Free and ester-bound triterpene alcohols and sterols in cellular subfractions of Calendula officinalis flowers

In the chromoplast fraction and in the chromoplast-free fraction, obtained from Calendula officinalis ligulate flowers, the contents of individual free and ester-bound triterpene alcohols and sterols as well as the fatty acid components of the ester form were determined. It was shown that all sterols and triterpene monols in both forms occur in the two subtractions investigated, whereas all diols are localized only in the chromoplast fraction. The compositions of the fatty acids esterifying monols and sterols were similar to those esterifying diols in the chromoplasts. However, the fatty acids esterifying extra-chromoplast monols and sterols were different. This result indicates that triterpene monol esters are substrates for the biosynthesis of 3-monoesters of diols.     

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.     

Nanoencapsulation of triterpene 3β,6β,16β-trihydroxylup-20(29)-ene from Combretum leprosum as strategy to improve its cytotoxicity against cancer cell lines

The pentacyclic triterpene 3β,6β,16β-tri-hydroxilup-20(29)-ene is a natural product produced by the Brazilian medicinal plant Combretum leprosum. Its cytotoxicity has been previously reported against breast cancer cell lines. The low water solubility of this natural product, that hampers its bioavailability, motivated the investigation of a new nanoparticle formulation containing the triterpene in order to improve its bioactivity. The triterpene was encapsulated in polycaprolactone (PCL) polymer by nanoprecipitation, producing homogenic nanoparticles with nanometer sizes (122.7 ± 2.06 nm), which were characterized by FT-IR, SEM imaging and DSC. The cytotoxicity (MTT method) of the nanoparticle containing the triterpene 1, besides the free natural product and the nanoparticle control (without 1), was assayed against three human tumor cell lines [human colon carcinoma line (HCT116), prostate (PC3) and glioblastoma (SNB19)] and the normal epithelial embryo kidney human cell line (Hek293T). The nanocarrier produced a significative effect in the cytotoxicity of the natural product in the nanoformulation (IC₅₀ 0.11–0.26 µg mL⁻¹) when compared with its free form (IC₅₀ 1.07–1.44 µg mL⁻¹). Additionally, higher selectivity of the triterpene to the tumor cells was found when it was encapsulated (SI 1.92–4.54) than in its free form (SI 0.42–0.56). In this case, the nanoencapsulated triterpene was more selective to PC3 (SI 3.33) and SNB19 (SI 4.54) tumor cells.     

Latex extractables of Calotropis gigantea

The hexane and methanol soluble extract of the latex coagulum of Calotropis gigantea afforded two new triterpene esters, viz. 3′-methylbutanoates of α-amyrin and ψ-taraxasterol, besides the known 3′-methylbutanoates of three triterpene alcohols. The compositions of the alkane fraction, total triterpene alcohol fraction, and free, acetyl and 3′-methylbutanoyl triterpene alcohol fractions of the extract were determined.     

Epicuticular wax of Cirsium arvense

Epicuticular wax of Cirsium arvense contains hydrocarbons (12%), esters (35%), free acids (3%), free alcohols (10%), triterpene acetates (8%) and 1,3-ditetradecanoyl-2-hexanoylglycerol (8%) as major components. Minor components are triterpene alcohols (3%) and nonacosan-10-ol (2%). The esters contain triterpene alcohol esters (19%) as well as esters of alkanols.     

Eudesmane triols from Verbesina virgata

Chemical study of Verbesina virgata collected in several localities afforded two eudesmane triols as 4-cinnamates. Their structures were determined by chemical and spectroscopic means. The structure and stereochemistry of one of the triols was confirmed by X-ray crystallography.     

Cellulose esters from waste cotton fabric via conventional and microwave heating

The esterification of cellulose from waste cotton fabric in a N,N-dimethylacetamide/lithium chloride solvent system was carried out using different types of fatty acid chloride including butyryl chloride, capryloyl chloride, and lauroyl chloride as esterifying agents, and N,N-dimethyl 1-4-aminopyridine as a catalyst under conventional and microwave activation. Microwave esterification was performed under 2.45 GHz with power varying from 90 to 450 W. The optimum conditions for esterification of cotton cellulose with various esterifying agents were investigated in terms of reaction time and temperature to attain appropriate %weight increase and degree of substitution of esterified-cellulose. The degree of substitution, functional group and chemical structure, and thermal stability of cellulose ester powder were characterized by ¹H NMR, FTIR, and TGA/SDTA analysis. Morphologies, crystallinity, and solubility of modified cellulose by two different heating methods were compared.     

Long-chain fatty acid esters of 3α-hydroxy-lup-20(29)-ene-23,28-dioic acid and other triterpenoid constituents from the bark of Schefflera octophylla

From the bark of Schefflera octophylla was isolated a series of triterpene fatty acid esters with the carbon numbers 16–21 and 23–29 in the fatty acid part. Oleanolic acid and 3α-hydroxy-lup-20(29)-ene-23,28-dioic acid were also identified.     

