Assessment of Biological Activity of 28-Homobrassinolide via a Multi-Level Comparative Analysis.

Brassinosteroids (BRs) play vital roles in the plant life cycle and synthetic BRs are widely used to increase crop yield and plant stress tolerance. Among them are 24R-methyl-epibrassinolide (24-EBL) and 24S-ethyl-28-homobrassinolide (28-HBL), which differ from brassinolide (BL, the most active BR) at the C-24 position. Although it is well known that 24-EBL is 10% active as BL, there is no consensus on the bioactivity of 28-HBL. A recent outpouring of research interest in 28-HBL on major crops accompanied with a surge of industrial-scale synthesis that produces mixtures of active (22R,23R)-28-HBL and inactive (22S,23S)-28HBL, demands a standardized assay system capable of analyzing different synthetic "28-HBL" products. In this study, the relative bioactivity of 28-HBL to BL and 24-EBL, including its capacity to induce the well-established BR responses at molecular, biochemical, and physiological levels, was systematically analyzed using the whole seedlings of the wild-type and BR-deficient mutant of Arabidopsis thaliana. These multi-level bioassays consistently showed that 28-HBL exhibits a much stronger bioactivity than 24-EBL and is almost as active as BL in rescuing the short hypocotyl phenotype of the dark-grown det2 mutant. These results are consistent with the previously established structure-activity relationship of BRs, proving that this multi-level whole seedling bioassay system could be used to analyze different batches of industrially produced 28-HBL or other BL analogs to ensure the full potential of BRs in modern agriculture.

Phytosteroid 28-homobrassinolide targets cholesterol and glucose homeostasis implicating ABCA1 and SREBP role in regulation.

Oxysterols are natural ligands of certain nuclear receptors known as liver X receptors (LXR). LXRs are regulators of fatty acid, cholesterol, and glucose homeostasis. Dietary phyto-oxysterol 28-homobrassinolide (28-HB) has been demonstrated to transactivate rat LXR α and ß. In this study we assessed the potential of 28-HB to effect such changes in - (1) human HepG2 cancer cell line, (2) isolated perfused goat liver, and (3) high-fat diet-fed C57BL/6J mice. Serum and perfusate marker levels along with hexokinase activity were determined through enzyme assays. Fat deposition was studied by Oil Red O staining, ATP-binding cassette transporter (ABCA1), and sterol regulatory element-binding transcription factor 2 (SREBP2) protein expression by Western blot and their mRNA expression through real-time PCR. In HepG2 cells, 28-HB (5-20 µM) treatment indicated a 2-fold increase in glucose utilization and ABCA1 and SREBP2 protein expression within 12 h. Tissue glucose and cholesterol levels decreased in 28-HB perfused goat liver within 2 h, whereas cholesterol level increased 54% in the perfusate (p < 0.05) and tissue hexokinase activity increased 23% (p < 0.05). Glucokinase, ABCA1, and SREBF1 gene expression increased 2.6, 5.37, and 2.85 fold respectively in the perfused tissue after 4 h. High-fat diet-fed C57BL/6J mice when treated with 28-HB (1-20 µg/day) for 6 weeks exhibited a marked decrease in aortic fat deposit and serum marker levels. Our study suggests that 28-HB modulates cholesterol and glucose homeostasis in animal cells through activation of LXR involving ABCA1 and SREBP-1 and 2 augmentations.

Study on synthesis of key squalamine intermediate 7α, 24R-dihydroxy-5α-cholestan-3-one.

A facile novel strategy has been developed to obtain a key intermediate of squalamine, 7α, 24R -dihydroxy-5α-cholestan-3-one, starting from methyl Δ5-3ß-hydroxycholanate. The pure product was successfully synthesized and separated from the C-24 position epimers in good purity, d.e.% and yield.

Comparative effect of 28-homobrassinolide and 24-epibrassinolide on the performance of different components influencing the photosynthetic machinery in Brassica juncea L.

