Improvement and prediction of the extraction parameters of lupeol and stigmasterol metabolites of Melia azedarach with response surface methodology.

BACKGROUND: Melia azedarach is known as a medicinal plant that has wide biological activities such as analgesic, antibacterial, and antifungal effects and is used to treat a wide range of diseases such as diarrhea, malaria, and various skin diseases. However, optimizing the extraction of valuable secondary metabolites of M. azedarach using alternative extraction methods has not been investigated. This research aims to develop an effective, fast, and environmentally friendly extraction method using Ultrasound-assisted extraction, methanol and temperature to optimize the extraction of two secondary metabolites, lupeol and stigmasterol, from young roots of M. azedarach using the response surface methodology. METHODS: Box-behnken design was applied to optimize different factors (solvent, temperature, and ultrasonication time). The amounts of lupeol and stigmasterol in the root of M. azedarach were detected by the HPLC-DAD. The required time for the analysis of each sample by the HPLC-DAD system was considered to be 8 min. RESULTS: The results indicated that the highest amount of lupeol (7.82 mg/g DW) and stigmasterol (6.76 mg/g DW) was obtained using 50% methanol at 45 °C and ultrasonication for 30 min, and 50% methanol in 35 °C, and ultrasonication for 30 min, respectively. Using the response surface methodology, the predicted conditions for lupeol and stigmasterol from root of M. azedarach were as follows; lupeol: 100% methanol, temperature 45 °C and ultrasonication time 40 min (14.540 mg/g DW) and stigmasterol 43.75% methanol, temperature 34.4 °C and ultrasonication time 25.3 min (5.832 mg/g DW). CONCLUSIONS: The results showed that the amount of secondary metabolites lupeol and stigmasterol in the root of M. azedarach could be improved by optimizing the extraction process utilizing response surface methodology.

Bacterial stigmasterol degradation involving radical flavin delta-24 desaturase and molybdenum-dependent C26 hydroxylase.

Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyzes the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one to a conjugated (22,24)-diene. We suggest an α4ß4 architecture of the 440 kDa enzyme, with each subunit covalently binding an flavin mononucleotide cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by stigmast-1,4-diene-3-one but cannot be oxidized even with strong oxidizing agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2. In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.

Stigmasterol Protects Against Steatohepatitis Induced by High-Fat and High-Cholesterol Diet in Mice by Enhancing the Alternative Bile Acid Synthesis Pathway.

BACKGROUND: Hepatic cholesterol accumulation is a significant risk factor in the progression of nonalcoholic fatty liver disease (NAFLD) to steatohepatitis. However, the precise mechanism by which stigmasterol (STG) mitigates this process remains unclear. OBJECTIVES: This study aimed to investigate the potential mechanism underlying the protective effect of STG in mice with NAFLD progressing to steatohepatitis while being fed a high-fat and high-cholesterol (HFHC) diet. METHODS: Male C57BL/6 mice were fed an HFHC diet for 16 wk to establish the NAFLD model. Subsequently, the mice received STG or a vehicle via oral gavage while continuing the HFHC diet for an additional 10 wk. The study evaluated hepatic lipid deposition and inflammation as well as the expression of key rate-limiting enzymes involved in the bile acid (BA) synthesis pathways. BAs in the colonic contents were quantified using ultra-performance liquid chromatography-tandem mass spectrometry. RESULTS: Compared with the vehicle control group, STG significantly reduced hepatic cholesterol accumulation (P < 0.01) and suppressed the gene expression of NLRP3 inflammasome and interleukin-18 (P < 0.05) in the livers of HFHC diet-fed mice. The total fecal BA content in the STG group was nearly double that of the vehicle control group. Additionally, the administration of STG increased the concentrations of representative hydrophilic BAs in the colonic contents (P < 0.05) along with the upregulation of gene and protein expression of CYP7B1 (P < 0.01). Furthermore, STG enhanced the α-diversity of the gut microbiota and partially reversed the alterations in the relative abundance of the gut microbiota induced by the HFHC diet. CONCLUSIONS: STG mitigates steatohepatitis by enhancing the alternative pathway for BA synthesis.

