Evaluation On Pharmacodynamics And Safety Of Active Fractions Extract From "Antipyretic Analgesics Powder" And Mechanism Study

"Fever" and "pain" is a common clinical symptom for which the non-steroidal anti-inflammatory drug(NSAID) as the representative of antipyretic analgesics are applied largestly and widely in clinical application.However,there will be lots of adverse reactions after a long-term use,such as damage to gastrointestinal system, cardiovascular system damage,liver damage,kidney damage,central nervous system damage.Nowadays,because of fewer side-effects the naturally occurring drugs represented by Chinese traditional medicine become more and more acceptable to the Chinese medicine sector and attention,so it is necessary to select Chinese medicine for the research and development ofantipyretic analgesic.At present,the antipyretic analgesic efficacy and mechanism research about single natural herbs have been reported substantial,in which the methods applied are the troditional solvent extraction,such as water extraction,ethanol extraction or water extraction combining alcohol precipitation methods etc.These methods based on the ideas of the natural medicine research can not embody the characteristics of traditional Chinese medicine.The studies of the Chinese herbs compound are limited to domestic reports,most of which adopt the traditional solvent extraction method to extract the entire compound,explore the efficacy and mechanism.The extracts from compound throgh this method should not be the best active ingredients,not achieve to the best pharmacological effect and that isn't conducive to the mechanism study.As we all know,the composed of components in Chinese compound medicine are complex and the pharmacodynamic mechanism about it is not clear,so it can't represent the traditional Chinses medicine's characteristic through the traditional research ideas.We will adopt a new methodology in which we establish a pathological model corresponding to some a disease based on clinical efficacy,determine the one or more efficacy as the observation and evaluation indicator,then track the activity of the entire compound and various parts separated,explore the effective parts producing pharmacological effects from compound medicine and the effective chemical constituents,analyze its changes in quality and quantity and effect relationship,and interpret the mechanism of Chinese compound medicine to some extent."Antipyretic analgesics powder" is an old famous medicine of a Professor named Zang Kun-Tang in my school.It had a significant effect for headache,fever and other sympotoms caused by upper respiratory tract infection of exopathic typhoid through clinical testing for many years.But for the application in clinical in the form of powder,patients need a large dose,bad taste,get inconvenient to use and carry,at the same time learn from the health point of view does not conform to norms,so we should make secondary development using the new research methods.We choose the supercritical fluid extraction-Molecular Distillation(SFE-MD) technology,using orthogonal design test methods to search for the pharmacodynamic active fractions by the analgesia experiments for track targets.And we use the gas chromatography-mass spectrometry(GC-MS) method to analysis the active site extracted and isolated from compound.We get its main components of cinnamaldehyde(content 48.00%) and phenacetin(content 4.343%).To its active site of the antipyretic and analgesic efficacy studies,we explore the explore the development of the mechanism and safety of the initial evaluation,This thesis based on the prophase research,we have get a systematic evaluation on the antipyretic analgesic efficiency and safety of the active site,and carry out a preliminary exploration of its related mechanism for the next step.The next step in the drug-based lay the foundation for the development of new drugs.The purpose of this study was designed to increase the technology content of traditional Chinese midicine,and tap its potential effects to targeted enhanced and further improve clinical efficacy.The quality standards are more secure stable and reliable,so as to offer the better needs of more patients.Objective:Based on the work of the extraction and separation from "Antipyretic analgesics powder",the preliminary studies have been completed,we have get its active fractions.And there are three main purposes on this subject of the study:1,Evaluation on the pharmacodynamics of active fractions from its extraction.2,Study on its mechanism of Antipyretic and Analgesics.3,Evaluation on the safety of its active fractions.Methods:1.pharmacodynamics activity1.1 analgesic activityGlacial acetic acid induced writhing test:Forty seven mice were divided into five groups,three groups received the extracts of high doses,middle doses and low doses,while the remaining two groups were positive control and blank control.Every group was administered intragastrically for three consecutive days.One hour after the last administration,the