Study Of Herbal Extraction On Immune Regulation And Extraction, Isolation, Identification And Determination Of Molecular Weight Of Polysaccharide

Poultry diseases, especially viral diseases, remain an obstacle to the development of animal husbandry. Vaccine is the most effective way to prevent disease. However, because of immune suppressive diseases prevalence in recent years, prevention has become exceptionally difficult. Scholars have been working to improve the immune-enhancing role of vaccine. Herbal medicine is the crystallization of Chinese traditional culture and the cultural treasures of the Chinese nation. Herbal medicine have played an important role in the process of livestock and poultry diseases prevention. The immunoenhancer of herbal medicine enhance or adjust the body cellular and humoral immunity function and enhance the body's resistance to disease, indicating a significant role in enhancing immune. reticuloendotheliosis virus (REV) infected in chickens usually in clinical practice, and resulted in a certain degree of immune suppression. In present study, animal model was established, and to compare the immunoregulatory of herbal extractions in the different immune status of chickens. The polysaccharide as a major components were isolated, identificated and determinate the molecular weight, the structure and function will be found in further study.1 Immune regulation of herbal extraction1.1 Effectiveness of herbal extraction and levamisole hydrochloride on immune regulation in chickensThe immune regulation of herbal extraction and levamisole hydrochloride was investigated in health chickens and immune suppressive chickens infected with REV in 1-day-old. The results showed that levamisole hydrochloride added 50mg/kg in water did not effect hemagglutination inhibition (HI) antibody titers both in the health chicken and the immune suppressive chickens after immunization with Newcastle disease virus (NDV) and H5 subtype of avian influenza virus (H5-AIV) vaccine(p>0.05), the same results was showed in body weight and the relative weight of immune organs. Herbal extraction composed of Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae did not effect HI antibody titers in the health chicken after immunization with NDV and H5-AIV vaccine (p>0.05), but it can significantly improve the HI antibody titers, body weight and the relative weight of immune organs in the immunosuppression chickens infected with REV(p<0.01), although the datas were still lower than the health chickens. The study showed that herbal extraction composed of Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae had immune enhancement effects in chickens immnunosuppressed by REV, and levamisole hydrochloride no effects.1.2 Effectiveness of different combinations herbal extraction on immune regulation of chickensThe immune regulation of herbal extraction of different concentraction Radix Astragali and different combination composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae was investigated in health chickens and immune suppressive chickens infected with REV in 1-day-old. The results showed that herbal extraction of different concentraction Radix Astragali was not effect on the NDV and H5-AIV of HI antibody titers in different immune status chickens, and not effect on the body weight and the relative weight of immune organs as well(p>0.05). The immune regulation of herbal extraction composed of Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae did not effect HI antibody titers in the health chicken immuned with NDV and H5-AIV vaccine(p>0.05), but it can significantly improve the HI antibody titers in the immunosuppression chickens infected with REV(p<0.01), although the datas were still lower than the health chickens. The herbal extraction could also increase the relative weight of immune organs. The study showed that a single Radix Astragali could not increase HI antibody titers in immunosuppressive chickens infected with REV. The herbal extraction composed of Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae had immune-enhancement in immunosuppressive chickens infected with REV.1.3 Effectiveness of herbal extraction extracted by different methods on immune regulation in chickensThe immune regulation of herbal extraction extracted by different methods was investigated in health chickens and immune suppressive chickens infected with REV in 1-day-old and that of water-soluble herbal extraction was researched in different origin chickens. The results showed that the herbal extraction composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae was added in water, no matter the herbal extraction was water-soluble extraction (water extraction and water extraction after extracted with ethyl ether) or fat-soluble extraction (ethyl ether extraction), did not effect HI antibody titers in health chicken after immuned with NDV and H5-AIV vaccine (p>0.05). The water-soluble extraction could increase the HI antibody titers of the chickens infected with REV, but the fat-soluble extraction the same composition had not immune-enhancement (P>0.05). The water-soluble extraction did not effect HI antibody titers both in the health chicken and the SPF chickens feeded in FPF environment (p>0.05), but it can significantly improve the HI antibody titers in the immunosuppressive chickens and SPF chickens feeded in non-SPF environment infected with REV(p<0.01), although the datas were still significantly lower than the health chickens.1.4 Effectiveness of herbal extraction of different molecular weights on immune regulation in chickensThe immune regulation of herbal extraction and different molecular weght of herbal extraction was investigated in different immune status chickens and SPF chickens infected with REV in 1-day-old. The results showed that the herbal extraction and the 1-10 million Dalton molecular weight of herbal extraction composed of Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae, no matter the herbal extraction was mixed firstly, extracted, ultrafiltered and concentrated or extracted, ultrafiltered, concentrated respectively and then mixed, can significantly improve the H5-AIV and NDV of HI antibody titers in immunosuppressive chickens induced with REV(p<0.01), although the datas were still lower than the control without REV-infected chickens. However, less than 10,000 Dalton and greater than 100,000 Dalton