Conjugates of 1,3- and 1,2-Acylglycerols with Stigmasterol: Synthesis, NMR Characterization, and Impact on Lipid Bilayers.

The main aim of research was synthesis and spectroscopic characterization of new conjugates in which stigmasterol was linked via carbonate or succinyl linker with 1,3- and 1,2-acylglycerols of palmitic and oleic acid. Acylglycerols containing stigmasterol residue at internal position have been synthesized from 2-benzyloxypropane-1,3-diol or dihydroxyacetone. Their asymmetric counterparts containing stigmasterol residue attached to sn-3 position have been obtained from (S)-solketal. Eight synthesized conjugates were used to create the liposomes as nanocarriers of phytosterols to increase their stability and protect them from degradation during thermal-oxidative treatments. Fluorimetric and ATR-FTIR methods were used to determine the impact of synthesized conjugates on the physicochemical properties of the lipid bilayer. The results indicate that conjugates with palmitic acid are better candidates for use as the potential stigmasterol nanocarriers compared to those with oleic acid because they increase the stiffness of the lipid bilayer and temperature of the main phase transition. The obtained results are the first step in designing of stigmasterol-enriched liposomal carriers with higher thermo-oxidative stability for their potential use in the food industry.

Stigmasterol isolated from Azadirachta indica flowers attenuated glutamate-induced neurotoxicity via downregulation of the Cdk5/p35/p25 signaling pathway in the HT-22 cells.

BACKGROUND: Glutamate, an excitatory neurotransmitter, was elevated in the brain of neurodegenerative disease (ND) patients. The excessive glutamate induces Ca2+ influx and reactive oxygen species (ROS) production which exacerbates mitochondrial function, leading to mitophagy aberration, and hyperactivates Cdk5/p35/p25 signaling leading to neurotoxicity in ND. Stigmasterol, a phytosterol, has been reported for its neuroprotective effects; however, the underlying mechanism of stigmasterol on restoring glutamate-induced neurotoxicity is not fully investigated. PURPOSE: We investigated the effect of stigmasterol, a compound isolated from Azadirachta indica (AI) flowers, on ameliorating glutamate-induced neuronal apoptosis in the HT-22 cells. STUDY DESIGN: To further understand the underlying molecular mechanisms of stigmasterol, we investigated the effect of stigmasterol on Cdk5 expression, which was aberrantly expressed in glutamate-treated cells. Cell viability, Western blot analysis, and immunofluorescence are employed. RESULTS: Stigmasterol significantly inhibited glutamate-induced neuronal cell death via attenuating ROS production, recovering mitochondrial membrane depolarization, and ameliorating mitophagy aberration by decreasing mitochondria/lysosome fusion and the ratio of LC3-II/LC3-I. In addition, stigmasterol treatment downregulated glutamate-induced Cdk5, p35, and p25 expression via enhancement of Cdk5 degradation and Akt phosphorylation. Although stigmasterol demonstrated neuroprotective effects on inhibiting glutamate-induced neurotoxicity, the efficiency of stigmasterol is limited due to its poor water solubility. We conjugated stigmasterol to soluble soybean polysaccharides with chitosan nanoparticles to overcome the limitations. We found that the encapsulated stigmasterol increased water solubility and enhanced the protective effect on attenuating the Cdk5/p35/p25 signaling pathway compared with free stigmasterol. CONCLUSION: Our findings illustrate the neuroprotective effect and the improved utility of stigmasterol in inhibiting glutamate-induced neurotoxicity.

Computational Analysis of Bacopa monnieri (L.) Wettst. Compounds for Drug Development against Neurodegenerative Disorders.

