Optimization of Metronidazole Emulgel.

The purpose of the present study was to develop and optimize the emulgel system for MTZ (Metronidazole), a poorly water soluble drug. The pseudoternary phase diagrams were developed for various microemulsion formulations composed of Capmul 908 P, Acconon MC8-2, and propylene glycol. The emulgel was optimized using a three-factor, two-level factorial design, the independent variables selected were Capmul 908 P, and surfactant mixture (Acconon MC8-2 and gelling agent), and the dependent variables (responses) were a cumulative amount of drug permeated across the dialysis membrane in 24 h (Y 1) and spreadability (Y 2). Mathematical equations and response surface plots were used to relate the dependent and independent variables. The regression equations were generated for responses Y 1 and Y 2. The statistical validity of the polynomials was established, and optimized formulation factors were selected. Validation of the optimization study with 3 confirmatory runs indicated a high degree of prognostic ability of response surface methodology. Emulgel system of MTZ was developed and optimized using 2(3) factorial design and could provide an effective treatment against topical infections.

A novel flavonoid isolated from the steam-bark of Ulmus wallichiana planchon stimulates osteoblast function and inhibits osteoclast and adipocyte differentiation.

(2S,3S)-Aromadendrin-6-C-ß-d-glucopyranoside (AG) is a novel flavonol isolated from the extract of Ulmus wallichiana (Himalayan Elm). Extract of U. wallichiana is used as a traditional medicine for rapid fracture repair in India. We characterized the mechanism of action of AG in mouse bone cells by investigating its effect on the precursors of osteoblasts, osteoclasts and adipocytes. At nanomolar concentrations, AG increased differentiation of preosteoblasts obtained from neonatal mouse calvaria. The gene expression of osteogenic markers, including runt-related transcription factor 2 (Runx-2), bone morphogenetic protein-2 (BMP-2), type I collagen and osteocalcin were elevated in the preosteoblasts. The extracellular matrix mineralization was higher in preosteoblast and bone marrow cells when AG was present in the medium. Furthermore, AG protected the differentiated osteoblasts from serum deprivation-induced apoptosis, and increased the expression of the anti-osteoclastogenic cytokine, osteoprotegerin. It inhibited osteoclast differentiation of bone marrow precursor cells to osteoclasts in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and monocyte/macrophage-colony stimulating factor (M-CSF). Additionally, in 3T3-L1 preadipocytes, AG decreased the expression of genes involved in adipogenesis, including peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element binding protein (SREBP) and CCAAT/enhancer-binding protein alpha (CEBP/α), and induced apoptosis of differentiated adipocytes. Induction of adipogenic differentiation was also inhibited in the presence of AG. AG exhibited no estrogenic/antiestrogenic effect. Together, our data show that AG has potent osteogenic, anti-osteoclastogenic and anti-adipogenic effects, which may translate to a better skeletal outcome in postmenopausal osteoporosis.

Aza-annulation on the 16-dehydropregnenolone, via tandem intermolecular aldol process and intramolecular Michael addition.

16-Dehydropregnenolone undergoes a smooth annulation with propan-1-amine and aromatic aldehydes. Several amine derivatives of 16- dehydropregnenolone were synthesized and evaluated as inhibitors of DPP-IV. The structures of compounds were confirmed by (1)H, (13)C, NMR and mass spectral analysis. Among 17 compounds evaluated only five compounds 1, 9, 13, 15 and 16 demonstrated significant inhibition of DPP. This study suggest that introduction of appropriate substituents in the 16-dehydropregnenolone plays an important role in DPP-IV inhibitory activity.

One step synthesis of 2-hydroxymethylisoflavone and their osteogenic activity.

