Anthocyanins from Cornus kousa ethanolic extract attenuate obesity in association with anti-angiogenic activities in 3T3-L1 cells by down-regulating adipogeneses and lipogenesis

Cornus kousa the Korean dogwood has been traditionally used in East Asia as therapeutic traditional medicine however biological activities of Cornus kousa have not been investigated previously. The aim of the present study was to evaluate anti-obesity activities coupled with anti-angiogenic activities of anthocyanins rich fraction of ethanolic leaf extract of Cornus kousa (ELECk) in HUVECs and 3T3- L1 cells. Dried plants leaves were extracted with 70% ethanol and anthocyanin fraction (AnT Fr) was obtained by eluting the ethanolic extract through non-polar macroporous resin and further purification by HPLC. Antiangiogenic activities were determined by antiproliferative effect of AnT Fr on HUVECs. In the presence of various concentrations of AnT Fr, 3T3-L1 preadipocytes were induced to differentiate. Lipid accumulation in differentiated adipocytes were quantified by Oil-Red O staining. AnT Fr significantly suppressed angiogenesis by inhibiting proliferation and tube formation of HUVECs via downregulating VEGRF 2, PI3K, β‐catenin, NF‐kB, and Akt1 in a dose dependent manner. AnT Fr inhibited lipid accumulation by down-regulating adipogenesis and lipogenesis promoting signaling proteins, PPARγ, CCAAT, C/EBPα, aP2, FAS, and LPL, however enhanced AMPK activation to p-AMPK in 3T3 cells quantified and expressed by western blotting. AnT Fr inhibit lipid accumulation by regulating adipogenesis and lipogenesis related genes and signaling proteins. The anti-obesity activities exerted by Cornus kousa are associated with antiangiogenic activities of anthocyanins rich fraction of Cornus kousa. Hence the presence of bioactive anthocyanins, Cornus kosa, is a good candidate for nutraceutical and pharmaceutical formulation for treating or controlling obesity.

Introduction the present study was to examine antiangiogenic effects and anti-obesity activities of anthocyanins present in Cornus kousa ethanolic leaf extract.

Preparation of plant extracts
Plant materials used in this study were bought from a local food shop located in the open food and vegetables market of Yeosu (Dun-Duk Dong, Yeosu, Chonnam, 550-749, Korea) The plants materials were consisted of leaves (Turned red because of accumulation of anthocyanins). It was freshly harvested in autumn from a farm (Myeongin Shin Kwang-Soo's tea farm Suncheon, Jeonnam chonnam republic of Korea) and were packed in polyethene bags (1 Kg/ bag) in natural form and have not undergone any pre-treatment or processing. The plant is well known and native to Korea that's why plant materials were easily identified by Human Herb Research Center (Korea). It was washed thoroughly with distilled water, dried, and ground into pomace powder. The powder (500 g) was then extracted with 70% ethanol by refluxing in heating mantles (Global Lab GLHMP-F500) at 60˚C for 8 hours. The resulting extract was filtered with membrane filtration system. The filtrate was then concentrated with a rotary vacuum evaporator (EYELA N-1000) at 50-60˚C. Concentrated extracts were dried by lyophilization. After storing portions of the extract at -20˚C, the rest was subjected to further purification with non-polar macroporous resin (D101), first eluted with 0.3N NaoH and then eluted with 100% ethanol. The fraction obtained was purified further by HPLC and was subjected to GC-Ms for analysis and quantification of the present anthocyanins compounds.

