Self Emulsifying Delivery System of Cissus quadrangularis: Evidence of Enhanced Efficacy and Promising Pharmacokinetic Profile in the Management of Osteoporosis.

Treatment therapies used to manage osteoporosis are associated with severe side effects. So worldwide herbs are widely studied to develop alternative safe & effective treatments. Cissus quadrangularis (CQ) has a significant role in bone health and fracture healing. It is documented that its extracts increase osteoblastic differentiation & mineralization. Currently, Cissus quadrangularis is available in the form of tablets in the market for oral delivery. But these conventional forms are associated with poor bioavailability. There is a need for a novel drug delivery system with improving oral bioavailability. Therefore, a Cissus quadrangularis-loaded self-emulsifying drug delivery system (CQ-SEDDS) was developed which disperses rapidly in the gastrointestinal fluids, yielding nano-emulsions containing a solubilized drug. This solubilized form of the drug can be easily absorbed through lymphatic pathways and bypass the hepatic first-pass effect. The emulsification efficiency, zeta potential, globule size, in-vitro dissolution, ex-vivo, in-vivo and bone marker studies were performed to assess the absorption and permeation potential of CQ incorporated in SEDDS. CQ-SEDDS with excipients Tween 80, Cremophor RH40, Transcutol HP & α-Tocopherol acetate had shown about 76% enhancement in the bioavailability of active constituents of CQ. This study provided the pre-clinical data of CQ-SEDDS using osteoporotic rat model studies.

Cissus quadrangularis L extract-loaded tricalcium phosphate reinforced natural polymer composite for guided bone regeneration.

Natural medicines plants are significant considerable attention as potential therapeutic agents for bone tissue engineering. Cissus quadrangularis L (CQ). is a potent therapeutic plant known for its own osteogenic properties. In this research work, a phytoconstituents-filled composite was produced by incorporating CQ extract with gelatin (Gel) and pectin (Pec) polymers collective through ß- tricalcium phosphate (ß-TCP) bioceramic via a green template method. The effect of CQ-filled composite morphology and chemical structural properties, in vitro cytotoxicity, cell proliferation, and differentiation was investigated. FTIR spectroscopic results indicated the prepared materials' structural confirmation. The CQ extract was the alcoholic -OH merge with the hydroxyl and -NH groups in the range of 3000 cm-1 to 3500 cm-1. Scanning electron microscopy images showed that the ß-TCP ceramic was perfectly embedded in Gel-Pec polymeric matrix, which is important for bone regeneration. In vitro cell culture results indicated that ß-TCP/Gel-Pec/CQ composite provided 92.0% of a favorable substrate for mesenchymal stem cell viability. The gene expression and RT-PCR studies represent the materials with good osteogenic expression, especially the ß-TCP/Gel-Pec/CQ composite is observed at 168.0% and 188.0% for RUNx2 and OCN, respectively. The result of the physicochemical characterizations and cell viability studies suggest that CQ-loaded ß-TCP/Gel-Pec composite can serve as a potential biomaterial for bone tissue repair and regeneration.

A New Ceramide from Cissus Aralioides Baker (Vitaceae) and its Antimicrobial Activity.

Purification through repeated column chromatography over silica gel and Sephadex LH-20 of the ethanol extract of the stems of Cissus aralioides (Baker) Planch. resulted in the isolation of a new ceramide, aralioidamide A (1), along with five known compounds (2-6). Their structures were determined by the extensive analyses of their spectroscopic (1D and 2D NMR) and spectrometric data, and comparison with those reported in the literature. Aralioidamide A (1) displayed weak antibacterial activity (MIC=256 µg/mL) against Bacillus subtilis, Staphylococcus aureus and Shigella flexneri and was inactive (MIC>256 µg/mL) against the tested fungi.

Phytochemical Profiling and Compound Isolation of Cissus rhombifolia Vahl. Leaves Aqueous Methanolic Extract with the Evaluation of Its Anti-Inflammatory Effect Using Lipopolysaccharide-Induced Inflammation in RAW 264.7 Cells.

