Suniti Misra

Pentacyclic triterpenoids and sterols from seven species of mangrove

Abstract The isolation of pentacyclic triterpenoids from seven species of fresh mangrove leaves using a simple and rapid method is described. The leaves were homogenized using chloroform—methanol and the extract was diluted with water to precipitate out triterpenoids which were separated into neutral and acidic fractions. These were analysed by gas-liquid chromatography as acetyl and trimethylsilyl ether derivatives on a 3% OV-17 column. Sterols were isolated from the chloroform layer by preparative thin layer chromatography and were analysed by gas-liquid chromatography as their trimethylsilyl ether derivatives on a 3% OV-17 column. The triterpenoids found were α-amyrin, β-amyrin, lupeol, oleanolic acid and ursolic acid in most of the samples. Sterols found in all the samples were cholesterol, campesterol, stigmasterol, sitosterol and stigmast-7-en-3β-ol. Retention indices of the triterpenoids and sterols have been determined.

LIPID CHANGES OF GOAT SPERM PLASMA MEMBRANE DURING EPIDIDYMAL MATURATION

Highly purified plasma membranes of maturing goat caput-, corpus- and cauda-epididymal spermatozoa were isolated by aqueous two-phase polymer methods and their lipid constituents were analysed. Phospholipid (approx. 75% w/w), neutral lipid (approx. 15% w/w) and glycolipid (approx. 10% w/w) were the major sperm membrane lipids. There was a significant decrease in the total lipids (approx. 25% w/w), phospholipid (approx. 30% w/w) and glycolipid (approx. 80% w/w) contents of sperm membrane during epididymal maturation. On the contrary, the mature cauda-sperm membrane showed greater (approx. 50% w/w) neutral lipid content than that of the immature caput sperm. Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin were the phospholipids of the sperm membrane, the former two being the major lipids. Both PC and PE fractions consisted of three species--diacyl, alkylacyl and alkenylacyl forms, the last one being the dominant species in both PC and PE. Of all the phospholipids, diacyl PE decreased most strikingly (approx. 65% w/w) during sperm maturation. The neutral lipid fraction contained sterols, wax esters, 1-O-alkyl-2,3-diacylglycerol, triacylglycerol and fatty acids. Sterols represented nearly 75% w/w of the neutral lipids and cholesterol was the major component (approx. 95% w/w) of the sterol fraction. The sperm maturity was associated with marked increase of sterol (approx. 60% w/w) and steryl ester (approx. 200% w/w) and decrease (approx. 50-65% w/w) of the other membrane-bound neutral lipids. The glycolipid was identified as monogalactosyldiacylglycerol. The fatty acid profile of the various membrane lipids underwent marked alteration during the epididymal transit of the male gametes. Cholesterol/phospholipid and saturated/unsaturated fatty acid ratios increased greatly in the maturing sperm membrane. The altered lipid profile of the mature sperm membrane leads to changes in its fluidity that play an important role in determining the structure and functions of the biomembrane.

Hydrocarbons and wax esters from seven species of mangrove leaves

Abstract Seven species of fresh mangrove leaves were found to contain saturated normal and branched chain hydrocarbons, mostly between C 16 and C 36 with both odd and even carbon numbers. Significant quantitative variations were found between species. Wax esters were found to contain fatty acids with chain lengths between C 12 and C 22 . Palmitic (16:0) and stearic (18:0) acids were the major component saturated fatty acids, whereas, oleic (18:1) and linolenic (18:3) acids were the major unsaturate α-acids. Chain lengths of the alcohols of wax esters were between C 14 and C 36 . Significant quantitative and minor qualitative differences were noted in the alcohol composition of wax esters. Hydrocarbon and wax ester compositions were characterised by the presence of low M , components in high proportions.

