CN108938724A - 一种印楝叶提取物及其制备工艺、应用 - Google Patents
一种印楝叶提取物及其制备工艺、应用 Download PDFInfo
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- CN108938724A CN108938724A CN201810972883.0A CN201810972883A CN108938724A CN 108938724 A CN108938724 A CN 108938724A CN 201810972883 A CN201810972883 A CN 201810972883A CN 108938724 A CN108938724 A CN 108938724A
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- Prior art keywords
- neem leaf
- leaf extract
- extract
- neem
- sodium
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Abstract
本发明公开了一种印楝叶提取物及其制备工艺、应用。本发明通过超临界二氧化碳法萃取获得印楝叶提取物,其主要药效成分为总黄酮20~35%,总萜类5~10%,总香豆素2~5%;该提取物对幽门螺杆菌抑制效果明显,与银杏提取物合用具有协同作用,故该提取物可用于制备抑制幽门螺杆菌活性的医药品、保健品、食品和日化用品,无需添加防腐剂、抑菌剂等,尤其适合与银杏叶提取物组合开发抑制幽门螺杆菌的牙膏。
Description
技术领域:
本发明涉及生物技术及医药技术领域,具体地,本发明涉及一种印楝叶提取物及其制备工艺、应用。
背景技术:
幽门螺杆菌是一种革兰氏阴性菌,主要存在于口腔及胃部,是引发胃部炎症及胃癌的关键因素之一。目前治疗胃部幽门螺杆菌感染的方法主要是1~3种抗生素的三联或四联疗法。该疗法虽是经口服药,却无法应对存在于牙菌斑、口腔粘膜、牙结石、龈袋等常见的口腔聚居地的幽门螺杆菌。同时,幽门螺杆菌的抗生素疗法疗程有限,容易产生细菌耐药性。
已有众多学者将研究目光转移到植物提取物中,如韩艳等报道了20种药用植物提取物的抗幽门螺杆菌的活性,发现丁香和云木香乙醇提取物显示了较强的抗幽门螺杆菌活性,为进一步确定抗幽门螺旋杆菌的有效成分和寻找先导化合物奠定了良好基础。其他一些传统药用植物也有类似报道,如印楝Azadirachta india被誉为“可以解决全球性问题的树”,在印度具有上千年的使用历史,而且在牙齿及口腔健康方面,一直为印度人所推崇。但目前对印楝的研究主要集中在具有杀虫作用的三萜类成分上。有研究表明,印楝种仁中含多种化合物,包括萜类、硫化物、黄酮类以及香豆素等,以萜类的含量较多。因其具有抑菌、抗疟、拒食杀虫等作用,常用于治疗疟疾、麻风和胃肠道疾病感染。而印楝叶的研究较少,文献报道其含有黄酮类、萜类、甾醇、生物碱以及香豆素等,以黄酮类的含量较多。因其具有降压、抑菌、杀虫等作用,用于治疗水痘,皮肤感染,麻风病,肠道感染,呼吸系统疾病等。Werner等发表的《Activity ofeastAfrican medicinal plantsagainstHelicobacterpylori》中提到,采用甲醇索氏提取的印楝叶提取物对幽门螺杆菌的MIC50值>4000μg/mL,抗菌效果不强。该甲醇索式回流提取法会破坏印楝叶的热敏成分(四环三萜类、部分多酚和黄酮类、蛋白质、有机酸等),提取得到的物质以非热敏性极性成分为主,包括碳水化合物、内酯类等。该甲醇索式回流提取物的抗菌效果不强意味着印楝叶中抑制幽门螺杆菌有效成分的提取方式有待改进。
