CN110368408A - 一种诃子糅质茶多酚的复合抑菌物及制备方法 - Google Patents
一种诃子糅质茶多酚的复合抑菌物及制备方法 Download PDFInfo
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- CN110368408A CN110368408A CN201910771746.5A CN201910771746A CN110368408A CN 110368408 A CN110368408 A CN 110368408A CN 201910771746 A CN201910771746 A CN 201910771746A CN 110368408 A CN110368408 A CN 110368408A
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Abstract
本发明公开了一种诃子糅质茶多酚的复合抑菌物,所述的复合抑菌物按照重量包括：诃子糅质30‑70份、茶多酚30‑70份、水溶性膳食纤维粉5‑10份、氯化钾溶液10‑20份、水适量；本发明还公开了一种诃子糅质茶多酚的复合抑菌物的制备方法，该方法包括：步骤一、将诃子果实粉碎后，用纯净水水煮浸提1‑3次，合并三次水煮液、过滤、浓缩成诃子糅质；步骤二、从茶叶中提取茶多酚；步骤三、取上述诃子糅质30‑70份，用水溶解，之后用过滤膜除菌；步骤四、加入氯化钾溶液10‑20份，加入水溶性膳食纤维粉5‑10份、茶多酚30‑70，充分搅拌；步骤五、用调节上述溶液的PH值为6‑8。
Description
技术领域
本发明涉及植物有效成份领域，具体来说是一种诃子糅质茶多酚的复合抑菌物及制备方法，更具体的涉及一种抑制口腔、肠道及其他外伤微生物感染的中药有效部位组合物及其制备和应用。
背景技术
诃子对常见的食源性致病微生物具有明显的抑制作用，可表现出广谱的抗菌作用。体外实验证明了成熟诃子果实的皮对某些痢疾杆菌的抑菌效果也十分显著，其原理可能为糅质可使菌体内的蛋白质凝结。有报道研究发现诃子的水提取物在121℃的高压灭菌条件下半小时后仍然具有抑菌活性，浓度在约2mg/ml范围里的依旧可以抑制幽门螺杆菌的生长。
Sato等人研究发现从诃子果实对金黄色葡萄球菌、绿脓杆菌、痢疾杆菌的有效浓度分别为1:128；1:64；1:32。并且诃子的提取物通过口腔抑菌测试发现具有明显抑制链球菌等微生物的生长以及微生物所引起的载附现象和糖酵解作用。Malekzadeh F等人研究了诃子水提物对幽门螺旋杆菌的抑制作用，结果表明抑菌效果良好，最低抑菌浓度(MIC为150mg/ml)。同时研究了诃子提取物中主要成分多酚类物质对十余种胃肠病致病菌都具有抑制活性，尤其对气单胞菌、枯草芽胞杆菌、霍乱型弧菌的抑制作用较强。
根据Sing P等人的研究结果：诃子对植物病原菌也有一定的抑制作用，研究显示诃子对7种真菌和霉菌也具有一定的抑制效果，因此发现可用于生物环保防治植物病原菌等。康帅等人用体外实验方法测定了诃子对放线杆菌的抑菌能力强弱，采用了二倍稀释法，结果表明在体外条件下，其最小抑菌浓度是一个范围，约在6mg-50mg/mL之间，说明诃子果实具有良好的抗菌活性。刘伟等利用诃子提取物对三种食源性微生物的抑菌能力进行了研究，结果表明诃子水提物对三种食源性微生物均有明显的抑菌作用，最低抑菌浓度(MIC)均为0.625g/mL,抑菌圈直径为0.68cm。
在诃子水提物中分离出的诃子酸，鞣花酸，1,3,6-三-o-没食子酰基-β-D-葡萄糖等糅质类成分都有很好的抑菌活性。使用传统的中医疗法治疗痢疾等微生物感染疾病一直是日常使用的良方。
