Identification of three light sensitive chemicals in a sponge, Phakellia flabellata, from the Central Section of the Great Barrier Reef

Samples of the sponge Phakellia flabellata were collected from the central section of the Great Barrier Reef, Australia during both winter and summer months and transported to AIMS frozen (-20°C).Three extraction and isolation procedures were adopted:Procedure 1:The finely-cut wet sponge (1 kg) was homogenised with water (1 litre) in a blender to give a fine slurry. After stirring for 48 h at 25°C, the slurry was centrifuged 5000 g, 30 min) and the residue re-extracted with water (2 x 500 mL). Combined water was removed under reduced pressure and purified on a column of Sephadex G -10. The column was eluted with deionised water and the fractions having the absorption maximum at lambda max 348 nm were freeze-dried and repeated chromatography of residue on Sephadex G-10 with water as eluent afforded the compound, Debromohymenialdisine hydrochloride, as a pale yellow amorphous solid (10 mg).Procedure 2:The wet sponge (1 kg) was extracted with refluxing acetone (3 x 1L) and after evaporation of extracting solvent, the remaining aqueous slurry was extracted with diethyl ether (2 x 250 mL) and then with n-butanol (2 x 150 mL) in a separatory funnel. The butanolic extract was suspended in methanol (150 mL) and the methanol insoluble fraction was filtered and the residue purified by repeated chromatography on Sephadex LH-20 (chloroform and mixtures of chloroform and methanol with increment of methanol) afforded the compound, Hymenialdisine, as a pale yellow amorphous solid (5 mg). Procedure 3:The finely-cut wet sponge (1 kg) was homogenised as before with 10% aqueous methanol and extracted with refluxing 10% aqueous methanol (3 x 1L). Removal of the solvent under reduced pressure gave an orange-yellow residue (80 g). The residue was further extracted with refluxing methanol. The residue (10 g) obtained after evaporation of hot methanol soluble fraction was a complex mixture (TLC) and was not investigated further. The residue (80 g) from 10% aqueous methanol was treated with 90% aqueous methanol (3 x 500 mL) at room temperature (48 h) and removal of the solvent from the soluble portion furnished a yellow residue (28 g). Purification of this residue (10 g) by repeated chromatography on Sephadex LH-20 with chloroform and mixtures of chloroform and methanol furnished two major fractions. The first fraction was an inseparable mixture of two components Debromohymenialdisine hydrochloride and Hymenialdisine. The second fraction was pre-absorbed onto Kieselgel and gradient elution with dichloromethane and mixtures of dichloromethane and methanol provided compound, Debromohymenialdisine, as a pale yellow amorphous solid (3 mg). The sponge, Phakellia flabellata, from the Great Barrier Reef was investigated for the presence of UV-absorbing metabolites. Interest in this animal focused on the fact that sponge extracts exhibit strong absorption in the UV A region. This research was part of a program searching for biologically active compounds and novel UV-absorbing compounds of lambda max 290-360 nm, for use as UV blockers in skin care preparations.

本研究从澳大利亚大堡礁中部海域采集扇形扁海绵（Phakellia flabellata）样本，分别于冬季和夏季开展采样，并以-20℃冷冻状态运送至澳大利亚海洋科学研究所（AIMS）。

本研究采用3种提取分离流程：
流程1：将1kg切细的湿海绵与1L去离子水在匀浆器中混匀，得到细腻匀浆。于25℃搅拌48h后，以5000g离心30min，残渣再用500mL水重复萃取2次。合并所得水相并经减压浓缩除去溶剂，随后通过Sephadex G-10凝胶柱进行纯化：以去离子水作为洗脱剂，收集λmax为348nm处具有最大吸收峰的馏分，经冷冻干燥后，将残渣以水为洗脱剂在Sephadex G-10柱上进行重复色谱分离，得到脱溴海膜菌素盐酸盐（Debromohymenialdisine hydrochloride），为淡黄色无定形固体，产量10mg。
流程2：将1kg湿海绵用回流丙酮（acetone）萃取3次，每次1L。蒸除萃取溶剂后，剩余水相匀浆在分液漏斗中依次用250mL乙醚（diethyl ether）萃取2次，再用150mL正丁醇（n-butanol）萃取2次。将丁醇相提取物悬浮于150mL甲醇（methanol）中，过滤除去甲醇不溶组分，随后以氯仿（chloroform）以及氯仿与甲醇的梯度混合溶剂（甲醇比例逐级递增）为洗脱剂，在Sephadex LH-20柱上进行多次色谱纯化，得到海膜菌素（Hymenialdisine），为淡黄色无定形固体，产量5mg。
流程3：将1kg切细的湿海绵按前述方法用10%甲醇水溶液匀浆，再用回流的10%甲醇水溶液萃取3次，每次1L。减压蒸除溶剂后得到橙黄色残渣（80g）。将该残渣用回流甲醇进一步萃取，蒸除热甲醇可溶组分后得到的残渣（10g）经薄层色谱（TLC）检测为复杂混合物，未做进一步研究。将前述10%甲醇水溶液萃取得到的80g残渣用90%甲醇水溶液在室温下萃取48h，重复3次，每次500mL，蒸除可溶部分的溶剂后得到黄色残渣（28g）。取该残渣10g，以氯仿以及氯仿与甲醇的混合溶剂为洗脱剂，在Sephadex LH-20柱上进行多次色谱纯化，得到两个主要馏分：第一馏分为脱溴海膜菌素盐酸盐与海膜菌素的不可分离混合物；第二馏分经预先吸附于硅胶（Kieselgel）上，以二氯甲烷（dichloromethane）以及二氯甲烷与甲醇的梯度混合溶剂进行洗脱，得到脱溴海膜菌素，为淡黄色无定形固体，产量3mg。

本研究针对大堡礁海域的扇形扁海绵（Phakellia flabellata）展开研究，旨在探寻其紫外吸收代谢产物。该海绵提取物在紫外线A（UV-A）区域具有强吸收特性，这也是本研究关注的核心要点。本研究隶属于一项旨在筛选具有生物活性的化合物以及λmax为290~360nm的新型紫外吸收化合物的项目，这类化合物可作为护肤制剂中的紫外防护剂。