|Budget Amount *help
¥7,800,000 (Direct Cost : ¥7,800,000)
Fiscal Year 1997 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1996 : ¥5,400,000 (Direct Cost : ¥5,400,000)
Forskol in, a labdane diterpenoid, was isolated from the tuberous roots of Coleus foreskohlii Briq. (Lamiaceae). C.forskohlii has been used as an important folk medicine in India, and has been found to be a potent activator of adenylate cyclase, leading to an increase of c-AMP.
The full measuring range of the assay extends from 5ng/ml to 5mug/ml of forskolin.
The concentration of forskolin in the stem base (4.04mug/mg dwt) exceeded that found in both the upper stem (1.58mug/mg dwt) and near the shoot tip (0.34mug/mg). This tendency to accumulate forskolin near the base stem was observed in individual branches, except for the lowest branch base which contained the highest amount of forskolin (5.97mug/mg dwt). In order to confirm the purity of the IgG.the MALDI mass spectrum was measured.
The molecular weight was 148600, which is in good agreement with that of human IgG being detaemined as 146000.
Since forskolin was almost insoluble in water, various buffer solutions were tested for the dis
solution of forskolin. It became evident that 6% of CH_3OH in PBS was necessary to dissolve forskolin. The forskolin concentrations eluted increased rapidly from 20% CH_3OH to the maximum at 45%. The crude diethyl ether extracts od tuberous root of C.forskohlii were loaded on the immunoaffinity column, washed 5 times with PBS containing 6% of CH_3OH,and eluted with PBS containing 45% CH_3OH.Fractions 2 to 8 conteined 45 mug of forskolin, which is greater than the column capacity, together with the related compounds, 1-deoxyforskolin, 1.9-dideoxyforskolin, 7-deacety forskolin and 6-acetyl-7-deacetyl forskolin, and other unknown compounds which were deteced by TLC.
The peak-of fractions 26 to 30 shows the elution of forskolin (21mug) eluted with the PBS containing 45% CH_3OH.Forskolin eluted by the washing solution (fraction 2 to 8) was repeatedly loaded and finally isolated. However, forskoklin purified in this way by the immunoaffinity column was still contaminated by a small amount of 7-deacetyl forskoklin because this compound has 5.5% of cross-reactivity against MAb as previously indicated. Therefore, the mixture was treated with pyridine and acetic anhydride at 4ﾟC for 2h to give forskolin. Less