Therapeutic importance of Kigelia africana subsp. africana: an alternative medicine.

AIM: To summarise a detailed up-to-date review of the traditional uses, phytoconstituents, and pharmacological activities of various parts of Kigelia africana. MATERIALS AND METHODS: Google Scholar, PubMed, PubChem, Elsevier, King Draw, indianbiodiversity.org. RESULT: The phytochemical analysis of Kigelia africana subsp. africana has revealed the presence of approximately 145 compounds extracted from different parts of the plant. These bioactive extracts of the plant possess anti-inflammatory, antioxidant, antimicrobial, antidiabetic, antineoplastic, and anti-urolithic activities. Due to its anti-inflammatory, antioxidant, and immune-booster properties, Kigelia can prove to be an essential source of drugs for treating various disorders. CONCLUSION: Knowledge of the phytoconstituents, non-medicinal and medicinal traditional uses, pharmacological activities, and products obtained from Kigelia is described in this review with the hope that the updated findings will promote research on its biological pathways.

Traditional medicinal importance of Kigelia africana subsp. africanaPhytoconstituents present in extracts from different parts of the plantPharmacological activities of phytochemicals extracted from KigeliaAnti-inflammatory and antioxidant role in preventing oxidative stressPotential as ethnopharmacological therapeutic in treating respiratory ailmentsToxicity evaluation of Kigelia africana subsp. africana.

Expression of nutrient transporter genes in response to dietary rice gluten meal and protease enzyme supplementation and the consequent effects on growth, nutrient digestibility, immunity and jejunum histomorphometry in chicken.

The objective of this study was to investigate the effects of feeding rice gluten meal (RGM) as an alternative protein source along with protease enzyme supplementation on growth performance, expression of nutrient transporter genes, nutrient digestibility, immune response and gut histomorphometry of broiler chicken. Proximate analysis of RGM revealed 923 g dry matter (DM), 500 g crude protein (CP), 69.2 g ether extract, 94.7 g crude fiber, 215.4 g nitrogen-free extract, 43.7 g ash, 6.20 g calcium, 7.80 g total phosphorus, 18.99 MJ gross energy and 12.68 MJ metabolizable energy per kg diet. Significant upregulation of nutrient transporter genes (PepT1, EAAT3 and mucin) and better growth performance was observed in the birds fed control diet which was statistically similar to the birds fed 150 g RGM compared to birds fed higher RGM levels. Histomorphometry of jejunum, nutrient digestibility, and immune response of birds did not reveal any significant effect of RGM or protease enzyme supplementation. However, the inclusion of RGM up to 150 g/kg diet resulted in significant decline of feed cost/kg live weight gain, dressed meat yield and eviscerated meat yield by 13.13%, 12.99% and 13.36%, respectively compared to control. Thus, it was concluded that the inclusion of 150 g RGM/kg diet in broiler chicken ration has no adverse effects on the growth pattern of birds and can be used for least-cost feed formulation for chicken.

Effect of dietary mannan oligosaccharides and fructo-oligosaccharides on physico-chemical indices, antioxidant and oxidative stability of broiler chicken meat.

The objective of this present study was to investigate the potentiality of prebiotics (mannan oligosaccharides-MOS and fructo-oligosaccharides-FOS) in replacement of antibiotic growth promoter and their relationship with physico-chemical indices, antioxidant and oxidative stability and carcass traits of broiler chickens meat. Accordingly, 240 day-old broiler chicks of uniform body weight divided in 6 treatment groups with 5 replicate each (5 × 6 = 30) having 8 birds in each replicate. Six corn based dietary treatments were formulated viz. T1 (control diet), T2 (T1 + Bacitracin methylene di-salicylate @ 0.002%), T3 (T1 + 0.1% MOS), T4 (T1 + 0.2% MOS), T5 (T1 + 0.1% FOS), and T6 (T1 + 0.2% FOS). Significant (p < 0.05) increase in cut up part yields (%) and reduction in cholesterol and fat content in T4 (0.2% MOS) group. The water holding capacity (WHC) and extract release volume (ERV) were increase (p < 0.05) in 0.1 or 0.2% MOS supplemented group. DPPH (1, 1-diphenyl-2-picrylhydrazy) was higher (p < 0.05) and lipid oxidation (free fatty acid and thio-barbituric acid reactive substances) was lower (p < 0.05) in T4 group. The standard plate count (SPC), staphylococcus and coliform counts were decreased (p < 0.05) in T3 or T4 group. Thus, it can be concluded that mannan oligosaccharides (MOS) may be incorporated at 0.2% level in diet for improved physico-chemical indices, antioxidant and oxidative stability and carcass characteristics of broiler chickens meat and it may be suitable replacer of antibiotic growth promoter.

