The Comparative Study Of Hawthorn Leaf And Major Hawthorn Leaf From Chengde Based On HPLC Fingerprint

Objective:1. To establish fingerprints of Hawthorn leaf and Major Hawthornleaf,taken from different time in Chengde, respectively by HPLCchromatography. And then to develop a method for simultaneousdetermination of vitexin-2″-O-rhamnoside, glucosyl-vitexin,chlorogenic acid,vitexin, rutin,and hyperoside in Hawthorn leaf and Major Hawthornleaf,taken from different time in Chengde. And further more to establish acomparative study on the contents of six indicator compounds from theHawthorn leaf and Major Hawthorn leaf. Monitoring the change of thecontents of the indicator compounds as the time goes.2. To develop a method for determination of total flavonoids of Hawthornleaf and Major Hawthorn leaf,taken from different time in Chengde byUV-Vis Spectrophotometry.And further more to establish a comparative studyon the contents of total flavonoids from two crude drugs. Monitoring thechange of the contents of the total flavonoids as the time goes.3. To determine the contents of six indicator compounds and totalflavonoids in Hawthorn leaf and Major Hawthorn leaf,taken from differenttime in Chengde.To discover differential between Hawthorn leaf and MajorHawthorn leaf, and confirm the optimum harvest time for two crude drugs,To provide theoretical basis for the development and utilization of Hawthornleaf and Major Hawthorn leaf.Methods:Using the HPLC chromatography and the following chromatographicconditions to establish fingerprints of Hawthorn leaf and Major Hawthorn leafrespectively: Agilent ZORBAX SB-C18column （4.6mm×250mm,5μm）,0.1% formic acid （A）-acetonitrile（B）-tetrahydrofuran（C） were mobile phases to dothe gradient elution:0¹5²0³0³5⁶0min,A（90%～84%～84%～78%～74%）,B（8%～14%～14%～20%～24%）,C（2%～2%～2%～2%～2%）;The wavelength: the detection wavelength was340nm; The flow rate was1.0mL·min-1, the column temperature was30℃and the sample size was10μL. Using 《Traditional Chinese medicine chromatographic fingerprintsimilarity of evaluation system2004A version》 of State PharmacopeiaCommittee of China to evaluate the fingerprint similarity and confirm thecommon peaks’ number in each fingerprint.2. As establishing the above fingerprints, using the external standardmethod to develop a method for content determination ofvitexin-2″-O-rhamnoside, glucosyl-vitexin,chlorogenic acid, vitexin, rutin,andhyperoside in Hawthorn leaf and Major Hawthorn leaf respectively.3. Using the UV-Vis Spectrophotometry to develop a method fordetermination of flavonoids in Hawthorn leaf and Major Hawthorn leafrespectively by external standard method, rutin was reference substance,500nm was wavelength and NaNO₂-Al（NO₃）₃-NaOH was the colordevelopment reagent.Results:1.The fingerprints of Hawthorn leaf were marked11common peaks thesimilarity were greater than0.88; The fingerprints of Major Hawthorn leafwere marked12common peaks and the similarity were greater than0.86.2.The highest contents of six indicator compounds in Hawthorn leaf andMajor Hawthorn leaf was in May.The average contents（mg/g） of eachproducing area were as follows:Hawthorn leaf:chlorogenic acid:12.95, glucosyl-vitexin:0.29, vitexin-2″-O-rhamnoside:8.63, rutin:0.14, vitexin:0.44, hyperoside:1.53,Major Hawthorn leaf:chlorogenic acid:10.58, glucosyl-vitexin:6.06,vitexin-2″-O-rhamnoside:11.77, rutin:0.42, vitexin:0.23, hyperoside:0.82.3. The average contents（mg/g） of flavonoids indifferent month were asfollows: Hawthorn leaf:May:245.92,June:100.00,July:80.25,August:122.34,september:95.00,October:80.35,November:70.16,Major Hawthorn leaf:May:161.02,June:80.07,July:72.24,August:107.82,september:93.31,October:70.00,November:73.92.Conclusion:1.Under the same HPLC chrmatographic condition, having discovereddifferential between Hawthorn leaf and Major Hawthorn leaf.The contents ofchlorogenic acid and glucosyl-vitexin in Major Hawthorn leaf were more thanthat in Hawthorn leaf,and the contents of vitexinhy, peroside and totalflavonoids in Major Hawthorn leaf were less than that in Hawthorn leaf.Dueto different chemical composition, the clinical effect of the Hawthorn leaf andMajor Hawthorn leaf differs a lot.Hens,establishing fingerprints of Hawthornleaf provides information about rational usage for clinical.2.To determine the contents of six indicator compounds and totalflavonoids in Hawthorn leaf and Major Hawthorn leaf,taken from differenttime and producing area in Chengde. And discovered Hawthorn leaf andMajor Hawthorn leaf that have the highest contents of six indicatorcompounds and total flavonoids were from Xinglong,the the optimum harvesttimes were May and Auguest.Therefore,when taking Hawthorn leaf andMajor Hawthorn leaf as raw material to produce chemicals and drug products,the crude drug should be collected in May and Auguest.