Distribution of triterpene alcohols in subcellular fractions from Calendula officinalis flowers

The distribution of the triterpene mono- and dihydroxy alcohols was investigated in the subcellular fractions of the flowers of Calendula officinalis. The triterpene monols were found mainly in the chromoplast fraction (68% of total) with smaller amounts in the cell debris, microsomal and supernatant fractions, the mitochondrial fraction was almost devoid of these compounds. Triterpene diols were present exclusively in the chromoplast fraction, 98% in the form of the 3-monoesters and 2% in the form of diesters. It is suggested that the hydroxylation of the triterpene monols to the corresponding diols proceeds in the chromoplasts and the esterified form of the monols is probably the substrate for this reaction.     

The Changes of The Level of Triterpenoids in Calendula officinalis during Vegetation

Using thin-layer chromatography for separation and colorimetric test with CoClj for determination, qualitative and quantitative composition of triterpenoids was estimated in Calendula officinalis dining vegetation. It was found that sterols and oleanolic acid as well as trace amounts of triterpenic monols occur in all organs of the plant during the whole vegetative period. Triterpenic diols and greater quantities of triterpenic monols appear in flowers. The biosynthesis rate of triterpcnoids is highest in young organs of the plant that is in seedlings, young leaves and flower buds. During flowering the content of sterols and oleanolic acid increases in all plant organs except of old leaves. A high level of oleanolic acid in the root during the flowering period implies that oleanolic acid glycosides can be transported from older leaves to underground parts of the plant. Biosynthesis of triterpenic monols, Ψ-taraxasterol and taraxasterol, precedes the formation of the diols faradiol and arnidiol. It can be inferred from the course of accumulation of these compounds that monols are the precursors of diols and that hydroxylation occurs in flowers.     

Triterpenes and sterols of Buxus sempervirens and local variations in their levels

The leaves of Buxus sempervirens L. contain sitosterol, stigmasterol, cycloartenol, lupeol, germanicol and β-amyrin in the free state. All of these compounds, except stigmasterol, were also found in the esters fraction, as were obtusifoliol, 24-methylenelophenol, and 24-ethylidenelophenol, The triterpene diois betulin and moradiol were isolated, the latter for the first time from a plant source. In nineteen Buxus samples from England, Wales and Scotland, the sterol compositions were quite similar while those of the triterpene monols varied considerably.     

Studies on Fatty Acids and Unsaponifiable Constituents of Oil from Tubers of Cyperus esculentus L.

The fatty acids of the oil from tubers of Cyperus esculentus L. were determined by gas chromatography with DC-11 and DEGS stationary phases. Oleic, linoleic, palmitic and stearic acids are the major constituents in the fatty acid fraction, while lauric, myristic, linolenic, arachidic, dadoleic, behenic and tetracosanoic acids are the minor ones. The unsaponifiable matters of the oil were separated by column chromatography with silica gel and thin layer chromatography with silica gel G into three fractions: sterols, 4-methylsterols and triterpene alcohols. The acetates of sterols, 4-methylsterols and triterpene alcohols were separated by TLC with 20% silver nitrate impregnated silica gel G, using CH2Cl2-petroleum system as developing reagents. The identification of major components was carried out by TLC, mp, optical rotation, GLC, IR spectrum and GC-MS. It was found that β-sitosterol, stigmasterol and campesterol were present in large amounts in the unsaponifiable fraction, β-sitosterol, stigmasterol, △5-and △7-avenasterol, 24- methylenecholesterol and 24-methylenecholest-7-enol in the sterol fraction, obtusifoliol, gramisterol and citrostadienol in the 4-methylsterol fraction, and cycloartanol, cydoartenol, 24- methylenecydoartanol and cyclobranol in the triterpene alcohol fraction were isolated and identified, while campesterol, campestanol, stigmastanol, △7-stigmastenol, △7-campestenol and △7-cholestenol were identified only. We found no evidence of the occurence of nonedibles in this oil.     

