芦荟预防与治疗慢传输型便秘的作用及机制研究
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摘要
目的慢传输型便秘(slow transit constipation,STC)是临床常见的消化道病症。随着现代人们生活节奏加快,生活水平提高,饮食结构改变以及社会人口老龄化,STC的患病率呈逐渐上升趋势,且随年龄的增长而增加。便秘不仅严重影响患者的生存质量,还与急性心脑血管疾病、痴呆、结直肠癌等有着密切的关系。芦荟中含有丰富的蒽醌类化合物、多糖、氨基酸、多种微量元素等有效化学成分,尽管芦荟用于治疗便秘已有悠久的历史,但对于芦荟润肠通便作用的机制研究很少,其促进肠运动的机制目前尚未阐明,尤其是对于调节胃肠激素方面,目前尚未见报道。基于以上几点,本课题首先测定芦荟的有效成分——芦荟苷的含量,然后用复方地芬诺酯诱发STC动物模型,用芦荟进行干预,观察其预防和治疗作用,并在此基础上研究芦荟能否改变STC动物模型中胃肠激素的紊乱,探讨其发挥药理学作用的机制,并为芦荟进一步应用于预防和治疗STC提供理论依据。
方法
1芦荟制品中芦荟苷含量测定
采用HPLC分别测定库拉索芦荟全叶干粉、精制干粉、全叶冻干粉、全叶喷干粉、纯化药用级干粉及市售复方芦荟胶囊六种芦荟制品中芦荟苷含量。测定条件:ZORBAX SB-18色谱柱(4.6mm×150mm,5μm),检测波长355nm,流动相为乙腈:水(25:75),流速:1ml/min,外标法测定不同芦荟制品中芦荟苷含量。
2芦荟制品通便功能筛选
采用复方地芬诺酯(10mg/kg·bw)诱发动物便秘模型,给予以上芦荟制品(1.0g/kg·bw),观察各组小鼠首次排黑便时间、6h排便粒数、粪便性状、粪便干湿重。
3芦荟对小鼠排便及肠运动功能的影响
3.1芦荟对正常小鼠排便功能的影响
雄性昆明小鼠50只,随机分为5组,空白对照组、芦荟低剂量组(0.17 g/kg·bw)、芦荟中剂量组(0.33 g/kg·bw)、芦荟高剂量组(1.0 g/kg·bw)及阳性对照组西沙必利(2.5mg/kg·bw),给药后连续观察每只动物首次排黑便时间、6h排便粒数、粪便湿重,计算粪便含水率。
3.2芦荟对便秘小鼠排便功能的影响
雄性昆明小鼠60只小鼠随机分为6组,空白对照组、芦荟低剂量组(0.17 g/kg·bw)、芦荟中剂量组(0.33 g/kg·bw)、芦荟高剂量组(1.0 g/kg·bw)、便秘模型对照组和阳性对照组西沙必利(2.5mg/kg·bw),连续灌胃7天,第8天便秘模型对照组、阳性对照组和芦荟低、中、高剂量组给予复方地芬诺酯(5 mg/kg·bw),空白对照组给予等体积的蒸馏水。给复方地芬诺酯0.5h后,芦荟低、中、高剂量组及阳性对照组分别给予含受试样品的指示墨汁、空白和便秘模型对照组只给墨汁。观察记录每只动物首次排黑便时间、6h排便粒数及重量,并计算粪便含水率。
3.3芦荟对便秘小鼠肠运动功能的影响
动物分组及给药方法同3.2,给予墨汁0.5h后,摘眼球取血,脱臼处死动物,迅速取出整个肠道,计算墨汁推进率,采用硝酸还原酶法测定血清中NO含量。
4芦荟对大鼠慢传输型便秘的预防与治疗作用
4.1预防作用:48只雄性Wistar大鼠随机分为空白对照组、STC模型组、芦荟小剂量组(0.33g/kg·bw),芦荟大剂量组(0.66g/kg·bw)共4组,每组12只。模型组,芦荟小剂量组及芦荟大剂量组的大鼠每天给予8 mg/kg.bw的复方地芬诺酯,空白对照组给予同体积蒸馏水;间隔2h后芦荟小剂量组给予芦荟混悬液,空白对照组及模型组给予同样体积的蒸馏水。连续给药8周,每两周测定肠道传输速度及粪便称重。8周后断头处死每组8只大鼠用于采集血浆及组织标本,测定血浆及肠组织中胃动素(motilin,MTL)、P物质(substance P,SP)、血管活性肠肽(vasoactiveintestinal peptide,VIP)含量,4只大鼠用于病理观察。
4.2治疗作用:48只Wistar大鼠随机分为空白对照组(12只)、STC模型组(36只)。STC模型组每天给予复方地芬诺酯灌胃,剂量8mg/kg.bw,灌胃体积0.5ml/100g·bw,空白对照组给予同体积蒸馏水。连续给药8周后,停药1周,测定大鼠的肠道传输速度。将STC模型组大鼠随机分为3组:自然恢复组、芦荟小剂量组(0.33g/kg·bw)、芦荟大剂量组(0.66g/kg·bw),每组12只。自第10周,各芦荟组分别经灌胃给予不同剂量芦荟,空白对照组及模型恢复组给予同体积蒸馏水,给药2周后测定大鼠肠道传输速度及粪便称重,8只大鼠用于采集血浆及组织标本,测定血浆及肠组织中胃动素(motilin,MTL)、P物质(substance P,SP)、血管活性肠肽(vasoactive intestinal peptide,VIP)含量,4只大鼠用于病理观察。
结果
1芦荟制品中芦荟苷含量:库拉索芦荟全叶干粉、精制干粉、全叶冻干粉、全叶喷干粉、纯化药用级干粉及市售复方芦荟胶囊六种芦荟制品的芦荟苷含量依次为0.088%、0.266%、0.908%、6.71%、13.97%和3.41%。
2芦荟精制干粉、芦荟纯化药用级干粉能够缩短便秘小鼠的首便时间,增加其所排粪便的粒数和重量;芦荟全叶冻干粉能够缩短便秘小鼠的首便时间,增加其所排粪便重量;芦荟全叶喷干粉能够增加排出粪便的重量,芦荟全叶干粉未见明显通便作用。芦荟精制干粉对便秘的效果较好。
3芦荟对小鼠排便及肠运动功能的影响
3.1芦荟对正常小鼠排便功能的影响高剂量的芦荟能缩短正常小鼠的排便时间、增加排便粒数、重量及粪便含水率,对正常小鼠具有润肠通便作用。
3.2芦荟对便秘小鼠排便功能的影响与空白对照组相比,便秘模型对照组小鼠的首便时间显著延长,6h黑便粒数减少。与便秘模型对照组相比,芦荟低、中、高剂量组均能不同程度的缩短便秘小鼠的首便时间,增加6h黑便粒数及粪便干、湿重,并能增加粪便含水率。小鼠排便干、湿重随着芦荟浓度的增加而增加,呈现明显的剂量效应关系。与便秘模型对照组相比较,芦荟各剂量组小鼠粪便含水率均有显著增加。阳性对照组小鼠的首便时间明显缩短,6h黑便粒数增加,但粪便干、湿重及粪便含水率与模型对照组小鼠相比,无明显差别。
3.3芦荟对小鼠肠推进率的影响
便秘模型对照组小鼠的墨汁推进率明显低于空白对照组。芦荟中、高剂量组均能提高小鼠的小肠墨汁推进率,且芦荟给药剂量与小鼠的小肠墨汁推进率成剂量-效应关系,表明芦荟能增加小鼠的肠推进率,促进肠道运动。便秘模型对照组的NO含量均值比空白对照组升高了64.6%,与便秘模型对照组比较,芦荟低、中、高剂量组的NO水平分别降低了4.33%、17.00%、21.80%,且高剂量组与便秘模型对照组相比有明显差异(P＜0.05)。相关性分析表明血清NO含量与小肠墨汁推进率呈负相关,r=-0.346(P＜0.05)。阳性对照组小鼠血清NO水平与模型对照组小鼠相比,无明显差别。
4芦荟对大鼠慢传输型便秘的预防与治疗作用
4.1芦荟对大鼠慢传输型便秘的预防作用及对胃肠激素的影响
肠道传输功能测定结果表明,与正常对照组相比,STC模型组的首次排便时间延长,排便粒数减少,粪便含水率降低,模型成功。与STC模型组相比,芦荟预防组大鼠首便时间缩短,排便粒数增加的粪便干、湿重及粪便含水率均增加。
