Quality Analysis Studies On The Antineoplastic Constituents From Bugula Neritina Growing In The South China Sea

Bryostatins are a series of macrolides compounds isolated from marine animal bugula neritina, which exhibited potential antinoeplastic activities. It is the low quantity of bryostatin that limit the development of the bryostatin 1 as a new drug candidate. We have found that bugula neritina distributed widely in South China Sea. We have isolated and identified several bryostatins from bugula neritina in South China Sea including bryostatin 4, 5, 6, 8, 10, 16,17,18 and 19. Of all the eight compounds bryostatin 4, 5, 8 and 10 were the prominent constituents.A. The studies of extraction and sample pre-treatmentThe bryostatins could hardly be detected in the crude extract of bugula neritina because of not only its low content but also serious interference. Of all the extraction methods CH2C12-CH3OH(1 '. 1) together with supersonic extraction was the most efficient. We have developed a sample pre-treatment procedure prior to the HPLC assay. The crude extract was successively purified by a silica column and an ODS column. The assay results showed that the concentration of the bryostatins has been increased and a great deal of interference has been eliminated after the pre-treatment.B. The studies on assay of bryostatins by HPLCThe extracts of bugula neritina were assayed by HPLC with good peak shape and resolution. The separation was carried out on a CIS 150mm *4.6 mm i.d. column and with a mobile phase comprised of methanol and water in the ratio of 80 to 20. Bryostatin 8 and 10, whose polarity was similar, were well separated. The method had been validated. Each compound showed good linearity. The average recovery of all these compounds was within the range of 98.4%~100.9% (n=3). The RSD of inter-day accuracy was less than 1.69% and the RSD of intra-day accuracy was less than 2.09%. To further optimize the chromatographic conditions, we have studied the effect of different columns, component of mobile phase and temperature on the resolution of bryostatin 8 and 10.C. Quantitative studies on bryostatins from bugula neritina inhibiting in different regions of South China SeaWe initiated an extensive investigation of antineoplastic constituents from this animal inhabiting in three different regions of South China Sea. The investigationfocused in some main harbors in Hainan, Guangdong and Zhejiang provinces. The result of the investigation came out extremely encouraging. All the bugula neritina collected from three regions contained the antinoeplastic constituents, of which Dayawan (Shenzhen, Zhejiang province) was the best product region.D. Quantitative studies of bryostatins from bugula neritina by supercritical fluid extraction -HPLCConsidering the polarity of bryostatins, we have tried supercritical fluid extraction (SFE) to extract the bryostatins. It is necessary that the chloroform modifier was added to improve the extraction efficiency. We have studied the effect of different factors on the extraction of bryostatins. The result showed that the parameters of optimum extraction condition were: .chloroform in the ratio of 1ml to 2g raw bugula neritina; the temperature of extraction was 45℃ and the pressure was 40MPa; the volume of dynamic extraction was 15ml. The SFE resulted in good recovery and selectivity.