银杏Ginkgo biloba L.有“活化石”之称,银杏叶入药始于明代,含有黄酮类、萜类内酯类及少量多酚类、生物碱、微量元素等;现代药理研究显示,银杏叶提取物具有保护心血管系统、保护中枢神经系统、抗氧化等作用。也有诸多文献报道银杏叶提取物具有抗菌消炎作用,如陈妍等报道了添加银杏叶提取物的牙膏对口腔主要致龋菌——变异链球菌和粘性放线菌等具有体外抗菌活性,其最小抑菌浓度(MIC)和-最小杀菌浓度(MBC)范围分别为0.31~1.25mg/mL和0.31~2.5mg/mL。Mahady等发表的《In vitro susceptibilityofHelicobacterpylori to botanical extracts used traditionally for thetreatment of gastrointestinal disorders》指出,银杏叶甲醇提取物抑制幽门螺杆菌的MIC>100μg/mL,其抑菌效果不强。梅林等发表的《银杏叶含药血清的抑菌活性实验》指出,银杏叶50%乙醇提取物的幽门螺杆菌MIC为6%。按银杏叶提取率为10%计算,银杏叶50%乙醇提取物的幽门螺杆菌MIC值为24mg/mL。
目前国内外耐药菌的报道日益增多,开发天然植物提取物抑菌是当前医药领域研发的热门领域,具有极高的医药价值。
发明内容
本发明提供一种印楝叶提取物,其具有很好的抑制幽门螺杆菌作用,可用于制备抑制幽门螺杆菌活性的食品、药品、保健品和日化用品等,尤其适用于牙膏等用于治疗或者辅助治疗牙龈炎、牙周炎、口腔溃疡等口腔疾病,其可以清洁口腔,改善口腔环境,维持口腔正常菌群。
其中,印楝叶提取物的主要药效成分为总黄酮20~35%,总萜类5~10%,总香豆素2~5%,余量为辅料。所述印楝叶提取物的制备工艺如下:
(1)印楝叶原料的预处理:将干燥的印楝叶原料粉碎,过80~100目筛,备用;
(2)采用超临界二氧化碳法萃取制备印楝叶提取物:萃取压力25~50MPa,萃取温度30~50℃,萃取时间60~180min,3~5mL/g乙醇为夹带剂;
(3)将印楝叶提取液浓缩、喷雾干燥;
(4)将干燥的印楝叶提取物粉碎,过180~200目筛备用。
进一步,其特征是步骤(2),采用超临界二氧化碳法萃取制备印楝叶提取物:萃取压力30MPa,萃取温度40℃,萃取时间120min,4mL/g乙醇为夹带剂。
本发明的印楝叶提取物可用于制备具有抑制幽门螺杆菌活性的医药品、保健品、食品和日化用品。
此外,本发明还提供一种含有印楝叶、银杏叶提取物的牙膏,其组成物及重量百分比为:银杏叶提取物0.05~2%,印楝叶提取物0.10~2%,余量为辅料,所有组分总量为100%。
其中,银杏印楝牙膏中银杏叶提取物添加量为0.3%,印楝叶提取物添加量为0.5%。
进一步,银杏印楝牙膏中辅料组成及其重量百分比为:保湿剂10~70%、摩擦剂10~30%、粘合剂0.1~5%、发泡剂0.1~10%、甜味剂0.1~5%、香精0.3~0.7%,余量为去离子水。
进一步,保湿剂为山梨醇、山梨糖醇、丙二醇中的一种或几种任意比的混合物,摩擦剂为碳酸钙、二氧化硅、磷酸氢钙中的一种或几种任意比的混合物,粘合剂为纤维素钠、黄原胶中的一种或两种任意比的混合物,发泡剂为月桂醇硫酸酯钠、焦磷酸钠中的一种或两种任意比的混合物,甜味剂为糖精钠、木糖醇中的一种或两种任意比的混合物。
进一步,所述辅料组成及其重量百分比为:山梨醇34%、丙二醇14%、二氧化硅28%、月桂醇硫酸酯钠0.7%、焦磷酸钠0.7%、纤维素钠0.8%、黄原胶0.9%、糖精钠0.4%、香精0.6%,余量为去离子水。
进一步,所述辅料组成及其重量百分比为:山梨糖醇28%、丙二醇17%、磷酸氢钙25%、月桂醇硫酸酯钠0.9%、焦磷酸钠0.7%、纤维素钠0.8%、黄原胶0.9%、木糖醇0.7%、香精0.5%,余量为去离子水。
本发明的优点和技术效果如下:
