16-Hydroxy-ent-halima-5(10),13-dien-15,16-olide from Polyalthia longifolia targets adipogenesis by inhibiting mitotic clonal expansion and ameliorates dyslipidemia.

Obesity-related metabolic disorders are increasing at an alarming rate worldwide. The FDA has approved many molecules for weight loss therapy; most of them act on the gut level by inhibiting lipid uptake or on the central nervous system by controlling appetite. Limitations and drawbacks have propelled the search for new pharmacophores exhibiting favourable metabolic alteration at adipocytes, and natural products have always been there to prove their worth. In our efforts, we have identified 16-hydroxy-ent-halima-5(10),13-dien-15,16-olide (PLH), a halimane diterpene isolated from Polyalthia longifolia, demonstrating anti-adipogenic and anti-dyslipidemic activity. It inhibited adipogenesis in 3T3-L1 preadipocyte and C3H10T1/2 mesenchymal stem cell lines. Furthermore, it decreased set of adipogenic markers at transcript and protein levels. Cell cycle studies indicated that PLH halts the mitotic clonal expansion. Mechanistic studies shows that PLH activate Wnt/ß-catenin signaling pathway to inhibit the adipogenesis. The study suggested that PLH inhibited adipogenesis during the early phase of differentiation by targeting mitotic clonal expansion and arresting the cell cycle in the G1 phase of the cell cycle. It improved the dyslipidemic condition in HFD-fed hamsters by decreasing the body weight, fat mass, eWAT weight and improving the serum lipid profile. Overall, PLH has been found as a potential drug candidate and a pharmacophore for combating metabolic disorders including obesity and dyslipidemia.

Antileishmanial evaluation of triazole-butenolide conjugates: design, synthesis, in vitro screening, SAR and in silico ADME predictions.

In the quest to discover novel scaffolds with leishmanicidal effects, a series of 23 compounds containing the most promising 1,2,3-triazole and highly potent butenolide in one framework were synthesized. The synthesized conjugates were screened against Leishmania donovani parasite; five of them showed moderate antileishmanial activity against promastigotes (IC50 30.6 to 35.5 µM) and eight of them exhibited significant activity against amastigotes (IC50 ≤12 µM). Compound 10u was found to be the most active (IC50 8.4 ± 0.12 µM) with the highest safety index (20.47). The series was further evaluated against Plasmodium falciparum (3D7 strain) and seven compounds were found to be moderately active. Among them, again 10u emerged as the most active compound (IC50 3.65 µM). In antifilarial assays against adult female Brugia malayi, five compounds showed grade II inhibition (50-74%). Structure-activity relationship (SAR) analysis suggested a substituted phenyl ring, triazole and butenolide as essential structural features for bioactivity. Moreover, the results of in silico ADME parameter and pharmacokinetic studies indicated that the synthesized triazole-butenolide conjugates abide by the required criteria for the development of orally active drugs, and thus this scaffold can be used as a pharmacologically active framework that should be considered for the development of potential antileishmanial hits.

Design, synthesis and evaluation of novel pyrrole-hydroxybutenolide hybrids as promising antiplasmodial and anti-inflammatory agents.

In the pursuance of novel scaffolds with promising antiplasmodial and anti-inflammatory activity, a series of twenty-one compounds embraced with most promising penta-substituted pyrrole and biodynamic hydroxybutenolide in single skeleton was designed and synthesized. These pyrrole-hydroxybutenolide hybrids were evaluated against Plasmodium falciparum parasite. Four hybrids 5b, 5d, 5t and 5u exhibited good activity with IC50 of 0.60, 0.88, 0.97 and 0.96 µM for chloroquine sensitive (Pf3D7) strain and 3.92, 4.31, 4.21 and 1.67 µM for chloroquine resistant (PfK1) strain, respectively. In vivo efficacy of 5b, 5d, 5t and 5u was studied against the P. yoelii nigeriensis N67 (a chloroquine-resistant) parasite in Swiss mice at a dose of 100 mg/kg/day for 4 days via oral route. 5u was found to show maximum 100% parasite inhibition with considerably increased mean survival time. Simultaneously, the series of compounds was screened for anti-inflammatory potential. In preliminary assays, nine compounds showed more than 85% inhibition in hu-TNFα cytokine levels in LPS stimulated THP-1 monocytes and seven compounds showed more than 40% decrease in fold induction in reporter gene activity analyzed via Luciferase assay. 5p and 5t were found to be most promising amongst the series, thus were taken up for further in vivo studies. Wherein, mice pre-treated with them showed a dose dependent inhibition in carrageenan induced paw swelling. Moreover, the results of in vitro and in vivo pharmacokinetic parameters indicated that the synthesized pyrrole-hydroxybutenolide conjugates abide by the required criteria for the development of orally active drug and thus this scaffold can be used as pharmacologically active framework that should be considered for the development of potential antiplasmodial and anti-inflammatory agents.

