| Natural product is an important source for drug. Previous researches indicated the close relationship among oxidative damage, immune imbalance and AIDS which suggested that antioxidants might play an important role in the treatment of AIDS. In this study, antioxidation was the breakthrough point to select natural ingredients from Nelumbo nucifera Gaertn. The macroporous resin named NKA was used for chromatography of the aqueous crude extract L. Fraction L2 showed the highest antioxidative activity in the three ingredients named L1, L2 and L3 from L. Chromatographed on Sephadex LH-20, L2 was separated into seven fractions named L2a to L2g. Antioxidative ingredients L2c and L2f were collected from them. High Performance Liquid Chromatography was applied to purify L2c-3 from L2c, while L2f-2 and L2f-3 from L2f. Identified by MS, FTIR and NMR, L2c-3 was tryptophan, L2f-2 was (+/-)-gallocatechin and L2f-3 was (-)-catechin. The inhibitions of L2c-3, L2f-2 and L2f-3 were 7539%,80.26% and 79.09% on erythrocyte hemolysis at 250μg/ml. L2f-2 and L2f-3 exhibited strong antioxidative activities on lipid peroxidation. The inhibitory rates were 75.42% and 74.48% at 250μg/ml. Furthermore, L2f-2 could eliminate superoxide anion and hydroxy radical with the IC50 values of 126.2μM and 102.0μM, while the IC50 values of L2f-3 were 204.8μM and 234.4μM. The crude aqueous extract L was precipitated with 50%(v/v) and then 67%(v/v) ethanol to collect fractions LA and LB. Antioxidative fraction LB2 was extracted from LB on Sephadex-G75 column. LB2 is a polysaccharide-protein complex with a molecular weight of 18.8 kDa. HPLC data showed that LB2 was composed of mannose, rhamnose, glucose, galactose and xylose with a mol ratio 2:8:7:8:1. LB2 was identified as a polysaccharide sulfate containingα/β-pyranoid and a-furanoid monose by FTIR. The antioxidative capability of LB2 on erythrocyte hemolysis assay was 72.2%(125μg/ml) and 83.2%(250μg/ml) on lipid peroxidation.HIV-1 integrase (IN) is a critical enzyme of HIV-1. It is an ideal target for anti-HIV therapy because previous researches have not found any functional integrase analogue in human bodies. But the agents against integrase were developed so slow that there was only one drug against IN named Raltegravir approved by FDA in 32 anti-HIV drugs. Proper screening model is the most important of drug development. In the present studies, two novel drug screening models named 3'-process model and double-plasmid model were established for IN inhibition screening. Two mutant sites F185K and C280S were introduced into cDNA of IN, which could improve the solubility of IN. In the 3'-process model, soluble IN was purified by NTA-Ni2+ from the lysate of pET-IN (BL21) induced by IPTG. Recombinant IN was identified by Western Blot. It showed high 3'-process activity on substrate plasmid pLTRs to make them linearization in vitro. The ratio of linear DNA to total DNA reflected the activity of IN. LB2 exhibited the highest inhibitory activity on IN with an IC50 value of 5.28μM. The inhibitory activities of L2f-2 (IC50: 412.45μM) and L2f-3 (IC50:432.40μM) on IN were higher than that of Raltegravir (IC50:686.17μM). Double-plasmid screening system contained two recombinant plasmids named pET-IN and pLTR in E.coli BL21. IN expressed by pET-IN acted on pLTR in bacteria which could inhibit their growth and samples with potential IN inhibition could recover the growth of bacteria. LB2 showed higher inhibitory rate (47.48%) on IN than Raltegravir (42.92%) at 10μM. The inhibitory rate of L2f-3 on IN was 48.78% at 100μM. The survival rates of splenic cells incubated with 1mM L2f-3 and LB2 were 100% when tested with MTT method, which exhibited that L2f-3 and LB2 did not show any cytotoxicity on normal cells. Raltegravir showed higher cytotoxicity than L2f-3 and LB2. Only 20% cells survived after incubated with 1mM Raltegravir.It was the first time that multiple effects of compounds from Nelumbo nucifera Gaertn were studied. HIV-1 reverse transcriptase (RT) and protease (PR) are other two critical enzymes of HIV-1. L2f-2, L2f-3 and LB2 could strongly inhibit RT with the IC50 values of 10.41μM,9.58μM and 33.70μM. The inhibitory rates of L2f-2 and L2f-3 on PR were 17.61% and 15.66% at 70μg/ml, while the inhibitory rate of Pepstatin A (standard PR inhibition) was only 7.36%. Immune reconstitution of HIV-infected patients was the most important part of immune function recovery. Cytokines were critical regulatory factors and played important roles in immune system. Real-time PCR was used to test the expressive levels of cytokines. The results demonstrated that both L2f-2 and L2f-3 could up-regulate the gene expressive levels of IL-2 and IL-6, while down-regulate IL-10. These regulations would be helpful to increase the organic immunity and balance immune system. Moreover, L2f-3 could inhibit HIV-1 directly by down-regulating TNF-αand inhibiting the activation of Tat on LTR. LB2 exhibited positive regulation on IL-2, IL-4 and IL-10. It could improve the inhibitory state of cytokines in HIV-1 sufferer and balance the immune system.In the present researches, two novel IN inhibitor screening models were established for quick and large scale drug screening. Natural products from Nelumbo nucifera Gaertn were examined in antioxidation, inhibition on the key enzymes of HIV-1 and immune regulation. Most chemically synthetic HIV-1 inhibitor could not deal with mutant virus. Natural products with multiple anti HIV-1 effects would overcome this disadvantage. Therefore, this research is significant for anti HIV-1 drug development and application of natural products to HIV-1 therapy. |
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