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EGb 761 promotes osteoblastogenesis, lowers bone marrow adipogenesis and atherosclerotic plaque formation.

ARTICLE INFO

Keywords: Ginkgo extract Flavonoids Osteoblastogenesis Adipogenesis Atherosclerosis Micro-CT

ABSTRACT

Aim of the study: Our earlier study has demonstrated that EGb 761 (standardized extract of Ginkgo) has the bone sparing effect on the estrogen deficiency induced bone loss model. In the present study, we have addressed the question whether treatment of osteoporosis benefits arterial calcification or vice versa, because both adipocyte and osteoblast originate from the same mesenchymal cell of the bone marrow cell (BMC) population.

Materials and methods: Bone marrow cells were isolated to study the effect of EGb 761 on osteoblast and adipocytes. For in vivo effect hamsters were fed high fat diet and the effect of EGb 761 studied on atherosclerotic plaque formation and endothelial function.

Results: BMC's undergoing induced osteogenic or adipogenic differentiations in the presence of EGb 761 show increase and decrease in mineralization and adipogenesis respectively. Osteogenic and adipogenic mRNAs, reveal lineage dependent expression patterns. Runx-2 (osteoblast transcription factor) showed a progressive increase, whereas PPAR--y (adipogenic regulator) was attenuated, with same pattern of expression being for late osteogenic and adipogenic genes. EGb 761 led to increase in apoptotic cells and ROS, an important upstream signal. In vivo experiments in hamsters after induction with high cholesterol diet (HCD) show improvement in endothelial function by EGb 761 with lowering in total plasma cholesterol levels. EGb 761 led to vascular preservation of the aortic lumen with impairment of the endothelium dependent relaxation which was corroborated by micro-CT and histological sections of the thoracic region of the aorta.

Conclusion: From this data, it can be implied that EGb 761 controls bone loss, adiposity and lowers atherogenic risk factor after HCD induction.

[c] 2012 Elsevier GmbH. All rights reserved.

Introduction

Osteoporosis and vascular calcification were considered to be independent processes that occur with aging (Anagnostis et al. 2009; Eastell et al. 2010). However, biological observations and epidemiological evidence demonstrate co-existence of these two diseases. Studies establish that vertebral fractures commonly coexist with aortic calcification (Naves et al. 2008; Szulc et al. 2008). This coexistence is built from data acquired from various epidemiological studies but the most convincing evidences relates to its underlying patho-physiology. The first association relates to the mechanism of mineralization of the arterial wall, which ascertained that there are many parallels between the mineralization of the bone tissue and the calcification of the blood vessel wall (Greenhill et al. 1985). The second line of evidence was demonstrated through gene deletion of certain proteins in the mouse giving rise to both vascular calcification and osteoporosis (Luo et al. 1997). The third link relates to clinical trials of drugs common for both osteoporosis and coronary artery diseases including lasofoxifene and beta blockers (Anagnostis et al. 2009; Cummings et al. 2010). These drugs developed for osteoporosis not only reduce incidence of vertebral and non-vertebral fractures but also decrease risk of stroke and coronary artery disease. Most of the studies conducted Cross-sectionally have shown an association between vascular damage, vascular calcification and osteoporosis. However, very few studies have examined the progression of vascular calcification with associated bone loss (Naves etal. 2008). Our study with EGb 761 (a standardized, concentrated extract of ginkgo leaves containing 24% flavonoids, 5-7% terpene lactones, 5-10% organic acids and other constituents) demonstrated that oral administration of EGb 761 restores bone mass in aged ovariectomized rats (Adis International 2003) (Trivedi et al. 2009). As the coexistence of bone loss and artery diseases have been demonstrated, we aimed to test a unifying hypothesis addressing the question, whether EGb 761 that is beneficial for the treatment of postmenopausal bone loss also modulates arterial calcification. In this study, we have examined the role of EGb 761 on the inverse relationship between bone marrow derived adipocytes and osteoblasts (Atmani et al, 2003). The study was further extended to test the effect of EGb 761 on atherosclerosis in a hamster model which shows susceptibility to atherosclerosis after introduction of high cholesterol diet (HCD). We have examined whether EGb 761 impairs endothelium-dependent relaxation in a dietinduced experimental model as endothelial dysfunction is one of the earliest manifestations of atherosclerosis and improvement in endothelium the earliest clinical marker after atherogenic risk factor modification (Haynes 2002). The study clearly suggests that EGb 761 in addition to controlling bone loss reduces adiposity and lowers atherogenic risk factor.

Materials and methods

Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA) and Sigma Aldrich (USA). All fine chemicals were purchased from Sigma Aldrich (St. Louis, MO). EGb 761 (trade name Biloban), was a generous gift from CIPAN (Companhia Industrial Produtora de Antibioticos SA) Lisbon, Portugal.