mice were given an intraperitoneal injection(ip) of glacial acetic acid to induce the characteristic writhings.The number of writhings occurring among 20 minutes after acetic acid injection was recorded.Inhibitory writhings rate(%) was expressed as[(the mean number of writhings in blank control group -the mean in test group)/value of the control]×100.Hot plate test:The fifty mice used for this study were divided into five groups,the same condition with the above groups.The animals were each placed on a hot plate maintained at 55℃.Reaction time was taken as the latency index between the instant the animal reaches the hot plate till the moment the animal licks its forepaws.Before administration,record the latency two times and get the mean as the ground of latency.Then measurements were carried out 30min,60min,90min and 120min after intragastric administration(ig) of the extract.1.2 Antipyretic activityThe forty eithgt rats were divided into five groups,three groups received the extracts of high doses,middle doses and low doses,while the remaining two groups were positive control(aspirin group) and blank control(NS group).The tempreture was taken two times before model building,the average of its taken as the basic.Rats models of fever were established with lipopolysaccharide(LPS),which were proved to be succeed when the temperature rose after intraperitoneal injection(ip) 30 minutes later.The body tempreture was measured at 30min,60min,90rain,120min,150min and 180min after intragastrical administration(ig) of extracts to each group,and the changes were recorded.2.Testing the transmitters of antipyretic and analgesic2.1 Testing the transmitters of analgesic in miceAfter the mice models have being established with glacial acetic acid,pick the eye blood at once.The brain was decapitated as soon as that the work of picking blood was finished.The plasma and the tissue fluid were centrifuged from 10%tissue homogenate fluid prepared by the separated hypothalamus.Then the supernatant was obtained.The content of NO in peripheral blood was measured by NO kit's nitrate reduce method;theβ-endorphin(β-EP) of hypothalamic was by radioimmunoassay according to the prescription in kit.2.2 Testing the transmitters of antipyretic in ratsRat models of fever were established with lip polysaccharide.When the rats were in the peak of fever,pick the blood from post-orbital vein.The hypothalamus and VSA were isolated from the whole brain after the head being decapitated for preparing the tissue homogenate solution.The supematant was taken by centrifugating the plasma and tissue homogenate.Then according to the prescription in kit,the content of AVP in ASA,peripheral blood and the content of PEG₂ in hypothalamus were measured by radioimmunoassay.3.Evaluation of the safety Because of the limited volume of the target extracts,the preliminary test failed to detect the LD₅₀.With the greatest test to be administered orally in mice can withstand dose(0.8ml/20g).In the greatest resistance test,forty mice were divided into four groups three of which were received the extracts disposable by ig,while the remaining group was the bank control.The animal response was recorded detailed among seven days' conventional feeding.In 8^th day,at the base of autopsy,the main organs'(heart,liver,stomach,lung,kidney,etc.) general form got observed through naked eyes.After the pathological sections finished,they were placed under the light microscope for morphological examination.4.Statistical methodsIn this study data were expressed with mean±standard deviation((?)±s),analyzed through SPSS 13.0 for windows statistical software.Applied statistical methods included:1,Completely randomized design data.Use the One-way ANOVA statistical method,LSD will be applied to all groups;significant level ofα=0.05.2,The data of many levels and two factors repeated measure analysis of variance statistical method,applied multiple comparison of the mean analysis of variance, significant level ofα=0.05.Results:1.Analgesic efficacy1.1 Glacial acetic acid induced writhing testIntraperitoneal injection of glacial acetic acid could induce the mice' writhing. Three experimental groups decrease the number of mice' writhing significantly (F=98.838,P=0.000);all drug groups compared to negative control group,there is a significant difference(P=0.000).With the dose increasing,four groups'(from positive control and low to high dose) inhibitory writhing rate were 62.14%,58.15%,57.76% and 59.86%respectively.1.2 Hot plate testStatistical analysis showed that all experimental groups increased the mice's latency to hot plate in comparison with administration of the formers and the negative control group.Every dose group showed a significant elevation in the pain threshold (F=16.319,P=0.000);the difference of all the time after administration have statistic significance(F=56.554,P=0.000);Compared with each dose group and all the time the difference were significant statistically(F=9.139,P=0.000);The differences of the remaining groups was statistically