| Objective:Rhynchophylla is a member of rubiaceae. It is cool in nature, sweet and bitter in taste.Its hooks, branches and stems can be used as medicine to clear away pathogenic heat,soothe liver, tranquilize endogenous wind and arrest convulsion. And as a kind oftraditional Chinese medicine it has experienced a long history. Researches by far find thatrhynchophylla alkaloid has pharmacologic effects on cardiovascular system like reducingblood pressure, antiarrhythmia and relaxing vascular smooth muscles as well as other rolesin central nerves such as sedation, anti-convulsion, anti-epilepsy and anxiolytic, etc.Moreover, the current domestic researches on rhynchophylla alkaloid mainly focus onpharmacologic effects of rhynchophyline and isorhynchophyline such as reducing pressureand cerebral ischemia protection. Considering the function of uncaria alkaloids on thecentral nervous system effects, and has high natural ingredient safety, which is necessary todeeply research its anxiolytic activity.The study firstly built the high-efficient, easily operable and safe RTA extractionprocess through optimization and assay, which provides material basis for investigatinganxiolytic efficacy and its mechanism. At the same time, we established high performanceliquid chromatography-ultraviolet (HPLC-UV) for faster detection and better separation,which lays quantitative basis for RTA extraction, content determination and the examinationof stability. Then we made different anti-anxiety animal models according to theinternational criteria, to observe the anxiolytic effect of self-extracted RTA on animalbehaviors. Finally, enzyme-linked immunosorbent assay (ELISA) was used to detect thecontent of monoamine transmitters, like norepinephrine (NE), dopamine (DA), and 5-hydroxy tryptamine (5-HT) in cerebral tissues before and after the interference of RTA inEPM and know the possible related mechanism as well. On the whole, this study isconducted for exploring natural anxiolytic drugs with high efficacy and fewer side effectsas well as providing the experimental basis for traditional Chinese medicine made ofrhynchophylla.Methods:1. Investigation on RTA assayWe analyzed and extracted the main components of RTA-the four membered ringoxindole alkaloids including rhynchophyline, isorhynchophyline, corynoxeine,isocorynoxeine by high performance liquid chromatography (HPLC); at the same timeHPLC-UV with faster detection and better efficiency was examined and built; next examinedand determined the linear range, quantitative lower limits, degree of accuracy, reproducibilityand stability of this analysis method.2. Investigation on the effect of single-factors on optimizing the RTA extractionprocessWe qualitatively differentiated and analyzed the purchased rhynchophylla whichcomes from different areas by thin-layer chromatography, and then screened out thematerial which has high extraction efficiency. After the pre-stage work, taking productionratio of RTA as response value and ethanol-ultrasound extraction method as the basis, weapplied the next several single-factors to examine the extraction process.3. Investigation on the effect of orthogonal experiment on optimizing the RTAextraction processBased on the previous examination effect of the single-factors, next we did aorthogonal experiment with three factors and three levels which are about three materialratios, that is,1:5,1:10,1:15, three frequencies of extraction:1time,2times and3times,the time of ultrasound:1h,2h,3h. This is done to further optimize RTA extraction process.4. Investigation on the preliminary pharmacodynamics of RTA anxiolytic action.Taking DiaZepatn as a positive control, we carried out the primary pharmacodynamicstudy on anxiolytic effects of self-extracted RTA in different anti-anxiety animal models:5. Investigation on preliminary mechanism of anxiolytic action of RTA.We adopted ELISA to measure the content of three kinds of monoamine transmitters- norepinephrine (NE), dopamine (DA), and5-hydroxy tryptamine (5-HT) in cerebral tissuesof the rat models before and after the intervention of RTA in EPM to investigate its possiblerelated mechanism.Results：1. Study on content determination of rhynchophylla total alkaloidsThe UV detection wavelength is determined as245.4nm by applying sample injectionfrom the high performance liquid chromatography (HPLC-UV). The content of majoreffective components of samples of rhynchophylla total alkaloids, rhynchophylline,isorhynchophylline, corynoxeine and isocorynox-eine, is up to69%. Through separatingexaminations of specificity, HPLC-UV chromatographic method has been established withquick detection and good separation effect. With the mobile phase being0.01mol/Lmonopotassium phosphate (HCl pH2.85): acetonitrile (70:30), column temperature being25oC and the flow rate being1.0mL/min, elution is conducted and the proportion of itreduces linearly from70:30to65:35in0to10minutes. Results of the analysis have shownlinear regression equations respectively. rhynchophylline:y=6.17×104x-2.17×104(0,52~66μg/ml,r2=0.998);Isorhynchophylline:y=5.77×104x-1.20×104(0.46~59μg/ml,r2=0.997);Corynoxeine:y=6.38×104x–1.45×104(0.48~61μg/ml,r2=0.998);Isocorynox-eine: y=5.54×104x-1.86×104(0.45~58μg/ml,r2=0.998)。