与正常对照组相比,STC模型组大鼠肠组织中MTL含量降低(P＜0.05);与模型组相比,芦荟大剂量预防组MTL含量升高(P＜0.05)。
与正常对照组相比,STC模型组大鼠血浆及肠组织中SP含量均降低;与模型组相比,芦荟小剂量预防组血浆SP含量升高(P＜0.05),芦荟大剂量预防组血浆及肠组织中SP含量均升高(P＜0.01)。
与正常对照组相比,STC模型组大鼠血浆及肠组织中VIP含量均降低(P＜0.01);与模型组相比,芦荟小剂量组肠组织中的VIP含量升高(P＜0.05),芦荟大剂量组血浆及肠组织中的VIP含量均升高(P＜0.05)。
4.2芦荟对大鼠慢传输型便秘的治疗作用及对胃肠激素的影响
肠道传输功能测定结果表明,与正常对照组相比,STC模型恢复组的首次排便时间延长(P＜0.01),排便粒数减少(P＜0.01);与STC模型恢复组相比,芦荟大、小剂量治疗组的首便时间缩短,且差异有统计学意义(P＜0.01)。
与正常对照组相比,STC模型恢复组大鼠肠组织中MTL含量降低,但差异无统计学意义;与模型组相比,芦荟大剂量组MTL含量升高(P＜0.05)
与正常对照组相比,STC模型恢复组大鼠血浆及肠组织中SP含量均降低(P＜0.05,P＜0.01);与模型恢复组相比,芦荟大剂量组血浆及肠组织中SP含量均升高(P＜0.01)。
与正常对照组相比,STC模型恢复组大鼠血浆及肠组织中VIP含量均降低,肠组织中的降低具有统计学意义(P＜0.01);与模型恢复组相比,芦荟小剂量和大剂量预防组血浆及肠组织中VIP含量均有升高,其中芦荟大剂量组肠组织中VIP含量升高具有统计学意义(P＜0.05)
结论
1芦荟制品具有润肠通便作用,其作用大小可能与加工方式有关,其中芦荟精制干粉通便作用较好。
2 1.0g/kg.bw的芦荟对正常小鼠具有润肠通便作用,而0.17g/kg.bw的芦荟对便秘小鼠即呈现明显的润肠通便作用。
3芦荟能明显改善复方地芬诺酯诱导的肠蠕动抑制小鼠的肠道运动功能,并呈剂量效应关系。此作用可能与芦荟增加粪便含水率、降低血清NO有关。
4芦荟对复方地芬诺酯诱导的大鼠STC具有一定的预防和治疗作用,可能与升高血浆和肠组织MTL、SP、VIP含量有关。
Objectives
Slow transit constipation(STC)is a common clinical gastrointestinal disease attributed to ineffective colonic propulsion and/or increased resistance to propagation of colonic contents.The gradually rising prevalence rate of STC,which increases with age,is due in large part to the changes of people's life style.Constipation not only seriously affects the quality of life,and also has a close positive relationship with acute cardiovascular and cerebrovascular disease,dementia,colorectal cancer and so on.Aloe is rich in anthraquinone compounds,polysaccharides,amino acids,trace elements,and other variety of chemical constituents.Although aloe for the treatment of constipation have a long history,the mechanism of promotion of intestinal movement mechanism still remains unclear,particularly regulating gastrointestinal hormones have not yet reported.Based on the above points,aloin-the active ingredients of aloe was determined by HPLC,then slow transit constipation animal model was induced by Compound Diphenoxylate and the mechanism of preventive and the therapeutic effect of aloe on STC was observed.We also focus on the different changes of gastrointestinal hormone between STC animal and those treated with aloe in this study.These results might be beneficial to the measures of prevent and treat slow transit constipation.
Methods
1 Determination of aloin level in different aloe product
The aloin level in different aloe product were determined by HPLC with the following determination conditions:ZORBAX SB-18 column(4.6mm×150mm,5μm),the detection wavelength was 355 nm,the mobile phase of acetonitrile:water (25:75),flow rate:1ml/min,use external reference method to determine aloin in different aloe products by HPLC.
2 Screening on facitating feces excretion functions of different aloe products
The aloe products(1.0 g/kg·bw)were given mice in advance,then Compound Diphenoxylate(10 mg/kg·bw)was used to induce constipation animal model.The first dejection time,stool amouts within six hours,feces traits,wet and dry faeces weight were observed.
3 Effects of aloe on defecation and intestinal motor function of mice
