Studies On Processing Mechanism Of Cornus Officinalis

The item came from Subject of Nation’s Tackle Key Problen in the tenth Five-year. Theprocessing mechanism of comus officinalis was mainly discussed in two aspects ofchemistry and pharmacology. We studied some components that can increase the efficacy orchange the character and function of the comus officinalis by comparing component andpharmacological index before and after processing to acquired active composition,summarized processing principle and provide the theoretical basis of quality standard andclinical application of comus officinalis.This dissertation illustrated the research of chemical active composition andprocessing mechanism of comus officinalis. It can be divided into four parts.PartⅠReview of Comus officinalis literatureIt included processing history, clinical application, modem chemistry andpharmacology of Comus officinalis. The work of this part paved the way for thedevelopment of research program.PartⅡThe chemical research of Comus officinalis include following contents.1. Comparative study of chemical constituents of Comus officinalis before and afterprocessing.The chemical components in the water and alcohol extraction and other extractionparts of organic solution（petroleum ether, dichloromethane, ethyl acetate, n-butanol andwater ） were analyzed by HPLC-DAD and HPLC-ELSD. The results showed that throughprocessing a new substance was produced, which was confirmed to be 5-HMF. Manycomponents changed largely. Gallic acid increased remarkably, while other chemicalcomponents such as loganin and Morroniside decreased. Besides, the chemicalcompositions that were very different from crude samples and processed samples.2. A method was built up to quantify the amount of 5-hydroxymethylfurfural, Gallicacid, loganin and Morroniside from Comus officinalis by HPLC. The result showed thatwithin the range of 30.9～309 g·mL^-1, Gallic acid had a good linear relationship, with r=0.9995; within the range of 25～250g·mL^-1, 5-hydroxymethylfurfural had a good linearrelationship, with r=0.9998; within the range of 14.6～292 g·mL^-1, Loganin had a goodlinear relationship, with r=0.9992; within the range of 47.4～948 g·mL^-1, Morroniside had agood linear relationship, with r=0.9999; The average recovery rate of Gallic acid was99.12%, RSD was 1.68%; 5-hydroxymethylfurfural was 99.12%,RSD was 2.11%;Morroniside was 100.2%, RSD was 2.42%; Loganin was 99.83%,RSD was 1.88%.Thecontents of Gallic acid were 0.081%（ n=5, RSD=0.78%） and 0.28 % （n=5, RSD=1.73%）respectively in Comus officinalis before and after processing; 5-hydroxymethylfurfural was0.78% （ n=5, RSD=0.69%） in processed sample; Morroniside were 1.91%（n=5,RSD=2.15%） and 1.38%（n=5, RSD=3.31%） respectively; Loganin were 1.41%（n=5,RSD=0.65%） and 1.27% （ n=5, RSD=1.54%） respectively.3. The contents of total Polysaccharides, flavonoids, saponins and Tannins weredetermined by Vis-UV spectrophotometer. The results showed that if Glucose was served asa reference, the average amount of total Polysaccharides were 7.72% （n=3, RSD 0.58%）and5.59% （n=3, RSD 0.72%） respectively; If Rutin was served as a reference, the averageamount of total Flavones were 3.09%（n=5, RSD 2.16%） and 1.42%（n=5, RSD 4.49%）respectively; If Ursolic acid was served as a reference, the average amount of totalSaponins were 3.88%（n=5, RSD 0.64%）and 2.81% （n=5, RSD 0.41%） respectively; IfGallic acid was served as a reference, the average amount of total Tannins were 4.02% （n=5,RSD 0.96%）and 3.01%（n=5, RSD 0.73%） respectively.4. Titration analysis was employed to determined the amount of total organic acidfrom Comus officinalis before and after processing. The results showed that the averageamount of total organic acid were 7.83% （n=3, RSD 1.83%） and 8.96% （n=3, RSD 2.52%）respectively. The contents of Ursolic acid and Oleanolic acid were detected by HPLC. Thecontents of Ursolic acid were 0.234% （n=3, RSD 1.40%）and 0.221% （n=3, RSD 2.70%）respectively, The contents of Oleanolic acid were 0.0644%（n=3, RSD 1.44%） and0.0622%（n=3, RSD 1.38%） respectively.5. The dynamic change of chemical component was observed by HPLC in theprocessing process. The results showed that there produced 5-hydroxymethylfurfural byprocessing and Gallic acid increased within 10 hours, but their contents were almoststeadiness between 10 hours and 12 hours. After processing, morroniside and loganindecreased. Morroniside was steadiness between 4 hours and 12 hours, loganin wassteadiness between 4 hours and 10 hours. After 12 hours, their contents decreased ulteriorly. 6. 