Antiangiogenetic assays
To determine the anti-angiogenic activity of anthocyanins rich fraction (AnT Fr) of ethanolic leaf extract of Cornus kousa (ELECk), first toxicity of AnT Fr was checked by MTT (dimethyl thiazol diphenyl tetrazolium bromide) assay. After determining the safe doses(concentrations), anti-angiogenic activities were determined by its inhibitory potential on HUVECs (Humen umbilical vein endothelia cell) proliferation and tube formation.
Cell viability essay. Stock solution of AnT Fr was prepared as 1mg/ml and then diluted to various concentrations. Human umbilical vein endothelial cell (HUVEC) line was purchased from the Korean Cell Line Bank (Seoul, Korea). HUVECs were cultured in EBM-2 media (Clonetics, USA) supplemented with EGM-2 Single Quot Kit (Clonetics, USA) at 37˚C in a humidified 5% CO 2 incubator. Cell viability was determined by MTT assay. HUVECs (1 × 10 4 cells/ well) were seeded into a 96-well plate, incubated for 24 h, and then treated with various concentrations (5,10,15,20,25,30,50,100, and 200 μg/ml) of AnT Fr for another 24 h at 37˚C in a humidified 5% CO 2 incubator. Control groups of HUVECs were grown in the absence of AnT Fr. Then 0.5% MTT solution (Sigma, MO, USA) was added to the medium and incubated for 4 h at 37˚C. MTT medium was removed by aspiration and DMSO (Sigma, MO, USA) was added to each well and keep for 15 min to dissolve formazan crystals. Absorbance was measured at wavelength of 540 nm using a microplate reader (Biochrom Ltd., Cambridge, UK). The experiments were run in triplicate.
Anti-angiogenic activities/inhibition of tubes formation. To determine anti-angiogenic activities of AnT Fr, 24-well culture plates were used for this assay. They were coated with Matrigel (BD Bioscience, MA, US) at 150 μl/well that was then allowed to solidify at 37˚C for 1 hr. HUVECs suspended in medium (2.5 × 10 4 cells/well) were added to Matrigel-coated wells and incubated at 37˚C for 4 h. Various concentrations (5, 20, 50, and100 μg/ml) of AnT Fr were then added to wells and incubated at 37˚C for 4 h. Tube formation was observed and photographed using a phase contrast inverted microscope (Nikon, Tokyo, Japan). Tube lengths in five photographs were obtained from random fields in each well. They were analyzed using Scion Image software (NIH, ML, USA). The experiment of the inhibitory effects of AnT Fr on angiogenesis were performed more than 2 times.
Analysis of gene expression level. To elucidate the effect of AnT Fr on the expression of angiogenic proteins (signaling molecules), RT-PCR analysis was performed using gene-specific primers. Expression (mRNA) levels of angiogenesis supporting signaling proteins in HUVECs were quantified to determine the effect of AnT Fr on angiogenesis. HUVECs were cultured in EBM2 media supplemented with 0.3% FBS in 6-well plate and incubated at 37C in CO 2 incubator. Cells were then treated with different concentrations (5,20,50, and 100 μg/ml) of AnT Fr and incubated in the CO 2 incubator for 24 hours. Total RNA was isolated from HUVECs using TRI reagent (Sigma Aldrich). Then 2 μg of total RNA was reverse transcribed to cDNA for RT-PCR using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Gene expression level of the mRNA of the related signaling molecules were cheeked more than Two times.

