| Objectives1.The comparative study of different concentrations of ethanol extracts of Baqia(Smilax china)was conducted in mice ear edema model and rats cotton ball-induced granuloma model to evaluate pharmacological effect,so it would to provide a reference for determining the best extraction process of Baqia.2.The comparative study of different polar organic solvent extracts of Baqia was conducted in mice ear edema model and rats cotton ball-induced granuloma model to evaluate pharmacological effect,to determine the bioactive fraction for anti-inflammatory and anti-CPID.So that we can provide scientific reference to the purification process of the Baqia preparation.3.The pharmacodynamic study of Baqia bioactive fraction extracted with modem extraction,separation and purification process was conducted in mice ear edema model and rats granuloma model.lt is to determine the pharmacological effect for anti-inflammatory and anti-CPID.So we can provide a reference for the secondary development pilot production process of Baqia preparation.4.The acute toxicity study of Baqia bioactive fraction with gavage to mice which will make the safety evaluation of those trials drug and will provide a scientific basis for clinical trial.Methods1.The pharmacodynamic screening study of different concentrations of ethanol extracts of Baqia1.1 The two hundred and thirty-eight female Kunming mice were randomly divided into seventeen groups,fourteen in each group.Baqia water extract,30%,50%,70%,95%ethanol extract at high(46.8 g·kg-1),medium(23.4 g·kg-1)and low(11.7 g·kg-1)dose groups were gavaged to mice(as crude drug meter,those are equivalent to 4,2,1 times of the clinical equivalent dose);The Jingangteng capsule group was gavaged 23.4 g·kg-1 to mice(as crude drug meter,those are equivalent to 2 times of the clinical equivalent dose);The model control group was given an equal volume of distilled water.q.d for three days continuous administration.Ear edema model in mice caused by xylene were produce for observing the left and right ear piece quality changes in each group and calculating ear swelling.The mice were midline cut in abdomen.The spleen were removal and weigh for calculated organ index.1.2 The one hundred and seventy female SD rats were randomly divided into seventeen groups of ten.Baqia water extract,30%,50%,70%,95%ethanol extract at high(32.4 g·kg-1),medium(16.2 g·kg-1)and low(8.1g·kg-1)dose groups were gavaged to mice(as crude drug meter,those are equivalent to 4,2,1 times of the clinical equivalent dose);The Jingangteng capsule group was gavaged 16.2 g·kg-1 to rats(as crude drug meter,those are equivalent to 2 times of the clinical equivalent dose);The model control group was given an equal volume of distilled water.Cotton ball-induced granuloma model in rats were produced,q.d for seven days continuous administration.after eight days of the first administration,3%sodium pentobarbital intraperitoneal anesthesia,the cotton balls were removed with the around proliferation of connective tissue from the original incision,which were excluded adipose tissue,placed oven of 60℃ for twenty-four hour and weighed by precision electronic balance.The original quality of cotton minuse the drying quality of cotton was granulomatous quality.2.The pharmacodynamic screening study of different polar organic solvent extracts of Baqia2.1 The two hundred and thirty-eight female Kunming mice were randomly divided into fourteen in seventeen groups.Baqia chloroform fraction,ethyl acetate fraction,n-butanol fraction,water,liquid fraction,fraction of ethyl acetate and n-butanol at high(46.8 g·kg-1),medium(23.4 g·kg-1)and low(11.7 g·kg-1)dose groups were gavaged to mice;The Jingangteng capsule group was gavaged 23.4 g·kg-1 to mice;The model control group was received an equal volume of distilled water,q,d for three days continuous administration.Ear edema model in mice caused by xylene were produce for observing the effects of different polar organic solvent extracts of Baqia.2.2 The one hundred and seventy female SD rats were randomly divided into seventeen groups of ten.Baqia chloroform fraction,ethyl acetate fraction,n-butanol fraction,water,liquid fractions,fraction of ethyl acetate and n-butanol at high(32.4 g·kg-1),medium(16.2 g·kg-1)and low(8.1g·kg-1)dose groups were gavaged to mice;The Jingangteng capsule group was gavaged 16.2 g·kg-1 to rats;The model control group was given an equal volume of distilled water.Cotton ball-induced granuloma model in rats were produced,q.d for seven days continuous administration.after eight days of the first administration,the effects of different polar organic solvent extracts of Baqia in rats granuloma model were observed.3.Baqia bioactive fraction Pharmacodynamic Study3.1 The seventy female mice were randomly divided into five groups of fourteen.Baqia bioactive fraction at high,medium and low dose groups were gavaged 46.8,23.4,11.7 g·kg-1 to mice;The Jingangteng capsule group was gavaged 23.4 g·kg-1 to mice;The model control group was received an equal volume of distilled water.q.d for three days continuous administration.Ear edema model in mice caused by xylene were produce for observing the effects of Baqia bioactive fraction.3.2 The fifty female SD rats were randomly divided into five groups of ten.Baqia bioactive fraction at high,medium and low dose groups were gavaged 32.4,16.2,8.1 g·kg-1 to rats;The Jingangteng capsule group was gavaged 26.2 g·kg-1 to rats;The model control group were received an equal volume of distilled water.Cotton ball-induced granuloma model in rats were produced,q.d for seven days continuous