An efficient one step synthesis of new 2-hydroxymethylisoflavone is reported. A series of deoxybenzoin was subjected to cyclization with glyoxal in the presence of basic condition (KOH/EtOH) to afford the 2-hydroxymethyl isoflavone. The structures of compounds 5a-g were confirmed by NMR experiments including (1)H, (13)C, HMBC, HSQC and COSY. These compounds were assessed for stimulation of osteoblast function using primary culture of rat calvarial osteoblasts in vitro. Compounds 5a, 5d, 5f and 5g were potent in stimulating differentiation of osteoblasts as assessed by measuring alkaline phosphatase (ALP) activity. Besides, effect of these analogs was also seen on the transcript levels of osteogenic genes like Runx-2, osteocalcin and Bone morphogenetic protein-2 (BMP-2), involved in osteoblast differentiation and mineralization. Based on quantitative PCR data, compound 5f was found to be the potent followed by 5d. Compound 5f robustly increased the mRNA levels of Runx-2 (8.0 fold), BMP-2 (∼2 fold) and osteocalcin (∼2.0 fold) in osteoblasts. Collectively, we demonstrate osteogenic activity of the novel 2-hydroxymethyl isoflavones with 5f having the most potent activity.

Differential effects of formononetin and cladrin on osteoblast function, peak bone mass achievement and bioavailability in rats.

Dietary soy isoflavones including genistein and daidzein have been shown to have favorable effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of cladrin and formononetin, two structurally related methoxydaidzeins found in soy food and other natural sources. Cladrin, at as low as 10 nM, maximally stimulated both osteoblast proliferation and differentiation by activating MEK-Erk pathway. On the other hand, formononetin maximally stimulated osteoblast differentiation at 100 nM that involved p38 MAPK pathway but had no effect on osteoblast proliferation. Unlike daidzein, these two compounds neither activated estrogen receptor in osteoblast nor had any effect on osteoclast differentiation. Daily oral administration of each of these compounds at 10.0 mg kg(-1) day(-1) dose to recently weaned female Sprague-Dawley rats for 30 consecutive days, increased bone mineral density at various anatomic positions studied. By dynamic histomorphometry of bone, we observed that rats treated with cladrin exhibited increased mineral apposition and bone formation rates compared with control, while formononetin had no effect. Cladrin had much better plasma bioavailability compared with formononetin. None of these compounds exhibited estrogen agonistic effect in uteri. Our data suggest that cladrin is more potent among the two in promoting parameters of peak bone mass achievement, which could be attributed to its stimulatory effect on osteoblast proliferation and better bioavailability. To the best of our knowledge, this is the first attempt to elucidate structure-activity relationship between the methoxylated forms of daidzein and their osteogenic effects.

Synthesis and antihyperglycemic activity of phenolic C-glycosides.

Various phenolic C-glycosides were evaluated for their in vitro and in vivo antihyperglycemic activity employing glucose uptake by rat muscle cell lines (L-6) and low dosed-streptozotocin-induced diabetic rats, respectively. Some of phenolic C-glycosides were isolated from Pterocarpus marsupium and Ulmus wallichiana and other were synthesized by unprotected sugar and phloroacetophenone using Sc(OTf)(3) in aqueous ethanol. Eight among tested compounds showed significant lowering of blood glucose level on low dosed-streptozotocin-induced diabetic rats. The compound 24 lowered the blood glucose levels by 34.9% and 33.6% during 0-5h and 0-24h, respectively, at the dose of 25mg/kg body weight which is comparable to standard antidiabetic drug metformin.

Quercetin-6-C-ß-D-glucopyranoside isolated from Ulmus wallichiana planchon is more potent than quercetin in inhibiting osteoclastogenesis and mitigating ovariectomy-induced bone loss in rats.