The inflammatory disorders represent a serious health issue. Certain Cissus species possess anti-inflammatory effect. Cissus rhombifolia Vahl. leaves' anti-inflammatory activities and phytoconstituents are poorly characterized. In this study, 38 constituents were tentatively characterized in Cissus rhombifolia Vahl. leaves' aqueous methanolic extract (CRLE) using high-performance liquid chromatography combined with mass spectrometry (HPLC/MS) and Proton Nuclear Magnetic Resonance (1 H-NMR). Myricetin, ß-amyrin, and alliospiroside A, were isolated from CRLE using column chromatography. The anti-inflammatory effect of CRLE and its isolated compounds were studied in lipopolysaccharide (LPS)-induced RAW 264.7 cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) was used to assess how CRLE and its isolated compounds affected cell viability. Further, its effects on the production of intracellular NO, and inflammatory cytokines cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) were assessed by the Griess test, and cytokine enzyme-linked immunosorbent assays, respectively. CRLE and its isolated compounds, myricetin, ß-amyrin, and alliospiroside A decreased the NO production. Western blotting was performed to assess the protein expression levels of the inflammatory cytokines inducible nitric oxide synthase (iNOS). Alliospiroside A downregulated IL-6, TNF-α, and COX-2 and inhibited the expression of iNOS. CRLE and its compounds represent effective alternative candidate to treat inflammatory diseases.

Simultaneous Estimation of Quercetin and trans-Resveratrol in Cissus quadrangularis Extract in Rat Serum Using Validated LC-MS/MS Method: Application to Pharmacokinetic and Stability Studies.

Cissus quadrangularis is a nutrient-rich plant with a history of use in traditional medicine. It boasts a diverse range of polyphenols, including quercetin, resveratrol, ß-sitosterol, myricetin, and other compounds. We developed and validated a sensitive LC-MS/MS method to quantify quercetin and t-res biomarkers in rat serum and applied this method to pharmacokinetic and stability studies. The mass spectrometer was set to negative ionization mode for the quantification of quercetin and t-res. Phenomenex Luna (C18(2), 100 A, 75 × 4.6 mm, 3 µ) column was utilized to separate the analytes using an isocratic mobile phase consisting of methanol and 0.1% formic acid in water (82:18). Validation of the method was performed using various parameters, including linearity, specificity, accuracy, stability, intra-day, inter-day precision, and the matrix effect. There was no observed significant endogenous interference from the blank serum. The analysis was completed within 5.0 min for each run, and the lower limit of quantification was 5 ng/mL. The calibration curves showed a linear range with a high correlation coefficient (r2 > 0.99). The precision for intra- and inter-day assays showed relative standard deviations from 3.32% to 8.86% and 4.35% to 9.61%, respectively. The analytes in rat serum were stable during bench-top, freeze-thaw, and autosampler (-4 °C) stability studies. After oral administration, the analytes showed rapid absorption but underwent metabolism in rat liver microsomes despite being stable in simulated gastric and intestinal fluids. Intragastric administration resulted in higher absorption of quercetin and t-res, with greater Cmax, shorter half-life, and improved elimination. No prior research has been conducted on the oral pharmacokinetics and stability of anti-diabetic compounds in the Ethanolic extract of Cissus quadrangularis EECQ, making this the first report. Our findings can provide the knowledge of EECQ's bioanalysis and pharmacokinetic properties which is useful for future clinical trials.

Cissus quadrangularis extract mitigates diabetic cardiomyopathy by inhibiting RAAS activation, inflammation and oxidative stress.

BACKGROUND: Diabetic cardiomyopathy (DCM) is an age-related disease, and its progression is accompanied by hyperglycaemia, cardiac dysfunction, and myocardial structural and functional abnormalities. Cissus quadrangularis, a traditional medicinal plant, contains polyphenols, flavonoids, phytosterols, carbohydrates and ascorbic acid. It is used to treat osteoporosis, asthma, haemorrhoids and menstrual disorders.Objective: In the current research, we have investigated the effect of ethanolic extract of C. quadrangularis (EECQ) against a high-fat diet (HFD)/streptozotocin-induced DCM by estimating cardiac biomarkers, inflammatory markers and Reactive oxygen species (ROS) production. MATERIAL AND METHODS: Rats were fed with an HFD for 12 weeks, followed by single-shot low-dose streptozotocin (35 mg/kg; i.p.). The treatment was performed by EECQ (200 mg/kg/day, orally) for six weeks. RESULTS: The extract EECQ improves glucose, insulin tolerance tests and hypercholesteremia. DCM is characterized by cardiac dysfunction, cardiac biomarkers CKMB and LDH, which were attenuated by the EECQ treatment. The hypertrophic biomarker ANP, BNP expression and cardiomyocyte surface area were decreased by EECQ. Moreover, EECQ also alleviated the biomarkers Angiotensin II and renin level. EECQ also reduced oxidative stress, ROS production and cardiac inflammation. CONCLUSIONS: Thus, these findings suggested that EECQ could be used as a possible therapeutic regiment to treat DCM.

