Structure analysis of complex carbohydrate exhibiting IL-12 inducing activity derived from mineral water in natural leaf soil

Mineral water, named Catalyser 21, prepared by the fermentation of ancient natural leaf soil, is often used as health-beneficial drinking water to enhance the immune system of cancer patients. The mineral water was dialyzed and fractionated by using gel chromatography. Fraction 4, containing carbohydrates, was further purified by using negative ion chromatography, obtaining a fraction named Fr4 (1-2). The Fr4 (1-2) was analyzed using the ABME marker method, carbohydrate analysis. It was composed of nine carbohydrates. The NMR spectra showed some specific complex carbohydrates pattern in the 1H-NMR and 2D-NMR HMQC methods. There were many cross peaks in the HMQC spectra, which displayed 13C-1H relationships of the anomeric region of complex carbohydrates Macrophages secrete interleukin-12 (IL-12), which activate the tumor immune system. The induction activity of the Fr4 (1-2) for IL-12 on the murine macrophage cell line, RAW264.7, was tested using the RT-PCR method. The Fr4 (1-2) induced the gene expression of IL-12, suggesting that polysaccharides in mineral water are responsible for the IL-12 induction activity.