CN113082104B - 一种含石榴皮水提物和花椒籽水提物的组合抑菌物 - Google Patents
一种含石榴皮水提物和花椒籽水提物的组合抑菌物 Download PDFInfo
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Abstract
本发明提供一种含石榴皮水提物和花椒籽水提物的组合抑菌物，属于抑菌剂技术领域。本发明石榴皮和花椒籽水提物通过热回流法获得，滤液冷冻干燥，即得，其中石榴皮水提物的收率为45‑50％，花椒籽水提物的收率为20‑25％。含石榴皮水提物和花椒籽水提物的组合抑菌物中的石榴皮和花椒籽水提物的质量比为1：1。上述的一种含石榴皮水取物和花椒籽水取物的组合抑菌物，对金黄色葡萄球菌具有良好的协同抑菌效果，最低3.125mg/mL即可起到抑菌效果。
Description
技术领域
本发明属于抑菌剂技术领域，具体涉及石榴皮和花椒籽水提物的制备及抑菌组合应用。
背景技术
随着现代化程度的提高，无论是生产还是生活中的菌落生长大肆蔓延，细菌真菌引起了一些或大或小的公共卫生安全问题，不容小觑。
目前世界上的抑菌剂可分为有机抑菌剂、无机抑菌剂和天然抑菌剂，有机和无机抑菌剂伤害力大，对环境易产生污染，天然抑菌剂又分为植物源抑菌剂、动物源抑菌剂和微生物源抑菌剂，植物源抑菌剂来源广，无毒无污染。
石榴皮(学名：grenadine pericarpium)是石榴科植物石榴(学名：Punicagranatum L.)的干燥果皮，据《中国药典》记载，具有涩肠止泻，止血，驱虫之功效。常用于久泻，久痢，便血，脱肛，崩漏，带下，虫积腹痛。
花椒(学名：Zanthoxylum bungeanum Maxim.)是芸香科、花椒属落叶小乔木，秋季釆收成熟果实，晒干，除去种子和杂质成干燥成熟果皮，据《中国药典》记载，花椒具有温中止痛，杀虫止痒。用于脏腹冷痛，呕吐泄泻，虫积腹痛；外治湿疹，阴痒等作用。
石榴皮中含有大量鞣质，花椒中含有的黄酮等活性物质。目前，石榴皮和花椒籽提取物中具有低抑菌浓度的主要为甲醇、乙醇等有机溶剂提取得到，植物提取中用到的溶剂量大，一些有机溶剂毒性大，对人体容易造成伤害，所以水提取要比有机溶剂提取经济、绿色环保，适合工业化生产。
其中虽然在现有技术中分别研究了花椒、石榴皮的提取物具有抗菌性，如“石榴皮提取物的抑菌作用及应用研究进展”中显示石榴皮水提物对金黄色葡萄球菌的最小抑菌浓度要大于12.5mg/mL，“茂县花椒化学成分分析及抑菌活性研究”中显示花椒籽水提物对金黄色葡萄球菌的最小抑菌浓度均大于100mg/mL，而已知的溶剂提取法得到的花椒提取物的最小抑菌浓度为7.5mg/mL。在抑菌效果上难以进一步提高。
而本发明将石榴皮水提物与花椒籽水提物联用作为抑菌物，虽然往往认为单宁酸和生物碱共存时会产生沉淀，但在单宁与共存物质相互作用的效果和生物碱生成沉淀及使沉淀溶解，且不同的单宁酸和生物碱的种类和比例会极大程度的影响沉淀的生成，且该复合物在溶液中的结合能力远强于在沉淀中，甚至会产生不可预料的协同作用。本发明发现将石榴皮水提物与花椒籽水提物联用后，相互之间产生协同作用，组合抑菌物中联用最小抑菌浓度较两种水提物分别单用的最小抑菌浓度均要更低。
发明内容
本发明的目在于提供一种含石榴皮水提物和花椒籽水提物的组合抑菌物，是一种基于沸水热回流法提取具有抑菌活性石榴皮和花椒籽的提取物，将石榴皮水提物与花椒籽水提物联用后，相互之间产生协同作用，抑菌能力较强。且该提取工艺中不涉及有机试剂的使用，所得的产品为橙黄色，具有环保，无污染，得率高，用量小，具有广泛的应用前景。
所述的石榴皮和花椒籽水组合抑菌提取物具体通过以下步骤获得：
(1)将石榴皮和花椒籽自然干燥，分别干燥后粉碎，过40～100目筛得到分别得到石榴皮和花椒籽粗粉；
(2)将石榴皮/花椒籽粗粉置于烧瓶，以电热器为加热装置，加入粗粉质量5～7倍的蒸馏水，通过热回流的方法，在90℃～100℃下热回流提取50分钟～70分钟，过滤滤液后再加等质量蒸馏水溶剂提取50分钟～70分钟，再过滤出滤液，两次滤液经离心机8000r/min～10000r/min转速下离心5～7分钟，取上层澄清液。
(3)将澄清提取液用瓷坩埚在水浴85℃～95℃中浓缩至原体积的10％～15％。
