| Pomegranate Peel Polyphenols (PPP) is a complex phenolic secondary metabolites in theplant, which has the structure of polyphenol, and mainly presented in fruits, roots, leaves, seeds,and the highest content in pomegranate peel. Previous studies showed that the PPP has a varietyof physiological and pharmacological activities, such as bactericidal, antianaphylaxis, immuneregulation, protecting stomach, lowering blood pressure, anti-aging, and the protection of theheart. However, it has not been reported The Regulating Mechanism in the Central NervousSystem of Pomegranate Peel Polyphenols on the Gastric Motility in Restraint andWater-Immersion Stress Rats. In this study, to observe RWIS on the distribution of PPP in themajor organ of rats through intracerebroventricular injection, finally the prevention effect of PPPon RWIS induceing gastric mucosal injury, the regulation of gastric motility, the influence of thebrainstem neurons activities was studied. The central effect and mechanism of PPP wereexplored in order to offer a new idea on PPP medicinal value evaluation.The research was divided into five parts:1. Effect of restraint and water-immersion stress on distribution of pomegranate peelpolyphenol in the major organ of rat. The polyphenol content of rat organ was determined bythe colorimetric method. The method involved the use of polyphenol and ferric chloride to obtainprussian blue color. Wistar male rats were randomly divided into control group Ⅰ(2mLdeionized water+RWIS,n=5);control group Ⅱ(1000mg Aqueous extraction of pomegranatepeel(AEPP), without stress)and experimental groups Ⅰ、Ⅱ、Ⅲ (10、100、1000mg AEPP+RWIS).Rats were RWIS3hours after drugs administration for an hour. Rats were killed by anoverdose of anesthetic and then removed blood through heart perfusion. The brain, heart,stomach, lung, liver, kidney, ileum and colon were harvested, pounded to homogenate,centrifuged, then they were blended the supernate and ferric chloride together, color-reaction. The results showed that PPP of AEPP was detected best with9%ferric chloride. The responsewas obviously with AEPP of0.2～0.5mg/mL. The supernate of brain, heart, lungs, liver, andkidney of the rats were not observed in a color reaction. The supernate of stomach, colon withferric chloride were positive reacted in experimental group Ⅲ; And the control group Ⅱileum,colon, also showed positive reaction, which was higher than the experimental group Ⅲ. Theseresults suggested that9%ferric chloride can sensitively detected the content of PPP in0.2～0.5mg/mL AEPP. The PPP in gastrointestinal wall was distributed more than other organs. RWISwere contributed to PPP enrichment at stomach wall, but reduced the PPP content at ileum walland colon wall in rats.2. Effects of PPP on RWIS induced gastric mucosal damage index in rats. To study theprotective effect of PPP on the RWIS inducing gastric mucosal damage in rats byintracerebroventricular injection, the following measures were taken. Wistar male rats wererandomly divided into control group Ⅰ(Intracerebroventricular injection of saline solution5μL,n=5);control group Ⅱ(Intracerebroventricular injection of EPP100μg)and experimentalgroups Ⅰ、Ⅱ、Ⅲ、Ⅳ(Intracerebroventricular injection of PPP1μg、10μg、50μg、100μg).Ratswere RWIS3hours after drugs administration half hour. Rats were killed by an overdose ofanesthetic and then removed blood through heart perfusion. The stomach were harvested, andwere fixed for30min in4%paraformaldehyde, cut opened along the greater curvature of thestomach, detected gastric mucosal damage. The results were as follow:1～10μg PPP group,gastric mucosal damage index was significantly less than the saline group and EPP group (p<0.05， p<0.01)， but50μg PPP group was not significant （p>0.05）. In addition,Intracerebroventricular injection of a high concentration of PPP100μg group, gastric mucosaldamage index was significantly higher than the saline group (p <0.01) and the EPP group (p<0.01). The above results suggested PPP in certain dosage range(1～10μg) had the effects ofprevention. But50μg PPP effect was not significant. And the high concentration (100μg) of thePPP had promoted the effect of stress inducing gastric mucosal damage.3. Effects of PPP on RWIS rats gastric contraction waves. Animal division was as thesame as the second part. Intracerebroventricular buried pipe of rats were recovery for3days,fasted for24hours, lightly anesthetized with ether, a latex balloon implanted proventriculus, andthe stomach keep original position. Sutured and sealed the abdominal wall wound. After rats regained consciousness, RWIS, recorded gastric contraction waves. The amplitude, duration,frequency, and motility index of gastric contraction waves within10minutes beforemicroinjection and0-10min,10-20min,20-30min,30-60min (randomly taken10min),60-90min(randomly taken10min) after microinjection were measured. The results showed that comparedwith after microinjection,10μg-100μg PPP can increased amplitude, duration, frequency, andmotility index of gastric contraction waves with varying degrees of the RWIS rats（p<0.01, but1μg PPP group, EPP groups and saline group had not diversity（p>0.05）. It showed thatintracerebroventricular injection of10μg-100μg PPPhave the effect of the gastric motility,and1μg PPP group,100μg EPP group did not show obvious effect. The results indicated thatpolyphenols had the active ingredient.4. Effects of PPP on RWIS dorsal motor nucleus of the vagus (DMV), nucleus ofsolitary tract (NTS), nucleus ambiguous (NA), Locus Coeruleus (LC) c-Fos expression inrats. Using Fos protein as neuronal excitability markers with the method of singleimmunohistochemistry, the study observed different nucleis Fos protein expression levelschanges, in order to understand the impact of PPP on the RWIS inducing brainstem neuronalactivity. Intracerebroventricular injection of different concentrations of PPP can be induced Fosprotein expression was significantly increased in LC (p <0.01), however, Fos protein expressionwas significantly decreased in the DMV, NTS and NA (p <0.01). These results showed that PPPcan enhance the activity of neurons in the LC and recede the activity of neurons in the DMV,NTS and NA.5. Effects of PPP on RWIS dorsal motor nucleus of the vagus (DMV), nucleus ofsolitary tract (NTS), nucleus ambiguous (NA), Locus Coeruleus (LC) neuronal activity ofcatecholamines in rats. It was determined by a single immunohistochemistry method forTyrosine hydroxylase and a double immunohistochemical method for collocations of Fos withone of Tyrosine hydroxylase (TH), in order to observed Catecholamine neuron activity indifferent nucleis. Compared with saline group, it was found that intracerebroventricular injectionof different concentrations of PPP, TH-IR increased in both LC and substantia nigra, and theimplied PPP can promote catecholamine neurons synthesis in the LC and substantia nigra whiledecreased in the DMV, NTS and NA TH-IR. It suggested that PPP can inhibit the synthesis ofcatecholamine neurons. In a word, this study confirmed that PPP can affect catecholamine neuron activity in thebrain, and regulate the gastric motility, so PPP had the effects of prevention on RWIS induceinggastric mucosal damage index in rats. The experimental results of this study will providepreliminary experimental basis for future research and development of PPP, and it will be a goodidea for the research and development of other plant polyphenols. Nevertheless, PPP regulatedcatecholamine neuron activity on pharmacological targets still need further study. |
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