Mineralization of bone marrow cells

Bone marrow cells from 4 to 6 weeks old female balb/c mice were isolated and cultures prepared according to a previously published protocol (Maniatopoulos et al. 1988; Trivedi et al. 2009). Briefly, the femora were excised aseptically, cleaned of soft tissues, and washed 3 times, 15 min each, in a culture medium. The epiphyses of femora were cut off and the marrow flushed out in 20 ml of [alpha]-MEM (minimum essential medium). Released bone marrow cells were collected and plated (2 x [10.sup.6] cells/well) in 12-well plate in the culture medium, consisting of a-MEM, supplemented with 10% fetal bovine serum, 100 nM dexamethasone, 10 mM 13-glycerophosphate and 50 [micro]g/m1 ascorbic acid.

An MIT assay was done before starting experiment to check toxicity with various concentrations of EGb 761 (supplementary data Fig. S1). Cells were cultured with and without EGb 761 (concentration 0.01% and 0.001%) for 21 days at 37[degrees]c in a humidified atmosphere of 5% [CO.sub.2] and 95% air, and the medium was changed every 48 h. After 21 days, the differentiated cells were fixed in 4% formaldehyde for 20 min at room temperature and rinsed once in PBS. After fixation, the specimens were processed for staining with 40 mM Alizarin Red S, which stains areas rich in nascent calcium (Santiago-Mora et al. 2011). The stain was extracted and quantified by the ELISA plate reader at 570 nm.

Induction of adipogenic differentiation in bone marrow cells

For adipogenic differentiation, 1 x [10.sup.7] BMC's were seeded in 24-well plates and cultured in an adipogenic medium [Dul-becco's modified Eagle's medium (DMEM)] containing 1.0 [micro]M dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX), 100 [micro]M indomethacin, 10% FBS and insulin (10 [micro]g/m1), for 7 days. On day 3, medium was replaced with complete growth medium containing only insulin (10 ig/m1). Treatment was continued for 21 days and the medium changed every third day. The treatment group contained similar medium with concentration of EGb 761 being 0.01% and 0.001%. Cells were fixed after 21 days in 4% paraformaldehyde and stained with Oil Red 0. To quantify the incorporation of lipid, the area stained with Oil Red 0 was measured by taking photomicrographs (Heim et al. 2002; Santiago-Mora et al. 2011; Trivedi et al. 2008). For extraction of Oil Red 0 stain, 500 [micro]l of 70% isopropanol was added to the stained cells and left at 37[degrees]C for 20 min (Heim et al. 2002). The extracted stain was quantified by the ELISA plate reader at 490 nm.

Real time PCR

qPCR was performed for assessing the expression of adipogenic and osteoblast specific genes from bone marrow cells differentiated to adipocytes and osteoblast respectively, following our optimized protocol (Bandyopadhyay et al. 2006; Trivedi et al. 2009). The house keeping gene GAPDH was used as the internal control in this study. Primers were designed using the Universal Probe Library (Roche Applied sciences) for genes as described in Table 1. cDNA was synthesized with a revert aid cDNA synthesis kit (Fermentas, Austin, USA). SYBR green chemistry was used to perform quantitative determination of relative expression of transcripts for all genes. All genes were analyzed using the Light Cycler 480 (Roche Molecular biochemicals, Indianapolis, Indiana, USA) real time PCR machine.

Table 1

Q-PCR primers.

Gene           Primer sequence           Accession number

OCN            F-TGAGGACCATCTTCTGCTCA         NM_01032298
               R-TGGACATGAAGGCTTTGTCA

Col-1          F-CATGTTCAGCTTTGTGGACCT        NM_007742.3
               R-GCAGCTGACITCAGGGATGT

Runx-2         F-CCCGGGAACCAAGAAATC            AF053956.1
               R-CAGATAGGAGGGGTAAGACTGG

PPAR-[gamma]   F-GAAAGACAACCGACAAATCACC       NM_011146.3
               R-GGGGGTGATATGTITGAACTTG

C/EBP-[alpha]  F-AAACAACCCAACCIGGAGA           BC051102.1
               R-GCGGTCATTGTCACTGGTC

SREBP-1c       F-GCTITTGAACGACATCGAAGA        NM_011480.3
               R-CGGGAAGTCACTCTCTTGGT

GAPDH          F-AGMGTCATCAACGGGAAG            DQ403054.1
               R-TTTGATGTI-AGTGGGGICTCG


Apoptosis detection

Differentiated bone marrow adipocytes were serum starved in the presence or absence of EGb 761 at the concentration of 0.01% and 0.001% for 48h. After the cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature they were rinsed with PBS, pH 7.4, and permeabilized with 1% Triton X-100 in 0.01 M citrate buffer, pH 6.0. DNA fragmentation was detected using TUNEL detection kit (In Situ Cell Death Detection Kit, Fluorescein, Roche), which specifically labeled 3'-hydroxyl termini of DNA strand breaks using fluorescein isothiocyanate (FITC)-conjugated dUTP. The percentage of apoptotic cells was calculated as the number of TUNEL positive cells per field (Elbaz et al. 2010; Swarnkar et al. 2011).

