五倍子单宁的提取纯化及其抗菌、抗突变作用研究
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摘要
五倍子(Galla chinensis)为倍蚜科昆虫角倍蚜或蛋倍蚜在其寄主漆树科植物盐肤木(Rhus chinensis)、青麸杨(Rhus.potaninii)或红麸杨(Rhus.punjabensis)等树上形成的干燥虫瘿。五倍子是中国的一种传统中药,其化学成分主要包括单宁、五倍子油、树脂、蛋白质、脂肪物质、淀粉、蜡质、矿物质等。五倍子单宁(Chinese Gallotannins,CGTs)是五倍子的主要功能成分,并且是五倍子中含量最多的组分。CGTs的研究涉及到提取纯化、抗氧化、抑菌、组成成分分析、结构鉴定等领域。但有关CGTs抗突变作用的报道非常少,而对于CGTs提取方面的研究主要集中在不同提取溶剂的选择上。本文以五倍子为研究对象,采用正交法对超声波辅助提取五倍子单宁的提取溶剂体积分数、液料比、超声时间、提取温度、超声功率等进行试验,同时还进行了CGTs纯化试验;采用Ames试验对五倍子单宁的致突变性进行了评价,同时对其抗突变作用机理加以研究；以茶多酚和青霉素为阳性对照,采用牛津杯法测定五倍子单宁的抑菌效果,通过二倍稀释法确定了五倍子单宁对七种常见食品腐败菌的最小抑菌浓度(MIC),同时观察了温度、PH对五倍子单宁抑菌效果的影响。
主要研究成果如下：
(1)通过正交试验法优化了五倍子单宁的超声波提取条件,同时对五倍子单宁进行了纯化试验。CGTs的最佳提取条件为,超声功率200w,丙酮体积分数60%,液料比24：1,超声时间30min,提取温度40。C；最佳条件下,提取2次就可以达到CGTs的最大提取率92.27%。有机溶剂法纯化CGTs后纯度达到92.89%。
(2)采用Ames试验对五倍子单宁进行致突变性和抗突变机理的研究。结果表明：CGTs对Ames试验无致突变性。CGTs各剂量组均能不同程度地抑制由致突变物引起的TA97、TA98、TA100和TA102回变菌落数的增加。实验剂量200-3500μg/皿的范围内,CGTs抑制强度与剂量呈相关趋势。CGTs具有显著的抗突变作用。
(3)牛津杯法测定五倍子单宁抑菌活性结果表明：CGTs对绿脓假单胞菌、鼠伤寒杆菌、李斯特杆菌、白色念珠球菌、苹果青霉、耐热菌繁殖体、巨大芽孢杆菌有显著的抑菌作用,对于巨大芽孢杆菌,CGTs与对照组抑菌活性大小：青霉素>CGTs=茶多酚(TP)；对于其他六种供试菌：青霉素>CGTs>TP。确定CGTs对供试菌的最小抑菌浓度值分别为：白色念珠球菌5.00g/L,绿脓假单胞菌2.50g/L,巨大芽孢杆菌5.00g/L,鼠伤寒杆菌2.50g/L,耐热菌繁殖体5.00g/L,李斯特杆菌5.00g/L,苹果青霉2.50g/L.当温度在45℃以下时,五倍子单宁能发挥较好的抑菌效果,但温度超过50℃,CGTs对所有供试菌丧失抑菌活性。PH值在3-5的范围内,CGTs对七种菌能完全抑制,酸性条件更有利于CGTs的抑菌活力。
Galla chinensis is the gall produced by some parasitic aphids on the Rhus leaves, P.potaninii leaves or R.punjabensis leaves of the family Anacardiaceae. Galla chinens is a traditional Chinese herb. Its'main chemical compositions are gallotannins, fatty oil, resin, protein, fat, starch, waxiness, and mineral substance, etc. Chinese gallotannins(CGTs) are the main bioactive and the most compounds, which have a research area including extraction, purification, antioxidant, bacteriostat, component analysis, and structuralidentification. few research are related to the antimutigenicity effect of CGTs, and in respects of extraction, studies have focused on the selection of different solvent.