3.1 Effects of aloe on defecation function of normal mice:50 male Kunming mice were randomly divided into five groups,i.e.control group,aloe low,middle and high dose group(0.17 g/kg·bw,0.33 g/kg.bw,1.0 g/ kg·bw)and the positive control group(cisapride,2.5 mg/kg·bw).The first dejection time,stool amours within six hours,feces traits,fresh and dry faeces weight were observed after administration,and then fecal water content was calculated.
3.2 Effects of aloe on defecation function of costive mice:60 male Kuning mice were randomly divided into 6 groups,aloe low,middle and high dose group(aloe 0.17 g/ kg·bw,0.33 g/kg·bw,1.0 g/kg·bw),the positive control group(cisapride,2.5 mg/ kg·bw),the blank control group and the model control group.Mice of Aloe low, middle and high dose group and the positive control group were given the corresponding dose aloe by gavage for seven days,the mice in blank control group and the model control group given the same volume of distilled water.On the 8~(th)day the mice in model group,positive control group and aloe low,middle and high dose groups were treated with Compound Diphenoxylate(5 mg/kg·bw),mice of the blank control group with the same volume of distilled water.After treating with Compound Diphenoxylate 0.5 hours,aloe low,middle and high dose group and the positive control group were given the ink mixed with corresponding tested samples,while other mice only ink.The first dejection time,stool amours within six hours,feces traits,fresh and dry faeces weight were observed and recorded after Diphenoxylate administration,then fecal water content were calculate.
3.3 Effects of aloe on intestinal motor function of mice:Animal groups and the administration method were same as 3.2 After giving ink 0.5 hour,blood were collected from inner canthus and content of NO were determined by using method of nitrate reductase,then mice were sacrificed,the entire intestine were quickly removed, and the intestinal transit ratio were calculated.
4 Effects of aloe on prevention and treatment of slow transit constipation of rats
4.1 Effects of aloe on prevention of slow transit constipation of rats:48 male Wistar rats were randomly divided into four groups:control group,model group,low-dose aloe group and high-dose aloe group,with each 12.Model group,low-dose aloe group and high-dose group rats were given Compound Diphenoxylate 8 mg/ kg·bw by gavage,blank control group given the same volume of distilled water.Two hours later, the low-dose aloe group rats were given aloe 0.33 g / kg·bw,and high-dose group 0.66 g / kg·bw,while model group and the blank control group given the same volume of distilled water.Administration lasted eight weeks,gastrointestinal transit time and faeces weight were observed and recorded every fortnight.Eight weeks later, 8 rats in each group were killed and plasma and tissue specimens were collected in order to determine motilin(MTL),substance P(SP),vasoactive intestinal peptide (VIP)in plasma and colon.The rest rats were perfusion-fixed with paraformaldehyde for pathological examination.
