Synthesis of a Side Chain Alkyne Analogue of Sitosterol as a Chemical Probe for Imaging in Plant Cells.

Clickable chemical tools are essential for studying the localization and role of biomolecules in living cells. For this purpose, alkyne-based close analogs of the respective biomolecules are of outstanding interest. Here, in the field of phytosterols, we present the first alkyne derivative of sitosterol, which fulfills the crucial requirements for such a chemical tool as follows: very similar in size and lipophilicity to the plant phytosterols, and correct absolute configuration at C-24. The alkyne sitosterol FB-DJ-1 was synthesized, starting from stigmasterol, which comprised nine steps, utilizing a novel alkyne activation method, a Johnson-Claisen rearrangement for the stereoselective construction of a branched sterol side chain, and a Bestmann-Ohira reaction for the generation of the alkyne moiety.

Preparation of phytosteryl ornithine ester hydrochloride and improvement of its bioaccessibility and cholesterol-reducing activity in vitro.

In this work, synthesis of phytosteryl ornithine ester hydrochloride was studied for the first time using an intermediate phytosteryl N,N'-bis[tert-butoloxycarbonyl(BOC)]-ornithine ester. This method also involved esterification of phytosterols with N,N'-bis(BOC)-ornithine and deprotection. The maximum yield was 90% and deprotection of BOC group was more than 99% using the HCl/ethyl acetate method. As a result, thermal stability and water solubility as well as emulsifying activity and stability of phytosterols were improved through coupling with ornithine, which is favorable for their application in water-based food systems. We also observed increased bioaccessibility of phytosteryl ornithine hydrochloride (4.5%) and 65% of phytosteryl ornithine hydrochloride was hydrolyzed in vitro. These results indicated that ornithine phytosteryl ester hydrochloride can reduce dissolution capacity of cholesterol in vitro, representing improved cholesterol-reducing activity, which will further expand the applications of phytosteryl ornithine ester hydrochloride.

Optimization and modeling for the synthesis of sterol esters from deodorizer distillate by lipase-catalyzed esterification.

In this paper, cotton seed oil deodorizer distillate (CSODD), was recovered to obtain fatty acid sterol ester (FASE), which is one of the biological activated substances added as human therapeutic to lower cholesterol. Esterification reactions were carried out using Candida rugosa lipase as a catalyst, and the conversion of phytosterol was optimized using response surface methodology. The highest conversion (90.8 ± 0.4%) was reached at 0.84 wt% enzyme load, 1:25 solvent/CSODD mass ratio, and 44.2 °C after 12 H reaction. A kinetic model based on the reaction rate equation was developed to describe the reaction process. The activation energy of the reaction was calculated to be 56.9 kJ/mol and the derived kinetic parameters provided indispensable basics for further study. The optimization and kinetic research of synthesizing FASE from deodorizer distillate provided necessary information for the industrial applications in the near future. Experimental results showed that the proposed process is a promising alternative to recycle sterol esters from vegetable oil deodorizer distillates in a mild, efficient, and environmental friendly method.

Structural Modifications of Deoxycholic Acid to Obtain Three Known Brassinosteroid Analogues and Full NMR Spectroscopic Characterization.

An improved synthesis route for obtaining known brassinosteroid analogues, i.e., methyl 2α,3α-dihydroxy-6-oxo-5α-cholan-24-oate (11), methyl 3α-hydroxy-6-oxo-7-oxa-5α-cholan-24-oate (15) and methyl 3α-hydroxy-6-oxa-7-oxo-5α-cholan-24-oate (16), from hyodeoxycholic acid (4) maintaining the native side chain is described. In the alternative procedure, the di-oxidized product 6, obtained in the oxidation of methyl hyodeoxycholate 5, was converted almost quantitatively into the target monoketone 7 by stereoselective reduction with NaBH4, increasing the overall yield of this synthetic route to 96.8%. The complete ¹H- and (13)C-NMR assignments for all compounds synthesized in this work have been made by 1D and 2D heteronuclear correlation gs-HSQC and gs-HMBC techniques. Thus, it was possible to update the spectroscopic information of ¹H-NMR and to accomplish a complete assignment of all (13)C-NMR signals for analogues 5-16, which were previously reported only in partial form.

Immobilization of Yarrowia lipolytica lipase Ylip2 for the biocatalytic synthesis of phytosterol ester in a water activity controlled reactor.