(4)将浓缩液转移至容器中，放入-70℃～-80℃冷冻柜冷冻1小时，再于真空冷冻干燥机中，温度条件-40℃，压力条件<100Pa下干燥24h～36小时，干燥后研磨，密封保存，分别得到石榴皮水提物和花椒籽水提物。
本发明采用冷冻干燥机对提取物进行干燥，使物料原有结构和形状得到最大程度保护。
将石榴皮水提物和花椒籽水提物按质量比混合，得到组合抑菌物。
进一步，组合抑菌物中石榴皮水提物和花椒籽水提物冷冻干燥粉的质量比为16：1～1：16。
作为优选，组合抑菌物中石榴皮水提物和花椒籽水提物冷冻干燥粉的质量比为1：1，水溶液浓度均为1.56mg/mL时，可达到协同抑菌效果。
本发明提供了一种含石榴皮水提物和花椒籽水提物的组合抑菌物，石榴皮水提物和花椒籽水提物具有较高的收率，通过将不同浓度石榴皮水提物和花椒籽水提物进行复配，对金黄色葡萄球菌有良好的协同抑菌作用，联用时的最小抑菌浓度要小于两种水提物单用，在制备抑制金黄色葡萄球菌药物中，有一定的应用价值。
附图说明
图1为实验例1对金黄色葡萄球菌的抑菌圈结果图。
图2为实验例2对金黄色葡萄球菌的抑菌圈结果图。
图3为实验例1对金黄色葡萄球菌的最小抑菌浓度实验结果图。
图4为实验例2对金黄色葡萄球菌的最小抑菌浓度实验结果图。
图5为MIC的实验例1和MIC、1/2MIC、1/4MIC、1/8MIC以及1/16MIC的实验例2混合后对金黄色葡萄球菌的最小抑菌浓度实验结果图(编号为11、12、14、18，116)。
图6为1/2MIC的实验例1和MIC、1/2MIC、1/4MIC、1/8MIC以及1/16MIC的实验例2混合后对金黄色葡萄球菌的最小抑菌浓度实验结果图(编号为21、22、24、28，216)。
图7为1/4MIC的实验例1和MIC、1/2MIC、1/4MIC、1/8MIC以及1/16MIC的实验例2混合后对金黄色葡萄球菌的最小抑菌浓度实验结果图(编号为41、42、44、48，416)。
图8为1/8MIC的实验例1和MIC、1/2MIC、1/4MIC、1/8MIC以及1/16MIC的实验例2混合后对金黄色葡萄球菌的最小抑菌浓度实验结果图(编号为81、82、84、88，816)。
图9为1/16MIC的实验例1和MIC、1/2MIC、1/4MIC、1/8MIC以及1/16MIC的实验例2混合后对金黄色葡萄球菌的最小抑菌浓度实验结果图(编号为161、162、164、168，1616)。
具体实施方式
实验例1
一种石榴皮水提物，包括以下步骤
(1)将石榴皮粉碎，过80目筛得到石榴皮粗粉；
(2)将100g石榴皮粗粉置于烧瓶，以电热器为加热装置，加入粗粉6倍质量的蒸馏水，通过热回流的方法，在90℃下热回流提取60分钟，过滤滤液后再加等质量蒸馏水溶剂提取60分钟，再过滤出滤液，两次滤液经离心机9000r/min转速下离心5分钟，取上层澄清液；
(3)将澄清提取液用瓷坩埚在水浴90℃中浓缩至原体积的10％；
(4)将浓缩液转移至容器中，放入-80℃冷冻柜冷冻1小时，再于真空冷冻干燥机中，温度条件-40℃，压力条件11Pa下干燥24小时，研磨，密封保存即可。
实验例2
一种花椒籽水提物，包括以下步骤
(1)将花椒籽粉碎，过80目筛得到花椒籽粗粉；
(2)将100g花椒籽粗粉置于烧瓶，以电热器为加热装置，加入粗粉6倍质量的蒸馏水，通过热回流的方法，在90℃下热回流提取60分钟，过滤滤液后再加等质量蒸馏水溶剂再提取60分钟，再过滤出滤液，两次滤液经离心机9000r/min转速下离心5分钟，取上层澄清液；
(3)将澄清提取液用瓷坩埚在水浴90℃中浓缩至原体积的10％；
(4)将浓缩液转移至容器中，放入-80℃冷冻柜冷冻1小时，再于真空冷冻干燥机中，温度条件-40℃，压力条件11Pa下干燥24小时，研磨，密封保存即可。
实验例1和实验例2的提取质量和提取率计算公式：
其中w为提取率，m为水提物冷冻干燥粉的质量，M为石榴皮或花椒籽植物粗粉的质量(100g)。
表1实验例的提取质量和提取率
| 实验例1 | 实验例2 | |
| 提取质量(g) | 45.17 | 20.20 |
| 提取率(％) | 45.17 | 20.20 |
对照例1
无菌水。
对比例2
营养肉汤。
抑菌实验1：抑菌圈试验