AIM: With several experimental studies establishing the role of Bacopa monnieri as an effective neurological medication, less focus has been employed to explore how effectively Bacopa monnieri brings about this property. The current work focuses on understanding the molecular interaction of the phytochemicals of the plant against different neurotrophic factors to explore their role and potential as potent anti-neurodegenerative drugs. BACKGROUND: Neurotrophins play a crucial role in the development and regulation of neurons. Alterations in the functioning of these Neurotrophins lead to several Neurodegenerative Disorders. Albeit engineered medications are accessible for the treatment of Neurodegenerative Disorders, due to their numerous side effects, it becomes imperative to formulate and synthesize novel drug candidates. OBJECTIVE: This study aims to investigate the potential of Bacopa monnieri phytochemicals as potent antineurodegenerative drugs by inspecting the interactions between Neurotrophins and target proteins. METHODS: The current study employs molecular docking and molecular dynamic simulation studies to examine the molecular interactions of phytochemicals with respective Neurotrophins. Further inspection of the screened phytochemicals was performed to analyze the ADME-Tox properties in order to classify the screened phytochemicals as potent drug candidates. RESULTS: The phytochemicals of Bacopa monnieri were subjected to in-silico docking with the respective Neurotrophins. Vitamin E, Benzene propanoic acid, 3,5-bis (1,1- dimethylethyl)- 4hydroxy-, methyl ester (BPA), Stigmasterol, and Nonacosane showed an excellent binding affinity with their respective Neurotrophins (BDNF, NT3, NT4, NGF). Moreover, the molecular dynamic simulation studies revealed that BPA and Stigmasterol show a very stable interaction with NT3 and NT4, respectively, suggesting their potential role as a drug candidate. Nonacosane exhibited a fluctuating binding behavior with NGF which can be accounted for by its long linear structure. ADME-Tox studies further confirmed the potency of these phytochemicals as BPA violated no factors and Vitamin E, Stigmasterol and Nonacosane violated 1 factor for Lipinski's rule. Moreover, their high human intestinal absorption and bioavailability score along with their classification as non-mutagen in the Ames test makes these compounds more reliable as potent antineurodegenerative drugs. CONCLUSION: Our study provides an in-silico approach toward understanding the anti-neurodegenerative property of Bacopa monnieri phytochemicals and establishes the role of four major phytochemicals which can be utilized as a replacement for synthetic drugs against several neurodegenerative disorders.

Tomato Sterol 22-desaturase Gene CYP710A11: Its Roles in Meloidogyne incognita Infection and Plant Stigmasterol Alteration.

Sterols are isoprenoid-derived lipids that play essential structural and functional roles in eukaryotic cells. Plants produce a complex mixture of sterols, and changes in plant sterol profiles have been linked to plant-pathogen interactions. ß-Sitosterol and stigmasterol, in particular, have been associated with plant defense. As nematodes have lost the ability to synthesize sterols de novo, they require sterols from the host. Tomato (Solanum lycopersicum) plants infected by the plant parasitic nematode Meloidogyne incognita show a reduced level of stigmasterol and a repression of the gene CYP710A11, encoding the sterol C-22 desaturase that is responsible for the conversion of ß-sitosterol to stigmasterol. In this study, we investigated the role of the tomato sterol C-22 desaturase gene CYP710A11 in the response to infection by M. incognita. We explored the plant-nematode interaction over time by analyzing the plant sterol composition and CYP710A11 gene regulation in S. lycopersicum after M. incognita infection. The temporal gene expression analysis showed that 3 days after inoculation with M. incognita, the CYP710A11 expression was significantly suppressed in the tomato roots, while a significant decrease in the stigmasterol content was observed after 14 days. A cyp710a11 knockout mutant tomato line lacking stigmasterol was analyzed to better understand the role of CYP710A11 in nematode development. M. incognita grown in the mutant line showed reduced egg mass counts, presumably due to the impaired growth of the mutant. However, the nematodes developed as well as they did in the wild-type line. Thus, while the suppression of CYP710A11 expression during nematode development may be a defense response of the plant against the nematode, the lack of stigmasterol did not seem to affect the nematode. This study contributes to the understanding of the role of stigmasterol in the interaction between M. incognita and tomato plants and shows that the sterol C-22 desaturase is not essential for the success of M. incognita.

The Influence of Exogenous Jasmonic Acid on the Biosynthesis of Steroids and Triterpenoids in Calendula officinalis Plants and Hairy Root Culture.

