ABSTRACT
Objective: To compare the flavonoids contained in different parts of different botanical origins of Epimedii Folium, and provide a basis for the quality evaluation of Epimedii Folium and the reasonable selection of medicinal parts. Methods: The aerial parts of 13 batches of Epimedii Folium were collected and divided into three parts: leaf, petiole and stem. The HPLC fingerprint and content of five flavonoids, including epimedin A, epimedin B, epimedin C, icariin and baohuoside I, were analyzed. Then the analysis of variance and the similarity evaluation software of traditional Chinese medicine chromatographic fingerprint were used. Combined with cluster analysis (HCA), the content differences of flavonoids in leaf, petiole and stem of Epimedii Folium were evaluated. Results: The fingerprints showed that the chemical constituents in Epimedii Folium leaves were richer than stems and petioles, and the chemical constituents in petioles and stems were basically the same. The content of five components in leaves was significantly higher than that of petiole and stem, even up to 10 times. Cluster analysis also showed that the leaves were obviously distinct from the petiole and stem. Conclusion: The quality differences of Epimedii Folium leaves, petioles and stems were clarified, and this study can provide the scientific evidence for the selection of medicinal parts and quality control of Epimedii Folium.
ABSTRACT
Objective: To investigate the best technological conditions for the purification of epimedin and icariin from Epimedii Folium by macroporous resin, and preliminarily characterize the purification fraction of the best technological conditions. Methods: Five kinds of macroporous resins were screened by static adsorption experiment with the content of epimedin A1, epimedin A, epimedin B, epimedin C and icariin as indexes. The best purification conditions were optimized by the concentration of upper column solution, the maximum sample volume, the upper column flow rate, the volume of water washing, the concentration of removing impurity ethanol and elution ethanol, the volume of removing impurity ethanol and elution ethanol, the column diameter-height ratio through dynamic adsorption experiment. Finally, UPLC-Q-TOF/MS, HPLC and ultraviolet spectrophotometry were used to characterize the purification fraction of the best technological conditions. Results: The best macroporous resin was AB-8, column diameter-height ratio was 1:7, 6 BV of upper column solution (crude drug 0.5 g/mL) was used for dynamic adsorption at a flow rate of 6 BV/h, 5 BV of water and 5 BV of 20% ethanol were used for impurity removal, and 6 BV of 50% ethanol was used for elution. The flow rate of impurity removal and elution was 6 BV/h. After purification, the total flavonoids content was 63.29%, the total content of epimedin A1, A, B, C and icariin was 40.48%, the content of epimedin A1, epimedin A, epimedin B, epimedin C and icariin was 1.63%, 2.52%, 16.36%, 5.51% and 14.46%, respectively. Conclusion: The purification process of epimedin and icariin from Epimedii Folium by AB-8 macroporous resin is stable, reasonable and feasible. The chemical characterization indicated that the purification fraction was mainly flavonoids, mainly consisting of epimedin and icariin. The optimized purification process can be used for the purification and enrichment of such ingredients.
ABSTRACT
Objective: In order to provide a scientific basis for the quality control of Kunxian Capsules (KC), HPLC characteristics chromatogram combined with multi-components determination was established. Methods: The analysis was performed on Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm), using acetonitrile and 0.1% phosphoric acid solution as the mobile phase at a flow rate of 0.8 mL/min for gradient elution, the column temperature was 33 ℃, and the detection wavelength was 270 nm. The fingerprints of 15 batches of KC were established and evaluated by the similarity evaluation system of TCM (2012A version), hierarchical cluster analysis and discriminant analysis of partial least squares. Furthermore, the content of hyperoside, epimedin A, epimedin B, epimedin C, icariin and baohuoside Ⅰ were determined. Results: The HPLC fingerprint with 21 common peaks of KC was established, and the similarities of samples were over 0.9. The linearity relationships separated with hyperoside, epimedin A, epimedin B, epimedin C, icariin and baohuoside Ⅰ were good, and the contents of the above-mentioned components in 15 batches of preparations were 2.817-7.527, 7.287-9.103, 8.730-18.675, 33.377-70.371, 35.297-50.291 and 4.059-9.079 mg/g, respectively. Conclusion: The combination methods of HPLC characteristic chromatograms and simultaneous determinations of multiple components are rapid, simple and reproducible, which can provide methodological reference for the quality control of KC.
