Unusual withanolides from aeroponically grown Withania somnifera

In an attempt to maximize production and the structural diversity of plant metabolites, the effect of growing the medicinal plant Withania somnifera under soil-less aeroponic conditions on its ability to produce withaferin A and withanolides was investigated. It resulted in the isolation and characterization of two compounds, 3α-(uracil-1-yl)-2,3-dihydrowithaferin A (1) and 3β-(adenin-9-yl)-2,3-dihydrowithaferin A (2), in addition to 10 known withanolides including 2,3-dihydrowithaferin A-3β-O-sulfate. 3β-O-Butyl-2,3-dihydrowithaferin A (3), presumably an artifact formed from withaferin A during the isolation process was also encountered. Reaction of withaferin A with uracil afforded 1 and its epimer, 3β-(uracil-1-yl)-2,3-dihydrowithaferin A (4). The structures of these compounds were elucidated on the basis of their high resolution mass and NMR spectroscopic data.     

The withanolides of Withania somnifera chemotypes I and II

Seven steroidal lactones of the withanolide series have been isolated as minor constituents of the leaves of Withania somnifera Dun. (Solanaceae) chemotype I, along with the major component withaferin A. Structures have been assigned to the new compounds: withanolide N (17α,27-dihydroxy-1-oxo-20R,22R-witha-2,5,14,24-tetraenolide) (6a) and withanolide O (4β,17α-dihydroxy-1-oxo-20R,22R-witha-2,5,8(14),24-tetraenolide) (7a). Similarly the leaves of W. somnifera chemotype II afforded three new withanolides along with the major component withanolide D (9a) and trace amounts of withanolide G (10). The new compounds are: 27-hydroxywithanolide D(4β,20α,27-trihydroxy-1-oxo-5β,6β-epoxy-20R,22R-witha-2,24-dienolide) (11a), 14α-hydroxywithanolide D (4β,14α,20α-trihydroxy-1-oxo-5β,6β-epoxy-20R,22R-witha-2,24-dienolide) (12a) and 17α-hydroxywithanolide D (4β,17β,20α-trihydroxy-1-oxo-5β,6β-epoxy-20S,22R-witha-2,24-dienolide) (13a). Whereas all the withanolides of chemotype I are unsubstituted at C-20 (20α-H), those of chemotype II possess an OH at this position (20α-OH).     

Unusually sulfated and oxygenated steroids from Withania somnifera

Four (1, 8-10) and six known (2-7) withanolides were isolated from the leaves of Withania somnifera. Among the new compounds, 10 possessed the rare 3-O-sulfate group with the saturation in A ring and 9 contained unusual 1,4-dien-3-one group. Compound 8 did not have usual 2,3 unsaturation in A ring while 1 had the rare C-16 double bond. The structures of all the compounds were elucidated by spectroscopic methods and chemical transformation.     

Structural characterization of a flavonoid glycosyltransferase from Withania somnifera

Medicinal plants are extensively utilized in traditional and herbal medicines, both in India and around the world due to the presence of diverse low molecular weight natural products such as flavonoids, alkaloids, terpenoids and sterols. Flavonoids which have health benefits for humans are the large class of phenylpropanoid-derived secondary metabolites and are mostly glycosylated by UDP-glycosyltransferases (UGTs). Although large numbers of different UGTs are known from higher plants, very few protein structures have been reported till now. In the present study, the three-dimensional model of flavonoid specific glycosyltransferases (WsFGT) from Withania somnifera was constructed based on the crystal structure of plant UGTs. The resulted model was assessed by various tools and the final refined model revealed GT-B type fold. Further, to understand the sugar donors and acceptors interactions with the active site of WsFGT, docking studies were performed. The amino acids from conserved PSPG box were interacted with sugar donor while His18, Asp110, Trp352 and Asn353 were important for catalytic function. This structural and docking information will be useful to understand the glycosylation mechanism of flavonoid glucosides.

Abbreviations

DOPE - Discrete Optimized Potential Energy, PDB - Protein Data Bank, PSPG - Plant Secondary Product Glycosyltransferase, RMSD - Root Mean Squared Deviation, UDP - Uridine diphosphate, UGT - UDP-glycosyltransferases.     

