| In this study, through the combination of single factor and orthogonal experiments to selectthe best extraction solvent, the best method of extracting chlorogenic acid and to establish thefingerprint of Eucommia ulmoides Oliv leaves by HPLC.Test results were as follows.(1)The standard curve of Chlorogenic acid obtained byultraviolet spectrophotometric method was obtained as follows: y=22.6442x-0.2039r=0.9999(n=6).y was chlorogenic acid concentrations(μg/mL),x was the absorbance value.The chlorogenicacid concentration within5~40μg/mL had good linear relationship; Standard curve of HPLC: y=0.001x+0.0022, r=0.999(n=6)y was chlorogenic acid mass (μg), x was the peak area ofchlorogenic acid.It showed that chlorogenic acid quality within the range of0.075~0.6μg hadgood linear relationship.(2)When the methanol, ethanol, acetone as chlorogenic acid extractionsolvent, their best volume fraction were20%,30%and40%, and the corresponding yield ofchlorogenic acid were0.8471%,0.9056%,0.9430%.Water as extraction solvent,chlorogenic acidyield was0.7032%. Through comparing with the fingerprint peak shape of20%methanol,30%ethanol and20%acetone extraction solvent could be concluded that their peak shapes wereconsistent, and chlorogenic acid peak were respectively took81.583,81.168,80.424percents, thedifferences among these percentages were very small.(3)When30%ethanol (volume fraction)as chlorogenic acid extraction solvent, the best conditions of eatch extraction method just asfollows:①Ultrasonic method,the solvent pH was4,the liquid to solid ratio was14,the ultrasonicfrequency was40KHz,the ultrasonic time was80min,the extraction rate of chlorogenic acidreached as high as0.9032%.Orthogonal experiment results showed that various factors on theimpact of chlorogenic acid yield the sequence was: ultrasonic power>ultrasonic time>solid toliquid ratio, namely the ultrasonic power influenced on the yield of chlorogenic acid was thelargest;②The conditions of microwave method,the solvent pH was4,the liquid to solid ratio was18, the microwave fire was medium heat, the microwave time was60s,the extraction rate ofchlorogenic acid reached as high as0.9313%.Orthogonal experiment results showed that variousfactors influenced chlorogenic acid yield, the descending order as follows: microwavefire>irradiation time>liquid ratio, namely the microwave fire influenced on the yield ofchlorogenic acid was the largest;③The conditions of enzymatic method,the liquid to solid ratiowas14, reaction time was1.5h, the solvent pH was4.5, enzyme dosage was0.35%,enzymolysistemperature was40℃,the chlorogenic acid yield as high as0.9508%.Orthogonal experimentresults showed that various factors influenced on the yield of chlorogenic acid from big to smallorder: pH>reaction temperature>enzyme dosage, pH had the largest influence on the yield of chlorogenic acid;④The conditions of heat immersion method,the solvent pH was4, heatimmersion temperature was70℃, reaction time was0.5h,the liquid to solid ratio was18, thechlorogenic acid yield as high as0.9654%.Orthogonal experiment results showed that variousfactors influenced on the yield of chlorogenic acid from big to small order: liquid to solidratio>heat immersion temperature>reaction time, liquid to solid ratio had the largest influence onthe yield of chlorogenic acid.(4)Thought analyzed the chromatogram could be concluded that thebest extraction method of chlorogenic acid was heat immersion method.(5)With the bestextraction process to make the HPLC fingerprint of10batches of eucommia ulmoides olivleaves, fingerprint similarity were above0.9.The study ultimately concluded that the Volume fraction of30%ethanol solution was thebest solvent for extracting the chlorogenic acid in eucommia ulmoides oliv leaves. Heatingimmersion method was the optimal process for extracting chlorogenic acid in eucommiaulmoides oliv leaves.Tests established HPLC fingerprint of eucommia ulmoides oliv leavesunder the best extraction techonolygy. |
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