茶叶中的有效成分——茶多酚，作为一种天然的抗氧化剂，具有很好的应用价值。茶多酚又名茶单宁、茶鞣质，是茶叶中所含有的一类多羟基酚类化合物的总称，简称为TP。其中主体儿茶素类为2－苯基苯并吡喃的衍生物，包括表儿茶素(EC)、没食子儿茶素(GC)、表没食子儿茶素(EGC)、表儿茶素没食子酸酯(ECG)、没食子儿茶素没食子酸酯(GCG)、表没食子儿茶素没食子酸酯(EGCG)。现代研究已证实，茶多酚的抑菌谱广，对革兰氏阳性和阴性细菌均有抑制作用，如对肉制品中常见的致病菌金黄色葡萄球菌、大肠杆菌和沙门氏菌都有抑菌活性。茶多酚对金黄色葡萄球菌、枯草芽孢杆菌和大肠杆菌的最低抑菌浓度分别为6.25％、12.5％、25％；茶多酚的浓度越高与菌种作用时间越长，抑菌活性越强；在弱碱性环境中的抑菌活性最强；一定温度内处理不影响茶多酚的抑菌活性；氯化钠能增强茶多酚的抑菌活性。
发明内容
本发明的目的在是提供增效的抑菌组合，使诃子鞣质与茶多酚的抑菌强度和范围更广，更有利于体外和肠道抑菌。
本发明针对诃子糅质已知抑菌活性结合茶多酚已知抑菌活性，提出两种物质组合增强抑菌效果的假设，并添加带有氯离子的盐溶液以及可溶性膳食纤维作为抗菌辅助剂，并通过一系列实验分析和综合评估、对比论证，结果证明抑菌有显著疗效的实验结果为由中药有效部位提取一定比例组合的组合物。本发明涉及有效部位的提取及其抑菌生物活性的增效对比，它作为外伤口感染外用涂抹或是口腔抑菌和治疗痢疾均具有良好的应用前景。
具体来说，本发明公开了一种诃子糅质茶多酚的复合抑菌物,所述的复合抑菌物按照重量包括：诃子糅质30-70份、茶多酚30-70份、水溶性膳食纤维粉5-10份、氯化钾溶液10-20份、氯化钠溶液5-10份、氯化钙溶液5-10份，水适量。
实验发现，添加氯离子、和钾、纳以及钙离子和可溶性膳食纤维作为抗菌辅助剂，能够提高诃子糅质和茶多酚的抗菌协同效果，制备的复合物的抗菌性明显优于单个抗菌物。
在一些优选的实施例中，各组分包括：诃子糅质30份、茶多酚70份、2％的氯化钾溶液10份、水溶性膳食纤维粉10份，5％的氯化钙5份、5％的氯化钠5份、水适量。
在一些优选的实施例中，水溶性膳食纤维粉为低聚果糖、低聚异麦芽糖、低聚乳糖、低聚木糖、大豆低聚糖、琼脂粉、羧甲基纤维素的一种或多种的混合物。
本发明还公开了一种诃子糅质茶多酚的复合抑菌物的制备方法，该方法包括：
步骤一、将诃子果实粉碎后，加入水，加入纳米铜，形成纳米铜悬浮液，用微波辐射，纳米铜在水中形成悬浮液，用纯净水水煮浸提1-3次，合并三次水煮液、过滤、浓缩成诃子糅质；
步骤二、从茶叶中提取茶多酚；
步骤三、取上述诃子糅质30-70份，用水溶解，之后用过滤膜除菌；
步骤四、加入氯化钾溶液10-20份，加入水溶性膳食纤维粉5-10份、茶多酚30-70，氯化钙5份，氯化钠5份，充分搅拌；
步骤五、用调节上述溶液的PH值为6-8。
在本发明中施加微波一方面能够使得纳米金属碰撞植物细胞，使得溶解的更加充分，另一方面微波辐射会导致溶液温度过高，纳米金属粒子能够在一定程度提高散热效果，防止溶液温度过高。在一些情况下，纳米铜能够改变溶剂对有效成份的提取性能，进一步的提高提取率，提取率能够提高20％以上。
在一些优选的实施例中，所述的步骤一包括：选用水做为提取溶剂，将3.5kg的诃子果实粉碎后40目筛，再将粉末置于大锅内，加入纳米铜形成纳米铜悬浮液，用粉末三倍的纯净水水煮浸提三次，微波辐射，每次30min，合并三次水煮液，用棉纱布过滤，在转蒸发仪上浓缩成浸膏。
在一些优选的实施例中，所述的水溶性膳食纤维粉为低聚果糖、低聚异麦芽糖、低聚乳糖、低聚木糖、大豆低聚糖、琼脂粉、羧甲基纤维素的一种或多种的混合物。
在一些优选的实施例中，所述的氯化钾溶液的浓度为2％-4％。
在一些优选的实施例中，诃子鞣质与茶多酚的重量比例为1:(0.3～5)。
在一些优选的实施例中，诃子鞣质与茶多酚的重量比例为1:(0.5～3)。
在一些优选的实施例中，各原料包括：诃子糅质30份、茶多酚70份、2％的氯化钾溶液10份、羧甲基纤维素10份、5％的氯化钙5份、5％的氯化钠5份、适量水以使得上述物质溶解进而制备抑菌物。
具体实施方式
下面对本发明作详细的说明。