(1)本发明中印楝叶提取物的总黄酮、总萜类和总香豆素具有很好的抑制幽门螺杆菌活性,其MIC是25.5mg/mL,与文献报道中索式提取法获得的印楝叶索式提取物相比,其抑制幽门螺杆菌活性更显著。以0.5%的比例与0.3%的银杏叶提取物混合使用时,印楝叶提取物MIC值降低至12.75mg/mL,即印楝叶提取物与银杏叶提取物在抑制幽门螺杆菌过程中,产生了协同效应,在产生抑菌效应的同时,降低了印楝叶提取物的MIC值与银杏叶提取物的MIC值(银杏叶提取物MIC值由单用时的10.5mg/mL降至合用时的5.25mg/mL,远低于献报道的银杏叶提取物MIC值24mg/mL),减少了用药量。
(2)本发明除了具有抑制幽门螺杆菌的活性外,还具有抑制环境中常见细菌和真菌的活性,制备食品、药品、保健品和日化用品,可避免添加防腐剂、抑菌剂等,尤其是应用于日常使用的牙膏,其能维护口腔正常菌群,长期使用而无副作用。
(3)本发明所用的印楝叶提取物采用超临界二氧化碳方法制备,与文献报道的索氏提取法相比,获得的总黄酮、总香豆素、总萜类提取效率更高,抑制幽门螺杆菌效果更好;同时大大减少有机溶剂的使用,既保护环境又避免提取物有机溶剂残留的问题,适于工业化大生产。
(4)本发明综合利用了印楝叶,其可以充分利用资源,降低生产成本。
具体实施方式
下面通过具体实施方式对本发明做进一步说明,但本发明保护范围不局限于所述内容。下面提到的印楝叶提取物即采用超临界法从印楝叶中获得的提取物,与印楝叶索式提取物有所区分。
实施例1:印楝叶提取物体外抑菌实验
1体外抑菌实验
1.1实验细菌:幽门螺杆菌株(NCTC11637)、大肠埃希菌药敏标准株(ATCC25922)、金黄色葡萄球菌药敏标准株(ATCC25923)、酵母菌(ATCC90028)、毛霉菌(ATCC40241)。
1.2含供试品溶液的药敏纸片制备
将直径6mm的空白药敏纸片放入培养皿中,120℃高压蒸气灭菌45min,60℃烘干备用。使用灭菌枪头吸取20μL(2.50g/mL,浓度以提取物质量计)供试品溶液加在灭菌滤纸片上。另制备灭菌双蒸水纸片作为对照。
1.3受试菌液制备
1.3.1幽门螺杆菌
1.3.1.1幽门螺杆菌菌种静置培养:将幽门螺杆菌株接种于哥伦比亚琼脂培养基,置于微需氧发生袋中密封,放于37℃厌氧培养箱中静置培养72h。
1.3.1.2幽门螺杆菌菌液制备:用L型灭菌玻棒刮取菌苔,放入灭菌生理盐水中,采用比浊法将幽门螺杆菌配制成1*108CFU·mL-1的菌液。
1.3.2大肠埃希菌
1.3.2.1大肠埃希菌菌种培养:将大肠埃希菌株密集划线接种于MH琼脂培养基,在37℃培养箱中静置培养24h。
1.3.2.2大肠埃希菌菌液制备:按1.3.1.2的方法,将大肠埃希菌配制成1*106CFU·mL-1的菌液。
1.3.3金黄色葡萄球菌
1.3.3.1金黄色葡萄球菌菌种培养:将金黄色葡萄球菌株密集划线接种于MH琼脂培养基,在37℃培养箱中静置培养24h。
1.3.3.2金黄色葡萄球菌液制备:按1.3.1.2的方法,将金黄色葡萄球菌配制成1*106CFU·mL-1的菌液。1.3.4酵母菌
1.3.4.1酵母菌菌种培养:将酵母菌株密集划线接种于YPD琼脂培养基,在25℃培养箱中静置培养72h。
1.3.4.2酵母菌菌液制备:按1.3.1.2的方法,将酵母菌配制成1*109CFU·mL-1的菌液。1.3.5毛霉菌
1.3.5.1毛霉菌菌种培养:将毛霉菌密集划线接种于YPD琼脂培养基,在25℃培养箱中静置培养72h。
1.3.5.2毛霉菌菌液制备:按1.3.1.2的方法,将毛霉菌配制成1*109CFU·mL-1的菌液。
1.4药物敏感实验操作
1.4.1幽门螺杆菌实验操作:将0.1mL幽门螺杆菌液加入哥伦比亚琼脂培养基,立即用L型玻棒均匀涂布,将含供试品溶液的药敏纸片置于上述培养基中,另将灭菌双蒸水纸片置于哥伦比亚琼脂培养基上,作为阴性对照,放于37℃厌氧培养箱中静置培养72h,观察抑菌环大小。