In this paper, CGTs was taken to be the research object. Orthogonal array design was employed to optimize ultrasonic extraction condition. the effects of volume fraction of solvent, liquid-to-solild ratio, ultrasonic time, extractive temperature, ultrasonic power on the yield of CGTs was investigated, the purification process of CGTs was also studied. The effects of CGTs on mutagenicity and antimutagenicity were studied by Ames test; the antibacterial activity of CGTs was studied by oxford cup method compared with TP and penicillin.their minimum inhibitory concentration(MIC) against seven common food spoilage microbes was established by double broth dilution method. what is more, the effects of temperature and pH on the antibacterial activity of CGTs was observed and researched.
Principal research achievements are as follows:
(1) Orthogonal array design was employed to optimize the ultrasonic extraction of Chinese Gallotannins(CGTs),and the purification process of CGTs was also studied in this paper. The optimal extraction condition of CGTs are as follows:ultrasonic power is 200 W,60% acetone as extractant, liquid-to-solid ratio is 24:1, extraction time is 30 min, temperature is 40℃; The CGTs can obtain maximum extraction efficiency as 92.27% in two times extraction.The purification degree of CGTs was up to 92.89% with organic solvents. (2) The effects of CGTs on mutagenicity and antimutagenicity were studied by Ames test. The results showed that no mutagenicity was observed with CGTs. Comparing with positive groups, CGTs groups could decrease the colonies in TA97,TA98,TA100 and TA102 strains. Moreover, the antimutigenicity was correlated to the applied dosage(200～3500μg/dish) in Ames test. we demonstrated that CGTs possessed significant antimutagenic properties.
(3) the antibacterial activity of Chinese Gallotannins(CGTs) was studied by oxford cup method.the results showed that CGTs had prominent antimicrobial activities on Bacillus pyocyaneus, Bacullus typhi murium, Lister mocytogenes, Candida albicans, Penicilliumexpansum, Alicyclobacillus acidoterrestris, and Bacillus megaterium. Their MIC values were 5.0000g/L, 2.50g/L,5.00g/L,2.50g/L,5.00g/L,5.00g/L and 2.50g/L. to Bacillus megaterium, inhibition effect is:penicillin>CGTs=TP, to the other bacteria, inhibition effect is:penicillin> CGTs>TP. CGTs can attain perfect bacteriostatic efficacy when the temperature is below 45℃,but when the temperature is higher than 50℃,CGTs completely loose antibacterial activity. CGTs can inhibit the bacteria completely when the pH was 3-5, so acidiccondition is more beneficial to the antibacterial activity of CGTs.
主要研究成果如下：
(1)通过正交试验法优化了五倍子单宁的超声波提取条件,同时对五倍子单宁进行了纯化试验。CGTs的最佳提取条件为,超声功率200w,丙酮体积分数60%,液料比24：1,超声时间30min,提取温度40。C；最佳条件下,提取2次就可以达到CGTs的最大提取率92.27%。有机溶剂法纯化CGTs后纯度达到92.89%。
(2)采用Ames试验对五倍子单宁进行致突变性和抗突变机理的研究。结果表明：CGTs对Ames试验无致突变性。CGTs各剂量组均能不同程度地抑制由致突变物引起的TA97、TA98、TA100和TA102回变菌落数的增加。实验剂量200-3500μg/皿的范围内,CGTs抑制强度与剂量呈相关趋势。CGTs具有显著的抗突变作用。
(3)牛津杯法测定五倍子单宁抑菌活性结果表明：CGTs对绿脓假单胞菌、鼠伤寒杆菌、李斯特杆菌、白色念珠球菌、苹果青霉、耐热菌繁殖体、巨大芽孢杆菌有显著的抑菌作用,对于巨大芽孢杆菌,CGTs与对照组抑菌活性大小：青霉素>CGTs=茶多酚(TP)；对于其他六种供试菌：青霉素>CGTs>TP。确定CGTs对供试菌的最小抑菌浓度值分别为：白色念珠球菌5.00g/L,绿脓假单胞菌2.50g/L,巨大芽孢杆菌5.00g/L,鼠伤寒杆菌2.50g/L,耐热菌繁殖体5.00g/L,李斯特杆菌5.00g/L,苹果青霉2.50g/L.当温度在45℃以下时,五倍子单宁能发挥较好的抑菌效果,但温度超过50℃,CGTs对所有供试菌丧失抑菌活性。PH值在3-5的范围内,CGTs对七种菌能完全抑制,酸性条件更有利于CGTs的抑菌活力。
