scholarly journals The Role of Semipurified Fractions Isolated from Quercus infectoria on Bone Metabolism by Using hFOB 1.19 Human Fetal Osteoblast Cell Model

2018 ◽  
Vol 2018 ◽  
pp. 1-13 ◽  
Author(s):  
Amira Raudhah Abdullah ◽  
Hermizi Hapidin ◽  
Hasmah Abdullah
Keyword(s):  
Gene Expression ◽  
Bone Formation ◽  
Column Chromatography ◽  
Cell Model ◽  
Osteoblast Cell ◽  
Isolation And Purification ◽  
Traditional Medicines ◽  
Phytochemical Compounds ◽  
Layer Chromatography ◽  
Quercus Infectoria

Background. Quercus infectoria (QI) is a plant used in traditional medicines in Asia. The plant was reported to contain various active phytochemical compounds that have potential to stimulate bone formation. However, the precise mechanism of the stimulation effect of QI on osteoblast has not been elucidated. The present study was carried out to isolate QI semipurified fractions from aqueous QI extract and to delineate the molecular mechanism of QI semipurified fraction that enhanced bone formation by using hFOB1.19 human fetal osteoblast cell model. Methods. Isolation of QI semipurified fractions was established by means of column chromatography and thin layer chromatography. Established QI semipurified fractions were identified using Liquid Chromatography-Mass Spectrometry (LC-MS). Cells were treated with derived QI semipurified fractions and investigated for mineralization deposition and protein expression level of BMP-2, Runx2, and OPN by ELISA followed gene expression analysis of BMP-2 and Runx2 by RT-PCR. Results. Column chromatography isolation and purification yield Fractions A, B, and C. LC-MS analysis reveals the presence of polyphenols in each fraction. Results show that QI semipurified fractions increased the activity and upregulated the gene expression of BMP-2 and Runx2 at day 1, day 3, and day 7. OPN activity increased in cells treated with QI semipurified fractions at day 1 and day 3. Meanwhile, at day 7, expression of OPN decreased in activity. Furthermore, the study showed that combination of Fractions A, B, and C with osteoporotic drug (pamidronate) further increased the activity and upregulated the gene expression of BMP-2 and Runx2. Conclusions. These findings demonstrated that polyphenols from semipurified fractions of QI enhanced bone formation through expression of the investigated bone-related marker that is its potential role when combined with readily available osteoporotic drug.

scholarly journals ID: 1008 Quercus infectoria semi-purified fractions promoted BMP-2, Runx2 and osteopontin expression in human fetal osteoblastic cell line

2017 ◽  
Vol 4 (S) ◽  
pp. 85
Author(s):  
Amira Raudhah Abdullah ◽  
Hermizi Hapidin ◽  
Hasmah Abdullah
Keyword(s):  
Bone Formation ◽  
Protein Expression ◽  
Cell Line ◽  
Osteoblastic Cell ◽  
Osteoblastic Cell Line ◽  
Traditional Medicines ◽  
In Series ◽  
Phytochemical Compounds ◽  
Layer Chromatography ◽  
Quercus Infectoria

Quercus infectoria (QI) is traditional medicines in Asia which contain many active phytochemical compounds that have the potential to stimulate bone formation. However, the precise mechanism of the therapeutic effect of QI on osteoblast has not been elucidated. The present study was carried out to extract the active compound of QI through a series of purification in order to obtain semi-purified fractions of QI followed by examining its phytochemical profile through liquid chromatography mass spectrometry (LC-MS) as well as to delineate the molecular mechanism of QI semi-purified fraction-enhanced bone formation by investigating the protein expression of bone morphogenic protein-2 (BMP-2), Runx2 and osteopontin (OPN) in the hFOB 1.19 human fetal osteoblastic cell line. The semi-purified fractions of QI were produced in series of column chromatography and thin layer chromatography (TLC) technique. The obtained fractions were then tested in a series of bio-guided assay through MTT assay. Three most potent fractions with lowest EC50 value (Fraction A-10.85 µg/ml, Fraction B-12.00 µg/ml and Fraction C-11.60 µg/ml) were selected to be treated with hFOB1.19 cell followed by quantification of BMP-2, Runx2 and OPN protein expression at day 1, 3 and 7. Compound profiling through LC-MS analysis showed that the chosen fractions consist of main active component known as gallic acid that has been showed previously to promote bone formation. Results show that QI semi-purified fractions increased the activity of BMP-2, Runx2 and OPN at day 1 and day 3. Meanwhile as the expression of BMP-2 and Runx2 continue to increase at day 7, the expression of OPN shows a down regulation at day 7. Furthermore, the study showed that combination of Fraction A, B and C wit osteoporotic drug (pamidronate) further increase the expression of BMP-2 and Runx2 contrary to OPN expression. These finding demonstrated that semi-purified fraction of QI enhanced bone formation through expression of investigated bone-related marker.