A standardized extract of Coleus forskohlii root protects rats from ovariectomy-induced loss of bone mass and strength, and impaired bone material by osteogenic and anti-resorptive mechanisms.

Introduction: In obese humans, Coleus forskohlii root extract (CF) protects against weight gain owing to the presence of forskolin, an adenylate cyclase (AC) activator. As AC increases intracellular cyclic adenosine monophosphate (cAMP) levels in osteoblasts that has an osteogenic effect, we thus tested the skeletal effects of a standardized CF (CFE) in rats. Methods: Concentrations of forskolin and isoforskolin were measured in CFE by HPLC. CFE and forskolin (the most abundant compound present in CFE) were studied for their osteogenic efficacy in vitro by alkaline phosphatase (ALP), cAMP and cyclic guanosine monophosphate (cGMP) assays. Femur osteotomy model was used to determine the osteogenic dose of CFE. In growing rats, CFE was tested for its osteogenic effect in intact bone. In adult ovariectomized (OVX) rats, we assessed the effect of CFE on bone mass, strength and material. The effect of forskolin was assessed in vivo by measuring the expression of osteogenic genes in the calvarium of rat pups. Results: Forskolin content in CFE was 20.969%. CFE increased osteoblast differentiation and intracellular cAMP and cGMP levels in rat calvarial osteoblasts. At 25 mg/kg (half of human equivalent dose), CFE significantly enhanced calcein deposition at the osteotomy site. In growing rats, CFE promoted modeling-directed bone formation. In OVX rats, CFE maintained bone mass and microarchitecture to the level of sham-operated rats. Moreover, surface-referent bone formation in CFE treated rats was significantly increased over the OVX group and was comparable with the sham group. CFE also increased the pro-collagen type-I N-terminal propeptide: cross-linked C-telopeptide of type-I collagen (PINP : CTX-1) ratio over the OVX rats, and maintained it to the sham level. CFE treatment decreased the OVX-induced increases in the carbonate-to-phosphate, and carbonate-to-amide-I ratios. CFE also prevented the OVX-mediated decrease in mineral crystallinity. Nanoindentation parameters, including modulus and hardness, were decreased by OVX but CFE maintained these to the sham levels. Forskolin stimulated ALP, cAMP and cGMP in vitro and upregulated osteogenic genes in vivo. Conclusion: CFE, likely due to the presence of forskolin displayed a bone-conserving effect via osteogenic and anti-resorptive mechanisms resulting in the maintenance of bone mass, microarchitecture, material, and strength.

Furostanol saponins from Asparagus racemosus as potential hypoglycemic agents.

Bioactivity guided phytochemical investigation led to isolation of six undescribed furostanol saponins, furoasparoside A-F along with five known compounds, gallic acid, methyl gallate, quercetin-3-O-ß-glucopyranoside, liquiritigenin 4׳-O-ß-apiofuranosyl-(1 â†’ 2)-ß-glucopyranoside and ß-glucogallin for the first time from the roots of Asparagus racemosus. Isolated saponins were screened for their antidiabetic potential in L6-GLUT4myc myotubes in vitro followed by an in vivo evaluation in streptozocin-induced diabetic rats and db/db mice. Furoasparoside E produced a notable decrease in the postprandial blood glucose profile, in leptin receptor-deficient db/db mice, type 2 diabetes model. The effect of furoasparoside E on GLUT4 translocation was found to be mediated by the AMPK-dependent signaling pathway in L6-GLUT4myc myotubes. Moreover, it emerged as a stable plant metabolite with higher bioavailability and efficacy in in vivo pharmacokinetic studies. Therefore, these studies indicated that furoasparoside E may serve as a propitious lead for the management of type 2 diabetes and its secondary complications from natural source.

