木香、马兜铃、赤芍、稻芽粗提物对变异链球菌作用的体外实验研究
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摘要
龋病是一种细菌感染性疾病,其中变异链球菌(streptococcus mutans, S.mutans)是最主要的病原菌。本研究在前期100种天然药物抗龋筛选试验的基础上,选择木香、马兜铃、赤芍、稻芽这四种抑菌效果较好的天然药物粗提物,研究它们对S. mutans的体外抑菌、黏附、产酸、产胞外多糖的作用,并分析木香内的有效药物成分及该成分的抑菌效果,为天然药物防龋的进一步开发应用提供科学依据。
目的:在前期体外初筛抗S. mutans药物的基础上,选取出对口腔致龋菌的生长有较明显抑制功效的天然药物,通过细菌体外药物实验,观察其对变异链球菌的的抑制效果,为进一步研究天然药物在口腔疾病治疗中的作用奠定基础。
方法:提取同一批次不同时间以及不同批次的木香、马兜铃、赤芍、稻芽的药物粗提物,重复实验,采用倍比稀释法测定不同浓度药物粗提物对S.mutans的抑制效果,确定最低抑制浓度(minimum inhibitory concentration, MIC),并测定各种药物粗提物对S.mutans产酸、黏附及产胞外多糖能力的影响。利用高效液相色谱法(High Performance Liquid Chromatography ,HPLC)分析木香粗提物中的药物成分及其含量,再使用该成分的标准品进行S.mutans抑菌试验,观察其
对细菌的抑制效果。结果:1.应用不同浓度木香、马兜铃、赤芍、稻芽粗提物进行变异链球菌的生长抑制试验,得出当木香、马兜铃、赤芍粗提物药液浓度≥12.50mg/ml、稻芽粗提物药液浓度≥6.25mg/ml时,对变异链球菌的生长具有抑制作用(P<0.05)。
2.应用此四种药物粗提物进行变异链球菌黏附作用的影响实验,发现当木香、马兜铃、赤芍、稻芽粗提物浓度分别≥6.25mg/ml、3.13mg/ml、3.13 mg/ml、0.78mg/ml时,与对照组相比具有明显的抑制变异链球菌的黏附作用(P<0.05)。
3.应用此四种药物粗提物进行抑制变异链球菌产酸作用的实验,结果得当木香、马兜铃、赤芍、稻芽粗提物浓度分别≥3.13mg/ml、1.56mg/ml、3.13mg/ml、0.39mg/ml时,与对照组相比具有明显的抑制变异链球菌产酸的作用(P<0.05)。
4.应用此四种药物粗提物进行抑制变异链球菌合成胞外可溶性及不溶性多糖作用的实验发现:当木香、赤芍粗提物浓度分别≥6.25mg/ml、3.13mg/ml时,与阳性对照组相比具有明显的抑制变异链球菌产生可溶性胞外多糖的作用(P<0.05);当马兜铃、赤芍、稻芽粗提物浓度分别≥0.78mg/ml、6.25mg/ml、0.39mg/ml时,与阳性对照组相比具有明显的抑制变异链球菌产生不溶性胞外多糖的作用(P<0.05 )。
5.将木香粗提物样品用HPLC进行分析,色谱图可得木香粗提物样品中含有去氢木香内酯及木香烃内酯两种成分,将不同浓度的两种标准品用以变异链球菌的生长抑制试验,结果发现,当去氢木香内酯标准品浓度≥0.441mg/ml、木香烃内酯浓度≥0.227mg/ml时,两种标准品对变异链球菌的生长具有抑制作用(P<0.05)。
结论:1.木香、马兜铃、赤芍、稻芽粗提物对变异链球菌的生长有抑制作用,并可不同程度的降低变异链球菌的黏附、产酸、产胞外多糖的作用。
2.木香粗提物内含有去氢木香内酯及木香烃内酯两种药物成分,这两种成分均可对变异链球菌的生长起到一定的抑制作用。
Dental caries is one kind of infectious diseases. It was found that S.mutans was the main cariogenic bactera of caries. The study was based on the exsisting experiment of selecting effectively anti-carious ones from 100 natural medicines. The extractives of Radix Aucklandiae, Aristolochia debilis, radix paeoniae rubra and fructus oryzae germinates were chosen as four well-effective medicines to investigate their effects on anti-carious performance, adherence, acid generation, and glucan production of S.mutans .Then the effective ingredients in Radix Aucklandiae and their inhibition of S.mutans were analyzd.The results of this study could provide scientific evidence for further development and enhance the application of natural medicines on caries prevention.
