In Vitro Analysis of Immune+ from Chinese Herbal USA, Inc. by the School of Medicine at University of California, Davis:
Treatment Preparation: Ten mg of Immune+ was suspended in 1 ml RPMI-1640 with 10% heat inactivated fetal bovine serum (media). The suspension was shaken vigorously on a vortex mixer. The insoluble components were quickly pelleted and the remaining suspension filtered twice: first through a 0.45-micron syringe filter followed by a 0.2-micron syringe filter. This filtrate was used as our stock (approx. 10 mg/ml) from which dilutions were made for the incubation with PBMC. The stock solution was diluted in media prior to treatment with PBMC.
PBMC Isolation: Peripheral blood from healthy volunteers was collected and the peripheral blood mononuclear cells (PBMC) were isolated over a density gradient via centrifugation. The cell were resuspended in RPMI-1640 containing 10% fetal bovine serum and supplemented with 0.1% of a 50-mg/ml gentamicin solution (Gibco BRL). PBMC concentration was adjusted to 2 x 10 (6) viable cells/ml after estimation of viability by trypan blue exclusion assay. Viability was consistently greater than 96%.
Culture of PBMC with Immune+: Five hundred ml of a 1.0 x 10 (6) cell suspension were cultured with equal volume of the Immune+ treatments at 37/C with 5% CO2 in 48-well plates. PBMCs were exposed to a final concentration of 100, 10, 1, 0.1 mg/ml of Immune+. In addition, the Immune+ was incubated in the presence of PHA at 10 mg/ml. Culture supernatant fractions were harvested after 72 hr and were stored at –20/C until analysis by ELISA.
ELISA Analysis of Cytokines: Levels of IL-1b and IFN-g were measured in supernatant from 1.0 x 10 (6) cells/ml stimulated with the herbal products in the presence or absence of PHA. Standard ELISA kits were used to quantitate IL-1b and IFN-g (R&D Systems, Minneapolis, MN) with detection limits of 3.9 and 15.6 pg/ml respectively.
Results: Immune+ considerably augmented production of IL-1b at the highest concentration (100mg/ml); showing a 413+/-102 percent increase, when compared to media control. At a treatment of 10mg/ml, Immune+ moderately enhance IL-1b secretion (44+/-18%). In our analysis of IFN-g, Immune+ , as well as media control were unable to induce its protein secretion. However, PHA control stimulated production of IFN-g to a concentration of 866=/-286 pg/ml. Importantly: In the presence of PHA, Immune+ at 0.1,1,10, and 100 mg/ml cause a –28+/-17, 15+/-34, 36+/-33, and 91+/-35 percentage changed from PHA control.
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