| Objective:In this study,the extraction and purification process of total alkaloids from malt was studied on the basis of previous experiments.The use of modern pharmacology research methods,replication of high prolactin hyperlipidemia animal disease model,and different polar parts of different chemical fractions and malt,purified alkaloid substances in pharmacodynamic test,screening of effective parts of malt treatment HPRL,and clarify its mechanism.Methods:(?)take an appropriate amount of malt extracted with 70% ethanol extract,water dissolved made into suspension followed by adding petroleum ether,ethyl acetate,n-butanol extracts from different polar fractions,and chemical composition of the various parts of the content determination.According to the related literature,selection of water as a solvent,40? water bath,extracted total malt extract;extraction with 70% ethanol,then by polyamide resin column simple purification,obtained total flavonoids malt extracts;The crude extract of total malt phenol was obtained by ultrasonic extraction with 70% ethanol,and then purified by HPD-600 macroporous resin column.The preparation and content determination method of crude extract of malt alkaloid were carried out according to the experimental method in the earlier stage of this research group.(?)According to the experiment of single factor extraction solvent selection of alkaloid substances in malt,then the orthogonal test,the total alkaloids and malt alkali extraction comprehensive score quantity as the evaluation index,the optimum extraction process of malt alkaloid substances.With the transfer rate and purity of total alkaloids as the evaluation index,to compare the effect of purification by column chromatography and solvent extraction method of total alkaloids of malt,the degreasing solvent extraction solvent,water bath temperature and aqueous ammonia solution to different pH were investigated.The optimum purification technology of total alkaloids from selected malt.(?)The establishment of HPRL model: in addition to the rats in the normal group,the other rats were injected into the back of the Metoclopramide Dihydrochloride Injection 50mg/kg weight,every morning and afternoon were regular injections of 1 times,5 consecutive days.Packet delivery: 120 female non pregnant SD rats were randomly divided into 12 groups: normal group,model group,petroleum ether and ethyl acetate group,n-butanol group,water group,raw material water extract group,total alkaloid extract and the total polysaccharide fractions of crude extract fractions,total flavonoids,total phenolic extract group of alkaloids part group,low concentration group,high concentration of alkaloids group,10 rats in each group.Preparation of the model after administration of petroleum ether and ethyl acetate group was administered at a dose of 114.70mg·kg-1·d-1;n-butanol group was administered at a dose of 124.17mg·kg-1·d-1;the water fractions of the dosage was 645.96mg·kg-1·d-1;raw material water extract group dosage was 4.74g·kg-1·d-1,the total alkaloid extract fractions of the dosage was 592.43mg· kg-1·d-1;the total extracting polysaccharide fractions of the dosage was 1.57g·kg-1·d-1;total flavonoids crude extract fractions of the dosage was 57.83mg·kg-1·d-1;total phenolic extract fractions of the dosage was 103.39mg·kg-1·d-1;alkaloids in low concentration group was administered at a dose of 0.87 mg· kg-1·d-1;alkaloids in high concentration group was administered at a dose of 1.74 mg·kg-1·d-1.The normal group and model group were given the same volume of distilled water,continuous intragastric 30 d.After the last administration of 24 h,all rats were fasted for 12 h.The rats in each group were collected blood 2ml in the next morning,and the blood was set at a speed of 3000 rpm /min 5 min.Enzyme linked immunosorbent assay(ELISA)was used to determine the contents of prolactin(PRL),estrogen(E2)and progesterone(P)in the serum of rats in each group,and strictly in accordance with the operating procedures of the ELISA kit.Effect of hyperprolactinemia rat pituitary prolactin content of positive cells and expression of mRNA: normal group,model group,total alkaloids,total crude extract fractions of extracting polysaccharide and flavonoids crude extract fractions of total phenolic extract part group,part group,low concentration group and high concentration group of alkaloids rat 24 h after the last administration,the next morning with 20% urethane anesthesia from the pituitary gland.1/2 rats were fixed in the tissue fixative for 24 h.After paraffin embedded sections,immunohistochemical staining was performed to determine the positive light density(IOD)of PRL cells.Take another 1/2 rat pituitary gland to carry on the PRL mRNA fluorescence quantitative PCR experiment,calculate the expansion multiple,and carry on the statistical analysis.The influence of hyperprolactinemia rat mammary hyperplasia: normal group,model group,herbs group of water extract group,total alkaloids,total crude extract fractions of extracting polysaccharide and flavonoids crude extract fractions of total phenolic extract part group,part group,low concentration group and high alkaloids the concentration of rats in group 24 h after the last administration,the next morning with 20% urethane anesthesia after the second of breast tissue in 4% paraformaldehyde fixed tissue fluid in fixed 24 h after embedding,sections were stained with HE,observed in breast tissue of rats in the microscope,including breast lobule the number,shape and acinar ductal epithelial cells,acinar and ductal secretion in.Results:(?)Parts of water extract were obtained,followed by n-butanol,the total alkaloid substance concentration in the water and butanol fractions,flavonoids in the main parts of petroleum ether and ethyl acetate in the highest content of total phenolics content in three different polar parts in little difference.The content of total polysaccharide in crude extract was 98.09%,and the content of total flavonoids in crude extract was about 9.83%,and the content of total phenol in crude extract was about 4.84%.