| Objective: To study the immunological activity of Rehmannia glutinosa Libosch water extract (RGLE) in BALB/c mice. The present study was conducted to: screen out the best one of RGLE from three producing areas with two decoctions in five concentrations, that stimulates proliferation of spleen cells, prove the effect of RGLE on IgM production response by using plague forming cell (PFC) assay and examine the production of cytokine TNF-α from macrophages using biological test in vitro; determine the production of IgG from B cells in vivo; analyze the chemical ingredients of RGLE by sieve chromatography. Methods: 1. Preparation of RGLE 100g Dried Radix Rehmanniae (DRR) and Radix Rehmanniae glutinosa Libosch (RRGL) from three producing areas were taken and marinated in 1000ml distilled water respectively. One night later, all above the traditional Chinese medicine were boiled with mild fire for 60 minutes and filtered as the first decoction (10%). As the same process for the second decoction (10%). All decoctions were double diluted with distilled water into the proportion of 5%, 2.5%, 1.25%, 0.625% and sterilized, then stored at 4℃. 2. Lymphocytes proliferation test 1x106/ml of spleen cells from mice were prepared in RPMI 1640 medium containing 10% FCS. It was incubated with 5μg/ml of ConA or 5μg/ml of LPS and twelve decoctions in five concentrations respectively in 96 well culture plate under condition of 5% CO2, 37℃ for three days. Lymphocyte proliferation were measured by MTT colorimetry technique at 492nm and the rate of proliferations were calculated to screen out the best decoction. All results were repeated for three times. 3. Plaque forming cell assay Slide room were prepared withdegrease slides. Sterile and defibrin SRBC was washed with saline and made up into 15% suspension. Serum from over three cavies was diluted by 1:3 with Hanks. Preparation of immuned spleen cells suspension: sixteen mice were divided into two groups randomly: the treatment group and control group. The treatment group were femented decoction of RGLE 5000mg/(kg*d) (for 8d). The control group were femented equal quantity water for 8d. Four days before test ip 0.4ml 5% fresh SRBC to sixteen mice respectively. The eighth day (the fourth day after being immuned), all the mice were killed and spleen cells suspensions were made up. 1x106/ml spleen cells suspension 20μl, 15% suspension of SRBC 50μl, 1:3 diluted complement 50μl, and Hanks 180μl were mixed up and affused into slide room. They were incubated at 37℃. 1.5 hours later, they were taken out. The amount of plaques was counted. 4. Test for determining activity of monocyte Suspension of spleen cells was taken into plastic plate and incubated for two hours at 37℃. Adherent cells was harvested and made up into 1x106/ml. Then adherent cells suspension was incubated with 2.5μl, 5μl, 10μl, 20μl of RGLE and 10μl LPS (positive contrast) and 10μl PBS (negative contrast) respectively in 96 well culture plate at 37℃. 24h later, the supernatant was harvested for using. L929 cells were cultivated and made up into 2x105/ml and incubated in 96 well plate at 37℃ for 24h. The supernatant was discarded. Prepared supernatant 50μl was diluted with medium containing 50μl actinomycin D (final concentration as 1μg/ml) and put into 96 wells containing L929 cells. Incubated at 37℃ for 24h. The absorbances were read at 492nm by MTT colorimetry technique. TNF-α content was expressed as units comparing with recombinant human TNF-α. 5. Experiment in vivo Sixteen mice were divided into two groups randomly. The treatment group were femented decoction of RGLE 5000mg/(kg*d) (for 9d). The control group were femented equal quantity water for 9d. After 9 days, all mice were killed and taken their blood and spleen. The spleens were weighed and made up to cell suspension. Lymphocytes proliferation was determined by MTTcolorimetry technique. The content of IgG of mice serum were determined by using single diffusion method. 6. Analysis of chemical ingredients of RGLE 1ml R...
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