| It has recently attracted considerable scientific interest and attention to promote human health by enhancing natural immune protection via the potential of bioactive compound derived from the plant, with recent advances in the hightech instruments for isolation and characterisation of plant natural products, and the developments in the biological assay systems, there has been a growing attention to the importance of plants as a source of biologically-active substances. Betulinic acid (BA) is a pentacyclic triterpene which was found in the stem bark of the plant white birch, and also in various other plants widespread in tropical regions, such as Tryphyllum peltaum, Ancistrocladus heyneaus, Diospyoros leucomelas, Tetracera boliviana, and Syzygium formosanum. BA and its derivatives have been the subject of intense studies with focuses on their anti-cancer effects, anti-HIV, anti-bacterial, anti-inflammatory, antimalarial, as well as anti-helminthic and other pharmaceutical properties. These effects may be due to their ability to modulate immune function rather than having a direct effect on infections and on cancer cells. In addition, various bioactive materials derived from plants exhibit the immunomodulatory ability. Therefore, we proposed that BA may be another valuable immunomodulator. In the present study, the immunomodulatory effects of BA on mice were demonstrated by using the purified BA.1. BA was synthesized by oxidizing betulin from the bark of the white birth with Jone's reagent, followed by selective oxidation of the resulting betulonic acid with NaBH4. IR, HPLC-MS,1H-NMR and 13C-NMR were used identified the structure of betulin and betulinic acid. To compare extraction efficiency several solvents were evaluated, also extraction time and extraction temperature were optimized, and a quantitative analysis method of BA by HPLC was formed with ethanol-water (4g/L ammonium formate) as the mobile phase on Zorbax Eclipse XDB-C8 column (4.6 mm×150 mm,5μm). The flow-rate was 1 mL/min and the UV detection was set at 210 nm, with a column temperature of room temperature. The results showed that the optimal experimental conditions were as follows:the white birch bark was extracted 3 h with methanol under reflux at 70℃, and the ratio of the bark to the extraction solvent was 15/200(g/mL), and the ratio of betulin to Jone's reagent was 1:6 and the ratio of betulonic acid to NaBH4; was 1:1. A good linearity of betulinic acid was in the range of 0.03125 mg/mL to 0.5mg/mL (Y=379381x-2397, R2=0.9988), the recovery rate was 97.97%and the RSD (n=9) was 1.64%, and the content of betulinic acid was 96.53%.2. Female mice were orally administered with BA for 14 days at the dose of 0,0.25, 0.5, and 1 mg/kg body weight. We found that BA significantly enhanced the thymus and spleen indices and stimulated lymphocyte proliferation induced by Concanavalin A and lipopolysaccharide as shown by MTT assay. Flow cytometry revealed that BAincreased the percentage of CD4+in thymus as well as the percentage of CD19+B cells and the ratios of CD4+/CD8+cells in spleens. BA increased the number of plaque forming cell and the macrophage phagocytic activity as indicated by neutral red dye uptake assay, and the peritoneal macrophages levels of TNF-a were also increased by BA. In contrast, the serum level of anti-Sheep red blood cell antibodies (IgG and IgM) and the serum concentration of cytokine (IL-2 and IL-6) were decreased significantly in BA-treated mice compared to the control as assayed by haemagglutination test and ELISA assays, respectively. At the same time, BA significantly increased GSH-Px, SOD, T-AOC, LSZ activity and decreased MDA content. Considered together, these results suggest that BA enhances mouse cellular immunity, humoral immunity, and activity of macrophages and antioxidant. Thus, BA is a potential immune stimulator and may strengthen the immune response of its host.3. To investigate immune function and antioxidant activity effects of BA on mouse peritoneal macrophages. The effects of BA on the phagocytosis capability, energy metabolism level, nitrous oxide (NO) release, the product of tumor necrosis factor-α(TNF-α), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and lysozyme (LSZ) activity were assayed by adding different concentration of BA into cultured mouse peritoneal macrophages in vitro. The results showed that phagocytosis capability was increased at the dose of 2.5～10μg/ml BA and energy metabolism level was significantly enhanced at the dose of 5～20μg/ml BA, and the NO release, GSH-Px, SOD, LSZ activity were significantly higher at the dose of 1.25～10μg/ml BA, and the product of TNF-αwas augmented at the dose of 1.25～20μg/ml betulinic acid, compared to the control group. Thus, BA is a possible immune stimulant because it can activate macrophages, enhance its oxidation resistance.4. The protective effects of BA against the Dexamethasone (Dex) induced apoptosis of murine lymphocytes were investigated in vivo and in vitro. Morphological assessment of apoptosis was performed with fluorescence microscope and transmission electron microscope microscope. DNA fragmentation was visualized by agarose gel electrophoresis. The amount of apoptotic cells was measured by flow cytometry. The results showed that in the test of in vivo, apoptotic cells in spleen and thymus of mice treated with BA and Dex (25mg/kg) were less than those in Dex treatment alone. DNA fragmentation assay showed that BA (0.25,0.5, 1mg/mL) obviously reduced Dex-induced ladder bands. Flow cytometry analysis showed that BA decreased the ratio of apoptosis, Thus, BA reduced Dex-induced apoptosis in a dose dependent manner, In the test of in vitro,5μg/mL BA reduced Dex-induced apoptosis, the protective activity of BA was decreased in a dose dependent manner (5,10,20μg/mL BA). Thus, BA can reduced Dex-induced apoptosis, and protect lymphocytes, but the protective activity of preventive was in relation to dosage of BA.
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