In this work, phytosterol ester was synthesized using Yarrowia lipolytica lipase Ylip2 that had been immobilized on inorganic support in a solvent-free system and reacted in a computer-aided water activity controlled bioreactor. The immobilization of Ylip2 on celite led to a remarkable increase in the phytosterol conversion compared to that of free lipase. An investigation of the reaction conditions were oleic acid as the fatty acid variety, 10,000U/g substrate, and a temperature of 50°C for phytosterol ester synthesis. Controlling of the water activity at a set point was accomplished by the introduction of dry air through the reaction medium at a digital feedback controlled flow rate. For the esterification of phytosterol ester, a low (15%) water activity resulted in a considerable improvement in phytosterol conversion (91.1%) as well as a decreased reaction time (78h). Furthermore, Ylip2 lipase immobilized on celite retained 90% esterification activity for the synthesis of phytosterol oleate after reused 8 cycles, while free lipase was only viable for 5 batches with 90% esterification activity remained. Finally, the phytosterol oleate space time yield increased from 1.65g/L/h with free lipase to 2.53g/L/h with immobilized lipase. These results illustrate that the immobilized Yarrowia lipolytica lipase Ylip2 in a water activity controlled reactor has great potential for the application in phytosterol esters synthesis.

Novel Synthesis of Phytosterol Ester from Soybean Sterol and Acetic Anhydride.

Phytosterols are important bioactive compounds which have several health benefits including reduction of serum cholesterol and preventing cardiovascular diseases. The most widely used method in the synthesis of its ester analogous form is the use of catalysts and solvents. These methods have been found to present some safety and health concern. In this paper, an alternative method of synthesizing phytosterol ester from soybean sterol and acetic anhydride was investigated. Process parameters such as mole ratio, temperature and time were optimized. The structure and physicochemical properties of phytosterol acetic ester were analyzed. By the use of gas chromatography, the mole ratio of soybean sterol and acetic anhydride needed for optimum esterification rate of 99.4% was 1:1 at 135 °C for 1.5 h. FTIR spectra confirmed the formation of phytosterol ester with strong absorption peaks at 1732 and 1250 cm(-1) , which corresponds to the stretching vibration of C=O and C-O-C, respectively. These peaks could be attributed to the formation of ester links which resulted from the reaction between the hydroxyl group of soybean sterol and the carbonyl group of acetic anhydride. This paper provides a better alternative to the synthesis of phytosterol ester without catalyst and solvent residues, which may have potential application in the food, health-care food, and pharmaceutical industries.

Oxyphytosterols as active ingredients in wheat bran suppress human colon cancer cell growth: identification, chemical synthesis, and biological evaluation.

Consumption of whole grains has been reported to be associated with a lower risk of colorectal cancer. Recent studies illustrated that phytochemicals in wheat bran (WB) may protect against colorectal cancer. There is a growing interest in the phytosterol contents of foods as either intrinsic or added components due to their beneficial health effects. However, little is known whether phytosterols in WB contribute the observed chemopreventative activity of the grain. In the present study, we directly purified and identified four oxyphytosterols 1-4 from sterol-enriched fraction of WB, and also successfully synthesized five sterol oxides 5-8 and 13. Using these nine compounds as references, we outlined a comprehensive profile of steroids in WB using tandem liquid chromatography mass spectrometry with electrospray ionization (LC-ESI/MS(n), n = 2-3) techniques for the first time. Among them, three sterol oxides 13, 14, and 18 are novel compounds, and 14 compounds 3, 4, 6-11, 13, 14, 16, and 18-20 were reported in WB for the first time. Our results on the inhibitory effects of available sterol oxides 1-8 and 13 against the growth of human colon cancer cells HCT-116 and HT-29 showed that compounds 2-8 exerted significant antiproliferative effects, with oxysterol 8 being the most active one in both cells. We further demonstrated that four most active sterol oxides 5-8 could induce cell death through the apoptosis pathway. Our results showed that phytosterols, particularly oxyphytosterols, in WB possess significant antiproliferative properties, and thereby may greatly contribute the observed chemoprevention of the whole grain wheat.

An expeditious synthesis of spinasterol and schottenol, two phytosterols present in argan oil and in cactus pear seed oil, and evaluation of their biological activities on cells of the central nervous system.

Spinasterol and schottenol, two phytosterols present in argan oil and in cactus pear seed oil, were synthesized from commercially available stigmasterol by a four steps reactions. In addition, the effects of these phytosterols on cell growth and mitochondrial activity were evaluated on 158N murine oligodendrocytes, C6 rat glioma cells, and SK-N-BE human neuronal cells with the crystal violet test and the MTT test, respectively. The effects of spinasterol and schottenol were compared with 7-ketocholesterol (7KC) and ferulic acid, which is also present in argan and cactus pear seed oil. Whatever the cells considered, dose dependent cytotoxic effects of 7KC were observed whereas no or slight effects of ferulic acid were found. With spinasterol and schottenol, no or slight effects on cell growth were detected. With spinasterol, reduced mitochondrial activities (30-50%) were found on 158N and C6 cells; no effect was found on SK-N-BE. With schottenol, reduced mitochondrial activity were revealed on 158N (50%) and C6 (10-20%) cells; no effect was found on SK-N-BE. Altogether, these data suggest that spinasterol and schottenol can modulate mitochondrial activity and might therefore influence cell metabolism.