BRs are polyhydroxylated sterol derivatives, classified as phytohormones. Plants of Brassica juncea var. Varuna were grown in pots and an aqueous solution (10-8 M) of two brassinosteroid isomers 28-homobrassinolide (HBL) and 24-epibrassinolide (EBL) of same concentration (10-8 M) was applied to their leaves. The treatment up-regulated the photosynthetic machinery directly by enhancing water splitting activity, photochemical quenching, non-photochemical quenching, maximum PSII efficiency, actual PSII efficiency, electron transport rate, stomatal movement, stomatal conductance, internal CO2 concentration, transpiration rate, net photosynthetic rate and carbohydrate synthesis. Moreover, the level of biochemical enzymes (carbonic anhydrase and nitrate reductase), reactive oxygen species (superoxide and hydrogen peroxide) generation, antioxidant enzyme activity and mineral status (C, N, Mg, P, S, K), which indirectly influence the rate of photosynthesis, also improved in the treated plants. Out of the two BR analogues tested, EBL excelled in its effects over HBL.

28-homobrassinolide regulates antioxidant enzyme activities and gene expression in response to salt- and temperature-induced oxidative stress in Brassica juncea.

Brassinosteroids (BRs) are a group of naturally occurring plant steroid hormones that can induce plant tolerance to various plant stresses by regulating ROS production in cells, but the underlying mechanisms of this scavenging activity by BRs are not well understood. This study investigated the effects of 28-homobrassinolide (28-HBL) seed priming on Brassica juncea seedlings subjected to the combined stress of extreme temperatures (low, 4 °C or high, 44 °C) and salinity (180 mM), either alone or supplemented with 28-HBL treatments (0, 10-6, 10-9, 10-12 M). The combined temperature and salt stress treatments significantly reduced shoot and root lengths, but these improved when supplemented with 28-HBL although the response was dose-dependent. The combined stress alone significantly increased H2O2 content, but was inhibited when supplemented with 28-HBL. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) increased in response to 28-HBL. Overall, the 28-HBL seed priming treatment improved the plant's potential to combat the toxic effects imposed by the combined temperature and salt stress by tightly regulating the accumulation of ROS, which was reflected in the improved redox state of antioxidants.

28-Homocastasterone down regulates blood glucose, cholesterol, triglycerides, SREBP1c and activates LxR expression in normal & diabetic male rat.

Plant steroids are being recognized as influential secondary bio factors, assimilating in animal tissues through diet and affecting their cellular metabolic function to varying degree. They modulate catalytic and signaling functions in mammalian cells, affecting cellular homeostasis. The effect of phyto brassinosteroid ketoisoform 28-homocastasterone (28-HC), was assessed for its influence on blood glucose, plasma lipid and selective signal marker levels in normal and diabetic male wistar rat models. A 15 day oral feed regimen employing the experimental rat, noted that circulating blood glucose, cholesterol and triglyceride level in diabetic rat were markedly reduced by this compound. This study confirmed that the keto form had anti-hyperglycemic and anti-lipidemic potency associated with it and was available to man and animals in their diet. Western blots of marker protein, PCR amplicons of marker mRNA expressions and In Silico studies suggested that 28-HCeffect is being mediated through LxR molecular operatives in the rat cell.

Enantiospecific tritium labeling of 28-homocastasterone.

A regiospecific and enantiospecific synthesis of tritium-labeled 28-homocastasterone is reported. Appropriate chlorocarbonate, efficiently synthesized from the starting 28-homocastasterone in an overall yield of 46%, undergoes catalytic tritium dechlorination by the T2 /Pd[0]/Et3 N system, providing 28-[3ß-3 H]homocastasterone, in a good yield, radiochemical purity (>97%), and with a high specific activity (5.8 Ci/mmol).

Synthesis and preliminary evaluation of dimeric-28-homobrassinosteroids for plant growth regulators.