Anti-obesity activity
Anti-obesity activities were determined using mouse embryo 3T3-L1 preadipocytes. First, cell viability was determined by MTT assay. Lipid droplets in 3T3-L1 cells were then differentiated under various doses of AnT Fr Adipocytes were extracted by Oil Red O staining and quantified with a spectrophotometer.
MTT assay. Toxicity of AnT Fr to 3T3-L1 Cells was determined by MTT assay. 3T3-L1 cells were cultured in 96-well plate at density of 1 × 10 4 cells/well. These 96-well plates were kept in a humidified 5% CO2 incubator at 37˚C for 24 hours. Cells were treated with different concentrations (5,10,15,20,25,30,50, 100 and 200 μg/ml) of AnT Fr. After 24 hours of treatment with AnT Fr, MTT solution (0.5%) was added to each well of the plate containing cells and incubated for another 4 h. After aspiring the media, each well was added with dimethyl sulfoxide (DMSO) (Sigma) to dissolve formazan crystals. Absorbance was then read at wavelength of 540 nm on a microplate reader (Biochrom Ltd., UK). Cell viability (percentage) was calculated and compared to the control. Experiment was Two times repeated.
Oil Red O staining assay for anti-obesity determination. 3T3-L1 cells were cultured in 6-well plates. After two days or when cell confluency reached 80%, media were replaced with differentiation induction media (10 μg/mL insulin, 1 mM dexamethasone, 0.5 mM Isobutyl-1-methylxanthine). Cells were kept in this state for 24 h to arrest cell division. After 2 days, culture media were replaced with maintenance media (DMEM media supplemented with 10 μg/ insulin). These cells were then feed with maintenance media every 2 days up to 8 days. Samples of AnT Fr were added during the addition of induction media and maintenance media. GW9662 (10 μg/mL) was added as positive control as it is a potent antagonist of PPARγ inhibits adipogenesis. After 8 days of feeding phase, cells were washed with PBS and then fixed with formalin (10%) for 1 h. After cells were again washed with 60% iso-propanol, Oil Red O solution was added. Cells were then incubated at 37˚C for 3 h. After cells were washed 4 times with distilled water and drying, they were washed with isopropanol to extract the staining dye. Effects of AnT Fr on lipids accumulation in cells were determined by photographing extracted Oil Red O solution of each group with a phase contrast microscope at 5 random sites. The absorbance of the extracted Oil Red O solution was then measured at wavelength of 520 nm using with a spectrophotometer. Experiments of the quantification of lipid droplets(triglycerides) accumulation in the 3T3-L1 deposits were performed 2 times.
Analysis of gene expression level. The inhibitory effect of AnT Fr on mRNA expression levels of adipogenesis and lipogenesis related gens and signal molecules was investigated by RT-PCR analysis using gene-specific primers. 3T3-L1 cells were cultured in DMEM media supplemented with 10% FBS in 6-well plate and incubated at 37˚C in a CO 2 incubator. These cells were then treated with different concentrations of AnT Fr and incubated in the CO 2 incubator for 24 hours. Total RNA was isolated from these 3T3-L1 cells using TRI reagent (Sigma Aldrich) followed by DNase treatment. It was then quantified on a NanoDrop 2000 spectrophotometer. cDNA was then made from 500 ng of total RNA using Revert Aid Premium First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). cDNA was quantified by checking absorption on a Nano Drop spectrophotometer. cDNA was then used for real-time qPCR with gene-specific primers. DNA bands were obtained on agarose gel after electrophoresis. RT-PCR analysis experiments were repeated more than 2 times.

Western blotting
3T3-L1 adipocytes after differentiation were harvested at sub confluency, washed with PBS buffer and lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich, MO, USA) contained phenylmethylsulphonyl fluoride and protease inhibitor cocktail. Cell lysates were sonicated, centrifuged and supernatant was collected. protein concentration of the supernatant was measured by BCA protein Assay Reagent (Pierce, Rockford, IL, USA). 30 μg protein for each sample was separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis (PAGE) and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were then blocked in Tris-buffered saline (TBS)-Tween 20 solution containing 3% bovine serum albumin. After binding the membranes was then incubated overnight with AMPK, phospho-AMPKα (Cell Signaling Technology, Danvers, MA) and β-actin (Santa Cruz, CA) primary antibodies at 4˚C. Secondary antibody was conjugated to horseradish peroxidase and enhanced chemiluminescence (Amersham Bioscience, UK) for visualizing protein bands. Experiments were repeated 2 times.

Statistical analysis
Experiments were performed at least three times. Data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed with analysis of variance (ANOVA) using SPSS followed by post-hoc comparison with Tukey's test at α = 0.05. Statistical significance was considered at p < 0.05.