Synthesis of steryl ferulates with various sterol structures and comparison of their antioxidant activity.

Steryl ferulates synthesised from commercial sterols as well as commercial oryzanol were used to better understand how structural features affect antioxidant activity in vitro by the ABTS(+) radical decolorization assay, by oxidative stability index (OSI) of soybean oil, and by analysis of antioxidant activity during frying. Steryl ferulates inhibited the ABTS(+) radical by 6.5-56.6%, depending on their concentration, but were less effective, especially at lower concentrations, than ferulic acid. Ferulic acid and steryl ferulates had either no effect, or lowered the OSI of soybean oil by up to 25%, depending on the concentration. In their evaluation as frying oil antioxidants, steryl ferulates with a saturated sterol group had the best antioxidant activity, followed by sterols with one double bond in the C5 position. The results indicate that a dimethyl group at C4 as well as a C9,C19 cyclopropane group, as found in oryzanol, negatively affects antioxidant activity in frying oils.

Production of the anti-inflammatory compound 6-O-palmitoyl-3-O-ß-D-glucopyranosylcampesterol by Callus cultures of Lopezia racemosa Cav. (Onagraceae).

Lopezia racemosa Cav. is a plant used in Mexican traditional medicine to heal inflammatory diseases. From this plant we isolated the novel compound 6-O-palmitoyl- 3-O-ß-D-glucopyranosylcampesterol (1) and 6-O-palmitoyl-3-O-ß-D-glucopyranosyl-ß-sitosterol (2), previously reported to have cytotoxic activity on several cancer cell lines. We evaluated the anti-inflammatory activity of 1 in vivo by mouse ear edema induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and 57.14% inhibition was observed. The aim of our study was to obtain callus cultures derived from this plant species with the ability to produce the compounds of interest. Callus cultures were initiated on MS basal medium amended with variable amounts of naphthaleneacetic acid (NAA), or 2,4-dichlorophenoxyacetic acid (2,4-D), combined or not with 6-benzylaminopurine (BAP). Ten treatments with these growth regulators were carried out, using in vitro germinated seedlings as source of three different explants: hypocotyl, stem node, and leaf. Highest yield of 1 was observed on callus derived from leaf explants growing in medium containing 1.0 mg/L 2,4-D and 0.5 mg/L BAP. Selected callus lines produced less 1 than wild plants but the in vitro cultured seedlings showed higher production. So we conclude that it could be attractive to further investigate their metabolic potential.

Design and straightforward synthesis of novel galloyl phytosterols with excellent antioxidant activity.

Novel galloyl phytosterols were rationally designed by incorporation of gallic acid into phytosterols through straightforward esterification. The esterification was successfully achieved by coupling of gallic acid and phytosterols through a mild chemical Steglich esterification reaction that is more straightforward than the enzymatic method. The identity of the newly synthesized galloyl phytosterols was confirmed by NMR, HPLC-MS and IR spectroscopies. Further evaluation of the novel galloyl phytosterols with radical scavenging, ferrous ion chelating, and Rancimat methods revealed its excellent antioxidant activities that are comparable to the most potent fat-soluble antioxidants. This novel antioxidant offers an intriguing solution for naturally derived antioxidants and will have great potential application as antioxidant in food industry. The methods developed in this study will be valuable for development of other phenolic phytosterols.

Synthesis of phytosteryl ester containing pinolenic acid in a solvent-free system using immobilized Candida rugosa lipase.

Phytosteryl ester synthesized with pinolenic acid (PLA) from pine nut oil is expected to have features of both phytosterol and PLA. In this study, lipase from Candida rugosa (CRL) was immobilized and then used to optimize conditions for synthesis of phytosteryl ester containing PLA. Lewatit VP OC 1600, a macroporous hydrophobic resin, was selected as the best carrier, and the optimum condition for the immobilization of CRL was established. With immobilized CRL prepared, synthesis of phytosteryl ester with fatty acid from pine nut oil was carried out. Parameters investigated were temperature, molar ratio (phytosterol to fatty acid), enzyme loading, and vacuum. Optimum conditions for synthesis of phytosteryl ester were a temperature of 60 °C, molar ratio of 1:4, enzyme loading of 10% (based on the total weight of the substrate), and pressure of 80 kPa. The maximum conversion of phytosteryl ester was ca. 93 mol % at the optimum condition.