scholarly journals Bone Formation Ability and Cell Viability Enhancement of MC3T3-E1 Cells by Ferrostatin-1 a Ferroptosis Inhibitor of Cancer Cells

2021 ◽  
Vol 22 (22) ◽  
pp. 12259
Author(s):  
Alireza Valanezhad ◽  
Tetsurou Odatsu ◽  
Shigeaki Abe ◽  
Ikuya Watanabe
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Bone Formation ◽  
Cell Viability ◽  
Osteoblast Cell ◽  
Osteoblast Cells ◽  
Alizarin Red ◽  
Activity Test ◽  
Osteoblast Cell Line ◽  
Regulated Necrosis

Recently, ferroptosis has gained scientists’ attention as an iron-related regulated necrosis. However, not many reports have investigated the effect of ferroptosis on bone. Therefore, with the present study, we assessed the effect of ferroptosis inhibition using ferrostatin-1 on the MC3T3-E1 pre-osteoblast cell. Cell images, cell viability, alkaline phosphatase activity test, alizarin red staining, and RUNX2 gene expression using real-time PCR were applied to investigate the effects of ferrostatin and erastin on MC3T3-E1 osteoblast cells. Erastin was used as a well-known ferroptosis inducer reagent. Erastin with different concentrations ranging from 0 to 50 µmol/L was used for inducing cell death. The 25 µmol/L erastin led to controllable partial cell death on osteoblast cells. Ferrostatin-1 with 0 to 40 µmol/L was used for cell doping and cell death inhibition effect. Ferrostatin-1 also displayed a recovery effect on the samples, which had already received the partially artificial cell death by erastin. Cell differentiation, alizarin red staining, and RUNX2 gene expression confirmed the promotion of the bone formation ability effect of ferrostatin-1 on osteoblast cells. The objective of this study was to assess ferrostatin-1’s effect on the MC3T3-E1 osteoblast cell line based on its ferroptosis inhibitory property.

scholarly journals Gene expression signatures of a fibroblastoid preosteoblast and cuboidal osteoblast cell model compared to the MLO-Y4 osteocyte cell model

Bone ◽  
2009 ◽  
Vol 44 (1) ◽  
pp. 32-45 ◽  
Author(s):  
Wuchen Yang ◽  
Marie A. Harris ◽  
Jelica Gluhak Heinrich ◽  
Dayong Guo ◽  
Lynda F. Bonewald ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Model ◽  
Osteoblast Cell ◽  
Gene Expression Signatures

Isolation of Sinapic Acid from Habenaria intermedia D. Don: A New Chemical Marker for the Identification of Adulteration and Substitution

2020 ◽  
Vol 6 (4) ◽  
pp. 380-387
Author(s):  
Jaswinder Kaur Virk ◽  
Vikas Gupta ◽  
Mukesh Maithani ◽  
Ravindra K. Rawal ◽  
Sanjiv Kumar ◽  
...  
Keyword(s):  
Column Chromatography ◽  
Sinapic Acid ◽  
Phytochemical Screening ◽  
Chemical Marker ◽  
Reference Compound ◽  
Crystalline Powder ◽  
Chemical Test ◽  
Herbal Plants ◽  
Single Compound ◽  
Layer Chromatography

Background: Vriddhi is one of the Rasayana herbs in Ayurveda broadly used in vitality, strengthening Ayurvedic formulations. To fulfill steeply increased demand and declined supply, tubers have been collected in destructive manner resulting in reduced plant population and pushing the plant in Red list of IUCN endangered species. However, manufacturers are using substitutes and other substandard drugs leading to adulteration which puts the importance of therapeutically rich herbal plants at stake. Lack of chemical markers is the main inability of regulatory authorities for not taking any action against this adulteration. Objective: Isolation of chemical marker of plant that can be used as a reference compound for identification of unauthorized substitution. Methods: Preliminary phytochemical screening of methanolic and toluene extract of H. intermedia D. Don was done using standard methods followed by column chromatography for the isolation of phytoconstituents. A total of 3004 fractions were collected with Thin Layer Chromatography (TLC) profiling and different fractions were pooled. A single compound was isolated and confirmed by chemical test, melting point, spectral analysis and compared with the literature. Results: Phytochemical screening of extracts shows the presence of alkaloids, carbohydrates, steroids, terpenoids, flavonoids, tannins and phenolics. A pure white crystalline powder was isolated by column chromatography which was characterized as 3,5-dimethoxy-4- hydroxycinnamic acid (Sinapic acid) with the help of IR and Mass spectroscopy. Conclusion: This is the first report of Sinapic acid as a novel compound from Vriddhi, Habenaria genus and Orchidaceae family. It can be used as a marker for the identification of unauthorized substitution and adulteration claiming the use of Vriddhi.