Hepatic transcriptome analysis reveals altered lipid metabolism and consequent health indices in chicken supplemented with dietary Bifidobacterium bifidum and mannan-oligosaccharides.

This study investigated the role of dietary prebiotic mannan-oligosaccharides (MOS), and probiotic Bifidobacterium bifidum (BFD) in lipid metabolism, deposition, and consequent health indices in broiler chicken. The supplementation of 0.2% MOS along with either 106 or 107 CFU BFD/g feed resulted in downregulation of Acetyl-CoA carboxylase, fatty acid synthase, sterolregulatory element binding protein-1, and apolipoprotein B100; and up-regulation of peroxisome proliferator activated receptor-α AMP-activated protein kinase α-1, and stearoyl CoA (∆9) desaturase-1 hepatic expression in broiler chicken. The birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed depicted lower body fat percentage, palmitic acid, stearic acid, and saturated fatty acid contents, whereas, higher palmitoleic acid, oleic acid, and MUFA contents were observed. The ∆9-desaturase indices of chicken meat have shown higher values; and elongase index (only thigh) and thioesterase index have shown lower values in birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed. The meat health indices such as Polyunsaturated fatty acids (PUFA)/Saturated fatty acids (SFA) ratio, Mono-saturated fatty acids (MUFA)/SFA ratio, unsaturated fatty acids (UFA)/SFA ratio, hypocholesterolemic/hypercholesterolemic fatty acid ratio, saturation index, atherogenic index, thrombogenic index, and hypercholesterolemic fatty acid content were positively improved in birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed. Similarly, the birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed have shown lower serum triglyceride and total cholesterol levels along with higher high density levels and improved serum health indices cardiac risk ratio, atherogenic coefficient, and, atherogenic index of plasma.

ß-Sitosterol-D-Glucopyranoside Mimics Estrogenic Properties and Stimulates Glucose Utilization in Skeletal Muscle Cells.

Estrogenic molecules have been reported to regulate glucose homeostasis and may be beneficial for diabetes management. Here, we investigated the estrogenic effect of ß-sitosterol-3-O-D-glucopyranoside (BSD), isolated from the fruits of Cupressus sempervirens and monitored its ability to regulate glucose utilization in skeletal muscle cells. BSD stimulated ERE-mediated luciferase activity in both ERα and ERß-ERE luc expression system with greater response through ERß in HEK-293T cells, and induced the expression of estrogen-regulated genes in estrogen responsive MCF-7 cells. In silico docking and molecular interaction studies revealed the affinity and interaction of BSD with ERß through hydrophobic interaction and hydrogen bond pairing. Furthermore, prolonged exposure of L6-GLUT4myc myotubes to BSD raised the glucose uptake under basal conditions without affecting the insulin-stimulated glucose uptake, the effect associated with enhanced translocation of GLUT4 to the cell periphery. The BSD-mediated biological response to increase GLUT4 translocation was obliterated by PI-3-K inhibitor wortmannin, and BSD significantly increased the phosphorylation of AKT (Ser-473). Moreover, BSD-induced GLUT4 translocation was prevented in the presence of fulvestrant. Our findings reveal the estrogenic activity of BSD to stimulate glucose utilization in skeletal muscle cells via PI-3K/AKT-dependent mechanism.