OBJECTIVE: The aim of this study was to determine the skeletal effect of quercetin-6-C-ß-D-glucopyranoside (QCG) isolated from the extract of Ulmus wallichiana and compare this effect with quercetin (Q) in a rat model of postmenopausal bone loss. METHODS: Murine bone marrow cells were used to study the effect of QCG or Q on osteoclast differentiation. QCG or Q (1.0 and 5.0 mg kg(-1) d(-1) doses) was administered orally to ovarietomized (OVx) rats for 12 weeks. Sham-operated + vehicle and OVx + vehicle groups served as positive and negative controls, respectively. Bone mineral density, bone microarchitecture, biomechanical strength, bone turnover markers, and uterotrophic effect were studied. One-way analysis of variance was used to test significance of effects. RESULTS: QCG at 1.0 nM significantly inhibited differentiation of multinucleated osteoclasts and expression of osteoclastogenic genes from bone marrow cells, whereas Q at 10.0 µM had comparable results. OVx rats treated with QCG exhibited significantly higher bone mass and better microarchitecture in trabecular and cortical bones compared with OVx + vehicle. QCG treatment of OVx rats had better functional impact than did Q-treated OVx rats, evident from increased bone biomechanical strength. Serum osteocalcin and urinary fragments of type 1 collagen were significantly lower in QCG-treated OVx rats compared with OVx + vehicle group. The protective effect of QCG under ovariectomy-induced bone loss setting was found to be significantly better than Q. Uterine histomorphometry parameters of OVx rats did not change with QCG treatment. CONCLUSIONS: QCG improves bone biomechanical quality more effectively than Q through positive modifications of bone mineral density and bone microarchitecture without a hyperplastic effect on the uterus.

Anti-osteoporotic constituents from Indian medicinal plants.

The objective of this study was to determine the in vitro osteogenic activities of selected medicinal plants used traditionally in India. The compounds isolated from three plants viz. Allophylus serratus, Cissus quadrangularis and Vitex negundo were evaluated for their in vitro osteogenic activities. Primary cultures of osteoblasts were used to determine the effects of these components on osteoblast functions. Five of the fourteen compounds isolated led to increase in osteoblast differentiation and mineralization. These findings lend support to the use of Allophylus serratus, Cissus quadrangularis and Vitex negundo in traditional medicine.

Total extract and standardized fraction from the stem bark of Butea monosperma have osteoprotective action: evidence for the nonestrogenic osteogenic effect of the standardized fraction.

OBJECTIVE: The aim of this study was to determine the skeletal effects of Butea total extract (BTE) and its acetone soluble fraction (ASF) from Butea monosperma, which is rich in methoxyisoflavones, in ovariectomized (OVx) rats, a model for postmenopausal bone loss. METHODS: BTE (1.0 g kg d) and ASF (100 mg kg d) were given orally for 12 weeks to adult OVx rats. The sham-operated and ovariectomy + vehicle groups served as controls. Bone mineral density, osteoid formation (mineral apposition rate and bone formation rate), bone microarchitecture, and bone turnover/resorption markers were studied. Phytoestrogens in rats given BTE and ASF were analyzed by high-performance liquid chromatography. One-way analysis of variance was used to test significance of effects. RESULTS: OVx rats treated with either BTE or ASF exhibited increased bone mineral density in trabecular bones and improved trabecular microarchitecture compared with the ovariectomy + vehicle group. ASF treatment was more efficient than BTE treatment in maintaining trabecular microarchitecture. Serum osteocalcin and urinary type 1 collagen levels in OVx rats treated with either BTE or ASF were significantly lower than those of the ovariectomy + vehicle group. ASF treatment led to increased mineral apposition rate and bone formation rate compared with ovariectomy + vehicle, whereas BTE had no such effect. In the uterotropic assay, BTE was mildly estrogenic in adult OVx rats. In immature rats, BTE exhibited both estrogenicity and antiestrogenicity. ASF had neither uterine estrogenicity nor antiestrogenicity. Analysis of phytoestrogens revealed significant enrichment of cladrin, isoformononetin, and medicarpin in ASF over BTE. CONCLUSIONS: Derived from B monosperma, ASF at a 10-fold lower dose than that of BTE was effective in preventing OVx-induced bone loss and stimulated new-bone formation.

Tectone, a new antihyperglycemic anthraquinone from Tectona grandis leaves.