ABSTRACT
Objective To establish quantitative analysis of multi-components by single marker (QAMS) method for simultaneous determining the content of ten flavonoids in Kunxian Capsules (KC), and evaluate the adaptation and application of QAMS method in the quality control of KC. Methods The relative factor (fs/i) of epimedin A, epimedin C, epimedin B, icariin, luteolin, quercetin, nobiletin, kaempferol and baohuoside I were established by HPLC method with hyperoside as internal standard, which were used to calculate the content of ten flavonoids in the samples of KC. Meanwhile, external standard method (ESM) was used to calculate the content of ten flavonoids. The difference between QAMS and ESM were analyzed to evaluate the accuracy of QAMS. T-test was used to compare the content of ten flavonoids in KC. Results The fs/i of epimedin A, epimedin C, epimedin B, icariin, luteolin, quercetin, nobiletin, kaempferol, and baohuoside I were 0.756 5, 2.199 4, 1.232 7, 1.008 5, 0.635 7, 0.576 0, 0.487 5, 0.545 6, 0.675 8. The content determination results of ten batches of KC samples were calculated by the method of QAMS and ESM, with no significant difference in RSD < 2.0%. Conclusion The fs/i established in the QAMS method with hyperoside as the internal reference substance is accurate and feasible. The QAMS method can be used for the quality evaluation of KC.
ABSTRACT
Objective: To compare the neuroprotective effect of different elution fractions and compounds of Epimedium brevicormon on SH-SY5Y cells injury induced by Aβ25-35 so as to screen pharmacologically fraction and compounds. Methods: The isolation and purification of extract from E. brevicormon were performed on D101 macroporous resin column chromatography, silica gel column chromatography, Sephadex LH-20 column chromatography, and preparative liquid chromatography. Hoechst 33342/PI double staining method was used to screen neuroprotective fractions and components. Results: Compared to model group, all fractions and kaempferol-3-O-[α-L-rhamnopyranosyl (1→6)-β-D-galactoside]-7-O-α-L-rhamnopyranoside, icariin, epimedin A, and epimedin B from E. brevicormon could significantly increase the cells viability, except of 95% ethanol fraction. Conclusion: E. brevicormon show some protective effect against Aβ25-35 induced SH-SY5Y cells injury. The CH2CI2-MeOH (5:1) extract from E. brevicormon has the most pharmacologically activity, followed by 30% ethanol and 50% ethanol extracts from E. brevicormon.
ABSTRACT
OBJECTIVE:To establish a method for the simultaneous determination of psoralen,isopsoralen,epimedin B,epi-medin C and icariin in Chanlong dingchuan mixture. METHODS:HPLC was performed on the column of Welch Materials C18 with mobile phase of acetonitrile- methanol(1∶1,V/V)-water(gradient elution)at a flow rate of 0.9 ml/min,the detection wavelength was 246 nm(psoralen,isopsoralen)and 270 nm(epimedin B,epimedin C and icariin),column temperature was 30 ℃,injection volume was 20 μl. RESULTS:The linear range was 8.24-164.80 μg/ml for psoralen(r=0.999 7),5.15-103.00 μg/ml for isopso-ralen(r=0.999 3),4.06-81.20 μg/ml for epimedin B(r=0.999 6),5.88-117.60 μg/ml for epimedin C(r=0.999 5)and 4.90-98.00μg/ml for icariin(r=0.999 8);the limits of quantitation were 0.385 μg/ml,0.179 μg/ml ,0.124 μg/ml,0.218 μg/ml and 0.348 μg/ml, limits of detection were 0.127μg/ml,0.059μg/ml ,0.041μg/ml,0.072μg/ml and 0.115μg/ml;RSDs of precision,stability and re-producibility tests were lower than 2.0%;recoveries were 97.93%-100.06%(RSD=0.80%,n=6),96.91%-100.16%(RSD=1.37%,n=6),96.95%-99.63%(RSD=0.98%,n=6),96.69%-99.33%(RSD=1.03%,n=6) and 96.76%-98.53%(RSD=0.70%,n=6),respectively. CONCLUSIONS:The method is simple and reliable,and suitable for the simultaneous determination of psoralen,isopsoralen,epimedin B,epimedin C and icariin in Chanlong dingchuan mixture.