Major Extractable Components in Asclepias linaria (Asclepiadaceae) and Ilex verticillata (Aquifoliaceae), Two Potential Hydrocarbon Crops

The major extractable components of two species identified as having high oil or polyphenol contents were characterized in detail.Asclepias linaria, a desert milkweed, contains 30.3% extractable material on a dry-weight basis, andIlex verticillata contains 41.5% extractable material on a dry-weight basis. Important components inA. linaria oil fractions are triterpene alcohols and esters, wax, and natural rubber; fatty acid triglycerides were nearly absent.Ilex verticillata oil fractions were predominantly triglycerides with some triterpene fatty acid esters. The more polar polyphenol fraction contained sugars and sugar esters of fatty acids and triterpene acids. The polyphenol fraction from these plants is better described as a saponin fraction. Because the crude saponin fraction represents 10.7% of the dry weight of A. linaria and 18.9% of the dry weight ofI. verticillata and because the saponin fractions showed good emulsifying properties, the refined extract of these plants might be used as a biodegradable surfactant.     

Stereospecificity of biosynthesis of triterpene alcohols in Calendula officinalis flowers

Flowers of Calendula officinalis were incubated with mevalonic acid doubly labelled with ¹⁴C in position 2 and ³H in positions 2R, 2S, 4R or 5R,S and the [³H/¹⁴C] ratios determined in squalene and pentacylic mono- and dihydroxy-triterpene alcohols and also in some derivatives prepared from the triterpene alcohols. ³H atoms were located in positions 3, 12, 16, 21, 29, 30 of the ursane skeleton, positions 3, 12, 29, 30 of the lupane skeleton and positions 3, 11, 12, 18 of the oleanane skeleton. Stabilization of α- and β-Amyrins, ω-taraxasterol and lupeol occurs with the elimination of a proton from positions 12, 21 and 29 (or 30) respectively. In addition, during hydroxylation of triterpene monols to the corresponding diols a proton is substituted by the hydroxyl group.     

The Sterol,Fatty Acid,and Hydrocarbon Composition of Globodera solanacearum

Globodera solanacearum females were found to have less than 0.01% of dry wt as sterols. Seven sterols were detected in the nematode, with stanols (campestanol and stigmastanol) making tip more than 50% of the total sterols present. Lipid amounted to 29.4% of the dry weight of the nematode. Triglyceride, free fatty acid, and phospholipid classes were composed predominantly of 20:4, 20:1, and 18:1 fatty acids. Of the total weight of fatty acids found in G. solanacearum females, the greatest portion occurred in the triglyceride fraction, followed by the free fatty acid fraction then the phospholipid fraction. Several unidentified hydrocarbons were detected in the nematode. Paraffinic hydrocarbons detected ranged in carbon length from C15 to C29. Total concentration of hydrocarbon composed 0.20% of the dry wt.     

Effects of natural and artificial defoliations on the content and composition of extractive substances in birch leaves

Qualitative and quantitative compositions of extracts of birch (Betula pendula Roth.) leaves after natural and artificial defoliations were studied. Composition of the fraction of total lipids was determined. Overall, 11 fatty acids were identified. Differences between the fatty acid compositions of total lipids in the trees subjected to defoliation, consisting in the increase in quantities of short-chain saturated fatty acids and trienoic acids, were detected. Nine individual compounds--six flavones, two flavanones, and one flavanonol--were isolated from the flavonoid fraction by column chromatography. It was found that the total content of extractive substances in birch leaves as well as amounts of free sterols, triterpene compounds, and flavones decreased 1 year after an artificial defoliation and 1 month after depredation of 75% of birch stands by gypsy moth. On the contrary, the contents of flavanones and flavanonol increased. The assay method proposed may be used for studying the compositions of plant extracts.     