Antiglycation activity of ß-glucogallin from Asparagus racemosus.

The increased formation and accumulation of advanced glycation end products (AGEs) has been implicated in pathogenesis of various chronic ailments, including diabetes-associated secondary complications, atherosclerosis, aging, inflammatory and neurodegenerative diseases. Therefore, inhibition of AGEs formation is an imperative strategy for alleviating diverse pathologies. Here, we have demonstrated the AGEs inhibitory activity of ß-glucogallin, isolated for the first time from the roots of Asparagus racemosus. ß-glucogallin significantly mitigated fructose-, glucose- and methylglyoxal-induced glycation of bovine serum albumin (BSA). Also, the presence of ß-glucogallin decreased fructosamine and protein carbonyls content, and increased thiol group content in the fructose-BSA system. These activities of ß-glucogallin from Asparagus racemosus underscore its likely pharmacological potential for impeding AGEs-related metabolic disorders.

Synthesis and Evaluation of Galloyl Conjugates of Flavanones as BMP-2 Upregulators with Promising Bone Anabolic and Fracture Healing Properties.

The molecular hybridization concept led us to design a series of galloyl conjugates of flavanones that have potent osteoblast differentiation ability in vitro and promote bone formation in vivo. An array of in vitro studies, especially gene expression of osteogenic markers, evinced compound 5e as the most potent bone anabolic agent, found to be active at 1 pM, which was then further assessed for its osteogenic potential in vivo. From in vivo studies on rat calvaria and a fracture defect model, we inferred that compound 5e, at an oral dose of 5 mg/(kg day), increased the expression of osteogenic genes (RUNX2, BMP-2, Col1, and OCN) and the bone formation rate and significantly promoted bone regeneration at the fracture site, as evidenced by the increased bone volume/tissue fraction compared with vehicle-treated rats. Furthermore, structure-activity relationship studies and pharmacokinetic studies suggest 5e as a potential bone anabolic lead for future osteoporosis drug development.

A clerodane diterpene inhibit adipogenesis by cell cycle arrest and ameliorate obesity in C57BL/6 mice.

A clerodane diterpene, 16α-Hydroxycleroda-3, 13 (14) Z-dien-15, 16-olide (compound 1) isolated from Polyalthia longifolia had previously been reported as a new structural class of HMG-CoA reductase inhibitor apart from statins. Statins are known to be anti-adipogenic in nature. The distant structural similarity between compound 1 and lovastatin (polyketide class of compound) prompted us to investigate effects of diterpene compound 1 on adipogenesis and thereby obesity. High content microscopy proved diterpene compound 1 exhibits better anti-adipogenic activity and less toxicity in differentiating adipocytes. Moreover, it reduced expression levels of PPARγ, C/EBPα and GLUT4 during differentiation in a time and concentration dependent manner. Diterpene compound 1 during early differentiation reduced MDI induced-Akt/mTOR phosphorylation and expression of cell cycle proteins, and thereby halted mitotic clonal expansion, the decisive factor in early adipogenesis. Further, its anti-adipogenic activity was validated in murine mesenchymal cell-line C3H10T1/2 and human mesenchymal stem cell models of adipogenic differentiation. When compound 1 was administered along with HFD, for another 8 weeks in 2 month HFD fed overweight mice (with BMI > 30 and impaired glucose tolerance), it attenuated weight gain and epididymal fat accumulation. It improved body glucose tolerance, reduced HFD induced increase in total cholesterol and leptin/adiponectin ratio. All these effects were comparable with standard anti-obesity drug Orlistat with added edge of potently decreasing circulating triglyceride levels comparable with normal chow fed group. Histological analysis shows that compound 1 inhibit adipocyte hypertrophy and decreased steatosis in hepatocytes. Both in vivo and in vitro results demonstrate a potential value of compound 1 as a novel anti-adipogenic and anti-obesity agent.

Induction of mitochondrial dysfunction and oxidative stress in Leishmania donovani by orally active clerodane diterpene.