1.4.2大肠埃希菌和金黄色葡萄球菌实验操作:分别将0.1mL大肠埃希菌液、0.1mL金黄色葡萄球菌液加入MH琼脂培养基,立即用L型玻棒均匀涂布,将含供试品溶液的药敏纸片置于上述培养基中,另将灭菌双蒸水纸片置于MH琼脂培养基上,作为阴性对照,在37℃培养箱中静置培养24h,观察抑菌环大小。
1.4.3酵母菌和毛霉菌实验操作:分别将0.1mL酵母菌液、0.1mL毛霉菌液加入到YPD琼脂培养基,立即用L型玻棒均匀涂布,将含供试品溶液的药敏纸片置于上述培养基中,另将灭菌双蒸水纸片置于YPD琼脂培养基上,作为阴性对照,在25℃培养箱中静置培养72h,观察抑菌环大小。
2印楝叶原料预处理
将干燥的印楝叶粉碎,过80目筛,备用;
3单因素实验考察超临界二氧化碳萃取制备印楝叶提取物最佳工艺条件
3.1萃取压力对萃取效果的影响
以4mL/g乙醇为夹带剂,萃取温度40℃,萃取2h,考察萃取压力分别为20、25、30、35、40MPa对萃取效果的影响,以“1.4.2”和“1.4.3”中的体外抑菌实验为评价指标。结果如表1所示。随着萃取压力的提高,四种菌的抑菌环均增大,可以认为,萃取压力提高,印楝叶中抑菌类有效成分的提取率增大;当萃取压力大于30MPa时,抑菌环直径最大,且之后变化幅度较小,可以认为,当萃取压力达到30MPa时,印楝叶中抑菌类有效成分的提取效果最佳。
表1萃取压力的影响结果(n=3)
3.2萃取温度对萃取效果的影响
以4mL/g乙醇为夹带剂,萃取压力30MPa,萃取2h,考察萃取温度分别为30、35、40、45、50℃对萃取效果的影响,以“1.4.2”和“1.4.3”中的体外抑菌实验为评价指标。结果如表2所示。随着温度的提高,四种菌的抑菌环直径出现先增大后减小的现象,当萃取温度为40℃时,印楝叶提取物的抑菌效果最佳。这可能是因为,随着温度的提高,超临界二氧化碳对印楝叶中抑菌类有效成分的溶解度增大。当温度过高时,存在高温对有效成分的破坏、超临界二氧化碳对有效成分的溶解度减小等问题,导致抑菌效果反而降低。
表2萃取温度的影响结果(n=3)
3.3萃取时间对萃取效果的影响
以4mL/g乙醇为夹带剂,萃取压力30MPa,萃取温度40℃,考察萃取时间分别为30、60、90、120、150、180min对萃取效果的影响,以“1.4.2”和“1.4.3”中的体外抑菌实验为评价指标。结果如表3所示。随着萃取时间的延长,四种菌的抑菌环直径出现先增大后基本稳定的现象。这可能是因为,随着萃取时间的延长,印楝叶中的抑菌类有效成分浸出率提高,但是,萃取时间的延长也会导致杂质浸出率的提高,或者长时间受热产生的成分破坏等现象,因而出现萃取时间延长,而抑菌环直径反而减小的情况。当萃取时间为120min时,抑菌效果最佳。
表3萃取时间的影响结果(n=3)
3.4夹带剂用量对萃取效果的影响
萃取压力30MPa,萃取温度40℃,萃取时间为120min,考察夹带剂乙醇的添加量分别为2.5、3、3.5、4、4.5、5mL/g对萃取效果的影响,以“1.4.2”和“1.4.3”中的体外抑菌实验为评价指标。结果如表4所示。随着夹带剂用量的增加,四种菌的抑菌环直径逐渐增大后基本稳定,当夹带剂用量为4mL/g时,抑菌环最大,即抑菌效果最佳。二氧化碳为弱极性物质,在萃取过程中,加入一定量的乙醇,能够提高有效成分在超临界二氧化碳里的溶解度。
表4夹带剂用量对结果的影响(n=3)
4工艺验证
以最佳萃取工艺条件:4mL/g乙醇为夹带剂,萃取压力30MPa,萃取温度40℃,萃取120min,以“1.4.2”和“1.4.3”中的体外抑菌实验为评价指标,开展3组平行实验,结果如表5所示。该工艺稳定可行。
表5超临界萃取工艺验证
实施例2:印楝叶超临界萃取与索氏提取的提取物区别
1有效成分含量区别
1.1超临界萃取印楝叶
根据实施例1中“4工艺验证”的最佳工艺条件进行印楝叶的提取得到印楝叶提取物。
1.2索氏提取法提取印楝叶
取40~50g印楝叶细粉,在65℃下用甲醇索氏提取10h,提取液在50℃下旋蒸得到粗提物。用无水硫酸铜真空干燥得到固体或膏状的印楝叶索式提取物。