molecular weight of the herbal extraction had not immune-enhancement in immunosuppressive chickens infected with REV (p>0.05). This results also showed that the herbal extraction and the 1-10 million Dalton molecular weight of herbal extraction of had immune-enhancement in immunosuppressive SPF chickens infected with REV feeded in non-SPF environment (p<0.05), but had not immune-enhancement in the same chickens feeded in SPF environment(p>0.05). The research showed that the molecular weight of the herbal extraction with immune-enhancement composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae was between 1-10 million Dalton, and had not immunoenhancement in the SPF chickens feeded in SPF environment.1.5 Effectiveness of herbal extraction on peripheral lymphocyte proliferation in chickensThe study was conducted to detect the effects of herbal extraction on peripheral blood, thymus and spleen lymphocytes proliferation in chicken infected with REV. MTT method was used to observe lymphatic cell proliferation. The results showed that the blood, thymus and spleen lymphocyte proliferation were immune-suppressived in chickens infected with REV (p<0.05). Herbal extraction had no effects on the lymphocytes proliferation of blood, thymus and spleen in health chicken (p>0.05), but it can increased that of the blood, thymus before 35-day-old and the spleen before 21-day-old in chickens infected with REV(p<0.05), which has not effects the lymphocyte proliferation of blood, thymus after the 49-day-old and the spleen after 35-day-old in chickens(p> 0.05).2 Extracting, isolating, identifing and determining of molecular weight of polysaccharide2.1 The chemical constituents identification of herbal extractionThe mixture components of herbal extraction composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae was identified by the different motheds of chemical constituents identification. The results showed that herbal medicine was distilled by water contains tannin, sugar and their glycosides, saponins, amino acids, peptides and proteins, was extracted with ethyl ether contains alkaloids, flavonoids and their glycosides, volatile oil, fat class. The herbal medicine was distilled by water after extracted with ethyl ether firstly and was distilled by water was the same composition, after filtered with ultrafiltration membrane, the molecular weight of less than 10,000 Dalton of the herbal extraction contains tannic acid, amino acids, peptides and proteins,1-10 million Dalton contains sugars, polysaccharides, glycosides, saponins, peptides and proteins, more than 100,000 Dalton contains sugar, polysaccharides and their glycosides, saponins, peptides and proteins. The difference of fat-soluble components and water-soluble components of herbal extraction is that water-soluble components contain sugar, polysaccharides, saponins and fat-soluble components do not contain that. polysaccharides and saponins and their glycosides have a role in immune-enhancing.2.2 The study of extracting parameters of herbal extraction polysaccharideThe effect was investigated by a four-factor three-level orthogonal design of the extraction temperature (A), ratio of herb to liquid (B), extraction time (C) and extracting times (D)of on the yield of crude polysaccharide. The best extract parameters was A3B1C3D3, that the extraction temperature was 80℃, ratio of herba to liquid was 1:10, extraction time was 3h, and extract times were 3 times. The compound polysaccharide content of GCPS was 48.45% measured by phenol-sulfuric acid method. The test results were stable and reliable. Phenol-sulfuric acid method was more reliable determination of the polysaccharide content of the traditional methods. This method was simple, rapid, high sensitivity and good reproducibility.2.3 The study of purification and molecular weight identification of herbal extraction polysaccharidesThe compound polysaccharide(GCPS) was obtained from the herbal extraction composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae removaled of protein by Sevag method. The three polysaccharide (GCPS-1, GCPS-2 and GCPS-3) was obtained from GCPS by precipitation, centrifugation and drying using ethanol of final concentration 50%,60% and 70%. The three major polysaccharide(GCPS-1A, GCPS-2A, GCPS-3A) derived from above polysaccharide(GCPS-1, GCPS-2 and GCPS-3) was obtained with DEAE-cellulose column chromatography respectively, which were chromatographyed with TOYOPEARL HW-55F gel column and then getting the three major components of a GCPS-1A I, GCPS-2A II, GCPS-3AⅢ. After identified by Sephadex G-200 gel column chromatography, UV spectrophotometry and paper chromatography the composition (GCPS-1, GCPS-2 and GCPS-3) was homogeneous, does not contain nucleic acid and protein impurities. The polysaccharide contents of homogeneous composition (GCPS-1, GCPS-2 and GCPS-3) was 77.6%,95.2% and 87.3% respectively. The standard curve equation is obtained from series elution volume of known molecular weight dextran Dextran:Y=-0.1734X+0.8676, R=0.9993, and measured the molecular weigh(Mr) of GCPS-1AI, GCPS-2AII and GCPS-3AIII according to the standard equation:301254, 94541 and 12453. 3 InclusionThe experimental animal model was established firstly by REV as immunosuppressive factors to study the immune enhancement of herbal extraction composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae in different immune status chickens or SPF chicken, different molecular weight extraction, extraction with different methods, different combinations of extraction and compared with levamisole hydrochloride. Further the herbal extraction was used to research the regulation of cell-mediated immunity in experimental animals. The experimental animal model was established by REV as immunosuppressive factors to determinate the immune enhancement of herbal extraction, confirme the immune enhancement of compound herbal extraction. And the active components of the herbal extraction were water-soluble and that molecular weight is between 1-10 million Dalton. However, the specific chemical composition has yet to be studied in the future.The polysaccharide as a major components of immune enhancement was isolated, identificated and determinated the molecular weight and content of uniformity polysaccharides for further study of the structure and function. The best extraction conditions of polysaccharide is investigated also.