Identification and characterization of an acyl-CoA:triterpene acyltransferase activity in rabbit and human tissues.

Rabbit and human tissues contain substantial amounts of an unusual lipid, a fatty acid ester of a pentacyclic triterpene, that is a potent in vitro inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). A possible origin of the triterpene ester is via dietary absorption of plant triterpenes (which have a similar structure to the triterpene moiety of the animal triterpene ester), followed by fatty acid esterification of the triterpene in animal tissues. To support this idea, homogenates of rabbit and human enterocytes and liver are now shown to contain an acyl-CoA:triterpene acyltransferase activity (ATAT) which esterifies triterpene to a fatty acid. The enzyme activity was stimulated by exogenous triterpene and required ATP and coenzyme A when fatty acid was used as substrate; ATP and coenzyme A were not required when fatty acyl-CoA was used. ATAT was not inhibited by two structurally different ACAT inhibitors, which may indicate that ACAT and ATAT are different enzymes. Rat enterocytes and liver contained very little ATAT activity, consistent with the finding that rat liver contained very little triterpene ester. To establish that triterpene esterification occurs in vivo, [3H]triterpene was shown to be incorporated into triterpene ester in several organs and tissues from a rabbit given a gastric bolus of the labeled triterpene. These data provide support for the hypothesis that triterpene esters in animal tissues arise from the dietary absorption of triterpenes followed by the esterification of the triterpenes by an enzymatic activity in the animal tissues.     

Conjugated fatty acids from latex of Euphorbia lathyris

The fatty acids esterified with triterpene and diterpene alcohols from the latex of Euphorbia lathyris were analyzed. 77% of the fatty acids esterified with triterpenes were conjugated, the main components of which were decadienoic and decatrienoic acids. Of the acids esterified with diterpenes, 81% were decatrienoic and dodecatrienoic acids.     

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.     

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.     

Isolation and characterization of natural products from plant tissue cultures of Maytenus buchananii

Explants of Maytenus buchananii were induced to form a callus aid subsequently to form suspension cultures on a wide variety of media. Culture extracts showed cytotoxic activity, but examination by TLC did not indicate the presence of maytansine. Isolation of natural products from a large scale suspension culture led to the identification of polpunonic acid, sitosterol and the cytotoxic triterpene quinone-methides, tingenone and 22β-hydroxytingenone. Possible biosynthetic relationships of these and other triterpene quinone-methides are discussed.     

A gas chromatographic-mass spectrometric analysis of the esterifying alcohols of pumpkin seed protochlorophylls

The esterifying alcohols of protochlorophyll a and 4-vinyl-(4-desethyl)-protochlorophyll a (purified as the respective pheophytins) from pumpkin seeds were examined by gas chromatography-mass spectrometry. The results of the analysis suggested that pumpkin seed protochlorophyll a is esterified with all possible C₂₀ isoprenoid alcohols between and including geranylgeraniol and phytol, phytol comprising 90% or more of the mixture of esterifying alcohols, and that the 4-vinyl-(4-desethyl)-protochlorophyll a is esterified with farnesol and all possible C₂₀ isoprenoid alcohols between and including geranylgeranoid and phytanol, phytol comprising 50% or more of the mixture of esterifying alcohols. The 4-vinyl-(4-desethyl)-protochlorophyll a from a sample of older mature pumpkin seeds was found to be richer in esterifying alcohols corresponding to isoprenoid precursors of phytol then was the 4-vinyl-(4-desethyl)-protochlorophyll a from a sample of younger mature seeds. Other isoprenoid alcohols may have been present in very minor quantities in the mixtures of esterifying alcohols from the pumpkin seed protochlorophylls but were not looked for in this study. These results are discussed in terms of a biosynthetic accumulation of 4-vinyl-(4-desethyl)-protochlorophyll a in pumpkin inner seed-coat tissue.     

Formation of pregnenolone- and dehydroepiandrosterone-fatty acid esters by lecithin-cholesterol acyltransferase in human plasma high density lipoproteins

