Characterization of the cholesterol biosynthetic pathway in Dioscorea transversa.

Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1ß-hydroxyprotoneogracillin and protoneogracillin. Here, we used D. transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D. transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28 double bonds required for phytosterol biogenesis as well as Δ24,25 double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that the D. transversa sterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites.

Construction of a Pickering interfacial biocatalysis system in skim milk and enzymatic transesterification for enhancement of flavor and quality.

Despite considerable research efforts, lipase catalysis in a fluid milk system with aqueous multi-component mixtures containing multiple microphases, remains challenging. Pickering interfacial biocatalysis (PIB) platforms are typically fabricated with organic solvents/lipids and water. Whether a PIB with excellent catalytic performance can be constructed in complex milk mixtures remains unknown. Here, we challenged PIB with skim milk, and a small amount of flaxseed oil, and phytosterols as a model system for transesterification and lipolysis to enhance quality and flavor. The amino-modified mesoporous silica spheres (MSS-N) were employed as an emulsifier and carrier of lipase AYS (AYS@MSS-N). The conversion of phytosterol esters reached 75.5% at 1.5 h and prepared phytosterol ester-fortified milk with a content of 1.0 g/100 mL. The relative conversion rate remained above 70% after 6 cycles. In addition, the fortified milk showed an intensified and favorable effect on sensory traits through volatile flavor composition analysis. The findings provide a versatile alternative for PIB applications in complex environments, i.e., milk, which might inspire a new bioprocess strategy for dairy products.

Efficient Production of 9,22-Dihydroxy-23,24-bisnorchol-4-ene-3-one from Phytosterols by Modifying Multiple Genes in Mycobacterium fortuitum.

C19 steroids and C22 steroids are vital intermediates for the synthesis of steroid drugs. Compared with C19 steroids, C22 steroids are more suitable for synthesizing progesterone and adrenocortical hormones, albeit less developed. 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one(9-OHBA), due to its substituents at positions C-9 and C-22, is a beneficial and innovative steroid derivative for synthesizing corticosteroids. We focused on the C22 pathway in Mycobacterium fortuitum ATCC 35855, aiming to develop a productive strain that produces 9-OHBA. We used a mutant strain, MFΔkstD, that knocked out kstds from Mycobacterium fortuitum ATCC 35855 named MFKD in this study as the original strain. Hsd4A and FadA5 are key enzymes in controlling the C19 metabolic pathway of steroids in Mycobacterium fortuitum ATCC 35855. After knocking out hsd4A, MFKDΔhsd4A accumulated 81.47% 9-OHBA compared with 4.13% 9-OHBA in the strain MFKD. The double mutant MFKDΔhsd4AΔfadA5 further improved the selectivity of 9-OHBA to 95.13%, and 9α-hydroxy-4-androstenedione (9-OHAD) decreased to 0.90% from 4.19%. In the end, we obtained 6.81 g/L 9-OHBA from 10 g/L phytosterols with a molar yield of 80.33%, which showed the best performance compared with formerly reported strains.

An Unprecedented 4,8-Cycloeudesmane, Further New Sesquiterpenoids, a Triterpene, Steroids, and a Lignan from the Resin of Commiphora myrrha and Their Anti-Inflammatory Activity In Vitro.

Myrrh has a long tradition in the treatment of inflammatory diseases. However, many of its (active) constituents are still unknown. In the present study, secondary metabolites were isolated from an ethanolic extract by various separation methods (liquid-liquid partition, silica and RP18 flash chromatography, CPC, and preparative HPLC), their structures were elucidated with NMR spectroscopy and mass spectrometry, and the selected compounds were tested for their effect on LPS-induced NO production by RAW 264.7 murine macrophages. Among the isolated substances are 17 sesquiterpenes (1-17) including the first 4,8-cycloeudesmane (1), a triterpene (38), two phytosterols (39, 40) and one lignan (43), which were previously unknown as natural products. Numerous compounds are described for the first time for the genus Commiphora. Eight of the eleven compounds tested (1, 29, 31, 32, 34-37) showed a statistically significant, concentration-dependent weak to moderate anti-inflammatory effect on NO production in the LPS-stimulated RAW 264.7 macrophages in vitro. For the reference substance, furanoeudesma-1,3-diene, an IC50 of 46.0 µM was determined. These sesquiterpenes might therefore be part of the multi-target molecular principles behind the efficacy of myrrh in inflammatory diseases.