Cissus quadrangularis ameliorates hyperglycaemia, hyperinsulinemia and hyperlipidaemia.Cissus quadrangularis mitigates cardiac dysfunction.Cissus quadrangularis decreases RAAS activation, thereby down-regulates ANP, BNP expression.Cissus quadrangularis alleviates ROS propagated oxidative stress and apoptosis.

Ethanolic extract of Cissus quadrangularis improves vasoreactivity by modulation of eNOS expression and oxidative stress in spontaneously hypertensive rats.

BACKGROUND: Endothelial dysfunction is related to the reduced bioavailability of nitric oxide (NO) and plays a significant role in developing hypertension. The intake of a diet rich in antioxidants decreases the threat of hypertension. Cissus quadrangularis possesses antioxidant, anti-inflammatory, and hypocholesterolemic activities. However, to date, no studies have been performed to explore this plant's antihypertensive and vasorelaxant activity. Herein, we investigated the chronic effect of C. quadrangularis on blood pressure as well as vascular function in hypertensive rats. METHODS: Male spontaneously hypertensive rats (SHR) were randomly divided into two groups. Normotensive Wistar rats were taken as the control group. The treatment was done using ethanolic extract of C. quadrangularis (EECQ) at a dose of 200 mg/kg. RESULTS: The administration of EECQ for six weeks reduced the systolic blood pressure, mean arterial blood pressure, and heart rate. It also alleviated the cardiac and renal hypertrophy indices. Supplementation of EECQ improved the endothelium-dependent aortic vasodilation induced by acetylcholine. It restored the NO level and endothelial NO synthase expression in the aorta. Subsequently, the extract alleviates the oxidative stress and inflammatory markers in SHR rats. CONCLUSION: Thus, in the present study, the chronic treatment of EECQ to genetically hypertensive rats improved endothelium-dependent relaxation in addition to its antihypertensive effect by eNOS activation and inhibition of ROS production, inflammation.

Biocompatibility and antioxidant activity of a novel carrageenan based injectable hydrogel scaffold incorporated with Cissus quadrangularis: an in vitro study.

BACKGROUND: Over the past years, polysaccharide-based scaffolds have emerged as the most promising material for tissue engineering. In the present study, carrageenan, an injectable scaffold has been used owing to its advantage and superior property. Cissus quadrangularis, a natural agent was incorporated into the carrageenan scaffold. Therefore, the present study aimed to assess the antioxidant activity and biocompatibility of this novel material. METHODS: The present in vitro study comprised of four study groups each constituting a sample of 15 with a total sample size of sixty (n = 60). The carrageenan hydrogel devoid of Cissus quadrangularis acted as the control group (Group-I). Based on the concentration of aqueous extract of Cissus quadrangularis (10% w/v, 20% w/v and 30% w/v) in carrageenan hydrogel, respective study groups namely II, III and IV were considered. Antioxidant activity was assessed using a 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay, whereas the biocompatibility test was performed using a brine shrimp lethality assay. The microstructure and surface morphology of the hydrogel samples containing different concentrations of Cissus quadrangularis aqueous extract was investigated using SEM. One-way ANOVA with the post hoc tukey test was performed using SPSS software v22. RESULTS: A significant difference (P < 0.05) in the antioxidant activity was observed among the study groups. Group III reported the highest activity, whereas the control group showed the least antioxidant activity. Additionally, a significant (P < 0.01) drop in the antioxidant activity was observed in group IV when compared with group III. While assessing the biocompatibility, a significant (P < 0.001) dose-dependent increase in biocompatibility was observed with the increasing concentration of aqueous extract of Cissus quadrangularis. SEM analysis in group III showed even distribution throughout the hydrogel although the particles are close and densely arranged. Reduced antioxidant activity in group IV was probably due to clumping of the particles, thus reducing the active surface area. CONCLUSION: Keeping the limitations of in vitro study, it can be assumed that a carrageenan based injectable hydrogel scaffold incorporated with 20% w/v Cissus quadrangularis can provide a favourable micro-environment as it is biocompatible and possess better antioxidant property.