Hexane soluble non-volatiles in flowering to fruiting stages of Fatsia japonica

The n-hexane soluble non-volatile fraction of the acetone extracts from the flower buds, the flowers and the immature and the mature fruits of Fatsia japonica were all found to contain fatty acids, fatty acid methyl esters, squalene, β-amyrin and sterols. At all the stages between budding to the mature fruit, the major fatty acids were palmitic and linoleic acids and the major phytosterol was stigmasterol. In addition steryl and β-amyrenyl esters were found in the flowers and the immature and the mature fruits, but these esters were not present in the flower buds. Sitosteryl ester was the major constituent of the steryl ester fraction in the fruiting stages. Phytol was found in only the flowering stage and triglycerides in only the mature fruits. The variations in the lipid constituents is discussed in relation to the stages from budding to the mature fruit.     

Sterols and fatty acids of aflatoxin and non-aflatoxin producing isolates of Aspergillus

The fatty acids and sterols present in 5 isolates of Aspergillus flavus and 3 isolates of A. parasiticus were determined; 2 isolates within each species were aflatoxin producers. The 4 major fatty acids were 16:0, 18:0, 18:1 and 18:2 with a trace of 15:0 in one isolate and traces of 17:0 in 3 other isolates. Cholesterol, ergosterol and 5, 7-ergostadienol were present in all isolates; the 5 isolates of A. flavus could be identified on the basis of retention times of minor sterols present. There was no correlation of total lipids, fatty acids or sterols with the production of aflatoxins. Water soluble complexes of sterols were not detected.     

Sterols,sterol esters and fatty acids of Botrydium granulatum, Tribonema aequale and Monodus subterraneus

The composition of the sterols, sterol esters and fatty acids has been determined in 8-, 11- and 14-day cultures of three members of the Xanthophyceae, Botrydium granulatum, Tribonema aequale and Monodus subterraneus. The main sterols, whether esterified or unesterified, were cholesterol and clionasterol, whose proportions do not vary with age of culture. Much smaller quantities of cycloartenol and 24-methylenecycloartanol were also found in all three algae. The C₁₆ fatty acids are the most common fatty acids in all three algae with C_16:1 being particularly abundant. B. granulatum and T. aequale, however, differ from M. subterraneus in having polyunsaturated C₁₆ fatty acids and a smaller proportion of C_20:5.     

Sterols and fatty acids from three species of mangrove

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.     

Hydrocarbons,phenols and sterols of the testa and pigment strand in the grain of Hordeum distichon

Material from the testa of decorticated barley grains contained hydrocarbons, esters, triglycerides, free sterols, 5-n-alkylresorcinols, and traces of free alcohols, carbonyl compounds, and various polar, acidic materials. The hydrocarbon fraction was mainly a series of n-alkanes, extending at least from C₁₁ to C₃₆, in which the C₂₉ and C₃₁ components were prominent. Two minor series of alkanes were also present. Sometimes a trace of an unsaturated hydrocarbon was detected. The ester fraction contained sterols and alkanols esterified by fatty acids, which differed in relative amounts from the fatty acids found in the triglycerides. The triglycerides were thought to have leached from within the grain. At least five free sterols were present, including sitosterol and campesterol. The 5-n-alkylresorcinols were at least twelve members of a homologous series, of which four, C₂₅, C₂₇, C₂₉, and C₃₁, made 98% of the total. Members of the series with even numbers of carbon atoms were also present. It is suggested that they are partly responsible for excluding microorganisms from the interior of the grain. The testa membrane, with the associated pigment strand, contained an estolide of fatty acids and various hydroxyacids, a polysaccharide component, and uncharacterized material.     

The utilization of sterols and other lipids by insect cells grown in vitro

Cells derived from Antheraea eucalypti were grown in vitro in a medium containing triglycerides, diglycerides, free fatty acids, and sterols as the main ‘neutral’ lipids. The sterol content of the medium was derived chiefly from the haemolymph component. The ‘neutral’ lipids in the cells were triglycerides, free fatty acids, and sterols. During growth over 6 days there was a quantitative balance between cholesterol and β-sitosterol gained by the cells and those sterols removed from the medium when allowance was made for losses from sterile medium. Cells metabolized more triglycerides and free fatty acids than they incorporated.     