Measurement of intracellular ROS generation

Bone marrow cells were differentiated to adipocytes as described earlier (Atmani et al. 2003; Santiago-Mora et al. 2011). After 21 days cells were treated with or without EGb 761 (0.01% and 0.001% concentration) and with lipopolysaccharide (LPS) at the concentration of 100 ng/ml for 4 h (Lee et al. 2009). The determination of ROS was based on the oxidation of the non-fluorescent 2,7-dichlorodihydroflourescein diacetate into a fluorescent dye, 2,7-dichloroflourescein, by peroxide or nitric oxide production. Control cells and cells treated with EGb 761 were analyzed for changes in fluorescence. Cells were washed twice with PBS and incubated for 30 min at 37 [degrees]C in dark with the oxidation-sensitive probe, 2,7-dichlorodihydroflourescein diacetate (10 [micro]gimp (Molecular Probes, Eugene, OR). Lysates were collected in 0.1% triton X-100 and production of ROS measured by changes in fluorescence at an excitation wavelength of 495 nm and an emission wavelength of 525 nm (Ambati et al. 2009; Woo et al. 2003; Yang et al. 2006).

Animals and diet

Golden Syrian hamster (Mesocricetus auratus), male 12-week old, 110-120g body weight were taken for the study (Dillard et al. 2010). Three groups with 5 animals in each were housed at 25-26[degrees]C, relative humidity 60-80% and 12/12h light/dark cycle (light from 8.00 a.m. to 8.00 p.m.). Experimental protocols were approved by our institutional ethical committee, which follows guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), which complies with international norms of 'NSA. 1-1FD was prepared by mixing normal pellet diet with groundnut oil, cholesterol (Sigma), deoxycholesterol (Sigma), and fructose (Sigma) in a ratio of 610 g:300 m1:5 g:5 g:100 g, respectively, to a final weight of 1.0 kg (Rizvi et al. 2003). This homogenous soft cake was molded in the shape of pellets of about 3 g each. Animals of all the groups except control group were fed with HFD for 30 days before EGb 761 treatment. The animals had free access to 1-IFD and water.

EGb 761 was constituted with the vehicle (carboxy-methyl cellulose) and given orally at a dose of 250 mg/kg body weight, once daily for 30 days. At the end of the experiment hamsters were anaesthetized, blood withdrawn by cardiac puncture and aorta perfused with cold PBS containing 5 mM EDTA. Aorta were carefully dissected out, gently blotted, and fixed in 4% paraformaldehyde for histology and micro-CT experiments.

Plasma biochemistry

Plasma total cholesterol (TC), was measured enzymatically using commercial kits from Pointe Scientific (USA) (Alexaki et al. 2004; Allain et al. 1974). In brief, fresh plasma was isolated from citrated blood and treated with kit reagents as per Manufacturer's protocol and color produced was measured by ELISA plate reader at parameter specific wave lengths (Powerware XS, Biotek, USA).

Vascular ring preparation and vascular reactivity

Transverse 4 mm wide rings of thoracic aorta were cut and mounted in 10 ml organ baths containing Krebs solution. After equilibration, the aortic rings were exposed to KC1Krebs buffer (80 mM) in order to assess the maximum tissue contractility. Cumulative concentration dependent contraction responses to Phenylephrine (PE) (10 pIVI-100 [micro]M) was assessed. The presence of a functional endothelium was assessed by measuring relaxation to ACh (3 nM to 30 mM) in PE (1 [micro]M) precontracted rings. Finally tissue contractility and viability were assessed by exposing the rings to KCI Krebs buffer (80 mM) in all the groups (Zakaria el et al. 2005; Zhang et al. 2010) Experiments were standardized using standard controls ezitimide and fenofibrate (supplementary data Fig. S3).

Cholesterol measurement

Total cholesterol was measured using Amplex red cholesterol assay kit from Molecular Probes (Invitrogen, USA) according to Manufacturer's protocol.

Histological analysis

Aorta were carefully dissected out, gently blotted, and fixed in 4% paraformaldehyde for histology, following previously published papers (Benetos et al. 1997). About 5.0 mm pieces from the middle segment of each aorta were dehydrated in ascending grades of ethanol, cleared in xylene, and embedded in paraffin wax using standard procedures. Representative transverse sections (5.0 [micro]m) were cut, stained with haematoxylin and viewed by light microscopy.

