Major Sterol Research Articles

STEROLS AND VOLATILE COMPOUNDS OF THE FRESHWATER SPONGETROCHOSPONGILLA PAULULA(BOWERBANK, 1863)

The Neotropical region has the highest freshwater sponge diversity in the world, with the presence of species of the families Metaniidae, Potamolepidae, and Spongillidae. Due to the remarkable lipid diversity in these organisms, this study aimed to characterize the sterols and volatile compounds of the sponge Trochospongilla paulula collected in the Tapajos River. Seven volatile compounds were identified, with the long-chain alcohols tetradecanol, pentadecanol, and hexadecanol representing 51.65% of this fraction. Cholesterol is the major sterol species, as reported for other species of the family Spongillidae; however, T. paulula may be distinguished by a chromatographic profile of its sterols.

The composition of the lipoidal matter of the seeds of Pleiogynium timorense (DC.) Leenh

Background and objectives Nothing dealing with the chemistry of the seeds of Pleiogynium timorense (DC.) Leenh. could be traced in the available literature. The objective of the present work was to investigate the lipoidal matter from P. timorense seeds and to isolate and identify the major sterols and triterpenes in the petroleum ether extract. Materials and methods The petroleum ether extract of P. timorense seeds was prepared. The extract was subjected to column chromatography using silica gel (type 60-230 mesh) as an adsorbent. The unsaponifiable matter and fatty acid methyl esters were analyzed by GC/MS. Results and conclusion GC/MS analysis of the unsaponifiable matter from the petroleum ether extract of P. timorense seeds revealed the identification of 70.04% of unoxygenated compounds and 23.34% oxygenated compounds. Hydroxylated compounds, ketones, and steroidal compounds represent 23.24, 0.20, and 3.19%, respectively. 1-Heptene (66.47%) was the major compound in the USM, followed by butylated hydroxy toluene (21.07%). GC/MS analysis of fatty acid methyl esters showed that nine fatty acid methyl ester derivatives could be identified, representing 95.84% of the total composition. The major fatty acid was 9,12-octadecadienoic acid (linoleic acid) (33.8%). Saturated fatty acids represent 36.93% of the total fatty acid content, whereas monounsaturated and diunsaturated fatty acids represent 25.11 and 33.8% of the total fatty acid content, respectively. Column chromatography of the petroleum ether extract led to the isolation of two compounds [α-amyrin and 5,24(28)-cholestadien-24-methylen-3β-ol].

Study and Valorization of Dovyalis Caffra Seeds

In this present work, the characterization of Dovyalis caffra seeds (region Rabat-Morocco) was done through the identification and quantification of all components containing in the oil extracted from Dovyalis Caffra seeds including Fatty acid and sterol composition. This characterization was carried out using a technical instruments gas chromatography, In one hand the mean values of fatty acid composition found in the oil extracted from Dovyalis caffra seeds was: Linoleic acid (C18:2) with 54.98 %, Palmitic acid (C16:0) with 21.36% and oleic acid (C18:1) with 12.17%, In the other hand, β-sitosterol is the major sterol in the oil. The mean objective of this work is the valorization of Dovyalis caffra seeds.

Scarcity of the C30 sterane biomarker, 24-n-propylcholestane, in Lower Paleozoic marine paleoenvironments

24-n-Propylcholestane (24-npc), a C30 sterane compound derived from sterol precursors which are the major sterol constituents of modern pelagophyte microalgae, occurs in certain Neoproterozoic rocks and oils and throughout the Phanerozoic rock record. This broad distribution leads 24-npc to be widely considered a reliable indicator of open to partially restricted marine depositional conditions for source rocks and oils. Here we report two significant hiatuses in the occurrences of 24-npc in the Lower Paleozoic marine rock record: the first in the Middle–Late Cambrian and the second in the Late Ordovician–early Silurian transition for a range of lithofacies (carbonates and siliciclastic rocks), organic carbon contents (both organic-lean and organic-rich), and paleoceanographic environments (shelf and deeper water marine settings) and observed offshore of two paleocontinents, Laurentia and Baltica. The Ordovician–Silurian gap is at least 9millionyears, and possibly up to 20millionyears, in duration. Robust older occurrences of 24-npc steranes in some Neoproterozoic rocks and oils suggest that oceanographic conditions in our intervals of Lower Paleozoic time were unfavorable for the proliferation of pelagophyte algae as phytoplankton. Caution should therefore be applied when interpreting a lacustrine versus marine depositional environmental setting for source rocks and oils in these intervals of Early Paleozoic time using lipid biomarker assemblages.