Lipids in fruiting bodies of the basidiomyceteOudemansiella mucida

Dried fruiting bodies of the basidiomyceteOudemansiella mucida contain 29.1% total lipids. Their qualitative analysis revealed the presence of mono-, di-, triglycerides, sterols, free fatty acids and sterolesters. Quantitatively most significant were triglycerides (37.9%) and free fatty acids (29.7%). The phospholipid fraction contained phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid. Gas chromatography showed the presence of a broad spectrum of fatty acids. The ratio between the neutral and polar fractions was 6: 1, both having linoleic acid as the main component.     

Effects of natural and artificial defoliation on the content and composition of extractive substances in birch leaves

Qualitative and quantitative compositions of extracts of birch (Betula pendula Roth.) leaves after natural and artificial defoliation were studied. Composition of the fraction of total lipids was determined. Over-all, 11 fatty acids were identified. Differences between the fatty acid compositions of total lipids in the trees subjected to defoliation, consisting in the increase in quantities of short-chain saturated fatty acids and trienoic acids, were detected. Nine individual compounds—six flavones, two flavanones, and one flavanonol—were isolated from the flavonoid fraction by column chromatography. It was found that the total content of extractive substances in birch leaves as well as amounts of free sterols, triterpene compounds, and flavones decreased 1 year after artificial defoliation and 1 month after depredation of 75% of birch stands by gypsy moths. On the contrary, the contents of flavanones and flavanonol increased. The assay method proposed may be used for studying the compositions of plant extracts.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 107–112.Original Russian Text Copyright © 2005 by Shults, Bakhvalov, Martemyanov, Petrova, Syromyatnikova, Shakirov, Tolstikov.     

Lipids in roots of Pinus sylvestris seedlings and in mycelia of Pisolithus tinctorius during ectomycorrhiza formation: changes in fatty acid and sterol composition

The composition of fatty acids and sterols in soil lipid fractions is often used as a global indicator for the status and changes of soil microbial communities. In order to validate such analyses in the context of ectomycorrhizal communities, an experiment was performed in which seedlings of Pinus sylvestris and the fungus Pisolithus tinctorius were grown separately, or combined to form ectomycorrhiza under axenic conditions. Fatty acids of the neutral lipid fraction (NLFAs) and the phospholipid fraction (PLFAs) as well as sterols were identified and quantified by gas chromatography–mass spectrometry. When grown separately, the two organisms differed strongly with respect to the sterol composition. Sterols had a much higher relative abundance in the fungus in comparison with the plant, and the two main fungal sterols, ergosterol and 24‐ethyllanosta‐8,24(24′)‐diene‐3beta,22zeta‐diol (Et lano 8,24), as well as six minor fungal sterols were not found in the plant. On the other hand, the three sterols found in plant roots were absent from the fungus. With regard to fatty acids, the lipids of both organisms contained the same three major PLFAs, namely n16:0, 18:2–9,12c, and 18:1–9c. However, plant lipids contained, in addition, eight PLFAs and five NLFAs that were not present in the fungus. On the other hand, the fungus contained two PLFAs and two NLFAs that were not present in the plant. When the fungus and the plant were brought together, there was a drastic change in the lipid composition of the root: within a day, all the saturated fatty acids in the NLFA fraction increased very strongly and then slowly decreased but remained at an elevated level throughout the experiment. All these saturated fatty acids also started to appear in the extraradical fungal mycelium; they increased steadily and reached their highest levels at the end of the experiment. These results indicate that in symbiosis, the fungus transports plant lipids from the symbiotic interface to the extraradical mycelium. Concerning sterols, the extraradical mycelium acquired only a small amount of plant‐specific sterols. However, its ergosterol content steadily decreased whereas the content of Et lano 8,24 remained high, causing the ratio of these two sterols to decrease from 1 : 7 to 1 : 20, whereas in the ectomycorrhizal root, the opposite phenomenon occurred, so that the ratio increased to a value of almost 1 : 1. The marked changes in the composition of the extraradical mycelium were well reflected in a principal component analysis of all lipid components. The present results show that a given ectomycorrhizal fungus may display markedly different lipid compositions in its intraradical and extraradical parts. In addition, they highlight a potential role of plant lipid transfer from the root to the fungus in the functioning of the ectomycorrhizal symbiosis.     