Phenanthrenoid Coelogin Isolated from Coelogyne cristata Exerts Osteoprotective Effect Through MAPK-Mitogen-Activated Protein Kinase Signaling Pathway.

Osteoporosis is a major health problem in postmenopausal women globally. This study determined the mechanism through which coelogin stimulates osteoblastogenesis and its osteoprotective and bone regenerating potential. Coelogin effect on primary calvarial osteoblast cells was determined by measuring alkaline phosphatase activity, mineralization, osteoblast survival, and apoptosis and protein expression studies. The osteoprotective effect of coelogin was also evaluated on osteopenic adult female Swiss mice. At autopsy, bones were collected for dynamic and histomorphometry studies. Serum samples were also collected for assessment of serum parameters. Coelogin treatment led to increased osteoblast proliferation, survival, differentiation, and mineralization in osteoblast cells. Coelogin supplementation to Ovx mice promoted new bone formation, prevented Ovx-induced deterioration of bone microarchitecture, and enhanced bone regeneration. In addition, signaling studies revealed that coelogin treatment activates the ER-Erk and Akt-dependent signaling pathways which stimulate the osteoblastogenesis in osteoblast cells.

Dietary Mannan-oligosaccharides potentiate the beneficial effects of Bifidobacterium bifidum in broiler chicken.

This study investigated the effects of dietary Bifidobacterium bifidum (BFD) and mannan-oligosaccharide (MOS), as a synbiotic, on the production performance, gut microbiology, serum biochemistry, antioxidant profile and health indices of broiler chicken. Six dietary treatments were T1 (negative control), T2 (positive control-20 mg antibiotic BMD kg-1 diet; BMD: bacitracin methylene disalicylate), T3 (0·1% MOS + 106  CFU BFD per g feed), T4 (0·1% MOS + 107  CFU BFD per g feed), T5 (0·2% MOS + 106  CFU BFD per g feed) and T6 (0·2% MOS + 107  CFU BFD per g feed). Significantly (P < 0·01) better growth performance and efficiency was observed in birds supplemented with 0·2% MOS along with 106  CFU BFD per g of feed compared to BMD and control birds. Supplementation with 0·2% MOS along with either 106 or 107  CFU BFD per g feed reduced (P < 0·01) the gut coliform, Escherichia coli, total plate count, and Clostridium perfringens count and increased the Lactobacillus and Bifidobacterium count. Significantly (P < 0·01) higher serum and liver antioxidant enzyme pool, serum HDL cholesterol and lower serum glucose, triglyceride, total cholesterol, cardiac risk ratio, atherogenic coefficient and atherogenic index of plasma were observed in birds supplemented with 0·2% MOS along with 106  CFU BFD per g of feed compared to control or BMD supplemented birds. Better production performance, gut microbial composition, serum biochemistry, antioxidant profile and health indices were depicted by broiler chicken supplemented with 0·2% MOS and 106  CFU BFD per g of feed.

Comparative study on the responses of broiler chicken to hot and humid environment supplemented with different dietary levels and sources of selenium.

Present study was carried out with the objective of investigating the role of green synthesized nano Se (GNS) in growth performance, digestibility of minerals, immunity, stress alleviation, antioxidant status, and body Se content of broiler chicken raised under hot and humid environment with respect to market nano Se (MNS) and inorganic Se. The experimental design was 3 × 3 factorial, in which three levels (0.15, 0.20, and 0.25 ppm) and three sources (inorganic, green nano, and market nano) of Se resulted in nine treatments viz. IS-0.15, GNS-0.15, MNS-0.15, IZ-0.20, GNS-0.20, MNS-0.20, IS-0.25, GNS-0.25, and MNS-0.25 (IS: inorganic Se, GNS: green nano Se, MNS: market nano Se). A total of 432 broiler chicken were divided among nine treatments with six replicates of birds per treatment (8 birds/replicate). Results of present study revealed significantly better growth performance of birds supplemented with 0.25 ppm nano Se. The supplementation of 0.25 ppm nano Se improved the immune response and lymphoid organ development of birds. Significantly higher Se and nitrogen digestibility coefficients, serum antioxidant activity and decline of Heterophil: Lymphocyte ratio and expression of HSP70 gene were observed in birds supplemented with 0.25 ppm Se and nano source of Se compared to inorganic Se. Significantly higher Se concentration in liver and breast muscle and higher serum Se concentration were observed in birds fed 0.25 ppm nano Se. The liver Se concentration was much higher than that of breast muscle. However, the nano Se synthesized by green method in this study did not differ significantly from the chemically synthesized nano Se. It was concluded that 0.25 ppm Se and nano form of Se are superior to lower levels and inorganic form of Se, respectively, in improving the immunity, growth, antioxidant status, and in stress alleviation of broiler chicken. However, GNS is equally efficient as chemically synthesized MNS.