Objective:The purpose of this research is to choose well-effective natural medicines from the results of the exsisting experiment, then the medicine experiment in vitro was performed to observe their effects on the growing of S.mutans’s for the further study of the effects of natural medicine on caries prevention.
Methods: The extractives of Radix Aucklandiae, Aristolochia debilis, radix paeoniae rubra, fructus oryzae germinates from the same batch but different time or different batches were abstracted and tested repeatedly. Coubling dilution method was applied to test the effects of extractives with different concentrations on inhibition of S. mutans and to ascertain the minimum inhibitory concentration (MIC) as well as testing the effect of all the extractives on acid generation, adherence and glucan production of S. mutans. High Performance Liquid Chromatography (HPLC) was applied to analyze the medical ingredients and their contents in Radix Aucklandiae. Then the standard ingredients were tested to observe their effects on the inhibition of S.mutans.
Results: 1. Extractives of different concentrations were used to test the inhibition of the growing of S.mutans. It was found that when the extractives of Radix Aucklandiae, Aristolochia debilis, radix paeoniae rubra were no less than 12.5 mg/ml and the extractive of fructus oryzae germinates no less than 6.25 mg/ml, The growing of S.mutans cound be inhibited(P<0.05).
2. Extractives of different concentrations were used to test the inhibition of adherence of S.mutans. Results showed that when the separate concentration of the extractives of Radix Aucklandiae, Aristolochia debilis, radix paeoniae rubra, fructus oryzae germinates were no less than 6.25mg/ml, 3.13mg/ml, 3.13 mg/ml and 0.78mg/ml, the adherence of S.mutans were inhibited which had statistical significant difference(P<0.05)with the negative contrast.
3. Extractives of different concentrations were used to test the inhibition of acid generation of S.mutans. It was found that when the concentration of the extractives of Radix Aucklandiae and radix paeoniae were no less than 3.13mg/ml, the extractive of Aristolochia debilis rubra no less than 1.56mg/ml and the extractive of fructus oryzae germinates no less than 0.39mg/ml ,the acid generation of S.mutans were inhibited which had statistical significant difference(P<0.05)with the negative contrast.
4. Extractives of different concentrations were used to test the inhibition of glucan and insoluble glucan production of S.mutans. Results showed that the extractives of Radix Aucklandiae and radix paeoniae rubra could inhibit the glucan production. When the separate concentration of the extractives of Radix Aucklandiae and radix paeoniae rubra were no less than 6.25mg/ml and 3.13mg/ml, the glucan production of S.mutans were inhibited which had statistical significant difference(P<0.05)with the negative contrast. It was found that the extractives of Aristolochia debilis, radix paeoniae rubra and fructus oryzae germinates can inhibit the insoluble glucan production When the separate concentrations of the extractives of Aristolochia debilis, radix paeoniae rubra and fructus oryzae germinates were not less than 0.78mg/ml, 6.25mg/ml and 0.39mg/ml, the insoluble glucan production of S.mutans were inhibited which had statistical significant difference(P<0.05)with the negative contrast.
5. HPLC was applied to analyze the extractive of Radix Aucklandiae. It was found that dehydrocostuslactone (70.6043μg/ml) and Costunolide (36.3432μg/ml) were contained in the extractive of Radix Aucklandiae (2mg/ml) by regression equation. Standard dehydrocostuslactone and Costunolide with different concentrations were used to test the inhitbitive rate of the growing of S.mutans. Results showed that when the MIC of dehydrocostuslactone was 0.441 mg/ml and the MIC of Costunolide was 0.227mg/ml, the growing of S.mutans could be inhibited.