administration.after eight days of the first administration,the effects of Baqia bioactive fraction in rats granuloma model were observed.3.3 The seventy female SD rats CPID model which were successfully established were randomly divided into seven groups of ten.Baqia bioactive fraction of high,medium and low dose groups were gavaged 32.4,16.2,8.1 g·kg-1 to rats;The Jingangteng capsule group was gavaged 16.2 g·kg-1 to rats;The model control group,the normal control group and the Sham-operated group were gavaged to an equal volume of distilled water.Seven days after modeling at the beginning administration,q.d for fourteen day continuous administration.In the first fifteen days,The rats were injected to intraperitoneal anesthesia with 3%sodium pentobarbital.Abdominal aortic blood which were earried on detecting the blood routine and the blood rheology;The rats uterine with model side which were visual observed its change by anatomy morphology;The left and right side of the uterus were removal to weigh and calculate the rate of uterine swelling and swelling inhibition rate;The right side of the model uterus were soaked in formalin.HE conventional staining.The degree of pathological changes in rats uterus were observed under an optical microscope and the lesions were graded judge.4.The acute toxicity test of Baqia bioactive fractionThe forty Kunming mice(male and female)were randomly divided into two groups of ten.The Baqia bioactive fraction 748.8 g·kg-1 group(as crude drug meter,those are equivalent to sixty-four times of the clinical equivalent dose)was formulated into the suspension with the largest concentration of 37.44g·mL-1(Standard by the liquid through gavage needle and does not affect the normal gavage),disposable gavaged to 0.8ml.The normal control group were received an equal volume of distilled water.Mice were not provided food but given to water twelve hour before gavage to liquid.conventional breeding after administration.Observing the acute toxicity in mice consecutive for next fourteen days.5.StatisticalAll datas was dealed with by SPSS 16.0 statistical software.The measurement data was expressed as mean±standard deviation(x±s),One-way ANOVA were used to deal with the multiple samples comparation of completely randomized design data;LSD and Dunnett’s T3 test were took to compare the two groups when the data meeted homoscedasticity or not.Pathology score was dealed with by Rank sum test;Paired-samples T test was used to compare the body weight data with before and after in mice,weight data was dealed with by the variance analysis of repeated measures data.P<0.05 were considered statistically significant.Results1.The pharmacodynamic screening study that observed different concentrations of ethanol extracts of Baqia1.1 Compared with the model control group,70%ethanol extracts of Baqia at three dose groups have obvious inhibitory effect in mice ear swelling caused by xylene and the splenomegaly(P<0.05,P<0.01);it have good resistance to acute inflammation.Baqia water extract,30%,50%,95%ethanol extracts at three dose groups have no significant inhibition in mice ear swelling caused by xylene(P>0.05).Baqia 30%ethanol extracts at high and medium dose groups,50%ethanol extracts at medium and low dose groups and 95%ethanol extract at the low dose group can significantly inhibit the mice spleen enlargement(P<0.05);Baqia 50%ethanol extract at high dose group,95%ethanol extract at high and medium dose groups of splenomegaly have a significant inhibitory effect on splenomegaly(P<0.01);Baqia water extracts at three dose groups and 30%ethanol extract at low dose group have no significant inhibitory effect on splenomegaly(P>0.05)which have no statistically significant difference.1.2 Compared with the model control group,Baqia 70%ethanol extract at three dose groups have significant inhibition in rats granuloma model of cotton ball-induced proliferation(P<0.01);it have good resistance to chronic inflammation.Baqia 50%ethanol extracts at three dose groups,95%ethanol extracts at three dose groups have significant inhibition in rats granuloma model of cotton ball-induced proliferation P<0.01(;and the effect of Baqia 70%ethanol extracts at three dose groups are better than 50%ethanol extracts at three dose groups,Baqia 30%ethanol extracts at three dose groups and 95%ethanol extract at the low dose group can inhibit the eotton ball-induced proliferation in rats granuloma model(P<0.05),Baqia water extract at three dose groups have no significant difference for the cotton ball-induced hyperplasia in rats granuloma model(P>0.05).2.The pharmacodynamic screening study that observed different polar organic solvent extracts of Baqia2.1 Compared with the model control group,Baqia ethyl acetate fraction at three dose groups,fraction of ethyl acetate+n-butanol at three dose groups,n-butanol fraction at high dose groups have obvious inhibitory effect in mice ear edema caused by xylene(P<0.01);and Baqia ethyl acetate fraction at three dose groups are superior to fractions of ethyl acetate+n-butanol efficacy at three dose groups which are better than n-butanol fractions.Baqia n-butanol fractions at mediun and low dose groups have inhibition effect in mice ear edema caused by xylene(P<0.05);Baqia chloroform fraction and liquid fractions of Baqia at three dose groups have no significant inhibition in mice ear edema caused by xylene(P>0.05).The spleen index of mice compared with the model control group,Baqia ethyl acetate fraction at three dose groups,fractions of ethyl acetate+n-butanol at three dose groups,n-butanol fractions at high and medium dose