ABSTRACT
Objective: In order to evaluate the quality of Epimedium sagittatum and to provide the references for screening germplasm as well as for the utilization of resources. The concentration variations of main flavonoids, epimedins A-C, and icariin among 15 representative populations of E. sagittatum and one population of E. sagittatum var. glabratum were assessed by HPLC. Methods: The chromatographic separation was performed on a Zorbax SB-C8 column (250 mm × 4.6 mm, 5 μm). The mobile phase was a mixture of acetonitrile-1.44% acetic acid aqueous solution in gradient elution. The flow rate was 1.0 mL/min, the detection wavelength was 272 nm, and the column temperature was 25 ℃. Results: Remarkable variations within and among the populations were observed in epimedin A (1.00 - 16.64 mg/g), epimedin B (1.00 - 17.21 mg/g), epimedin C (1.00 - 76.21 mg/g), and icariin (1.00 - 46.19 mg/g). As far as the content of icariin concerned, only five populations (HBHF, HBLT, HNLS, HNZJ, and JXWN) had acceptable quality recorded in Chinese Pharmacopoeia, four populations were lower than the standard, and seven populations were even lower than the detectability. Conclusion: The present study suggests that E. sagittatum should not be recorded in Chiese Pharmacopoeia indiscriminately, but the populations of HNZJ, HBLT, HBHF, and HNLS are outstanding and excellent, which were the best candidates for germplasm selection. Meanwhile, their habitat can also provide the reference for cultivation.
ABSTRACT
OBJECTIVE: Prednisolone-induced osteoporosis model using zebrafish was used to evaluate the antiosteoporotic activity of micro amount icariin and epimedin B. METHODS: Zebrafish larvae were co-exposed with 25 μmol·L-1 prednisolone and a series of icariin and epimedin B solutions with a range of concentration (0.2, 1, 5 and 10 μmol·L-1) from 4 to 9 days post fertilization(dpf), 25 μmol·L-1 prednisolone was selected as model group, 15 μg·mL-1 etidronate disodium with 25 μmol·L-1 prednisolone as positive group, 0.5% DMSO as the vehicle control group, the medium as a negative blank control group, all groups were incubated in 24-well plates (28.5°C) until 9 dpf. The medium/solution was changed half every day. Zebrafish skeleton at 9 dpf were 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 zebrafish head skeleton mineralization. RESULTS: The results indicated head skeleton mineral aera and integrated optical density (IOD) of 25 μmol·L-1 prednisolone model group were significantly decreased when compared with vehicle control group and negative control group; and 15 μg·mL-1 etidronate disodium can increase the mineralized matrix of zebrafish head skeleton when compared with model group and prevent osteoporosis induced by prednisolone; head skeleton mineral area and IOD of 10 and 5 μmol·L-1 icariin groups and 1μmol·L-1 epimedin B groups were significantly increased when compared with model group, which indicated that icariin and epimedin B can prevent zebrafish osteoporosis induced by prednisolone. CONCLUSION: This novel osteoporosis model using zebrafish has advantages of simple, high efficiency, far less amount of compound needed and easy to perform, and it was successfully applied in quickly evaluating the antiosteoporosis activity of micro amount compounds such as icariin and epimedin B.
ABSTRACT
Objective: To investigate the influence of microwave radiation on the extraction of epimedin B from Epimedii Folium. Methods: The effects of extraction solvent, ratio of material to liquor, and duration of microwave radiation on the extraction yield of epimedin B were studied by single factor tests. The influence of microwave radiation on the stability of epimedin B was assessed by UV-Vis spectrophotometeric analysis. The impacts of microwave radiation on leaf sample were observed using paraffin section method. Results: The contributions of ethanol concentration and duration of microwave radiation for the extraction yield of epimedin B are significant, whereas ratio of material to liquor is not a significant factor. Microwave radiation could result in the disruptions of leaf tissues and other parts, but it did not affect the stability of epimedin B. Conclusion: Microwave radiation could facilitate the extraction of epimedin B, which might result from the enhancement of the mass transfer of ethanol solution into leaf tissues and dissolution of epimdin B from the matrix.