scholarly journals Isolation and Purification of Fucoxanthin from Brown Seaweed Sargassum horneri Using Open ODS Column Chromatography and Ethanol Precipitation

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3777
Author(s):  
Yuemei Ye ◽  
Jingwen Sun ◽  
Liting Wang ◽  
Junwang Zhu ◽  
Wei Cui ◽  
...  
Keyword(s):  
Column Chromatography ◽  
Recovery Rate ◽  
Biological Activities ◽  
Brown Seaweed ◽  
Sargassum Horneri ◽  
Scale Production ◽  
Water Mixture ◽  
Isolation And Purification ◽  
Ethanol Precipitation ◽  
Water Solvent

As an abundant marine xanthophyll, fucoxanthin (FX) exhibits a broad range of biological activities. The preparation of high-purity FX is in great demand, however, most of the available methods require organic solvents which cannot meet the green chemistry standard. In the present study, a simple and efficient purification approach for the purification of FX from the brown seaweed Sargassum horneri was carried out. The FX-rich ethanol extract was isolated by octadecylsilyl (ODS) column chromatography using ethanol–water solvent as a gradient eluent. The overwhelming majority of FX was successfully eluted by the ethanol–water mixture (9:1, v/v), with a recovery rate of 95.36%. A parametric study was performed to optimize the aqueous ethanol precipitation process by investigating the effects on the purity and recovery of FX. Under the optimal conditions, the purity of FX was 91.07%, and the recovery rate was 74.98%. Collectively, the eco-friendly method was cost-efficient for the purification of FX. The developed method provides a potential approach for the large-scale production of fucoxanthin from the brown seaweed Sargassum horneri.

Gene Expression Analysis of Ectopic Bone Formation Induced by Electroporatic Gene Transfer of BMP4

2006 ◽  
Vol 111 (2) ◽  
pp. 231-242 ◽  
Author(s):  
Satoshi Kotajima ◽  
Koshi N. Kishimoto ◽  
Munenori Watanuki ◽  
Masahito Hatori ◽  
Shoichi Kokubun
Keyword(s):  
Gene Expression ◽  
Gene Transfer ◽  
Bone Formation ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Ectopic Bone Formation ◽  
Ectopic Bone

Isolation of the antidiarrhoeal tiliroside and its derivative from Waltheria indica leaf extract

2021 ◽  
Vol 25 (1) ◽  
pp. 86-92
Author(s):  
B.A. Ayinde ◽  
J.O. Owolabi ◽  
I.S. Uti ◽  
P.C. Ogbeta ◽  
M.I. Choudhary
Keyword(s):  
Silica Gel ◽  
Column Chromatography ◽  
Methanol Extract ◽  
Preparative Thin Layer Chromatography ◽  
Inhibitory Effects ◽  
Aqueous Fraction ◽  
Vacuum Liquid Chromatography ◽  
Ileum Contraction ◽  
Layer Chromatography ◽  
Dose Dependent