Effect of Cissus quadrangularis Hydrogel on Enhancing Osseointegration of Titanium Implant to Bone: An In Vivo Study.

AIM: The aim of the study was to evaluate the osteogenic potential of Cissus quadrangularis (CQ) hydrogel in enhancing the osseointegration of titanium to the bone in an experimental rabbit model. MATERIALS AND METHODS: Six adult male New Zealand white rabbits were used in this study. A total of 24 implants (12 coated test implants and 12 uncoated control implants) were placed in these 6 rabbits. A polyethylene glycol (PEG) hydrogel was prepared with the C. quadrangularis hydrogel in which the test implants were coated. Each rabbit was operated on both hind legs and one implant, each, was placed in the femur and tibia. Hence, one rabbit received four implants [two test implants (HG coated) and two control implants (uncoated)]. The animals were sacrificed after 4 weeks, and the specimens were histomorphometrically analyzed. The bone-to-implant contact (BIC) and the bone area fraction occupancy (BAFO) were calculated using Image J analysis. RESULTS: The statistically analyzed values which were obtained by paired t-test, revealed that the average mean values were higher in the test implants (coated) than the control implants (uncoated). The BIC values of the test implants were not significantly different from the control implants in the case of both femur and tibia (p >0.05). The test implants showed significantly increased BAFO values in femur (p <0.05). However, the BAFO values of test implants in tibia did not vary significantly from the control implants. CONCLUSION: Based on the findings of the study, the authors conclude that the coating of C. quadrangularis hydrogel enhances the osseointegration of titanium implants to bone. The further studies need to be designed to check the osseointegrative potential of C. quadrangularis. CLINICAL SIGNIFICANCE: The findings of this study suggest that the C. quadrangularis hydrogel is a potent osteogenic material that can reduce the osseointegration period and thus enhance the patient compliance toward implant treatment.

Molecular docking, SAR analysis and biophysical approaches in the study of the antibacterial activity of ceramides isolated from Cissus incisa.

The developing of antibacterial resistance is becoming in crisis. In this sense, natural products play a fundamental role in the discovery of antibacterial agents with diverse mechanisms of action. Phytochemical investigation of Cissus incisa leaves led to isolation and characterization of the ceramides mixture (1): (8E)-2-(tritriacont-9-enoyl amino)-1,3,4-octadecanetriol-8-ene (1-I); (8E)-2-(2',3'-dihydroxyoctacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-II); (8E)-2-(2'-hydroxyheptacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-III); and (8E)-2-(-2'-hydroxynonacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-IV). Until now, this is the first report of the ceramides (1-I), (1-II), and (1-IV). The structures were elucidated using NMR and mass spectrometry analyses. Antibacterial activity of ceramides (1) and acetylated derivates (2) was evaluated against nine multidrug-resistant bacteria by Microdilution method. (1) showed the best results against Gram-negatives, mainly against carbapenems-resistant Acinetobacter baumannii with MIC = 50 µg/mL. Structure-activity analysis and molecular docking revealed interactions between plant ceramides with membrane proteins, and enzymes associated with biological membranes of Gram-negative bacteria, through hydrogen bonding of functional groups. Vesicular contents release assay showed the capacity of (1) to disturb membrane permeability detected by an increase of fluorescence probe over time. The membrane disruption is not caused for ceramides lytic action on cell membranes, according in vitro hemolyticactivity results. Combining SAR analysis, bioinformatics and biophysical techniques, and also experimental tests, it was possible to explain the antibacterial action of these natural ceramides.

Bioassay-Guided Identification of the Antiproliferative Compounds of Cissus trifoliata and the Transcriptomic Effect of Resveratrol in Prostate Cancer Pc3 Cells.

The bioassay-guided fractionation of a CHCl3-MeOH extract from the stems of Cissus trifoliata identified an active fraction against PC3 prostate cancer cells. The treatment for 24 h showed an 80% reduction in cell viability (p ≤ 0.05) by a WST-1 assay at a concentration of 100 µg/mL. The HPLC-QTOF-MS analysis of the fraction showed the presence of coumaric and isoferulic acids, apigenin, kaempferol, chrysoeriol, naringenin, ursolic and betulinic acids, hexadecadienoic and octadecadienoic fatty acids, and the stilbene resveratrol. The exposure of PC3 cells to resveratrol (IC25 = 23 µg/mL) for 24 h induced significant changes in 847 genes (Z-score ≥ ±2). The functional classification tool of the DAVID v6.8 platform indicates that the underlying molecular mechanisms against the proliferation of PC3 cells were associated (p ≤ 0.05) with the process of differentiation and metabolism. These findings provide experimental evidence suggesting the potential of C. trifoliata as a promising natural source of anticancer compounds.