1.3总黄酮、总萜类和总香豆素的含量检测实验
采用Al(NO3)3-NaNO2-NaOH体系比色法测定提取物中总黄酮含量,以芦丁为对照品。取2.5mL不同浓度的芦丁标准品溶液,加入一定体积的5%亚硝酸钠溶液,混匀,静置6min,加入相同体积的10%(m/V)硝酸铝溶液,混匀,静置,再加入一定体积的4%(m/V)氢氧化钠溶液,定容至25mL,充分混匀,静置一定时间,在510nm下测定吸光度,绘制芦丁标准曲线。取提取物进行Al(NO3)3-NaNO2-NaOH体系比色法分析,根据标准曲线换算得到提取物中总黄酮的含量,分别取5批次印楝叶提取物和5批次印楝叶索氏提取物进行检测。
采用比色法检测提取物中总萜类含量,在萜类物质中加入硫酸的香兰素甲醇溶液进行显色反应后绘制标准曲线,标准品为印楝素。取不同质量的印楝素溶于二氯甲烷配制成不同浓度,取1.4mL加入试管中,再加入香兰素乙醇溶液(0.02g/mL)0.4mL,振荡混合均匀,静置2min分3次加入浓硫酸(98%)0.6mL,每次加入后迅速振荡10s。然后加入1.4mL甲醇溶液,注意小心振荡均匀,溶液变为蓝绿色。静置10min后,用1cm比色皿测其光吸收值(577nm),绘制印楝素标准曲线。取提取物进行比色法分析,根据标准曲线换算得到提取物中总萜类的含量,分别取5批次印楝叶提取物和5批次印楝叶索氏提取物进行检测。
采用比色法检测提取物中总香豆素含量,以东莨菪内酯为对照品。称取东莨菪内酯对照品8.6mg,置100mL量瓶中,加乙醇溶解并稀释至刻度,精密量取该溶液5mL置10mL量瓶中,加乙醇稀释至刻度,得对照品溶液。取不同浓度的对照品溶液,在348nm下测定吸光度,绘制东莨菪内酯标准曲线。取提取物进行比色法分析,根据标准曲线换算得到提取物中总香豆素的含量,分别取5批次印楝叶提取物和5批次印楝叶索氏提取物进行检测。
1.4总黄酮、总萜类和总香豆素的含量检测结果
对超临界萃取法和索氏提取法获得的提取物成分进行对比,结果如表6所示。超临界萃取法获得的印楝叶提取物中,总黄酮、总萜类和总香豆素均比索氏提取法获得的要高。这也意味着这两种方法获得的提取物可能有不同的作用效果。
表6两种提取方法获得的提取物中总黄酮、总萜类和总香豆素的含量对比
2印楝叶提取物与印楝叶索氏提取物的抗幽门螺杆菌效果对比
取本实施例1.1印楝叶提取物和本实施例1.2中的印楝叶索氏提取物,按实施例1中“1.4.1幽门螺杆菌实验操作”进行幽门螺杆菌抑菌实验。根据实施例1中“1.2含供试品溶液的药敏纸片制备”的方法制备浓度梯度的药敏纸片,实验结果如表7所示。
表7两种萃取方法获得的印楝叶提取物抗幽门螺杆菌效果对比
实施例3:牙膏中印楝叶提取物与银杏叶提取物组方实验
1银杏叶提取物制备
银杏叶提取物按照《中国药典》(2015版)“银杏叶提取物”项下规定方法制得。
2印楝叶提取物制备
根据实施例1中“4工艺验证”的最佳工艺条件进行印楝叶的提取得到粗提物。
3印楝叶提取物与银杏叶提取物的最小抑菌浓度(MIC)测定
使用试管倍比稀释法,分三组进行MIC值测定,第一组测定印楝叶提取物,第二组测定银杏叶提取物,第三组测定混合提取物(印楝叶提取物与银杏叶提取物混合,初始浓度分别为25.5mg/mL、10.5mg/mL)。取无菌试管17支,每支试管中加入布氏肉汤1mL,供试药液1mL,其中,第一组5支试管,编号1~5,加入印楝叶提取物浓度分别为6.375mg/mL、12.75mg/mL、25.5mg/mL、51.0mg/mL、102.0mg/mL;第二组5支试管,编号5~10,加入银杏叶提取物浓度分别为2.625mg/mL、5.25mg/mL、10.5mg/mL、21.0mg/mL、42.0mg/mL;第三组4支试管,编号11~14,分别加入倍比稀释后的混合提取物溶液,各编号试管对应浓度分别为:11-(银杏叶提取物2.625mg/mL、印楝叶提取物6.375mg/mL)、12-(银杏叶提取物5.25mg/mL、印楝叶提取物12.75mg/mL)、13-(银杏叶提取物10.5mg/mL、印楝叶提取物25.5mg/mL)、14-(银杏叶提取物21.0mg/mL、印楝叶提取物51.0mg/mL)、15-(银杏叶提取物42.0mg/mL、印楝叶提取物102.0mg/mL);编号16、17的2支试管为对照组,不加供试药液。