Galla chinensis is the gall produced by some parasitic aphids on the Rhus leaves, P.potaninii leaves or R.punjabensis leaves of the family Anacardiaceae. Galla chinens is a traditional Chinese herb. Its'main chemical compositions are gallotannins, fatty oil, resin, protein, fat, starch, waxiness, and mineral substance, etc. Chinese gallotannins(CGTs) are the main bioactive and the most compounds, which have a research area including extraction, purification, antioxidant, bacteriostat, component analysis, and structuralidentification. few research are related to the antimutigenicity effect of CGTs, and in respects of extraction, studies have focused on the selection of different solvent.
In this paper, CGTs was taken to be the research object. Orthogonal array design was employed to optimize ultrasonic extraction condition. the effects of volume fraction of solvent, liquid-to-solild ratio, ultrasonic time, extractive temperature, ultrasonic power on the yield of CGTs was investigated, the purification process of CGTs was also studied. The effects of CGTs on mutagenicity and antimutagenicity were studied by Ames test; the antibacterial activity of CGTs was studied by oxford cup method compared with TP and penicillin.their minimum inhibitory concentration(MIC) against seven common food spoilage microbes was established by double broth dilution method. what is more, the effects of temperature and pH on the antibacterial activity of CGTs was observed and researched.
Principal research achievements are as follows:
(1) Orthogonal array design was employed to optimize the ultrasonic extraction of Chinese Gallotannins(CGTs),and the purification process of CGTs was also studied in this paper. The optimal extraction condition of CGTs are as follows:ultrasonic power is 200 W,60% acetone as extractant, liquid-to-solid ratio is 24:1, extraction time is 30 min, temperature is 40℃; The CGTs can obtain maximum extraction efficiency as 92.27% in two times extraction.The purification degree of CGTs was up to 92.89% with organic solvents. (2) The effects of CGTs on mutagenicity and antimutagenicity were studied by Ames test. The results showed that no mutagenicity was observed with CGTs. Comparing with positive groups, CGTs groups could decrease the colonies in TA97,TA98,TA100 and TA102 strains. Moreover, the antimutigenicity was correlated to the applied dosage(200～3500μg/dish) in Ames test. we demonstrated that CGTs possessed significant antimutagenic properties.
(3) the antibacterial activity of Chinese Gallotannins(CGTs) was studied by oxford cup method.the results showed that CGTs had prominent antimicrobial activities on Bacillus pyocyaneus, Bacullus typhi murium, Lister mocytogenes, Candida albicans, Penicilliumexpansum, Alicyclobacillus acidoterrestris, and Bacillus megaterium. Their MIC values were 5.0000g/L, 2.50g/L,5.00g/L,2.50g/L,5.00g/L,5.00g/L and 2.50g/L. to Bacillus megaterium, inhibition effect is:penicillin>CGTs=TP, to the other bacteria, inhibition effect is:penicillin> CGTs>TP. CGTs can attain perfect bacteriostatic efficacy when the temperature is below 45℃,but when the temperature is higher than 50℃,CGTs completely loose antibacterial activity. CGTs can inhibit the bacteria completely when the pH was 3-5, so acidiccondition is more beneficial to the antibacterial activity of CGTs.
引文
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