Study On Extraction And Isolation Of The Chlorogenic Acid In Honeysuckle Flowers And Its Bacteticidal Activity

Because the Clorogenic acid(CHA) has a significant effect to many diseases, academic medicine attention and enormous social needs is increased, but the current domestic chlorogenic acid research is still very weak, there is not satisfactory production process of the CHA. So the purpose of this study is to establish a stable and effective identification and detection methods of CHA in honeysuckle, analyze chemical stability of the CHA for the production and storage of it, find more efficient economy extraction and purification methods by the optimization process, further explore the CHA antibacterial activity and the chlorogenic acid antimicrobial spectrum.The best flowing ratio of the thin lyar chromatograph (TLC) was abtained:ethyl acetate / water / ethanoic acid (v/v,10/3/2) , the retention facter(Rf) value achieved 0.68; CHA had the biggest absorption in the ultraviolet 327 nm by the ultra-violet spectrograph(UV) scanning during 400-200 nm wave length, the specification curve regression equation: y =0.0553x-0.0034,R=0.9996, it's good in 1~15μg/ml scope; after the high performance liquid chromatograph(HPLC) analyzes experiment, we obtained the baseline separating parameter: column ZOBAX SB (150 mm×4.6 mm,5μm), the flowing ratio was: methyl alcohol/water/ethanoic acid (v/v,18/81/1), the speed of flowing: 1 mL/min, detection wave length: 327 nm, oven tempreature: 30℃, injecting volum: 20μL, these conditions reached; the curve equation: y = 51813 x-5832, R=0.9991, it's good 1~13μg /ml scope. The CHA retention time was 8.832 min, the CHA content of the raw material for extraction was 3.3 % (n=3, RSD=2.0 %), the recovery-ratio was 98 %; the CHA content of the material for purification was 10.15 % (n=3, RSD=1.8 %)The CHA stability trial indicated that: under the same store condition:30 days later, the CHA pH 3.0 was stabler than pH 5.6; under the same resolver condition: the CHA in 4℃temperature refrigerators was stabler than in 25℃natural light laying storeroom. After the single factor Duncan multiple function examination comparison in the confidence interval 5 % levels the performance was remarkable difference.This article compared ethylalcohol reflux condensation(ERC), ultrasonic wave extraction (UE), Microwave extraction (ME), microwave-ultrasonic wave extraction (MUE) . MUE's CHA extraction rates was the highest. We made the CHA breaking through cell wall by microwave heating, then used ultrasonicwave(20 Hz), 2 times. Under this condition:chlorogenic acid's extraction rates achieved 92.66 %, the chlorogenic acid's extraction rate of MUE (92.66 %) was higher than UE or ME (50 %) levels and ERC (71.15 %), the average growth rate was more than 30 %. The MUE orthogonal experiment obtained the best extaction parameter: microwave pre-treated material under 250 W,3 times, 30 s/per time,then joined 70 % ethyl alcohol according to the material/fluid (v/v,1/20), ultrasonic wave extracted 20 min under 18 W. Under this condition:the extraction rate reached above 73 % by the first time extracting, Various factors' influence decrease is: ultrasonic power > resolver quantity > ultrasonic time.With the UV and the HPLC detecting the content of CHA, the metallic ion precipitation isolation and the column chromatographic (three kind of padding: gathers amide, D101, silica gel G)pre-isolation CHA acid was compared, then ethyl acetate extraction further purification was used. The results showed that:the gathers amide column chromatographic was the best isolation method, the content of CHA increased from 15 % to 45.2 %, the growth rate is 30 %,the CHA recovery-ratio was 81.6 %; the silica gel G column chromatographic raw product rate was high, achieved 60.6 %, but the CHA content of the product to be lowest, only 18.8 %; both the D101 chromatographic and the Zn ion precipitation method chromatographic achieved 31 %, but the CHA recovery-ratio of Zn ion was low, only 50 %. After overall evaluation , we selected gathers amide column chromatographic to pre-isolate the CHA, and used the ethyl acetate further extracting 4 times ,then collected the crystallization completely.After the product detected by HPLC, the content of CHA achieved 92 %, the average recovery -ratio of the CHA was 88.4 % (RSD=2.15 %) and the absolute recovery-ratio was 72.1 % (81.6 %×88.4 %=72.1 %).This article studied the CHA bacteriostasis activeness of different kinds of Gram-negative bacteria (Shigella, Salmonella) and the Gram-positive bacteria (golden yellow Staphylococcus and the Lister), the results showed that: compared to the Gram-positive bacteria, the CHA to Gram-negative bacteria's bacteriostasis activeness was stronger;the minimum inhibitory concentration of CHA to shigella and salmonella was 0.125 mg/ml, almost the same to 0.1 mg/ml kanamycin.