各管内分别加入实施例1中“1.3.1”制备的幽门螺杆菌菌液0.1mL,混匀后置微需氧发生袋中,连同产气袋一起放置于适当大小的烧杯中,保持试管直立状态,放于37℃厌氧培养箱中震荡培养72h观察结果。液体清澈为有抑菌作用,混浊为有细菌生长。无细菌生长的最小浓度为MIC值。
MIC值检测结果:印楝叶提取物MIC值为25.5mg/mL;银杏叶提取物MIC值为10.5mg/mL;两者混合使用时,印楝叶提取物MIC值为12.75mg/mL、银杏叶提取物MIC值为5.25mg/mL。
4牙膏中印楝叶提取物与银杏叶提取物组方抑菌实验
以牙膏基础配方为起点,分别添加不同比例的银杏叶提取物和印楝叶提取物,以实施例1中“1药物体外抑菌实验”为评价指标,考察提取物最佳添加量。结果如表8~9所示。
牙膏的基础配方主要由保湿剂、摩擦剂、粘合剂、发泡剂、甜味剂、香精、水组成。这类辅料物质基本没有抑菌效果。从表8可知,添加银杏叶提取物、印楝叶提取物后,膏体体现出抑菌效果,对大肠埃希菌和金黄色葡萄球菌均有抑制效果,但是单独添加任何一种提取物,其抑菌效果增强并不显著,且对于幽门螺杆菌的抑制效果基本不体现。当膏体中同时添加银杏叶提取物和印楝叶提取物后,膏体对大肠埃希菌和金黄色葡萄球菌的抑制作用明显增强,增强了近1倍;对幽门螺杆菌也表现出了显著的抑制作用。说明,银杏叶提取物和印楝叶提取物在抑制幽门螺杆菌方面体现出了协同作用。
进一步对银杏叶提取物和印楝叶提取物的添加量及比例进行了研究,结果如表9所示。当银杏叶提取物添加量从0.3%降低到0.1%后,无论印楝叶提取物同比例添加(添加量为0.1%)还是提高比例(添加量为0.5%),抑菌效果都会明显降低;当银杏叶提取物添加比例为0.3%时,膏体表现出较强的抑菌效果,以幽门螺杆菌抑制效果的增强为突出表现;随着印楝叶提取物添加比例的提高,抑菌环直径逐渐增加,并趋于稳定。综合比较,以银杏叶提取物添加量0.3%,印楝叶提取物添加量0.5%为抑菌效果最佳。
表8添加提取物种类比较研究(n=3)
表9提取物添加比例考察(n=3)
实施例4:印楝叶提取物牙膏制备
1银杏叶提取物制备
按照实施例3中“1银杏叶提取物制备”方法制得。
2印楝叶提取物制备
(1)将干燥的印楝叶粉碎,过80目筛,备用;
(2)将步骤(1)中的印楝叶粉投入萃取釜,选择萃取温度40℃,萃取压力为30MPa,以4mL/g乙醇为夹带剂,萃取120min;含印楝叶成分的二氧化碳流体进入分离釜,降低压力和升高温度,使CO2气化,收集萃取液,CO2气体经冷凝后循环使用;
(3)将步骤(2)中的萃取液减压浓缩至相对密度1.15的浸膏,喷雾干燥得到粗粒的印楝叶提取物。
(4)将步骤(3)中的粗粒的印楝叶提取物粉碎,过180目筛即得印楝叶提取物。
3印楝叶提取物牙膏-1
配方:按膏体重量比例加入0.3%的银杏叶提取物,0.5%的印楝叶提取物;辅料重量比例分别为:山梨醇34%、丙二醇14%、二氧化硅28%、月桂醇硫酸酯钠0.7%、焦磷酸钠0.7%、纤维素钠0.8%、黄原胶0.9%、糖精钠0.4%、香精0.6%,余量为去离子水,总量为100%。
(1)将银杏叶提取物、印楝叶提取物、山梨醇、丙二醇、月桂醇硫酸酯、焦磷酸钠、糖精钠、香精和适量去离子水混合搅拌均匀后,与纤维素钠、黄原胶混合,再加入二氧化硅混合,经搅拌均匀,制成具有一定粘性、稀稠适当的膏体;