Analysis and quantifications of anthocyanins rich fraction (AnT Fr)
The HPLC purified fraction of anthocyanins (AnT Fr) from ethanol leaf extract of Cornus kusa (ELECk) was subjected to GC-MS for identification and quantification of the present anthocyanins compounds (S1 Fig). 3 compounds, representing about 90.05% of the anthocyanins fraction AnT Fr of ELECk. The major compounds that were identified and quantified by GC-MS were cyanidin 3-glucoside, delphinidin 3-glucoside and pelargonidin 3-glucoside with the percent quantities as 27.23%, 36.54% and 26.32% respectively along with some other minor components presented in trace amounts.

Anti-angiogenic activities
MTT assay. Anti-angiogenic potential of AnT Fr was determined using HUVECs. First, cell viability was determined by MTT assay and safe doses were determined. HUVCEs were allowed to proliferate in the presence of different concentrations of AnT Fr in 96-well microtiter plate with equal number of cells. Wells of the plate containing HUVCEs without treatment with AnT Fr were used as negative control. EGCG (Epigallocatechin gallate) was used as positive control (50 μg/ml). Toxicity of EGCG to HUVECs was first determined by MTT assay (S2 Fig). MTT assay results shown that AnT Fr do not pose any toxicity to HUVECs up to concentration of 100 μg/ml (Fig 1).
Only safe (nontoxic) doses of AnT Fr were selected for further experiments to determine its anti-angiogenesis activities. Effect of AnT Fr of ELECk on human erythrocytes was evaluated in order to determine its hemolysis effects. The used dosses of AnT Fr in experiments did not show hemolytic effects after incubation for 240 minutes indicating the safety of the anthocyanins rich fraction (AnT) to normal cells (S3 Fig). Anti-angiogenic/tube formation-inhibiting activities. Antiangiogenic effect of AnT Fr was explored by determining its inhibitory effect on the formation of tubular structure by HUVECs in Matrigel. HUVECs were grown in 96-well microtiter plate. After reaching confluency, cells were treated with various concentrations of AnT Fr. They were allowed to proliferate and make tubular structures in Matrigel under the influence of AnT Fr. HUVECs of each group proliferated under AnT Fr were photographed at 5 random sites with a phase contrast inverted microscope. HUVECs in the negative and positive control group was also photographed randomly (S4 Fig). The effect of different concentrations of AnT Fr on HUVECs proliferation in Matrigel was determined (Fig 2). Total tube length was calculated for each concentration and compared with the control. Results showed that AnT Fr significantly inhibited HUVECs proliferation in a dose-dependent manner. Total tube length was found to be decreased with increasing concentrations of AnT Fr (Fig 3). Total tube length was the highest in the control. It was the lowest in the group treated with the highest concentration (i.e., 100 μg/ml) of ELECk, indicating that suppression of angiogenesis in HUVECs by ELECk was in a dose-dependent manner.
RT-PCR analysis. The mechanisms of the antiangiogenic activities of AnT Fr in HUVECs was analyzed by RT-PCR. Total RNA was extracted from the treated and control cells and reverse transcribed to cDNA. The resulted cDNA was subjected to Real time PCR using gen specific primers (Table 1). The product was then visualized by obtaining bands by Gel doc after gel electrophoresis. AnT Fr significantly decreased expression levels of angiogenic signal molecules such as VEGFR2, PI3k, Akt, and Nf-Kb in a dose-dependent manner (Fig 4). The highest safe dose of AnT Fr (i.e., 100 μg/ml) showed the highest inhibition on angiogenesis process in HUVECs by suppressing signaling proteins and transcriptional factors of angiogenesis.

