Chemical Constituents Of Pithecellobium Clypearia Benth.and Its Effects On The Psoriasis Mice Model Induced By Imiquimod

Purpose:Pithecellobium clypearia Benth.had long been used clinically as a heat-clearing and detoxifying Chinese medicine.Based on the determination of the antioxidant and anti-complementary active fractions of P.clypearia,this paper clarified the small molecule active ingredients and verified the effect of the active fractions of P.clypearia in vivo on the psoriasis mice model induced by imiquimod.Furthermore,the ethanol extraction process of P.clypearia was optimized.The purpose of this study is to reveal the pharmacodynamic basis of the heat-clearing and detoxification of P.clypearia from the perspective of anti-inflammatory and immune regulation,and provide experimental pharmacological evidence for systematically elucidating the biological essence of P.clypearia for clearing away heat and detoxification and grasping the scientific connotation of its property flavors attributive channel（xing wei gui jing in Chinese medicine）.Based on the basis,it provides a theoretical basis for the rational development and application of P.clypearia drug resources.Method:1 DPPH free radical scavenging method and cell hemolysis method were used to determine the antioxidant and anti-complementary activity of the ethanol extract and the different polarity part（ethyl acetate fraction,n-butanol fraction and water layer）,the water extract and the different part（ethanol precipitation and ethanol supernatant）from P.clypearia,respectively.2 Silica gel column chromatography,Sephadex LH-20 column chromatography and TLC were applied to separate the chemical constituents of ethyl acetate fraction of ethanol extract from P.clypearia.The structures of the compounds were identified by nuclear magnetic resonance.3 The antioxidant and anti-complementary activities of the compounds were determined by DPPH free radical scavenging method and cell hemolysis method.4 The safe dosage of medicine for P.clypearia was determined by the maximum tolerated amount experiment.5 The psoriasis mice model induced by imiquimod was established.PASI score,HE staining to detect morphological changes of skin tissue,measurement of organ index（spleen index and thymus index）and immunohistochemistry for detection of PCNA in skin tissue were used to investigate the intervention effect of ethanol extract and the ethanol supernatant of water extract from P.clypearia.6 The optimum ethanol extraction process of P.clypearia was investigated by orthogonal tests with integrated indicators of chemical analysis and anti-inflammatory activity.The extraction rate of extractum,total flavonoids and gallic acid from crude materials,antioxidant activities(IC₅₀value)and anti-complementary activities(CH₅₀value)in vitro were chosen as the assessment guideline.Ethanol volume fraction（%）,extraction time（h）,the amount of ethanol（fold）and the number of extractions（times）were investigated and the orthogonal extraction test L₉（3⁴）was used to determine the optimal ethanol extraction process.Results:1 Ethanol extract and water extract of P.clypearia showed strong antioxidant and anti-complementary activities,IC₅₀values were 0.22±0.01 mg/m L and 0.52±0.01mg/m L,CH₅₀values were 0.06±0.00 mg/m L and 0.04±0.00 mg/m L,respectively.Ethyl acetate fraction of ethanol extract showed the strongest activity,IC₅₀and CH₅₀values were 0.29±0.01 mg/m L and 0.06±0.00 mg/m L,respectively.Ethanol supernatant of water extract showed the strongest activity,IC₅₀and CH₅₀values were 0.37±0.01mg/m L and 0.06±0.00 mg/m L,respectively.2 Seven compounds were isolated from the ethyl acetate fraction of P.clypearia,includingβ-sitosterol（HEH-1）,ethyl gallate（HEH-2）,gallic acid（HEH-3）,quercetin（HEH-4）,kaempferol（HEH-5）,myricetin（HEH-6）,7-O-galloyltricetiflavan（HEH-7）.Kaempferol（HEH-5）and myricetin（HEH-6）were isolated from P.clypearia for the first time.3 Ethyl gallate（HEH-2）,gallic acid（HEH-3）,quercetin（HEH-4）,kaempferol（HEH-5）,myricetin（HEH-6）,7-O-galloyltricetiflavan（HEH-7）showed antioxidant activity(IC₅₀values varying from 0.22 to 0.64 m M).Ethyl gallate（HEH-2）and gallic acid（HEH-3）had no anti-complementary effect.Quercetin（HEH-4）,kaempferol（HEH-5）,myricetin（HEH-6）,7-O-galloyltricetiflavan（HEH-7）showed anti-complementary activity(CH₅₀values varying from 0.35 to 1.03 m M).7-O-galloyltricetiflavan（HEH-7）had the strongest antioxidant activity as IC₅₀values of 0.22±0.01 m M and myricetin（HEH-6）had the strongest anti-complementary activity as CH₅₀values of 0.35±0.01 m M.The structure-activity relationship analysis showed that the more phenolic hydroxyl groups of flavonoids,the antioxidant activity was stronger and there is no correlation between the number of phenolic hydroxyl groups and anti-complementary activity.4 The results of acute toxicity pre-experiment showed that the ethanol extract and water extract of P.clypearia had no acute toxicity.The maximum tolerated amounts of ethanol extract and water extract of P.clypearia were 15 g/kg and 27 g/kg,respectively.5 Ethanol extract of P.clypearia had good effect on the psoriasis mice model induced by imiquimod.The PASI score indicated that the experimental model was successful,but ethanol extract of P.clypearia had no dose-dependent effect.The results of HE staining showed that ethanol extract of P.clypearia had significantly good effect on the acanthosis of the lesion tissue,reducing the thymus index,dose-dependent inhibition of the spleen index.Moreover,ethanol extract of P.clypearia also inhibited the expression of PCNA and had a good dose-activity relationship,but had no effect on the production of CD3.6 The ethanol supernatant of water extract from P.clypearia had good effect on the psoriasis mice model induced by imiquimod.The PASI score indicated that the experimental model was successful,but the ethanol supernatant of water extract of P.clypearia had no dose-dependent effect.The results of HE staining showed that the ethanol supernatant of water extract of P.clypearia had significantly good effect on the acanthosis of the lesion tissue and had a good dose-activity relationship,reducing the thymus index,dose-dependent inhibition of the spleen index.Moreover,the ethanol supernatant of water extract of P.clypearia inhibited also the expression of PCNA and had a good dose-activity relationship.7 The optimum ethanol extraction process conditions were as follows:10 fold of 50%ethanol was added,to be extracted 2 times by reflux and 1 h per time.Conclusions:1 Antioxidant and anti-complementary fractions of P.clypearia were determined in this study.The chemical constituents of ethyl acetate fraction in ethanol extract were investigated.The antioxidant and anti-complementary activities of compounds were evaluated in vitro,indicating that flavonoids had certain antioxidant and inhibitory activities.Structure-activity relationship of compounds was analyzed.7-O-galloyltricetiflavan（HEH-7）and myricetin（HEH-6）could be used as natural antioxidants and complement inhibitors,and were the main active ingredients of P.clypearia for anti-inflammatory and immunomodulatory activities.2 Ethanol extract and ethanol supernatant of water extract of P.clypearia had significantly good effects on the skin lesions of psoriasis induced by imiquimod in Balb/c mice,by inhibiting the proliferation of keratinocytes,reducing the spleen index,reducingthe thickness of the spinous layer.