4.2 Effects of aloe on treatment of slow transit constipation of rats:48 Wistar rats were randomly divided into 2 groups:blank control group(n=12),STC model group (n=36).The rats of STC model group were given Compound Diphenoxylate 8 mg/ kg·bw by gavage every day for 8 weeks,blank control group given the same volume of distilled water.Eight weeks later,gastrointestinal transit time and faeces weight were observed and recorded.STC model rats were randomly divided into 3 groups: natural recovery group,low-dose aloe group,high-dose aloe group,with 12 each group.The 10~(th)week,the low-dose aloe group rats were given aloe 0.33 g/kg·bw, and high-dose group 0.66 g / kg·bw,while model group and the blank control group mice given the same volume of distilled water.Two weeks later,8 rats in each group were killed and plasma and tissue specimens were collected in order to determine motilin,substance P,vasoactive intestinal peptide in plasma and colon.The rest rats were perfusion-fixed with paraformaldehyde for pathological examination.
Results
1 Determination of aloin level in different aloe product:the aloin level in Aloe vera whole leaf powder is 0.088%,refined powder 0.266%,lyophilized powder leaves 0.908%,the whole foliar spray powder 6.71%,purified aloe powder for medicine 13.97%,aloe capsule 3.41%.
2 Screening on facitating feces excretion functions of different aloe products:Aloe refined powder and purified aloe powder for medicine could shorten the first dejection time,increase stool amouts and weight within six hours.Aloe leaves freeze-dried powder could shorten the first dejection time,increase stool weight.Aloe leaves sprayed powder could increase the weight of the stool.The whole aloe leaf powder has no obvious effects on the constipation.Among all the aloe products that were tested,aloe refined powder was found to be the most effective treatment on constipation.
3 Effects of aloe on defecation and intestinal motor function of mice
3.1 Effects of aloe on defecation function of normal mice:Aloe could shorten the first dejection time,increase stool amouts and weight,also could increase fecal water content.There were no significant effects on intestinal motor function to be observed in mice treated with Cisapride.
3.2 Effects of aloe on defecation function of mice with constipation:aloe could improve stool traits.Compared with constipation model group,aloe in from 0.17g / kg·bw to 1.0 g / kg·bw dose could shorten the first black stool time and increase stool grains and weight in 6 hours of mice with constipation.
3.3 Effects of aloe on intestinal motor function of mice:Compared with constipation model group,the propulsion rate of intestines in the aloe group was significantly higher than that of model group as well.The NO level in high-dose aloe group decreased obviously compared with model group(P＜0.05),and there was a negative correlation between the NO level of serum and propulsion rate of intestines in mice (r=-0.346,P＜0.05).
4 Effects of aloe on prevention and treatment of slow transit constipation of rats
4.1 Effects of aloe on prevention of slow transit constipation of rats
Compared with normal control group,STC model group rats had a longer first black stool time,less stool grains and decreased fecal water content,which showed STC model is induced successful.Compared with the STC model group,aloe prevention group could shorten the first black stool time and increase stool grains and fecal water content.
Compared with normal control group,MTL decreased in intestinal tissue in the rats of STC model group(P＜0.05).Compared with the STC model group,high dose aloe could increase MTL content(P＜0.05).Compared with normal control group,SP content decreased in plasma and intestinal tissue in the rats of STC model group. Compared with the STC model group,low dose aloe could increase plasma SP content(P＜0.05),high-dose aloe could increase plasma and intestinal tissue SP content(P＜0.01).Compared with normal control group,VIP decreased in plasma and intestinal tissue in the rats of STC model group(P＜0.01).Compared with the STC model group,the low-dose aloe could increase intestinal tissue VIP content(P＜0.05), high-dose aloe could increase the plasma and intestinal tissues VIP content(P＜0.05).
4.2 Effects of aloe on treatment slow transit constipation of rats.
The results showed that compared with normal control group,STC model extent first black stool time(P＜0.01),decrease defecation grains(P＜0.01).Compared with the STC model group,aloe group could shorten the first black stool time and increase stool grains and fecal water content(P＜0.01).
Compared with STC model group,MTL content increased in the high-dose aloe group(P＜0.05).Compared with normal control group,SP decreased in plasma and intestinal tissue in the rats of STC model group(P＜0.05,P＜0.01).Compared with STC model group,high-dose aloe could increase the plasma and intestinal tissues SP content(P＜0.01).Compared with normal control group,VIP decreased in plasma and intestinal tissue in the rats of STC model group(P＜0.01).Compared with STC model group,VIP levels in the intestinal tissue and plasma both in low-dose and high-dose aloe group increase,and high-dose aloe group has a statistically significant(P＜0.05).
