| Objective:To establish the fingerprints that can reflect the global features of propolis from different places using fingerprint technology and modern analysis methods.Determine the main active ingredient combined with the measurement of effective parts and active components to investigation the spectrum-effect relationship between HPLC fingerprints and in vitro antioxidant activity,in order to establish a comprehensive and effective way to control the quality of propolis,and provide the scientific basis for comprehensive development and utilization of propolis.Methods:1 Infrared Fingerprints of Propolis AssayThe IR fingerprints of propolis from different places were established using FTIR,and main absorption peaks were mapped using the OMNIC software preprocessing,the IR fingerprints of 26 batches of propolis were analyzed by dual-index and cluster analysis,and the difference of fingerprints of different sample were compared using the second derivative.2 HPLC Fingerprints of Propolis AssayTo establish fingerprints of propolis by HPLC-UV.Analyzed the fingerprints of 26 batches of propolis by "Similarity Evaluation System for Chromatographic Fingerprint of TCM 2004A",and identified the common peaks and the similarity,used statistical method for cluster analysis.3 Determinate of Effective parts and Active ingredient contents in PropolisTo determine total flavonoids in propolis with rutin as reference substance,the NaNO2-Al(NO3)3 chromogenesis,measured at 500 nm wavelength by UV-visible spectrophotometry.The determination of pinocembrin、chrysin and galangin were carried out by UPLC.ACQUITY UPLC BEH C18(2.1 mm× 50 mm,1.7 μm)column was used at 32℃.The mobile phase consisted of methanol-0.4%phosphoric acid aqueous solution with the gradient elution,at the flow rate of 0.3 mL · min-1,and the detection wavelength were set at 290nm、268nm、265nm.4 Study the Spectrum-effect Relationship between HPLC fingerprints and in vitro Antioxidant activityThe antioxidant activity of propolis were measured by DPPH free radical scavenging capacity,with ascorbic acid as positive control drug.The spectrum effect relationship between HPLC fingerprints and DPPH free radical scavenging capacity was analyzed by using statistical method.Results:1 Infrared Fingerprints of Propolis AssayThe FTIR fingerprints of 26 batches of propolis were established,a total of 11 common peaks were identified,the FTIR spectra from different origins were basically the same,and the correlation coefficient(median)with common pattern between 0.9522 to 0.9964.But in 1800 to 600 cm-1 band,there were some differences of absorption peaks in the number and intensity.The result of common and variation peak ratio in FTIR fingerprints of propolis by dual-index sequential analysis have found that the common peak rate of propolis from similar origin was higher,and the variation peak rate of propolis from distant origin was higher.There were obvious differences in wave number 1711 cm-1、1562 cm-1、1250 cm-1、910 cm-1 between fj19 and f j23 whose common peak ratio was highest by calculating the second derivative spectra.26 batches of propolis can be divided into nine categories by clustering analysis,including fj1、fj2、fj3、fj9、fj11、fj13、fj16、fj17、fj18、fj21、fj22、fj23 and fj24 gathered for a class,fj7、fj8、and fj19 gathered for a class,fj12 and fj26 gathered for a class,fj4 and fj6 gathered for a class,fj5、f j10、fj20 and fj25 were respectively clustered into one category.2 HPLC Fingerprints of Propolis AssayThe HPLC fingerprints of 26 batches of propolis were established,a total of 22 common peaks were identified,and the known compounds were caffeic acid、p-coumaric acid、apigenin、chrysin、pinocembrin and galangin.The RSD of relative peak in propolis from different regions were greater The similarities of each HPLC fingerprint with the reference spectrum were between 0.745 to 0.990,26 batches of propolis can be divided into six categories by cluster analysis of peak area,including fj12、fj13、fj16、fj17、fj20、fj21、fj25 and fj26 gathered for a class,fj1、fj3、fj4、fj6、fj7、fj8 and fj19 gathered for a class,fj10、fj18、fj22 and fj24 gathered for a class,fj2、fj9、fj11 and fj23 gathered for a class,fj14 and fj15 gathered for a class,fj5 was respectively divided into one category.3 Determinate of