Triterpenoid Hydrocarbons Research Articles

Using Compound-Specific Carbon Stable Isotope Analysis of Squalene to Establish Provenance and Ensure Sustainability for the Deep-Water Shark Liver Oil Industry

Deep-water dogfish (sharks) are caught on Australia’s continental shelf as by-products to other deep-water species with revenue derived from fillets for human consumption and from the livers which are sold for their oil content. Deep-water dogfish utilise a large oil-rich liver for buoyancy, which may account for 20–25% of their body weight. An important constituent of certain dogfish liver oil is squalene, a highly unsaturated triterpenoid (C30H50) hydrocarbon which in some species can be up to 90% of the oil, though in the Australian commercial species it is typically around 50%. Squalene (and deep-water dogfish liver oil in general) has a long-standing high value in products, such as cosmetics and nutraceuticals. Manufacturers are increasingly required to demonstrate the sustainability of products, and this is integral to the importance of demonstrating product provenance. Australia’s mid-slope deep-water dogfish fishery is recognised globally as well managed and sustainable; therefore, it is important to be able to distinguish products derived from these regions from other unregulated, unsustainable and cheaper sources in order to protect Australia’s competitive advantages and ensure sustainability. In this study, we have sourced deep-water dogfish liver oil samples originating from Southeast Australia, New Zealand, India, Northeast Africa and the Arabian Sea. The squalene was isolated by commercial or laboratory processing. A compound-specific carbon stable isotope analysis of the derived squalene was then used to determine isotopic resolution and assign the likely origins of a variety of commonly available off-the-shelf nutraceuticals in Australian outlets. Squalene sourced and produced from Southeast Australian and New Zealand dogfish liver oils showed δ13C values in the range of −22.1 to −24.2‰, with all other squalene samples distinguishable at −19.9 to −20.7‰. Many of the off-the-shelf squalene products claiming to be of Australian origin showed δ13C values very distinct from the range of the genuine Southeast Australian- and New Zealand-produced squalene.

Towards the commercialization of Botryococcus braunii for triterpenoid production

Botryococcus braunii can accumulate unusually high levels of triterpenoid hydrocarbons making it a potential source of high value chemicals. However, its commercial application is hampered by its slow growth and lack of large-scale studies of triterpenoid hydrocarbon production. This study investigated hydrocarbon production in two race B of B. braunii strains, Overjuyo-3 and Kossou-4, at 25 °C in 500 L open tanks under artificial lighting in modified BG11 medium over 60 days. Maximum growth was reached by 40 days with Overjuyo-3 producing more biomass (3.05 g L(-1)) than Kossou-4 (2.55 g L(-1)). However, Kossou-4 produced more oil (0.75 g L(-1)) and triterpenoid hydrocarbons (C30-C34; 50 % of oil weight) compared to 0.63 g L(-1) of oil in Overjuyo-3 with triterpenoid hydrocarbons making up 29 % of oil weight. This research demonstrates for the first time that large-scale production of high value triterpenoid hydrocarbon for commercial application is feasible with Kossou-4 strain.

Road tunnel, roadside, and urban background measurements of aliphatic compounds in size-segregated particulate matter

Particulate matter samples were collected in a road tunnel in Lisbon (PM0.5, PM0.5–1, PM1–2.5, and PM2.5–10) and at two urban locations representing roadside and background stations (PM2.5 and PM2.5–10). Samples were analysed for organic and elemental carbon (OC and EC), n-alkanes, n-alkenes, hopanes, some isoprenoid compounds, and steranes. Particulate matter concentrations in the tunnel were 17–31 times higher than at roadside in the vicinity, evidencing an aerosol origin almost exclusively in fresh vehicle emissions. PM0.5 in the tunnel comprised more than 60% and 80% of the total OC and EC mass in PM10, respectively. Concentrations of the different aliphatic groups of compounds in the tunnel were up to 89 times higher than at roadside and 143 times higher than at urban background. Based on the application of hopane-to-OC or hopanes-to-EC ratios obtained in the tunnel, it was found that vehicle emissions are the dominant contributor to carbonaceous particles in the city but do not represent the only source of these triterpenic compounds. Contrary to what has been observed in other studies, the Σhopane-to-EC ratios were higher in summer than in winter, suggesting that other factors (e.g. biomass burning, dust resuspension, and different fuels/engine technologies) prevail in relation to the photochemical decay of triterpenoid hydrocarbons from vehicle exhaust.

