PHYCOCHEMICAL STUDIES OF EIGHTEEN SPECIES OF RED ALGAE FROM THE COAST OF KARACHI

Eighteen commonly occurring species of marine benthic algae i.e. Asparagopsis taxiformis (Delile) Trevisan, Bangia atropurpurea (Roth) C. Agardh, Centroceras clavulatum (c. Agardh) Montagne, Calliblepharisfimbriata (Greville) Kützing, Coelarthrum muelleri (Sonder) PǾgesen, Cottoniella filamenotsa (Howe) BØorgesen, Gracilaria .froliifera (Forsskål) BØorgesen, Halymenia porphyriaeformis (BØorgesen) Parkinson, Hypnea musciformis (Wulfen) Lamouroux, Hypnea valentiae (Turner) Montagne, Laurencia obtusa (Hudson) Lamouroux, Melanothamnus somalensis. Bornet et Falkenberg in Falkenberg, Porphyra vietnamensis Tanaka et Pham-hoáng Hô, Sarconema filiforme (Sonder) Kylin, Sebdenia flabellata (BØorgesen) Parkinson, Scinaia .fascicularis (BØorgesen) Huisman, Scinaia hatei BØorgesen and Solieria robusta (Greville) Kylin have been collected from the coastal areas near Karachi, Pakistan. These red algae, which belong to 13 families and 6 orders being distributed under both the classes of Rhodophyta, have been investigated taxonomically as well as phycochemically. All the investigated seaweeds are taxonomically known species. During this study Melanothamnus somalensis has been reported for the first time from this region and Asparagopsis taxiformis, Bangia atropurpurea, Cottoniella filamentosa, Gracilaria foliifera, Halymenia porphyriaeformis, Melanothamnus somalensis, Sarconema fi/!{orl17e, Sebdenia flabellata, Scinaia .rascicularis and Solieria robusta taxonomically described for the first time from the coast of Pakistan.A variety of saturated and unsaturated fatty acids were detected in the extracts of these algal species and analysed as methyl esters qualitatively as well as quantitatively through GLC and GC-MS techniques. Apart from that several sterols, a few fatty acid derivatives, carbohydrates, glycosidic and chamigrene compounds have been detected and isolated from these seaweeds, purified and their structures elucidated chemically through standard chemical methods, e.g. IR & UV, EI-, FAB-, FD-, GC- & HR-MS and 1H- & 13C-NMR. Altogether 26 different fatty acids (12 saturated & 14 unsaturated), I fatty-acid derivative, I fatty alcohol, 18 sterols, 2 carbohydrates, 1 glycoside and I chamigrene have been extracted from them. All natural products described, have been reported for the first time from the corresponding algal species. Certain new and novel compounds have been isolated from them: e.g 7-hydroxy-tctrmlcc-4-cnoic acid from Hypnea valentiae, foliiferol (2-mcthyl-trincont-5, 10, 16,22,27-pcntncn-1S-oJ) and foliiferoside (G-O-dodecyl-a-D-glucpyranosyl-(1→2)-β-D-fructofuranoside) from Gracilaria foliifera, somalenone from Melanothamnus somalensis, cholesta-S,20,24-trien-3-3-01, ergost-S-en-3,25-diol and lanostanol from Scinaia .fascicularis, cholestan-3B-ol from Sarconemafiliforme, asparasterol and taxiforminone (4,10-dibromo-3-chloro-7-3,8-3-epoxy-a.-chamigrene) from Aspcragopsis taxiformis, 7-oxocholesterol from Halymenia porphyriaeformis and Melanothamnus somalensis. Some of the extracted compounds are new to the chemical world and were not known previously from any living source, e.g. a C26 sterol: Somalenone (cholest-S-en-3,7-dione) and a C27 sterol: Asparasterol (3-oxocholesta-4,23- dien-2-23-01).The saturated fatty acids were present in overwhelming quantity (56-86%) and this tendency was exhibited by 18 seaweeds of both the classes. The fatty acid composition of the investigated seaweeds varied not only from order to order but also from species to species, and no generalization may be made. The investigated species also behaved differently in tee diversity of their fatty acid compositions. Bangia atropurpurea appeared to be the least diverse species with seven fatty acids only, while Halymel1ia porphyriaeformis exhibited the greatest diversity with 16 fatty acids. Porphyra vietnamensis and Centroceras clavulatum were the next diverse seaweeds with IS fatty acids followed by Scinaia hatei and Melanothamnus somalensis with 13 fatty acids. Palmitic acid appeared to be the most predominant fatty acid. It was not only detected in all the investigated species but was found to be present as the predominant fatty acid in nine species. Pentadecylic acid was also detected in all the investigated species but was present in three species as a predominant fatty acid.The saturated fatty acids showed a range between C 12 and C25 chain lengths, except C21:0 and C24:0 all other 12 acids of this range investigated species. Pentadecylic (C 15 :0) and palmitic (C 16:0) commonly occurring saturated acids, as they were found in Myristie (C 14:0) and margaric (C 17:0) acids were the ext cc were present in 16-17 species. Pentacosanoic acid (C25:0) was the least common acid being present in only three species, while lauric (C12:0), arachidic (('20:0) and trichosanoic (C23 :0) acids were detected in 4-5 species.The unsaturated fatty acids included nine monoenoic, two dienoic and three trienoic acids. The monoenoic acids exhibited a range between CI4 and C26 chain lengths, except C21: 1 and C23: I to C25: 1 