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.

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.

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.

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.

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.

Protection of growth in response to 28-homobrassinolide under the stress of cadmium and salinity in wheat.

This study examines the impact of 28-homobrassinolide (HBL) in alleviating the effects of cadmium (Cd) and salinity (NaCl) on wheat plants solely and against the synergy. The surface sterilized seeds of Triticum aestivum cv. PBW-373 were sown in the soil amended either with salinity or cadmium or both. The foliage in was sprayed at 20d after sowing (DAS) both in stressed and non-stressed plants. The spray of HBL at 20 DAS increased almost all the parameters while decreased the H2O2 content and lipid peroxidation in the leaves. The presence of cadmium and/or salinity decreased the values for all the growth and photosynthetic parameters but improved the activity of antioxidant enzymes and proline content in 30d old plants. However, the ill effects observed under NaCl and/or Cd treatment were completely overcomed by the spray of HBL to the plants at 20d stage. The spray of HBL to stressed plants further increased the antioxidant enzyme activities and proline content thereby giving tolerance to the plants against the stress.

A phytooxysterol, 28-homobrassinolide modulates rat testicular steroidogenesis in normal and diabetic rats.

Steroidogenesis in testicular cells depends upon the availability of cholesterol within testicular mitochondria besides the activities of 3ß-hydroxysteroid dehydrogenase (3ß-HSD, 17ß-hydroxysteroid dehydrogenase [17b-HSD]), and the tissue levels of steroidogenic acute regulatory protein (StAR), androgen-binding protein (ABP), and testosterone (T). Cellular cholesterol biosynthesis is regulated by endogenous oxycholesterols acting through nuclear hormone receptors. Plant oxysterols, such as 28-homobrassinolide (28-HB), available to human through diet, was shown to exhibit antihyperglycemic effect in diabetic male rat. Its role in rat testicular steroidogenesis and lipid peroxidation (LPO) was therefore assessed using normal and streptozotocin-induced diabetic male rats. Administration of 28-HB (333 µg/kg body weight) by oral gavage for 15 consecutive days to experimental rats diminished LPO, increased antioxidant enzyme, 3ß-HSD and 17ß-HSD activities, and elevated StAR and ABP expression and T level in rat testis. We report that 28-HB induced steroidogenesis in normal and diabetic rat testis.

Mechanisms of natural brassinosteroid-induced apoptosis of prostate cancer cells.

Brassinosteroids (BRs) are a group of polyhydroxylated sterol derivatives with important regulatory roles in various plant physiological processes. The aim of this study was to examine the mechanism of the antiproliferative activity of natural BRs 28-homocastasterone (28-homoCS) and 24-epibrassinolide (24-epiBL) in hormone-sensitive and -insensitive (LNCaP and DU-145, respectively) human prostate cancer cell lines. The effects of BRs on prostate cancer cells were surveyed using flow cytometry, Western blotting, TUNEL, DNA ladder assays and immunofluorescence analyses. The studied BRs inhibited cell growth and induced G(1) blocks in LNCaP cells accompanied by reductions in cyclin D(1), CDK4/6 and pRb expression. Following BR treatment of DU-145 cells, increases in proportions of cells in the G(2)/M phase of cell cycle were observed, accompanied by down-regulation of cyclins A and B(1). Changes in AR localization patterns in LNCaP cells treated with BRs were shown by immunofluorescence analysis. Furthermore, apoptotic detection methods demonstrated induction of apoptosis mediated by BRs in both cell lines, although changes in the expression of apoptosis-related proteins were modulated differently by 28-homoCS and 24-piBL in each cell line. The studied BRs seem to exert potent growth inhibitory and pro-apoptotic effects and could be therefore highly valuable new candidates for prostate anticancer drugs.

Cellular glutathione redox homeostasis plays an important role in the brassinosteroid-induced increase in CO2 assimilation in Cucumis sativus.

Brassinosteroids (BRs) play a vital role in plant growth, stress tolerance and productivity. Here, the involvement of BRs in the regulation of CO(2) assimilation and cellular redox homeostasis was studied. The effects of BRs on CO(2) assimilation were studied in cucumber (Cucumis sativus) through the analysis of the accumulation of H(2)O(2) and glutathione and photosynthesis-related enzyme activities using histochemical and cytochemical detection or a spectrophotometric assay, and Rubisco activase (RCA) using western blot analysis and immunogold labeling. Exogenous BR increased apoplastic H(2)O(2) accumulation, the ratio of reduced to oxidized glutathione (GSH:GSSG) and CO(2) assimilation, whereas a BR biosynthetic inhibitor had the opposite effects. BR-induced CO(2) assimilation was decreased by a H(2)O(2) scavenger or inhibition of H(2)O(2) generation, GSH biosynthesis and the NADPH-generating pentose phosphate pathway. BR-, H(2)O(2) - or GSH-induced CO(2) assimilation was associated with increased activity of enzymes in the Benson-Calvin cycle. Immunogold labeling and western blotting showed that BR increased the content of RCA and this effect was blocked by inhibitors of redox homeostasis. These results strongly suggest that BR-induced photosynthesis involves an H(2)O(2) -mediated increase in the GSH:GSSG ratio, which may positively regulate the synthesis and activation of redox-sensitive enzymes in carbon fixation.

Comparative effect of 28 homobrassinolide and salicylic acid in the amelioration of NaCl stress in Brassica juncea L.