Preparation of synthetic analogues of 28-homobrassinosteroids is reported. Also, the addition of the 28-homocastasterone at the C6 carbonyl group via allyl Gringard reagent followed by olefin cross metathesis resulted in dimeric analogues. Rice lamina inclination assay showed that the replacement of the C6 carbonyl group by 6α-allyl and 6ß hydroxyl groups led to a decrease in bioactivity, whereas the dimeric analogues showed a reduced but significant bioactivity when compared to the 28-homocastasterone.

A plant oxysterol, 28-homobrassinolide binds HMGCoA reductase catalytic cleft: stereoselective avidity affects enzyme function.

Understanding the influence of ubiquitously present plant steroids on mammalian cell biology is currently of interest. Feedback inhibition of HMGCoA reductase (HMGCR) catalytic activity in the transformation of HMG-CoA to mevalonate is a significant regulatory step in sterol biosynthetic pathway. To assess the role of dietary steroids in this biochemical transformation, the phytosteroid isoform 28-homobrassinolide (28-HB), 90 % pure, obtained from Godrej Agrovet (India) was used to determine its effect on mammalian HMG-CoA reductase. Photometric assay of pure human and select rat tissue HMGCR post 28-HB oral feed, PCR-HMGCR gene expression, and in silico docking of 28-HB and HMGCoA on HMGCR protein template were carried out. Using an oral feed regimen of pure 28-HB, we noted a decrease of 16 % in liver, 17.1 % in kidney and 9.3 % in testicular HMGCR enzyme activity, 25 % in HMGCR gene expression and 44 % in the activity of pure human HMGCR due to this plant oxysterol. In silico docking studies yielded binding metrics for 28-HB-HMGCR lower than for HMGCoA-HMGCR, indicating stronger binding of HMGCR by this ligand. 28-HB exerts differential effects on rat tissue HMGCR, down regulates liver HMGCR gene expression and significantly inhibits HMGCR activity.

Functional characterization of CYP71D443, a cytochrome P450 catalyzing C-22 hydroxylation in the 20-hydroxyecdysone biosynthesis of Ajuga hairy roots.

20-Hydroxyecdysone (20HE), a molting hormone of insects, is also distributed among a variety of plant families. 20HE is thought to play a role in protecting plants from insect herbivores. In insects, biosynthesis of 20HE from cholesterol proceeds via 7-dehydrocholesterol and 3ß,14α-dihydroxy-5ß-cholest-7-en-6-one (5ß-ketodiol), the latter being converted to 20HE through sequential hydroxylation catalyzed by four P450 enzymes, which have been cloned and identified. In contrast, little is known about plant 20HE biosynthesis, and no biosynthetic 20HE gene has been reported thus far. We recently proposed involvement of 3ß-hydroxy-5ß-cholestan-6-one (5ß-ketone) in 20HE biosynthesis in the hairy roots of Ajuga reptans var. atropurpurea (Lamiaceae). In this study, an Ajuga EST library was generated from the hairy roots and P450 genes were deduced from the library. Five genes with a high expression level (CYP71D443, CYP76AH19, CYP76AH20, CYP76AH21 and CYP716D27) were screened for a possible involvement in 20HE biosynthesis. As a result, CYP71D443 was shown to have C-22 hydroxylation activity for the 5ß-ketone substrate using a yeast expression system. The hydroxylated product, 22-hydroxy-5ß-ketone, had a 22R configuration in agreement with that of 20HE. Furthermore, labeling experiments indicated that (22R)-22-hydroxy-5ß-ketone was converted to 20HE in Ajuga hairy roots. Based on the present results, a possible 20HE biosynthetic pathway in Ajuga plants involved CYP71D443 is proposed.

Homobrassinolide induced conformational changes in hexokinase: a possible mechanism for its antidiabetic potential.

