Annual variation and cellular source of renieramycins from the blue sponge Xestospongia sp.

Abstract

The Thai blue sponge Xestospongia sp. was found to produce large amount of the highly potent cytotoxic bistetrahydroisoquinolines, named renieramycins. Pretreatment of the sponge with potassium cyanide before extraction yielded a mixture of stabilized renieramycins, of which renieramycin M was predominant. The purpose of this study was to determine the annual variation in quantity and to localize the cellular source of renieramycin M within the sponge. The sponge Xestospongia sp. samples were monthly collected from Sichang Island, Cholburi Province, in the Gulf of Thailand during August 2005 to July 2006. A high performance liquid chromatography (HPLC) method, which was developed to analyze renieramycins, consisted of a RP-18e column (125×4 mm, 5 μm), the isocratic solution of methanol/water (7:3) as the mobile phase at flow rate 1.0 mL/min, a photodiode array detector at wavelength 270 nm and acenaphthene as the internal standard. The HPLC method was reliable in terms of linearity (R² = 0.9999), accuracy (%recovery of 103.10-107.14%), and precision (%RSD of 0.39-0.95%). The HPLC analyses showed that renieramycin M was normally detected as the major component at retention time 8.9 min and the annual variation of renieramycin M quantity (%w/w, semi-dried weight sponge) was significantly different (ANOVA, p < 0.05) with the average of 0.13%. However, the sponge produced the highest amount of renieramycin M at 0.20% w/w in April and October and the lowest at 0.08% w/w in January and at 0.09% w/w in July. Following the Pearson correlation coefficient (r), the relationships between renieramycin M annual content and other environment factors were found to have the positive correlations with total dissolved solids and chlorophyll-a. Fractionation of the sponge cells was performed by differential centrifugation and Ficoll density gradient centrifugation. The result showed that choanocytes and archaeocytes were the major cell types of the sponge. The HPLC analyses of cell fractions demonstrated that renieramycin M was associated with both sponge cells but not with the bacterial fraction