Dietary flaxseed and turmeric is a novel strategy to enrich chicken meat with long chain ω-3 polyunsaturated fatty acids with better oxidative stability and functional properties.

The purpose of the present study was to elucidate the effects of feeding flaxseed meal (FSM) and turmeric rhizome powder (TRP) supplementation on tissue lipid profile, lipid metabolism, health indices, oxidative stability, and physical properties of broiler chicken meat. The 100 g FSM along with 10.0 g TRP supplementation significantly increased the ω-3 PUFA, particularly ALA, EPA, DPA, and DHA of broiler chicken meat due to the corresponding increase ∆9 and Δ5 + Δ6 desaturase activities. The increased activities of the desaturases resulted in significantly better health indices of the broiler chicken meat. The feeding of 100 g FSM along with 10.0 g TRP supplementation reduced the atherogenic and thrombogenic indices of broiler chicken meat. The 100 g FSM feeding reduced the oxidative stability, water holding capacity, extract release volume of broiler chicken meat and increased drip loss, whereas, 10.0 g TRP supplementation reversed these negative effects of FSM.

Feeding value of rice distiller's dried grains with solubles as protein supplement in diet of laying hens.

A feeding trial of 10 weeks duration was undertaken on laying hens (n = 240) to evaluate feeding value of rice distiller's dried grains with soluble (rDDGS) with or without enzyme supplementation (α-amylase, ß-glucanase, xylanase, carboxymethylcellulase, pectinase, proteinase, α-galactosidase, ß-galactosidase, lipase, and phytase), following 4 × 2 factorial design, on egg production, nutrient utilization, and cost economics of egg production. The birds were randomly assigned to eight dietary treatments with 30 birds/treatment. The birds were housed individually in layer cages and each bird was taken as an experimental unit. Eight experimental diets were prepared by incorporating four levels (0, 50, 75, and 100 g/kg) of rDDGS with and without enzyme supplementation. The results revealed a significant (P < 0.01) increase of egg mass, feed intake, egg production, and body weight gain in dietary treatments with up to 75 g rDDGS though the values were statistically similar to the hens fed 100 g rDDGS. Enzyme supplementation resulted in significant (P < 0.01) improvement of egg mass, egg production, feed conversion ratio (FCR) per dozen eggs, FCR per kilogramme egg mass, and net FCR. The significantly (P < 0.01) higher yolk index was observed at 100 g rDDGS level, while shell thickness improved significantly (P < 0.01) up to 75 g rDDGS level. No significant effect of rDDGS inclusion was observed on shape index, albumin index, and Haugh unit. Enzyme supplementation significantly improved the shell thickness and yolk colour of eggs. Nitrogen, calcium, and phosphorus retention and dry matter metabolizability did not show any significant treatment effects. There was significant (P < 0.01) reduction in feed-cost per kilogramme egg mass or per dozen eggs with the increased DDGS levels and dietary enzyme supplementation. It was concluded that rDDGS can be used up to 100 g/kg diet of laying hens along with enzyme supplementation for better productivity of layer hens.

New lignan glycosides from Cissus quadrangularis stems.