Conclusions
1 Aloe products had the facitating feces excretion function,and which might be related to the processing methods and other factors.Refined aloe powder has a better role.
2 To the normal mice,given the aloe of 1.0g/kg·bw to had the facitating feces excretion function,while to the constipation mice,0.17g / kg·bw had the same effect.
3 Aloe could promote the mobility of intestine and ameliorate the constipation induced by Compound Diphenoxylate of mice,which might attribute to the increase of fecal water content and decrease of the serum NO level.
4 Aloe had effect in the prevention and treatment of STC developed by Compound Diphenoxylate,which may be related to raise the MTL,SP,VIP content in plasma and intestinal tissue.
方法
1芦荟制品中芦荟苷含量测定
采用HPLC分别测定库拉索芦荟全叶干粉、精制干粉、全叶冻干粉、全叶喷干粉、纯化药用级干粉及市售复方芦荟胶囊六种芦荟制品中芦荟苷含量。测定条件:ZORBAX SB-18色谱柱(4.6mm×150mm,5μm),检测波长355nm,流动相为乙腈:水(25:75),流速:1ml/min,外标法测定不同芦荟制品中芦荟苷含量。
2芦荟制品通便功能筛选
采用复方地芬诺酯(10mg/kg·bw)诱发动物便秘模型,给予以上芦荟制品(1.0g/kg·bw),观察各组小鼠首次排黑便时间、6h排便粒数、粪便性状、粪便干湿重。
3芦荟对小鼠排便及肠运动功能的影响
3.1芦荟对正常小鼠排便功能的影响
雄性昆明小鼠50只,随机分为5组,空白对照组、芦荟低剂量组(0.17 g/kg·bw)、芦荟中剂量组(0.33 g/kg·bw)、芦荟高剂量组(1.0 g/kg·bw)及阳性对照组西沙必利(2.5mg/kg·bw),给药后连续观察每只动物首次排黑便时间、6h排便粒数、粪便湿重,计算粪便含水率。
3.2芦荟对便秘小鼠排便功能的影响
雄性昆明小鼠60只小鼠随机分为6组,空白对照组、芦荟低剂量组(0.17 g/kg·bw)、芦荟中剂量组(0.33 g/kg·bw)、芦荟高剂量组(1.0 g/kg·bw)、便秘模型对照组和阳性对照组西沙必利(2.5mg/kg·bw),连续灌胃7天,第8天便秘模型对照组、阳性对照组和芦荟低、中、高剂量组给予复方地芬诺酯(5 mg/kg·bw),空白对照组给予等体积的蒸馏水。给复方地芬诺酯0.5h后,芦荟低、中、高剂量组及阳性对照组分别给予含受试样品的指示墨汁、空白和便秘模型对照组只给墨汁。观察记录每只动物首次排黑便时间、6h排便粒数及重量,并计算粪便含水率。
3.3芦荟对便秘小鼠肠运动功能的影响
动物分组及给药方法同3.2,给予墨汁0.5h后,摘眼球取血,脱臼处死动物,迅速取出整个肠道,计算墨汁推进率,采用硝酸还原酶法测定血清中NO含量。
4芦荟对大鼠慢传输型便秘的预防与治疗作用
4.1预防作用:48只雄性Wistar大鼠随机分为空白对照组、STC模型组、芦荟小剂量组(0.33g/kg·bw),芦荟大剂量组(0.66g/kg·bw)共4组,每组12只。模型组,芦荟小剂量组及芦荟大剂量组的大鼠每天给予8 mg/kg.bw的复方地芬诺酯,空白对照组给予同体积蒸馏水;间隔2h后芦荟小剂量组给予芦荟混悬液,空白对照组及模型组给予同样体积的蒸馏水。连续给药8周,每两周测定肠道传输速度及粪便称重。8周后断头处死每组8只大鼠用于采集血浆及组织标本,测定血浆及肠组织中胃动素(motilin,MTL)、P物质(substance P,SP)、血管活性肠肽(vasoactiveintestinal peptide,VIP)含量,4只大鼠用于病理观察。
4.2治疗作用:48只Wistar大鼠随机分为空白对照组(12只)、STC模型组(36只)。STC模型组每天给予复方地芬诺酯灌胃,剂量8mg/kg.bw,灌胃体积0.5ml/100g·bw,空白对照组给予同体积蒸馏水。连续给药8周后,停药1周,测定大鼠的肠道传输速度。将STC模型组大鼠随机分为3组:自然恢复组、芦荟小剂量组(0.33g/kg·bw)、芦荟大剂量组(0.66g/kg·bw),每组12只。自第10周,各芦荟组分别经灌胃给予不同剂量芦荟,空白对照组及模型恢复组给予同体积蒸馏水,给药2周后测定大鼠肠道传输速度及粪便称重,8只大鼠用于采集血浆及组织标本,测定血浆及肠组织中胃动素(motilin,MTL)、P物质(substance P,SP)、血管活性肠肽(vasoactive intestinal peptide,VIP)含量,4只大鼠用于病理观察。
结果
1芦荟制品中芦荟苷含量:库拉索芦荟全叶干粉、精制干粉、全叶冻干粉、全叶喷干粉、纯化药用级干粉及市售复方芦荟胶囊六种芦荟制品的芦荟苷含量依次为0.088%、0.266%、0.908%、6.71%、13.97%和3.41%。
2芦荟精制干粉、芦荟纯化药用级干粉能够缩短便秘小鼠的首便时间,增加其所排粪便的粒数和重量;芦荟全叶冻干粉能够缩短便秘小鼠的首便时间,增加其所排粪便重量;芦荟全叶喷干粉能够增加排出粪便的重量,芦荟全叶干粉未见明显通便作用。芦荟精制干粉对便秘的效果较好。
3芦荟对小鼠排便及肠运动功能的影响
3.1芦荟对正常小鼠排便功能的影响高剂量的芦荟能缩短正常小鼠的排便时间、增加排便粒数、重量及粪便含水率,对正常小鼠具有润肠通便作用。
3.2芦荟对便秘小鼠排便功能的影响与空白对照组相比,便秘模型对照组小鼠的首便时间显著延长,6h黑便粒数减少。与便秘模型对照组相比,芦荟低、中、高剂量组均能不同程度的缩短便秘小鼠的首便时间,增加6h黑便粒数及粪便干、湿重,并能增加粪便含水率。小鼠排便干、湿重随着芦荟浓度的增加而增加,呈现明显的剂量效应关系。与便秘模型对照组相比较,芦荟各剂量组小鼠粪便含水率均有显著增加。阳性对照组小鼠的首便时间明显缩短,6h黑便粒数增加,但粪便干、湿重及粪便含水率与模型对照组小鼠相比,无明显差别。