Chitosan-coated soybean protein isolate/lecithin nanoparticles for enhancing the stability and bioaccessibility of phytosterol.

BACKGROUND: Phytosterols (PS) have various beneficial effects on human health, especially the property of reducing blood cholesterol. However, the low solubility and bioaccessibility of PS have greatly limited their application in functional food ingredients. RESULTS: To improve the bioaccessibility and stability of PS, chitosan-coated PS nanoparticles (CS-PNP) were successfully prepared by self-assembly. The properties of CS-PNP, including size, zeta potential, encapsulation efficiency (EE) and loading amount (LA) were characterised. The optimisation of CS concentration (0.4 mg mL-1) and pH (3.5) resulted in the formation of CS-PNP with an EE of over 90% and a particle size of 187.7 nm. Due to the special properties of CS chitosan, the interaction between CS and soybean protein isolate (SPI)/lecithin (SL) led to the formation of a soluble complex. CS-PNP exhibited good stability to temperature variations but was more sensitive to salt ions. During in vitro digestion, CS efficiently maintained the stability of nanoparticles against the hydrolysis of SPI by pepsin under acidic conditions. However, these nanoparticles tended to aggregate in a neutral intestinal environment. After 3 h of in vitro digestion, the bioaccessibility of PS increased from 18.2% of free PS to 63.5% of CS-PNP. CONCLUSION: Overall, these results highlight the potential of chitosan-coated nanoparticles as effective carriers for the oral administration of PS. This multilayer construction may serve as a promising for applications in food products as delivery vehicles for nutraceuticals. © 2024 Society of Chemical Industry.

Improving the production of 9α-hydroxy-4-androstene-3,17-dione from phytosterols by 3-ketosteroid-Δ1-dehydrogenase deletions and multiple genetic modifications in Mycobacterium fortuitum.

BACKGROUND: 9α-hydroxyandrost-4-ene-3,17-dione (9-OHAD) is a significant intermediate for the synthesis of glucocorticoid drugs. However, in the process of phytosterol biotransformation to manufacture 9-OHAD, product degradation, and by-products restrict 9-OHAD output. In this study, to construct a stable and high-yield 9-OHAD producer, we investigated a combined strategy of blocking Δ1­dehydrogenation and regulating metabolic flux. RESULTS: Five 3-Ketosteroid-Δ1-dehydrogenases (KstD) were identified in Mycobacterium fortuitum ATCC 35855. KstD2 showed the highest catalytic activity on 3-ketosteroids, followed by KstD3, KstD1, KstD4, and KstD5, respectively. In particular, KstD2 had a much higher catalytic activity for C9 hydroxylated steroids than for C9 non-hydroxylated steroids, whereas KstD3 showed the opposite characteristics. The deletion of kstDs indicated that KstD2 and KstD3 were the main causes of 9-OHAD degradation. Compared with the wild type M. fortuitum ATCC 35855, MFΔkstD, the five kstDs deficient strain, realized stable accumulation of 9-OHAD, and its yield increased by 42.57%. The knockout of opccr or the overexpression of hsd4A alone could not reduce the metabolic flux of the C22 pathway, while the overexpression of hsd4A based on the knockout of opccr in MFΔkstD could remarkably reduce the contents of 9,21 ­dihydroxy­20­methyl­pregna­4­en­3­one (9-OHHP) by-products. The inactivation of FadE28-29 leads to a large accumulation of incomplete side-chain degradation products. Therefore, hsd4A and fadE28-29 were co-expressed in MFΔkstDΔopccr successfully eliminating the two by-products. Compared with MFΔkstD, the purity of 9-OHAD improved from 80.24 to 90.14%. Ultimately, 9­OHAD production reached 12.21 g/L (83.74% molar yield) and the productivity of 9-OHAD was 0.0927 g/L/h from 20 g/L phytosterol. CONCLUSIONS: KstD2 and KstD3 are the main dehydrogenases that lead to 9-OHAD degradation. Hsd4A and Opccr are key enzymes regulating the metabolic flux of the C19- and C22-pathways. Overexpression of fadE28-29 can reduce the accumulation of incomplete degradation products of the side chains. According to the above findings, the MF-FA5020 transformant was successfully constructed to rapidly and stably accumulate 9-OHAD from phytosterols. These results contribute to the understanding of the diversity and complexity of steroid catabolism regulation in actinobacteria and provide a theoretical basis for further optimizing industrial microbial catalysts.