Identification of a Cholesterol-Binding Pocket in Inward Rectifier K+ (Kir) Channels

Cholesterol is the major sterol component of all mammalian plasma membranes. Recent studies have shown that cholesterol inhibits both bacterial (KirBac1.1 and KirBac3.1) and eukaryotic (Kir2.1) inward rectifier K+ (Kir) channels. Lipid-sterol interactions are not enantioselective, and the enantiomer of cholesterol (ent-cholesterol) does not inhibit Kir channel activity, suggesting that inhibition results from direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionary conserved cholesterol-binding pocket, which we aimed to identify. Computational experiments were performed by docking cholesterol to the atomic structures of Kir2.2 (PDB: 3SPI) and KirBac1.1 (PDB: 2WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. The stability of cholesterol in each of these poses was then confirmed by molecular dynamics simulations. Finally, mutation of key residues (S95H and I171L) in this putative binding pocket found within the transmembrane domain of Kir2.1 channels were shown to lead to a loss of inhibition by cholesterol. Together, these data provide support for this location as a biologically relevant pocket.

Changes in Sterol Composition of Hazelnut During Fruit Development

Hazelnut is a valuable source of fat-soluble bioactive components. Among the fat-soluble bioactive components, phytosterols have gained much interest due to their cholesterol reducing properties. This study presents the evaluation of changes in the sterol composition of Turkish hazelnut cultivars (Tombul, Palaz, and Sivri) during fruit development. Three major sterols (β-sitosterol, campesterol, and Δ5-avenasterol), and five minor sterols (clerosterol, brassicasterol, stigmasterol, Δ7-stigmastenol, and Δ7-avenasterol) were detected in all hazelnut cultivars during all stages. The total sterol contents of Turkish hazelnut cultivars reached the maximum values at the early stage and a significant reduction in the total sterol content occurred at the harvest stage.

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S-Adenosylmethionine:Sterol C-24 Methyltransferase.

The AIDS-associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S-adenosylmethionine:sterol C24-methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C-24 position of the sterol side chain producing both C28 and C29 24-alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild-type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy ((1)H-NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ(24(28)) -sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii.

Determination of sterols using liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry

A new method, reversed phase liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry (RPLC–SALDI MS) for the determination of brassicasterol (BR), cholesterol (CH), stigmasterol (ST), campesterol (CA) and β-sitosterol (SI) in oil samples has been developed. The sample preparation consisted of alkaline saponification followed by extraction of the unsaponificable fraction with diethyl ether. The recovery of the sterols ranged from 91 to 95% with RSD less than 4%. Separation of the five major sterols on a C18 column using methanol-water gradient was achieved in about 10min. An on-line UV detector was employed for the initial sterol detection prior to effluent deposition using a laboratory-built spotter with 1:73 splitter. Off-line SALDI MS was then applied for mass determination/identification and quantification of the separated sterols. Ionization of the nonpolar analytes was achieved by silver ion cationization with silver nanoparticles used as the SALDI matrix providing limits of detection 12, 6 and 11fmol for CH, ST and SI, respectively. Because of the incorporated splitter, the effective limits of detection of the RPLC–SALDI MS analysis were 4, 3 and 4pmol (or 0.08, 0.06 and 0.08μg/mL) for CH, ST and SI, respectively. For quantification, 6-ketocholestanol (KE) was used as the internal standard. The method has been applied for the identification and quantification of sterols in olive, linseed and sunflower oil samples. The described off-line coupling of RPLC to SALDI MS represents an alternative to GC–MS for analysis of nonpolar compounds.

Role of membrane contact sites in PM ‐ ER sterol transport (606.7)

Intracellular trafficking of sterols occurs largely by non‐vesicular processes that are poorly understood. Cytoplasmic sterol transport proteins (STPs) are presumed to ferry sterols between membranes and this may be enhanced in regions where the participating membranes are closely apposed (membrane contact sites or MCSs). The major sterol trafficking pathway in the yeast Saccharomyces cerevisiae involves direct transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). Yeast STPs have not been identified, although the cytoplasmic protein Kes1/Osh4 has been implicated in the phosphoinositide‐driven cycling of ergosterol between the ER and TGN. We considered whether MCSs between the ER and PM might play a role in sterol transport between the two organelles. We tested this hypothesis by analyzing sterol traffic in Saccharomyces cerevisiae cells that were largely devoid of ER ‐ PM contact sites (∆tether cells). We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the PM to the ER and lipid droplets, and high performance liquid chromatography to quantify, in parallel, the transport‐coupled formation of DHE esters. We found that sterol transport from PM to ER is unaffected in ∆tether cells. We are currently generating strains based on ∆tether cells in which all residual ER‐PM MCSs are eliminated, and also testing whether vesicular pathways might play a redundant role in the absence of MCSs.