The interplay between steroids and triterpenoids, compounds sharing the same biosynthetic pathway but exerting distinctive functions, is an important part of the defense strategy of plants, and includes metabolic modifications triggered by stress hormones such as jasmonic acid. Two experimental models, Calendula officinalis hairy root cultures and greenhouse cultivated plants (pot plants), were applied for the investigation of the effects of exogenously applied jasmonic acid on the biosynthesis and accumulation of steroids and triterpenoids, characterized by targeted GC-MS (gas chromatography-mass spectroscopy) metabolomic profiling. Jasmonic acid elicitation strongly increased triterpenoid saponin production in hairy root cultures (up to 86-fold) and their release to the medium (up to 533-fold), whereas the effect observed in pot plants was less remarkable (two-fold enhancement of saponin biosynthesis after a single foliar application). In both models, the increase of triterpenoid biosynthesis was coupled with hampering the biomass formation and modifying the sterol content, involving stigmasterol-to-sitosterol ratio, and the proportions between ester and glycoside conjugates. The study revealed that various organs in the same plant can react differently to jasmonic acid elicitation; hairy root cultures are a useful in vitro model to track metabolic changes, and enhanced glycosylation (of both triterpenoids and sterols) seems to be important strategy in plant defense response.

Structural Analysis of Cholesterol Binding and Sterol Selectivity by ABCG5/G8.

The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.

24(R, S)-Saringosterol - From artefact to a biological medical agent.

Enhancing the cholesterol turnover in the brain via activation of liver x receptors can restore memory in a mouse model for Alzheimer's disease. The edible Asian brown alga Sargassum fusiforme (Hijiki) contains high amounts of oxysterols such as (3ß, 24ξ)-stigmasta-5, 28-dien-3, 24-diol (24[R, S]-saringosterol) that are a potent liver x receptor agonists. We aimed to find native European seaweed species with contents of 24(R, S)-saringosterol that are comparable to those found in Sargassum fusiforme. Additionally, we hypothesize that seasonal variations modify the amount of 24(R, S)-saringosterol in seaweeds. Sterols and oxysterols were extracted with chloroform/methanol from various seaweed species harvested in the Eastern Scheldt in different seasons between October 2016 and September 2017. Identification and quantification of the lipids was performed by gas chromatography- mass spectrometry and gas chromatography- flame ionization detection. We confirmed that brown algae Undaria pinnatifida harvested in February and Sargassum muticum harvested in October contained the highest amounts of 24(R, S)-saringosterol (32.4 ± 15.25 µg/g, mean ± S.D. and 32.95 ± 2.91 µg/g, respectively) and its precursor fucosterol (1.48 ± 0.11 mg/g), higher than Sargassum fusiforme (20.94 ± 3.00 µg/g, mean ± S.D.), while Ascophyllum nodosum and Fucus vesiculosus and Fucus serratus contained amounts of 24(R, S)-saringosterol (22.09 ± 3.45 µg/g, 18.04 ± 0.52 µg/g and 19.47 ± 9.01 µg/g, mean ± S.D., respectively) comparable to Sargassum fusiforme. In other algae only minor amounts of these sterols were observed. The green algae Ulva lactuca contained only 0.29 mg/g fucosterol and 10.3 µg/g 24 (R, S)-saringosterol, while all investigated red algae did not contain any 24(R, S)-saringosterol or fucosterol. In the Eastern Scheldt algae harvested in September/October delivered the highest yield for 24(R, S)-saringosterol, with the exception of Undaria pinnatifida that showed the highest levels in February. We showed that exposure of lipid extracts of Ulva lactuca to sunlight at room temperature or in the presence of oxygen to UV-C light lead to the quantitative conversion of fucosterol into 24(R, S)-saringosterol. Exposing pure fucosterol to UV-light did not convert any fucosterol into 24(R, S)-saringosterol underscoring the requirement of seaweed constituents in the conversion of fucosterol into 24(R, S)-saringosterol. In conclusion, we showed that brown seaweeds harvested from the Eastern Scheldt contain amounts of 24(R, S)-saringosterol comparable to Sargassum fusiforme, varying per season and showing the highest amounts in spring. In accordance with these observations the amount of 24(R, S)-saringosterol in the brown seaweeds can be modulated by light.