Conclusions: 1. The growing of S.mutans could be inhibited by the extractive of Radix Aucklandiae、Aristolochia debilis、radix paeoniae rubra、fructus oryzae germinates .These extractives had different inhibitory effects on adherence、acid generation and glucan production of S mutans.
2. Dehydrocostuslactone and Costunolide were found in the extractive of Radix Aucklandiae, which both had inhibitory effect on the growing of S.mutans.
目的:在前期体外初筛抗S. mutans药物的基础上,选取出对口腔致龋菌的生长有较明显抑制功效的天然药物,通过细菌体外药物实验,观察其对变异链球菌的的抑制效果,为进一步研究天然药物在口腔疾病治疗中的作用奠定基础。
方法:提取同一批次不同时间以及不同批次的木香、马兜铃、赤芍、稻芽的药物粗提物,重复实验,采用倍比稀释法测定不同浓度药物粗提物对S.mutans的抑制效果,确定最低抑制浓度(minimum inhibitory concentration, MIC),并测定各种药物粗提物对S.mutans产酸、黏附及产胞外多糖能力的影响。利用高效液相色谱法(High Performance Liquid Chromatography ,HPLC)分析木香粗提物中的药物成分及其含量,再使用该成分的标准品进行S.mutans抑菌试验,观察其
对细菌的抑制效果。结果:1.应用不同浓度木香、马兜铃、赤芍、稻芽粗提物进行变异链球菌的生长抑制试验,得出当木香、马兜铃、赤芍粗提物药液浓度≥12.50mg/ml、稻芽粗提物药液浓度≥6.25mg/ml时,对变异链球菌的生长具有抑制作用(P<0.05)。
2.应用此四种药物粗提物进行变异链球菌黏附作用的影响实验,发现当木香、马兜铃、赤芍、稻芽粗提物浓度分别≥6.25mg/ml、3.13mg/ml、3.13 mg/ml、0.78mg/ml时,与对照组相比具有明显的抑制变异链球菌的黏附作用(P<0.05)。
3.应用此四种药物粗提物进行抑制变异链球菌产酸作用的实验,结果得当木香、马兜铃、赤芍、稻芽粗提物浓度分别≥3.13mg/ml、1.56mg/ml、3.13mg/ml、0.39mg/ml时,与对照组相比具有明显的抑制变异链球菌产酸的作用(P<0.05)。
4.应用此四种药物粗提物进行抑制变异链球菌合成胞外可溶性及不溶性多糖作用的实验发现:当木香、赤芍粗提物浓度分别≥6.25mg/ml、3.13mg/ml时,与阳性对照组相比具有明显的抑制变异链球菌产生可溶性胞外多糖的作用(P<0.05);当马兜铃、赤芍、稻芽粗提物浓度分别≥0.78mg/ml、6.25mg/ml、0.39mg/ml时,与阳性对照组相比具有明显的抑制变异链球菌产生不溶性胞外多糖的作用(P<0.05 )。
5.将木香粗提物样品用HPLC进行分析,色谱图可得木香粗提物样品中含有去氢木香内酯及木香烃内酯两种成分,将不同浓度的两种标准品用以变异链球菌的生长抑制试验,结果发现,当去氢木香内酯标准品浓度≥0.441mg/ml、木香烃内酯浓度≥0.227mg/ml时,两种标准品对变异链球菌的生长具有抑制作用(P<0.05)。
结论:1.木香、马兜铃、赤芍、稻芽粗提物对变异链球菌的生长有抑制作用,并可不同程度的降低变异链球菌的黏附、产酸、产胞外多糖的作用。
2.木香粗提物内含有去氢木香内酯及木香烃内酯两种药物成分,这两种成分均可对变异链球菌的生长起到一定的抑制作用。
Dental caries is one kind of infectious diseases. It was found that S.mutans was the main cariogenic bactera of caries. The study was based on the exsisting experiment of selecting effectively anti-carious ones from 100 natural medicines. The extractives of Radix Aucklandiae, Aristolochia debilis, radix paeoniae rubra and fructus oryzae germinates were chosen as four well-effective medicines to investigate their effects on anti-carious performance, adherence, acid generation, and glucan production of S.mutans .Then the effective ingredients in Radix Aucklandiae and their inhibition of S.mutans were analyzd.The results of this study could provide scientific evidence for further development and enhance the application of natural medicines on caries prevention.