试验方法依据：美国临床实验室标准化协会(CLSI)标准；
试验样品：实验例1，实验例2，对比例1；
菌种：金黄色葡萄球菌；
培养基：营养琼脂培养基；
方法：利用无菌水，将实验例配成200mg/mL的溶液，并以无菌水作为对照，涡旋搅拌溶解，药敏纸片(直径6mm)分别在溶液中浸泡30分钟，取出，无菌环境风干，在营养琼脂培养基上均匀涂布106CFU/mL金黄色葡萄球菌菌悬液，将实验例和对照例1的纸片贴在平板上，37℃培养18小时，结果如表1所示。
表2抑菌圈试验结果
| 抑菌圈/mm | 实验例1 | 实验例2 | 对照例1 |
| 金黄色葡萄球菌 | 25 | 11 | 0 |
抑菌实验2：最小抑菌浓度(MIC)测试
试验方法依据：美国临床实验室标准化协会(CLSI)标准；
试验样品：实验例1，实验例2，对比例2；
菌种：金黄色葡萄球菌；
培养基：营养肉汤液体培养基；
方法：利用96孔板，通过微量肉汤二倍稀释法，称取一定量的提取物，用无菌水配置成25mg/mL，用液体培养基稀释成12.5～0.39mg/mL，每种浓度的药液中加入等量一定浓度的菌液，于37℃细菌恒温培养箱培养24小时，培养结束，将各培养液划线接种在培养基上，二次培养24小时，结果如表2所示。
表3提取物的最小抑菌浓度(mg/mL)
根据结果图3、4所示，当石榴皮和花椒籽水提物处于最小抑菌浓度(MIC)时，培养基上只有少数可数菌落，而当浓度为1/2MIC时，菌落铺满平面，不可计数，以菌落可数的浓度作为水提物的最小抑菌浓度。
抑菌实验3：联用最小抑菌浓度测试
试验方法依据：美国临床实验室标准化协会(CLSI)标准，抑菌实验2结果；
试验样品：实验例1，实验例2；
菌种：金黄色葡萄球菌；
培养基：营养肉汤液体培养基；
方法：以抑菌实验2的结果为依据，采用协同指数法和微量肉汤稀释法，将石榴皮水提物和花椒籽水提物分别配置成MIC、1/2MIC、1/4MIC、1/8MIC及1/16MIC浓度，取两种各浓度提取液按体积比1:1复配，在混合提取液中含有的石榴皮水提物和花椒籽水提物的质量比为16：1～1：16。再加等量105CFU/mL的菌液，37℃恒温培养箱培养24小时，培养结束，将各培养液划线接种在培养基上，二次培养24小时，复配液的杀菌结果如表4所示。
表4提取物联用抑菌效果
-为没有菌生长；
+为少量菌生长，菌落可数；
++为有一定量菌生长；
+++为有大量菌生长。
由表4可知，本发明提供的组合物，实验例1的浓度为1/4MIC，实验例2的浓度为1/4MIC，实验例1和实验例2的质量比为1:1时，即实验例1和实验例2在溶液中的浓度都为1.56mg/mL时，协同抑菌指数FIC达到最低值。协同抑菌指数FIC计算公式：
表5石榴皮和花椒籽水提物的FIC指数
判断标准：
FIC≤0.5，两种药为协同作用；
0.5<FIC≤1，两种药为相加作用；
1<FIC≤2，两种药为无关作用；
2<FIC，两种药为拮抗作用。
如表5所示，协同抑菌指数FIC为0.5≤0.5，根据判断标准可知这两种水提物联用具有协同的抑菌作用；
石榴皮水提物和花椒籽水提物质量比为1：1混合时，组合物的最小抑菌浓度为3.125mg/mL，小于石榴皮水提物和花椒籽水提物分别单用时的最小抑菌浓度6.25mg/mL，抑菌效果良好，两者之间具有协同作用。
Claims (3)
1.一种含石榴皮水提物和花椒籽水提物的组合抑菌物，其特征在于：组合抑菌物中由石榴皮水提物和花椒籽水提物组成；
具体为：将石榴皮和花椒籽分别自然干燥，粉碎成植物粗粉，过筛，在90℃~100℃水中回流提取后过滤，分别得到滤液和滤渣，滤渣继续用水在90℃~100℃中回流提取，过滤，得到滤液，将滤液合并离心，取上层澄清液，浓缩后的提取液干燥，分别得到石榴皮水提物和花椒籽水提物，质量比为1：1混合后得组合抑菌物；
所述组合抑菌物在制备用于抑制金黄色葡萄球菌药物中的应用。
2.如权利要求1所述的石榴皮水提物和花椒籽水提物的组合抑菌物，其特征在于，石榴皮或花椒籽粗粉与水的固液质量比为1：5~1：7。
3.如权利要求1所述的石榴皮水提物和花椒籽水提物的组合抑菌物，其特征在于，浓缩后的提取液用冷冻干燥机干燥得到粉末状提取物。
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