Pregnenolone- (PREG-), and dehydroepiandrosterone- (DHEA-) fatty acid esters (FA) are present in human plasma, where they are associated with lipoproteins. Because plasma has the ability to form PREG-FA and DHEA-FA in vitro from their unconjugated steroid counterparts, we postulated that the LCAT enzyme might be responsible for their formation. Here we show that lecithin-cholesterol acyltransferase (LCAT) has PREG and DHEA esterifying activities. First, VLDL, IDL, LDL, and HDL were isolated by the sequential ultracentrifugation micromethod from the plasma of fasting men and women and tested for their ability to form PREG-FA, DHEA-FA, and cholesteryl esters in vitro from their respective unconjugated counterparts. The results showed that the three steroids were esterified only in HDL subfractions. The rate of tritiated PREG esterification was clearly higher than that of tritiated cholesterol and DHEA, both in total plasma and isolated HDL, and no gender difference was observed. Second, human and guinea pig LCAT were purified and used in phosphatidylcholine-reconstituted vesicles containing human apoAI to show their ability to esterify tritiated cholesterol, PREG, and DHEA in the absence of unlabeled steroid. The amount of cholesteryl ester, PREG-FA, and DHEA-FA increased after incubation as a function of time and amount of purified LCAT, showing that PREG is preferentially acylated by LCAT compared to cholesterol and DHEA. The PREG and DHEA esterifying activities of LCAT were cofactor-dependent, as shown by the absence of acylation without apoAI. Finally, we determined by HPLC the fatty acid moiety of PREG-GA and DHEA-FA formed in human plasma and guinea pig and rat sera in vitro after incubation with unconjugated tritiated PREG and DHEA. We showed that the fatty acid moieties of newly formed tritiated PREG-FA and DHEA-FA were similar to that reported for cholesteryl esters in the plasma of the three species. We conclude that LCAT has a lecithin-steroid acyltransferase activity and that PREG is probably the preferential substrate of this enzyme. In addition, the fact that the differences in the fatty acid moieties of cholesteryl esters of human, guinea pig, and rat plasmas are also observed for PREG-FA and DHEA-FA suggests that the LCAT is the sole circulating enzyme that has PREG and DHEA esterifying activities.     

The pentacyclic triterpene Esters and the free,esterified and glycosylated sterols of Sorghum vulgare grain

The free, esterified and glycosylated sterols and triterpene esters of Sorghum vulgare grains were analysed by GLC. 4-Demethylsterols were found free, esterified and glycosidated. 4-Monomethyl-sterols were found completely free whilst the triterpenes were completely esterified. Three quarters of the total sterols in the grain were located in the embryo whilst the triterpenes were equally distributed between the embryo and endosperm.     

Variations in sterol and triterpene contents of developing Sorghum bicolor grains

The free, esterified and glycosylated sterols and the pentacyclic triterpene esters of developing Sorghum bicolor grains were analysed by GLC and GC-MS. All the pentacyclic triterpenes were completely esterified but were not detected until 24 days after anthesis. Lupanol, multiflorenol, α-amyrin and isoarborinol were identified in the mature grains as components of the triterpene fraction but no 4,4-dimethylsterols could be found at any stage of development. A sixfold increase in total sterol per grain occurred during development. At 8 days after anthesis, 28-isofucosterol was found to be the second most abundant steryl ester. Campesterol was the major steryl glycoside and obtusifoliol was the major 4-monomethylsterol.     

Terpenoid biosynthesis in Euphorbia latex

The latex of Euphorbia lathyris can utilize acetate, pyruvate and mevalonate for triterpene synthesis in vitro. Acetyl-CoA, hydroxymethylglutarate, hydroxymethylglutaryl-CoA and isopentenyl pyrophosphate were not effective as precursors for triterpene biosynthesis. Acetate is utilized only by the terpenoid pathway and by the tricarboxylic acid cycle; it is not used for fatty acid synthesis in this system. However, phospholipids were found to be efficient acyl donors for triterpene ester synthesis. The observed selectivity of precursor utilization as well as the observed rates for product formation indicate separate sites for triterpenol and triterpene ester synthesis and that one is not precursor for the other.     

Intracellular localization of metabolism of lupeol and its palmitate in Calendula officinalis flowers

In the non-fractionated flowers and in the chromoplast and extra-chromoplast fractions obtained from Calendula officinalis flowers the incorporation of radioactivity after incubation with [3-³H]lupeol and [3-³H]lupeyl[¹⁴C]palmitate were determined. It was shown that both precursors were metabolized. Monol esters are the main precursors of diol 3-monoesters, whereas free monols are hydroxylated to the diols and triols. The shape and course of the dynamic curves suggest that the process of hydroxylation of free monols, free diols and monol esters is situated only inside chromoplasts. On the other hand the hydrolysis of monol esters and esterification of free monols proceeds both outside and inside the chromoplasts.     

Enzymatic esterification of vitamin a in the pigment epithelium of bovine retina

The kinetic properties and subcellular distribution of an esterifying enzyme in the pigment epithelium of bovine retina have been studied using both [1-³H]retinol and [³H]retinol bound to cellular retinol-binding protein as substrates. The most active esterifying fraction in pigment epithelial cell preparations was the microsomes, but the lysosome plus mitochondria fraction also showed some activity, probably due to endoplasmic reticulum present as an impurity. The microsomal enzyme showed optimum activity at pH 7.5, and the reaction was linear up to 30 μg protein and for the first 10–15 min. The apparent K_m values were 16.6 · 10^?6 and 5.5 · 10^?6 M for [³H]retinol and bound [³H]retinol, respectively. This is the first time that retinol bound to cellular retinol-binding protein has been shown to undergo metabolic stransformation. The microsomal esterifying activity was destroyed by boiling for 1 min, or after freezing for 2 months. No clear requirement for ATP, CoA or fatty acid could be demonstrated.Of all the other tissues examined under the same experimental conditions as those used for the pigment epithelium, onlt intestine showed measurable activity. With larger amounts of tissue protein and longer incubation periods, activity was also detectable in microsomes of liver, testis and retina