Phytosterol conversion into C9 non-hydroxylated derivatives through gene regulation in Mycobacterium fortuitum.

Androst-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) are important drug intermediates that can be biosynthesized from phytosterols. However, the C9 hydroxylation of steroids via 3-ketosteroid 9α-hydroxylase (KSH) limits AD and 4-HBC accumulation. Five active KshAs, the oxidation component of KSH, were identified in Mycobacterium fortuitum ATCC 35855 for the first time. The deletion of kshAs indicated that the five KshA genes were jointly responsible for C9 hydroxylation during phytosterol biotransformation. MFKDΔkshA, the five KshAs deficient strain, blocked C9 hydroxylation and produced 5.37 g/L AD and 0.55 g/L 4-HBC. The dual function reductase Opccr knockout and 17ß-hydroxysteroid dehydrogenase Hsd4A enhancement reduced 4-HBC content from 8.75 to 1.72% and increased AD content from 84.13 to 91.34%, with 8.24 g/L AD being accumulated from 15 g/L phytosterol. In contrast, hsd4A and thioesterase fadA5 knockout resulted in the accumulation of 5.36 g/L 4-HBC from 10 g/L phytosterol. We constructed efficient AD (MFKDΔkshAΔopccr_hsd4A) and 4-HBC (MFKDΔkshAΔhsd4AΔfadA5) producers and provided insights for further metabolic engineering of the M. fortuitum ATCC 35855 strain for steroid productions. KEY POINTS: • Five active KshAs were first identified in M. fortuitum ATCC 35855. • Deactivation of all five KshAs blocks the steroid C9 hydroxylation reaction. • AD or 4-HBC production was improved by Hsd4A, FadA5, and Opccr modification.

Heterologous expression of Chlorophytum borivilianum Squalene epoxidase in tobacco modulates stigmasterol production and alters vegetative and reproductive growth.

KEYMESSAGE: CbSE overexpression increased stigmasterol levels and altered plant morphology. The genes upstream and downstream of CbSE were found to be upregulated, which confirms its regulatory role in the saponin biosynthetic pathway. Chlorophytum borivilianum is a high-value medicinal plant with many promising preclinical applications that include saponins as a major active ingredient. Squalene epoxidase (SE) is one of the major rate-limiting enzymes of the saponin biosynthetic pathway. Here, we functionally characterized C. borivilianum SE (CbSE) by over-expressing heterologously in Nicotiana tabacum. The heterologous expression of CbSE resulted in stunted pant growth with altered leaf and flower morphology. Next, RT-qPCR analysis of transgenic plants overexpressing CbSE revealed increased expression levels of Cycloartenol synthase (CAS), Beta amyrin synthase (ßAS), and cytochrome P450 monooxygenase 51 (CYP51) (Cytochrome P450), which encode key enzymes for triterpenoid and phytosterol biosynthesis in C. borivilianum. Further, Methyl Jasmonate (MeJa) treatment upregulated Squalene synthase (SQS), SE, and Oxidosqualene cyclases (OSCs) to a significant level. GC-MS analysis of the leaf and hairy roots of the transformants showed an increased stigmasterol content (0.5-1.0 fold) compared to wild type (WT) plants. These results indicate that CbSE is a rate-limiting gene, which encodes an efficient enzyme responsible for phytosterol and triterpenoid production in C. borivilianum.

Identification of Incomplete Annotations of Biosynthesis Pathways in Rhodophytes Using a Multi-Omics Approach.