(2)研磨:膏体经过研磨机研磨,可将过粗、过硬的颗粒磨细,使膏体细腻、均匀、稳定,研磨可分两次进行,第一次研磨后贮存,第二次研磨后要进行真空脱气;
(3)陈化:将膏体打入储存锅进行陈化,使物料自然冷却至常温,同时使物料充分膨胀形成均相的粘合体,提高物料的弹性;
(4)灌装:脱气后的膏体经静置后即可通过自动灌装入定制的软管内;
(5)封口。
4印楝叶提取物牙膏-2
配方:按膏体重量比例加入0.3%的银杏叶提取物,0.5%的印楝叶提取物;所述辅料组成及其重量百分比为:山梨糖醇28%、丙二醇17%、磷酸氢钙25%、月桂醇硫酸酯钠0.9%、焦磷酸钠0.7%、纤维素钠0.8%、黄原胶0.9%、木糖醇0.7%、香精0.5%,余量为去离子水。
(1)将银杏叶提取物、印楝叶提取物、山梨糖醇、丙二醇、月桂醇硫酸酯钠、焦磷酸钠、木糖醇、香精和适量去离子水混合搅拌均匀后,与纤维素钠、黄原胶混合,再加入磷酸氢钙混合,经搅拌均匀,制成具有一定粘性、稀稠适当的膏体;
(2)研磨:膏体经过研磨机研磨,可将过粗、过硬的颗粒磨细,使膏体细腻、均匀、稳定,研磨可分两次进行,第一次研磨后贮存,第二次研磨后要进行真空脱气;
(3)陈化:将膏体打入储存锅进行陈化,使物料自然冷却至常温,同时使物料充分膨胀形成均相的粘合体,提高物料的弹性;
(4)灌装:脱气后的膏体经静置后即可通过自动灌装入定制的软管内;
(5)封口。
5印楝叶提取物牙膏-3
配方:按膏体重量比例加入0.3%的银杏叶提取物,0.5%的印楝叶提取物;所述辅料组成及其重量百分比为:山梨醇38%、丙二醇17%、碳酸钙27%、焦磷酸钠1.6%、纤维素钠1.7%、木糖醇2.4%、香精0.5%,余量为去离子水。
(1)将银杏叶提取物、印楝叶提取物、山梨醇、丙二醇、焦磷酸钠、木糖醇、香精和适量去离子水混合搅拌均匀后,与纤维素钠混合,再加入碳酸钙混合,经搅拌均匀,制成具有一定粘性、稀稠适当的膏体;
(2)研磨:膏体经过研磨机研磨,可将过粗、过硬的颗粒磨细,使膏体细腻、均匀、稳定,研磨可分两次进行,第一次研磨后贮存,第二次研磨后要进行真空脱气;
(3)陈化:将膏体打入储存锅进行陈化,使物料自然冷却至常温,同时使物料充分膨胀形成均相的粘合体,提高物料的弹性;
(4)灌装:脱气后的膏体经静置后即可通过自动灌装入定制的软管内;
(5)封口。
实施例5:印楝叶提取物牙膏的防腐与抗幽门螺杆菌作用
1印楝叶提取物牙膏防腐作用
1.1牙膏抑菌性能测试方法:试验菌为大肠埃希菌、金黄色葡萄球菌、酵母菌和毛霉菌;其中大肠埃希菌与金黄色葡萄球菌采用连续转种24h新鲜细菌培养物,培养基为MH琼脂培养基;酵母菌和毛霉菌采用连续转种72h新鲜真菌培养物,培养基为YPD琼脂培养基。牙膏基础配方作阴性对照,随机购买2个含防腐剂的品牌牙膏为阳性对照,将这些牙膏的抑菌结果与银杏印楝牙膏进行对照。
1.2操作步骤
取被测牙膏样品3g于无菌平皿中,涂成3.0cm×3.0cm,各制2个平皿。取实施例1中的“1.3受试菌液制备”得到的菌悬液,分别在每个试验平皿上滴加0.1mL,涂布均匀,开始计时,于20min后用3mL无菌PBS(0.03mol·L-1,pH 7.2),作适当稀释,然后取其中2~3个稀释度,分别吸取1.0mL置于2个平行平皿,用凉至40~50℃的营养琼脂15mL作倾注,使其充分混合均匀,凉至琼脂凝固翻转平板,大肠埃希菌与金黄色葡萄球菌置于37℃培养24h,作菌落计数为回收菌数。酵母菌与毛霉菌置于25℃培养72h,作菌落计数为回收菌数。取无菌平皿为空白对照,按照上述步骤进行处理,分别记录四种菌的菌落数为平板实验起始菌落。
1.3计算
抑菌率=[(A-B)/A]×100%。
式中:A——用于平板实验起始菌落;
B——试验样品平均回收菌落。
1.4实验结果
表10牙膏抑菌实验结果
2印楝叶提取物牙膏临床抗幽门螺杆菌实验
2.1唾液样本获取