Phytochemical investigation of Cissus quadrangularis stems led to the isolation of one new phenolic glycoside (1) and two new lignan glycosides (7 & 8) along with twelve known compounds (2-6 & 9-15). Their chemical structures were determined on the basis of extensive spectroscopic analysis using 1D, 2D NMR, and mass spectrometric analysis. Among the known compounds, 4-6, 9 and 12 were isolated for the first time from the genus Cissus whereas compounds 10, 11 and 13 for the first time from this plant.

Detection of flavonoids from Spinacia oleracea leaves using HPLC-ESI-QTOF-MS/MS and UPLC-QqQLIT-MS/MS techniques.

Spinacia oleracea L. (Spinach) is a leafy vegetable which is considered to have a high nutritional value. Flavonoids in spinach were reported to act as antimutagenic property. Rapid detection of these flavonoids in Spinach was achieved by using HPLC-ESI-QTOF-MS/MS. Thirty six compounds were tentatively identified based on their retention times, accurate mass and MS/MS spectra. The fragmentation patterns of known compounds were applied to elucidate the structure of their corresponding derivatives having the same basic skeleton. Out of thirty six peaks, three peaks were assigned as patuletin and six peaks were assigned as spinacetin derivatives. Twelve compounds were first time identified following the fragmentation pattern of known compounds. Five of the identified compounds i.e., spinacetin, 5,3',4'-trihydroxy-3-methoxy-6,7-methylenedioxyflavone, protocatechuic acid, ferulic acid and coumaric acid were simultaneously quantified in spinach leaves by a validated UPLC-ESI-MS/MS method under MRM mode.

Spinacia oleracea extract attenuates disease progression and sub-chondral bone changes in monosodium iodoacetate-induced osteoarthritis in rats.

BACKGROUND: Spinacia oleracea is an important dietary vegetable in India and throughout the world and has many beneficial effects. It is cultivated globally. However, its effect on osteoarthritis that mainly targets the cartilage cells remains unknown. In this study we aimed to evaluate the anti-osteoarthritic and chondro-protective effects of SOE on chemically induced osteoarthritis (OA). METHODS: OA was induced by intra-patellar injection of monosodium iodoacetate (MIA) at the knee joint in rats. SOE was then given orally at 250 and 500 mg.kg- 1 day- 1 doses for 28 days to these rats. Anti-osteoarthritic potential of SOE was evaluated by micro-CT, mRNA and protein expression of pro-inflammatory and chondrogenic genes, clinically relevant biomarker's and behavioural experiments. RESULTS: In vitro cell free and cell based assays indicated that SOE acts as a strong anti-oxidant and an anti-inflammatory agent. Histological analysis of knee joints at the end of the experiment by safranin-o and toluidine blue staining established its protective effect. Radiological data corroborated the findings with improvement in the joint space and irregularity of the articular and atrophied femoral condyles and tibial plateau. Micro-CT analysis of sub-chondral bone indicated that SOE had the ability to mitigate OA effects by increasing bone volume to tissue volume (BV/TV) which resulted in decrease of trabecular pattern factor (Tb.Pf) by more than 200%. SOE stimulated chondrogenic marker gene expression with reduction in pro-inflammatory markers. Purified compounds isolated from SOE exhibited increased Sox-9 and Col-II protein expression in articular chondrocytes. Serum and urine analysis indicated that SOE had the potential to down-regulate glutathione S-transferase (GST) activity, clinical markers of osteoarthritis like cartilage oligometric matrix protein (COMP) and CTX-II. Overall, this led to a significant improvement in locomotion and balancing activity in rats as assessed by Open-field and Rota rod test. CONCLUSION: On the basis of in vitro and in vivo experiments performed with Spinacea oleracea extract we can deduce that SOE has the ability to alleviate the MIA induced deleterious effects.

Formononetin, a methoxy isoflavone, enhances bone regeneration in a mouse model of cortical bone defect.

