Study On The Preparation And Immunoenhancement Of CHBG

Traditional Chinese medicine(TCM)for tonifying qi has unique effects on improving immunity of normal animals and restoring the immunity of immunosuppressed animals.In this study,Two kinds of medicinal herbs for tonifying the Astragali Radix and Atractylodis Macrocephalae Rhizoma were mixed together to prepare compound huangqi&baizhu granules(CHBG)by water extraction and alcohol precipitation,granulation,drying,and other processes.In order to acquire a new veterinary immunopotentiator,the studies of pharmaceutics,toxicology and pharmacodynamics were carried out.Then the experimental results are summarized below:1.Screening proportion Astragali Radix and Atractylodis Macrocephalae Rhizoma in CHBGIn order to obtain CHBG having better curative effect,chicks were used as experimental animals,and granules prepared in different proportions of Astragali Radix and Atractylodis macrocephalae were used as experimental drugs,at the same time vaccine immunization control group was set up.The compatibility ratio of Astragali Radix and Atractylodis macrocephalae was screened by investigating the effect of experimental drugs on ND vaccine antibody level.The results showed that when the ratio of Astragali Radix and Atractylodis macrocephalae was 1:1,ND antibody level in chicks was significantly higher than that in vaccine-immunized control group(P<0.05),and it’s ND antibody level was the highest among all the drug test groups.The results indicated that when the ratio of Astragali Radix and Atractylodis macrocephalae was 1:1,the granule had better immune enhancement effect,so CHBG was prepared on the basis2.Preparation and stability of CHBGIn order to prepare GHBG with stable and controllable quality,the quality of Astragali Radix and Atractylodis Macrocephalae Rhizoma were determined by TLC and HPLC.On this basis,the extract from Astragali Radix and Atractylodis macrocephalae was prepared by Ca(OH)2 basic water and alcohol precipitation.CHBG could be obtained by adding excipients,wet granulation and drying,and then the CHBG’s stability was measured by accelerated testing and long-term testing.The results showed that:(1)In the TLC of Astragali Radix and Atractylodis macrocephalae,the same fluorescent spots of the samples and their corresponding control herbs were displayed at the same position.The similarity between the characteristic chromatograms of the two herbs and the corresponding control herbs were above 0.90,which indicated that the two herbs selected were qualified.(2)The average yield of polysaccharides in three batches was 7.58%,and the average content of polysaccharide was 73.83%.Each 1 gram of the finished product corresponded to 2 grams of the crude drug.(3)While CHBG was sealed in a commercial aluminum-plastic package and was placed in the environment with 40±2 0C,75±5%relative humidity for 6 months and in the environment with 25±2℃,60±10%relative humidity for 18 months,but its appearance,identification and content and other quality indicators all were in line with the requirements of CHBG quality standard.The results indicated that CHBG prepared by extracting with Ca(OH)2,alcohol precipitation,spray drying and wet granulation was stable,and its term of validity could be up to 18 months.The quality standard established could be used to control for quality of CHBG.3.Quality standards of CHBGIn order to control the quality of CHBG,the quality standard of CHBG was established by using Pharmacopoeia method and TLC and HPLC methods.The results showed that:According to the Chinese Veterinary Pharmacopoeia,the determination method of CHBG content was established,and the inspection items of CHBG,such as appearance,water content,granularity,solubility and microbial limit were determined.The methods of qualitative identification of polysaccharides,Astragaloside iv and Atractylodis macrocephalae were established by TLC;The methods for the determination of monosaccharides and disaccharides in CHBG by HLPC were established according to Chinese Veterinary Pharmacopoeia,and the detection limits of monosaccharides and disaccharides were specified.At the same time,specific chromatograms of 9 batches of CHBG were investigated by HLPC,and the fingerprint of CHBG was established.The results indicated that the quality standard established in the experiment could be used for the quality control of CHBG.4.Safety evaluation of CHBGIn order to evaluate the safety of CHBG and provide the reference for security of drug usage in clinical practice by the methods of Modified Kohl’s or maximum tolerance dose(MTD).The LD50 of CHBG orally administered to mice could not be determined by modified Karber’s method,however the MTD of CHBG in mice was 180 g/(kg·bw)measured by the MTD test.In addition,there was no significant change in body mass and organ index in mice at MTD.The results indicated that CHBG is safe for experimental mice.5.Immunoregulatory effect of CHBGIn order to investigate the immunoregulatory effect of CHBG on immunodeficient animalsmice was used as experimental animals in the study,and