Cissus subtetragona Planch. Ameliorates Inflammatory Responses in LPS-induced Macrophages, HCl/EtOH-induced Gastritis, and LPS-induced Lung Injury via Attenuation of Src and TAK1.

Several Cissus species have been used and reported to possess medicinal benefits. However, the anti-inflammatory mechanisms of Cissus subtetragona have not been described. In this study, we examined the potential anti-inflammatory effects of C. subtetragona ethanol extract (Cs-EE) in vitro and in vivo, and investigated its molecular mechanism as well as its flavonoid content. Lipopolysaccharide (LPS)-induced macrophage-like RAW264.7 cells and primary macrophages as well as LPS-induced acute lung injury (ALI) and HCl/EtOH-induced acute gastritis mouse models were utilized. Luciferase assays, immunoblotting analyses, overexpression strategies, and cellular thermal shift assay (CETSA) were performed to identify the molecular mechanisms and targets of Cs-EE. Cs-EE concentration-dependently reduced the secretion of NO and PGE2, inhibited the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells, and decreased NF-κB- and AP-1-luciferase activity. Subsequently, we determined that Cs-EE decreased the phosphorylation events of NF-κB and AP-1 pathways. Cs-EE treatment also significantly ameliorated the inflammatory symptoms of HCl/EtOH-induced acute gastritis and LPS-induced ALI mouse models. Overexpression of HA-Src and HA-TAK1 along with CETSA experiments validated that inhibited inflammatory responses are the outcome of attenuation of Src and TAK1 activation. Taken together, these findings suggest that Cs-EE could be utilized as an anti-inflammatory remedy especially targeting against gastritis and acute lung injury by attenuating the activities of Src and TAK1.

Evaluation of osteogenic potential of Cissus quadrangularis on mandibular alveolar ridge distraction.

BACKGROUND: This randomized clinical trial was designed to evaluate osteogenic potential of Cissus quadrangularis in alveolar distraction to facilitate implant installation. MATERIAL AND METHODS: Twenty patients with atrophic ridge were treated by alveolar distraction. After completing distractor activation, patients were randomly divided into two equal groups according to administered drug (placebo and Cissus quadrangularis group). After a consolidation period, distractors were removed and implants were inserted. Clinical evaluation was done to assess wound healing, and distractor and implant stability. Histological evaluation was performed at time of implant insertion. Radiographic evaluation was performed to assess bone volume and density after distraction, as well as, density and bone loss around implant. RESULTS: Radiographic and histological results showed that bone formation and maturation of study group were faster than that of control group. There was a significant increased bone density in distracted area and around implant in study group than control group. A significant bone loss at end of consolidation period, and around implant at end of the study was reported in control group than study group. CONCLUSION: Cissus quadrangularis administration during the consolidation period is associated with increased osteogenic potential of distracted bone. The histological and radiographic findings of current study proved that Cissus quadrangularis not only enhances rate of new bone formation, but also bone density to withstand the biomechanical requirements of implant placement in a shorter time. Trial registration This study was retrospectively registered on www.ClinicalTrial.gov : NCT04669795-17\12\2020.

Bioactivity-Guided Isolation and Identification of Anti-adipogenic Constituents from the n-Butanol Fraction of Cissus quadrangularis.

Obesity is marked by the buildup of fat in adipose tissue that increases body weight and the risk of many associated health problems, including diabetes and cardiovascular disease. Treatment options for obesity are limited, and available medications have many side effects. Thus there is a great need to find alternative medicines for treating obesity. This study explores the anti-adipogenic potential of the n-butanol fraction of Cissus quadrangularis (CQ-B) on 3T3-L1 mouse preadipocyte cell line. The expression of various lipogenic marker genes such as adiponectin, peroxisome proliferator-activated receptor gamma, leptin, fatty acid-binding proteins, sterol regulatory element-binding proteins, fetal alcohol syndrome, steroyl-CoA desaturase-1, lipoproteins, acetyl-CoA carboxylase alpha, and acetyl-CoA carboxylase beta were variously significantly downregulated. After establishing the anti-adipogenic potential of CQ-B, it was fractionated to isolate anti-adipogenic compounds. We observed significant reduction in neutral lipid content of differentiated cells treated with various fractions of CQ-B. Gas chromatography-mass spectrometry analysis revealed the presence of thirteen compounds with reported anti-adipogenic activities. Further studies to purify these compounds can offer efficacious and viable treatment options for obesity and related complications.

