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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.
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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.
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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.
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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.
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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.
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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.