all other nine acids of this range were present in the investigated species. Palmitoleic acid (C 16: 1) appeared to be the most commonly occurring monoenoic fatty acid as it was found in 13 species, myristoleic (CI4: 1) and heptadecenoic (CI7:1) acids were the next common ones as they could be detected in 11 or 12 species. Gadoleic (C20: I), cetoleic (C22: I) and hexacosenoic (C26: I) were the least common acids as they were present in only one of the investigated species. Oleic acid (C 18: I) was not very common being present in 10 species and was detected as the most abundant monoenoic fatty acid in seven species.Only two dienoic fatty acids were found to be present, heptaecadienoic acid (C 17:2) in two and linoleic acid (C 18:2) in three species. They were present in more or less similar amount (5-6%) except in Melanothamnus .somalensis where they were in very small quantity (1%). Three trienoic acids could be detected in four species, ranging from CI4 to CI7 except CI5:3. They were present in small quantity (2-4%). Tetradecatrienoic (C14:3) was the only trienoic acid present in two species, while others were detected singly in one species. The investigated species did not reveal the presence of any acid. more unsaturated than trienoic ones.Eighteen different sterols have been isolated and chemically elucidated from 11 species, including one C26 sterol, seven C27 sterols, four C28, four C29 and two C30 sterols. Cholesterol appeared to be the most common sterol, as it was present in nine species. It was also the most predominant sterol, being present in the predominant quantity in seven species. Its proportion varied from 41-74%. Desmosterol was found as predominant sterol in two species and its varied from 28-46%. It is followed by 22-dehydrocholesterol, which, in eight species and was predominant sterol in two species; its proportion varied from 4-43%. Ostreasterol and 24-methyl sterol were the next common sterols in occurrence. They were present in six species and varied in proportion from 5-17%.Among C29 sterols, fucosterol was the most commonly occurring sterol, it was present in small proportion (2-6%) in five species. Other sterols of this series found in the investigated species were stigmasterol, J3-sitosterol and nor 31-cycloartanol, they occurred in small proportion (3-7%) in one, two or three species only. Two C30 sterols were detected in very small proportions (3-4%) viz. cycloartanol and lanostanol, the former was found in three species while the latter in only one species. Occurrence of lanostanol in a red alga is a very unique feature, it was present in an amount of 4% in Scinaia fascicufaris.Scinaia fascicufaris appeared to be very rich in sterol composition, it possessed eight different sterols belonging to all types except C26 ones. It was followed by Solieria ,robusta and Hypnea valentiae, they contained seven different sterols each. The next group was of Porphyra vietnamensis and Halymenia porphyriaeformis, they showed the presence of six different sterols each. Bangia atropurpurea and Gracilaria foliifera were very poor in sterol composition, they exhibited the presence of only three sterols each. It was observed that various species of the same genus differ in their sterol composition.Apart from fatty acids and sterols six other natural products belonging to different chemical groups have been isolated from four species, viz. Asparagopsis taxiformis, Gracilaria foliifera, Sarconema filiforme and Hypnea valentiae. The first and last mentioned species contained only one natural product the third one showed the presence of two secondary metabolites of the same chemical group, while G. foliifera G exhibited two different natural products. These metabolites include one fatty acid derivative, one fatty alcohol, two carbohydrates, one glycoside and one chamigrene.The methanolic extract of Hypnea musciformis was tested against three species of Gram positive and seven of Gram negative bacteria by disc diffusion technique. The former bacterial organisms were found to be more sensitive than the latter ones. Bacillus .subtilis appeared to be the most sensitive bacterium in this regard, while Escherichia coli was the least sensitive one. The crude methanolic extract of Porphyra vietnamensis exhibited a strong antifungal activity against nine species of common pathogenic fungi.Six different sterols extracted from P. vietnamensis were tested against nine pathogenic fungi. Cycloartanol appeared to be the most active sterol, while stigmasterol was the least active one. Cholesterol was found to be more active than its 24-methylene and 24-methyl derivatives. Various sterols exhibited different patterns of antifungal activity. No fungal species was found sensitive against all the sterols, seven species belonging to the same genus showed differences in their sensitivity against extracted sterols. Aspergillus flavus and Trichophyton rubrum were found to be most resistant fungal species, while Aspergillus niger and Penicillium funiculosum appeared to be the most sensitive fungi. Various fungal species showed different modes of sensitivity against isolated sterols.