石榴皮提取物抗兔球虫作用及其机制的研究
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摘要
石榴皮中生物活性成分较多，以鞣质类（多酚）、黄酮类、生物碱类、有机酸类为主。作为传统中药，石榴皮有多种药用功效。药典（2010版）明确指出石榴皮具有涩肠止泻、止血、驱虫的功效。兔球虫病是家兔最常见的一种寄生虫病，感染率高、患病幼兔死亡率高达40％～70％，对养兔业危害较大。石榴皮的提取物对家兔球虫的杀灭作用，对家兔球虫病的防治作用及其作用途径未见报道。本文通过研究石榴皮提取物对离体培养兔球虫卵囊和家兔粪便中球虫卵囊检出数量，以及家兔肝、肠病理切片中球虫数量等的影响，探索石榴皮提取物防治家兔球虫病的药用价值及机理，以期为球虫病的防治提供参考。
本研究分为以下四个部分：
一、石榴皮黄酮的制备及含量测定
选用枣庄甜石榴的干燥果皮，粉碎，过60目筛，以70%乙醇作为溶剂，料液比1g﹕20ml混合。80℃水浴浸提3小时，过滤。滤液再加4%明胶溶液沉淀，过滤。将滤液干燥即可制得石榴皮黄酮粗提物粉末，得率约为25％左右。利用分光光度法测得黄酮粗提物粉末中总黄酮的含量为117mg/g。结果提示：该法能简便高效地提取石榴皮黄酮，得率和含量都较好，可以应用于下面的研究。
二、石榴皮提取物对离体培养兔球虫卵囊的杀灭作用
采集已感染球虫的18只家兔（体重2.0～2.5kg）的新鲜粪便（24h内，35g/只），用饱和盐水漂浮法分离、提取球虫卵囊。球虫卵囊分别在蒸馏水、石榴皮水提物、石榴皮黄酮、石榴皮多酚、盐酸氯苯胍中培养24、48、72h，光镜下观察不同浓度的各种药物对兔球虫卵囊数量的影响。结果显示，与蒸馏水组比较，5、10mg/mL剂量组培养72h，四种药物均使兔球虫卵囊数量明显减少(P＜0.05)。球虫卵囊的减少率石榴皮水提物组大于石榴皮黄酮组，石榴皮黄酮组大于石榴皮多酚组和盐酸氯苯胍组；起效时间48～72h。1mg/mL剂量组四种药物虽可使兔球虫卵囊数量减少，但无显著差异(P＞0.05)。结果提示：石榴皮提取物对体外培养的兔球虫卵囊有较强的杀灭作用；其中石榴皮水提物对兔球虫卵囊杀灭作用最强，石榴皮黄酮和石榴皮多酚次之；其杀灭作用呈一定的正相关量效关系。
三、口服石榴皮提取物对家兔粪便、肝肠中球虫卵囊检出率的影响
选取体重2.0kg左右、已患球虫病的家兔，随机分为空白对照组（1mL双蒸水口服/㎏体重/d），石榴皮水提取物组和盐酸氯苯胍（市售药物）组，二者的给药剂量均为25㎎/㎏体重/d。石榴皮水提取物组和盐酸氯苯胍组均给药3天、6天、9天、12天、15天，每天观察其粪便中球虫卵囊数的变化。在不同时间，分别过量麻醉处死动物，取其肝脏、结肠、回肠、盲肠进行石蜡切片，H.E染色，光镜比较观察。结果发现随着给药时间延长，石榴皮水提物组和盐酸氯苯胍组粪便中检出的卵囊数总趋势均减少，且卵囊数量的消长周期较双蒸水组缩短。卵囊最大减少率，石榴皮组整体优于盐酸氯苯胍组。病理切片中斯氏艾美耳球虫和黄艾美耳球虫卵囊的检出率表明，石榴皮水提物和盐酸氯苯胍对肝内的斯氏艾美耳球虫均有杀灭作用；对肠内的黄艾美耳球虫的杀灭效果均不显著。研究结果提示：石榴皮水提物对肝内斯氏艾美耳球虫的杀灭作用，以及对肠内黄艾美耳球虫的杀灭作用与盐酸氯苯胍类似；对其他球虫的杀灭作用石榴皮水提物优于盐酸氯苯胍。
四、石榴皮水提物对患球虫病腹泻幼兔的治疗作用
观察不同剂量（剂量组）石榴皮水提物对2月龄内腹泻幼兔的治疗作用，与药物吡哌酸作对比，并设置空白对照组。结果发现高剂量的石榴皮水提物对患球虫病轻度腹泻幼兔有较好治疗作用，所需时间较药物吡哌酸长。结果显示：石榴皮水提物对患球虫病家兔腹泻有一定的治疗作用，机理可能与其对兔球虫的杀灭作用有关。
Pomegranate peel is traditional Chinese medicine and contains many active ingredients suchas tannins (polyphenols), flavonoids, alkaloids and organic acids. Pharmacopoeia (2010), reportsthat pomegranate peel can“antidiarrheal, hemostasis and expelling parasite”. Rabbits coccidianis a common parasite, has a high infection rate ranging from 40% to 70%. To explore thetherapentic properties of the pomegranate peel extracts on rabbit coccidian and its mechanism,we studied its effect on rabbit coccidian oocysts in vivo and in vitro; including the number ofcoccidian oocysts in the liver and intestines. All of these are useful to cure rabbit coccidian.