Lipid and sterol composition of the pollen of the west african oilpalm, Elaeis guineensis

The lipid classes, fatty acid methyl esters and the sterols of oilpalm pollen were analysed. The neutral lipid fraction consisted of triglycerides, esterified and free sterols and trace amounts of hydrocarbons. Monogalactosyl and digalactosyl diglycerides, phosphatidyl choline, phosphatidyl inositol and phosphatidyl ethanolamine represented the polar lipids. The major fatty acids were linoleic, palmitic and linolenic acids together with small to trace amounts of oleic, stearic, arachidic, myristic, lauric, palmitoleic and margaric acids. Unsaturated fatty acids predominated over saturated ones in the ratio of 3:2. The 4-desmethyl sterols were the major phytosterols in the free form but they constituted a lower proportion of the sterols in the esterified state. 28-Isofucosterol was isolated and characterized as the principal sterol.     

Intracellular distribution and origin of sterols in Calendula officinalis leaves

In Calendula officinalis leaves 66% of all steryl forms are present in the ‘microsomal fraction’ (IV), 24% in the mitochondrial and Golgi membranes (III), 5% in the ‘chloroplast’ (II), 4% in the ‘cell wall and membrane’ (I) fraction and 1%. in the cytosol. Free sterols, their esters, glycosides and acylated glycosides are present in varying proportions in all cellular subtractions. Mevalonate-[2¹⁴C] labelling of sterols derived from various steryl forms showed that free sterols and all their derivatives, i.e. steryl esters and glucosides, are formed in fraction IV and are then translocated to other organelles. Fraction III is the main site of glycosylation of transported sterols as well as of acylation of steryl glycosides.     

Identification of lipids in the cuticle of the parasitic nematode Anisakis simplex and the somatic tissues of the Atlantic cod Gadus morhua

The main aim of this work was to assign the cuticular lipids identified in a parasitic nematode and to distinguish those originating from its host. The hypothesis that long-chained fatty acids and sterols are imported by the parasite in the absence of certain enzymes was also tested. The organisms (Anisakis simplex and Gadus morhua) were extracted in petroleum ether and dichloromethane. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was used to identify unknown components, and electrospray ionization mass spectrometry (ESI/MS) to verify recognized groups of lipids. The lipid classes identified in the surface layer were free saturated and unsaturated fatty acids, triacylglycerols, sterols and non-polar sphingolipids (ceramides, sphingoid bases). The most abundant fraction consisted of fatty acids. The predominant saturated acids were tetradecanoic acid in the petroleum ether extract of A. simplex, hexadecanoic acid in the dichloromethane extract of A. simplex, and also the polyunsaturated octadecahexaenoic and octadecatrienoic acids in both extracts of the parasitic nematode. The mass spectrum revealed the presence of fatty acids with different numbers of carbons, and with odd and even numbers of unsaturated bonds. The MALDI-TOF mass spectrum also identified triacylglycerols (TAGs). The dominant short-chain TAGs were CoCoCy:₁, CoCoPg and Bu0:0B:₆. The majority of TAGs were found in the ether and dichloromethane extracts of A. simplex. Sterols were the least common class of lipids found in the nematode extracts; most likely, this is the fraction that is entirely incorporated from the host organism because of the parasite’s inability to synthesize them. MALDI-TOF also identified non-polar sphingolipids - ceramides and sphingoid bases. The signals due to N-octanoyl-d-erythro-octasphinganine (m/z 288.3) and N-tetranoyl-d-erythro-tetradecasphinganine (m/z 316.4) were dominant on the mass spectra; quite a large number of short-chain non-polar sphingolipids were also identified.