度下,IL-6和IL-1β分别显著降低(*p<0.05和**p<0.01)。
进行抗炎实验之前,人参皂苷化合物K和BTEX-K两个样品均已溶解在乙醇中,因此,先对用于抗炎实验的Raw264.7细胞在乙醇中的细胞活力进行了确认。结果证实,在乙醇达到约1%之前,对细胞活力的影响较小,但,高于该浓度值,就会影响细胞的活力。因此,可知乙醇的浓度不宜超过1%。
另外,根据人参皂苷化合物K和BTEX-K的浓度确认了Raw264.7细胞的活力,并确定了要在实验中使用的适当浓度。结果证实,人参皂苷类化合物K浓度达到20ug/ml时,Raw264.7细胞的活力降低,BTEX-K浓度达到30ug/ml时,Raw264.7细胞的活力开始降低。因此,在随后进行的抗炎实验中,将人参皂苷化合物K的适当温度固定在1、5、10ug/ml,将BTEX-K的适当浓度固定在5、10、20ug/ml。
另外,通过NO生成确认了LPS诱发炎症的最佳浓度,结果证实,LPS浓度在100ng/ml时NO生成最佳。因此,在Raw264.7细胞中,按上述确定的浓度对人参皂苷化合物K和BTEX-K两种样品进行处理后,进行100ng/ml的LPS处理,以确认NO生成,结果证实,BTEX-K浓度达到20ug/ml时,NO生成显著减少。
另外,在细胞因子生成实验中,处理BTEX-K 20ug/ml时也证实,IL-6和IL-1β的细胞因子显著减少。然而,在人参皂苷化合物K中,NO和细胞因子的生成分别表现出降低和增加的趋势且并不显著。
工业实用性
本发明的复合人参皂苷组合物可应用于抗炎药学组合物、口腔炎症预防或治疗用牙膏组合物、包括口腔清洁剂在内的口腔用组合物、皮肤炎症治疗用皮肤外用制剂组合物、化妆品组合物和食品组合物等。
Claims (10)
1.一种抗炎组合物,包含复合人参皂苷组合物作为有效成分,其通过以下步骤获得:
步骤1,对红参进行提取、浓缩,获得红参浓缩物;
步骤2,溶解部分红参浓缩物后,使其穿过吸附树脂柱完成吸附,再用乙醇解析,获得包含人参皂苷Rb1、Rb2、Rc和Rd的复合人参皂苷;
步骤3,用α-半乳糖苷酶处理人参皂苷,获得发酵复合人参皂苷;及
步骤4,在通过所述步骤1获得的红参浓缩物中,添加通过步骤3获得的发酵复合人参皂苷,
其中,以100重量份的人参皂苷化合物K为准,分别包含7重量份以上的人参皂苷Rg3(R+S)、人参皂苷F2,人参皂苷Rh2和人参皂苷Rb1。
2.如权利要求1所述的抗炎组合物,其特征在于,所述复合人参皂苷组合物以100%重量份的人参皂苷化合物K为准,包含20重量份以上的人参皂苷Rg3(R+S)、8重量份以上的人参皂苷F2,12重量份以上的人参皂苷Rh2及8重量份以上的人参皂苷Rb1。
3.如权利要求1所述的抗炎组合物,其特征在于,所述复合人参皂苷组合物包含100mg/g以上的人参皂苷化合物K,且分别包含8mg/g以上的人参皂苷Rg3(R+S),人参皂苷F2,人参皂苷Rh2及人参皂苷Rb。
4.如权利要求1所述的抗炎组合物,其特征在于,所述步骤1、2和4中的红参浓缩物为粉末状态或液态。
5.一种皮肤炎症治疗用皮肤外用制剂组合物,其包含:如权利要求1至3任一项所述的抗炎组合物。
6.一种化妆品组合物,其包含:如权利要求1至3任一项所述的抗炎组合物。
7.一种药学组合物,其包含:如权利要求1至3任一项所述的抗炎组合物。
8.一种口腔炎症预防或改善用牙膏组合物,其包含:如权利要求1至3任一项所述的抗炎组合物。
9.一种口腔炎症预防或改善用口腔清洁剂组合物,其包含:如权利要求1至3任一项所述的抗炎组合物。
10.一种食品组合物,其包含:如权利要求1至3任一项所述的抗炎组合物。
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