Phytosteryl sinapates and vanillates: chemoenzymatic synthesis and antioxidant capacity assessment.

Phytosterols and their derivatives have attracted much attention because of their health benefits to humans and are widely used in food, pharmaceuticals, and cosmetics in the past decades. While most of the research has focused on free phytosterols and phytosteryl esters of fatty acids, few researches reported on phytosteryl phenolates, the esters of phytosterols with phenolic acids. Two novel group phytosteryl phenolates, namely phytosteryl sinapates and vanillates, were successfully chemoenzymatically synthesised in this work and their structures confirmed. Fourier transform infrared (FTIR) and high performance chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) using atmospheric pressure chemical ionisation (APCI) under both positive and negative ion modes were employed for this purpose. High antioxidant capacity of phytosteryl sinapates was observed using both oxygen radical absorbance capacity (ORAC) assay and cooked ground meat model system. Although phytosteryl vanillates showed lower antioxidant capacity than phytosteryl sinapates, they were stronger antioxidants than vanillic acid and vinyl vanillate in both assays employed. Conjugation of phytosterols with sinapic or vanillic acid rendered higher antioxidant capacity. Further studies on health benefits of phytosteryl sinapates and vanillates are necessary.

Novel synthesis strategy for the preparation of individual phytosterol oxides.

Sterols (cholesterol and phytosterols) are important structural components of cell membranes and major constituents of lipid metabolism. Research on their oxides, such as the factors affecting oxidation, oxides' structures, and qualitative and quantitative analysis, aroused more attention in this decade. However, the biological roles of individual phytosterol oxides are still unclear because no commercial individual phytosterol oxide standards are available. Different from the traditional chemical synthesis, in the present study, chemical synthesis from a starting phytosterol mixture followed with a semipreparative HPLC separation produced individual oxides. TLC and analytical HPLC were used here to not only monitor the reaction process but also specifically analyze the synthetic intermediates and oxides. The chromatographic results exhibited strict rules and similar characteristics. Finally, for the first time, four individual phytosterol oxides were successfully separated and collected by a semipreparative HPLC system, thus providing a novel strategy for the preparation of individual phytosterol oxides.

Synthesis and assessment of the relative toxicity of the oxidised derivatives of campesterol and dihydrobrassicasterol in U937 and HepG2 cells.

The cytotoxic effects of the oxidised derivatives of the phytosterols, stigmasterol and ß-sitosterol, have previously been shown to be similar but less potent than those of the equivalent cholesterol oxides in the U937 cell line. The objective of the present study was to compare the cytotoxic effects of the oxidised derivatives of synthetic mixtures of campesterol and dihydrobrassicasterol in both the U937 and HepG2 cell lines. The parent compounds consisted of a campesterol: dihydrobrassicasterol mix at a ratio of 2:1 (2CMP:1DHB) and a dihydrobrassicasterol:campesterol mix at a ratio of 3:1 (3DHB:1CMP). The 2CMP:1DBH oxides were more cytotoxic in the U937 cells than the 3DBH:1CMP oxides but the difference in cytotoxicity was less marked in the HepG2 cells. The order of toxicity of the individual oxidation products was found to be similar to that previously observed for cholesterol, ß-sitosterol and stigmasterol oxidation products in the U937 cell line. There was an increase in apoptotic nuclei in U937 cells incubated with the 7-keto and 7ß-OH derivatives of both 2CMP:1DHB and 3DHB:1CMP and also in the presence of 3DHB:1CMP-3ß,5α,6ß-triol and 2CMP:1DHB-5ß,6ß-epoxide. An additional oxidation product synthesised from 2CMP:1DHB, 5,6,22,23-diepoxycampestane, was cytotoxic but did not induce apoptosis. These results signify the importance of campesterol oxides in the overall paradigm of phytosterol oxide cytotoxicity.

Efficient solvent-free synthesis of phytostanyl esters in the presence of acid-surfactant-combined catalyst.

