Natural Sterols Research Articles

Natural product for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is related to increasing age. It is mainly characterized by progressive neurodegenerative disease, which damages memory and cognitive function. Natural products offer many options to reduce the progress and symptoms of many kinds of diseases, including AD. Meanwhile, natural compound structures, including lignans, flavonoids, tannins, polyphenols, triterpenes, sterols, and alkaloids, have anti-inflammatory, antioxidant, anti-amyloidogenic, and anticholinesterase activities. In this review, we summarize the pathogenesis and targets for treatment of AD. We also present several medicinal plants and isolated compounds that are used for preventing and reducing symptoms of AD.

Synthesis of side-chain oxysterols and their enantiomers through cross-metathesis reactions of Δ22 steroids

A synthetic route that utilizes a cross-metathesis reaction with Δ22 steroids has been developed to prepare sterols with varying C-27 side-chains. Natural sterols containing hydroxyl groups at the 25 and (25R)-26 positions were prepared. Enantiomers of cholesterol and (3β,25R)-26-hydroxycholesterol (27-hydroxycholesterol) trideuterated at C-19 were prepared for future biological studies.

Obtaining of 11α-Hydroxyandrost-4-ene-3,17-dione from Natural Sterols.

Two-step one-pot microbial transformation enables obtaining of valuable steroids that are difficult to produce chemically. Here we describe a method for obtaining 11α-hydroxyandrost-4-ene-3,17-dione (11α-HAD) from cheap and available natural sterols (phytosterols or cholesterol).11α-HAD is a primary adrenal steroid in mammals and also a key precursor in the syntheses of halogenated corticoids. Conventional routes for its obtaining are based on chemical synthesis, or microbial hydroxylation of androst-4-ene-3,17-dione (AD). AD in turn is produced primarily with microbial biotransformation of natural sterols by some actinobacteria.Consequent bioconversions of sterols using two microbial strains in one bioreactor vessel without separation and purification of AD provides high yield of 11α-HAD. At the first fermentation step, phytosterol is converted to AD with Mycobacterium neoaurum NRRL 3805B, or relative strains, to yield about 70% (mol/mol). At the second step, AD is almost fully (98%) hydroxylated at the position 11α with Aspergillus ochraceus VKM F-830, or other suitable organisms, in the same bioreactor. At the average, 30% (w/w) of the high-purity crystalline 11α-HAD can be obtained.The method can be exploited for production of 11α-HAD for practical use.

Engineering Mycobacterium smegmatis for testosterone production.

SummaryA new biotechnological process for the production of testosterone (TS) has been developed to turn the model strain Mycobacterium smegmatis suitable for TS production to compete with the current chemical synthesis procedures. We have cloned and overexpressed two genes encoding microbial 17β‐hydroxysteroid: NADP 17‐oxidoreductase, from the bacterium Comamonas testosteroni and from the fungus Cochliobolus lunatus. The host strains were M. smegmatis wild type and a genetic engineered androst‐4‐ene‐3,17‐dione (AD) producing mutant. The performances of the four recombinant bacterial strains have been tested both in growing and resting‐cell conditions using natural sterols and AD as substrates respectively. These strains were able to produce TS from sterols or AD with high yields. This work represents a proof of concept of the possibilities that offers this model bacterium for the production of pharmaceutical steroids using metabolic engineering approaches.

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

SummaryA number of pharmaceutical steroid synthons are currently produced through the microbial side‐chain cleavage of natural sterols as an alternative to multi‐step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc2155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc2155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3‐ketosteroid 9α‐hydroxylase (KshAB) and a ketosteroid Δ1‐dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, we have constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4‐androstadiene‐3,17‐dione. In addition, we have demonstrated that a double deletion mutant M. smegmatis MS6039‐5941 [ΔMSMEG_6039 (ΔkshB1) and ΔMSMEG_5941 (ΔkstD1)] transforms natural sterols into 4‐androstene‐3,17‐dione with high yields. These findings suggest that the catabolism of cholesterol in M. smegmatis mc2155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids.

The Role of fadD19 and echA19 in Sterol Side Chain Degradation by Mycobacterium smegmatis.

Mycobacteria are able to degrade natural sterols and use them as a source of carbon and energy. Several genes which play an important role in cholesterol ring degradation have been described in Mycobacterium smegmatis. However, there are limited data describing the molecular mechanism of the aliphatic side chain degradation by Mycobacterium spp. In this paper, we analyzed the role of the echA19 and fadD19 genes in the degradation process of the side chain of cholesterol and β-sitosterol. We demonstrated that the M. smegmatis fadD19 and echA19 genes are not essential for viability. FadD19 is required in the initial step of the biodegradation of C-24 branched sterol side chains in Mycobacterium smegmatis mc2155, but not those carrying a straight chain like cholesterol. Additionally, we have shown that echA19 is not essential in the degradation of either substrate. This is the first report, to our knowledge, on the molecular characterization of the genes playing an essential role in C-24 branched side chain sterol degradation in M. smegmatis mc2155.