The bone regeneration and healing effect of formononetin was evaluated in a cortical bone defect model that predominantly heals by intramembranous ossification. For this study, female Balb/c mice were ovariectomised (OVx) and a drill-hole injury was generated in the midfemoral bones of all animals. Treatment with formononetin commenced the day after and continued for 21 d. Parathyroid hormone (PTH1-34) was used as a reference standard. Animals were killed at days 10 and 21. Femur bones were collected at the injury site for histomorphometry studies using microcomputed tomography (µCT) and confocal microscopy. RNA and protein were harvested from the region surrounding the drill-hole injury. For immunohistochemistry, 5 µm sections of decalcified femur bone adjoining the drill-hole site were cut. µCT analysis showed that formononetin promoted bone healing at days 10 and 21 and the healing effect observed was significantly better than in Ovx mice and equal to PTH treatment in many aspects. Formononetin also significantly enhanced bone regeneration as assessed by calcein-labelling studies. In addition, formononetin enhanced the expression of osteogenic markers at the injury site in a manner similar to PTH. Formononetin treatment also led to predominant runt-related transcription factor 2 and osteocalcin localisation at the injury site. These results support the potential of formononetin to be a bone-healing agent and are suggestive of its promising role in the fracture-repair process.

Alcoholic Extract of Eclipta alba Shows In Vitro Antioxidant and Anticancer Activity without Exhibiting Toxicological Effects.

As per WHO estimates, 80% of people around the world use medicinal plants for the cure and prevention of various diseases including cancer owing to their easy availability and cost effectiveness. Eclipta alba has long been used in Ayurveda to treat liver diseases, eye ailments, and hair related disorders. The promising medicinal value of E. alba prompted us to study the antioxidant, nontoxic, and anticancer potential of its alcoholic extract. In the current study, we evaluated the in vitro cytotoxic and antioxidant effect of the alcoholic extract of Eclipta alba (AEEA) in multiple cancer cell lines along with control. We have also evaluated its effect on different in vivo toxicity parameters. Here, we found that AEEA was found to be most active in most of the cancer cell lines but it significantly induced apoptosis in human breast cancer cell lines by disrupting mitochondrial membrane potential and DNA damage. Moreover, AEEA treatment inhibited migration in both MCF 7 and MDA-MB-231 cells in a dose dependent manner. Further, AEEA possesses robust in vitro antioxidant activity along with high total phenolic and flavonoid contents. In summary, our results indicate that Eclipta alba has enormous potential in complementary and alternative medicine for the treatment of cancer.

Guava fruit extract and its triterpene constituents have osteoanabolic effect: Stimulation of osteoblast differentiation by activation of mitochondrial respiration via the Wnt/ß-catenin signaling.

The aim of this study was to evaluate the skeletal effect of guava triterpene-enriched extract (GE) in rats and identify osteogenic compounds thereof, and determine their modes of action. In growing female rats, GE at 250 mg/kg dose increased parameters of peak bone mass including femur length, bone mineral density (BMD) and biomechanical strength, suggesting that GE promoted modeling-directed bone growth. GE also stimulated bone regeneration at the site of bone injury. In adult osteopenic rats (osteopenia induced by ovariectomy, OVX) GE completely restored the lost bones at both axial and appendicular sites, suggesting a strong osteoanabolic effect. Serum metabolomics studies showed changes in several metabolites (some of which are related to bone metabolism) in OVX compared with ovary-intact control and GE treatment to OVX rats reversed those. Out of six abundantly present triterpenes in GE, ursolic acid (UA) and 2α-hydroxy ursolic acid (2α-UA) induced osteogenic differentiation in vitro as did GE by activating Wnt/ß-catenin pathway assessed by phosphorylation of GSK-3ß. Over-expressing of constitutively active GSK-3ß (caGSK-3ß) in osteoblasts abolished the differentiation-promoting effect of GE, UA and 2α-UA. All three increased both glycolysis and mitochondrial respiration but only rotenone (inhibitor of mitochondrial electron transfer) and not 2-deoxyglucose (to block glycolysis) inhibited osteoblast differentiation. In addition, caGSK-3ß over-expression attenuated the enhanced mitochondrial respiration caused by GE, UA and 2α-UA. We conclude that GE has osteoanabolic effect which is contributed by UA and 2α-UA, and involve activation of canonical Wnt signaling which in turn modulates cellular energy metabolism leading to osteoblast differentiation.