An efficient approach based on the synthesis of phytostanyl esters with an acid-surfactant-combined catalyst in a solvent-free system was developed. The effect of catalyst dose, substrate molar ratio, reaction temperature, and acyl donor was considered. The reaction conditions were further optimized by response surface methodology, and a high yield of phytostanyl laurate (>92%) was obtained under optimum conditions: 3.17:1 molar ratio of lauric acid to plant stanols, 4.01% catalyst dose (w/w), 119 °C, and 4.1 h. FT-IR, MS, and NMR were adopted to confirm the chemical structure of phytostanyl laurate. Meanwhile, the physiochemical properties of different phytostanyl esters were investigated. Compared with phytostanols, the prepared phytostanyl esters had much lower melting temperature and higher oil solubility. There was no obvious difference in melting and solidification properties between sunflower oil with phytostanyl laurate (<5%) or oleate (<10%) and the original sunflower oil, suggesting that the esterification of phytostanols greatly facilitated their corporation into oil-based foods.

Novel human umbilical vein endothelial cells (HUVEC)-apoptosis inhibitory phytosterol analogues: insight into their structure-activity relationships.

Design, synthesis and insight into the structure-activity relationships (SAR) of phytosterol analogues as novel antiapoptotic agents are described. In particular, the non-branched alkyl chain at C24 and the pseudosugar moiety at C3 hydroxyl group turned out crucial for the inhibition of human umbilical vein endothelial cells (HUVEC) apoptosis.

Lipase-mediated synthesis of water-soluble plant stanol derivatives in tert-butanol.

The effects of solvents with different log P values, and of lipases on the synthesis of water-soluble plant stanol derivatives were investigated. Results showed that conversion in solvents with log P<0.37 was mainly controlled by the hydrophobicity of the solvent and subsequent complete or partial deactivation of the enzyme. The solubility of substrate was the leading factor for the conversion in solvents with log P>0.37. Lipozyme RM IM and tert-butanol was the most suitable biocatalyst and solvent, respectively. The highest yield (>51%) of plant stanyl sorbitol succinate was obtained under the selected conditions: 50 µmol/mL plant stanyl hemisuccinate, 1:3 molar ratio of plant stanyl hemisuccinate to d-sorbitol, 80 mg/mL 3 Å molecular sieves and 100mg/mL Lipozyme RM IM in tert-butanol, 150 r/min and 55 °C. Fourier transform infrared spectroscopy, mass spectroscopy and nuclear magnetic resonance spectroscopy were adopted to determine the structure of product, suggesting that water-soluble plant stanol derivatives were successfully synthesized.

Enzymatic synthesis of phytosteryl docosahexaneates and evaluation of their anti-atherogenic effects in apo-E deficient mice.

Phytosterols have attracted much attention in recent years due to their health benefits, such as cholesterol lowering, anti-inflammatory, anti-atherogenicity, and anti-cancer potential. Docosahexaenoic acid (DHA) has been demonstrated to possess cardioprotective and immune-enhancing effects. Esterification of phytosterols with DHA may render improved physiochemical properties such as solubility, miscibility, oxidative stability and hence bioactivity and bioavailability. Thus, phytosteryl docosahexaneates (PS-DHA) may offer both the benefits of phytosterols and DHA, possibly in a synergistic manner. Here, we describe a method for enzymatic synthesis of phytosteryl docosahexaneates and evaluation of metabolic and cardiovascular benefits in apo-E deficient (apo E-KO) mice. The structures of phytosteryl docosahexaneates were confirmed by infrared (IR) and high performance liquid chromatography-mass spectrometry (HPLC-MS) using both normal and reverse phase chromatography. Apo E-KO mice were fed with an atherogenic diet containing 2% (w/w) PS-DHA for 7 weeks. Plasma lipid levels and the extent and complexity of atherosclerotic lesions were examined and compared with those in the control group. The PS-DHA-treated mice had significantly lower plasma cholesterol levels and three times smaller atherosclerotic lesions in the aortic roots. This pilot study suggests cardiovascular benefits for PS-DHA. Further experimental and clinical studies are needed to confirm such benefits of PS-DHA.

Enzymatic synthesizing of phytosterol oleic esters.

A method of synthesizing the phytosterol esters from oleic acid and sterols was studied, using immobilized lipase Candida sp. 99-125 as catalyst. Molar ratio (oleic acid/phytosterols), temperature, reaction period, organic solvents, catalyst, and silica-gel drier were optimized, and the result showed that 93.4% of the sterols had been esterified under the optimal synthetic condition: the molar ratio of oleic acid/phytosterol is 1:1 in 10 mL iso-octane, immobilized lipase (w, 140% of the sterols), incubated in an orbital shaker (200 rpm) at a temperature of 45 °C for 24 h. The immobilized lipase could be reused for at least 13 times with limited loss of esterification activity. The conversion still maintained up to 86.6%. Hence, this developed process for synthesizing phytosterol esters could be considered as simple and low-energy consumption compared to existing chemical processes.