16 compounds in the petroleum ether active part were identified by HPLC-MS.Theyare Sitosterol, Vitamin e, Amyrin, 9,12-Octadecadadienoic acid; Dibutyl phthalate, and soon. After processing, Vitamin e increased 46.6 %, 9,12-Octadecadadienoic acid increased18.3 %, Amyrin increased 16.5%, 9,12,15-Octadecatrien-1-ol increased 30.9% inwine-baked sample. 25 compounds in dichloromethane active site were identified byHPLC-MS. They are 5-HMF, dibutyl phthalale, and so on. After processing the content of5-HMF increased by 80%, nearly two-fold. Amino acids and trace elements were detectedby ICP-AES and HPLC. There were 16 and 15 kinds of free aa, 15 and 14 kinds of total aarespectively found in water extraction of comus officinalis.7. The new compound was separated by using D₁₀₁ macroporous resin columnchromatography and organic solvent extraction. Its structure was identified by IR, UV,GC-MS and compared with standard sample. It is 5-HMF, which was separated for thepharmacological experiment.8. Two purifier polysaccharides were acquired by using DEAE-52 and Sephadexcolumn chromatography. Their chemical composition and molecular weight weredetermined with HPLC. Molecular weight of SZYS is 4.29x10⁴, SZYP is 1.38×10⁴. SZYSand SZYP are composed of Glu, Ara and Rha, but they are different in proportion. InSZYS,Glu:Ara:Rha was 1:1.51:1.17,while in SZYP, Glu:Ara:Rha was 1:8.05:1.78. Twopurifier polysaccharides were separated for the pharmacological experiment.9. Determining the technological parameters of the enrichment purification of iridoidlgycoside with SP825-Macroporous resin. The optimum process was 2 times macroporousresin which was absorbed two dynamicly with columns of SP825-macroporous resin andthe column washed with 7 column volume of 50% ethanol.10. Fingerprints of comus officinalis before and after processing were establishedfrom 13 raw and 11 samples processed by HPLC. Gradient mobile system was applied withacetonitrile-phosphoric acid and the method of logical experiments showed high precisionand good repeatability. There were 26 common fingerprint peaks dentified in many samplesprocessed. There are 23 common fingerprint peaks were identified in raw samples. Bycomparing with standard samples, some primary peaks were identified. The fingerprintsshowed unitary character of comus officinalis before and after processing in chemicalcomposition kinds and quantities.PartⅢ: The chemical and pharmacodynamic effect on active ingredient tonifyingliver and kidney was studied and processing mechanism was summed-up as follows: 1. Summing up the abstract of the results of research on pharmacological experimentsof the filter of active parts and comparative study between sample before and afterprocessing.2. Pharmacodynamic experiments demonstrated that 5-HMF had protective effect onacute liver injury in mice and can protect human vein epidermal cell against H₂O₂ andglucose. The antioxidant effect may be one of the mechanisms to protect liver andendothelium.3. Pharmacodynamic experiments demonstrated that crude polysaccharides had effecton tonifying humoral immunity and cell immunity. The effect of sample processed excelledone of crude sample.4. Comparative analysis of results and the literature study of Comus officinalis,explored the material basis increasing ability of tonifying liver and kidney and processingmechanism of Comus officinalis was summed up. The results showed that 5-HMF hasprotective effects on liver and vascular endothelial cell and polysaccharides could enhancethe immune efficiency. Both 5-HMF and polysaccharide were the material basis oftonifying liver and kidney. After processing, the structure and composition change ofpolysaccharide was to enhance effect. Aspartic acid K and Mg has positive effects onmitochondria, cell metabolism, ammonia metabolism in liver and ion transportation ofcytomembrane. Comus fruit contains large quantity of Asp and K and Mg, which mayexplain why it has protective effect on acute liver injury in mice and tonifying liver andkidney. The increasing of the amount of vitamin E and polyunsaturated fatty acidreinforced the tonifying function, which had certaint effect on processing synergism. Thesignificant increase of Leu, Ile, La, Ce, and so on, may contribute to tonifying liver andkidney. Tannins and iridoid glucosides decreased, which may also play a role in increasedfunction.All the literature and experimental study showed that Comus officinalis played a roleof tonifying liver and kidney through a number of channels of singe active component or achannel or a number of channels of many ones. The function increase was implemented by5-HMF increase, the change of polysaccharides structure and many compositions’ contents.PartⅣ: Discussion.