Dried and free flowing granules of Spinacia oleracea accelerate bone regeneration and alleviate postmenopausal osteoporosis.

OBJECTIVE: The aim of this study was to demonstrate the efficacy of extract derived from Spinacia oleracea extract (SOE) in reversing bone loss induced by ovariectomy and bone healing properties in a drill-hole fracture model in rats. METHODS: SOE was administered orally for 12 weeks in adult ovariectomized Sprague Dawley rats after inducing osteopenic condition. Bone micro-architecture, expressions of osteogenic and resorptive gene markers, biomechanical strength, new bone formation, and bone turnover markers were studied. Uterine histomorphometry was used to assess estrogenicity. Bone regeneration potential of SOE was assessed in a drill-hole fracture model. Fracture healing was assessed by calcein intensity and micro-CT analysis of callus at fracture region. RESULTS: SOE prevented ovariectomy-induced bone loss as evident from 122% increase in bone volume/tissue volume (BV/TV) and 29% decline in Tb.Sp in femoral trabecular micro-architecture. This was corroborated by the more than twofold stimulation in the expression of osteogenic genes runt-related transcription factor 2, osterix, osteocalcin, bone morphogenetic protein 2, collagen-1. Furthermore in the fracture healing model, we observed a 25% increase in BV/TV and enhancement in calcein intensity at the fractured site. The extract when converted into dried deliverable Spinaceae oleracea granule (SOG) form accelerated bone regeneration at fracture site, which was more efficient as evident by a 39% increase in BV/TV. Transforming SOE into dried granules facilitated prolonged systemic availability, thus providing enhanced activity for a period of 14 days. CONCLUSIONS: SOE treatment effectively prevents ovariectomy-induced bone loss and stimulated fracture healing in adult rats. The dried granular form of the extract of Spinaceae oleracea was effective in fracture healing at the same dose.

Methoxyisoflavones formononetin and isoformononetin inhibit the differentiation of Th17 cells and B-cell lymphopoesis to promote osteogenesis in estrogen-deficient bone loss conditions.

OBJECTIVE: Recent studies have shown that immune system plays a major role in pathophysiology of postmenopausal osteoporosis. Previously we have shown that phytoestrogens like daidzein and medicarpin exhibit immunoprotective effects, by virtue of which they alleviate bone loss. With this background, methoxyisoflavones like formononetin (formo) and isoformononetin (isoformo) that have been studied for preventing bone loss in ovariectomized rats were tested for their immunomodulatory effects in estrogen-deficient bone loss mice model. METHODS: Adult Balb/c mice (N = 8/group) were given oral dose of formo and isoformo at 10 mg/kg body weight, post ovariectomy (Ovx) daily for 6 weeks. Animals were autopsied and long bones were harvested to study bone microarchitecture. Peripheral blood mononuclear cells were isolated for fluorescence-activated cell sorting and RNA analysis. Serum was collected for enzyme-linked immunosorbent assay. RESULTS: It was observed that formo and isoformo treatment to Ovx mice led to significant restoration of Ovx-induced deterioration of trabecular microarchitecture. Pro-osteoclastogenic subset Th17 and B cells were decreased in formo/isoformo-treated Ovx mice in comparison with vehicle-treated Ovx group. Formo and isoformo treatment to Ovx mice also led to decreased expression of Th17 diffentiation factors and promoted T-regulatory cell differentiation. Formo was more effective in enhancing the FOXP3 expression compared with isoformo. IL-17A-induced osteoclastogenesis and inhibition of osteoblast apoptosis were also suppressed by formo and isoformo treatment, with formo having a more potent effect. CONCLUSIONS: Our study demonstrates the immunomodulatory activity of methoxyisoflavones, formo, and isoformo, which translate into improved skeletal parameters, thereby preventing Ovx-induced bone loss.

Osteogenic activity of natural diterpenoids isolated from Cupressus sempervirens fruits in calvarial derived osteoblast cells via differentiation and mineralization.