The recovery of the low, medium and high concentration ofrhynchophylline in recovery experiment results is103%,94.8%and103%respectively;recovery of the low, medium and high concentration of Isorhynchophylline is88%,90%,100%respectively; recovery of the low, medium and high concentration of corynoxeine is92%,88%,98%respectively; recovery of the low, medium and high concentration ofIsocorynox-eine is106%,113%,108%.RSD of the intra batch precision in repetitiveexperiment is1.52%,1.97%,1.81%,1.66%respectively. The inter batch precision is1.9%,1.2%,2.0%and1.9%respectively with good repeatability. Stability experiment resultsshow content RSD of extraction of total alkali solution at room temperature and coldstorage (4oC) on day1,2,3,4,5with good stability.2Single factor study on the extraction of rhynchophylla total alkaloidsAfter qualitative analysis and identification of TLC, uncaria rhynchophylla of differentorigins have been screened with the yield of them as the response value, results have shownhigh extraction rate of uncaria rhynchophylla in Hunan which has been determined as the basis of medicinal material. Through many of the above single factor examinations and acomprehensive evaluation, the best initial extraction is24hour quenching,80%ethanol,0.2%HCl acidification and triple amount of chloroform extraction for3times.3. Optimized study on the orthogonal experiment of the extraction of rhynchophyllatotal alkaloidsBased on the previous initial extraction of rhynchophylla total alkaloids, orthogonalexperiment (L933) optimization is conducted with three factors and three levels. Resultshave shown that based on optimum conditions of material ratio of1:10, twice extractionsand2hour ultrasonic, extraction of rhynchophylla total alkaloids, which involves materialratio of1:10,24hour quenching of0.2%HCl acidified80%ethanol, twice extractionsduring a2h exposure to ultrasonic, concentrated liquid ammonia with adapted pH10-11recycling from the extracting solution, triple amount chloroform extraction for3times,recycle, vacuum drying (<80oC), has been established with high extraction rate and simple,feasible and safe method. The extraction rate reaches0.2%.4. Preliminary pharmacodynamics study of anti-anxiety effect of rhynchophylla rotalalkaloidsSelf-made rhynchophylla total alkaloids have demonstrated different levels ofanti-anxiety efficacy on several different designed anxiety animal models and the efficacyis especially notable for the elevated plus-maze test of mice and rats and test of anticipatoryanxiety in mice: Compared with blank control group, the OE%and OT%of rats in RTA3dosage group is much higher (P<0.05). Compared with DZP positive control group, there isno statistical differences (P<0.05); increased body temperature caused by irritability hasshown in normal control group. However, this situation does not exist in positive controlgroup and3doses of RTA groups show the anti-anxiety effect. Results showed that RTAdemonstrated certain anti-anxiety effect.5. Preliminary mechanism of Rhynchophylla Total Alkaloids on anti-anxiety effectThrough ELISA determination of the content of three monoamine neurotransmitters,Norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in brain tissues of rats beforeand after EPM intervention, it is shown that with the comparison of RTA group and blankcontrol group, the content of NA, DA and5-HT in rat’s brain can be notably reduced, whichindicates that RTA has regulating effect on monoamine neurotransmitter after anxiety and it might be one of the anti-anxiety functional mechanism.Conclusion：1. With regard to the major effective components in the samples of rhynchophylla totalalkaloids (Rhynchophylline, Isorhynchophylline, Corynoxeine, isocorynox-eine), has builta HPLC-UV chromatographic conditions method with quick detection and good separatedeffect has been developed. The method in specificity, accuracy, sensitivity, reproducibility,etc all can meet the requirements of rhynchophylla total alkaloids measurement.2. Safe extraction of rhynchophylla total alkaloids with high extraction rate, simpleand feasible method has been developed: material ratio of1:10,24hour quenching of0.2%HCL acidified80%ethanol, twice extractions during a2hour exposure to ultrasonic,concentrated liquid ammonia with adapted pH10-11recycling from the extracting solution,triple amount chloroform extraction for3times, recovery, vacuum drying (<80oC). Theextraction yield is0.2%.3. Rhynchophylla rotal alkaloids have shown different levels of anti-anxiety efficacyon the animal models of experimental design and the efficacy is especially notable forthe elevated plus-maze test of mice and rats and test of anticipatory anxiety in mice. Theexperiments results indicate that rhynchophylla total alkaloids have certain anti-anxietyefficacy. They have regulating effect on monoamine neurotransmitter after anxiety model,which might be one of the anti-anxiety functional mechanisms. |
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