Fermentation of soybean oil deodorizer distillate with Candida tropicalis to concentrate phytosterols and to produce sterols-rich yeast cells

Phytosterols have been recovered from the deodorizer distillate produced in the final deodorization step of vegetable oil refining by various processes. The deodorizer distillate contains mainly free fatty acids (FFAs), phytosterols, and tocopherols. The presence of FFAs hinders recovery of phytosterols. In this study, fermentation of soybean oil deodorizer distillate (SODD) with Candida tropicalis 1253 was carried out. FFAs were utilized as carbon source and converted into cellular components as the yeast cells grew. Phytosterols concentration in SODD increased from 15.2 to 28.43% after fermentation. No significant loss of phytosterols was observed during the process. Microbial fermentation of SODD is a potential approach to concentrate phytosterols before the recovery of phytosterols from SODD. During SODD fermentation, sterols-rich yeast cells were produced and the content of total sterols was as high as 6.96%, but its major sterol was not ergosterol, which is the major sterol encountered in Saccharomyces cerevisiae. Except ergosterol, other sterols synthesized in the cells need to be identified.

Structure and dynamics studies of sterol 24-C-methyltransferase with mechanism based inactivators for the disruption of ergosterol biosynthesis

The enzyme sterol 24-C-methyltransferase (SMT) belongs to the family of transferases, specifically to the one-carbon transferring methyltransferases. SMT has been found playing a major role during the production of steroids, especially for the biosynthesis of ergosterol, which is the major membrane sterol in leishmania parasites, causing leishmaniasis. However, SMT and ergosterol are not found in mammals, so, an extensive study has been carried out over the susceptible SMT protein, which is found to be highly conserved among all the Leishmania species and holds a significant anti-leishmanial drug target. To date, there is no computational data available for SMT, due to its highly unexplored profile. In this work, a complete set of structural attributes have been examined through the available computational procedures, along with an attempt to characterize the most capable modeling server available. The exploration ranges from physicochemical characterization, pairwise alignment, secondary structure prediction, to active site detection. With this information, a docking study was carried out to find the compound that best binds into the active site. Moreover, molecular dynamics simulation was conducted to examine the stability of the homology modeled protein and the ligand-enzyme complex. The results indicate that the ligand-enzyme complex is more stable.

Biological activities of Schottenol and Spinasterol, two natural phytosterols present in argan oil and in cactus pear seed oil, on murine miroglial BV2 cells

The objective of this study was to evaluate the biological activities of the major phytosterols present in argan oil (AO) and in cactus seed oil (CSO) in BV2 microglial cells. Accordingly, we first determined the sterol composition of AO and CSO, showing the presence of Schottenol and Spinasterol as major sterols in AO. While in CSO, in addition to these two sterols, we found mainly another sterol, the Sitosterol. The chemical synthesis of Schottenol and Spinasterol was performed. Our results showed that these two phytosterols, as well as sterol extracts from AO or CSO, are not toxic to microglial BV2 cells. However, treatments by these phytosterols impact the mitochondrial membrane potential. Furthermore, both Schottenol and Spinasterol can modulate the gene expression of two nuclear receptors, liver X receptor (LXR)-α and LXRβ, their target genes ABCA1 and ABCG1. Nonetheless, only Schottenol exhibited a differential activation vis-à-vis the nuclear receptor LXRβ. Thus Schottenol and Spinasterol can be considered as new LXR agonists, which may play protective roles by the modulation of cholesterol metabolism.