A new anthraquinone (3,8-dihydroxy-2-methyl anthraquinone), named tectone (1), along with fourteen known compounds (2-15) comprised of five terpenoids (2-5, 15), four flavonoids (6-9), three flavone glycosides (10-12), and two phenolic glycosides (13-14) were isolated from the chloroform and n-butanol fractions of the ethanol extract of Tectona grandis leaves. Attempts were made to synthesize compound 1. This resulted in the synthesis of three additional anthraquinones (16-18), out of which compound 16 is new. The structures of all compounds were established by spectral analysis. The isolated and synthesized compounds were evaluated for their antihyperglycemic activity. Compounds 1, 2, 4 and 14 showed significant antihyperglycemic activity in streptozotocin-induced diabetic rats at a dose of 100 mg/kg body weight, which is comparable to the standard drug metformin.

8,8''-Biapigeninyl stimulates osteoblast functions and inhibits osteoclast and adipocyte functions: Osteoprotective action of 8,8''-biapigeninyl in ovariectomized mice.

8,8''-Biapigeninyl (BA), a condensation product of two apigenin molecules, is found abundantly in the nuts of Cupressus sempervirens. We investigated the effects of BA on murine bone cells in vitro and in ovariectomized (OVx) mice. BA at 10(-10)M and 10(-8)M, inhibited osteoclastogenesis of bone marrow cells (BMCs) and displayed concentration dependence. BA at 10(-8) M and 10(-6) M inhibited differentiation of 3T3-L1 and BMCs to mature adipocytes. BA (10(-10)M) stimulated osteoblast proliferation, differentiation and mineralization. In stimulating osteoblast function, BA was found to be 10(4)-fold more potent than apigenin. The effect of BA in osteoblasts appeared to be mediated via estrogen receptors (ER) as antiestrogen, ICI-182780 abolished BA-stimulated osteoblast differentiation. In OVx mice BA treatment (at 1.0-, 5.0- and 10.0 mg kg(-1) day(-1) doses) given orally for 30 days dose-dependently inhibited mRNA levels of osteoclastic genes including tartrate-resistant acid phosphatase, receptor activator of nuclear factor (RANK), tumor necrosis factor alpha, interleukin-6 and the ratio of RANK ligand/osteoprotegerin ratio in bones compared with OVx mice treated with vehicle. In addition, BA treatment to OVx mice dose-dependently stimulated production of osteoprogenitor cells in the bone marrow and increased mRNA levels of osteogenic genes core binding factor alpha-1, type I collagen and bone morphogenic protein-2 in bones compared with OVx+vehicle group. Microcomputed tomography revealed that BA treatment to OVx mice improved parameters of trabecular and cortical architecture. BA exhibited no uterine estrogenicity. From these data, we conclude that BA exerts osteoprotective effect in OVx mice by multiple beneficial effects on bone cells.

A novel flavonoid, 6-C-beta-d-glucopyranosyl-(2S,3S)-(+)-3',4',5,7-tetrahydroxyflavanone, isolated from Ulmus wallichiana Planchon mitigates ovariectomy-induced osteoporosis in rats.

OBJECTIVE: The aim of this study was to determine the skeletal effect of 6-C-beta-d-glucopyranosyl-(2S,3S)-(+)-3',4',5,7-tetrahydroxyflavanone (GTDF)/Ulmoside A, a new compound isolated from the extract of Ulmus wallichiana in a rat model of postmenopausal bone loss. METHODS: GTDF (1.0 and 5.0 mg kg d) was given orally to ovariectomized (OVx) rats (180-200 g) for 12 weeks. Sham operated + vehicle, ovariectomy + 17beta-estradiol (2.5 microg kg d), and ovariectomy + vehicle groups served as various controls. Bone mineral density (BMD), trabecular microarchitecture, bone biomechanical strength, levels of bone turnover/resorption markers, uterotropic effect, and plasma pharmacokinetics were studied. One-way analysis of variance was used to test significance of effects. RESULTS: OVx rats treated with both doses of GTDF exhibited significantly higher BMD in the trabecular (distal femur, proximal tibia, and vertebrae) and cortical (femur shaft) regions compared with the ovariectomy + vehicle group. Micro-CT demonstrated that OVx rats treated with 5.0 mg kg day of GTDF had better bone microarchitectural parameters compared with the ovariectomy + vehicle group. Serum osteocalcin and urinary C-terminal teleopeptide of Type I collagen levels in OVx rats treated with GTDF (at both doses) were significantly lower than those in the ovariectomy + vehicle group. At neither of the two doses did GTDF exhibit uterine estrogenicity. A pharmacokinetic study revealed that GTDF achieved maximum plasma concentration (40.67 ng mL) at approximately 1 hour, indicating its slow absorption. Its absolute bioavailability was found to be 1.04% with a plasma elimination half-life of approximately 5 hours. CONCLUSIONS: GTDF, a novel compound isolated from U wallichiana extract, improves bone biomechanical quality through positive modifications of BMD and trabecular microarchitecture without a hyperplastic effect on the uterus.