Glycosylated Phenols and an Unprecedented Diacid from the Saudi Plant Cissus rotundifolia.

Bioassay-guided investigation of the Saudi medicinal and edible plant Cissus rotundifolia yielded seven metabolites, including the new sucrose diester cissuxinoside (1) and the unprecedented cissoic acid (2), belonging to unusual classes of secondary metabolites. Their chemical structures were elucidated through a combination of HR-MS and NMR data. The absolute configuration of cissoic acid was assigned by comparison of experimental and TDDFT-calculated electronic circular dichroism spectra. In addition, three rare C-glycosyl flavones (3-5) were fully characterized, and for 3 and 4 NMR data are reported here for the first time. This study identified 1-O-(4-coumaroyl)-ß-d-glucopyranose (7) as the main compound responsible for the glucose uptake stimulation effect exerted by the extract.

Metabolic Profile and Evaluation of Biological Activities of Extracts from the Stems of Cissus trifoliata.

Cissus trifoliata (L.) L belongs to the Vitaceae family and is an important medicinal plant used in Mexico for the management of infectious diseases and tumors. The present study aimed to evaluate the metabolic profile of the stems of C. trifoliata and to correlate the results with their antibacterial and cytotoxic activities. The hexane extract was analyzed using gas chromatography coupled with mass spectrometry (GC-MS) and the CHCl3-MeOH and aqueous extracts by ultraperformance liquid chromatography quadrupole time of fly mass spectrometry (UPLC-QTOF-MS). The antibacterial activity was determined by broth microdilution and the cytotoxicity was evaluated using MTS cell proliferation assay. Forty-six metabolites were putatively identified from the three extracts. Overall, terpenes, flavonoids and stilbenes characterize the metabolic profile. No antibacterial activity was found in any extract against the fifteen bacteria strains tested (MIC >500 µg/mL). However, high cytotoxic activity (IC50 ≤ 30 µg/mL) was found in the hexane and aqueous extracts against hepatocarcinoma and breast cancer cells (Hep3B, HepG2 and MCF7). This is the first report of the bioactive compounds of C. trifoliata stems and their antibacterial and cytotoxic properties. The metabolic profile rich in anticancer compounds correlate with the cytotoxic activity of the extracts from the stems of C. trifoliata. This study shows the antitumor effects of this plant used in the traditional medicine and justifies further research of its anticancer activity.

Osteogenic potential of hexane and dichloromethane fraction of Cissus quadrangularis on murine preosteoblast cell line MC3T3-E1 (subclone 4).

In continuation of the investigation of osteogenic potential of solvent fractions of ethanolic extract of Cissus quadrangularis (CQ), an ancient medicinal plant, most notably known for its bone-healing properties, to isolate and identify antiosteoporotic compounds. In the current study, we report the effect of hexane fraction (CQ-H) and dichloromethane fraction (CQ-D) of CQ on the differentiation and mineralization of mouse preosteoblast cell line MC3T3-E1 (subclone 4). Growth, viability, and proliferation assays revealed that low concentrations (0.1, 1, and 100 ng/ml) of both solvent fractions were nontoxic, whereas higher concentrations were toxic to the cells. Differentiation and mineralization of MC3T3-E1 with nontoxic concentrations of CQ-D and CQ-H revealed that CQ-D delayed the mineralization of MC3T3-E1 cells. However, early and enhanced mineralization was observed in cultures treated with nontoxic concentrations of CQ-H, as indicated by Von Kossa staining and expression profile of osteoblast marker genes such as osterix, Runx2, alkaline phosphatase (ALP), collagen (Col1a1), integrin-related bone sialoprotein (IBSP), osteopontin (OPN), and osteocalcin (OCN). These findings suggest CQ-H as the most efficacious solvent fraction for further investigation to isolate and identify the active compounds in CQ-H.

Ethyl acetate and n-butanol fraction of Cissus quadrangularis promotes the mineralization potential of murine pre-osteoblast cell line MC3T3-E1 (sub-clone 4).