3.3芦荟对小鼠肠推进率的影响
便秘模型对照组小鼠的墨汁推进率明显低于空白对照组。芦荟中、高剂量组均能提高小鼠的小肠墨汁推进率,且芦荟给药剂量与小鼠的小肠墨汁推进率成剂量-效应关系,表明芦荟能增加小鼠的肠推进率,促进肠道运动。便秘模型对照组的NO含量均值比空白对照组升高了64.6%,与便秘模型对照组比较,芦荟低、中、高剂量组的NO水平分别降低了4.33%、17.00%、21.80%,且高剂量组与便秘模型对照组相比有明显差异(P＜0.05)。相关性分析表明血清NO含量与小肠墨汁推进率呈负相关,r=-0.346(P＜0.05)。阳性对照组小鼠血清NO水平与模型对照组小鼠相比,无明显差别。
4芦荟对大鼠慢传输型便秘的预防与治疗作用
4.1芦荟对大鼠慢传输型便秘的预防作用及对胃肠激素的影响
肠道传输功能测定结果表明,与正常对照组相比,STC模型组的首次排便时间延长,排便粒数减少,粪便含水率降低,模型成功。与STC模型组相比,芦荟预防组大鼠首便时间缩短,排便粒数增加的粪便干、湿重及粪便含水率均增加。
与正常对照组相比,STC模型组大鼠肠组织中MTL含量降低(P＜0.05);与模型组相比,芦荟大剂量预防组MTL含量升高(P＜0.05)。
与正常对照组相比,STC模型组大鼠血浆及肠组织中SP含量均降低;与模型组相比,芦荟小剂量预防组血浆SP含量升高(P＜0.05),芦荟大剂量预防组血浆及肠组织中SP含量均升高(P＜0.01)。
与正常对照组相比,STC模型组大鼠血浆及肠组织中VIP含量均降低(P＜0.01);与模型组相比,芦荟小剂量组肠组织中的VIP含量升高(P＜0.05),芦荟大剂量组血浆及肠组织中的VIP含量均升高(P＜0.05)。
4.2芦荟对大鼠慢传输型便秘的治疗作用及对胃肠激素的影响
肠道传输功能测定结果表明,与正常对照组相比,STC模型恢复组的首次排便时间延长(P＜0.01),排便粒数减少(P＜0.01);与STC模型恢复组相比,芦荟大、小剂量治疗组的首便时间缩短,且差异有统计学意义(P＜0.01)。
与正常对照组相比,STC模型恢复组大鼠肠组织中MTL含量降低,但差异无统计学意义;与模型组相比,芦荟大剂量组MTL含量升高(P＜0.05)
与正常对照组相比,STC模型恢复组大鼠血浆及肠组织中SP含量均降低(P＜0.05,P＜0.01);与模型恢复组相比,芦荟大剂量组血浆及肠组织中SP含量均升高(P＜0.01)。
与正常对照组相比,STC模型恢复组大鼠血浆及肠组织中VIP含量均降低,肠组织中的降低具有统计学意义(P＜0.01);与模型恢复组相比,芦荟小剂量和大剂量预防组血浆及肠组织中VIP含量均有升高,其中芦荟大剂量组肠组织中VIP含量升高具有统计学意义(P＜0.05)
结论
1芦荟制品具有润肠通便作用,其作用大小可能与加工方式有关,其中芦荟精制干粉通便作用较好。
2 1.0g/kg.bw的芦荟对正常小鼠具有润肠通便作用,而0.17g/kg.bw的芦荟对便秘小鼠即呈现明显的润肠通便作用。
3芦荟能明显改善复方地芬诺酯诱导的肠蠕动抑制小鼠的肠道运动功能,并呈剂量效应关系。此作用可能与芦荟增加粪便含水率、降低血清NO有关。
4芦荟对复方地芬诺酯诱导的大鼠STC具有一定的预防和治疗作用,可能与升高血浆和肠组织MTL、SP、VIP含量有关。
Objectives
Slow transit constipation(STC)is a common clinical gastrointestinal disease attributed to ineffective colonic propulsion and/or increased resistance to propagation of colonic contents.The gradually rising prevalence rate of STC,which increases with age,is due in large part to the changes of people's life style.Constipation not only seriously affects the quality of life,and also has a close positive relationship with acute cardiovascular and cerebrovascular disease,dementia,colorectal cancer and so on.Aloe is rich in anthraquinone compounds,polysaccharides,amino acids,trace elements,and other variety of chemical constituents.Although aloe for the treatment of constipation have a long history,the mechanism of promotion of intestinal movement mechanism still remains unclear,particularly regulating gastrointestinal hormones have not yet reported.Based on the above points,aloin-the active ingredients of aloe was determined by HPLC,then slow transit constipation animal model was induced by Compound Diphenoxylate and the mechanism of preventive and the therapeutic effect of aloe on STC was observed.We also focus on the different changes of gastrointestinal hormone between STC animal and those treated with aloe in this study.These results might be beneficial to the measures of prevent and treat slow transit constipation.