Anti-obesity activity
Anti-obesity effect of AnT Fr was determined by antihyperlipidemic activities via antiadipogenic and anti-lipogenic potential of AnT Fr. MTT assay. For antihyperlipidemic activities, first the cell viability assay was performed by MTT using 3T3-L1 cell line for determination of safe and toxic concentrations level. AnT Fr did not show any toxicity to 3T3 -L1 cells at concentration up to 100 μg/ml. At concentration higher than 100 μg/ml, AnT Fr deceased viability of 3T3 -L1 cells (Fig 5). Thus, safe doses of AnT Fr were determined as up to 100 μg/ml for further experiments.
Toxicity of the positive controls EGCG and GW9662 to 3T3-L1 cells was also determined by MTT assay before using for further experiments (S5 Fig). Oil Red O staining assay. Pre-mature 3T3-L1 cells were differentiated in the presence of various concentrations (nontoxic level) of AnT Fr. Lipid droplets accumulated in the cells were extracted with Oil Red O staining solution and was quantified with a spectrophotometer by checking the absorbance of the extracted Oil Red O staining. Photographs of lipid droplets in Oil Red O staining solution showed that lipid accumulation was significantly reduced with increasing concentrations of AnT Fr (Fig 6).
AnT Fr inhibited lipid accumulation in differentiated 3T3-L1 adipocytes in a dose-dependent manner as compared to controls. The highest concentration of AnT Fr supressed lipid accumulation in 3T3-L1 cells nearly the same as GW9662, the positive control (Fig 7)  RT-PCR. The mechanisms of the antihyperlipidimic activities of AnT Fr on 3T3-L1 proliferation and lipid accumulation was analyzed by RT-PCR. Total RNA was isolated from 3T3-L1 cells proliferated under the presence of various concentrations of AnT Fr. RNA was reverse transcribed to cDNA by reverse polymerase chain reaction and then converted to DNA by real time qPCR using gen specific primers ( Table 2).
The product was then visualized by obtaining bands by Gel doc after gel electrophoresis. RT-PCR rresults showed that AnT Fr significantly inhibited expression levels of adipogenesis and lipogenesis related genes and signal molecules such as PPARγ, C/EBPα, aP2, and SREBP-1c in a dose-dependent manner (Fig 8).

Effect of ELECk on activation of p-AMPK
Activation of p-AMPK by AnT Fr was determined by western blot analysis. 3T3-L1 adipocytes were differentiated under treatment with various concentrations of AnT Fr. Proteins were extracted with radioimmunoprecipitation assay buffer and concentration was measured by BCA protein Assay Reagent (Pierce, Rockford, IL, USA). Protein separated by SDS-PAGE were transferred onto nitrocellulose membranes was then incubated overnight with AMPK, phospho-AMPKα (Cell Signaling Technology, Danvers, MA) and β-actin (Santa Cruz, CA) primary antibodies at 4˚C. Secondary antibody was conjugated to horseradish peroxidase and enhanced chemiluminescence Amersham Bioscience, UK for visualizing protein bands. The result shown that AnT Fr has effect on activation of p-APMK in a dose dependent manner. The expression level of the p-APMK shows enhancement with increasing concentration of AnT Fr as compared to AMPK and beta actin level which was taken as control (Fig 9).