Lipid-lowering Activity of Natural and Semi-Synthetic Sterols and Stanols.

Consumption of plant sterols/ stanols has long been demonstrated to reduce plasma cholesterol levels. The objective of this review is to demonstrate the lipid-lowering activity and anti-atherogenic effects of natural and semi-synthetic plant sterols/ stanols based on evidence from cell-culture studies, animal studies and clinical trials. Additionally, this review highlights certain molecular mechanisms by which plant sterols/ stanols lower plasma cholesterol levels with a special emphasis on factors that affect the cholesterol-lowering activity of plant sterols/stanols. The crystalline nature and the poor oil solubility of these natural products could be important factors that limit their cholesterol-lowering efficiency. Several attempts have been made to improve the cholesterol-lowering activity by enhancing the bioavailability of crystalline sterols and stanols. Approaches involved reduction of the crystal size and/or esterification with fatty acids from vegetable or fish oils. However, the most promising approach in this context is the chemical modification of plant sterols /stanols into water soluble disodium ascorbyl phytostanyl phosphates analogue by esterification with ascorbic acid. This novel semi-synthetic stanol derivative has improved efficacy over natural plant sterols/ stanols and can provide additional benefits by combining the cholesterol-lowering properties of plant stanols with the antioxidant potential of ascorbic acid. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Mycobacterium smegmatissynthesizes in vitro androgens and estrogens from different steroid precursors

Fast-growing mycobacteria such as Mycobacterium sp. and Mycobacterium smegmatis degrade natural sterols. They are a model to study tuberculosis. Interestingly, M. smegmatis has been found in river effluents derived from paper production, and therefore, it would be important to gain further insight into its capacity to synthesize steroids that are potential endocrine disruptors affecting the development and reproduction of fishes. To our knowledge, the capacity of M. smegmatis to synthesize estrogens and even testosterone has not been previously reported. Therefore, the objective of this study was to investigate the capacity of M. smegmatis to synthesize in vitro testosterone and estrogens from tritiated precursors and to investigate the metabolic pathways involved. Results obtained by thin-layer chromatography showed that (3)H-progesterone was transformed to 17OH-progesterone, androstenedione, testosterone, estrone, and estradiol after 6, 12, or 24 h of incubation. (3)H-androstenedione was transformed into testosterone and estrogens, mainly estrone, and (3)H-testosterone was transformed to estrone and androstenedione. Incubation with (3)H-dehydroepiandrosterone rendered androstenediol, testosterone, and estrogens. This ability to transform less potent sex steroids like androstenedione and estrone into other more active steroids like testosterone and estradiol or vice versa suggests that M. smegmatis can influence the amount of self-synthesized strong androgens and estrogens and can transform those found in the environment.

A DSC and FTIR spectroscopic study of the effects of the epimeric cholestan-3-ols and cholestan-3-one on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes: Comparison with their 5-cholesten analogs

We present the results of a comparative differential calorimetric and Fourier transform infrared spectroscopic study of the effect of cholesterol and five analogs on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes. These sterols/steroids differ in both the nature and stereochemistry of the polar head group at C3 (β-OH, α-OH or CO) and in the presence or absence of a double bond in ring B. In both the Δ5 and saturated sterols/steroid series, the concentration of these compounds required to abolish the DPPC pretransition, inversely related to their relative ability to disorder gel state DPPC bilayers, decreases in the order β-OH>α-OH>CO. However, in the saturated series, these concentrations are much more similar, regardless of polar head group chemical structure. Similarly, the residual enthalpy of the DPPC main phase transition at 50mol% sterol/steroid, inversely related to the miscibility of these compounds in fluid DPPC bilayers, also increases in the order β-OH>α-OH>CO, but this effect is again attenuated in the saturated series. Moreover, replacement of the double bond at C5 with a saturated linkage also reduces sterol/steroid solubility in all cases. Interestingly, the C5 double bond has no effect on DPPC hydrocarbon chain ordering in the βOH sterol pair, considerably increases ordering in the αOH pair, and considerably reduces ordering in the CO pair. Moreover, the ability of these compounds to order the DPPC hydrocarbon chains decreases in the order β-OH>α-OH>CO in the Δ5 series of compounds, but in the order β-OH>CO>α-OH in the saturated series. Our results indicate that the effects of the presence or absence of a double bond at C5 of ring B on the thermotropic phase behavior and organization of DPPC bilayers are influenced by the nature and stereochemistry of the polar group present at C3 and vice versa. Nevertheless, the characteristic effects of sterols/steroids on fluid lipid bilayers are optimal when an OH group rather than CO group is present at C3, and when this OH group is in the equatorial (β) orientation. Moreover, the presence of a single double bond specifically at C5 is required to maximize sterol solubility in fluid DPPC bilayers, which is probably its primary function in natural sterols.