The antidiarrhoeal effect of Waltheria indica methanol extract and fractions have been reported earlier but, the present work examined the intestinal relaxant effects of two flavonoid-phenyl propanoids isolated from the methanol extract. The active aqueous fraction was subjected to vacuum liquid chromatography using dichloromethane with increasing concentration of ethyl acetate, and that of methanol and water successively. The ten (10) fractions obtained were combined to give seven (7). The fraction 2 (C, D) was subjected to preparative thin layer chromatography on silica gel GF254 (10-40μm) using CHCl3-CH3OH (8:2) to obtain compound coded F2. Fraction 4 (F) was subjected to column chromatography using silica gel (60-120μm mesh) and eluted with  dichloromethane with increasing concentrations of methanol. Fractions 9-28 were combined and subjected to column  chromatography using chloroform with increasing concentration of methanol. The fractions 1-16 of these were purified on Sephadex LH-20 to obtain compound BAA. The identities of the two compounds were established using spectroscopic methods. The  antidiarrheal effect of compound F2 was evaluated on mice using charcoal transit (100,200, 400mg/kg), castor oil (40, 60 mg/kg)  while the two compounds were examined for their inhibitory effects on Ach-induced ileum contraction. The effects of the  compounds were compared with loperamide (3mg/kg) and atropine (80μg). Compounds F2 and BAA were identified as tiliroside and 3’’’, 5’’’-dimethoxy tiliroside respectively. Tiliroside inhibited the charcoal transition in the animals in a dose dependent pattern with 400mg/ mL eliciting 63.41% inhibition compared to 59.23% produced by loperamide. The compound also elicited significantly (P<0.05) prolonged onset of stooling and reduced the number and weight of stools produced lower than the control. The two  compounds drastically inhibited the Ach-induced contractions of the ileum. The compound, tiliroside at 10mg, completely abolished  the contraction by Ach unlike 3’’’, 5’’’-dimethoxy tiliroside which reduced the contraction to 1.92% at 20mg. The identified compounds seem to be responsible for the ethnomedicinal use of the plant in treating diarrhea.

scholarly journals c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in αT3-1 Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 839-849 ◽  
Author(s):  
Buffy S. Ellsworth ◽  
Brett R. White ◽  
Ann T. Burns ◽  
Brian D. Cherrington ◽  
Annette M. Otis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Cell Model ◽  
Receptor Gene ◽  
Critical Role ◽  
Dominant Negative ◽  
Activator Protein 1 ◽  
Activator Protein

Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homologous regulation of the GnRHR is an important mechanism underlying gonadotrope sensitivity to GnRH. Previously, we have found that GnRH induction of GnRHR gene expression in αT3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH3). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in αT3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components.

scholarly journals Prolyl Hydroxylase Domain-Containing Protein 3 Gene Expression in Chondrocytes Is Not Essential for Bone Development in Mice

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2200
Author(s):  
Weirong Xing ◽  
Sheila Pourteymoor ◽  
Gustavo A. Gomez ◽  
Yian Chen ◽  
Subburaman Mohan
Keyword(s):  
Gene Expression ◽  
Bone Formation ◽  
Bone Mass ◽  
Trabecular Bone ◽  
Bone Volume ◽  
Endochondral Bone Formation ◽  
Chondrocyte Differentiation ◽  
Trabecular Bone Volume ◽  
Endochondral Bone ◽  
Trabecular Bone Mass

We previously showed that conditional disruption of the Phd2 gene in chondrocytes led to a massive increase in long bone trabecular bone mass. Loss of Phd2 gene expression or inhibition of PHD2 activity by a specific inhibitor resulted in a several-fold compensatory increase in Phd3 expression in chondrocytes. To determine if expression of PHD3 plays a role in endochondral bone formation, we conditionally disrupted the Phd3 gene in chondrocytes by crossing Phd3 floxed (Phd3flox/flox) mice with Col2α1-Cre mice. Loss of Phd3 expression in the chondrocytes of Cre+; Phd3flox/flox conditional knockout (cKO) mice was confirmed by real time PCR. At 16 weeks of age, neither body weight nor body length was significantly different in the Phd3 cKO mice compared to Cre−; Phd3flox/flox wild-type (WT) mice. Areal BMD measurements of total body as well as femur, tibia, and lumbar skeletal sites were not significantly different between the cKO and WT mice at 16 weeks of age. Micro-CT measurements revealed significant gender differences in the trabecular bone volume adjusted for tissue volume at the secondary spongiosa of the femur and the tibia for both genotypes, but no genotype difference was found for any of the trabecular bone measurements of either the femur or the tibia. Trabecular bone volume of distal femur epiphysis was not different between cKO and WT mice. Histology analyses revealed Phd3 cKO mice exhibited a comparable chondrocyte differentiation and proliferation, as evidenced by no changes in cartilage thickness and area in the cKO mice as compared to WT littermates. Consistent with the in vivo data, lentiviral shRNA-mediated knockdown of Phd3 expression in chondrocytes did not affect the expression of markers of chondrocyte differentiation (Col2, Col10, Acan, Sox9). Our study found that Phd2 but not Phd3 expressed in chondrocytes regulates endochondral bone formation, and the compensatory increase in Phd3 expression in the chondrocytes of Phd2 cKO mice is not the cause for increased trabecular bone mass in Phd2 cKO mice.

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