Rhodophytes (red algae) are an important source of natural products and are, therefore, a current research focus in terms of metabolite production. The recent increase in publicly available Rhodophyte whole genome and transcriptome assemblies provides the resources needed for in silico metabolic pathway analysis. Thus, this study aimed to create a Rhodophyte multi-omics resource, utilising both genomes and transcriptome assemblies with functional annotations to explore Rhodophyte metabolism. The genomes and transcriptomes of 72 Rhodophytes were functionally annotated and integrated with metabolic reconstruction and phylogenetic inference, orthology prediction, and gene duplication analysis to analyse their metabolic pathways. This resource was utilised via two main investigations: the identification of bioactive sterol biosynthesis pathways and the evolutionary analysis of gene duplications for known enzymes. We report that sterol pathways, including campesterol, ß-sitosterol, ergocalciferol and cholesterol biosynthesis pathways, all showed incomplete annotated pathways across all Rhodophytes despite prior in vivo studies showing otherwise. Gene duplication analysis revealed high rates of duplication of halide-associated haem peroxidases in Florideophyte algae, which are involved in the biosynthesis of drug-related halogenated secondary metabolites. In summary, this research revealed trends in Rhodophyte metabolic pathways that have been under-researched and require further functional analysis. Furthermore, the high duplication of haem peroxidases and other peroxidase enzymes offers insight into the potential drug development of Rhodophyte halogenated secondary metabolites.

Bioactives from Crude Rice Bran Oils Extracted Using Green Technology.

Crude rice bran oils from different rice cultivars and extraction methods bear different contents of nutraceuticals. The health benefits of lowering cholesterol activity of rice bran oil being confirmed by many reports are partly attributed to non-nutrient nutraceuticals, especially γ-oryzanol, phytosterols, and policosanols. As the world has been facing the global warming crisis, green extraction technology is gaining attention from many sectors. The current study aims to compare the nutraceutical composition with respect to γ-oryzanol, phytosterol, and policosanol content as well as the antioxidant properties of crude rice bran oils extracted from white and red rice bran using three green technologies, comparing with conventional hexane extraction. The data show that the traditional solvent extraction gave the highest oil yield percentage (26%), but it was not significantly different from subcritical liquefied dimethyl ether extraction (24.6%). Subcritical liquefied dimethyl ether extraction gave higher oil yield than supercritical CO2 extraction (15.5-16.2%). The crude rice bran oil extracted using subcritical liquefied dimethyl ether extraction produced the highest total phenolic contents and antioxidant activities. The highest γ-oryzanol content of the crude rice bran oil was found in oil extracted by conventional cold press (1370.43 mg/100 g). The γ-oryzanol content of the oil obtained via subcritical liquefied dimethyl ether extraction was high (1213.64 mg/100 g) compared with supercritical CO2 extraction. The red rice bran yielded the crude rice bran oil with the highest total phytosterol content compared with the white bran, and the oil from red rice bran extracted with subcritical liquefied dimethyl ether generated the highest total phytosterol content (1784.17 mg/100 g). The highest policosanol content (274.40 mg/100 g) was also found in oil obtained via subcritical liquefied dimethyl ether extraction.

Study on the synthesis of pine sterol esters in solvent-free systems catalyzed by Candida rugosa lipase immobilized on hydrophobic macroporous resin.

BACKGROUND: Pine sterol ester is a type of novel food source nutrient with great advantages in lowering blood cholesterol levels, inhibiting tumors, preventing prostate enlargement, and regulating immunity. Macroporous resins with large specific surface area, stable structures, and various functional groups (epoxy, amino, and octadecyl groups) have been selected for immobilization of Candida rugosa lipase (CRL) to improve its stability and efficiency in the synthesis of pine sterol esters. A solvent-free strategy using oleic acid (substrate) as an esterification reaction medium is an important alternative for avoiding the use of organic solvents. RESULTS: The immobilization conditions of CRL immobilized on several types of commercial macroporous resins were optimized. Fortunately, by adsorption (hydrophobic interaction), a high immobilization efficiency of CRL was obtained using macroporous resins with hydrophobic octadecyl groups with an immobilization efficiency of 86.5%, enzyme loading of 138.5 mg g-1 and enzyme activity of 34.7 U g-1 . The results showed that a 95.1% yield could be obtained with a molar ratio of oleic acid to pine sterol of 5:1, an enzyme amount of 6.0 U g-1 (relative to pine sterol mass) at 50 °C for 48 h. CONCLUSION: The hydrophobic macroporous resin (ECR8806M) with a large specific surface area and abundant functional groups was used to achieve efficient immobilization of CRL. CRL@ECR8806M is an efficient catalyst for the synthesis of phytosterol esters and has the potential for further large-scale applications. Therefore, this simple, green, and low-cost strategy for lipase immobilization provides new possibilities for the high-efficiency production of pine sterol esters and other food source nutrients. © 2023 Society of Chemical Industry.