Selective enrichment of steroid and triterpenoid hydrocarbons from crude oil using gel permeation chromatography for stable carbon isotope analysis

Gel permeation chromatography (GPC) using a high performance liquid chromatography (HPLC) system was studied for the separation and enrichment of steroid and hopanoid hydrocarbons from crude oil for stable carbon isotope analysis. A crude oil sample was pretreated using silica gel chromatography and 5A molecular sieve to remove polycyclic aromatic hydrocarbons and n-alkanes. The GPC behavior of both the pretreated saturated hydrocarbon fraction of the oil and standard steroid [5α(H), 14α(H), 17α(H) C27–C29 steranes], hopanoid [17α(H) C27 trisnorhopane, 17α(H), 21β(H) C29–C32 hopanes] and triterpenoid [18α(H)-oleanane, gammacerane] mixtures were examined. The results indicate that 17α(H), 21β(H) hopanes as well as steranes could be enriched efficiently using GPC and that they could be obtained without removing n-alkanes from the oil saturated hydrocarbon fraction. The GPC behavior of steroid and triterpenoid hydrocarbons was controlled by molecular size and shape.

The geochemistry of aliphatic and polar organic tracers in sediments from Lake Bera, Malaysia

Abstract The molecular distributions of homologous aliphatic lipid compounds ( n -alkanes, n -alkanoic acids, n -alkanols, n -alkanals and n -alkan-2-ones) were investigated in sediments of Lake Bera, Malaysia as well as in plant waxes from the vicinity using gas chromatography–mass spectrometry. In addition, triterpenoids of the oleanane, ursane, lupane and friedelane series, and their diagenetic products were also identified. Using the aquatic proxy P aq , it was found that the n -alkanes present in surface sediments S1, S5 and S6 correspond to an input from emergent macrophytes, whereas in sediments collected from open water (S2, S3 and S4) they correspond to input from submerged/floating macrophytes. The presence of n -alkan-2-ols, a commonly found homologous series in the sediments as well as in the plant wax, is novel here. In addition, the presence of 24-methylcycloart-22-enol and 24-ethylcycloart-22-enol in sediments as well as the plant wax of Pandanus helicopus , a dominant reed around the lake, also supports the input of plant biomarkers to the sediments. The occurrence of 5-membered ring triterpenoid derivatives as major diagenetic products of the triterpenoids identified (e.g. friedelan-2,3-dione) shows that the degradation of the organic matter in the sediments occurs mainly under oxic conditions. Also, the presence of ring-A degraded triterpenoids (dihydroputranjivic acid, 3-nor-2,4-seco-friedelane, 4α- and 4β-3-norfriedelanes, A-norfriedel-10-one, and A-norfriedel-8-en-10-one) and the aromatized triterpenoid hydrocarbons (mono- and triaromatic derivatives) as minor diagenetic products indicates photochemical and microorganism-mediated alteration reactions of natural products in the sediments. Additional novel polyfunctional friedelane derivatives, namely friedelane-2,3-dione, friedelane-1,3-diol-2-one and friedel-1(10)-ene-1,3-diol-2-one were identified for the first time in this study. These novel polyfunctional triterpenoids were inferred to derive from the oxidation of the triterpenoids from higher plants in this oxic tropical environment. For future reference purposes, the mass spectra of the identified polar triterpenoids are also presented here. This study provides important data on the biogeochemistry of organic matter in a tropical lake of SE Asia.