Complete Genome Sequence of Steroid-Transforming Nocardioides simplex VKM Ac-2033D.

Nocardioides simplex VKM Ac-2033D is an effective microbial catalyst for 3-ketosteroid 1(2)-dehydrogenation, and it is capable of effective reduction of carbonyl groups at C-17 and C-20, hydrolysis of acetylated steroids, and utilization of natural sterols. Here, the complete genome sequence is reported. An array of genes related to steroid metabolic pathways have been identified.

Microbial side-chain cleavage of phytosterols by mycobacteria in vegetable oil/aqueous two-phase system.

Microbial side-chain cleavage of natural sterols to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by Mycobacteria has received much attention in pharmaceutical industry, while low yield of the reaction owing to the strong hydrophobicity of sterols is a tough problem to be solved urgently. Eight kinds of vegetable oils, i.e., sunflower, peanut, corn, olive, linseed, walnut, grape seed, and rice oil, were used to construct oil/aqueous biphasic systems in the biotransformation of phytosterols by Mycobacterium sp. MB 3683 cells. The results indicated that vegetable oils are suitable for phytosterol biotransformation. Specially, the yield of AD carried out in sunflower biphasic system (phase ratio of 1:9, oil to aqueous) was greatly increased to 84.8 % with 10 g/L feeding concentration after 120-h transformation at 30 °C and 200 rpm. Distribution coefficients of AD in different oil/aqueous systems were also determined. Because vegetable oils are of low cost and because of their eco-friendly characters, there is a great potential for the application of oil/aqueous two-phase systems in bacteria whole cell biocatalysis.

Polyunsaturated fatty acids and not plant sterols naturally-occurring in food infl uence lipid profi le in obese women

The aim of this study was to assess the influence of the consumption level of plant sterols naturally occurring in diet on the lipid profile in obese women with dyslipidemia. The study population was primarily comprised of 120 obese women aged over 55 from Poland, with twenty seven subjects dropping out of the study due to the inclusion and exclusion criteria. Anthropometric appraisal included body height and weight, as well as waist and hip circumferences. Lipid profile was determined by commercially available enzymatic methods. Nutritional data, including plant sterols intake were assessed by 7-day food records. The multiple linear regression, adjusted for age, height, Body Mass Index and Waist to Hip Ratio, showed the relation of polyunsaturated fatty acids with LDL-cholesterol concentration (p < 0.05). Natural plant sterols intake was estimated at Median = 146 mg/day. The correlation between LDL-cholesterol concentration and plant sterols intake was not statistically significant (p > 0.05). The results of this study suggest that consumption of naturally-occurring polyunsaturated fatty acids and not plant sterols is connected with lower LDL-cholesterol concentration in obese women.

The regio- and stereo-selective reduction of steroidal 4-en-3-ones using Na2S2O4/NaHCO3 and CuCl/NaBH4

This paper describes the regio- and stereoselective reduction of △4-3-keto moiety in certain steroids using Na2S2O4/NaHCO3 and CuCl/NaBH4, respectively. Using either one of the two reduction agents in the reaction, the 17-substituents in the D ring were observed to have clearly influenced the stereoselective reduction of 4-ene in the A ring by the so-called conformational transmission effect. Na2S2O4/NaHCO3 regioselectively reduced CC at 4-position of 17-substituted-androst-4-en-3-one derivatives to 5α-H-3-one as the main isomer. And as an extended application, Epiandrosterone (11) was further synthesized from androst-4-en-3,17-dione (AD) via four steps. The total yield from this was about 45%. In the presence of CuCl/NaBH4, △4-3-keto conjugated reduction of 17-spirocyclic ethylene ketal protected androst-4-en-3-one derivatives mainly produced 3α-hydroxy-5β-H isomers, at a yield around 81%. Considering the scaffold configuration of 3α-hydroxy-5β-H moiety coincided with that of bile acid analogs, this selective reduction could also be used as an alternative method for the synthetic study of bile acids using AD and its derivatives, which are from the microorganism degradation of natural sterols, as the potential materials. Meanwhile, configurations of the reductive compounds 5b, 6b, 9, 10 and 17e were identified by X-ray diffraction.

