The Steroidal Saponins From Anemarrhena Asphodeloides Bag. And Their Pharmacological Effects On Hyperthyroidism In Rats

Anemarrhena asphodeloides(AA)is the dried roots of Anemarrhena asphodeloides Bge,which has the functions of reducing internal heat,producing saliva to embellish dryness.Attributive to the lung,stomach and kidney meridians.According to the basic theory and the new hypothesis of professor Kuang Hai-Xue,"one medicine has and Y natures(Y≤X)" of the traditional Chinese medicine,AA decoction was split into five components,namely volatile oil,polysaccharide fraction,ethyl acetate,timosaponin fraction a and rest ingredients component.And saponin components belongs to the material basis for the "Bitter" flavor,it plays a typical role in antipyretic,modulating hyperthyroidism,regulating immune function,anti-inflammatory and diuresis.In order to study the saponin constituents of AA,this paper under the guidance of the nature and flavor of traditional Chinese medicine,we studied the substance basis of the bitterness of the AA,and investigate the effects of saponin of AA used alone and combined with methimazole on hyperthyoidism in rats.The saponin constituents of AA were studied as follows:1.Chemical separation and structural identification of steroid saponins from Rhizoma Anemarrhena.Purpose:Chemical separation and structural identification of the main steroidal saponins from the n-butanol part of Rhizoma Anemarrhenae were carried out in this part.Methods:The water decoction of Rhizoma Anemarrhenae was precipitated by 80%alcohol,concentrated and purified by macroporous resin,extracted by ethyl acetate and n-butanol.The decoction was fractioned as polysaccharides,ethyl acetate fraction,n-butanol fraction and water fraction.And then,the n-butanol componds were isolated and purified by using different column chromatography,including D101 macroporous resin,silica gel and ODS as well as HPLC.These compounds were identified by 1H-NMR、13C-NMR and MS spectroscope methods.Results:Seven steroidal saponins were isolated,and identified as Timosaponin O(1)、Anemarsaponin BⅡ(2)、Timosaponin BⅠ(3)、Timosaponin J(4)、TimosaponinBV(5)、1,4-Bis(4-β-D-glucopyranosyloxybenzyl)(2R)-2-benzylmalate(7)、25R-26-O-β-D-glucopyranosyl-22-hydroxy-5β-furost-3-β,26-diol-3-O-β-D-glucopyranosyl(1→2)-β-D-galactopyranoside(6).2.Determination of major steroid glycosides in Rhizoma Anemarrhenae by HPLC-ELSD.Purpose:To establish an HPLC-ELSD method for simultaneous determination of three major steroid glycosides(Timosaponin O,Anemarsaponin BⅡ and Timosaponin BI).Method:The Diamonsil C18(2)(250 mm×4.6 mm,5 μm)column was used.The mobile phase consisted of methanol(A)and water(B)with gradient elution at a flow rate of 1.0 mL·min-1.The column temperature was set at 30℃.The ELSD conditions were as follows:the nebulizing gas flow rate was 2.0 L·min-1,drift tube temperature was 50℃.The volume was 10 μL.Result:The linearity was obtained over 64.13～1026 μg·mL-1(r=0.9989)for TimosaponinO,57.15～1028 μg·mL-1(r=0.9965)for Anemarsaponin BⅡ,67.32～1077μg·mL-1(r=0.9998)for Timosaponin BI.The mean recoveries were between 99.43%～102.8%,RSD was 2.28%～3.64%.3.Pharmacological effects of Steroidal saponin components used alone and combined with methimazole on Hyperthyroidism in Rats.Purpose:To investigate and analyze the effects of steroid saponins used alone and steroid saponins in combination with methimazoleon on hyperthyroidism in rats.Methods:Rats were randomly divided into the normal group,the model group,the methimazole group,the steroidal saponins groups(low and high dose),Low and High dose steroidal saponins combined with the methimazole group(n=7 animals in each group).In addition to the normal group,other groups were prepared by intragastric administration of euthyrox in the morning,with a dose of 500μg·kg-1.In the afternoon,except the normal group and the model group,the other groups were given drug treatment,administration 21 days in a row.The daily behavior of the rats were observed and body weight were detected regularly,and the peripheral blood and thyroid tissue were taken.The serum levels of FT3,FT4 and TSH were measured by ELISA methods.Thyroid tissue was used for HE staining and Western blotting test.Results:(1)Compared with the normal group,the rats in model group drunk more water,excessively excited,manic and irritable,and the bodyweight increasing was deferred obviously,and the serum levels of FT3 and FT4 were increased significantly(P<0.01),the level of TSH was declined(P<0.05).Compared with the model group,the levels of FT3 and FT4 were declined significantly(P<0.01)in the methimazole group and the combination high dose group,and the level of TSH was increased significantly in the methimazole group(P<0.01)and increased in the combination high dose group(P<0.05);after high dose of steroid saponins and the low dose combined with methimazole treatment,the levels of FT3 and FT4 were reduced(P<0.05),and the level of TSH was increased(P<0.05).(2)HE staining shows that the follicles in the model group were small and lined with hyperpllastic columnar epithelium and contained scant colloid that displayed much marginal scalloping and vacuolization,the nuclei was vesicular and basally located and exhibit occasional mitoses,papillary projections of the hyperplastic epithelium extended into the lumina of the follicle,Vascularity was increased,and there was a varying infiltration by lymphocytes and plasma cells;the methimazole group and the high dose combined with methimazole group put up significantly improvement the above pathological abnormalities,the above conditions were improved to different degrees in the high and the combination low dose groups.(3)In western blot,compared with the normal group,the protein expression of PI3K was increased in the model group(P<0.05),the protein expression of Akt,NF-κB was increased remarkablely(P<0.01).Compared with the model group,the protein expression of PI3K and NF-κB were significantly reduced in the methimazole group(P<0.01)and Akt were reduced in the methimazole group(P<0.05);in the combination high dose group,the protein expression of PI3K and Akt were reduced(P<0.05);the protein expression of NF-κB was significantly reduced(P<0.01);after the high and the combination low dose steroid saponins treatment,the protein expression of PI3K,Akt and NF-κB were.reduced(P<0.05).This experiment,guided by the theory of traditional Chinese medicine,used the research methods that property and flavor can be disassembled.And adopted the methods of chemical,pharmacology and molecular biology to study on the chemical characteristics of the steroid saponins of AA.Finally,we studied steroid saponins used alone and in combination with methimazoleon in the treatment of hyperthyroidism.To explore a new method for the preparation of saponin in AA and apply it to the development of new drugs,in order to expand the indications and applications.