RESUMEN
Cassia occidentalis Linn (CO) is an annual/perennial plant having traditional uses in the treatments of ringworm, gastrointestinal ailments and piles, bone fracture, and wound healing. Previously, we confirmed the medicinal use of the stem extract (ethanolic) of CO (henceforth CSE) in fracture healing at 250â¯mg/kg dose in rats and described an osteogenic mode of action of four phytochemicals present in CSE. Here we studied CSE's preclinical safety and toxicity. CSE prepared as per regulations of Current Good Manufacturing Practice for human pharmaceuticals/phytopharmaceuticals and all studies were performed in rodents in a GLP-accredited facility. In acute dose toxicity as per New Drug and Clinical Trial Rules, 2019 (prior name schedule Y), in rats and mice and ten-day dose range-finding study in rats, CSE showed no mortality and no gross abnormality at 2500â¯mg/kg dose. Safety Pharmacology showed no adverse effect on central nervous system, cardiovascular system, and respiratory system at 2500â¯mg/kg dose. CSE was not mutagenic in the Ames test and did not cause clastogenicity assessed by in vivo bone marrow genotoxicity assay. By a sub chronic (90 days) repeated dose (as per OECD, 408 guideline) study in rats, the no-observed-adverse-effect-level was found to be 2500â¯mg/kg assessed by clinico-biochemistry and all organs histopathology. We conclude that CSE is safe up to 10X the dose required for its osteogenic effect.
Asunto(s)
Fitoquímicos/toxicidad , Extractos Vegetales/toxicidad , Senna , Animales , Etanol , Ratones , Nivel sin Efectos Adversos Observados , Ratas , Roedores , Pruebas de ToxicidadRESUMEN
Fruit extract of Cupressus sempervirens (CS), which is used traditionally to treat Benign Prostatic Hyperplasia (BPH)-like urinary symptoms in patients, was scientifically validated for anti-BPH activity. The ethanolic fruit extract of CS inhibited proliferation of human BPH-stromal cells and the activity was localized to its chloroform-soluble, diterpene-rich fraction. Eight major diterpenes isolated from this fraction exhibited moderate to potent activity and the most active diterpene (labda-8(17),12,14-trien-19-oic acid) exhibited an IC50 of 37.5 µM (antiproliferative activity against human BPH-stromal cells). It significantly inhibited activation (phosphorylation) of Stat-3 in BPH-stromal cells and prevented transactivation of androgen sensitive KLK3/PSA and TMPRSS2 genes in LNCaP cells. Labda-8(17),12,14-trien-19-oic acid-rich CS fraction prevented prostatic hyperplasia in rat model and caused TUNEL labeling of stromal cells with lower expressions of IGF-I, TGF-ß and PCNA, and bcl-2/bax ratio. Human BPH tissues exhibited precise lowering of stromal component after incubation in labda-8(17),12,14-trien-19-oic acid, ex vivo. We conclude that labda-8(17),12,14-trien-19-oic acid contained in CS exhibits anti-BPH activity through inhibition of stromal proliferation and suppression of androgen action in the prostate, presenting a unique lead structure for further optimization of anti-BPH activity.
Asunto(s)
Andrógenos/metabolismo , Cupressus/química , Diterpenos/farmacología , Hiperplasia Prostática/tratamiento farmacológico , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Frutas/química , Humanos , Masculino , Fosforilación , Ratas , Ratas Sprague-Dawley , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Cassia occidentalis L., a synonym of Senna occidentalis (belongs to Caesalpiniaceae family) is an annual plant. Pursuing a lead from a folk practice prevalent since the late nineteenth century in Andhra Pradesh, a Southern state of India, of use of Cassia occidentalis leaf and stem for treating patients with fracture and bone diseases, we have not only confirmed its fracture healing activity but also demonstrated efficacy in preventing glucocorticoid-induced osteoporosis (GIO), the commonest form of medication-induced bone loss caused chiefly due to impairment of bone formation. AIM OF THE STUDY: In the present work, the effects of extract and fraction of leaf and stem of Cassia occidentalis was investigated in fracture healing and GIO models of rat. The study also aimed to identify osteogenic compounds from this plant. MATERIALS AND METHODS: Ethanolic extracts from leaf and stem of Cassia occidentalis were prepared and their efficacy tested in rat femur osteotomy (fracture healing) model. Subsequently, a butanolic fraction was prepared and osteogenic efficacy compared with the ethanolic extract, and upon finding the former to be more potent, its osteogenic effect was studied in details in GIO model. Chemical finger-printing and isolation of ten pure compounds were done to assess their osteogenic effect in rat primary osteoblast cultures. RESULTS: Ethanolic extract of stem was more effective than the leaf extract in enhancing bone regeneration at the site of osteotomy. Further, butanolic fraction of the ethanolic extract of stem was more effective than the later in bone regeneration at the femur osteotomy site and in preventing bone loss in GIO model. The mechanism of skeletal preservation involved stimulation of new bone formation and inhibition of bone resorption. As many as six osteogenic compounds were isolated out of which apigenin-6C-glucopyranoside was most effective in vitro. CONCLUSION: Our study found that a standardized extract of an ethanolic extract and its butanolic fraction from the stem of Cassia occidentalis has osteogenic as well as anti-resorptive effects, resulting in the protection against glucocorticoid-induced bone loss. Our results contribute towards validation of the traditional use of Cassia occidentalis in fracture healing and also suggest its beneficial use in GIO for which clinical trials are warranted.