groups,chloroform fraction at high dose group have obvious inhibitory effect in splenomegaly(P<0.01);while Baqia n-butanol fraction at low dose group,chloroform fractions at meditum and low dose groupsjiquid fractions of Baqia at three dose groups have not enlarged spleen inhibition(P>0.05)which no statistically significant difference.2.2 Compared with the model control group,Baqia ethyl acetate fraction at three dose groups,fractions of ethyl acetate+n-butanol at three dose groups,n-butanol fraction at high and medium dose groups have significantly inhibitory effect in the proliferation of cotton ball-induced granuloma model in rats(P<0.01);Baqia butanol fraction at low dose group has inhibited to the proliferation of cotton ball-induced granuloma model in rats(P<0.05);and Baqia ethyl acetate fraction at three dose groups are superior to fractions of ethyl acetate+n-butanol efficacy at three dose groups which are better than n-butanol fractions.Baqia chloroform fraction and liquid fraction of Baqia at three dose groups have no significant inhibition in the proliferation of cotton ball-induced granuloma model in rats(TP>O.05).3.Baqia bioactive fraction Pharmacodynamic Study3.1 Compared with the model control group,Baqia bioactive fraction at high and medium dose groups have significantly inhibitory effect in mice ear edema model caused by xylene(P<0.01);Baqia bioactive fraction at low dose group has also inhibited to mice ear edema model caused by xylene(P<0.05).The Jingangteng capsules group can significantly improve the degree of ear swelling in mice(P<0.01);it have good resistance to acute inflammation.The effect of Baqia bioactive fraction at high and medium dose groups are better than the Jingangteng capsule group,and following with those doses increases,the inhibition turn to stronger.Compared with the model control group,Baqia bioactive fraction at high and medium dose groups,The Jingangteng capsule group has significantly reduced to enlargement of the spleen(P<0.01);while Baqia bioactive fraction at low dose group has significant differences in splenomegaly(P<0.05).3.2 Compared with the model control group,Baqia bioactive fraction at three dose groups can significantly reduce to the proliferation of cotton ball-induced granuloma model in rats(P<0.01).It have good effect against chronic inflammation.The effect of Baqia bioactive fraction at high and medium dose groups are better than Jingangteng capsule group,And following with those doses increases,the inhibition turn to stronger.3.3 Compared with the model control group,Baqia bioactive fraction at three dose groups,The Jingangteng capsule group has significantly decreased to uterine swelling rate of rats(P<0.01).Baqia bioactive fraction at three dose groups,The Jingangteng capsule group has very significant difference in the whole blood viscosity of all the indexes(P<0.01).Baqia bioactive fraction at high and medium dose groups,The Jingangteng capsule group can very significantly reduce the number of white blood cells,lymphocytes percentage,neutrophil percentage,hematocrit,red blood cell count,hemoglobin levels(P<0.01);Baqia bioactive fraction at low dose group can reduce the number of white blood cells,lymphocytes percentage,neutrophil percentage,hematocrit,red blood cell count,hemoglobin levels(P<0.05).It have a good anti-inflammatory,anti-CPID effect.The effect of Baqia bioactive fraction at high and medium dose groups are better than the Jingangteng capsule group,And following with those doses increases,the inhibition turn to stronger.we can know that the rats uterine anatomic morphology are visual observed:compared with the model control group,Baqia bioactive fraction at three dose groups,The Jingangteng capsules group has different degrees reduced to uterine swelling and adhesion of pelvic connective tissue.The effect of Baqia bioactive fraction at high and medium dose groups are better than the Jingangteng capsule group.Pathology score shows as follows:compared with the model control group,Baqia bioactive fraction at three dose groups can very significantly improve the symptoms of uterine tissue lesions(P<0.01),reduce chronic inflammatory cell infiltration and improve congestion,edema and each layer uterine wall structure,the majority of glandular structures are clear and complete;The effect becomes more obvious as those doses increases.The Jingangteng capsule group has a very significant effect in the treatment(P<0.01)but the effect is not as good as Baqia at high and medium dose groups.4.The acute toxicity study of Baqia bioactive fractionThe results shows that Kunming mice have been fed with Baqia bioactive fraction with maximum tolerated dose of 748.8g·kg-1,those are equivalent to 64 times of the adult clinical equivalent dose.All mice have survived in the fourteen day test and do not occur any adverse reactions.Conclusions1.The effect anti-inflammatory activity of Baqia 70%ethanol extracts at three dose groups are the best.2.The effect anti-inflammatory activity of Baqia each chemical fraction at three dose groups are as follows:Baqia ethyl acetate fraction(flavonoids)>Baqia fraction of ethyl acetate+n-butanol>Baqia n-butanol fraction(saponins).3.Baqia bioactive fraction at three dose groups have anti-inflamnatory and anti-CP ID active effect.And the effect of Baqia bioactive fraction at high and medium dose groups are better than the Jingangteng capsule group.4.Baqia bioactive fraction within 748.5g·kg-1(as crude drug meter,those are equivalent to sixty-four times of the clinical equivalent dose)dose range is safe. |
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