Wavelength specificity of growth, photosynthesis, and hydrocarbon production in the oil-producing green alga Botryococcus braunii

The effect of monochromatic light on growth, photosynthesis, and hydrocarbon production was tested in Botryococcus braunii Bot-144 (race B), which produces triterpenoid hydrocarbons. The growth was higher in order of red, blue, and green light. The color of red light-grown cells became more orange–yellow and their shape dominantly changed to grape-like with long branches. Photosynthetic carbon fixation activity was higher in order of blue, red, and green light-grown cells, but photosystem activities showed no difference. In the pulse-chase experiments with 14CO2, no major difference was observed in the production of lipids, hydrocarbons, polysaccharides, or proteins among the three kinds of cells, although hydrocarbon production was slightly lower in green light-grown cells. These results indicate that blue and red light were more effective for growth, photosynthetic CO2 fixation, and hydrocarbon production than green light, and that red light is the most efficient light source when calculated based on photoenergy supplied.

ChemInform Abstract: Fern Constituent: A New Triterpenoid Hydrocarbon, Trisnorhopane, Isolated from the Leaves of Dryopteris crassirhizoma and Gleichenia japonica.

From the leaflets of Dryopteris crassirhizoma, a new triterpenoid hydrocarbon, trisnorhopane (1) was isolated together with fern-7-ene (2), neohop-12-ene (3), fern-9(11)-ene (4) and hop-22(29)-ene (5). Compound 1 was also isolated from the leaflets of Gleichenia japonica, together with 4, 5 and neohop-13(18)-ene (6). The structure of 1 was elucidated on the basis of spectral data.

A comparison of thermal maturity parameters between freely extracted hydrocarbons (Bitumen I) and a second extract (Bitumen II) from within the kerogen matrix of Permian and Triassic sedimentary rocks

In this study we compared various maturity dependent aromatic, steroid and triterpenoid hydrocarbon ratios in bitumens that are freely extractable from sedimentary rocks (Bitumen I) with those in second extracts that comprised hydrocarbons closely associated with the kerogen/mineral matrix (Bitumen II). Bitumen II was released through kerogen isolation and demineralisation using HCl and HF/H 3BO 3. The samples studied, although of similar age, come from different localities. They represent a range of facies and two kerogen types (II and III), and all were deposited under marine conditions. The results show that the more stable β isomers of methylnaphthalene (MN) and methylphenanthrene (MP) are relatively more abundant in Bitumen II. The difference between the methylnaphthalene ratio (MNR) of Bitumens I and II, when plotted against the ratio of clay to total organic carbon (clay/TOC), gives an excellent correlation ( R 2 = 0.88). The highest clay/TOC ratio corresponds with the biggest difference in MNR for the respective bitumens, consistent with the more stable β isomers being preferentially retained within the clay/kerogen matrix. Since the β MP isomers are higher in Bitumen II than in Bitumen I, it was anticipated that their methylphenanthrene index (MPI-1) values, a commonly used measure of thermal maturity, would also be different. For most samples, the measured MPI-1 in Bitumen I is generally higher than that in Bitumen II (except for three samples), thus showing a bias towards a lower thermal maturity for the second extract. The opposite is true of the β/α MP ratio and MNR. This may be attributed to the fact that phenanthrene (P), which is part of the denominator in MPI-1, is more stable than its methylated counterparts. Steroid and triterpenoid maturity parameters also showed differences between the two extracts, with C 27 diasterane/sterane and Ts/(Ts + Tm) being higher in Bitumen I. Only the former parameter is positively correlated with the clay/TOC ratio, suggesting that clay content probably influences the diagenetic transformation of steroid precursors to diasteranes. The observed differences between the maturity parameters of Bitumen I and Bitumen II may be especially important in sedimentary rocks that contain high percentages of clay minerals.