Methods
1 Determination of aloin level in different aloe product
The aloin level in different aloe product were determined by HPLC with the following determination conditions:ZORBAX SB-18 column(4.6mm×150mm,5μm),the detection wavelength was 355 nm,the mobile phase of acetonitrile:water (25:75),flow rate:1ml/min,use external reference method to determine aloin in different aloe products by HPLC.
2 Screening on facitating feces excretion functions of different aloe products
The aloe products(1.0 g/kg·bw)were given mice in advance,then Compound Diphenoxylate(10 mg/kg·bw)was used to induce constipation animal model.The first dejection time,stool amouts within six hours,feces traits,wet and dry faeces weight were observed.
3 Effects of aloe on defecation and intestinal motor function of mice
3.1 Effects of aloe on defecation function of normal mice:50 male Kunming mice were randomly divided into five groups,i.e.control group,aloe low,middle and high dose group(0.17 g/kg·bw,0.33 g/kg.bw,1.0 g/ kg·bw)and the positive control group(cisapride,2.5 mg/kg·bw).The first dejection time,stool amours within six hours,feces traits,fresh and dry faeces weight were observed after administration,and then fecal water content was calculated.
3.2 Effects of aloe on defecation function of costive mice:60 male Kuning mice were randomly divided into 6 groups,aloe low,middle and high dose group(aloe 0.17 g/ kg·bw,0.33 g/kg·bw,1.0 g/kg·bw),the positive control group(cisapride,2.5 mg/ kg·bw),the blank control group and the model control group.Mice of Aloe low, middle and high dose group and the positive control group were given the corresponding dose aloe by gavage for seven days,the mice in blank control group and the model control group given the same volume of distilled water.On the 8~(th)day the mice in model group,positive control group and aloe low,middle and high dose groups were treated with Compound Diphenoxylate(5 mg/kg·bw),mice of the blank control group with the same volume of distilled water.After treating with Compound Diphenoxylate 0.5 hours,aloe low,middle and high dose group and the positive control group were given the ink mixed with corresponding tested samples,while other mice only ink.The first dejection time,stool amours within six hours,feces traits,fresh and dry faeces weight were observed and recorded after Diphenoxylate administration,then fecal water content were calculate.
3.3 Effects of aloe on intestinal motor function of mice:Animal groups and the administration method were same as 3.2 After giving ink 0.5 hour,blood were collected from inner canthus and content of NO were determined by using method of nitrate reductase,then mice were sacrificed,the entire intestine were quickly removed, and the intestinal transit ratio were calculated.
4 Effects of aloe on prevention and treatment of slow transit constipation of rats
4.1 Effects of aloe on prevention of slow transit constipation of rats:48 male Wistar rats were randomly divided into four groups:control group,model group,low-dose aloe group and high-dose aloe group,with each 12.Model group,low-dose aloe group and high-dose group rats were given Compound Diphenoxylate 8 mg/ kg·bw by gavage,blank control group given the same volume of distilled water.Two hours later, the low-dose aloe group rats were given aloe 0.33 g / kg·bw,and high-dose group 0.66 g / kg·bw,while model group and the blank control group given the same volume of distilled water.Administration lasted eight weeks,gastrointestinal transit time and faeces weight were observed and recorded every fortnight.Eight weeks later, 8 rats in each group were killed and plasma and tissue specimens were collected in order to determine motilin(MTL),substance P(SP),vasoactive intestinal peptide (VIP)in plasma and colon.The rest rats were perfusion-fixed with paraformaldehyde for pathological examination.