Micro-CT analysis

uCT (Skyscan 1076) was used to study calcification of the arterial wall (Higgins et al. 2005; Langheinrich et al. 2004). It enables nondestructive visualization and localization of calcium hydroxyapatite (CHA) in very thin tissue slices. Calcification nodules localized mainly in the necrotic core or luminal surface, and calcification plates localized more to the medial layer, often extending around a substantial fraction of its circumference were observed. Paraffin embedded aorta were scanned at 18 [micro]m resolution with 0.4 rotation and 3 x frame averaging. The applied voltage was 501N and current 160 kV. The scanned images were reconstructed using Nrecon software and analyzed using CTscan software provided with the Skyscan (model) microCT instrument (Sharan et al. 2010).

Statistical analysis

The statistical analysis was carried out using the GraphPad Prism Software. All results are presented as the means [+ or -] S.D. of results from three or four cultures and the significance of differences was analyzed by Student's ttest. Groups were analyzed via ttests (two-sided) or ANOVA for experiments with more than two subgroups. Probability values of p< 0.05 were considered to be statistically significant.

Results

EGb 761 increases osteoblastogenesis versus adipogenesis in bone marrow cells

BMC cultures were differentiated using osteogenic supplements (Trivedi et al. 2008). Alizarin Red-S staining of mineralized nodules (for determining calcium deposition) was used for assessment of bone forming action of EGb 761 on BM cells differentiated to osteogenic lineage. EGb 761 treatment from 0.01% to 0.001% showed an increase in mineralization compared to control cells (receiving vehicle) (Fig. 1A). Consistent with mineralization data, EGb 761 significantly increased mRNA levels of osteoblast specific genes including Runx-2, Coll and OCN (Fig. 1B).

BMC cultures were differentiated to adipocytes and stained with Oil Red 0 to assess accumulation of intracellular lipid droplets (Fig. 2A). In comparison to the control, EGb 761 treatment resulted in significantly reduced lipid accumulation and led to fewer lipid-filled cells (Fig. 2A). Absorbance data of extracted Oil Red 0 stain from the cells show ~42% inhibition of adipogenesis of preadipocytes by EGb 761 treatment (panel below Fig. 2A). Inhibition of adipocytic differentiation by EGb 761 was further confirmed using qPCR determination of the expression of adipogenic genes C/EBP[alpha], FABP-4 and PPAR--[gamma]. Data shows that EGb 761 decreased the mRNA levels of all the genes (Fig. 2B).

EGb 761 induces apoptosis of mature adipocytes

Next, we studied the effect of EGb 761 on mature adipocytes in the paradigm of serum withdrawal-induced apoptosis. Presence of EGb 761 for 48h exhibited 3-4 fold increase in TUNEL positive adipocytes compared with controls (vehicle) (Fig. 3A). Some of the apoptotic cells have been marked with the arrows. Data below Fig. 3A shows quantitation of TUNEL positive cells representing that both concentration of 0.001and 0.01% induce apoptosis.

EGb 761 induces ROS generation

Previous reports show that polyphenols inhibit fat accumulation by modulating the ROS pathway (Ambati et al. 2009; Woo et al. 2003; Yang et al. 2006). As shown in Fig. 3B, ROS generation was rapidly increased in mature adipocytes by a positive control lipopolysaccharide (LPS) and EGb 761 treatment at 0.01 and 0.001% increased ROS levels as compared to the non treated cells.

EGb 761 treatment lowers cholesterol levels

HCD treatment to the hamsters for 30 days significantly increased total cholesterol levels. Treating EGb 761 to HCD fed animals significantly lowered the total cholesterol levels compared to the HCD fed group (control) (Fig. 4).

EGb 761 treatment relaxes the proximal segments of thoracic aorta in response to acetylcholine

In aortic vessels from hamsters fed normal chow diet, acetylcholine produced concentration dependent (3 nM-30 mM) relaxation (Fig. 5). Vaso-relaxation in response to acetylcholine was impaired in the proximal aortas of hamsters fed HCD that contained atherosclerotic lesions. HCD animals fed EGb 761 (250 mg/kg body weight) for 15 days had no significant effect on endothelium relaxation in the presence of acetylcholine up to 300 [micro]M but it was significantly greater than HCD group at 3 mM and 30 mM acetylcholine (Fig. 5).

EGb 761 improves vascular calcification assessment by histology and microcomputed tomography

Vascular response (development of calcification) to HCD and then its restoration by EGb 761 treatment was quantified by micro computed tomography (CT). As shown in (Fig. 6B, b) the aortas of animals being fed HCD appeared to be at the stage of calcification as observed by high contrast by micro-CT.

This was confirmed by staining the aortic sections with von Kossa that stains calcified nodules (data not shown). EGb 761 treatment for 15 days showed evidence of reduced calcification (Fig. 6B, c) and the effect was comparable to the control (chow diet) animals (Fig. 6B, a).