cyclophosphamide(Cy)was used to make the immune suppression model,5 groups including blank group,Cy group,Cy+CHBG3.75 group(Note:the dosage is 3.75 g/(kg-bw),the same below in this section),Cy+CHBG7.5 group and Cy+CHBG15.o group were set up respectively,and the effects of CHBG on immune organ index,carbon clearance function,spleen lymphocyte proliferation,serum lysozyme and cytokines such as TNF-α、IL-2、IL-4 and IFN-y in immunosuppressed mice were investigated.Meanwhile,blank group,Astragalus Polysacharin(APS)group,CHBG3.75 group,CHBG7.5 group and CHBG15.0 group were set up to determine the effect of CHBG on spleen lymphocyte subsets of normal mice by flow cytometry(FCM).The results showed that,(1)Cy treatment could decrease spleen index and thymus index,inhibit the proliferation of spleen lymphocyte,and decrease the levels of serum lysozyme and TNF-a,IL-2,IL-4,IFN-y Compared with the blank group,the levels of spleen index,serum lysozyme content and IFN-y decreased significantly(P<0.05),which indicated that Cy treatment caused significant immunosuppression.(2)Compared with the Cy group,CHBG15.0 could significantly increase the spleen index,the content of IFN-γ(P<0.05),and extremely increase the content of serum lysozyme(P<0.01).CHBG7.5 could extremely increase the spleen index,thymus index and the content of serum lysozyme(P<0.01).CHBG3.75 could significantly restore and promote the proliferation of spleen lymphocytes and the content of IL-4(P<0.05),and extremely increase the spleen index and the content of serum lysozyme(P<0.01).All dose groups of CHBG could increase the clearance index and phagocytosis index of carbon particles,but the difference were not significant(P>0.05).CHBG had no significant effect on TNF-a and IL-2.(3)Finally,CHBG3.75 could observably reduce the number of CD8+T cells in normal mice(P<0.05),and markedly increase the ratio of CD4+/CD8+(P<0.05).In addition,The number of CD4+T cells and the ratio of CD4+/CD8+in CHBG15.0 group were significantly lower than those in APS group(P<0.05),while the number of CD8+T cells in CHBG15.0 group was significantly higher than that in APS group(P<0.05),but The number of CD4+T cells,CD8+T cells and the ratio of CD4+/CD8+ in CHBG3.75 group and CHBG7.5 group were not significantly different from those in APS group(P>0.05).The results indicated that CHBG could restore the inhibition of immune organs inhibited by Cy,enhance proliferation of splenic lymphocyte induced by Con A,enhance the phagocytosis function of monocyte macrophage and promote lysozyme releasing,induce to produce IFN-y and increase the content of IL-4 in serum,and increase the ratio of CD4+/CD8+T lymphocyte cells.6.Effect of CHBG on Immunization Efficacy of Newcastle disease(ND)vaccineIn order to explore effect of CHBG on the immune efficacy of ND vaccine,we investigated the effects of different doses of CHBG on ND antibody level in chicks by oral doses of 0.5,1.0,2.0,3.0 and 5.0 g/L for 7 days after each immunization.Then we investigated the effects of different therapeutic schemes of CHBG on ND antibody level in chicks by three therapeutic schemes:continuous administration throughout the period of immunity,administration for one week after the 1st and 2nd immunization continually,and administration for one week after the 1st immunization.The results showed that:(1)when the chicks were given CHBG by different doses,compared with the vaccine immunization control group,CHBG1.0(Note:the dosage is 1 g/L for drinking water,the same bellow in this section)could increase the antibody levels of ND significantly on the 1st and 2nd week after the 1st immunization(P<0.05),and it could increase the antibody levels of ND extremely on the 1st and 2nd week after the 2nd immunization(P<0.01).Compared with the APS group,the level of ND antibody in the CHBG5.0 group at the 35th day was significantly lower than that of the APS group(P<0.05),but there were no significant difference between the other groups and the APS group(P>0.05).(2)when the chicks were given CHBG by different administration schemes,among these schemes,the scheme of 1.0 g/L dose of CHBG administered for 7 days continuously by drinking water after each immunization was better,because it could increase ND antibody level significantly on the 1st and 2nd week after the 1st immunization(P<0.05),and it could increase the antibody levels of ND extremely on the 1st and 2nd week after the 2nd immunization(P<0.01),and it also could save costs.In summary,CHBG prepared by using this method was safe and stable,and CHBG quality standard established in this study could be used for the quality control of CHBG because of the detection method is simple and feasible.It was confirmed that CHBG not only improved the non-specific immunity of chicks,but also enhanced the specific immunity of chicks.In addition,this study provided a therapeutic scheme of CHBG,namely,the drug at the dose of 1.0 g/L administered for 7 days continuously by drinking water after each immunization,which would be helpful to enhance the level of ND antibody in chickens and provide more protection for the chicks.