4.2 Effects of aloe on treatment of slow transit constipation of rats:48 Wistar rats were randomly divided into 2 groups:blank control group(n=12),STC model group (n=36).The rats of STC model group were given Compound Diphenoxylate 8 mg/ kg·bw by gavage every day for 8 weeks,blank control group given the same volume of distilled water.Eight weeks later,gastrointestinal transit time and faeces weight were observed and recorded.STC model rats were randomly divided into 3 groups: natural recovery group,low-dose aloe group,high-dose aloe group,with 12 each group.The 10~(th)week,the low-dose aloe group rats were given aloe 0.33 g/kg·bw, and high-dose group 0.66 g / kg·bw,while model group and the blank control group mice given the same volume of distilled water.Two weeks later,8 rats in each group were killed and plasma and tissue specimens were collected in order to determine motilin,substance P,vasoactive intestinal peptide in plasma and colon.The rest rats were perfusion-fixed with paraformaldehyde for pathological examination.
Results
1 Determination of aloin level in different aloe product:the aloin level in Aloe vera whole leaf powder is 0.088%,refined powder 0.266%,lyophilized powder leaves 0.908%,the whole foliar spray powder 6.71%,purified aloe powder for medicine 13.97%,aloe capsule 3.41%.
2 Screening on facitating feces excretion functions of different aloe products:Aloe refined powder and purified aloe powder for medicine could shorten the first dejection time,increase stool amouts and weight within six hours.Aloe leaves freeze-dried powder could shorten the first dejection time,increase stool weight.Aloe leaves sprayed powder could increase the weight of the stool.The whole aloe leaf powder has no obvious effects on the constipation.Among all the aloe products that were tested,aloe refined powder was found to be the most effective treatment on constipation.
3 Effects of aloe on defecation and intestinal motor function of mice
3.1 Effects of aloe on defecation function of normal mice:Aloe could shorten the first dejection time,increase stool amouts and weight,also could increase fecal water content.There were no significant effects on intestinal motor function to be observed in mice treated with Cisapride.
3.2 Effects of aloe on defecation function of mice with constipation:aloe could improve stool traits.Compared with constipation model group,aloe in from 0.17g / kg·bw to 1.0 g / kg·bw dose could shorten the first black stool time and increase stool grains and weight in 6 hours of mice with constipation.
3.3 Effects of aloe on intestinal motor function of mice:Compared with constipation model group,the propulsion rate of intestines in the aloe group was significantly higher than that of model group as well.The NO level in high-dose aloe group decreased obviously compared with model group(P＜0.05),and there was a negative correlation between the NO level of serum and propulsion rate of intestines in mice (r=-0.346,P＜0.05).
4 Effects of aloe on prevention and treatment of slow transit constipation of rats
4.1 Effects of aloe on prevention of slow transit constipation of rats
Compared with normal control group,STC model group rats had a longer first black stool time,less stool grains and decreased fecal water content,which showed STC model is induced successful.Compared with the STC model group,aloe prevention group could shorten the first black stool time and increase stool grains and fecal water content.
Compared with normal control group,MTL decreased in intestinal tissue in the rats of STC model group(P＜0.05).Compared with the STC model group,high dose aloe could increase MTL content(P＜0.05).Compared with normal control group,SP content decreased in plasma and intestinal tissue in the rats of STC model group. Compared with the STC model group,low dose aloe could increase plasma SP content(P＜0.05),high-dose aloe could increase plasma and intestinal tissue SP content(P＜0.01).Compared with normal control group,VIP decreased in plasma and intestinal tissue in the rats of STC model group(P＜0.01).Compared with the STC model group,the low-dose aloe could increase intestinal tissue VIP content(P＜0.05), high-dose aloe could increase the plasma and intestinal tissues VIP content(P＜0.05).
4.2 Effects of aloe on treatment slow transit constipation of rats.
The results showed that compared with normal control group,STC model extent first black stool time(P＜0.01),decrease defecation grains(P＜0.01).Compared with the STC model group,aloe group could shorten the first black stool time and increase stool grains and fecal water content(P＜0.01).
Compared with STC model group,MTL content increased in the high-dose aloe group(P＜0.05).Compared with normal control group,SP decreased in plasma and intestinal tissue in the rats of STC model group(P＜0.05,P＜0.01).Compared with STC model group,high-dose aloe could increase the plasma and intestinal tissues SP content(P＜0.01).Compared with normal control group,VIP decreased in plasma and intestinal tissue in the rats of STC model group(P＜0.01).Compared with STC model group,VIP levels in the intestinal tissue and plasma both in low-dose and high-dose aloe group increase,and high-dose aloe group has a statistically significant(P＜0.05).
Conclusions
1 Aloe products had the facitating feces excretion function,and which might be related to the processing methods and other factors.Refined aloe powder has a better role.
2 To the normal mice,given the aloe of 1.0g/kg·bw to had the facitating feces excretion function,while to the constipation mice,0.17g / kg·bw had the same effect.
3 Aloe could promote the mobility of intestine and ameliorate the constipation induced by Compound Diphenoxylate of mice,which might attribute to the increase of fecal water content and decrease of the serum NO level.
4 Aloe had effect in the prevention and treatment of STC developed by Compound Diphenoxylate,which may be related to raise the MTL,SP,VIP content in plasma and intestinal tissue.
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