Effects Of Mulberry Leaves Extracts On Isolated Intestinal Muscles In Guinea Pigs And Intestinal Function In Constipated Rats Induced By Loperamide

Part One: Effects of mulberry leaves extracts on isolated intestinal muscles tone of ileum and colon in guinea pigsConstipation is often accompanied by a drop in intestinal muscle tension,dry and hard stool and other characteristics.Studies have shown that mulberry leaves have pharmacological effects of lowering blood glucose and lipid,anti-inflammation,anti-oxidation,anti-tumor,improving immunity and regulating intestinal tract.The composition distribution of spring protein mulberry in water extract(ESML)and the extract of after frosted mulberry leaves(EFML)which prescribed by the pharmacopoeia were explored,and observed its effects on the changes of intestinal muscle tension to provide a basis for the full use of mulberry leaves resources and the development of products that improve constipation.Objective: To explore the composition distribution of ESML and EFML and their effects on guinea pig intestinal muscle tension,and to provide experimental basis for the application of mulberry leaves.Methods: 1.ESML and EFML were prepared,and the composition and content of the two extracts were compared by HPLC;the content of total flavonoids was compared by ultraviolet spectrophotometry.2.Intestinal segments of ileum and colon were collected from 10 healthy male guinea pigs,about 0.8cm long was took in each segment and were placed in a constant temperature water bath,which connected to a tension exchanger.The changes of muscle tension in vitro were recorded before and after administration,and the effects of different mulberry leaves extracts on spontaneous muscle tension and the contractile response of carbamoylcholine chloride(CCh)in isolated gut muscle were compared.Results: 1.The total flavonoids in ESML and EFML were 7.67% and 14.31% respectively by UV spectrophotometry.2.In vitro experiments results: 2.1.Effects on the contraction amplitude of intestinal muscle: ESML inhibited colon movement in low-dose(P<0.05)while promoted in high-dose(P<0.05),and promoted ileum movement in low-dose(P<0.01).EFML showed inhibitory effects on intestinal muscle contraction,among which low-dose administration in the colon(P<0.01)and high-dose administration in the ileum(P<0.05)had the most obvious effects.2.2.Influence on the contraction mean value: Different dose of ESML inhibited the contraction of colon(P<0.01)and promoted the ileum(P<0.05).Different dose of EFML significantly inhibited the contraction of ileum and colon(P<0.01).2.3.Influence on changes in intestinal muscle tension induced by CCh: ESML showed promotion contraction effects in low-dose(P<0.05)and inhibition effects in high-dose(P<0.05)on the ileum and colon.EFML showed inhibitory contraction effects on both the ileum and colon(P<0.01),and the higher dose was more significant.Conclusions: ESML has promotion effects on intestinal muscles tone,while EFML has significant inhibition effects on intestinal muscles tone.Part Two: Effects of water extract of mulberry leaves on intestinal tract of constipated rats and related mechanismAccording to the results of part one,the ESML have the effects of promoting intestinal muscle contraction,which may be suitable for the product research for treatment or prevention of constipation.The long-term constipation model and the initial constipation model were established by intragastric(i.g.)administration of loperamide to rats,to observe the effects of ESML on improving constipation.Objective: To investigate the effect of ESML on loperamide induced constipation rats,and explore the possible mechanism.Methods: 1.Treatment dosage regimen: 20 clean grade female Wistar rats were randomly divided into normal control group(NC,n=6),constipation model group(CM,n=7)and mulberry leaves group(ML,n=7),CM group and ML group was given loperamide(10 mg/kg/d),the NC group was given equal physiological saline solution(NS),ML group rats was given ESML(5 g/kg/d)for 7 days after 14 days of modeling,CM group and NC group rats were given equal NS;each group was i.g administrated one time everyday.2.Prevention dosage regimen: 32 clean grade female Wistar rats were randomly divided into NC group(n=6),3 days CM group(n=5),3 days ML group(n=5),7 days CM group(n=8)and 7 days ML group(n=8),ML groups was given ESML every morning and given loperamide every afternoon,CM group was given physiological saline solution every morning and given loperamide every afternoon,NC group was given equal physiological saline solution the same time,the total daily dose was the same as above;each group was i.g.administrated twice time everyday.3.Changes in fecal water content and body weight were recorded in each group.After the end of the experiment cycle,measure the percentage of pushing carbonic powder in small intestine.The transverse colon of rats was taken and paraffin sections were prepared for HE and immunohistochemical staining;the pathological changes and the expression of AQP3,e NOS,VIP and mucus quantity were observed.Results: 1.Change of fecal water content of rats: In treatment dosage regimen,fecal moisture content of CM group rats was significantly lower than NC group rats after modeling(P<0.01),and ML group was significantly higher than that in CM group rats after treatment(P<0.05).In the prevention dosage regimen,the fecal water content of CM group was lower than that of NC group,and the difference was significant on the 7th day(P<0.05).The fecal water content of ML group was significantly higher than that of NC group on the 3rd day(P<0.05),and the level of ML group was the same as that of NC group on the 7th day.2.Changes of small intestinal in propulsion rate of rats: The small intestinal propulsive rate in CM group was lower than that of NC group at different time of modeling,and the small intestinal propulsive rate of mulberry leaves treatment and prevention was significantly increased with significant difference(P<0.05).3.Changes in colon tissue morphology: Muscle thickness in the CM group was keep deceased with the extension of modeling time,compared with the NC group,and mucosal thickness in the CM group firstly increased and then decreased compared with the NC group,and all the corresponding ML groups showed corrective effects.4.Immunohistochemical results in colon tissue: After modeling,the expression levels of AQP3 and e NOS in CM group were higher than those in NC group(P<0.01,P<0.05),and the expression levels were decreased after intervention of mulberry leaves,even lower than that in NC group(P<0.01,P<0.05);and the effects of mulberry leaves prevention group for 3 days was the most significant.5.Changes of mucous in colon tissue: AB-PAS staining results showed that purplish-red neutral mucin appeared in the colon tissue sections of the CM group at different modeling times,and the mucus area decreased.The mulberry leaves had a improving effects on this change,and the mucus area increased significantly when the mulberry leaves were given for 3 days.Conclusions: The treatment administration using ESML significantly improve the long-term constipation model;and the preventive administration using ESML also have a good preventive effects on the chronic constipation model.The mechanism were related to increasing the intestinal muscle thickness,promoting the intestinal peristalsis,regulating the intestinal mucus secretion,and inhibiting the expression of AQP3,e NOS.