Protective effect of surfactant modified phytosterol oleogels on loaded curcumin.

BACKGROUND: Oleogels represent one of the most important carriers for the delivery of lipophilic nutraceuticals. Phytosterols (PS), plant-derived natural sterol compounds, are preferred for oleogel preparation due to their self-assembly properties and health function. However, the relationship between the physical properties of PS-based oleogels and the chemical stability of loaded bioactive compounds is still unclear. RESULTS: The influence of lecithin (LC) and glycerol monostearate (GMS) on the physical properties of PS-based oleogels made of liquid coconut oil and the stability of curcumin as a model bioactive loaded in the oleogels was investigated. Results showed that the flow consistency index was much higher for GMS-containing oleogels than that for LC-containing oleogels. The optical microscopy and X-ray scattering analysis showed that the addition of GMS in the PS oleogels promoted the formation of a crystal mixture with different crystal polymorph structures, whereas LC addition promoted the formation of needle-like crystals of PS. Using curcumin as a model lipophilic nutraceutical, the GMS-enriched PS oleogels with high crystallinity and flow consistency index exhibited a good retention ratio and scavenging activity of the loaded curcumin when stored at room temperature. CONCLUSION: This study shows that enhancing the firmness of oleogels made from PS and liquid coconut oil is beneficial to the retention and chemical stability of a loaded bioactive (curcumin). The findings of the study will boost the development of PS-based oleogel formulations for lipophilic nutraceutical delivery. © 2022 Society of Chemical Industry.

Soy proteins as vehicles for enhanced bioaccessibility and cholesterol-lowering activity of phytosterols.

BACKGROUND: The formulation of phytosterol (PS)-enriched functional foods has attracted increasing interest in the recent years, owing to its potential health effects. However, the poor solubility and bioavailability greatly limit PS application in this regard. This study investigated whether soy protein isolate (SPI) could effectively perform as a nanocarrier for improving the water solubility, bioaccessibility, and cholesterol-lowering activity of PSs. RESULTS: In this work, we fabricated SPI-PS nanocomplexes, which not only can enhance the stability and bioaccessibility of PS, but also improve the cholesterol-lowering ability of SPI. This improvement was mainly due to the formation of protein-active substance complexes, through hydrophobic interactions. The complexation with PSs resulted in formation of nanosized particles with greater sizes, lower ζ-potential, and higher surface hydrophobicity. The encapsulation efficiency, loading amount, and solubility of PS were closely related to the applied PS concentration in the mixed dispersions, and the maximal PS solubility in the aqueous phase reached about 1.63 mg mL-1 at the highest PS concentration (2.0 mg mL-1 ). The PS molecules in the nanocomplexes were mainly present in the amorphous form. The enhanced in vitro cholesterol-lowering activity of PS nanocomplexes relative to free PS seemed to be closely related to its higher bioaccessibility. CONCLUSION: The findings are of relevance for the development of food-grade PS ingredients suitable for the formulations of PS-enriched functional foods. © 2022 Society of Chemical Industry.

Replacement of soybean oil by Hermetia illucens larvae fat in broiler diets alters the breast muscle lipidome and reduces lipid oxidation of the breast muscle during heat-processing.