患者晨起空腹,用无菌生理盐水漱口2次,每次1min,取第二次生理盐水漱口液10mL,15000r·min-1离心20min,弃去上清液,作为0时刻样本,备用;患者立刻用1g上述方法制备的印楝叶提取物牙膏刷牙,刷牙保持3min,无菌纯水洗净口腔,不得有牙膏残留,再用无菌生理盐水漱口2次,每次1min,取第二次生理盐水漱口液10mL,15000r·min-1离心20min,弃去上清液,作为1时刻样本,备用;患者禁饮食2h,再用无菌生理盐水按上述方法制备唾液样本,作为2时刻样本,备用。另,随机购买2个品牌牙膏作为牙膏对照组,以不刷牙仅无菌纯水漱口3min为空白对照组,按上述方法留取唾液样本。每个实验组随机分配10名幽门螺杆菌患者。
幽门螺杆菌选择性培养基制备:在哥伦比亚琼脂培养基中加入幽门螺杆菌选择性添加剂和5%脱纤维绵羊血,混匀,备用。
将唾液样本接种于上述幽门螺杆菌选择性培养基中,置于微需氧发生袋中密封,放于37℃厌氧培养箱中培养72h。观察幽门螺杆菌生长情况,计培养皿上的幽门螺杆菌菌落数,实验结果见表11。牙膏幽门螺杆菌实验结果显示,使用印楝叶提取物牙膏能有效抑制口腔中幽门螺杆菌,且其抑菌效果能维持至少2h。
表11幽门螺杆菌抑菌实验结果
Claims (10)
1.一种印楝叶提取物,其特征在于,该提取物的主要药效成分为总黄酮20~35 %,总萜类5~10 %,总香豆素2~5 %。
2.一种权利要求1所述的印楝叶提取物的制备工艺,其特征在于包括以下步骤:
(1)印楝叶原料的预处理:将干燥的印楝叶原料粉碎,过80~100目筛,备用;
(2)采用超临界二氧化碳法萃取制备印楝叶提取物:萃取压力25~50 MPa,萃取温度30~50 ℃,萃取时间60~180 min,3~5 mL/g乙醇为夹带剂;
(3)将印楝叶提取液浓缩、喷雾干燥;
(4)将干燥的印楝叶提取物粉碎,过180~200目筛备用。
3.根据权利要求2所述的印楝叶提取物的制备工艺,其特征在于,步骤(2)采用超临界二氧化碳法萃取制备印楝叶提取物:萃取压力30 MPa,萃取温度40 ℃,萃取时间120 min,4mL/g乙醇为夹带剂。
4.根据权利要求1所述的印楝叶提取物在制备抑制幽门螺杆菌活性的医药品、保健品、食品和日化用品中的应用。
5.一种含有如权利要求1所述的印楝叶提取物的牙膏,其特征在于,牙膏组成物及重量百分比为:银杏叶提取物0.05~2 %,印楝叶提取物0.10~2 %,余量为辅料,所有组分总量为100 %。
6.根据权利要求5所述的印楝叶提取物牙膏,其特征在于,银杏叶提取物添加量为0.3%,印楝叶提取物添加量为0.5 %。
7.根据权利要求5所述的印楝叶提取物牙膏,其特征在于,所述辅料组成及其重量百分比为:保湿剂10~70 %、摩擦剂10~30 %、粘合剂0.1~5 %、发泡剂0.1~10 %、甜味剂0.1~5 %、香精0.3~0.7 %,余量为去离子水。
8.根据权利要求7所述的印楝叶提取物牙膏,其特征在于,保湿剂为山梨醇、山梨糖醇、丙二醇中的一种或几种任意比的混合物,摩擦剂为碳酸钙、二氧化硅、磷酸氢钙中的一种或几种任意比的混合物,粘合剂为纤维素钠、黄原胶中的一种或两种任意比的混合物,发泡剂为月桂醇硫酸酯钠、焦磷酸钠中的一种或两种任意比的混合物,甜味剂为糖精钠、木糖醇中的一种或两种任意比的混合物。
9.根据权利要求书7或8所述的印楝叶提取物牙膏,其特征在于,所述辅料组成及其重量百分比为:山梨醇34%、丙二醇14%、二氧化硅28%、月桂醇硫酸酯钠0.7%、焦磷酸钠0.7%、纤维素钠0.8%、黄原胶0.9%、糖精钠0.4%、香精0.6%,余量为去离子水。
10.根据权利要求书7或8所述的印楝叶提取物牙膏,其特征在于,所述辅料组成及其重量百分比为:山梨糖醇28%、丙二醇17%、磷酸氢钙25%、月桂醇硫酸酯钠0.9%、焦磷酸钠0.7%、纤维素钠0.8%、黄原胶0.9%、木糖醇0.7%、香精0.5%,余量为去离子水。
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