Hormonal regulation of cell growth and development, tissue morphology, metabolism and physiological function in animals and man is a well-established knowledge domain in modern biological science. The present study was carried out to investigate the structural stability of hexokinase when exposed to diabetic levels of glucose and its binding efficiency. The fluorescence study indicated that 28-homobrassinolide was able to protect or restore the native structure of hexokinase. Proteins are synthesized and fold into the native form to become active. The inability of a protein molecule to remain in its native form is called as protein misfolding and this is because of several factors. Protein aggregation and misfolding are known to play a critical role in several human diseases including diabetes. Homobrassinolide interaction with hexokinase was studied by UV-Vis spectrophotometer and fluorescence spectrophotometer. Results were suggested that the denatured hexokinase was renatured upon binding with homobrassinolide. In silico, docking study was performed to recognize the binding activity of homobrassinolide against a subunit of the glucokinase, and homobrassinolide was able to bind to the drug binding pocket of glucokinase. The glide energy is -7.1 kcal/mol, suggesting the high binding affinity of homobrassinolide to glucokinase. Overall, these studies predict that the phytohormone 28-homobrassinolide would function as an anti-diabetic when present in human and animal diet by augmenting the hexokinase enzyme activity in the animal cell. Copyright © 2015 John Wiley & Sons, Ltd.

Lycopersicon esculentum under low temperature stress: an approach toward enhanced antioxidants and yield.

Brassinosteroids (BRs) have been implicated to overcome various abiotic stresses, and low temperature stress poses a serious threat to productivity of various horticultural crops like tomato. Therefore, a study was conducted to unravel the possible role of BRs in conferring alleviation to low temperature stress in Lycopersicon esculentum. Twenty-day-old seedlings of tomato var. S-22 (chilling tolerant) and PKM-1 (chilling sensitive) were sown in earthen pots, and at 40 days stage of growth, plants were exposed to varied levels of low temperatures (10/3, 12/7, 20/14, or 25/18 °C) for 24 h in a growth chamber. At 50 days stage of growth, the foliage of plants were sprayed with 0 or 10(-8) M of BRs (28-homobrassinolide or 24-epibrassinolide), and 60-day-old plants were harvested to assess various physiological and biochemical parameters. Low temperatures induced a significant reduction in growth traits, chlorophyll content, and rate of photosynthesis in both the varieties differentially. Activities of antioxidant enzymes (catalase, peroxidase, and superoxide dismutase) and leaf proline content also increased substantially in both the varieties with decreasing temperature. On the other hand, treatment of BRs under stress and stress-free conditions significantly increased the aforesaid growth traits and biochemical parameters. Moreover, BRs further accelerated the antioxidative enzymes and proline content, which were already enhanced by the low temperature stress. Out of the two analogues of BRs tested, 24-epibrassinolide (EBL) was found more effective for both the varieties of tomato. EBL was found more potent stress alleviator against low temperature in both varieties of tomato.

Biosynthesis of 20-hydroxyecdysone in plants: 3ß-hydroxy-5ß-cholestan-6-one as an intermediate immediately after cholesterol in Ajuga hairy roots.

3ß-Hydroxy-5ß-cholestan-6-one was identified in the EtOAc extract of Ajuga hairy roots by micro-analysis using LC-MS/MS in the multiple reaction mode (MRM). Furthermore, administration of (2,2,4,4,7,7-(2)H6)- and (2,2,4,4,6,7,7-(2)H7)-cholesterols to the hairy roots followed by LC-MS/MS analysis of the EtOAc extract of the hairy roots indicated that cholesterol was converted to the 5ß-ketone with hydrogen migration from the C-6 to the C-5 position. These findings, in conjunction with the previous observation that the ketone was efficiently converted to 20-hydroxyecdysone, strongly suggest that the 5ß-ketone is an intermediate immediately formed after cholesterol during 20-hydroxyecdysone biosynthesis in Ajuga sp. In addition, the mechanism of the 5ß-ketone formation from cholesterol is discussed.

Biosynthetic relationship between C28-brassinosteroids and C29-brassinosteroids in rice (Oryza sativa) seedlings.