Among various environmental stresses, salt stress is extensively damaging to major crops all over the world. An experiment was conducted to explore the role of exogenously applied 28 homobrassinolide (HBL) and salicylic acid (SA) on growth, photosynthetic parameters, transpiration and proline content of Brassica juncea L. cultivar Varuna in presence or absence of saline conditions (4.2 dsm(-1)). The leaves of 29d old plants were sprayed with distilled water, HBL and/or SA and plant responses were studied at 30 days after sowing (24 h after spray) and 45 days after sowing. The salinity significantly reduced the plant growth, gas exchange parameters but increased proline content and electrolyte leakage in the leaves. The effects were more pronounced at 30 DAS than 45 DAS. Out of the two hormones (HBL/SA) HBL excelled in its effects at both sampling stages. Toxic effects generated by salinity stress were completely overcome by the combination of the two hormones (HBL and SA) at 45 DAS.

Polyhydroxylated steroids from the bamboo coral Isis hippuris.

In previous studies on the secondary metabolites of the Taiwanese octocoral Isis hippuris, specimens have always been collected at Green Island. In the course of our studies on bioactive compounds from marine organisms, the acetone-solubles of the Taiwanese octocoral I. hippuris collected at Orchid Island have led to the isolation of five new polyoxygenated steroids: hipposterone M-O (1-3), hipposterol G (4) and hippuristeroketal A (5). The structures of these compounds were determined on the basis of their spectroscopic and physical data. The anti-HCMV (human cytomegalovirus) activity of 1-5 and their cytotoxicity against selected cell lines were evaluated. Compound 2 exhibited inhibitory activity against HCMV, with an EC(50) value of 6.0 µg/mL.

28-Homobrassinolide mitigates boron induced toxicity through enhanced antioxidant system in Vigna radiata plants.

The objective of this study was to establish relationship between boron induced oxidative stress and antioxidant system in Vigna radiata plants and also to investigate whether brassinosteroids will enhance the level of antioxidant system that could confer tolerance to the plants from the boron induced oxidative stress. The mung bean (V. radiata cv. T-44) plants were administered with 0.50, 1.0 and 2.0 mM boron at 6 d stage for 7 d along with nutrient solution. At 13 d stage, the seedlings were sprayed with deionized water (control) or 10(-8) M of 28-homobrassinolide and plants were harvested at 21 d stage to assess growth, leaf gas-exchange traits and biochemical parameters. The boron treatments diminished growth, water relations and photosynthetic attributes along with nitrate reductase and carbonic anhydrase activity in the concentration dependent manner whereas, it enhanced lipid peroxidation, electrolyte leakage, accumulation of H(2)O(2) as well as proline, and various antioxidant enzymes in the leaves of mung bean which were more pronounced at higher concentrations of boron. However, the follow-up application of 28-homobrassinolide to the boron stressed plants improved growth, water relations and photosynthesis and further enhanced the various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase and content of proline. The elevated level of antioxidant enzymes as well as proline could have conferred tolerance to the B-stressed plants resulting in improved growth, water relations and photosynthetic attributes.

Anabolic effect of plant brassinosteroid.

Brassinosteroids are plant-derived polyhydroxylated derivatives of 5a-cholestane, structurally similar to cholesterol-derived animal steroid hormones and insect ecdysteroids, with no known function in mammals. 28-Homobrassinolide (HB), a steroidal lactone with potent plant growth-promoting property, stimulated protein synthesis and inhibited protein degradation in L6 rat skeletal muscle cells (EC(50) 4 µM) mediated in part by PI3K/Akt signaling pathway. Oral administration of HB (20 or 60 mg/kg/d for 24 d) to healthy rats fed normal diet (protein content 23.9%) increased food intake, body weight gain, lean body mass, and gastrocnemius muscle mass as compared with vehicle-treated controls. The effect of HB administration increased slightly in animals fed a high-protein diet (protein content 39.4%). Both oral (up to 60 mg/kg) and subcutaneous (up to 4 mg/kg) administration of HB showed low androgenic activity when tested in the Hershberger assay. Moreover, HB showed no direct binding to the androgen receptor in vitro. HB treatment was also associated with an improved physical fitness of untrained healthy rats, as evident from a 6.7% increase in lower extremity strength, measured by grip test. In the gastrocnemius muscle of castrated animals, HB treatment significantly increased the number of type IIa and IIb fibers and the cross-sectional area of type I and type IIa fibers. These findings suggest that oral application of HB triggers selective anabolic response with minimal or no androgenic side-effects and begin to elucidate the putative cellular targets for plant brassinosteroids in mammals.

Brassinosteroids protect photosynthetic machinery against the cadmium induced oxidative stress in two tomato cultivars.

The present study was conducted with an aim to gain better insight of brassinosteroid generated response on the effects of cadmium on photosynthetic machinery and active oxygen metabolism in two tomato cultivars (K-25 and Sarvodya). These tomato cultivars were subjected to graded cadmium levels in soil (0, 3, 6, 9 or 12 mg kg(-1) soil) with their foliage being sprayed with 0 or 10(-8) M of 28-homobrassinolide/24-epibrassinolide (HBL/EBL) at 59 d stage. The results suggested that photosynthetic parameters, leaf water potential and activity of several enzymes (nitrate reductase and carbonic anhydrase) decreased significantly in both the cultivars, to a lesser extent in K-25 than Sarvodya with the increasing levels of cadmium in the soil. However, the activity of antioxidant enzymes and proline content increased in response to metal treatment as well as the application of brassinosteroids (HBL/EBL). Overall, exogenous application of brassinosteroids improved the activity of photosynthetic machinery and that of antioxidant defense system in both the cultivars, and also nullified the damaging effect of metal on these parameters.