Effective parts and Active ingredient contents in PropolisThe content of total flavonoids in 26 batches of propolis were between 5.55%to 43.89%,there were significant differences between each samples with different regions(P<0.05),the content of fj14 propolis where the origin of Beijing was highest,and the content of f j26 propolis where the origin of Henan was lowest.The linear in the range of 0.0301～0.6020 mg·mL-1 for pinocembrin,regression equation was Y=6632175.9479X+44901.8611(r =0.9996),the average recovery of pinocembrin was 96.20%(RSD=1.65%).The content of pinocembrin in 26 batches of propolis between 0.41%to 3.84%,there were significant differences between each samples with different regions(P<0.05),the content of f j5 propolis where the origin of Xuanwei was highest,and the content of fj26 propolis where the origin of Henan was lowest.The linear in the range of 0.0361～0.9030 mg·mL-1 for chrysin,regression equation was Y=10453377.6566X+66397.6577(r=0.9998),the average recovery of chrysin was 97.13%(RSD=1.95%).The content of chrysin in 26 batches of propolis between 0.62%to 5.84%,there are significant differences between each samples with different regions(P<0.05),the content of fj18 propolis where the origin of Luzhong was highest,and the content of fj4 propolis where the origin of Sichuan was lowest.The linear in the range of 0.0117～0.5850 mg·mL-1 for galangin,regression equation was Y=6573583.6179X-3744.0261(r =0.9999),the average recovery of galangin was 98.57%(RSD=2.45%).The content of galangin in 26 batches of propolis between 0.47%to 4.10%,there were significant differences between each samples with different regions(P<0.05),the content of fj18 propolis where the origin of Luzhong was highest,and the content of fj26 propolis where the origin of Henan was lowest.4 Study the Spectrum-effect Relationship between HPLC fingerprints and in vitro Antioxidant activity26 batches of propolis have a certain scavenging on DPPH free raical,the clearance rate was obvious increased while the concentration increased,it showed a good dose-response relationship.The IC50 values of each propolis samples were between 16.23 to 166.81μg · mL-1,the scavenging of fj14 where the origin of Beijing was strongest,and the the scavenging of f j26 where the origin of Henan was weakest.The major peaks of scavenging DPPH free radicals were NO.1、2、3、10、11、14、15、17、19 common peak in propolis,there were also obvious correlation between each chromatographic peak.Conclusion1 Infrared Fingerprints of Propolis AssayThe infrared fingerprint technology combined with dual-index sequence or cluster analysis can be quickly and reliably identify the propolis with different origin,the second derivative spectra can further distinguish the differences between two or more propolis samples.The FTIR technology can provide a quick and simple method for quality control and provenance of propolis.2 HPLC Fingerprints of Propolis AssayThe HPLC fingerprint technology can reflect the overall characteristics of the chemical composition in propolis,it can guarantee the propolis were stable and consistency.The HPLC fingerprints also can be combined with chemical pattern recognition or pharmacodynamics activity in order to obtain more abundant information,this technology is an effective method for quality evaluation of propolis.3 Determinate of Effective parts and Active ingredient contents in PropolisIt can be fastly and accurately determine the connet of pinocembrin chrysin and galangin in propolis by UPLC,this method can greatly reduce the analysis time compare with HPLC.It can evaluate the quality of propolis in a certain extent by the determination of effective parts and active ingredient.4 Study the Spectrum-effect Relationship between HPLC fingerprints and in vitro Antioxidant activityThere were significant differences in antioxidant activity of propolis with different origins,also there were correlation between the antioxidant activity with the connet of total flavonoids、pinocembrin、chrysin and galangin.The preliminary study of the spectrum-effect relationship in propolis can expound the major chemical component of antioxidant activity,it can provide scientific basis for the quality control of propolis. |
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