Replacement of soybean oil by insect fat from Hermetia illucens (HI) has been reported to increase the proportions of saturated fatty acids (SFA) and decrease those of polyunsaturated fatty acids (PUFA) in total lipids of breast and thigh meat in broilers. Since the susceptibility of meat to oxidation is strongly dependent on its PUFA content, the present study hypothesised that replacement of soybean oil by HI larvae fat in broiler diets reduces the formation of lipid oxidation products, including oxidation products of cholesterol and phytosterols, in heat-processed breast muscle of broilers. To test this hypothesis, 100 male, 1-day-old Cobb 500 broilers were assigned to three groups and fed three different nutrient adequate diets, which varied only in the fat source (group HI-0: 0% HI larvae fat and 5% soybean oil; group HI-2.5: 2.5% HI larvae fat and 2.5% soybean oil; group HI-5.0: 5.0% HI larvae fat and 0% soybean oil), in a three-phase feeding system for 35 days. While the growth performance of the broilers was not different, the absolute and relative breast muscle weights were higher in group HI-5.0 than in group HI-0 (p < 0.05). The proportions of C12:0, C14:0, C14:1, C16:0, C16:1 and total SFA were higher and those of C18:1, C18:2 n-6, C18:3 n-3 and total PUFA were lower in breast muscle total lipids of group HI-5.0 than in groups HI-2.5 and HI-0 (p < 0.05). Lipidomic analysis of breast muscle revealed that the concentration of triacylglycerols was 46% and 53% lower in groups HI-2.5 and HI-5.0, respectively, than in group HI-0 (p < 0.05), whereas all other lipid classes detected did not differ among groups. Concentrations of thiobarbituric acid-reactive substances, 7α-hydroxycholesterol, 7ß-hydroxycholesterol and total cholesterol oxidation products in heat-processed breast muscle were lower in group HI-5.0 than in group HI-0 (p < 0.05). Concentrations of oxidation products of phytosterols in heat-processed breast muscle were generally much lower than those of cholesterol oxidation products and did not differ between the three groups of broilers. In conclusion, complete replacement of soybean oil with HI larvae fat in broiler diets strongly alters the fatty acid composition of breast muscle total lipids and reduce lipid oxidation of the breast muscle during heat-processing.

Triterpenoid esters from Capparis erythrocarpos (Isert), Capparaceae, root bark ameliorates CFA-induced arthritis.

The root bark of Capparis erythrocarpos (CERB) is employed to treat rheumatoid arthritis (RA) in Africa, particularly in Ghana. However, there was no isolation and characterization of the bioactive constituents responsible for the pharmacological actions of this plant. The aim of this study is to isolate, characterize and evaluate the anti-arthritic activity of the constituents of CERB. CERB was soxhleted and partitioned into various fractions. The constituents were isolated using column chromatography and characterized by 1D and 2D NMR spectroscopy. The precise carboxylic acid residues of the esters were determined using saponification, derivatization and GC-MS analysis. Anti-arthritic activity was evaluated in the CFA-induced arthritic model. Two triterpenoid esters namely, sitosterol 3-hexadecanoate or sitosterol 3-palmatate (1) and sitosterol 3-tetradecanoate or sitosterol 3-myristate (2) in addition to beta sitosterol (3) were isolated and characterized. Compounds 1 and 2 administered at 3 µmol/kg (p.o.) produced anti-inflammatory activity (P < 0.0001) of 31.02 and 39.14% respectively, in addition to arthritic score index (P < 0.0001) of 1.600 ± 0.2449 and 1.400 ± 0.2449 against CFA-induced arthritis which are equivalent to those of the standard drug, diclofenac sodium (DS), 3 µmol/kg (p.o.), (30.79% anti-inflammatory activity and 1.800 ± 0.3742 arthritic score index). The compounds produced similar anti-inflammatory effects as DS. Also, radiographical and histopathological studies showed that, the compounds and DS protected against bone destruction, inflammatory cells invasion into interstitial spaces and synovial liner hyperplasia of the joints. This is the first study to report the characterization of the constituents of C. erythrocarpos in addition to anti-arthritic activity of sitosterol 3-palmatate and sitosterol 3-myristate. These results provide the missing link between the chemistry and the pharmacological activities of C. erythrocarpos. The isolates also offer a different class of molecule which could provide alternative treatment for RA.

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation.

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph-mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

A review on chemical and physical modifications of phytosterols and their influence on bioavailability and safety.

Phytosterols have been shown to lower cholesterol levels and to have antioxidant, anti-inflammatory and other biological activities. However, the high melting point and poor solubility limit their bioavailability and practical application. It is advantageous to modify phytosterols chemically and physically. This article reviews and discusses the chemical and physical modifications of phytosterols, as well as their effects on the bioavailability and possible toxicity in vivo. The current research on chemical modifications is mainly focused on esterification to increase the oil solubility and water solubility. For physical modifications (mainly microencapsulation), there are biopolymer-based, surfactant-based and lipid-based nanocarriers. Both chemical and physical modifications of phytosterols can effectively increase the absorption and bioavailability. The safety of modified phytosterols is also an important issue. Phytosterol esters are generally considered to be safe. However, phytosterol oxides, which may be produced during the synthesis of phytosterol esters, have shown toxicity in animal models. The toxicity of nanocarriers also needs further studies.