Fomentarols A–D, sterols from the polypore macrofungus Fomes fomentarius

Four (1-4) hitherto unknown and seven (5-11) known ergostane-type sterols were isolated from the EtOH extract of the dried fruiting bodies of the polypore macrofungus Fomes fomentarius. On the basis of spectroscopic analysis, the structures of polyhydroxylated sterols 1-4 were elucidated to be (22E,24R)-3β,5α,6β,14α-tetrahydroxyergosta-7,9(11),22-triene (fomentarol A, 1), (22E,24R)-3β,5β,6α,7α-tetrahydroxy-8α,9α-dihydroergosta-14,22-diene (fomentarol B, 2), (22E,24R)-3β,5α-dihydroxy-6β-ethoxyergosta-7,22-diene (fomentarol C, 3), and (22E,24S)-3β,25-dihydroxy-15α-O-β-D-glucopyranosylergosta-7,22-dien-6-one (fomentarol D, 4), respectively. Rings A/B and B/C are in turn cis-fused in compound 2, which is uncommon in natural ergostane-type sterols. The potential biogenetic relationship of 2 and other ergostane-type sterols isolated from F. fomentarius was briefly discussed. Moderate cytotoxic effects of the isolated sterols against a small panel of human cancer cell lines were also established.

Reminiscences of research on the chemistry and biology of natural sterols in insects, plants and humans.

Natural sterols often occur as a heterogeneous mixture of homologs, which had disturbed the progress of steroid research. Development and application of GC methodology overcame this difficulty and enabled us to obtain detailed sterol profiles. Together, fine synthesis of stereo-defined isomers and homologs of steroids having oxygenated side chains allowed us to compare them with natural samples as well as to investigate structure-activity relationship. Advance of HPLC technology also facilitated the determination of the stereochemical structure of naturally occurring steroidal compounds, which were obtained only in minute amounts. This review highlights three topics out of our steroid research that have been performed mainly at Tokyo Institute of Technology around 1970–1990. These are sterol metabolism in insects focusing on the mechanism of the conversion of plant sterols to cholesterol and ecdysone biosynthesis, the synthesis and biochemical research of active forms of vitamin D3 derivatives, and the synthesis and microanalysis of plant hormone brassinosteroids.

22-NBD-cholesterol, a new fluorescent substrate of bacterial cholesterol-oxidases

Fluorescent analogs of sterols are convenient agents for studying the transport, distribution, and protein—ligand interaction of natural sterols in cells [1]. However, the effect of the fluorescent moiety on the interaction parameters with cholesterol-transforming enzymes such as bacterial cholesterol-oxidases or mammalian steroid-hydroxylating cytochrome P450 has not been addressed in the literature. Herein we report data for the interaction of the fluorescent cholesterol analog 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3 -ol [22-NBD-cholesterol (1)] with cholesteroloxidases (EC 1.1.3.6). Cholesterol-oxidases are FAD-dependent enzymes that catalyze the transformation of 3 -hydroxy5steroids and are widely used for laboratory and clinical cholesterol determinations [2]. We calculated the interaction of 1 with the enzyme based on the three-dimensional structure of Brevibacterium sterolicum cholesterol-oxidase (code PDB 1COY) using the Autodock 4.0 programs and MGLTools. It was found with the help of molecular docking that 1 can bind to the active center of cholesterol-oxidase such that its 3 -hydroxy group is situated in close proximity to the functionally important His447 and Glu361 residues [3] and the redox-active groups of the FAD molecule. The calculated binding energies for cholesterol and 1 were practically the same (–11.43 and –11.94 kcal/mol, respectively). Next selective transformation of 1 was demonstrated using cholesterol-oxidase in vitro. After biotransformation of fluorescent substrate 1 with purified cholesterol-oxidase, the formation of 2, which retained the fluorescent properties of 1 (Scheme 1) was detected by HPLC (Shimadzu LC10-AD chromatograph with an RF-10A 1 fluorimetric detector and SPD-10A spectrophotometric detector; CH3CN:H2O:i-PrOH, 84:16:5; LiChroCART C18 column, Merck, 250 4 mm).