This research is divided into four parts:
1. The preparation of pomegranate flavonoids and determination of the content. We crushedthe sweet pomegranate peel from Zaozhuang, extracted by 70% ETOH, then filtered with 4%gelatin solution. We could get the extraction yield of 25% after dried the filtrate. The totalflavonoids of pomegranate peel contained 46mg/g, determining by spectrophotometry. Theresults proved this method to be an efficient way to purify flavonoids.
2. The extermination effect of pomegranate peel extracts on rabbit coccidian oocystscultured in vitro. We collected the excrement (24h, 35g each rabbit) infected with coccidian fromeighteen rabbits weighing 2.0 to 2.5kg, separated and extracted the coccidia oocysts withsaturation salt water floating method. The coccidia oocysts were cultivated in distilled water,aqueous extract of pomegranate peel, pomegranate flavonoids, pomegranate polyphenols andROBENIDINE HCL for 24, 48 and 72h, respectively. The amount of rabbit coccidia oocysts wasobserved with the optical microscope. The effects of different concentrations of drugs anddifferent cultivated time on the amount of rabbit coccidia oocysts were estimated. The resultsshowed that comparing with distilled water group, the amount of rabbit coccidia oocystsseparately cultivated in 72h and 5, 10mg of the four drugs groups reduced significantly (P＜0.05), and the reducing number ranged in descending order was aqueous extract of pomegranatepeel group, pomegranate flavonoids group, pomegranate polyphenols group and ROBENIDINEHCL group. The amount of rabbit coccidia oocysts cultivated in 24, 48, 72h and 1mg of the four drugs groups not reduced significantly (P＞0.05). Besides, the higher the concentration of thesedrugs, the more coccidia oocysts reduced. The onset time of the extracts of pomegranate peelwas 48 and 72h. There were many notes which indicate that the extracts of pomegranate peel hadthe stronger exterminatory action to the rabbit coccidia oocysts cultured in vitro. Theexterminatory action of aqueous extract to the rabbit coccidia oocysts was the strongest,followed by pomegranate polyphenols and flavonoids in the 3 extractions from pomegranate peel.The exterminatory action had a certain positive correlation with the dose.
3. The effect of pomegranate peel extracts on rabbit coccidian oocysts detected in theexcrement. We collected the rabbits weighing 2.0 kg infected by coccidian. They were dividedinto control group(1mL distilled water/㎏weight/d), aqueous extract of pomegranate peel groupand ROBENIDINE HCL group, the doses were 25mg/㎏weight/d. Both aqueous extract ofpomegranate peel group and ROBENIDINE HCL group were dosed 3 -15 days, and tested theamount of coccidian oocysts in the excrement every day. Then we got the liver, colon, ileum andcaecum of different times, did the tissue section with paraffin, stained with HE, observed withthe optical microscope. The results showed that the coccidian amounts in rabbits dealed withaqueous extract of pomegranate peel and ROBENIDINE HCL reduced with the dosed timeextended. Compared with distilled water group, the coccidian oocysts cycle was shortened, theaqueous extract of pomegranate peel had a better rate in amount reduction. The photo of liverand intestines pathologic slices indicated that both aqueous extract of pomegranate peel andROBENIDINE HCL had a strong exterminatory action to Eimeria stiedai, but the exterminatoryaction to Eimeria flavescens was not significant. The results suggested that the exterminatoryaction of the extracts of pomegranate peel to Eimeria stiedai and Eimeria flavescens was similarto ROBENIDINE HCL, but had a better exterminatory action to other coccidians.