In a sequel to investigate osteogenic potential of ethanolic extract of Cissus quadrangularis (CQ), the present study reports the osteoblast differentiation and mineralization potential of ethyl acetate (CQ-EA) and butanol (CQ-B) extracts of CQ on mouse pre-osteoblast cell line MC3T3-E1 (sub-clone 4) with an objective to isolate an antiosteoporotic compound. Growth curve, proliferation, and viability assays showed that both the extracts were nontoxic to the cells even at high concentration (100 µg/ml). The cell proliferation was enhanced at low concentrations (0.1 µg/ml and 1 µg/ml) of both the extracts. They also upregulated the osteoblast differentiation and mineralization processes in MC3T3-E1 cells as reflected by expression profile of osteoblast marker genes such as RUNX2, Osterix, Collagen (COL1A1), Alkaline Phosphatase (ALP), Integrin-related Bone Sialoprotein (IBSP), Osteopontin (OPN), and Osteocalcin (OCN). CQ-EA treatment resulted in early differentiation and mineralization as compared with the CQ-B treatment. These findings suggest that low concentrations of CQ-EA and CQ-B have proliferative and osteogenic properties. CQ-EA, however, is more potent osteogenic than CQ-B.

Modulation of bone turnover by Cissus quadrangularis after ovariectomy in rats.

In women, age-related bone loss is associated with increased risk of bone fracture. Existing therapies are associated with severe side effects; thus, there is a need to find alternative medicines with less or optimal side effects. Cissus quadrangularis (CQ), an Ayurvedic medicine used to enhance fracture healing, was tested for its bone protective properties and studied to discern the mechanism by which it is beneficial to bone. Female Sprague Dawley rats were either sham operated or ovariectomized and were fed CQ for 3 months. Several biochemical markers, cytokines and hormones were assayed. Femur, tibia and lumbar vertebrae were subjected to pQCT and µCT densitometry. MC3T3 cells were cultured, treated with CQ and used to analyze miRNA content and subjected to qPCR for gene expression analysis related to bone metabolism. CQO rats showed protected bone mass and microarchitecture of trabecular bone in the distal femoral metaphysis and the proximal tibial metaphysis. The lumbar vertebrae, however, showed no significant changes. Serum protein expression levels of P1NP increased and Trap5b and CTX levels decreased with in vivo CQ treatment. Some influence on the anti- and pro-inflammatory markers was also observed. Significantly high level of estradiol in the CQO rats was observed. In vitro expression of a few genes related to bone metabolism showed that osteocalcin increased significantly. The other genes-collagen I expression, SPP1, BMP2, DCAT1-decreased significantly. Certain miRNA that regulate bone turnover using the BMP pathway and Wnt signaling pathways were upregulated by CQ. qPCR after acute treatment with CQ showed significantly increased levels of osteocalcin and decreased levels of Wnt/ß catenin antagonist DCAT1. Overall, CQ protected the microarchitecture of the long bones from ovariectomy-induced bone loss. This may be because of decreased inflammation and modulation through the BMP and Wnt signaling pathways. We conclude that CQ is a potential therapeutic agent to treat postmenopausal osteoporosis with no side effects.

Central nervous system depressant effects of fractions of methanol leaf extract of Cissus cornifolia (Baker) Planch.

Cissus cornifolia is an annual herb used in the treatment of mental derangement in the African Traditional Medicine. As part of a continuous research on this medicinal plant to scientifically validate its use in mental derangement, the fractions of the leaf extract were investigated for central nervous system (CNS) depressant effects. Successive fractionation of the methanol leaf extract of C. cornifolia was utilized to obtain the chloroform fraction (CLF), ethyl acetate fraction (EAF) and the residual aqueous fractions (RAF). These fractions were investigated for CNS-depressant effects in mice using diazepam-induced sleep, head-dip and motor-coordination tests. CLF, EAF and RAF significantly (p<0.01) prolonged the duration of sleep in mice. EAF significantly (p<0.05) reduced the mean head-dips in mice at 75 and 150 mg/kg. Similarly, a significant decrease in the mean head-dips (p<0.05, p<0.01 and p<0.005) was produced by RAF at 150, 300 and 600 mg/kg respectively. In the beam-walking assay test, all the fractions did not produce motor coordination deficit in mice. The data obtained revealed the fractions of methanol leaf extract of Cissus cornifolia possess remarkable central nervous system depressant effects.