Exploiting anti-obesity mechanism of Clerodendrum phlomidis against two different models of rodents

Roots of Clerodendrum phlomidis are used by the local people of Dibrugarh district of Assam state India as a dietary supplement for treating weight issues and are also mentioned in the traditional system of Indian medicine as a remedy for obesity. We examined the anti-obesity effect of Clerodendrum phlomidis (family Verbenaceae) L. roots against cafeteria diet (CD) and progesterone-induced obesity. In CD-induced model obesity was induced by feeding CD for 48-days and increase in body weight and fat storage was suppressed co-administration with methanolic extract of Clerodendrum phlomidis (MECP) at 400 mg/kg. Blood analysis showed that the levels of triglyceride and cholesterol were significantly lowered by MECP administration and there is subsequent rise in HDLcholesterol level. From this experiment, we demonstrated that MECP is effective in ameliorating the CD-induced hyperglycemia, hyperinsulimenia, dyslepidemia, increase in wet weight of white adipose tissue, and hypertrophy of fat cells. In drug induced obesity model hyperphagia was induced by progesterone (10 mg/kg s.c.) for 28 days and was suppressed by co-administration with MECP in dose dependent manner. It is tempting to speculate that these protective effects shown by Clerodendrum phlomidis is by multiple mechanisms. MECP contains β-sitosterol in the abundant quantity because of the structural similarity it do the physical competition with natural sterols while absorption of food stuffs from GIT and moreover the crude saponin and flavonoid has been reported for it’s the appetit suppressant property and hence reduces hyperphagia produced by progesterone. This is the first report demonstrating that Clerodendrum phlomidis is effective in ameliorating insulin resistance and visceral obesity induced by CD and Progesterone. Key words: Adipocyte, cafeteria diet, clerodendrum phlomidis, hyperphagia, hypertrophy, progesterone

Selective synthesis of phytosterol esters from natural sterols and fatty methyl esters over Mg-containing solid catalysts

Abstract Phytosterol esters are useful in food applications, as well as in cosmetics, while exhibiting an advantage over phytosterols naturally occurring antioxidants, with better fat solubility and compatibility. Here, a green chemical process based on the transesterification of fatty methyl esters with natural sterols in the absence of any solvent using basic solid reusable catalysts is described. Alumina-supported magnesium oxide proved to be particularly selective, affording the desired phytosterol esters with a far increased yield (93%), compared with that of other basic oxide catalysts previously used. The most relevant results were obtained when the alumina used as support for MgO was prepared through the sol-gel route, due to appropriate acid-base properties. The calcination temperature was shown to have a strong impact on the catalytic behavior. The study also demonstrated that the reuse characteristics of the solid catalyst are excellent.

Cholesterol-Lowering Effects of Plant Steryl and Stanyl Laurate by Oral Administration in Mice

The present study was conducted to investigate the efficacy of synthesized plant steryl and stanyl laurate in lowering the cholesterol level and to further examine the cholesterol-lowering potential of the free plant sterols and stanols dissolved in liquid emulsion on serum and liver lipids in mice by oral administration. Experimental results showed that both plant steryl and stanyl laurate could significantly decrease the serum levels of TC, LDL-C, LDL-C/HDL-C, and liver cholesterol contents and markedly increase fecal cholesterol concentrations but have no effect on serum TAG level, indicating that the produced plant steryl and stanyl laurate retained the cholesterol-lowering potential of natural plant sterols and stanols. However, no statistical difference in cholesterol-lowering efficacy was observed between plant steryl laurate and plant stanyl laurate, and free plant sterols and stanols dissolved in liquid emulsion could also significantly decrease serum cholesterol levels and markedly increase fecal cholesterol excretion. These results suggested that the esterified plant sterols/stanols had comparable effects to the free plant sterols/stanols in lowering serum TC levels but that they did gain a solubility advantage from the free plant sterols/stanols. Therefore, plant steryl/stanyl laurate could be considered as a potential nutraceutical or functional ingredient to reduce or prevent atherosclerosis and its related complications.

New cytotoxic oxygenated sterols from the marine bryozoan Cryptosula pallasiana.

Six new sterols (1-6), together with seven known sterols (7-13), were isolated from the CCl4 extract of the marine bryozoan Cryptosula pallasiana, four (3-6) of which have already been reported as synthetic sterols. This is the first time that these compounds (3-6) are reported as natural sterols. The structures of the new compounds were determined on the basis of the extensive spectroscopic analysis, including two-dimensional (2D) NMR and HR-ESI-MS data. Compounds 1-4, 7 and 10-13 were evaluated for their cytotoxicity against HL-60 human myeloid leukemia cell line, and all of the evaluated compounds exhibited moderate cytotoxicity to HL-60 cells with a range of IC50 values from 14.73 to 22.11 µg/mL except for compounds 12 and 13.