Characterization of Oilseed Lipids from “DHA-Producing Camelina sativa”: A New Transformed Land Plant Containing Long-Chain Omega-3 Oils

New and sustainable sources of long-chain (LC, ≥C20) omega-3 oils containing DHA (docosahexaenoic acid, 22:6ω3) are required to meet increasing demands. The lipid content of the oilseed of a novel transgenic, DHA-producing land plant, Camelina sativa, containing microalgal genes able to produce LC omega-3 oils, contained 36% lipid by weight with triacylglycerols (TAG) as the major lipid class in hexane extracts (96% of total lipid). Subsequent chloroform-methanol (CM) extraction recovered further lipid (~50% polar lipid, comprising glycolipids and phospholipids) and residual TAG. The main phospholipid species were phosphatidyl choline and phosphatidyl ethanolamine. The % DHA was: 6.8% (of total fatty acids) in the TAG-rich hexane extract and 4.2% in the polar lipid-rich CM extract. The relative level of ALA (α-linolenic acid, 18:3ω3) in DHA-camelina seed was higher than the control. Major sterols in both DHA- and control camelina seeds were: sitosterol, campesterol, cholesterol, brassicasterol and isofucosterol. C16–C22 fatty alcohols, including iso-branched and odd-chain alcohols were present, including high levels of iso-17:0, 17:0 and 19:0. Other alcohols present were: 16:0, iso-18:0, 18:0 and 18:1 and the proportions varied between the hexane and CM extracts. These iso-branched odd-chain fatty alcohols, to our knowledge, have not been previously reported. These components may be derived from wax esters, or free fatty alcohols.

Cholesterol Transport and Regulation in the Mammary Gland

The milk-producing alveolar epithelial cells secrete milk that remains after birth the principal source of nutrients for neonates. Milk secretion and composition are highly regulated processes via integrated actions of hormones and local factors which involve specific receptors and downstream signal transduction pathways. Overall milk composition is similar among mammalian species, although the content of individual constituents such as lipids may significantly differ from one species to another. The milk lipid fraction is essentially composed of triglycerides, which represent more than 95 % of the total lipids in human and commercialized bovine milk. Though sterols, including cholesterol, which is the major milk sterol, represent less than 0.5 % of the total milk lipid fraction, they are of key importance for several biological processes. Cholesterol is required for the formation of biological membranes especially in rapidly growing organisms, and for the synthesis of sterol-based compounds. Cholesterol found in milk originates predominantly from blood uptake and, to a certain extent, from local synthesis in the mammary tissue. The present review summarizes current knowledge on cellular mechanisms and regulatory processes determining intra- and transcellular cholesterol transport in the mammary gland. Cholesterol exchanges between the blood, the mammary alveolar cells and the milk, and the likely role of active cholesterol transporters in these processes are discussed. In this context, the hormonal regulation and signal transduction pathways promoting active cholesterol transport as well as potential regulatory crosstalks are highlighted.

The Major Cellular Sterol Regulatory Pathway Is Required for Andes Virus Infection

The Bunyaviridae comprise a large family of RNA viruses with worldwide distribution and includes the pathogenic New World hantavirus, Andes virus (ANDV). Host factors needed for hantavirus entry remain largely enigmatic and therapeutics are unavailable. To identify cellular requirements for ANDV infection, we performed two parallel genetic screens. Analysis of a large library of insertionally mutagenized human haploid cells and a siRNA genomic screen converged on components (SREBP-2, SCAP, S1P and S2P) of the sterol regulatory pathway as critically important for infection by ANDV. The significance of this pathway was confirmed using functionally deficient cells, TALEN-mediated gene disruption, RNA interference and pharmacologic inhibition. Disruption of sterol regulatory complex function impaired ANDV internalization without affecting virus binding. Pharmacologic manipulation of cholesterol levels demonstrated that ANDV entry is sensitive to changes in cellular cholesterol and raises the possibility that clinically approved regulators of sterol synthesis may prove useful for combating ANDV infection.

Zinc Finger Transcription Factors Displaced SREBP Proteins as the Major Sterol Regulators during Saccharomycotina Evolution

In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs), which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1) and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina.

Chemical compositions of the Tunisian Ziziphus jujuba oil

Thirteen fatty acids were identified from the pulps of four tunisian Ziziphus jujuba (Z. jujuba) ecotypes (Sfax, Choutrana, Mahres and Mahdia), using capillary gaseous chromatography method. These oils presented 8.31% to 12.35% of dry weights. Compared to the other ecotypes, Mahres and Choutrana were the richest of the oleic acid. The palmitic acid was the most important compound of the Sfax ecotype. Unsaturated fatty acids ranged from 62.63% to 72.40% of the total fatty acids of each ecotype. So a ratio of the unsaturated/saturated (U/S) varied from 1.68 to 2.37. The β-sitosterol and the Stigmasterol were identified as major sterols. The β-sitosterol was theprominent component in the all ecotypes and the highest level (10.65 mg/100g) was noted in Choutrana ecotype. Stigmasterol was more abundant in Sfax (16.12 mg/100g) and Choutrana (4.67 mg/100g) ecotypes. The campesterol sterol was identified only in Sfax and Choutrana ecotypes being more important (2.4 mg/100g) in Choutrana pulps.