Mono- and dicyclic unsaturated triterpenoid hydrocarbons in sediments from Lake Masoko (Tanzania) widely extend the botryococcene family

A mixture of C 33–C 37 botryococcenes and partially reduced derivatives was isolated from ca. 32,000 year old sediment from Lake Masoko, a freshwater crater lake in the Rungwe Range area (Tanzania). Botryococcenes and derivatives accounted for 246 μg/g dry sediment and for >92% of the hydrocarbon fraction; 1D and 2D nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry allowed the structure of the dominant botryococcene (43% of hydrocarbon fraction) to be established, after purification using high performance liquid chromatography (HPLC). The compound is a novel tetraunsaturated dicyclic C 34 botryococcene and is named C 34 masokocene. Overall, the structures of six other novel botryococcenes and four partially reduced derivatives were tentatively assigned. The structures of the new biomarkers, three dicyclic C 34–C 36 botryococcenes (or masokocenes) and seven monocyclic C 34–C 37 analogues are discussed along with their biosynthetic relationship. The high abundance of such polyunsaturated compounds preserved in 32,000 year old sediment from the lake indicates an aquatic ecosystem dominated at the time by the green alga Botryococcus braunii, as well very good preservation of the organic matter.

Occurrence of squalene and cholesterol in various species of Czech freshwater fish

The triterpenoid hydrocarbon squalene, C<sub>30</sub>H<sub>50</sub>, abundantly occurring in nature, is known as a substance with a high anti-tumour activity proven by a number of research studies. A high content of squalene was found mainly in shark liver oil and also in olive oil and amaranth seed oil. Our work was aimed at examining squalene contents in fresh water fish. Altogether 20 fish species were analysed. Squalene was determined in the unsaponifiable matter of muscular and visceral fat by a capillary gas chromatography method using a flame ionisation detector; the analysis of the unsaponifiable matter was augmented by cholesterol assay. The quantity of squalene found in muscular and visceral fat of individual fish averaged hundreds of mg/kg, ranging from 98.0 to 1536.8 mg/kg in muscular fat and from 70.1 to 1803.8 mg/kg in visceral fat. Muscular cholesterol amounted from 0.011% to 0.170% and visceral cholesterol from 0.104% to 0.297%.

Steroids, triterpenoids and molecular oxygen.

There is a close connection between modern-day biosynthesis of particular triterpenoid biomarkers and presence of molecular oxygen in the environment. Thus, the detection of steroid and triterpenoid hydrocarbons far back in Earth history has been used to infer the antiquity of oxygenic photosynthesis. This prompts the question: were these compounds produced similarly in the past? In this paper, we address this question with a review of the current state of knowledge surrounding the oxygen requirement for steroid biosynthesis and phylogenetic patterns in the distribution of steroid and triterpenoid biosynthetic pathways. The hopanoid and steroid biosynthetic pathways are very highly conserved within the bacterial and eukaryotic domains, respectively. Bacteriohopanepolyols are produced by a wide range of bacteria, and are methylated in significant abundance at the C2 position by oxygen-producing cyanobacteria. On the other hand, sterol biosynthesis is sparsely distributed in distantly related bacterial taxa and the pathways do not produce the wide range of products that characterize eukaryotes. In particular, evidence for sterol biosynthesis by cyanobacteria appears flawed. Our experiments show that cyanobacterial cultures are easily contaminated by sterol-producing rust fungi, which can be eliminated by treatment with cycloheximide affording sterol-free samples. Sterols are ubiquitous features of eukaryotic membranes, and it appears likely that the initial steps in sterol biosynthesis were present in their modern form in the last common ancestor of eukaryotes. Eleven molecules of O2 are required by four enzymes to produce one molecule of cholesterol. Thermodynamic arguments, optimization of function and parsimony all indicate that an ancestral anaerobic pathway is highly unlikely. The known geological record of molecular fossils, especially steranes and triterpanes, is notable for the limited number of structural motifs that have been observed. With a few exceptions, the carbon skeletons are the same as those found in the lipids of extant organisms and no demonstrably extinct structures have been reported. Furthermore, their patterns of occurrence over billion year time-scales correlate strongly with environments of deposition. Accordingly, biomarkers are excellent indicators of environmental conditions even though the taxonomic affinities of all biomarkers cannot be precisely specified. Biomarkers are ultimately tied to biochemicals with very specific functional properties, and interpretations of the biomarker record will benefit from increased understanding of the biological roles of geologically durable molecules.

