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Research On Transformation Rule, Biopharmaceutical Properties And Soft Capsule Initial State Of A Flavonoid Component Of Herba Epimedii

Posted on:2017-02-22Degree:MasterType:Thesis
Country:ChinaCandidate:B J ZhaoFull Text:PDF
GTID:2284330488995972Subject:Pharmacy
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Herba Epimedii is a traditional tonic medicine of Chinese traditional medicine and has the efficacy of kidney yang, strong bones and muscles, rheumatism. In this paper, we comprehensively reviewed relevant reports of pharmacological action of Herba Epimedii and found that it is widely used for anti-osteoporosis. The main bioactive components of this herb are prenylated flavonol glycosides, and the absorption of flavonol glycosides increasing with the number of glucoside decreased. According to the researches of our laboratory, we prepared a flavonoid fraction with less glucoside from Herba Epimedii and named it as HEFF. This study was conducted to explore the transformation rule of HEFF of different varieties Herba Epimedii and evaluate whether it has a better effect for anti-osteoporosis.In the second chapter of this study, four different varieties Herba Epimedii were refluxed with 18-fold 50% ethanol and 15-fold 50% ethanol (v/v) for 1 h, respectively. We used high performance liquid chromatography (HPLC) and ultraviolet spectrophotometry (UV) method and to analyze the difference of the content and ratio of prenylated flavonol glycosides in alcohol extract. All the extracts were combined together and removed ethanol under reduced pressure, then enzymolysis by cellulose (cellulose:total flavonoids=1:7). The enzymatic hydrolysate were precipitated by adding ethanol to a ratio of ethanol-water=60:40 (v/v) and filtered, then the supernatant of this extract was concentrated and loaded onto the macroporous resin AB-8 column. After adsorption overnight, the adsorbate-laden column was eluted with different concentrations of ethanol (0%,30%,60%,80%) at the flow rate of 2 BV/h, respectively. Then 60% and 80% ethanol eluent were combined and then concentrated, stewing overnight. The deposit was re-dissolved in 95% ethanol and centrifuged at 4000 rpm for 10 min while the supernatant was taken for concentration and dried at 50℃, and then HEFF were obtained. In order to identify the main compounds of HEFF, HPLC-ESI-MS assay was performed in this experiment. We speculated the eight main compounds according to the chromatographic retention time, relative molecular mass, fragment ion information, and the relevant literature data. They are epimedoside A, ikarisoside F, baohuoside Ⅱ, sagittatoside A, sagittatoside B, 2"-O-rhamnosyl icariside Ⅱ,7-O-rhamnosyl icariside Ⅱ and baohuoside Ⅰ, respectively. Then, we respectively hydrolyzed epimedin A, epimedin B, epimedin C and icariin to investigate the transformation rule. The result showed that the hydrolysates of epimedin A, epimedin B, epimedin C and icariin is sagittatoside A, sagittatoside B and baohuoside I,2"-O-rhamnosyl icariside II and baohuoside I, respectively.In the third chapter of this study, the MTT assay was used to detect the effect of HEFF on the proliferation of UMR-106 cells, the result showed that the HEFF made from Epimedium koreanu Nakai has the best effect on the proliferation of UMR-106 cells at 48 h. At the same time, the influence of HEFF to the UMR-106 cells differentiation was observed through testing the ALP activity of UMR-106 cells using biochemical assays. The HEFF significantly increased ALP activites of intracellular and extracellular. These results indicated that the HEFF could promote UMR-106 cells proliferation and differentiation.In the fourth chapter of this study, we evaluated the better effect of HEFF compare to alcohol extract (HEE).Firstly, we used the ovariectomized rats as model. Serum alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (StrACP), the femur and uteri wet weight, tibia biomechanical characteristics, parameters of trabecular bone structure, osteoprotegerin (OPG) and receptor activator of nuclear factor κ B ligand (RANKL) levels were measured, respectively. The results showed that both HEFF and HEE could significantly reversed the change of the indicators induced by osteoporosis. Unfortunately, compared to the same crude drug dosage HEE, HEFF did not have a better effect except HEFF-M group on the serum StrACP level and OPG protein content. This result may be caused by two factors:One reason is that rat intestinal bacteria could transform polyglycosides to low glucoside at these gluco sides; The other reason is that the equilibrium solubility is bad. In order to verify whether due to the first reason, then we used the zebrafish osteoporosis model to evaluate the better effect of HEFF compare to HEE.Zebrafish larva at 3 d post fertilization were divided in to blank control group,0.5% DMSO group, prednisolone group, etidronate disodium group, HEE(6.56,13.125,26.25,52.5,105 mg·L-1 crude drug) groups. HEFF(6.56,13.125,26.25,52.5,105 mg·L-1 crude drug) groups. All groups were incubated in 24-well plates for 6 d until execution, and then zebrafish skeleton was anesthetized and fixed for staining with alizarin red.Quantitative analysis of the stained area was performed by microscopic inspection and digital imaging methods to reflect the amount of bone mineralization. The results showed that head skeleton mineral area and IOD of the HEFF groups and HEE groups were significantly increased when compared to model group. HEFF groups significantly increased head skeleton mineral IOD compared to HEE groups at 13.125,26.25, 52.5,105 mg·L-1 crude drug.In the fifth chapter of this study, the equilibrium solubility and apparent oil-water partition coefficient in water and different pH value of solution of the eight compounds in the HEFF were determined. Similarity analysis of compounds in the HEFF were conducted based on Cosine method. Then, the weight coefficient method was adopted to integrate the quality characterization of biopharmaceutical properties of the HEFF. The equilibrium solubility of the HEFF was between 1.14~91.77 μgmL-1, which belonged to the very slightly soluble. While the oil/water partition coefficient values LogP ranged from 1.91~3.04, that shows a good absorptivity according to "wulv". It should been classified to Ⅱ type traditional Chinese medicine component on the basis of the BCS classification systems.To solve the problem of poor solubility of HEFF, following the principle of economy and convenience, in the sixth chapter of this study, we designed the HEFF as suspensions using soybean oil as substrate, beewax as suspending agent, soyabean lecithin as wetting agents. Formula was evaluated by mobility and sedimentation volume ratio optimized by single factor test and L9 (34) orthogonal test. The best formula is that the ratio of drug, substrate, beewax and soyabean lecithin is 1.0:1.5:0.1:0.04. This preparation is simple, stable and controllable. It can be applied to industrial production.In summary, the author successfully prepared HEFF and explored the transformation rule of HEFF. Then, we evaluated the better effect of HEFF compare to alcohol extract (HEE) using the ovariectomized rats and zebra fish as model, respectively. In the end, in order to improve the bioavailability, HEFF were prepared to suspensions combined its biopharmaceutical characteristics.
Keywords/Search Tags:Herba Epimedii, Flavonoid Fraction of Herba Epimedii(HEFF), osteoporosis, UMR-106 cells, zebrafish, suspension
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