The Open State Selectivity of the Bean Seed VDAC Depends on Stigmasterol and Ion Concentration.

The voltage-dependent anion channel (VDAC) is the major pathway for metabolites and ions transport through the mitochondrial outer membrane. It can regulate the flow of solutes by switching to a low conductance state correlated with a selectivity reversal, or by a selectivity inversion of its open state. The later one was observed in non-plant VDACs and is poorly characterized. We aim at investigating the selectivity inversion of the open state using plant VDAC purified from Phaseolus coccineus (PcVDAC) to evaluate its physiological role. Our main findings are: (1) The VDAC selectivity inversion of the open state occurs in PcVDAC, (2) Ion concentration and stigmasterol affect the occurrence of the open state selectivity inversion and stigmasterol appears to interact directly with PcVDAC. Interestingly, electrophysiological data concerning the selectivity inversion of the PcVDAC open state suggests that the phenomenon probably does not have a significant physiological effect in vivo.

Structural and functional analysis of tomato sterol C22 desaturase.

BACKGROUND: Sterols are structural and functional components of eukaryotic cell membranes. Plants produce a complex mixture of sterols, among which ß-sitosterol, stigmasterol, campesterol, and cholesterol in some Solanaceae, are the most abundant species. Many reports have shown that the stigmasterol to ß-sitosterol ratio changes during plant development and in response to stresses, suggesting that it may play a role in the regulation of these processes. In tomato (Solanum lycopersicum), changes in the stigmasterol to ß-sitosterol ratio correlate with the induction of the only gene encoding sterol C22-desaturase (C22DES), the enzyme specifically involved in the conversion of ß-sitosterol to stigmasterol. However, despite the biological interest of this enzyme, there is still a lack of knowledge about several relevant aspects related to its structure and function. RESULTS: In this study we report the subcellular localization of tomato C22DES in the endoplasmic reticulum (ER) based on confocal fluorescence microscopy and cell fractionation analyses. Modeling studies have also revealed that C22DES consists of two well-differentiated domains: a single N-terminal transmembrane-helix domain (TMH) anchored in the ER-membrane and a globular (or catalytic) domain that is oriented towards the cytosol. Although TMH is sufficient for the targeting and retention of the enzyme in the ER, the globular domain may also interact and be retained in the ER in the absence of the N-terminal transmembrane domain. The observation that a truncated version of C22DES lacking the TMH is enzymatically inactive revealed that the N-terminal membrane domain is essential for enzyme activity. The in silico analysis of the TMH region of plant C22DES revealed several structural features that could be involved in substrate recognition and binding. CONCLUSIONS: Overall, this study contributes to expand the current knowledge on the structure and function of plant C22DES and to unveil novel aspects related to plant sterol metabolism.

Carrier-Free Triterpene Prodrugs with Glutathione Response and Biosafety for Synergistically Enhanced Photochemotherapy.

Carrier-free pure drug self-assembled nanosystems have been proposed as a promising strategy for synergetic anticancer therapy. Herein, we purposefully designed and synthesized disulfide-modified glutathione (GSH)-responsive natural pentacyclic triterpene betulinic acid (BA) with better biodegradability and biocompatibility to construct carrier-free photosensitive prodrugs BA-S-S/Ce6 NPs for synergistically enhanced and biosafe photochemotherapy. The molecular dynamics simulation elucidates the possible coassembly mechanism that the coplanar arrangement of BA-S-S dimeric may be primarily responsible for the formation of a long lamella-like or spherical morphology. The density functional theory calculations demonstrate that the reduced energy gap (ΔEST) of Ce6 facilitates the improved singlet oxygen generation of BA-S-S/Ce6 nanoparticles (NPs). The assembled prodrugs exhibited remarkable GSH-responsive property and multiple favorable therapeutic features, leading to enhanced synergistic antitumor efficacy without noticeable toxicity. Additionally, evaluation of the antitumor efficacy of another tetracyclic triterpene stigmasterol (ST)-mediated ST-S-S/Ce6 NPs further confirmed the effectiveness of this rational design. This work provides a promising insight for exploring the pure drug self-assembly behavior and construction of GSH-responsive carrier-free triterpenoid prodrugs toward improved multiple combination antitumor therapies.

