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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: Based on UPLC-Q/TOF-MS technology, the fingerprint of Epimediumkoreanum before and after processing was established to analyze the whole composition and find out the iconic chemical composition to clarify the change rule of flavonoids. Methods: The data of E.koreanum raw and processed products were collected in positive ion mode using UPLC-Q/TOF-MS technology, and orthogonal partial least least squares-discriminant analysis (OPLS-DA) method was used to explore the differences in chemical composition of E.koreanum before and after processing in nine different origins and batches. Results: Nine iconic chemical constituents from E.koreanum raw and processed products were found and identified, including 8-ethylene-kaempferol, icaritin, icariin I, icartin-3-O-glucoside, isoamyl alcohol sagittatoside B,1,3-prenyl epimedin C, 1,3-prenyl-sagittatoside B-7-O-glucuronic acid, 3-O-((4-acetoxy) rhamnose-2-O-(m-2acetoxy) glucose) icariin and its isomers. Conclusion: The structures of E.koreanum's flavonoids changed after the processing, the secondary glycosides of flavonoids increased, the polyglycosides decreased, and the flavonoids were generally converted to low glycoside components, which further clarified the change rule of E.koreanum's flavonoids after processing.
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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 assay Icariine, Epimedin C, asperosaponin Ⅵ, psoralen and angelicin in Xianlinggubao capsules via multi-wavelength HPLC method.Methods Separation was carried out on Welch Ultimate○R XB-C18 column.The mobile phase was acetonitrile-water system and a linear gradient elution was used.The column temperature was 30 ℃.The detection wavelength for Icariine, Epimedin C,asperosaponin Ⅵ was set at 212 nm, psoralen and angelicin at 246 nm.Results Five components reached baseline separation, the linearity was good when sample size was in the range of 0.008 2-0.328 μg for Icariine(r=0.999 5), 0.055 6-2.224 μg for Epimedin C (r=0.999 6), 0.144 1-5.764 μg for asperosaponin Ⅵ(r=0.999 6), 0.005 4-0.215 2 μg for psoralen(r=0.998 0), 0.006 6-0.265 6 μg for angelicin(r=0.998 5).The average recoveries were 97.59％, 98.58％, 98.11％, 97.86％, 98.22％ respectively.The RSDs of recovery were all less than 2.0％.Conclusion This method is simple, accurate, with good separation, high sensitivity for the assay of multiple components in Xianlinggubao capsule.
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Objective To establish the method for simultaneous determination of epimedium glycoside and other 4 kinds of active ingredients in Xianling Gubao Capsules by three wavelength switching method. Methods An Waters Atlantis T3 C18 column (4.6 mm × 250 mm, 5 μm) was used with the mixture of acetonitrile-0.05％ formic acid solution as the mobile phase in gradient elution (0–5 min, 12％–20％ A; 5–15 min, 20％–55％ A; 15–35 min, 55％ A;35–55 min, 55％–76％ A; 55.1 min, 12％ A; 55.1–60 min, 12％ A). Detection wavelength was as follow: 0–30 min, 212 nm; 30–42 min, 246 nm; 42–60 min, 270 nm. The flow rate was 1.0 mL/min. The column temperature was 30 ℃. Results The calibration curves of asperosaponin Ⅵ, psoralen, angelicin, epimedin C, and icariin were in good linearity among the ranges of 101.6–3048 ng, 8.7–261 ng, 7.9–237 ng, 117.2–3516 ng, 78–2340 ng, respectively. In the instrument precision test, stability test, repeatability test, the RSD was less than 3％. The average recoveries were 99.83％, 100.35％, 100.59％, 100.60％, 99.72％, respectively, and all the RSD were less than 3％. Conclusion The method is sensitive, accurate, and separation effect is good, which can provide a basis for quality evaluation standard of Xianling Gubao Capsules.
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Objective To evaluate the anti-osteoporosis effect of the mixtures of epimedin C and icariin monomers with invariant molarity on zebrafish osteoporosis model. Methods The zebrafishes, fertilized 4 d ago, were exposed in 11 groups of nutrient solutions with prednisolone (25 μmol/L) as well as epimedin C and icariin (15 μmol/L) of various contents. The ratio of epimedin C and icariin in the 11groups were as follows: A (10:0), B (9:1), C (8:2), D (7:3), E (6:4), F (5:5), G (4:6), H (3:7), I (2:8), J (1:9), and K (0:10). Meanwhile, a negative control group with prednisolone (25 μmol/L) was prepare as S. The selected zebrafish fetus was put into the 24-hole culture plate, and ensure every 5 zebrafishes in 1 hole and 2 holes as a group. They were placed in incubator at 28.5 °C, and the daily changes of fluid were investigated until they were put to death on day 8 and then fixed. After dyeing with alizarin red, the segmental venter of zebrafish skulls was observed and quantitative analysis of dyed area was conducted. Results Compared with the negative control group S, the integrated optical density (IOD) values of cranial dyed area in all groups increased significantly (P < 0.05); Compared with group S, the IOD value of cranial dyed area in mixtures of epimedium monomers increased significantly (P < 0.05). The mixtures of epimedium monomers were all effective in facilitating zebrafish cranial mineralization and preventing prednisolone from inducing osteoporosis. According to mixtures of A-K groups, zebrafish cranial mineralization gradually decreased with gradually reduced content of epimedin C, with significant difference among groups (P < 0.05). Conclusion The higher the content of epimedin C in mixtures with invariant molarity is, the more active the anti-osteoporosis effect of epimedinC to zebrafish osteoporosis model is.