significant(P≤0.001).But between 60min and 120min there were no statistics differences It proved that three drug dose group may be a marked increase in pain threshold in mice.The analgesic effect is the same with the positive control.The extraction has a significant analgesic effect.2.Antipyretic efficacyThe model was proved to be succeeded when the body temperature of rats began to rise 30 minutes after the intraperitoneal injection of negative control group about lippolysaccharide(LPS),achieved the peak at 120 minute,got the degree of 1～1.5℃mean rised,rats' body temperature in three experimental groups rised 30 minutes after intraperitoneal injection about lipopolysaccharide(LPS),while droped obviously in comparison with negative control group after administrion of extract and got the minimum at 120 minute,the difference of all the time after administration have statistic significance,F statistic and P value of main effect(F=19.707,P=0.000). Compared with each dose group the difference was significant statistically,F statistic and P value of main effect(F=11.374,P=0.000).F statistic and P value of crossover effect(F=2.409,P=0.003).The differences compared with NS group the difference between the remaining groups were statistically(P≤0.001).It's proved that three doses administrated groups have the ability to degrade the temperature,however there were no clear dose-effect relationship among of them.3.Detection of pain transmitterIn pain model being established with glacial acetic acid,the extract at low,high doses and the positive drugs produced a lower content level of NO significantly in mice' peripheral blood in comparison with negative control group(F=5.417, P=0.001),so did the positive control group,the low dose group and the high group compared to the blank control,the difference was significant statistically(P＜0.01).The middle dose compared with the NS group has a significant difference(P＜0.05).The test drug can reduce NO content in peripheral blood of mice,it has a analgesic effect.The content ofβ-EP in mice' hypothalamic in positive control group increased evidently compared to the negative group(F=2.679,P=0.045).The efficacy in increasing the content ofβ-EP had statistical significance just in high dose group(P＜0.05),however neither low dose nor middle dose groups had this in comparison with negative control group.4.Detection of antipyretie transmitterThe content of PGE₂ in hypothalamic in high,middle dose drug groups and positive control group were the same,and significantly less than the blank control (F=2.749,P=0.040).So did the aspirin group,middle dose and high dose compared with the NS group,the PGE₂ in hypothalamic has a significant reduce(P＜0.05).In abdominal cavity,the content of AVP in three dose drug groups achieved the higher level than the blank control group's(F=2.887,P=0.033).So did the aspirin group compared to NS group the difference was significant statistically(P＜0.01);the each drug doses compared with NS group the difference was significant statistically(P＜0.05).Drugs can increase the AVP content of rat's centralIn the peripheral blood,the content of AVP in three dose drug groups achieved the higher level than the blank control group's(F=3.813,P=0.010);so did the aspirin group compared to NS group,the difference was significant(P＜0.01);content increased just in high and middle dose groups compared with NS group,the difference was significant statistically(P＜0.05);the difference in low dose group were not statistically significant(P=0.383).It's proved that extraction of the drug has the ability to degrade the temprature,it has a significant antipyretic effect.5.Evaluation on the safetyAt the beginning of 2 days,mice appeared anepithymia,low interesting on activities,accelerated breathing after administration.Since the 3^th day,they had been prone to normal in drinking water,activities and weight changing,color smoothness, and possessed no deaths.Mice were executed after 8 days,dissected for reviewing the function about heart,liver,stomach,lung,kidney.As the result,there were no abnormal among them.Then the organizational pathological sections made for examination under the light microscope displayed no pathological change.It is indicated that the maximal tolerance dose in mice is not less than 30g/kg(equivalent to 2143g original medicine/kg) which was 2571 times more than the volume of clinical pharmacology.Conclusions:1..Extracts of "Antipyretic Analgesics Powder" have antipyretic analgesic effects.2.The antipyretic effect of extracts was demonstrated by influencing cerebrum center for significantly reducing PGE₂ content of neurotransmitters,an increase of negative regulation of neurotransmitter AVP content,but also increasing peripheral AVP levels.The extracts showed analgesics effect via increasing the central transmitter ofβ-endorphin content and significantly decreasing transmitter NO content in peripheral blood.3.The extracts from "Antipyretic Analgesics Powder" are not toxic and can be safely used by humans at moderate doses.