This study was performed to investigate the mechanistic aspects of cell death induced by a clerodane diterpene (K-09) in Leishmania donovani promastigotes that was previously demonstrated to be safe and orally active against visceral leishmaniasis (VL). K-09 caused depolarization of the mitochondrion and the generation of reactive oxygen species, triggering an apoptotic response in L. donovani promastigotes. Mitochondrial dysfunction subsequently resulted in the release of cytochrome c into the cytosol, impairing ATP production. Oxidative stress caused the depletion of reduced glutathione, while pretreatment with antioxidant N-acetyl cysteine (NAC) was able to abrogate oxidative stress. However, NAC failed to restore the mitochondrial membrane potential or intracellular calcium homeostasis after K-09 treatment, suggesting that the generation of oxidative stress is a downstream event relative to the other events. Caspase-3/-7-like protease activity and genomic DNA fragmentation were observed. Electron microscopy studies revealed gross morphological alterations typical of apoptosis, including severe mitochondrial damage, pyknosis of the nucleus, structural disruption of the mitochondrion-kinetoplast complex, flagellar pocket alterations, and the displacement of organelles. Moreover, an increased number of lipid droplets was detected after K-09 treatment, which is suggestive of altered lipid metabolism. Our results indicate that K-09 induces mitochondrial dysfunction and oxidative stress-mediated apoptotic cell death in L. donovani promastigotes, sharing many features with metazoan apoptosis. These mechanistic insights provide a basis for further investigation toward the development of K-09 as a potential drug candidate for VL.

Antiplasmodial activity of artecyclopentyl mether a new artemisinin derivative and its effect on pathogenesis in Plasmodium yoelii nigeriensis infected mice.

In an effort to evaluate novel derivatives from artemisinin, possessing potential antimalarial activity, a new derivative artecyclopentyl mether (CPM-1) was derivatized and evaluated for its dose-dependent efficacy in Plasmodium yoelii nigeriensis infected mice. The survivability of mice at 7.5 mg/kg was >28 days with negligible parasitaemia and recovered anemia (66.16-72.62%). Artecyclopentyl mether was also found to modulate the pro- and anti-inflammatory cytokines (IFN-γ, 39.64-56.92%; TNF, 49.10-74.31%; IL-4, 11.53-43.22%; IL-10, 37.60-53.52%) favourably besides optimizing the oxidative stress to the infected subjects as evident by the nitric oxide (88.76-95.43%), lipid peroxidation (59.30-76.05%) and glycaemic data (62.70-76.66%). The results indicate the potentiality of the new derivative as an antimalarial against asexual stages of the parasite.

16alpha-Hydroxycleroda-3,13 (14)Z-dien-15,16-olide from Polyalthia longifolia: a safe and orally active antileishmanial agent.

BACKGROUND AND PURPOSE: New antileishmanials from natural products are urgently needed due to the emergence of drug resistance complicated by severe cytotoxic effects. 16alpha-Hydroxycleroda-3,13 (14)Z-dien-15,16-olide (Compound 1) from Polyalthia longifolia was found to be a potential antileishmanial and non-cytotoxic, as evidenced by long-term survival (>6 months) of treated animals. This prompted us to determine its target and, using molecular modelling, identify the interactions responsible for its specific antileishmanial activity. EXPERIMENTAL APPROACH: In vitro activity of compound was assessed using intracellular transgenic green fluorescent protein-stably expressed Leishmania donovani parasites. In vivo activity and survival of animals post-treatment were evaluated in L. donovani-infected hamsters. Known property of clerodane diterpenes as potent human DNA topoisomerase inhibitors led us to evaluate the inhibition of recombinant L. donovani topoisomerase I using relaxation assay. Mode of cell death induced by Compound 1 was assessed by phosphotidylserine exposure post-treatment. Molecular modelling studies were conducted with DNA topoisomerase I to identify the binding interactions responsible for its activity. KEY RESULTS: Bioassay-guided fractionation led to isolation of Compound 1 as a non-cytotoxic, orally active antileishmanial. Compound 1 inhibited recombinant DNA topoisomerase I which, ultimately, induced apoptosis. Molecular docking studies indicated that five strong hydrogen-bonding interactions and hydrophobic interactions of Compound 1 with L. donovani DNA-topoisomerase are responsible for its antileishmanial activity. CONCLUSIONS AND IMPLICATIONS: The data reveal Compound 1 is a potent and safe antileishmanial. The study further exploited the structural determinants responsible for its non-cytotoxic and potent activity, to raise the feasibility of specifically targeting the target enzyme responsible for its activity through rational drug design.