Occurrences of sesqui-, di-, and triterpenoid hydrocarbons in the Tertiary oils and sediments from the Pacific side of Japan

Occurrences of sesqui-, di-, and triterpenoid hydrocarbons in the Tertiary oils and sediments from the Pacific side of Japan

Relative hopane content confirming the mineral origin of hydrocarbons contaminating foods and human milk

Hopanes, triterpenoid hydrocarbons formed under geological conditions, were analysed to confirm the mineral origin of the unresolved complex mixtures of hydrocarbons observed in the gas chromatography with flame ionization detection chromatograms of human milk and certain foodstuffs. The ‘relative hopane content’ (RHC) is introduced, i.e. it is the area ratio of the sum of the hopanes and the paraffins in the same segment of the chromatogram. The RHC in various mineral oil products (motor oils, hydraulic oils, lubricating oils, Vaseline) was 3.4%, with a relative standard deviation of 19%. The RHC determined in samples of vegetable oils, mussels and clams as well as of human milk containing an unresolved complex mixture of hydrocarbons was in the same range, confirming that these samples were contaminated by mineral oil material.

Biosynthesis of the triterpenoids, botryococcenes and tetramethylsqualene in the B race of Botryococcus braunii via the non-mevalonate pathway

The green microalga Botryococcus braunii race B accumulates two types of triterpenoid hydrocarbons, botryococcenes and tetramethylsqualene. Both triterpenoids are synthesized via the non-mevalonate pathway.

Molecular Characterization of Squalene Synthase from the Green Microalga Botryococcus braunii, Race B

The green microalga Botryococcus braunii produces large amounts of liquid hydrocarbons and is classified into three races, depending on the type of the hydrocarbon produced. The B race produces two types of triterpenoid hydrocarbons, squalene and botryococcene, both of which are putative condensation products of farnesyl diphosphate. In an attempt to better understand the regulation involved in the production of squalene and botryococcene, we have isolated and characterized a squalene synthase (SS) gene from the B race of B. braunii. A 366-bp cDNA fragment was initially obtained from the B race utilizing a reverse transcription/polymerase chain reaction and degenerate primers based on conserved amino acid sequences found in all SS enzymes. Using this putative SS fragment as a probe, a 2632-bp cDNA clone was isolated from a cDNA library. This cDNA contained an open reading frame coding for a protein with 461 amino acids and a predicted molecular mass of 52.5 kDa. Comparison of the Botryococcus SS (BSS) with SS from different organisms showed 52% identity with Nicotiana tabacum, 51% with Arabidopsis thaliana, 48% with Zea mays, 40% with rat, 39% with yeast, and 26% with Zymomonas mobilis. Expression of full-length and carboxy-terminus truncated BSS cDNA in Escherichia coli resulted in significant levels of bacterial SS enzyme activity but no botryococcene synthase activity. RNA blot hybridization analysis of algal cultures during a culture cycle indicated that BSS gene expression is preferential during rapid growth. Given that the DNA blot analysis indicated only a single copy of the SS gene in the algal genome, these results suggest either that there exists coordinate expression of separate synthase genes for squalene and botryococcene biosynthesis or that there might be unique physiological conditions controlling the SS vs botryococcene synthase activity of a single peptide species.

