Study On Immunological Activity Of Dianthrone In Hypericum Perforatum And Development Of Dianthrone Albumin-IgG Antibody Conjugates

Hypericum extract contains numerous active compounds, each type of them have different pharmacological effects. Dianthrone is one of active compounds of Hypericum extract, including hypericin, pseudo hypericin, the original of hypericin, the original pseudo-hypericin, Central hypericin, etc. Dianthrone has many pharmacological effects such as anti-inflammatory, anti-depressants, anti-tumor and antiviral activity. There are several problems during the reasch,1. The anti-virus mechanism of Dianthrone is not clear.2. The low bioavailability of active ingredient.3. Site of action in the body is unknown. To the problems of the anti-virus mechanism, the roles of dianthrone on the immune organs, enzymes and cytokines were studied, the effects of dianthrone on the immune network were discussed, the conclusion of dianthrone antiviral mechanism was to be drawn, and which was to enhance the body's immunity. To increase the bioavailability, dianthrone was encapsulated in biodegradable materials bovine serum albumin (BSA) improving the bioavailability and effective content of dianthrone and also providing ideas for other similar active ingredients research. To clear the site of action in the body, dianthrone albumin clathrate and IgG antibodies were conjugated successfully, initial detection were conducted to provide a theoretical basis for targeted drug delivery systems. The research works were performd as follows.1. Effects of dianthrone on immune function and antioxidant capability in mice. The thymus index, the spleen index, the activities of six enzymes(T-AOC,T-SOD,MPO,XOD,GSH-PX,POD), and the level of MDA, H2O2and NO were measured. Histopathological sections of immune organs have been observed. The following conclusions to be drawn: dianthrone had a functions to increase the spleen index, the level of T-SOD, T-AOC, GSH-PX and POD, decrease the level of MDA, NO and XOD, have no influence on the level of H2O2. The findings suggested that dianthrone could play an antioxidation activity by increasing spleen index, decreasing the level of MDA and NO, regulating activities of enzymes involved in production and elimination of free radicals in mice. The results of histopathological sections of immune organs showed that dianthrone has a repair function on the immune suppressed mice liver and spleen, and have a certain recovery on the thymus, but the effect is not obvious.2. Effects of dianthrone on T cell surface film molecules and cytokines in serum in mice. The T cell surface film molecules(CD3,CD4,CD8,CD4/CD8) and cytokines (IL-4,IL-6,IL-10,IFN-γ,TNF-α) in serum were measured by ELISA method. The following conclusions to be drawn:dianthrone had functions to increase the T cell surface film molecules(CD3, CD4, CD8, CD4/CD8) and cytokines (IFN-γand TNF-α) significantly, decrease level of IL-4, IL-6, IL-10. The findings suggested1dianthrone can increase the number of mature T cells, promote T cells to release cytokines, and regu the activity of Thl cells and Th2cells to increase the body's immune function. 3. Effects of dianthrone on cAMP/cGMP signaling pathway in rats. The level of cAMP and cGMP were measured by ELISA method, and the ratios of cAMP and cGMP were calculated. The following conclusions to be drawn: dianthrone had functions to increase the level of cAMP significantly, and decrease the level of cGMP and the ratios of cAMP and cGMP. The findings suggested that dianthrone can enhance the differentiation and proliferation of B lymphocytes, and promote the antibody generated to enhance the humoral immune function.4. Effects of dianthrone on hypothalamic-pituitary-adrenal axis in rats. The level of ACTH and CORT were measured by ELISA method. The following conclusions to be drawn: dianthrone had functions to decrease the level of ACTH and CORT significantly. The findings suggested that dianthrone can decrease the level of ACTH and CORT to promote T cell transformation, reduce the hypothalamic-pituitary-adrenal axis hyperactivity to weak the dysfunction of hypothalamus, pituitary, adrenal and the effects of immunocompromised caused by HPA axis hyperactivity.5. Effects of dianthrone on Neuroendocrine-immune network-related factor in rats.The level of IL-1, DA, NA and5-HT were measured by ELISA method, the following conclusions to be drawn: dianthrone had functions to decrease the level of IL-1, DA, NA and5-HT significantly. The findings suggested that dianthrone can affect the role of vasopressin on adrenal gland, reduce the GC release to alleviate the inhibition of the immune process. Or affect the level of ACTH in pituitary by reducing the level of Adrenal cortical hormone to alleviate the immunosuppressive. Or inhibition the function of B cells and enhanced the by increasing the level of DA torecover the immune function.6. Preparation of dianthrone-loaded bovine serum albumin (BSA) clathrate. Used a desolvation-crosslinking method to prepare clathrate, and the prescription process was optimized. Its morphology, particle size distribution and potential distribution were estimated, the yield, encapsulation efficiency and drug loading were determined, the profile of in vitro drug release were assessed. The conditions for dianthrone-loaded bovine serum albumin clathrate preparation were optimized: the amount of albumin was1.5g, crosslinking time was24h, the amount of glutaraldehyde was15μL, pH was9, the dropping speed of ethanol was1.0mL/min. The surface morphology of the clathrate possessed smooth surfaces and spherical without any agglomeration. The mean size of the clathrate was122nm, and the zeta potential of the particles was greater than-50mV. The yield, encapsulation efficiency and drug-loading of the nanoparticles were95.5%,71.43%and4.13%, respectively. The drug release rate was greater than50%of entrapped in the BSA nanoparticles being released within4days, was10%-15%at4d to10d, and little change in drug concentration at10d to the25th day. The desolvation-crosslinking preparation method was a suitable preparation method for dianthrone albumin nanoparticles, the clathrate with a regular and uniform shape, high stability, yield, encapsulation efficiency and drug loading, and excellent controlled-release characteristics.7. The conjunction of dianthrone albumin clathrate with IgG antibodies. Dianthrone albui clathrate and IgG antibodies were conjugated by glutaraldehyde method and optimized the conjunct conditions. The results were detected by SDS-PAGE electrophoresis. The conditions for dianthrone albumin clathrate-IgG antibody conjugates preparation were optimized: IgG antibodies and clathrate in the proportion of1:1, amount of Glutaraldehyde were1/4of the total volume of solution. Coupling time were2h, temperature were25℃and pH were neutral. Dianthrone albumin clathrate-IgG antibody conjugates were detected by SDS-PAGE electrophoresis. The results showed that dianthrone albumin clathrate-IgG antibody conjugates had a band near the210kDa, IgG antibody had a band near the150kDa, and dianthrone albumin clathrate had a band near the66kDa. The results were consistent with the theoretically molecular weight of IgG antibodies and dianthrone albumin clathrate. The glutaraldehyde method was a suitable preparation method for dianthrone albumin clathrate-IgG antibody conjugates.