New antiproliferative and immunosuppressive withanolides from the seeds of Datura metel

Two new withanolides baimantuoluoside H (1) and baimantuoluoline K (2), and one known withanolide glycoside (3) were isolated and identified from the ethyl acetate-soluble fraction of ethanol extract of Datura metel seeds. The structures of the new compounds were determined using 1D and 2D NMR spectroscopy, and mass spectrometry. All isolated compounds were evaluated for their antiproliferative activity against human gastric adenocarcinoma (SGC-7901), human hepatoma (Hepg2), and human breast cancer (MCF-7) cells, as well as their immunosuppressive properties. It was determined that compounds 1–3 exhibited medium antiproliferative and potential immunosuppressive effects.     

Analysis of withanolides in root and leaf of Withania somnifera by HPLC with photodiode array and evaporative light scattering detection

A reversed-phase HPLC method for the simultaneous analysis of nine structurally similar withanolides, namely, 27-hydroxy withanone, 17-hydroxy withaferin A, 17-hydroxy-27-deoxy withaferin A, withaferin A, withanolide D, 27-hydroxy withanolide B, withanolide A, withanone and 27-deoxywithaferin A, has been developed using a linear binary gradient solvent system comprising methanol and water containing 0.1% acetic acid. Both photodiode array and evaporative light scattering detection were used to profile the extract compositions and to quantify the withanolides therein. Homogeneity and purity of each peak was ascertained by comparative evaluation of the on-line UV spectra of the eluted compounds with those of the reference compounds. The method has been validated with respect to various parameters of performance quality including computation regression analysis based on calibration curves, peak resolution factor, asymmetry factor, tailing factor, RSD (%) of retention time and peak area response, limit of quantivation, limit of detection, precision and recovery. The developed method has been applied to the analysis of leaf and root tissues of Withania somnifera for withanolide content.     

New withanolides from a cross of a South African chemotype by chemotype II (Israel) in Withania somnifera

Three new withanolides have been isolated from hybrids obtained by crossing a chemotype of Withania somnifera received from South Africa and chemotype II originating in Israel. The compounds have been characterized as 4β,20α-dihydroxy-1-oxo-5β,6β-epoxy-20R,22R-witha-24-enolide, 20α-hydroxy-1,4-dioxo-5β,6β-epoxy-20R,22R-witha-2,24-dienolide, and 20α-hydroxy-1,4-dioxo-5β,6β-epoxy-20R,22R-witha-2-enolide. The major steroid of the plant is withanolide D, while the other known withanolides present are 4β,20α-dihydroxy-1-oxo-5β,6β-epoxy-20R,22R,24S,25R-witha-2-enolide and withaferin A. The structures assigned to the new compounds are based on spectral evidence, analysis of their fragmentation under electron impact, and on chemical correlation with known compounds. The formation of these withanolides in this new hybrid is discussed briefly.     

Displacement of sterols from sterol/sphingomyelin domains in fluid bilayer membranes by competing molecules

The formation of sterol and palmitoyl sphingomyelin enriched ordered domains in a fluid bilayer was examined using domain selective fluorescent reporter molecules (cholestatrienol and trans-parinaric acid containing lipids) together with a quencher molecule in the fluid phase. The aim of the study was to explore how stable the ordered domains were and how different, biologically interesting, membrane intercalators could affect domain stability and sterol distribution between domains. We show that sterols easily can be displaced from ordered domains by a variety of saturated, single- and double-chain membrane intercalators with a small polar group as a common denominator. Of the two-chain intercalators examined, both palmitoyl ceramide and palmitoyl dihydroceramide were effective in displacing sterols from ordered domains. Of the single-chain intercalators, hexadecanol and hexadecyl amide displaced the sterol from sterol/sphingomyelin domains, whereas palmitic acid, sphingosine and sphinganine failed to do so. All molecules examined stabilized the sphingomyelin-rich domains, as reported by trans-parinaric-sphingomyelin and by scanning calorimetry. Parallels between the displacement of sterol from ordered domains in our model membrane system and the ability of the above mentioned molecules to alter the chemical activity and distribution of sterols in biological membranes are discussed.     