HCD treatment elicited significant increase in the aortic wall thickness with decreased lumen thickness (Fig. 6A, b) this was evident by the observations of the transverse section of aorta compared with the animals that were being fed normal chow diet (Fig. 6A, a). Treatment with EGb 761 to HCD animals resulted in significant decrease in the aorta wall thickness (Fig. 6A, c). Quantification of aorta wall thickness and calcification by micro-CT showed significant decrease in the above parameters as shown in Fig. 6C and D. Data suggests that EGb 761 treatment for 15 days to the HCD animals was able to restore the aortic morphology.

Discussion

A reciprocal association between osteoblasts and adipocytes that originate from the pluripotent mesenchymal progenitor stem cells prompted us to study the effect of EGb 761 in bone marrow cells (BMCs) (Di lorgi et al. 2008). Here we show that EGb 761 at physiological concentrations enhances the commitment and differentiation of BMC's toward osteoblast lineage, whereas adipogenic differentiation and maturation were hindered by lowering the number of adipocytes and decreasing lipid droplet size thus helping lower atherosclerotic plaque formation. Data was further corroborated at the transcript level by the differentiation of BMC cultures into osteogenic lineage. There was a significant reduction in the expression of FABP-4, C/EBP-[alpha] and PPAR--[gamma]. PPAR--[gamma] being a master adipogenic transcription factor regulates anabolic processes such as triacylglycerol synthesis by enhancing the transcription of genes encoding proteins such as aP2, PPAR--[gamma] and C/EBP[alpha] that act synergistically to promote adipogenesis (Brun and Spiegelman 1997; Santiago-Mora et al. 2011). Further, apoptosis was enhanced by EGb 761 during differentiation, which suggests that the anti-obesity ability of EGb 761 was exerted by increased levels of ROS. This mediates reduction in the adipose tissue mass by both inhibiting maturation of cells and increasing cell death (Babich et al. 2009; Hwang et al. 2007; Lin et al. 2007; Yang et al. 2006). This data is in line with many of the studies published with natural compounds where it has been demonstrated that ROS activation is critical for AMPK signaling and thus inhibition of fat accumulation (Babich et al. 2009; Hwang et al. 2007; Lin et al. 2007; Yang et al. 2006).

Having shown that EGb 761 alters the coupling of BMCs in favor of bone formation and attenuates lipid formation we aimed to test the epidemiological data providing evidence of an inverse relationship between atherosclerosis and antioxidant intake (Azen et al. 1996). A preliminary in vivo study was done to screen EGb 761's hypolipidemic action at various doses using Triton-WR-1339 (induces hyperlipidemia), EGb 761 inhibited serum cholesterol and triglyceride levels, thus decelerating lipid biosynthesis leading to lipid catabolism with the most effective dose being 250 [mg.sup.-1] [kg.sup.-1] (supplementary data Fig. S2). For further studies we used the dose of 250 [mg.sup.-1] [kg.sup.-1] to show that EGb 761 helps in vasodilatation by lowering cholesterol levels in the hamster model for atherosclerosis. In atherosclerosis, it is the endothelium dependent relaxation that is first impaired both in humans and in several animal models of atherosclerosis (Bonthu et at. 1997). Since, integrity of the arterial wall rests primarily on the intact endothelial cells, we set out to study if this function of endothelium is restored in the aorta in the presence of EGb 761. We tested this hypothesis in vivo in high cholesterol diet induced hamster model. Our study shows that in the presence of atherosclerotic lesions induced by high cholesterol diet, there is vascular remodeling and impairment of relaxation to receptor and non-receptor mediated endothelium dependent agonist (acetylcholine). Under HCD conditions, cells residing in the vascular wall (smooth muscle cells) or precursor cells with mesenchymal differentiation potential (calcifying vascular cells) acquire osteogenic properties, which may involve BMP and cbfal signaling pathways (Hofbauer et al. 2007; Vattikuti and Towler 2004). These osteoblast like cells then deposit bone matrix proteins that subsequently become mineralized and lead to impaired endothelium function.

Calcium and phosphorus are the most abundant minerals in the body involved in the formation of calcium hydroxyapatite i.e. calcification. Hydroxyapatite deposits not only in bone but along with other forms of calcium phosphate in the vasculature with adverse effects (Villa-Bellosta et al. 2011). Our micro CT data and histology sections assessed this effect quantitatively and demonstrated increase in the aortic wall thickness and aortic calcification in HCD fed hamsters.

EGb 761 treatment for 2 weeks reduced these cardiovascular events in part by improving the endothelial function albeit at higher concentrations of 3 mM and 30 mM of acetylcholine. Biochemical assessment of total and aortic cholesterol levels suggest its lowering although lowering of aortic cholesterol levels did not achieve significance. It is possible that prolonging the duration of treatment with EGb 761 or increasing the dose of EGb 761, could restore the impairment of vasodilation function effectively at lower doses (Pierre et al. 2008). It is seen that the circulating monocytes initiate fatty streak formation with further transformation of these cells by CSF-1 (colony stimulating factor) into macrophages that engorge oxidized LDL and become foam cells. Our data in monocytic macrophages derived from hematopoetic origin in the presence of MCSF and RANKL shows that EGb 761 which is an extract derived from GB inhibits osteoclast formation (data not shown). This suggests that EGb 761 by inhibiting macrophages into osteoclasts, decreases the number of these cells which in part could be the predominant mechanism underlying the beneficial effects of standardized EGb 761 extract (Gardner et al. 2007). In addition to several reported pharmacological activities, our results demonstrate that EGb 761 at cellular and whole organism levels displays health benefits for prevention of obesity and associated metabolic disorders by lowering vasodilation in endothelial cells in aorta, and down regulating preadipocyte differentiation. The mechanism by which EGb 761 modulates these processes needs to be further investigated.