Phytosterols and phytostanols and the hallmarks of cancer in model organisms: A systematic review and meta-analysis.

Phytosterols and phytostanols are natural products present in vegetable oils, nuts, and seeds, or added to consumer food products whose intake is inversely associated with incidence and prognosis of several cancers. Randomized cancer prevention trials in humans are unfeasible due to time and cost yet the cellular processes and signaling cascades that underpin anti-cancer effects of these phytochemicals have been explored extensively in vitro and in preclinical in vivo models. Here we have performed an original systematic review, meta-analysis, and qualitative interpretation of literature published up to June 2020. MEDLINE, Scopus, and hand-searching identified 408 unique records that were screened leading to 32 original articles that had investigated the effects of phytosterols or phytostanols on cancer biology in preclinical models. Data was extracted from 22 publications for meta-analysis. Phytosterols were most commonly studied and found to reduce primary and metastatic tumor burden in all cancer sites evaluated. Expression of pAKT, and markers of metastasis (alkaline phosphatase, matrix metalloproteases, epithelial to mesenchymal transcription factors, lung and brain colonization), angiogenesis (vascular endothelial growth factor, CD31), and proliferation (Ki67, proliferating cell nuclear antigen) were consistently reduced by phytosterol treatment in breast and colorectal cancer. Very high dose treatment (equivalent to 0.2-1 g/kg body weight not easily achievable through diet or supplementation in humans) was associated with adverse events including poor gut health and intestinal adenoma development. Phytosterols and phytostanols are already clinically recommended for cardiovascular disease risk reduction, and represent promising anti-cancer agents that could be delivered in clinic and to the general population at low cost, with a well understood safety profile, and now with a robust understanding of mechanism-of-action.

GC/EI-MS method for the determination of phytosterols in vegetable oils.

Sterols are a highly complex group of lipophilic compounds present in the unsaponifiable matter of virtually all living organisms. In this study, we developed a novel gas chromatography with mass spectrometry selected ion monitoring (GC/MS-SIM) method for the comprehensive analysis of sterols after saponification and silylation. A new referencing system was introduced by means of a series of saturated fatty acid pyrrolidides (FAPs) as internal standards. Linked with retention time locking (RTL), the resulting FAP retention indices (RIFAP) of the sterols could be determined with high precision. The GC/MS-SIM method was based on the parallel measurement of 17 SIM ions in four time windows. This set included eight molecular ions and seven diagnostic fragment ions of silylated sterols as well as two abundant ions of FAPs. Altogether, twenty molecular ions of C27- to C31-sterols with 0-3 double bonds were included in the final method. Screening of four common vegetable oils (sunflower oil, hemp oil, rapeseed oil, and corn oil) enabled the detection of 30 different sterols and triterpenes most of which could be identified.

The pharmacokinetic characteristics and excretion studies of fucosterol from Sargasssum fusiforme in rats.

Fucosterol is the main phytosterol in brown algae with various pharmacological effects such as cholesterol-lowering, anticancer, hepatoprotection and neuroprotection. Little is known about the pharmacokinetics and excretion characteristics of fucosterol. In this study, a GC-MS method was developed and validated for the determination of fucosterol in rat plasma, urine and feces. The method effectively avoids the interference of Δ5 -avenasterol, a cis-trans-isomer of fucosterol derived from feed, by using a TG-5 capillary column (a nonpolar column with 5% phenyl-methylpolysilicone as stationary phase material). The linearity ranges were fucosterol 0.300-18.0 µg/ml (R2 = 0.9960) for plasma, 0.0500-2.50 µg/ml for the urine sample (R2 = 0.9963) and 0.100-8.00 µg/mg (R2 = 0.9923) for the feces sample. With good extraction recoveries and stability, this rapid and sensitive method was successfully applied to the pharmacokinetic and excretion studies of fucosterol in Sprague-Dawley rats. Fucosterol from Sargassum fusiforme had poor absorption and slow elimination with an absolute oral bioavailability of 0.74%, and was mainly eliminated through fecal excretion.