Sterols and fatty acids from three species of mangrove

Abstract The hydrolysis of steryl esters on thin-layer chromatographic plates by porcine pancreatic lipase is described. The sterols and fatty acids produced were separated on the same plate, recovered, and analysed by gas-liquid chromatography for their compositions. Synthetic cholesteryl esters containing various saturated and unsaturated fatty acids and synthetic steryl oleates with various sterols were lipolysed along with steryl esters of Acanthus ilicifolius , Bruguiera gymnorhiza and Rhizophora mucronata mangrove leaves. The major sterol was sitosterol which was accompanied by cholesterol, campesterol, stigmasterol and 28-isofucosterol. In addition, stigmast-7-en-3β-ol was present in R. mucronata leaves. The component fatty acids found in all three species were 16:0, 18:0, 18:1, 18:2 and 18:3. The relative proportions of the sterols and fatty acids were significantly different from the chemotaxonomic standpoint. The results obtained by carrying out plate lipolysis for 45 min at 40° compared well with those produced by conventional chemical hydrolysis.

Phospholipid asymmetry of goat sperm plasma membrane during epididymal maturation

The phospholipids and their fatty acids of the inner and outer plasma membrane leaflets of the maturing goat caput-, corpus-and cauda-epididymal spermatozoa were analyzed by treating the intact spermatozoa with phospholipase C and trinitrobenzene sulphonate. The inner and outer membrane showed marked differences in the phospholipid composition at all stages of epididymal sperm maturation. The outer membrane was rich in phosphatidylcholine (PC) and sphingomyelin (SPH) whereas the inner leaflet was dominated by phosphatidylethanolamine (PE). Although the ratio of PE/PC in the inner membrane was similar in both the mature cauda sperm and the immature caput sperm, it decreased significantly in sperm undergoing maturation in the corpus-epididymis. The distribution of the saturated and unsaturated fatty acids in the phospholipid fractions of both the membrane leaflets underwent profound alterations during the epididymal maturation. The data demonstrate asymmetry of phospholipids and their fatty acids in the sperm inner and outer plasma membranes and this lipid asymmetry is greatly altered during epididymal maturity of the male gametes.

Effect on the leaf lipids of three species of mangrove of periodic submergence in tidal water

Abstract Mangrove plant leaves of Avicennia officinalis , Acanthus illicifolius and Bruguiera gymnorhiza , grown under tidal water stress, were found to biosynthesize higher proportions of hydrocarbons, wax esters, sterol esters, triglycerides, sterols and low- M , terpenes, compared to the plants grown normally. Hydrocarbons and n -alcohols of wax esters, with longer chain lengths were found in higher proportions in the plants grown normally. Higher proportions of unsaturated n -alcohols of wax esters were present in the leaves of periodically submerged plants. Proportions of unsaturated fatty acids of wax esters, sterol esters and triglycerides were also higher in the submerged plants. Sitosterol, stigmasterol and campesterol of the sterol esters, were higher in submerged plants, whereas 28-isofucosterol was higher in normal plants. Of the free sterols, sitosterol was higher in submerged plants, whereas stigmast-7-en-3β-ol and campesterol were higher in normal plants and 28-isofucosterol was found only in submerged plants. Pentacyclic triterpenoids were found only in normal plants, whereas, the submerged plants contained low- M , terpenes in higher proportions.

Lipids and fatty acids of the horseshoe crabsTachypleus gigas andCarcinoscorpius rotundicauda

Total lipids from hepatopancreas of the horseshoe crabs,Tachypleus gigas andCarcinoscorpius rotundicauda, obtained in 7.6 and 3.3% wet weight respective yields, were fractionated by various chromatographic techniques and identified by gas-liquid chromatography and spectroscopic methods. Fatty acid-containing lipids were rich in 16∶0 (8.0–25%), 18∶1ω9 (6.9–22%) and 18∶2ω6 (6.8–18.5%); appreciable amounts of 16∶1ω7, 18∶3ω3, 20∶5ω3 and 22∶6ω3 were also present. The level of 26∶0 in the hydrocarbon fractions was unusually high (64 and 68%). Carbon chain lengths of major wax esters were 44, 46 and 48 forT. gigas and 38, 40 and 42 forC. rotundicauda. 1-O-Alkyl diglycerides were 7.2 and 9.1% of the total lipids in the two species and contained 14∶0(20%), 16∶0(60%) and 18∶0(20%) alkyl chains along with a relatively higher percentage (32–35%) of saturated fatty acids. High levels of cholesterol (>50% of total sterol) in the free and combined state were encountered in both samples, phospholipid contents being 40 and 35%, respectively, and contained highest levels of unsaturated fatty acids.