The aim of the present study was to investigate the antiosteoporotic activity of four structurally related diterpenoids: sugiol (1), trans-communic acid (2), 15-acetoxy imbricatolic acid (3) and imbricatolic acid (4). Their osteogenic effect was evaluated by using validated models including alkaline phosphatase (ALP) assay, mineralization assay and expression of osteogenic genes-bone morphogenetic protein-2 (BMP-2) and osteoblast transcription factor (RUNX2) - in primary calvarial cultures harvested from neonatal mice. Among them, compound 1 at a dose of 1.0 mg/kg body weight exhibited significant osteoprotective effects and did not show uterine estrogenicity at the same dose. Additionally, compound 1 treatment led to improved biomechanical properties as exhibited by increased power, energy and stiffness in femoral bones compared to untreated Ovx animals. Since osteoporotic compression fracture correlates with the mechanical characteristics of trabecular bone, so that it could effectively reduce the risk of this type of fracture by improving trabecular micro architecture in postmenopausal women. Therefore, our findings proposed that diterpenoids may be useful new chemical agents in the treatment of diseases associated with bone loss.

Analysis of constituents of the eastern Nigeria mistletoe, Loranthus micranthus linn revealed presence of new classes of osteogenic compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Mistletoe extracts (decoctions) are used traditionally in eastern Nigeria for the management of bone pain, post menopausal syndrome and diabetes amongst several other ailments. While scientific evidence supporting its folkloric use as an antidiabetic agent has been documented, the age-long practice of its use in treatment of post menopausal syndrome has not been scientifically validated. Postmenopausal osteoporosis accounts for one of the prevalent disease conditions in aging population globally. This situation is exacerbated by the lack of osteogenic therapy. In search for plants of Nigerian origin with osteogenic potential, we evaluated eastern Nigerian mistletoe, having ethnotraditional claims of anti-diabetic, anti-hypertensive and anti-cancer activities as well as preventive effect in various post-menopausal syndromes. MATERIALS AND METHODS: Methanolic extracts of mistletoe leaves harvested from three host tress - Kola acuminata (KM), Citrus spp (CM) and Garcinia kola (GKM) - were evaluated for osteoblast viability and osteogenic activities using primary rat calvaria culture. Lupeol (1) was isolated from the stem bark of Bombax ciba and its congener, dihydoxylupeol palmitate (2) in addition to three other compounds; 3-methoxy quercetin (3), 3,4,5-trimethoxy gallate (4), and friedelin (5) were isolated from the leaves of mistletoes species. Following their chemical characterization, the compounds were evaluated for osteogenic potential using validated models including alkaline phosphatase (ALP) assay, mineralization assay and expression of osteogenic genes - bone morphogenetic protein-2 (BMP2) and osteoblast transcription factor (RUNX2) - in primary calvarial cultures harvested from neonatal rats. Uterine estrogenicity of the extracts was tested in adult female Sprague Dawley rats. RESULTS: Methanol extracts of mistletoe from three hosts exhibited increase in ALP activity (a marker of osteoblast differentiation) at lower concentrations (0.2-0.8 µg/ml) and either no or inhibitory effect at higher concentrations (1.6 and 3.2 µg/ml). None of the extract had cytotoxicity to osteoblasts at the concentrations tested. Five compounds viz. 1 from Bombax ciba, and 2-5 were isolated from the mistletoe leaves. Out of these, 5 exhibited significant loss of osteoblast viability and hence it was not considered further. All four compounds exhibited stimulatory effects on osteoblast differentiation as assessed by ALP assay and determination of osteogenic gene expression. Compound 2 was relatively more potent than its precursor, compound 1 in stimulating BMP2 upregulation. KM did not show uterine estrogenicity. CONCLUSION: Methanolic extracts from the three mistletoes species possess in vitro osteogenic activity, and from these extracts three new classes of compounds have been found to promote osteoblast differentiation in vitro. In light of these findings, we propose that mistletoe species may be developed as safer alternative(s) in the management of diseases where lack of bone formation is the pathology.