Funding

Ministry of Health and Family Welfare, Council of Scientific and Industrial Research, Indian Council of Medical Research, Department of Biotechnology (DBT), Department of Science and Technology (DST), Government of India.

Conflict of interest

Authors have no conflict of interest.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phymed.2012.07.005.

CDRI Communication number: 190/2012/RT.

* Corresponding author. Tel.: +91 522 2612411 18x4246; fax: +91 5222623938. E-mail address: ritu_rivedi@cdri.res.in (R. Trivedi).

(1.) Both the authors have contributed equally in the manuscript.

0944-7113/$--see front matter [c] 2012 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.phymed.2012.07.005

References

Adis International, 2003. ECM 761: Ginkgo biloba extract, Ginkor. Drugs in R&D 4, 188-193.

Alexaki, A., Wilson, T.A., Atallah, M.T., Handelman, G., Nicolosi, R.J., 2004. Hamsters fed diets high in saturated fat have increased cholesterol accumulation and cytokine production in the aortic arch compared with cholesterol-fed hamsters with moderately elevated plasma non-HDL cholesterol concentrations. Journal of Nutrition 134,410-415.

Allain, C.C., Poon, LS., Chan, C.S., Richmond, W., Fu, P.C., 1974. Enzymatic determination of total serum cholesterol. Clinical Chemistry 20,470-475.

Ambati, S., Yang, J.Y., Rayalam, S., Park, KJ., Della-Fera, M.A., Baile, C.A., 2009. Ajoene exerts potent effects in 3T3-L1 adipocytes by inhibiting adipogenesis and inducing apoptosis. Phytotherapy Research 23, 513-518.

Anagnostis, P., Karagiannis, A., Kakafika, A.I., Tziomalos, K., Athyros, V.G., Mikhai-lidis, D.P., 2009. Atherosclerosis and osteoporosis: age-dependent degenerative processes or related entities? Osteoporosis International 20, 197-207.

Atmani, H., Chappard, D., Basle, M.F., 2003. Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: effects of dexamethasone and calcitriol. Journal of Cellular Biochemistry 89, 364-372.

Azen, S.P., Qian, D., Mack, WJ., Sevanian, A., Selzer, R.H., Liu, C.R., Liu, C.H., Hodis, H.N., 1996. Effect of supplementary antioxidant vitamin intake on carotid arterial wall intima-media thickness in a controlled clinical trial of cholesterol lowering. Circulation 94, 2369-2372.

Babich, H., Ackerman, NJ., Burekhovich, F., Zuckerbraun, H.L., Schuck, A.G., 2009. Gingko biloba leaf extract induces oxidative stress in carcinoma HSC-2 cells. Toxicology In Vitro 23, 992-999.

Bandyopadhyay, S., Lion, J.M., Mentaverri, R., Ricupero, D.A., Kamel, S., Romero, IR., Chattopadhyay, N., 2006. Attenuation of osteoclastogenesis and osteoclast function by apigenin. Biochemical Pharmacology 72, 184-197.

Benetos. A., Lacolley, P., Safar, M.E., 1997. Prevention of aortic fibrosis by spironolactone in spontaneously hypertensive rats. Arteriosclerosis, Thrombosis, and Vascular Biology 17.1152-1156.

Bonthu, S., Heistad, D.D., Chappell, D.A., Lamping, K.G., Farad, F.M., 1997. Atherosclerosis, vascular remodeling, and impairment of endothelium-dependent relaxation in genetically altered hyperlipidemic mice. Arteriosclerosis, Thrombosis. and Vascular Biology 17, 2333-2340.

Brun, R.P., Spiegelman, B.M., 1997. PPAR gamma and the molecular control of adipogenesis. Journal of Endocrinology 155, 217-218.

Cummings, S.R., Ensrud, K., Delmas. P.D., LaCroix, A.Z., Vukicevic, S., Reid, D.M., Goldstein, S., Sriram, U., Lee, A., Thompson, J., Armstrong, R.A., Thompson, D.D., Powles, T., Zanchetta, J., Kendler, D., Neven, P., Eastell, R., 2010. Lasofoxifene in postmenopausal women with osteoporosis. New England Journal of Medicine 362, 686-696.