一、实验菌种
金黄色葡萄球菌(Staphylococcus aureus)
蜡样芽孢杆菌(Bacillusereu)
枯草芽孢杆菌(Bacillus subtilis)
沙门氏菌(Salmonelas Penteritidis)
志贺菌(Shigella)
弯曲空肠杆菌(Campylobacter jejuni)
白色念珠菌(Monilia albican)
大肠杆菌(Escherichia coli)。
二、实验材料
主要是诃子果实(西藏)、茶多酚、可溶性膳食纤维等。
三、实验方法
3.1提取物制备
1)诃子糅质提取物制备
选用水做为提取溶剂，将3.5kg的诃子果实粉碎后40目筛。再将粉末至于大锅内，加入水和纳米铜，纳米铜平均粒径平均粒径80nm、纯度99.9％，比表面积14.5，体积密度0.25g/cm3，密度8.9g/cm3，用微波辐射，微波频率为400MHz，功率为200w，在用水提取的时候，纳米铜在水中形成悬浮液，用柯子粉末三倍的纯净水水煮浸提三次，每次30min，合并三次水煮液，用棉纱布过滤，量取100ml水煮液旋转蒸发仪上浓缩成浸膏。
2)细菌液体培养基的配制
称取牛肉膏固体0.33g，Nacl粉末5g，蛋白胨粉末3.3g，置于1000ml三角瓶中，加入蒸馏水300ml，混匀微热溶解，用封口膜将瓶口封住后外包牛皮纸，置于121℃高压灭菌锅中灭菌约60min后，在超净台中吹无菌风放凉，现用。
3)细菌固体培养基的配制
称取牛肉膏0.15g，蛋白胨5g，葡萄糖12g，Nacl5g，琼脂粉12g，置于1000ml三角瓶中，加入蒸馏水300ml，混匀微热溶解，混匀微热溶解，用封口膜将瓶口扎住后再从外面包上牛皮纸，置于121℃高压灭菌锅中灭菌。约60min后，灭菌完毕[8]，在超净台中放凉，现用。
4)细菌的活化培养
将各供试菌分别接于牛肉膏蛋白胨液体培养基中，设置温度为30-37℃，转速为100r/min的恒定条件下进行细菌培养，利用分光光度计检测菌体浓度，待细菌培养到对数期生长期之后，用稀释涂布计数法将各种供试菌用无菌水配制成为浓度为107cfu/mL的菌悬液，放置于冰箱备用。
5)抑菌圈测定
抑菌圈的测定采用滤纸片扩散法，利用打孔器将滤纸片处理成的圆形滤纸片，灭菌后，将滤纸片浸入供试样品溶液后取出，自然沥干样品溶液后贴在已经涂布好各种供试菌的平板上，每个培养皿内放入3片滤纸片，并用无菌水和二甲亚枫作空白对照。将培养皿于恒温培养箱中，在37℃条件下培养24～36h，取出观察，用直尺测量各培养皿中的抑菌圈直径大小(mm)，每组测量三次求其平均值，作为抑菌效果的衡量标准，每组实验重复3次。
本发明方法采用水提法所提取的诃子糅质的含量大于30％(W/W)，诃子糅质中的主要成分为，诃子酸、鞣花酸、1,3,6-三-o-没食子酰基-β-D-葡萄糖，其中诃子酸含量约占总含量的17％(以含量计)以上，在3.125mg/ml的浓度下，此三种物质均对金黄色葡萄球菌(107cfu/ml)具有微弱的抑制作用(见表1)。
表1诃子糅质中的三种成分对金黄色葡萄球菌的抑制作用(cm)
3.2茶多酚的提取和检测
购买或者提取茶多酚；
其中茶多酚中的主要成分EGCG。此物质在6.25mg/ml时，对金黄色葡萄杆菌(107mg/ml)的抑菌能力(见表2)
表2 EGCE的抑菌活性
3.3组合物制备
取上述诃子糅质，用水溶解，之后用过滤膜除菌；加入氯化钾溶液，加入水溶性膳食纤维粉、茶多酚，充分搅拌；醋酸或碳酸氢钠调节上述溶液的PH值为8。其中，诃子糅质30g、茶多酚70g、氯化钾2g、氯化钙0.5g、氯化钠0.5g、羧甲基纤维素10g，用水将组合物浓度配为6.25mg/ml，对金黄葡萄球菌(107cfu/ml)的抑制作用(见表3)，其中组合物的浓度为组合物质量与体积的比，组合物质量为诃子糅质、茶多酚、氯化钾、羧甲基纤维素、氯化钙、氯化钠和氯化钾的质量之和。
从表3中可以看出，组合物具有明显的抑菌效果.
