The Study Of HPLC Fingerprint Of Mulberry Leaves And Its Application In The Quality Control Of Mulberry Leaf Preparations

Mulberry leaves are dried leaves from Morus alba L., recorded in the " Shen Nong’sHerbal Classic", with the effects of wind-heat evacuation, clearing the lungs and moistening,liver eyesight. Mulberry leaves contain polysaccharides, flavonoids, alkaloids, volatile oilcomponents, vitamins, amino acids and other active substances, with pharmacologicalactivities of hypoglycemic, lipid-lowering, scavenging oxygen free radicals and others. Typeand content of the chemical compositions of mulberry leaves are affected by species andplanting environmental conditions, thus they are affecting the qualities of mulberry leaves andrelevant products.In order to enhance the development and utilization of resources and control the qualityof mulberry leaf, this study mainly focused on fingerprint of21batches of different originMulberry herbs using the method of high performance liquid chromatography (HPLC)combined fingerprint and quantitative analysis of multi-component index, then an accurate andreliable chromatographic fingerprints was built. Optimizing the process of flavonoidsextraction from mulberry leaves by ultrasonic assisted extraction method; establish HPLCfingerprint of mulberry leaves and its conditions by trial and error experimentation. Combinedwith quantitative detection of some of the main active ingredients, the quality standards ofmulberry leaves was supplemented and improved, it may provide a reference for the controlstandards of mulberry quality. The main contents are as follows:(1)The optimum extraction of flavonoids Mulberry: flavonoids are the main activechemical ingredients in mulberry leaf; the method of extraction has a greater impact on theircontents. In order to improve the extraction rate of flavonoids, it takes a systematic studyabout the different effects of extraction, such as solvent, temperature, time and other factors.Using single-factor optimization and orthogonal experiment, it is to optimize the process offlavonoids extraction from mulberry leaves. The results showed that: the best extraction is that70%ethanol as extraction solvent,1:25(W/V) as solid-liquid ratio, using ultrasonic extraction,twice, each time120min.(2) The study of HPLC fingerprint of Mulberry leaves: The21batches of mulberry leaves were collected from pharmacy. The chromatographic conditions were optimized: Agilent1100high-performance liquid chromatography combined diode array detector, Agilent TC-C18column (250mm×4.6mm,5μm), mobile phase A:0.1%phosphoric acid, mobile phase B:methanol, using a binary gradient elution program:0-14-28-35-50-65-70min,15-30-40-45-60-70B%; Flow rate is1.0mL·min-1; the detection wavelength is340nm;column temperature is25℃; Injection volume is10μL. The result of methodology showedthat the method of mulberry leaves HPLC fingerprint is specific, stable, and it can be morefully reflect the characteristics of mulberry leaves.(3) The study of21batches of Mulberry leaves HPLC fingerprint: the result of21batchesof mulberry fingerprints using established method showed that21batches mulberry leaveshave25common peaks, and the common peaks’ areas are accounted for80%of all peak areas.Meanwhile, compared with flavonoid references, it can determine the peaks of Chlorogenicacid, Rutin, Quercetin. The result of HPLC fingerprint similarity calculation showed thatsimilarity of21batches of samples are more than0.70, the highest similarity is0.991.17samples’ similarity are greater than0.90, one is between0.85-0.90, there are three samplessimilarity of less than0.85.(4) The observation of mulberry leaves’ characters and analysis of the main activeingredients: traits of different batches of mulberry leaves are in line with description ofcharacter identification in Chinese Pharmacopoeia, but the traits have significant differences incolors, mulberry leaves size and thinness, thickness of petiole, etc. The contents ofChlorogenic acid, Quercetin, Rutin,1-deoxynojirimycin (DNJ) were determined by usingmethods of ultraviolet spectrophotometry, HPLC-DAD, HPLC-ELSD, then to analyze thecorrelation between the content of active chemical ingredient and the character of each of theactive ingredients. The results showed that there is a large difference between different batchesof mulberry leaves, with no apparent correlation with the traits. In the period of planting, thefactors such as the climate, soil, plucking time have a greater impact on medicinal propertiesand contents of active ingredients.(5) The fingerprint of raw herbs and intermedial of Sangyuan Zhike Capsule: Sangyuan Zhike Capsule is developed by General Hospital of Guangzhou Military Command of PLA,department of pharmacy; it is a unilateral formulation which is the non-standard preparationsin the military medical institution. Controlling the quality of the intermediate and evaluatingthe process using the method of fingerprint technology are the effective way to improve thequality of Sangyuan Zhike Capsule in the whole system. The result of similarity calculation ofintermediate and preparation’s fingerprints showed that the process of capsule is stable andfeasible. The result of correlation between mulberry herbs, capsuable intermediate andpreparation showed that the peaks of main chemical components in mulberry leaves arereflected in chromatogram of intermediate, and there is a less loss of chemical compositionduring the preparation process, it provides a good grarantee for finished product.The significance of this thesis is to study the differences between different origins ofmulberry leaves. A reliable analytical method has been established and it provides a qualitycontrol reference for health care products and Chinese herbal preparation which raw materialis mulberry leaves. At the same time, the result is also benefit for structure-activity researchand development comprehensively of mulberry leaves.