The potential LXRß agonist stigmasterol protects against hypoxia/reoxygenation injury by modulating mitophagy in primary hippocampal neurons.

BACKGROUND: Neuronal excitotoxicity induces a plethora of downstream signaling pathways, resulting in the calcium overload-induced excitotoxic cell death, a well-known phenomenon in cerebrovascular and neurodegenerative disorders. The naturally occurring phytosterol, stigmasterol (ST) is known for its potential role in cholesterol homeostasis and neuronal development. However, the ability of ST to protect against the induced excitotoxicity in hippocampal neurons has not been investigated yet. PURPOSE: The present study aimed to investigate whether ST could protect against hypoxia/reoxygenation (H/R)-induced excitotoxicity in hippocampal neurons. METHODS: After H/R, neurons were initially subjected to trypan blue exclusion assay for the assessment of cell viability. Live staining using fluorescence dyes namely JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide), DCFDA (2',7'-dichlorofluorescein diacetate) and FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) were used to measure MMP, ROS and synaptic vesicle pool size. Immunostaining was performed to analyze the expression levels of vesicular glutamate transporter 1 (VGLUT1), N-methyl-D-acetate receptor subunit 2B (GluN2B), LC3BII, p62, and PTEN induced protein kinase 1 (PINK1) in neuron after H/R. Western blotting was carried out to measure the protein expression of GluN2B. The molecular dynamics simulation was employed to elucidate the LXRß agonistic conformation of ST. RESULT: Pre-incubation of neuronal cultures with ST (20 µM) protected against excitotoxicity, and attenuated reactive oxygen species (ROS) generation, double-stranded DNA break, and mitochondrial membrane potential (MMP) loss. ST treatment also resulted in the downregulation of the expressions of VGLUT1 and GluN2B and the reduction of the size of recyclable synaptic vesicle (SV) pool. Like LXRß agonist GW3695, ST suppressed the expression of GluN2B. Furthermore, ST induced mitophagy through upregulating the expressions of LC3BII, p62, and PINK1. The molecular simulation study showed that ST interacted with the ligand binding domain of liver X receptor ß (LXRß), a known binding receptor of ST, through multiple hydrogen bonding. CONCLUSION: Collectively, these findings revealed that ST exhibited a promising neuroprotective effect by regulating both pre- and post-synaptic events following H/R, particularly, attenuation of GluN2B-mediated excitotoxicity and oxidative stress, and induction of mitophagy, and suggested that ST might be a therapeutic promise against ischemic stroke and its associated neurological disorders.

Characterization and overexpression of sterol Δ22-desaturase, a key enzyme modulates the biosyntheses of stigmasterol and withanolides in Withania somnifera (L.) Dunal.

Withanolides constitute an extensive and vital class of metabolites displaying wide array of structural and therapeutic properties with unique side-chain modifications. These show diversified scaffolds and are promising pharmaceutical molecules with well documented anti-inflammatory and anti-cancer properties. Sterols are dynamic class of compounds and essential molecules having structural and functional significance. These contribute to the synthesis of withanolides by providing structural precursors. In this context, we have characterized sterol Δ22-desaturase from Withania somnifera and also functionally validating it by confirming its desaturase nature in conjunction with quantitative real-time expression profiling and metabolite evaluation. Further, transgenic hairy roots of W. somnifera displayed a higher accumulation of stigmasterol and withanolides. The increase in chemical constituents was concomitant with an increased gene copy number predicted via Southern blotting. Additionally, transgenic lines of tobacco over-expressing WsCYP710A11 displayed a substantial increase in its expression, corroborating well with enhanced stigmasterol content. Characterization of CYP710A11 from W. somnifera and its homologous transgenic expression has demonstrated its role in the regulation of withanolides biosynthesis. It also exhibited a differential transcriptional profile in response to exogenous elicitations. These empirical findings suggest the crucial role of CYP710A11 in stigmasterol biosynthesis. This in turn has implications for the overproduction of withanolides via pathway channelling.