A crude enzyme solution was prepared from young rice seedlings, and the metabolism of C29-brassinosteroids identified from the seedlings was examined. When 28-homoteasterone was added as a substrate, 28-homotyphasterol, teasterone, and 26-nor-28-homoteasterone were characterized as enzyme products by GC-MS/SIM analysis. With 28-homotyphasterol, 28-homoteasterone, typhasterol, 28-homocastasterone, and 26-nor-28-homotyphasterol were formed and identified as products. When 28-homocastasterone was used, castasterone and 26-nor-28-homocastasterone were identified as products. Together with the reduced biological activity of C29-brassinosteroids and their metabolites in the rice lamina inclination assay, these metabolic studies suggest a biosynthetic sequence, 28-homoteasterone↔28-homotyphasterol→28-homocastasterone for C29-brassinosteroid biosynthesis is connected to the biosynthetic sequence teasterone↔typhasterol→castasterone for C28-brassinosteroids by C-28 demethylation, i.e., in order to increase biological activity in the rice plant. Additionally, the C29-brassinosteroids seem to bio-degrade their C-26 demethylated C28-brassinosteroid analogs to reduce brassinosteroid activity in planta. In conclusion, the biosynthesis of C29-brassinosteroids is a likely alternative route to the biologically-active brassinosteroid, castasterone, in rice.

Antiviral action of synthetic stigmasterol derivatives on herpes simplex virus replication in nervous cells in vitro.

Polyfunctionalized stigmasterol derivatives, (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3ß-bromo-5α,22,23-trihydroxystigmastan-6-one (compound 2), inhibit herpes simplex virus type 1 (HSV-1) replication and spreading in human epithelial cells derived from ocular tissues. Both compounds reduce the incidence and severity of lesions in a murine model of herpetic stromal keratitis when administered in different treatment modalities. Since encephalitis caused by HSV-1 is another immunopathology of viral origin, we evaluate here the antiviral effect of both compounds on HSV-1 infected nervous cell lines as well as their anti-inflammatory action. We found that both stigmasterol derivatives presented low cytotoxicity in the three nervous cell lines assayed. Regarding the antiviral activity, in all cases both compounds prevented HSV-1 multiplication when added after infection, as well as virus propagation. Additionally, both compounds were able to hinder interleukin-6 and Interferon-gamma secretion induced by HSV-1 infection in Neuro-2a cells. We conclude that compounds 1 and 2 have exerted a dual antiviral and anti-inflammatory effect in HSV-1 infected nervous cell lines, which makes them interesting molecules to be further studied.

28-Homobrassinolide: a novel oxysterol transactivating LXR gene expression.

Cholesterol is the template for steroid hormone biosynthesis. Cholesterol homeostasis is regulated by Cyt-P450 oxygenated cholesterols acting as ligands on LXR-α and LXR-ß transcription factors that are now emerging as drug targets. Heterodimerization of LXRs with retinoic acid receptor is considered a prerequisite for target gene activation. Dietary plant oxysterol 28-homobrassinolide (28-HB) is a proven antihyperglycemic and a pro-steroidogenic agent in the rat. Whether 28-HB has a role in LXR gene expression was therefore investigated using oral gavage (15 days) of 28-HB (333 µg/kg b w) to normal and diabetic rat. PCR amplified LXR-α and ß mRNA transcripts from treated rat liver and testis exhibited quantitative differences in their expression. Conformational differences in 28-HB docking to LXR-α and ß binding domains were also noted through in silico studies, LXR-ß adopting lesser specificity. We report that 28-HB transactivates LXR genes in the rat tissues.

Thunberol, a new sterol from the Chinese brown alga Sargassum thunbergii.

A new sterol, named thunberol (1), along with four known analogs, 24-ethylcholesta-4,24(28)-dien-3-one (2), stigmasta-5,28-dien-3ß-ol (3), cholesta-5,14-dien-3ß-ol (4), and cholesta-5,23-dien-3ß,25-diol (5), were isolated from the brown alga Sargassum thunbergii collected from East China Sea. The structures of these metabolites were elucidated on the basis of detailed spectroscopic analysis and by comparison with the literature data. Thunberol (1) exhibited significant inhibitory activity against protein tyrosine phosphatase 1B, a potential drug target for the treatment of Type-II diabetes and obesity, with an IC50 value of 2.24 µg/ml.