Reverse phase-HPLC method for determination of marker compounds in NP-1, an anti-osteoporotic plant product from Butea monosperma.

An isocratic, reversed phase, high-performance liquid chromatographic (HPLC) method was developed for the determination of the marker compounds K052 (iso-formononetin), K054 (methoxy derivative) and K080 (formononetin) in NP-1, an anti-osteoporotic plant product from Butea monosperma. The separation was achieved on a C18 column with a mobile phase consisted of a mixture of 0.05M potassium dihydrogen phosphate containing 0.1% v/v triethyl amine (pH adjusted to 2.5 with phosphoric acid) buffer and acetonitrile (70:30 v/v), at a flow rate of 1.5 mL/min. The retention times of K054, K080 and K052 were about 13, 21 and 23 mins, respectively. The effluents were monitored at 254 nm. The calibration curves were linear over the concentration ranges of 2.7-21.3, 2.1-33.6 and 2.8-22.4 microg/mL for K054, K052 and K080, respectively. The limits of detection were 0.42, 0.53 and 0.56 microg/mL, respectively. The accuracies and precisions in all cases were less than 5% in the calibration range.

Phenolic glycosides from Dodecadenia grandiflora and their glucose-6-phosphatase inhibitory activity.

Phytochemical investigation of Dodecadenia grandiflora leaves led to the isolation and identification of three phenolic glycosides, designated 1-[(4'-O-(E)-p-coumaroyl)-beta-D-glucopyranosyl]-oxy-2-phenol (1), 1-[(6'-O-(E)-p-coumaroyl)-beta-D-glucopyranosyl]-oxy-2-phenol (2) and 1-[O-beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl]-oxy-2-phenol (3), along with nine known compounds. Compounds 1, 2, 5 and 9 exhibited significant glucose-6-phosphatase inhibitory activity (63.7, 66.9, 82.9 and 85.4%) with IC(50) values of 88.5, 81.0, 51 and 50 microM respectively. On the basis of biological results, a structure-activity relationship has been discussed.

Antioxidant flavonoid glycosides from Evolvulus alsinoides.

Oxidative damage is an established outcome of chronic stress. Thus, the present study was designed to investigate the modulatory role of ethanolic extract of Evolvulus alsinoides (EA) in terms of oxidative alterations at peripheral and central level in rats subjected to chronic unpredictable stress (CUS). CUS exposure for 7 days reduced Cu, Zn superoxide dismutase and catalase activity with increase in glutathione peroxidase activity and lipid peroxidation, while decrease in reduced glutathione level in blood plasma, frontal cortex and hippocampus regions of brain. Oral administration of EA extract at 200mg/kg p.o. normalized these stress induced oxidative alterations with an efficacy similar to that of melatonin. Further, EA extract was taken up for detailed chemical investigation. Two new flavonol-4'-glycoside, kaempferol 4'-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside (3) and kaempferol 4'-O-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (5) were isolated, along with eight known compounds (1, 2, 4 and 6-10). The structures of new compounds were established by detailed spectroscopic studies, while known compounds were characterized by direct comparison of their reported NMR data. All these compounds were evaluated for their in vitro antioxidant activity. Compounds 3, 5, 9 and 10 at 100 and 200 microg/ml showed significant in vitro antioxidant activity. Therefore, EA may hold great potential in preventing clinical deterioration in stress induced oxidative load and related disorders.