Micropropagation of Withania somnifera from germinating seeds and shoot tips

Shoot multiplication was achieved in vitro from shoot tips of aseptically germinated seedlings of Withania somnifera L. using low concentrations of 6-benzyladenine (BA), viz. 2.2, 4.4 and 8.9 M. Maximum number of shoots were obtained when 2.3 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 2.5 M indolebutyric acid (IBA) was added to medium containing 4.4 M BA during initiation of shoot multiplication, but not when added later. Direct multiple shoot initiation was also obtained from germinating seeds in the presence of BA alone. Rooting was successful in excised shoots grown on growth regulator-free MS medium. Rooted shoots were successfully established in soil in a greenhouse.     

Purification and physico-kinetic characterization of 3beta-hydroxy specific sterol glucosyltransferase from Withania somnifera (L) and its stress response

Sterol glycosyltransferases catalyze the synthesis of diverse glycosteroids in plants, leading to a change in their participation in cellular metabolism. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a cytosolic sterol glucosyltransferase was purified 3406 fold to near homogeneity from W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme was active with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols and phytosterols with 3beta-OH group. It showed a low level of activity with flavonoids and isoflavonoids. The enzyme gave maximum K(cat)/K(m) value (0.957) for 24-methylenecholesterol that resembles aglycone structure of pharmacologically important sitoindosides VII and VIII from W. somnifera. The enzyme follows ordered sequential bisubstrate mechanism of reaction, in which UDP-glucose and sterol are the first and second binding substrates. This is the first detailed kinetic study on purified plant cytosolic sterol glucosyltransferases. Results on peptide mass fingerprinting and substrate specificity suggested that the enzyme belongs to the family of secondary metabolite glucosylating glucosyltransferases. The enzyme activity exhibited a rapid in vivo response to high temperature and salicylic acid treatment of plants, suggesting its physiological role in abiotic and biotic stress.     

Purification and characterization of a glycoprotein inhibitor of toxic phospholipase from Withania somnifera

A phospholipase inhibitor (WSG) has been purified from Withania somnifera using gel-filtration and ion-exchange chromatographies. The WSG is an acidic glycoprotein. Its molecular mass as determined by SDS-PAGE was 27kDa. It neutralized the enzyme activity and pharmacological properties such as cytotoxicity, edema, and myotoxicity of a multi-toxic Indian cobra venom phospholipase (NNXIa-PLA) but failed to neutralize the neurotoxicity. The glycan part of the molecule does not appear to be involved in any of the pharmacological properties studied. The results suggest that the neutralization of the pharmacological effects of the toxic phospholipase is brought about by inhibition of the enzyme activity by formation of a complex between the WSG and the toxic phospholipase. We report the purification and characterization of a glycoprotein phospholipase A inhibitor from Withania somnifera, medicinal plant.     

Purification, characterization and inhibition of sterol C24-methyltransferase from Candida albicans

Solubilized sterol C24-methyltransferase (24-SMT) was purified to homogeneity from a cell extract of the yeast Candida albicans (Ca) by anion exchange chromatography, gel permeation chromatography and fast performance liquid chromatography using a Mono Q column. The purified enzyme has an apparent molecular mass of 178 kDa on gel permeation chromatography and 43 kDa on SDS/PAGE, indicating that it is composed of four identical subunits. The substrate requirement of the native enzyme has an optimal specificity for zymosterol with associated kinetic constants of K_m 50 μM and k_cat of 0.01 s⁻¹. The product of the enzyme incubated with zymosterol was fecosterol. Inhibition of the catalyst was observed with substrate analogs designed as transition state analogs (25-azalanosterol, K_i = 54 nM and 24 (R,S),25-epiminolanosterol, K_i = 11 nM) or as mechanism-based inactivators (26,27-dehydrozymosterol, K_i 9 μM) and k_inact = 0.03 min⁻¹) of the C24-methylation reaction. Product analogs ergosterol and fecosterol, but neither cholesterol nor sitosterol, inhibited activity affording K_i values of 20 and 72 μM, respectively. Ammonium and thia analogs of the intermediates of the sterol C24-methyl reaction sequence were effective growth inhibitors exhibiting IC₅₀ values that ranged from 3 to 20 μM.     