Lipid composition of Porteresia coarctata from two different mangrove habitats in India

Abstract Porteresia coarctata collected, at Prentice and Chuksar Islands of the Sunderban mangrove ecosystem, was studied for its lipid composition. Hydrocarbon, wax ester, sterol esters, triacylglycerols, sterols and terpenoids, were all found in higher proportions in the Prentice Island sample. Palmitic acid was the major fatty acid in all fractions except for the steryl esters of P. coarctata collected from Prentice Island, where oleic and linoleic acids were the major components. Sterols found were, sitosterol, stigmasterol, campesterol and cholesterol, of which, sitosterol and stigmasterol were the major components. Terpenoids present were, β-amyrin, α-amyrin, lupeol, betulin, oleanolic acid and ursolic acid, of which, lupeol and oleanolic acid were the major components. Quantitative variations of the lipid constituents were found between the two samples studied.

Lipid contaminants in commercial lipases.

During lipolysis of wax esters on thin layer chromatography (TLC) plates (1), abnormally high errors for stearic (18:0), palmitic (16:0) and oleic (18: t ) acids were found in the hydrolysates. These anomalous results prompted us to examine the source of the contaminations, which was found to be the lipase used. We have analyzed 2 commercial lipase samples that were used for the lipolysis of wax esters and developed a procedure for the preparation of commercial lipase free of fatty acid (FA) contaminants. Pancreatic lipase from 300 mg each from Sigma Chemical Co. (St. Louis, MO, Type II, crude from porcine pancreas) and from Calbiochem (Los Angeles, CA, B grade from porcine pancreas) were mixed vigorously with 15 ml of redistilled diethyl ether and centrifuged. The supernatnats were carefully removed with a syringe and saved. The extract ion procedure with diethyl ether was repeated 6 times for each lipase sample. The pooled etherial extracts were dried over anhydrous sodium sulphate and the solvent was evaporated and weighed. The lipases were then washed 6 times with 15 ml protions of acetone. The acetone extracts were also pooled and the solvent was evaporated and weighed. The etherial extracts were spotted on preparative TLC plates that were developed using a solvent system of light petroleum ether (40 C60 C) diethyl ether/acetic acid (80:20:1.5, v/v). The bands were visualized by putting the plates in an iodine chamber. The bands were identified by comparing the Rf values with those of known standards. Finally, the various bands were scraped off the plates and the compounds were extracted using diethyl ether, the solvent was evaporated and then weighed. The free fatty acid (FFA) bands from the 2 lipase samples were methylated using diazomethane (2). To each of the methyl ester samples, 50 /~g of methyl pentadecanoate (15:0) were added and analyzed by gas liquid chromatography (GLC) using a 10% DEGS column. From the chromatograms, peak areas were determined and each of the components was identified and quantified. Beeswax and Avicennia officinalis leafwax were hydrolyzed with extracted and unextracted lipases on TLC plates, using 1 mg lipase and 1 mg wax esters, according to the method des-

Occurrence of wax esters and 1-O-alkyl-2,3-diacylglycerols in goat epididymal sperm plasma membrane

Two unusual lipid classes were detected by thin-layer chromatography in the neutral lipids derived from goat cauda-epididymal sperm plasma membrane. The lipids were identified as wax esters and 1-O-alkyl-2,3-diacylglycerols based on chromatographic properties, identity of their hydrolysis products, and infrared/1H nuclear magnetic resonance spectral evidence. The membrane containedca. 3 and 5 μg/mg protein of wax esters and alkyldiacylglycerols, respectively. The relative proportions of wax esters and alkyldiacylglycerols in the total neutral lipids were 1.5% and 2.4%, respectively. The lipids contained fatty acids with chain lengths of C14 to C22. The major fatty acids of the wax esters were 14∶0, 16∶0, 16∶1ω7, 18∶0 and 18∶1ω9. The fatty acids in alkyldiacylglycerol were 16∶0, 18∶0, 22∶5ω3 and 22∶6ω3. Alkyldiacylglycerol was particularly rich in docosahexaenoic acid 22∶6ω3) representing 30% of the total fatty acids. The alcohols of wax ester were all saturated with C20–C29 carbon chains. The deacylated products derived from alkyldiacylglycerols were identified as hexadecyl, octadecyl and octadec-9′-enyl glycerol ethers.