Di lorgi, N., Rosol, M., Mittelman, S.D., Gilsanz, V., 2008. Reciprocal relation between marrow adiposity and the amount of bone in the axial and appendicular skeleton of young adults. Journal of Clinical Endocrinology and Metabolism 93, 2281-2286.

Dillard, A., Matthan, N.R., Lichtenstein, A.H., 2010. Use of hamster as a model to study diet-induced atherosclerosis. Nutrition and Metabolism (London) 7, 89.

Eastell, R., Newman, C., Crossman, D.C., 2010. Cardiovascular disease and bone. Archives of Biochemistry and Biophysics 503, 78-83.

Elbaz, A., Wu, X., Rivas, D., Gimble, J.M., Duque, G., 2010. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro. Journal of Cellular and Molecular Medicine 14, 982-991.

Gardner, C.D., Zehnder, J.L., Rigby, A.J., Nicholus, J.R., Farquhar, J.W., 2007. Effect of Ginkgo biloba (EGb 761) and aspirin on platelet aggregation and platelet function analysis among older adults at risk of cardiovascular disease: a randomized clinical trial. Blood Coagulation and Fibrinolysis 18. 787-793.

Greenhill, N.S., Presland, M.R., Rogers, K.M., Stehbens, W.E., 1985. X-ray microanalysis of mineralized matrix vesicles of experimental saccular aneurysms. Experimental and Molecular Pathology 43, 220-232.

Haynes. W.G., 2002. ATVB in focus: noninvasive assessment of atherosclerosis-from structure to function. Arteriosclerosis, Thrombosis, and Vascular Biology 22, 1064.

Heim, M., Johnson, J., Boess, F., Bendik, I., Weber, P., Hunziker, W., Fluhmann, B., 2002. Phytanic acid, a natural peroxisome proliferator-activated receptor (PPAR) agonist, regulates glucose metabolism in rat primary hepatocytes. FASEB Journal 16,718-720.

Higgins, C.L., Marvel, S.A., Morrisett, J.D., 2005. Quantification of calcification in atherosclerotic lesions. Arteriosclerosis. Thrombosis, and Vascular Biology 25, 1567-1576.

Hofbauer, LC., Brueck, C.C., Shanahan, C.M., Schoppet, M., Dobnig, H., 2007. Vascular calcification and osteoporosis - from clinical observation towards molecular understanding. Osteoporosis International 18, 251-259.

Hwang, J.T., Kim, S.H., Lee, M.S., Kim, S.H., Yang, H.J., Kim, M.J., Kim, H.S., Ha, J., Kim, M.S., Kwon, D.Y., 2007. Anti-obesity effects of ginsenoside Rh2 are associated with the activation of AMPK signaling pathway in 313-L1 adipocyte. Biochemical and Biophysical Research Communications 364, 1002-1008.

Langheinrich. A.C., Bohle, R.M., Greschus, S., Hackstein, N., Walker, G., von Gerlach, S., Rau, W.S., Holschermann, H., 2004. Atherosclerotic lesions at micro CT: feasibility for analysis of coronary artery wall in autopsy specimens. Radiology 231, 675-681.

Lee, J.A., Song, H.Y., Ju, S.M., Lee, S.J., Kwon, H.J., Eum, W.S., Jang, S.H., Choi. S.Y., Park, J.S., 2009. Differential regulation of inducible nitric oxide synthase and cyclooxygenase-2 expression by superoxide dismutase in lipopolysaccharide stimulated RAW 264.7 cells. Experimental and Molecular Medicine 41,629-637.

Lin, C.L. Huang, H.C., Lin, J.K., 2007.Theaflavins attenuate hepatic lipid accumulation through activating AMPK in human HepG2 cells. Journal of Lipid Research 48, 2334-2343.

Luo, G., Ducy. P., McKee, M.D., Pinero, Loyer. E., Behringer, R.R., Karsenty, G., 1997. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 386, 78-81.

Maniatopoulos, C., Sodek, J., Melcher, A.H., 1988. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell and Tissue Research 254, 317-330.

Naves, M., Rodriguez-Garcia. M., Diaz-Lopez, J.B., Gomez-Alonso, C., Cannata-Andia. J.B., 2008. Progression of vascular calcifications is associated with greater bone loss and increased bone fractures. Osteoporosis International 19, 1161-1166.

Pierre, S.V., Lesnik, P., Moreau, M., Bonenllo, L., Droy-Lefaix, M.T., Sennoune, S., Duran, M.J., Pressley. T.A., Sampol, J., Chapman, J., Maixent, J.M., 2008. The standardized Ginkgo biloba extract Egb-761 protects vascular endothelium exposed to oxidized low density lipoproteins. Cell and Molecular Biology (Noisy-Le-Grand) 54 (Suppl.), 0L1032-0L1042.