4. The antidiarrheal effects of the aqueous extract of pomegranate peel on immaturerabbits infected by coccidian. The antidiarrheal effects of the aqueous extract of pomegranatepeel (10, 30, 40, 100, 200, 400mg/kg) were evaluated in diarrheal immature rabbits with twomonths old, comparing with Pipemidic Acid and distilled water. We found that higher doses ofthe extract had a better treatment, but required longer time than Pipemidic Acid. The resultsshowed that pomegranate peel extracts could cure the diarrhea of rabbits infected by coccidian,its reason might be related to suppression effect of the pomegranate peel extracts on coccidian.
本研究分为以下四个部分：
一、石榴皮黄酮的制备及含量测定
选用枣庄甜石榴的干燥果皮，粉碎，过60目筛，以70%乙醇作为溶剂，料液比1g﹕20ml混合。80℃水浴浸提3小时，过滤。滤液再加4%明胶溶液沉淀，过滤。将滤液干燥即可制得石榴皮黄酮粗提物粉末，得率约为25％左右。利用分光光度法测得黄酮粗提物粉末中总黄酮的含量为117mg/g。结果提示：该法能简便高效地提取石榴皮黄酮，得率和含量都较好，可以应用于下面的研究。
二、石榴皮提取物对离体培养兔球虫卵囊的杀灭作用
采集已感染球虫的18只家兔（体重2.0～2.5kg）的新鲜粪便（24h内，35g/只），用饱和盐水漂浮法分离、提取球虫卵囊。球虫卵囊分别在蒸馏水、石榴皮水提物、石榴皮黄酮、石榴皮多酚、盐酸氯苯胍中培养24、48、72h，光镜下观察不同浓度的各种药物对兔球虫卵囊数量的影响。结果显示，与蒸馏水组比较，5、10mg/mL剂量组培养72h，四种药物均使兔球虫卵囊数量明显减少(P＜0.05)。球虫卵囊的减少率石榴皮水提物组大于石榴皮黄酮组，石榴皮黄酮组大于石榴皮多酚组和盐酸氯苯胍组；起效时间48～72h。1mg/mL剂量组四种药物虽可使兔球虫卵囊数量减少，但无显著差异(P＞0.05)。结果提示：石榴皮提取物对体外培养的兔球虫卵囊有较强的杀灭作用；其中石榴皮水提物对兔球虫卵囊杀灭作用最强，石榴皮黄酮和石榴皮多酚次之；其杀灭作用呈一定的正相关量效关系。
三、口服石榴皮提取物对家兔粪便、肝肠中球虫卵囊检出率的影响
选取体重2.0kg左右、已患球虫病的家兔，随机分为空白对照组（1mL双蒸水口服/㎏体重/d），石榴皮水提取物组和盐酸氯苯胍（市售药物）组，二者的给药剂量均为25㎎/㎏体重/d。石榴皮水提取物组和盐酸氯苯胍组均给药3天、6天、9天、12天、15天，每天观察其粪便中球虫卵囊数的变化。在不同时间，分别过量麻醉处死动物，取其肝脏、结肠、回肠、盲肠进行石蜡切片，H.E染色，光镜比较观察。结果发现随着给药时间延长，石榴皮水提物组和盐酸氯苯胍组粪便中检出的卵囊数总趋势均减少，且卵囊数量的消长周期较双蒸水组缩短。卵囊最大减少率，石榴皮组整体优于盐酸氯苯胍组。病理切片中斯氏艾美耳球虫和黄艾美耳球虫卵囊的检出率表明，石榴皮水提物和盐酸氯苯胍对肝内的斯氏艾美耳球虫均有杀灭作用；对肠内的黄艾美耳球虫的杀灭效果均不显著。研究结果提示：石榴皮水提物对肝内斯氏艾美耳球虫的杀灭作用，以及对肠内黄艾美耳球虫的杀灭作用与盐酸氯苯胍类似；对其他球虫的杀灭作用石榴皮水提物优于盐酸氯苯胍。
四、石榴皮水提物对患球虫病腹泻幼兔的治疗作用
观察不同剂量（剂量组）石榴皮水提物对2月龄内腹泻幼兔的治疗作用，与药物吡哌酸作对比，并设置空白对照组。结果发现高剂量的石榴皮水提物对患球虫病轻度腹泻幼兔有较好治疗作用，所需时间较药物吡哌酸长。结果显示：石榴皮水提物对患球虫病家兔腹泻有一定的治疗作用，机理可能与其对兔球虫的杀灭作用有关。
Pomegranate peel is traditional Chinese medicine and contains many active ingredients suchas tannins (polyphenols), flavonoids, alkaloids and organic acids. Pharmacopoeia (2010), reportsthat pomegranate peel can“antidiarrheal, hemostasis and expelling parasite”. Rabbits coccidianis a common parasite, has a high infection rate ranging from 40% to 70%. To explore thetherapentic properties of the pomegranate peel extracts on rabbit coccidian and its mechanism,we studied its effect on rabbit coccidian oocysts in vivo and in vitro; including the number ofcoccidian oocysts in the liver and intestines. All of these are useful to cure rabbit coccidian.