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Objective: To develop a method for controlling the quality consistency of Qianlie Shule Capsule (QSC) by blending raw marerials. Methods: Blending raw materials were blended by medicine calculator V2. The content differences of epimedin C and icariine in QSC made from raw materials and their mixtures were compared. Results: The RSD of index component epimedin C from blended and raw materials were 11.2% and 82.5%, the RSD of icariine were 8.70% and 35.5%, the difference between index components decreased obviously, and it showed that the quality consistency in QSC was greatly increased. Conclusion: Using the method to control the quality coherence of Chinese patent medicine has the obvious results, it is easy to operate and suitable for actual production.
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Objective: To investigate the positioning based on the relative retention time of fingerprinting and to establish a new quality evaluation method for traditional Chinese medicine preparations, using one chemical reference substance to calcutate multi- components simultaneously. Methods: Employed icariin as the maker component, icariin relative correction factors (RCF) of epimedin C to icariin, asperosaponin VI to icariin, psoralen to icariin and angelicin to icariin were ealeatated in the chromatographic conditions for determination of the four components in Xianling Gubao Capsule (XGC). The contents of icariin were determined by external standard method, and those of epimedin C, asperosaponin VI, psoralen and angelicin were calculated by icariin and their RCF. The accuracy of the new method was evaluated by comparing the relative retention times and calculating the content which using different brands columns for determination. Results: The analysis methods were established,the linearity was good when sample volume was in the range of at 8.2—328.0 ng for icariine(r = 0.999 5), 0.055 6—2.224 μg for epimedin C (r = 0.999 6), 0.144 1—5.764 μg for asperosaponin VI (r = 0.999 6), 5.4—215.2 ng (r = 0.998 0) for psoralen, 6.6—265.6 ng (r = 0.998 5) for angelicin. The average recoveries of asperosaponin VI, psoralen and psoralen were 97.59%, 98.58%, 98.11%, 97.86%, 98.22%, respectively. The RSDs of recovery were all less than 2.0%; There has been no significant difference between the calculated contents and the determined contents, according to the angle cosine value. Conclusion: The method can control the components without providing epimedin C, asperosaponin VI, psoralen, and angelicin reference. The method is not only save reference substance and medicine resources, but also suitable quality evaluation pattern for TCM preparation. This new method made fingerprinting more meaningful in TCM quality control.
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Objective To optimize ultrasonic-assisted extraction Epimedin C and Icariin from Epimedii Wushanensis Herba by response surface methodology. Methods On the basis of single factor tests, Box-Behnken experimental design and response surface methodology were adopted to optimize extraction conditions with the concentration of ethanol, ultrasonic time and solid-liquid ratio as factors. HPLC was used to determine the content of Epimedin C and Icariin from Epimedii Wushanensis Herba. Results Optimal ultrasound-assisted extraction technology was as following: the concentration of ethanol was 73%; the ultrasonic time was 22 min; the ratio of liquid to material was 1:32. The contents of Epimedin C and Icariin from Epimedii Wushanensis Herba were 15.90% and 0.75%, respectively. Conclusion This extraction technology can improve the extraction efficiency of Epimedin C and Icariin from Epimedii Wushanensis Herba, which is in accordance with predicted value.
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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.
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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.
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Herba Epimedii (named Yinyanghuo in Chinese) of the family Berberidaceae is one of the most famous traditional Chinese medicines (TCMs) recorded in the pharmacopoeia of the People's Republic of China.Total flavonoids of Epimedium (TFE) are considered to be its active components.TFE content was measured by ultraviolet method and its representative constituents,icariin and epimedin C,were determined by high performance liquid chromatography.The test results from 36 samples of 4 species showed that the contents differed significantly in different species.The average data of the summation (icariin and epimedin C) content were 20.83,7.61,14.43,23.29 mg/g in Epimedium pubescens Maxim.,E.koreanum Nakai.,E.brevicornum Maxim.,E.sagittatum (Sieb.Et Zucc.) Maxim.,respectively.Both E.sagittatum and E.pubescens performed better than the other two batches.E.sagittatum hardly has any circulation in Chinese herbal medicine market.The results showed that E.pubescens,which had much more amounts of icariin,epimedin C and TFE than those of other species,has a better quality and may be considered as potential anti-osteoporosis drug.
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AIM:To set up a method for determining epimedin C and icariin in Xianling Guobao Capsules(Herba Epimedii,Radix et Rhizoma Salviae miltiorrhizae,Fructus Psoraleae,Radix Rehmanniae,etc.). METHODS:The chromatographic conditions included the column of Spherisorb C 18 (4.6 mm?250 mm,5 ?m),the mobile phase was acetonitrice and water as gradient eluent was at a flow rate of 1.0 mL/min,the detection wavelength was set at 270 nm and the column temperature was at 25 ℃. RESULTS:The linear range of epimedin C was 0.22-2.20 ?g and icariin was 0.04-0.40 ?g,respectively. The average recovery of epimedin C and icariin were 103.2% (RSD=3.1%) and 97.8% (RSD=3.2%),respectively. CONCLUSION:The method is reliable,stable and well reproducible,and can control the quality of Xianling Guobao Capsules.