Extract and fraction from Ulmus wallichiana Planchon promote peak bone achievement and have a nonestrogenic osteoprotective effect.

OBJECTIVE: This study aimed to determine the skeletal effects of total ethanolic extract (TEE) and its butanolic fraction (BF) from the stem-bark of Ulmus wallichiana, which is rich in C-glycosylated flavonoids, in growing rats (for peak bone [PB] achievement) and in ovariectomized (OVx) rats (for menopausal bone loss). METHODS: TEE (750 mg kg(-1) d(-1)) and BF (50 mg kg(-1) d(-1)) were given orally for 10 weeks to weaning female Sprague-Dawley rats and for 12 weeks to adult OVx rats of the same strain, respectively. In studies with OVx rats, sham operated + vehicle, OVx + 17beta-estradiol, and OVx + vehicle groups served as various controls. Bone mineral density (BMD), biomechanical strength, bone histology, formations of osteoprogenitor cells, osteoid formation, and bone turnover/resorption markers were studied. Bioactive marker compounds in TEE and BF were analyzed by high-performance liquid chromatography. One-way analysis of variance was used to test significance of effects. RESULTS: In growing rats, both TEE and BF increased BMD, bone strength, and bone formation rate, suggesting higher PB achievement. OVx rats treated with either TEE or BF exhibited increased BMD at various anatomical positions and improved bone strength and trabecular architecture compared with the OVx + vehicle group. Serum osteocalcin and urinary type 1 collagen degradation product levels in OVx rats treated with either TEE or BF were significantly lower than those of the OVx + vehicle group. Neither TEE nor BF exhibited uterine estrogenicity. Analysis of marker compounds revealed significant enrichment of two bioactive markers in BF over TEE. CONCLUSIONS: Derived from U wallichiana, BF at much a lower dose than TEE was effective in PB achievement and prevention of OVx-induced bone loss.

Constituents from fruits of Cupressus sempervirens.

Two new phenolic glycosides (1, 2), along with fourteen known compounds (3-16) have been isolated from the fruit of Cupressus sempervirens. The structures of these compounds were determined by spectroscopic analysis and were evaluated for their inhibitory activity against glycogen phosphorylase and glucose-6-phosphatase enzymes. Compounds 14 showed a moderate inhibition against glucose-6-phosphatase and 15 against glycogen phosphorylase enzymes.

Antihyperglycemic activity of phenylpropanoyl esters of catechol glycoside and its dimers from Dodecadenia grandiflora.

Bioactivity-guided separation of an antihyperglycemic extract from the leaves of Dodecadenia grandiflora afforded two phenylpropanoyl esters of catechol glycosides (1 and 4) and two lignane bis(catecol glycoside)esters (2 and 3). Their structures were established on the basis of extensive spectroscopic analysis (1D and 2D-NMR, MS). Compounds 2 and 3 are believed to be derived from dimerization via the two phenylpropanoid units of 1. Compounds 1-4 showed significant antihyperglycemic activity in streptozotocin-induced (STZ) diabetic rats, which is comparable to the standard drug metformin. Our results provide support to explain the use of D. grandiflora as antihyperglycemic agent by the traditional medical practitioners.

Constituents of Tinospora sinensis and their antileishmanial activity against Leishmania donovani.

Two new compounds 4-methyl-heptadec-6-enoic acid ethyl ester (2) and 3-hydroxy-2,9,11-trimethoxy-5,6-dihydro isoquino[3,2-a]isoquinolinylium (7) were isolated from an ethanolic extract of the stems of Tinospora sinensis, along with six known compounds (1, 3-6 and 8). The structures of new compounds were established on the basis of detailed spectroscopic studies. Compound 7 exhibited the highest in vitro antileishmanial activity against Leishmania donovani promastigotes and intracellular amastigotes, whereas compounds 2, 4, 5 and 6 demonstrated moderate activity. Other compounds were found to be inactive.