This research is divided into four parts:
1. The preparation of pomegranate flavonoids and determination of the content. We crushedthe sweet pomegranate peel from Zaozhuang, extracted by 70% ETOH, then filtered with 4%gelatin solution. We could get the extraction yield of 25% after dried the filtrate. The totalflavonoids of pomegranate peel contained 46mg/g, determining by spectrophotometry. Theresults proved this method to be an efficient way to purify flavonoids.
2. The extermination effect of pomegranate peel extracts on rabbit coccidian oocystscultured in vitro. We collected the excrement (24h, 35g each rabbit) infected with coccidian fromeighteen rabbits weighing 2.0 to 2.5kg, separated and extracted the coccidia oocysts withsaturation salt water floating method. The coccidia oocysts were cultivated in distilled water,aqueous extract of pomegranate peel, pomegranate flavonoids, pomegranate polyphenols andROBENIDINE HCL for 24, 48 and 72h, respectively. The amount of rabbit coccidia oocysts wasobserved with the optical microscope. The effects of different concentrations of drugs anddifferent cultivated time on the amount of rabbit coccidia oocysts were estimated. The resultsshowed that comparing with distilled water group, the amount of rabbit coccidia oocystsseparately cultivated in 72h and 5, 10mg of the four drugs groups reduced significantly (P＜0.05), and the reducing number ranged in descending order was aqueous extract of pomegranatepeel group, pomegranate flavonoids group, pomegranate polyphenols group and ROBENIDINEHCL group. The amount of rabbit coccidia oocysts cultivated in 24, 48, 72h and 1mg of the four drugs groups not reduced significantly (P＞0.05). Besides, the higher the concentration of thesedrugs, the more coccidia oocysts reduced. The onset time of the extracts of pomegranate peelwas 48 and 72h. There were many notes which indicate that the extracts of pomegranate peel hadthe stronger exterminatory action to the rabbit coccidia oocysts cultured in vitro. Theexterminatory action of aqueous extract to the rabbit coccidia oocysts was the strongest,followed by pomegranate polyphenols and flavonoids in the 3 extractions from pomegranate peel.The exterminatory action had a certain positive correlation with the dose.
3. The effect of pomegranate peel extracts on rabbit coccidian oocysts detected in theexcrement. We collected the rabbits weighing 2.0 kg infected by coccidian. They were dividedinto control group(1mL distilled water/㎏weight/d), aqueous extract of pomegranate peel groupand ROBENIDINE HCL group, the doses were 25mg/㎏weight/d. Both aqueous extract ofpomegranate peel group and ROBENIDINE HCL group were dosed 3 -15 days, and tested theamount of coccidian oocysts in the excrement every day. Then we got the liver, colon, ileum andcaecum of different times, did the tissue section with paraffin, stained with HE, observed withthe optical microscope. The results showed that the coccidian amounts in rabbits dealed withaqueous extract of pomegranate peel and ROBENIDINE HCL reduced with the dosed timeextended. Compared with distilled water group, the coccidian oocysts cycle was shortened, theaqueous extract of pomegranate peel had a better rate in amount reduction. The photo of liverand intestines pathologic slices indicated that both aqueous extract of pomegranate peel andROBENIDINE HCL had a strong exterminatory action to Eimeria stiedai, but the exterminatoryaction to Eimeria flavescens was not significant. The results suggested that the exterminatoryaction of the extracts of pomegranate peel to Eimeria stiedai and Eimeria flavescens was similarto ROBENIDINE HCL, but had a better exterminatory action to other coccidians.
4. The antidiarrheal effects of the aqueous extract of pomegranate peel on immaturerabbits infected by coccidian. The antidiarrheal effects of the aqueous extract of pomegranatepeel (10, 30, 40, 100, 200, 400mg/kg) were evaluated in diarrheal immature rabbits with twomonths old, comparing with Pipemidic Acid and distilled water. We found that higher doses ofthe extract had a better treatment, but required longer time than Pipemidic Acid. The resultsshowed that pomegranate peel extracts could cure the diarrhea of rabbits infected by coccidian,its reason might be related to suppression effect of the pomegranate peel extracts on coccidian.
引文
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