表3抑菌物的的组成
表4抑菌效果
从表4中可以看出，在相同的浓度下，诃子糅质和茶多酚的组合物表现出一定的抑菌增效作用，但效果不明显，但是在加了氯化钾、氯化钙、氯化钠之后，组合物的抑菌效果得到明显提高，钾元素、钙元素、钠离子、氯离子以及可溶性膳食纤维对两者组合物的抑菌效果具有较强的增强作用，对于开发对痢疾及其它伤口炎症等的发生与发展具有明显的治疗作用药物是可行的。
Claims (10)
1.一种诃子糅质茶多酚的复合抑菌物,其特征在于，所述的复合抑菌物按照重量包括：诃子糅质30-70份、茶多酚30-70份、水溶性膳食纤维粉5-10份、氯化钾溶液10-20份、氯化钠溶液5-10份、氯化钙溶液5-10份，水适量。
2.根据权利要求1所述的诃子糅质茶多酚的复合抑菌物，其特征在于，各组分包括：诃子糅质30份、茶多酚70份、2％的氯化钾溶液10份、水溶性膳食纤维粉10份，5％的氯化钙5份、5％的氯化钠5份、水适量。
3.根据权利要求2所述的诃子糅质茶多酚的复合抑菌物，其特征在于，水溶性膳食纤维粉为低聚果糖、低聚异麦芽糖、低聚乳糖、低聚木糖、大豆低聚糖、琼脂粉、羧甲基纤维素的一种或多种的混合物。
4.一种诃子糅质茶多酚的复合抑菌物的制备方法，其特征在于，
步骤一、将诃子果实粉碎后，加入水，加入纳米铜，形成纳米铜悬浮液，用纯净水水煮浸提1-3次，辅助微波辐射，合并三次水煮液、过滤、浓缩成诃子糅质；
步骤二、从茶叶中提取茶多酚；
步骤三、取上述诃子糅质30-70份，用水溶解，之后用过滤膜除菌；
步骤四、加入氯化钾溶液10-20份，加入水溶性膳食纤维粉5-10份、茶多酚30-70，氯化钙5份，氯化钠5份，充分搅拌；
步骤五、调节上述溶液的PH值为6-8。
5.根据权利要求4所述的诃子糅质茶多酚的复合抑菌物的制备方法，其特征在于，所述的步骤一包括：选用水做为提取溶剂，将3.5kg的诃子果实粉碎后40目筛，再将粉末置于大锅内，加入纳米铜形成纳米铜悬浮液，微波辐射，用果实粉末三倍的纯净水水煮浸提三次，每次30min，合并三次水煮液，用棉纱布过滤，在转蒸发仪上浓缩成浸膏。
6.根据权利要求5所述的诃子糅质茶多酚的复合抑菌物的制备方法，其特征在于，所述的水溶性膳食纤维粉为低聚果糖、低聚异麦芽糖、低聚乳糖、低聚木糖、大豆低聚糖、琼脂粉、羧甲基纤维素的一种或多种的混合物。
7.根据权利要求6所述的诃子糅质茶多酚的复合抑菌物的制备方法，其特征在于，所述的氯化钾溶液的浓度为2％-4％。
8.根据权利要求7所述的诃子糅质茶多酚的复合抑菌物的制备方法，其特征在于，诃子鞣质与茶多酚的重量比例为1:(0.3～5)。
9.根据权利要求8所述的诃子糅质茶多酚的复合抑菌物的制备方法，其特征在于，诃子鞣质与茶多酚的重量比例为1:(0.5～3)。
10.根据权利要求9所述的诃子糅质茶多酚的复合抑菌物的制备方法，其特征在于，各原料包括：诃子糅质30份、茶多酚70份、2％的氯化钾溶液10份、羧甲基纤维素10份、氯化钙5份、氯化钠5份、水适量制备抑菌物。
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| CN115887311A (zh) * | 2021-09-30 | 2023-04-04 | 伽蓝（集团）股份有限公司 | 一种皮肤外用组合物及其作为蓝光吸收剂和抗氧化剂的应用 |
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