Distribution Patterns of Pentacyclic Triterpenoid Hydrocarbons in Low‐Mature Source Rocks from Eastern China

Abstract: Four typical distribution patterns of pentacyclic triterpenoid hydrocarbons (types A‐D) are distinguished in the low‐mature source rocks from eastern China. Type A has a relatively high content of pentacyclic triterpenes. It exists in immature sediments and the distribution and abundance of triterpenes vary with the maturity of the sediments. An unknown C30 triterpene (UCT2) has also been detected in very shallow sediments. This compound is very unstable and disappears rapidly with the increase of depth. Type B is characterized by a relatively high amount of 17α(H), 21β(H)‐30‐homohopane. This kind of distribution pattern is common in coals and terrestrial sediments of low maturity. Type C has a relatively high content of diahopane and neohopane series. The analysis shows that this distribution pattern may have an indirect relationship with the input of higher plants despite its microbial source. There are C30—unconfirmed triterpane (UCT2) and a relatively high content of C35 hopane in type D. The distribution patterns of pentacyclic triterpenoids are varied under influences of biological sources, diagentic and sedimentary environments, thermal maturity etc. Therefore, they can provide useful information about the source rocks. Based on geochemical characteristics of the source rocks, the authors postulated that the type B and type C patterns are mainly related to suboxic‐anoxic environments. Type D indicates the reducing to anoxic environment, in which C30‐UCT3 may be related to some specific biological source in saline environments. The precursors and thermal evolution of some triterpenoid hydrocarbons are also discussed herein.

Fernenes and other triterpenoid hydrocarbons in Dicroidium-bearing Triassic mudstones and coals from South Australia

Tandem sector mass spectrometry of the aliphatic hydrocarbon fraction of selected Late Triassic sub-bituminous coals, mudstones and plant fossils from the Leigh Creek coalfield, Telford Basin, South Australia, has revealed three pseudohomologous series of triterpenoid hydrocarbons. Two of these series have been identified as fern-8-enes (C 29–C 31) and fern-9(11)-enes (C 29–C 30). The third series (C 29–C 31) comprises saturated hydrocarbons tentatively identified as fernanes. These hydrocarbons appear to be related to pteridosperms (seed ferns) of the genus Dicroidium, fronds of which are preserved throughout the sedimentary sequence.

Fern constituent: Preoleanatetraene, a novel bicyclic triterpenoid hydrocarbon from Polypodiodes formosana

A novel bicyclic triterpenoid hydrocarbon, preoleanatetraene ( 1) was isolated from the fresh rhizomes of Polypodiodes formosana and the structure of 1 was established by spectral data. A new biogenetic pathway for the oleanane triterpenoids in this fern is proposed.

Novel unsaturated triterpenoid hydrocarbons from sediments of Sacred lake, Mt.Kenya, Kenya

A novel family of three isoprenoidal hydrocarbons ( 1–3), a C 34H 64 and two C 33H 60, have been isolated from lacustrine sediment deposited 5,000–18,000 yrs BP ( 14C dating) and their structures and stereochemistry determined from the interpretation of 1D and 2D 1H and 13C NMR and mass spectral data as well as degradation by ozonolysis. These compounds are attributed to algae, based on their close structural relationship with botryococcenes and other isoprenoidal hydrocarbons produced by the green microalga Botryococcus braunii.

Anti-tumor-promoting activities of triterpenoids from ferns. I.

To search for possible anti-tumor-promoters (cancer chemopreventive agents), we carried out primary screening of 23 triterpenoid hydrocarbons (1-23) isolated from ferns using an in vitro synergistic assay system. Of these triterpenoids, hop-17(21)-ene (2), neohop-13(18)-ene (3), neohop-12-ene (4), taraxerane (17), multiflor-9(11)-ene (18), multiflor-8-ene (19), glutin-5(10)-ene (21) and taraxastane (23) exhibited remarkable inhibitory effects on Epstein-Barr virus (EBV) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Further, compounds 2 and 3 exhibited remarkable anti-tumorpromoting effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test using 7,12-dimethybenz[a]anthracene (DMBA) as an initiator and TPA as a promoter.