(?)The optimum extraction and purification process of total alkaloids: weigh malt coarse powder,adding 5 times amount of 80% alcohol,ultrasonic extraction 3 times,extraction time 45 min,HCl water solution,using PH 1~2 collected filtrate evaporated after filtration,the filtrate is added in petroleum ether extraction 3 times the amount of water collected.Solution with concentrated ammonia solution transfer PH=11,standing in the refrigerator,collecting precipitation,precipitation with ammonia solution is washed several times after PH=11 abandoned,with the filtrate in a separatory funnel,chloroform extraction with 3 times amount of 5 times,1h/ times,with the chloroform solution to 60? water bath dry solvent,dissolved in 0.03%HCl filtering,the total alkaloid content was 56.64%.Different origin of malt alkaloid substance content is large,the alkaloid substances content in Anhui Bozhou production of malt barley in the highest,students does not contain hordenine,low alkaloid content in the raw material of fried malt malt.(?)Influence on the content of PRL in serum: compared with the normal group(4.714±5.159 pg·ml-1),the content of PRL in serum of rats in model group increased significantly;(30.404±5.516 pg·ml-1,P<0.01)compared with the model group,different polar parts were serum PRL content in rats the lower,only crude parts of total alkaloid group had significant difference(11.051±5.516pg·ml-1,P<0.01),the group of rats serum PRL content was the lowest close to normal rats serum PRL content.Compared with the model group,the total alkaloid of crude alkaloid part group,low concentration group(11.571±4.263 pg·ml-1)and alkaloids in high concentration group(7.004±4.044 pg· ml-1)showed significant differences(P<0.01),the content of alkaloids in high concentration group rats serum PRL the lowest close to the normal value.Influence on the content of P in serum of different polar parts compared with the normal group(1.283±0.116 ng·ml-1)compared the content of P in serum of rats in model group increased significantly(1.993±0.116 ng·ml-1,P<0.01);compared with the model group,the content of all parts of petroleum ether and ethyl acetate group,n-butanol and water group group P increased in serum of rats.The content of P in serum of the crude extract,water extract group and crude alkaloid extract fraction group all decreased,and the total alkaloid extraction group had very significant difference(1.46±0.135ng· ml-1,P<0.01).Compared with the model group,the content of P in serum of each group was decreased,and the difference was not big.Among them,total alkaloids,crude extract fractions,total polysaccharides,crude extract fractions,total flavonoids,crude extract fractions,total phenols,crude extract fractions and alkaloid high concentration groups were significantly different.Influence on the content of E2 in serum of different polar parts compared with the normal group(69.919±5.986 pg·ml-1)compared the content of E2 in serum of rats in model group increased significantly(100.825±5.986 pg·ml-1,P<0.01);compared with the model group,raw material water extract group(68.949±6.309 pg· ml-1)and total alkaloid extract fractions(64.550±5.986 pg·ml-1)was significantly decreased(P<0.01),there were no significant differences between other groups.Compared with the model group,the content of E2 in the serum of each group was decreased.The positive expression of prolactin in cytoplasm is brownish yellow.The greater the cumulative optical density of prolactin,the greater the positive response.Compared with the normal group(0.0068±0.0016),the model group had significant difference(0.0553±0.0172,P<0.05),and the average optical density of the unit area of prolactin cells was significantly higher than that of the normal group.Compared with the model group,the total alkaloid extract fractions(0.0097±0.0026),from the low concentration group(0.0087±0.0028)and alkaloids in high concentration group(0.0070±0.0019)had significant difference(P<0.05),total alkaloid extract positive fractions of prolactin cells is smaller than the other three chemical site,but high concentration was greater than the alkaloid.PRL and mRNA were expressed in the pituitary tissues of rats in each group,and compared with the normal group(6.23±1.12),there was significant difference between the model group(22.11±3.86,P<0.05).Compared with the model group,the total alkaloid extract fractions(5.95±1.07),from the low concentration group(6.14±1.36)and alkaloids in high concentration group(4.32±1.08)had significant difference(P<0.05),the expression level of PRL mRNA was significantly lower than the model group.In normal group,the small and medium leaves of mammary gland were not hyperplasia,less acini,no dilated ducts,no secretions,and 0 grade hyperplasia.In the model group,the mammary glands of rats in the model group were partly dilated,the ducts dilated,and no secretions were found.There were two grades of hyperplasia,indicating that hyperprolactinemia accompanied by hyperplasia of mammary glands.The total alkaloids,crude extract fractions,total flavonoids,crude extract fractions and total phenols crude extract fractions of rats,mammary gland lobules,individual acini hyperplasia,ductal dilatation,no secretions,for primary hyperplasia.In the high concentration group of alkaloid,the small and medium leaves of the breast tissue were not proliferated,the acini were few,the ducts were not dilated,and there were no secretions,which was grade 0 hyperplasia.Conclusion:(?)The results showed that the content of alkaloid in malt was low,and the traditional water extraction method could not extract it in large quantities,which provided a material basis for the use of malt milk in the large clinical dosage of Chinese medicine.(?)After the optimized purification method,the total malt alkaloid fraction of purity greater than 50% can be obtained,which provides a sample for the pharmacodynamics experiment and the chemical component analysis study.(?)Confirmed the malt treatment efficacy of substance of hyperprolactinemia which contains alkaloids,the mechanism is by reducing the number of PRL positive cells in rat pituitary,pituitary down-regulation of PRL m RNA cells,thereby reducing the content of PRL in rats,to treat the HPRL. |
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