Objective:The purpose of this research is to choose well-effective natural medicines from the results of the exsisting experiment, then the medicine experiment in vitro was performed to observe their effects on the growing of S.mutans’s for the further study of the effects of natural medicine on caries prevention.
Methods: The extractives of Radix Aucklandiae, Aristolochia debilis, radix paeoniae rubra, fructus oryzae germinates from the same batch but different time or different batches were abstracted and tested repeatedly. Coubling dilution method was applied to test the effects of extractives with different concentrations on inhibition of S. mutans and to ascertain the minimum inhibitory concentration (MIC) as well as testing the effect of all the extractives on acid generation, adherence and glucan production of S. mutans. High Performance Liquid Chromatography (HPLC) was applied to analyze the medical ingredients and their contents in Radix Aucklandiae. Then the standard ingredients were tested to observe their effects on the inhibition of S.mutans.
Results: 1. Extractives of different concentrations were used to test the inhibition of the growing of S.mutans. It was found that when the extractives of Radix Aucklandiae, Aristolochia debilis, radix paeoniae rubra were no less than 12.5 mg/ml and the extractive of fructus oryzae germinates no less than 6.25 mg/ml, The growing of S.mutans cound be inhibited(P<0.05).
2. Extractives of different concentrations were used to test the inhibition of adherence of S.mutans. Results showed that when the separate concentration of the extractives of Radix Aucklandiae, Aristolochia debilis, radix paeoniae rubra, fructus oryzae germinates were no less than 6.25mg/ml, 3.13mg/ml, 3.13 mg/ml and 0.78mg/ml, the adherence of S.mutans were inhibited which had statistical significant difference(P<0.05)with the negative contrast.
3. Extractives of different concentrations were used to test the inhibition of acid generation of S.mutans. It was found that when the concentration of the extractives of Radix Aucklandiae and radix paeoniae were no less than 3.13mg/ml, the extractive of Aristolochia debilis rubra no less than 1.56mg/ml and the extractive of fructus oryzae germinates no less than 0.39mg/ml ,the acid generation of S.mutans were inhibited which had statistical significant difference(P<0.05)with the negative contrast.
4. Extractives of different concentrations were used to test the inhibition of glucan and insoluble glucan production of S.mutans. Results showed that the extractives of Radix Aucklandiae and radix paeoniae rubra could inhibit the glucan production. When the separate concentration of the extractives of Radix Aucklandiae and radix paeoniae rubra were no less than 6.25mg/ml and 3.13mg/ml, the glucan production of S.mutans were inhibited which had statistical significant difference(P<0.05)with the negative contrast. It was found that the extractives of Aristolochia debilis, radix paeoniae rubra and fructus oryzae germinates can inhibit the insoluble glucan production When the separate concentrations of the extractives of Aristolochia debilis, radix paeoniae rubra and fructus oryzae germinates were not less than 0.78mg/ml, 6.25mg/ml and 0.39mg/ml, the insoluble glucan production of S.mutans were inhibited which had statistical significant difference(P<0.05)with the negative contrast.
5. HPLC was applied to analyze the extractive of Radix Aucklandiae. It was found that dehydrocostuslactone (70.6043μg/ml) and Costunolide (36.3432μg/ml) were contained in the extractive of Radix Aucklandiae (2mg/ml) by regression equation. Standard dehydrocostuslactone and Costunolide with different concentrations were used to test the inhitbitive rate of the growing of S.mutans. Results showed that when the MIC of dehydrocostuslactone was 0.441 mg/ml and the MIC of Costunolide was 0.227mg/ml, the growing of S.mutans could be inhibited.
Conclusions: 1. The growing of S.mutans could be inhibited by the extractive of Radix Aucklandiae、Aristolochia debilis、radix paeoniae rubra、fructus oryzae germinates .These extractives had different inhibitory effects on adherence、acid generation and glucan production of S mutans.
2. Dehydrocostuslactone and Costunolide were found in the extractive of Radix Aucklandiae, which both had inhibitory effect on the growing of S.mutans.
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