Isoform-Dependent Cholesterol Regulation of HCN Channels

Cholesterol is the major sterol component of all mammalian plasma membranes, and has been shown to regulate numerous ion channels. The hyperpolarization current, Ih, generated by the hyperpolarization-activated cyclic AMP-dependent (HCN) ion channels is a major contributor to the pacemaking in cardiac and neuronal tissue. However, regulation of each human HCN channel isoform (HCN1-4) by membrane lipids, including cholesterol, has not been systematically examined. Cholesterol depletion in CHO-K1 cells by MsCD or Mevastatin or enrichment using a cholesterol-MsCD complex expressing HCN1 or HCN2 channels leads to a reduction in current densities as well as altering the kinetics of activation and deactivation. Immunohistochemistry suggests that there is no change in the distribution of these channels from lipid raft domains to non-raft domains as a result of cholesterol modification. Specific isoform differences between HCN1 and HCN2 could be detected in non-equilibrium state known as hysteresis, examined using ramp protocols. HCN1 demonstrates hysteresis when voltage ramps of 600mV/s are applied, whereas hysteresis is observed in HCN2 channels only at much slower ramp speeds (eg. 150mV/s). However, while the degree of hysteresis is unchanged in HCN1 channels when membrane cholesterol levels are modified, hysteresis in HCN2 channels begins to be apparent at faster ramps speeds (300 mV/s) upon cholesterol depletion. Such isoform dependent differences in cholesterol regulation of HCN channels may contribute to their differential distribution and function in cardiac and neuronal tissue.

Method development for forensic identification of biodiesel based on chemical fingerprints and corresponding diagnostic ratios

A forensic identification method based on the chemical fingerprinting of the first generation of biodiesel (fatty acid alkyl esters as effective components), and several corresponding diagnostic ratios was developed and validated. The distribution of major fatty acid methyl esters (FAMEs) and polar compounds (free fatty acids, glycerol, monoacylglycerides, and free sterols) in several representative above biodiesel products commercially available in Canada were positively quantified and compared, a number of cross-plots of diagnostic ratios of target FAMEs and sterols were developed for biofuel correlation and differentiation. It was found that the cross-plots of FAME ratios, for example, the sum of the di-unsaturated relative to saturated homologues of FAMEs (D/S) versus the sum of the mono-saturated to saturated FAMEs (M/S), and the sum of di-unsaturated to mono-saturated FAMEs (D/M) versus the sum of the mono-saturated to saturated FAMEs (M/S), could cluster samples clearly into their individual feedstock. The cross-plots of diagnostic ratios of individual major sterols (cholesterol, brassicasterol, campesterol, β-stiosterol and stigmasterol) to the total sterols were also developed and proved to be effective in identifying biodiesel sources due to their self-normalizing effect on sterol data. The case study of a mystery biodiesel spill using this method showed that the two real samples can be tightly clustered into biodiesel from animal fat (Ban) group. However, the significant discrepancy of free fatty acids, glycerol, monoacylglycerides and sterol concentrations between the two real samples indicated their different producing batches.

Simultaneous HPLC quantification of five major triterpene alcohol and sterol ferulates in rice bran oil using a single reference standard

A high performance liquid chromatography (HPLC) method was developed for simultaneous quantification of five major triterpene alcohol and sterol ferulates in rice bran oils (RBO) with a single internal standard, cycloartenyl ferulate. The five compounds are cycloartenyl ferulate (1), 24-methylene cycloartanyl ferulate (2), campesteryl ferulate (3), sitosteryl ferulate (4) and stigmastanyl ferulate (5). All five compounds had good linear concentration-measurement relationships (r2⩾0.9995) and possessed similar relative response factors. The relative deviation of this method was less than 2.5% for intra- and inter-day assays, and the average recovery varied from 95.1% to 99.4%. The new method was validated by comparing the amount of 24-methylene cycloartanyl ferulate (2) in 17 RBO samples obtained with this method and that with an external standard method. This method was also successfully applied to determine five major triterpene alcohol and sterol ferulates in 17 batches of RBO samples. The results demonstrated that the present method could be utilised for quality control of RBO since some of the reference standards are not commercially available.