Naturally occurring compounds elicit HIV-1 replication in chronically infected promonocytic cells.

Since antiretroviral therapy suppresses but does not eradicate HIV-1 infection, methods to purge viral reservoirs are required. Many strategies involve the reactivation of chronically HIV infected cells to induce the expression of integrated viral genome. In this study, five bioactive compounds, the plant derivatives 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), nordihydroguaiaretic acid (NDGA), and curcumin (Cur) and the synthetic stigmasterol analogs (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3 ß -bromo-5 α ,22,23-trihydroxystigmastan-6-one (compound 2), were evaluated for their ability to elicit HIV replication in promonocytic (U1) and lymphocytic (H9+) HIV-1 chronically infected cells. The results revealed that natural compounds CDM, NDGA, and Cur were able to increase HIV-1 p24 antigen, determined by ELISA, only in latently infected promonocytic cells. CDM would reactivate HIV from latency by modulating the release of IL-6 and TNF- α , since the amount of both cytokines measured through ELISA significantly increased in U1 treated cells. Besides, NDGA increased ROS production, which might be related to the increase on p24 level observed in NDGA treated U1. These findings suggest that CDM, NDGA, and Cur might be candidates for further studies on latency-reversing therapeutics to eliminate latently HIV-1 reservoirs.

Probing the selective antitumor activity of 22-oxo-26-selenocyanocholestane derivatives.

Diverse steroidal compounds have shown antiproliferative activity on certain tumor cell lines; however, their complete role on cancer cells has not been extensively established since the research is quite recent. Hence, deeper study in this field is required. Due to the importance of selenium in animal and human health; herein, we report the synthesis, characterization, and biological evaluation of two novel 22-oxo-26-selenocyanocholestanic steroids on cervicouterine cancer cells and non-tumor cells. The title compounds were straightforward prepared from diosgenin and hecogenin in excellent overall yields. We determined their effect on cell proliferation on HeLa, CaSki, and ViBo cell cultures. Their cytotoxic effect on tumor cells, as well as on peripheral blood lymphocytes was also evaluated. The increase in the expression of active caspase-3 along with the fragmentation of DNA confirm that the new 22-oxo-26-selenocyanocholestane frameworks potentiate apoptosis in tumor cells. The antiproliferative activity on tumor cells affects to some extent the proliferative potential of peripheral blood lymphocytes, so an immunosuppressive effect has also been established. The novel 22-oxo-26-selenocyanocholestane compounds show selective antitumor activity and therefore are promising lead candidates for further in vivo evaluation.

Synthesis of 5α-cholestan-6-one derivatives and their inhibitory activities of NO production in activated microglia: discovery of a novel neuroinflammation inhibitor.

Glial activation-mediated neuroinflammation plays a pivotal role in the process of several neuroinflammatory diseases including stroke, Alzheimer's diseases, Parkinson's diseases, multiple sclerosis and ischemia. Inhibition of microglial activation may ameliorate neuronal degeneration under the inflammatory conditions. In the present study, a number of 5α-cholestan-6-one derivatives were prepared and the anti-inflammatory effects of these compounds were evaluated in LPS-stimulated BV-2 microglia cells. Those derivatives were synthesized from readily available hyodeoxycholic acid (1). Among the tested compounds, several analogs (16-18, 25, 35, 38) exhibited potent inhibitory activities on nitric oxide production with no or weak cell toxicity. Compound 16 also significantly suppressed the expression of TNF-α, interleukin (IL)-1ß, cyclooxygenase (COX-2) as well as inducible nitric oxide synthase (iNOS) in LPS-stimulated BV-2 microglia cells. In addition, compound 16 markedly reduced infarction volume in a focal ischemic mice model.