| Objective:Ilicis Cornutae Folium is the dried mature leaves of Ilex cornuta Lindl. ex Paxt, which belongs to the Ilex Holly family. The young leaves of Ilicis Cornutae Folium can be used as Leaf of Chinese Hollym after steaming. Ilicis Cornutae Folium is bitter and cold, which could heat, benefit kidney and get rid of rheumatism. It’s always used for tuberculosis hemoptysis, Gu Zheng hot flashes, dizziness, rheumatism and other illnesses in clinical treatment. Character, identification checks and check items are mainly controled in The "Chinese Pharmacopoeia"(edition 2015). In order to establish a more comprehensive control of Ilicis Cornutae Folium, this reaserch studied its related-standards items, developed a better draft quality standards and provided the basis for a comprehensive quality control and identification of which.Method:To complete the pharmacognosy identification research of Ilicis Cornutae Folium, this research studied the Characters of herbs, organizational structure near the base of the cross-section and microscopic characteristics of powder; studied the TLC methods for the identification of Ilicis Cornutae Folium with chlorogenic acid and ursolic as the reference substances; determined the moisture content, the total ash content, the acid insoluble ash content and the extract; determined the content of chlorogenic acid, ursolic and oleanolic acid by HPLC method and validated the chromatographic condition; studied one method investigating the four kinds of flavonoids; for the first time studied the HPLC fingerprint chromatographic conditions, anddrawed the control fingerprint.Result:Established character identification, microscopic identification (sliced structure and powder characteristics) and other pharmacognosy identification methods of Ilicis Cornutae Folium; Established TLC methods of Ilicis Cornutae Folium with chlorogenic acid and ursolic as active ingredient, in which the tests and chlorogenic acid, ursolic had the same colored spots in the same Rf values, clear, round and separated.The contents of chlorogenic acid were determined by HPLC, the linear regression equation was y= 925.8x+1742, the correlation coefficient R2 was 0.9998, the linear relationship was good in the range of 0.98μg～6.86μg, the chlorogenic acid content of the nine batches of Ilicis Cornutae Folium which from different areas were 0.46%～2.01%, the average was 1.13%.The contents of ursolic and Oleanolic acid were determined by HPLC, the linear regression equation of ursolic was y= 412.7x-17.19, the correlation coefficient R2 was 0.9995, the linear relationship was good in the range of 0.88μg-4.40μg;the linear regression equation of Oleanolic acid was y= 240.8x-26.30, the correlation coefficient R2 was 0.9999, the linear relationship was good in the range of 0.54μg～2.7μg; the ursolic content of the nine batches of Ilicis Cornutae Folium which from different areas were 0.5169%-1.0548%and oleanolic acid were 0.0906%-0.1906%. The total amount were ranging from 0.6075%-1.2454%, with an average of 0.9264%. The rutin content was in the range of 1.5774%-6.9464%; the quercetin content was in the range of 0.2123%～2.6812%; the kaempferol content was in the range of 0%～0.0229%; the Isorhmnetin was in the range of 0.0698%-0.5840%.The optimal conditions of HPLC fingerprint were:colmn:Kromasil C-18 (250mm×4.6 mm,5um); Detection wavelength:210nm; Column temperature 30 ℃; Flow rate:1mL/min; mobile phase A (acetonitrile)-B (0.1% aqueous phosphoric acid solution) gradient elution programwas:0-25min,10%A-24%A,25-40min,24%-31%,40-43min,31%-35%, 43-60min,45%-55%. The fingerprints had a total of 20 characteristical peaks, in which identified 4 and No.6 was chlorogenic acid, No.8 was rutin, No.10 was quercetin, No.18 was Long stem holly glycosides.Conclusion:The research indicated that the proposed quality standards can control the quality of Ilicis Cornutae Folium better. It increased powder Identification items, ash limited, extract content, TLC and HPLC.For the first time it established the fingerprinting method of Ilicis Cornutae Folium which was in good precision, stability and reproducibility. This research could provide a basis for quality control and authentification of Ilicis Cornutae Folium. |
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