Asunto(s)
Enfermedades Óseas Metabólicas/prevención & control , Glucocorticoides/toxicidad , Extractos Vegetales/farmacología , Senna/química , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Etanol/química , Curación de Fractura/efectos de los fármacos , India , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Eclipta alba (Bhringraj) in ayurveda has been widely used as a traditional medicine for its multi-therapeutic properties for ages. Luteolin (LTL), wedelolactone (WDL) and apigenin (APG) are the three main bioactive phytochemicals present in Eclipta alba extract. However there was a lack of sensitive bioanalytical method for the pharmacokinetics of these free compounds in plasma which majorly contributes for their activities after oral administration of Eclipta alba. The present study aims to develop a sensitive, rapid and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous estimation of mice plasma concentrations of LTL, WDL and APG using quercetin as an internal standard for the pharmacokinetic analysis. Analytes were separated on Phenomenex Luna C18 (150â¯×â¯4.6â¯mm, 3.0⯵m) column with mobile phase containing methanol: acetonitrile (90: 10, v/v) and 0.1% formic acid in 10â¯mM ammonium formate buffer in the ratio of 70: 30 (v/v) in isocratic mode. Liquid-liquid extraction was optimized using Hansen solubility parameters and diethyl ether finalized as an extraction solvent for the recovery ranging from 61 to 76% for all analytes in mice plasma. The validated method has an accuracy and precision over the linearity range of 0.1-200â¯ng/mL with a correlation coefficient (r2) of ≥0.997. The intra and inter-day assay accuracy was between 98.17 and 107% and 95.83-107.89% respectively and the intra and inter day assay precision ranged from 0.37-6.05% and 1.85-10.76%, respectively for all the analytes. This validated method can be used for future clinical investigation studies of Eclipta alba extracts.
Asunto(s)
Apigenina/sangre , Cumarinas/sangre , Eclipta/química , Extracción Líquido-Líquido/métodos , Luteolina/sangre , Extractos Vegetales/farmacocinética , Animales , Apigenina/química , Apigenina/aislamiento & purificación , Apigenina/farmacocinética , Cloroformo , Cromatografía Liquida/métodos , Cumarinas/química , Cumarinas/aislamiento & purificación , Cumarinas/farmacocinética , Límite de Detección , Modelos Lineales , Luteolina/química , Luteolina/aislamiento & purificación , Luteolina/farmacocinética , Ratones , Extractos Vegetales/química , Reproducibilidad de los Resultados , Solubilidad , Espectrometría de Masas en Tándem/métodosRESUMEN
Major challenges for current therapeutic strategies against breast cancer are associated with drug-induced toxicities. Considering the immense potential of bioactive phytochemicals to deliver non-toxic, efficient anti-cancer therapeutics, we performed bio-guided fractionation of Eclipta alba extract and discovered that particularly the chloroform fraction of Eclipta alba (CFEA) is selectively inducing cytotoxicity to breast cancer cells over non-tumorigenic breast epithelial cells. Our unbiased mechanistic hunt revealed that CFEA specifically activates the intrinsic apoptotic pathway by disrupting the mitochondrial membrane potential, upregulating Hsp60 and downregulating the expression of anti-apoptotic protein XIAP. By utilizing Hsp60 specific siRNA, we identified a novel pro-apoptotic role of Hsp60 and uncovered that following CFEA treatment, upregulated Hsp60 is localized in the endoplasmic reticulum (ER). To our knowledge, this is the first evidence of ER specific localization of Hsp60 during cancer cell apoptosis. Further, our LC-MS approach identified that luteolin is mainly attributed for its anti-cancer activities. Moreover, oral administration of CFEA not only offers potential anti-breast cancer effects in-vivo but also mitigates tumor induced hepato-renal toxicity. Together, our studies offer novel mechanistic insight into the CFEA mediated inhibition of breast cancer and may potentially open up new avenues for further translational research.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Chaperonina 60/metabolismo , Eclipta/química , Retículo Endoplásmico/metabolismo , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Animales , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Chaperonina 60/genética , Cloroformo/química , Femenino , Humanos , Células MCF-7 , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos BALB C , Microscopía Confocal , Fitoterapia/métodos , Extractos Vegetales/química , Extractos Vegetales/farmacología , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
(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.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Flavonas/aislamiento & purificación , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Glicósidos/aislamiento & purificación , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Ulmus/química , Adipocitos/citología , Fosfatasa Alcalina/biosíntesis , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Flavonas/farmacología , Glicósidos/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , Osteoblastos/citología , Osteoblastos/fisiología , Osteoclastos/citología , Corteza de la Planta/química , Tallos de la Planta/químicaRESUMEN
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.
Asunto(s)
Glucosa-6-Fosfatasa/antagonistas & inhibidores , Glicósidos/aislamiento & purificación , Lauraceae/química , Fenoles/aislamiento & purificación , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Glicósidos/química , Concentración 50 Inhibidora , Estructura Molecular , Fenoles/química , Hojas de la Planta/químicaRESUMEN
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.
Asunto(s)
Abietanos/aislamiento & purificación , Cupressus/química , Diterpenos/aislamiento & purificación , Inhibidores Enzimáticos/aislamiento & purificación , Glucosa-6-Fosfatasa/antagonistas & inhibidores , Glucógeno Fosforilasa/antagonistas & inhibidores , Extractos Vegetales/química , Abietanos/química , Abietanos/farmacología , Diterpenos/química , Diterpenos/farmacología , Inhibidores Enzimáticos/farmacología , Frutas , Glicósidos/aislamiento & purificación , Glicósidos/farmacología , Estructura Molecular , Fenoles/aislamiento & purificación , Fenoles/farmacología , Extractos Vegetales/farmacología , Hojas de la PlantaRESUMEN
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.