Molecular docking analysis of hyperphosphorylated tau protein with compounds derived from Bacopa monnieri and Withania somnifera

Tau protein, the major player in Alzheimer’s disease forms neurofibrillary tangles in elderly people. Bramhi (Baccopa Monniera) is often used as an ayurvedic treatment for Alzheimer''s disease. Therefore it is of interest to study the interaction of compounds derived from Baccopa with the Tau protein involved in tangle formation. We show that compounds such as bacopaside II, bacopaside XII, and nicotine showed optimal binding features with the R2 repeat domain of hyperphosphorylated tau protein for further consideration in the context of Alzheimer''s disease (AD).     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biosorption of sterols from tobacco waste extract using living and dead of newly isolated fungus Aspergillus fumigatus strain LSD-1

ABSTRACT

Sterols are verified to be able to produce polycyclic aromatic hydrocarbons during its pyrolysis. In this study, a kind of Aspergillus fumigatus (LSD-1) was isolated from cigar leaves, and the biosorption effects on the stigmasterol, β-sitosterol, campesterol, cholesterol, and ergosterol by using living and dead biomass of LSD-1 were investigated. The results showed that both living and dead biomass could efficiently remove these sterols in aqueous solution and tobacco waste extract (TWE). Interestingly, compared with the living biomass of LSD-1, the dead biomass of LSD-1 not only kept a high adsorption efficiency but also did not produce ergosterol. Overall, dead biomass of LSD-1 was a more suitable biosorbent to sterols in TWE. Furthermore, Brunner-Emmet-Teller (BET), Fourier transformed infrared spectrometer (FTIR) and scanning electron microscope (SEM) analysis were used to explore the biosorption process of living and dead biomass and their differences, suggesting that the biosorption of sterols was a physical process.     

Conservation and cloning of CYP51: a sterol 14 alpha-demethylase from Mycobacterium smegmatis

The genetic locus encoding cytochrome P450 51 (CYP51; P450(14DM)) in Mycobacterium smegmatis is described here together with confirmation of activity in lanosterol 14 alpha-demethylation. The protein bound azole antifungals with high affinity and the rank order based on affinity matched the ranked order for microbiological sensitivity of the organism, thus supporting a possible role for CYP51 as a target in the antimycobacterial activity of these compounds. Non-saponifiable lipids were extracted from the bacteria grown on minimal medium. Unlike a previous report using growth on complex medium, no cholesterol was detected in two strains of M. smegmatis, but a novel lipid was detected. The genetic locus of CYP51 is discussed in relation to function; it is conserved as part of a putative operon in M. smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium bovis and consists of six open-reading frames including two CYPs and a ferredoxin under a putative Tet-R regulated promoter.     

Effects of two sterol biosynthesis inhibitor fungicides (fenpropimorph and fenhexamid) on the development of an arbuscular mycorrhizal fungus

The effects of different concentrations (0.2, 2, 20, 200 mg l⁻¹) of two sterol biosynthesis inhibitor (SBI) fungicides, i.e. fenpropimorph and fenhexamid, were evaluated on the spore germination, germ tube elongation, sporulation, and root colonization of Glomus intraradices grown monoxenically in association with transformed carrot roots. The percentage of germinated spores incubated on the SBI fungicides and the length of the germ tubes decreased with increasing concentrations of both fungicides. However, for spore germination this impact was fungistatic rather than fungicidal. Extraradical mycelium architecture and spore production in contact with the SBI fungicides were also strongly impacted at high concentration (20 mg l⁻¹). Conversely, the colonization of roots developing in the fungicide-free compartment, but interconnected with the extraradical mycelium developing on the SBI fungicides, appeared unaffected. Our results demonstrated that the monoxenic culture system could be used as a standardized, reproducible technique to compare the impacts of different molecules on arbuscular mycorrhizal fungi, and for the initial screening of new candidate molecules before registration.