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

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, ß-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.

Phytochemical investigation of Hyoscyamus albus.

The work presented in this paper illustrates the isolation and structure elucidation of secondary metabolites of Hyoscyamus albus. Two new natural source and three known compounds were isolated from the Hyoscyamus albus. Among the isolated compounds, grivilloside H (1) and betulaplatoside (2) were isolated for the first time while scopolamine (3), ß-sitosterol (4) and stigmasterol (5) have been reported previously from the same plant. The structures of all the isolated compounds were established by using modern spectroscopic technique (UV, IR, NMR, and EI-MS) and by comparing with those available in literature.

A Plant like Cytochrome P450 Subfamily CYP710C1 Gene in Leishmania donovani Encodes Sterol C-22 Desaturase and its Over-expression Leads to Resistance to Amphotericin B.

BACKGROUND: Leishmania donovani is a protozoan parasite, a primary causative agent of visceral leishmaniasis. Sterol produced via the mevalonate pathway, show differences in composition across biological kingdoms. The specific occurrence of Δ22-unsaturated sterols, containing a double bond at the C-22 position in the side chain occurs in fungi as ergosterol and as stigmasterol in plants. In the present study, we report the identification and functional characterization of a plant-like Cytochrome P450 subfamily CYP710C1 in L. donovani as the Leishmania C-22 desaturase. METHODOLOGY: In silico analysis predicted the presence of a plant like CYP710C1 gene that encodes a sterol C-22 desaturase, a key enzyme in stigmasterol biosynthesis. The enzymatic function of recombinant CYP710C1 as C-22 desaturase was determined. To further study the physiological role of CYP710C1 in Leishmania, we developed and characterized an overexpressing strain and a gene deletion mutant. C-22 desaturase activity and stigmasterol levels were estimated in the wild-type, overexpressing promastigotes and heterozygous mutants. CONCLUSION: We for the first time report the presence of a CYP710C1 gene that encodes a plant like sterol C-22 desaturase leading to stigmasterol biosynthesis in Leishmania. The recombinant CYP710C1 exhibited C-22 desaturase activity by converting ß-sitosterol to stigmasterol. Axenic amastigotes showed higher expression of CYP710C1 mRNA, protein and stigmasterol levels compared to the promastigotes. Sterol profiling of CYP710C1 overexpressing L. donovani and heterozygous mutant parasites demonstrated that CYP710C1 was responsible for stigmasterol production. Most importantly, we demonstrate that these CYP710C1 overexpressing promastigotes are resistant to amphotericin B, a drug of choice for use against leishmaniasis. We report that Leishmania sterol biosynthesis pathway has a chimeric organisation with characteristics of both plant and fungal pathways.

Heterologous expression and functional characterization of the ligand-binding domain of oxysterol-binding protein from Aspergillus oryzae.

Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (ß-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.

Frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor.

The Lemnaceae, known as duckweed, the smallest flowering aquatic plant, shows promise as a plant bioreactor. For applying this potential plant bioreactor, establishing a stable and efficient genetic transformation system is necessary. The currently favored callus-based method for duckweed transformation is time consuming and genotype limited, as it requires callus culture and regeneration, which is inapplicable to many elite duckweed strains suitable for bioreactor exploitation. In this study, we attempted to establish a simple frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor, one of the most widespread duckweed species in the world. To evaluate the feasibility of the new transformation system, the gene CYP710A11 was overexpressed to improve the yield of stigmasterol, which has multiple medicinal purposes. Three L. minor strains, ZH0055, D0158 and M0165, were transformed by both a conventional callus transformation system (CTS) and the simple frond transformation system (FTS). GUS staining, PCR, quantitative PCR and stigmasterol content detection showed that FTS can produce stable transgenic lines as well as CTS. Moreover, compared to CTS, FTS can avoid the genotype constraints of callus induction, thus saving at least half of the required processing time (CTS took 8-9 months while FTS took approximately 3 months in this study). Therefore, this transformation system is feasible in producing stable transgenic lines for a wide range of L. minor genotypes.