Discussion
Obesity is a multifactorial metabolic disorder and a risk factor for diabetes, cancer, cardiovascular diseases, and inflammatory diseases [22,23]. Lipid accumulation increases the size of cells, resulting in expansion of adipose tissues. Lipid synthesis occurs as a result of adipogenesis and lipogenesis regulated by a number of signaling pathways. These pathways involve many signal molecules and transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) that play a key role in adipocyte differentiation. PPARγ regulates the expression of adipogenesis promoting genes [10,11]. PPARγ assists C/EBPα expression and induces adipocyte differentiation by activating aP2 and LPL [12] (Song Y et al., 2013). SREBP-1c is crucial for lipogeneses promotion via activating transcriptional factors SCD-1, FAS, ACC, and PPARγ [24][25][26]. AMPK is a key factor that regulates glucose and lipids metabolism. Phosphorylated AMPK inactivates key enzymes involved in fatty acid and sterol synthesis such as fatty acid synthetase (FAS) and acetyl-CoA carboxylase (ACC-1). It enhances fatty acid oxidation [27]. AMPK controls the expression of PPARγ, C/EBPα, and SREBP [28,29]. AMPK activation and overexpression will downregulate adipogenesis and lipogenesis.
Natural products are significant therapeutic compounds with multiple molecular targets. They are preferred alternatives to synthetic chemical compounds [30,31]. Various natural products have been used to treat different diseases by modulating, enhancing, or suppressing transcriptional factors and signaling proteins. Anti-obesity activities of natural compounds are mediated by regulating various pathways (including lipid absorption, energy intake and expenditure), increasing lipolysis, and decreasing lipogenesis, differentiation, and proliferation of preadipocytes [30].
The genus Cornus commony known as dogwood consists of about 55 species and several species have been reported for analgesic, anti-malarial, diuretic anti-bacterial, anti-allergic anti-histamine and anti-microbial activities and are used in Chinese herbal medicine [32]. Cronus spp. Contained Anthocyanin, the bioactive flavonoids with therapeutic effects which have been reported to possess antioxidant, anti-inflammatory, anticancer and anti-diabetic activities [33,34]. Anthocyanin possess antihyperlipidemic effects and reduces serum cholesterol level [35]. It has been studied that anthocyanin effectively reduce fats level in C57BL/6J mice fed with high lipids diet [36]. Peng et al., [37] reported significant reduction of accumulated body fats by anthocyanins rich mulberry water extracts. Anthocyanins modulate obesity and reduce inflammation by regulating MAPK and NF-κB signaling pathways [38]. Elena et al., [39] reported anti-obesity activities of anthocyanin supplementation. In our study we Table 2. Gene-specific primers used for adipogenesis and lipogenesis related PCR along with the product size and gene bank accession number details.

Gene
Gene bank accession no. Primer sequence Size (bp) SREBP NM_033218. 1  F -TTG CAC CAG AGA GCA TTT TG-3'  R -GAA AAT GAG AGG CTG GTT GC-3  investigated the inhibitory effects of Cornus kusa extracted anthocyanins on angiogenesis, adipogenesis and lipogenesis. In this study, we experimentally proved that AnT Fr could inhibit lipid accumulation during differentiation of 3T3-L1 adipocytes without posing toxicity to normal cells. Its action of mechanisms was elucidated by measuring expression levels of adipogenesis and lipogenesis related genes and proteins in 3T3-L1 adipocytes by RT-PCR and western blot analysis. AnT Fr significantly suppressed expression levels of adipogenesis/lipogenesisassociated genes and proteins in 3T3-L1adipocytes. It also upregulated protein expression levels of p-AMPK. Treatment with AnT Fr decreased expression levels of signaling proteins PPARγ and C/EBPα and adipogenesis related gens aP2 and LPL in a dose-dependent manner. Similarly, mRNA expression levels of lipogenesis related genes (such as SCD-1, FAS, and ACC) and protein expression levels of SREBP-1c and FAS were decreased in 3T3-L1 cells after treatment with AnT Fr. Angiogenesis assists obesity by feeding adipose tissues. Inhibition or suppression of angiogenesis contributes to inhibition of fat accumulation in adipocytes, resulting in downregulation of obesity. We investigated the antiangiogenic potential of AnT Fr in HUVECs determined by the inhibition of tubular structures (i.e., vessels formation by HUVECs) in Matrigel. Our results showed that AnT Fr inhibited tube formation of HUVECs in a dose-dependent manner. RT-PCR analysis revealed that AnT Fr downregulated angiogenesis by suppressing expression levels of angiogenesis promoting signaling proteins. The anti-obesity and anti-angiogenic activities of AnT Fr might be due to the presence of anthocyanins i.e. delphinidin 3-glucoside, cyanidin 3-glucoside, pelargonidin 3-glucoside.