Rizvi, F., Puri, A., Bhatia, G., Khanna, A.K., Wulff, E.M., Rastogi, A.K., Chander, R., 2003. Anticlyslipidemic action of fenofibrate in dyslipiclemic-diabetic hamster model. Biochemical and Biophysical Research Communications 305, 215-222.

Santiago-Mora, R., Casado-Diaz, A., De Castro. M.D., Quesada-Gomez, J.M., 2011. Oleuropein enhances osteoblastogenesis and inhibits adipogenesis: the effect on differentiation in stem cells derived from bone marrow. Osteoporosis International 22, 675-684.

Sharan, K., Swarnkar, G., Siddiqui, J.A., Kumar, A., Rawat, P., Kumar, M., Nagar, G.K., Manickavasagam, L, Singh, S.P., Mishra, G., Wahajuddin Jain, G.K., Maurya. R., Chattopadhyay, N., 2010.A novel flavonoid, 6-C-beta-d-glucopyranosyl-(2S,3S)-(+)-3,4,5,7-tetrahyclroxyflavanone, isolated from Ulmus wallichiana Planchon mitigates ovariectomy-induced osteoporosis in rats. Menopause 17, 577-586.

Swarnkar. G., Sharan, K., Siddiqui, J.A., Chakravarti. B., Rawat, P., Kumar. M., Arya, K.R., Maurya, R., Chattopadhyay, N., 2011. A novel flavonoid isolated from the steam-bark of Ulmus Wall ichiana Planchon stimulates osteoblast function and inhibits osteoclast and aclipocyte differentiation. European Journal of Pharmacology 658, 65-73.

Szulc, P., Kiel, D.P., Delmas, RD., 2008. Calcifications in the abdominal aorta predict fractures in men: MINOS study. Journal of Bone and Mineral Research 23, 95-102.

Trivedi, R., Kumar, A., Gupta, V., Kumar, S., Nagar, G.K., Romero, J.R., Dwivedi, A.K., Chattopalhyay, N., 2009. Effects of Egb 761 on bone mineral density, bone microstructure. and osteoblast function: possible roles of quercetin and kaempferol. Molecular and Cellular Endocrinology 302, 86-91.

Trivedi, R., Kumar, S., Kumar, A., Siddiqui, J.A., Swarnkar, G., Gupta, V., Kendurker, A., Dwivedi, AK., Romero, J.R., Chattopadhyay, N., 2008. Kaempferol has osteogenic effect in ovariectomized adult Sprague-Dawley rats. Molecular and Cellular Endocrinology 289, 85-93.

Vattikuti, R., Towler, D.A., 2004. Osteogenic regulation of vascular calcification: an early perspective. American Journal of Physiology and Endocrinology Metabolism 286, E686-E696.

Villa-Bellosta, R., Millan, A., Sorribas, V., 2011. Role of calcium-phosphate deposition in vascular smooth muscle cell calcification. American Journal of Physiology and Cell Physiology 300, C210-C220.

Woo, C.H., Yoo, M.H., You, H.J., Cho, S.H., Mun, Y.C., Seong, C.M., Kim, J.H., 2003. Transepithelial migration of neutrophils in response to leukotriene B4 is mediated by a reactive oxygen species-extracellular signal-regulated kinase-linked cascade. Journal of Immunology 170, 6273-6279.

Yang, J.Y., Della-Fera, M.A., Nelson-Dooley, C., Baile, C.A., 2006. Molecular mechanisms of apoptosis induced by ajoene in 3T3-L1 adipocytes. Obesity (Silver Spring) 14, 388-397.

Zakaria el, R., Hunt, C.M., Li, N., Harris, P.D., Garrison, R.N., 2005. Disparity in osmolarity-induced vascular reactivity. Journal of the American Society of Nephrology 16.2931-2940.

Zhang, H.T., Wang, Y., Deng, XI., Dong, M.Q., Zhao, LM., Wang, Y.W., 2010. Daidzein relaxes rat cerebral basilar artery via activation of large-conductance Ca2.-activated lc channels in vascular smooth muscle cells. European Journal of Pharmacology 630, 100-106.

Jyoti Gautam (a), (1), Priyanka Kushwaha (a), (1), Gaurav Swarnkar (a), Vikram Khedgikar (a), Geet K. Nagar (a), Divya Singh (a), Vishal Singh (b), Manish Jain (b), Manoj Barthwal (b), Ritu Trivedi* (a)

(a) Division of Endocrinology, (SIR-Central Drug Research Institute, Chattar Manzil, Lucknow, India

(b) Division of Pharmacology, CSIR-Central Drug Research Institute. Chattar Manzil, Lacknow, India
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Title Annotation:extract of ginkgo biloba 761
Author:Gautam, Jyoti; Kushwaha, Priyanka; Swarnkar, Gaurav; Khedgikar, Vikram; Nagar, Geet K.; Singh, Divya
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Sep 15, 2012
Words:5639
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