Screening of Crude Plant Extracts for anti-adipogenesis activity in 3T3-L1 cells.

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1 Research Article ISSN: Geetha Kodali et al. / Journal of Pharmacy Research 2014,8(1), Available online through Screening of Crude Plant Extracts for anti-adipogenesis activity in 3T3-L1 cells. Geetha Kodali *1, Sreekanth Kakarla 2 and Ganapaty Seru 1 1 Department of Pharmacognosy and Phytochemistry, University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India. 2 Department of Pharmacy, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India. *Corresponding author. Geetha Kodali Department of Pharmacognosy and Phytochemistry, University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India. Received on: ; Revised on: ; Accepted on: ABSTRACT Objective: Hyperlipidemia is the major risk factor contributing to prevalence and severity of cardiovascular complications like atherosclerosis. The present study was aimed to screen the selected crude plant extracts for antiadipogenesis activity in-vitro in 3T3-L1 cells. Methodology: Four herbal drugs which were used traditionally in the treatment of several disorders including obesity were selected namely, Commiphora caudata (leaves, bark), Boswellia ovalifoliolata (leaves, gum), Saccharum spontaneum (whole plant) and Garcinia mangostana (fruit pericarp). Ethanolic extracts of the selected plants were screened for their cytotoxicity and anti-adipogenic activity in-vitro. For testing the in-vitro cytotoxicity of the selected plant extracts, an MTT assay was performed with concentrations of test extracts of the selected plants ranging from to 1000 µg/ml. Effect of the plant extracts on lipid accumulation in cultured 3T3-L1 adipocytes was determined by measuring Oil Red O staining. The 3T3-L1 adipocytes were cultured and differentiated in a Dulbecco s Modified Eagle s Medium containing 10% fetal bovine serum for 6 to 8 days in the absence and presence of plant extracts. Results: Among the six plant extracts tested, ethanolic extract of B.ovalifoliolata gum showed higher cytotoxicity with a lower CTC50 value of 100 µg/ml and the remaining extracts showed CTC50 ranging from µg/ml. Adipogenesis was substantially inhibited by the test extracts and among the six extracts screened, C. caudata leaves, B.ovalifoliolata Gum, S. spontaneum whole plant and G. mangostana fruit pericarp significantly reduced the lipid accumulation with 51.5, 46.1, 26 and 56.3 (%) over control at 100, 700, 250 and 250µg/mL respectively through the quantification method of Oil Red O staining. Other two extracts namely C. caudata bark (4.8) and B.ovalifoliolata leaves (8.3)) did not show significant inhibition of fat accumulation at the tested concentration. Conclusion: The results suggest that these extracts may be useful in the treatment of Hyperlipidemia and its associated cardiovascular diseases like atherosclerosis. Keywords: Adipogenesis, Oil red O, 3T3 L1 cell line, MTT assay, Plant extracts 1. INTRODUCTION: Many natural extracts have been found to have beneficial effects on health, and these extracts have drawn attention because of their relative safeness, low cost as compared to synthetic drugs and accumulated evidence of physiological properties such as anti-obesity and anti-diabetic effects. 1 Decreased adipocytic lipogenesis is one of the proposed mechanisms for anti-obesity. 2 Capsaicin in red pepper and Red yeast rice extracts have been reported to suppress adipogenesis in 3T3-L1 adipocytes 3, 4, and pine needle extract suppresses differentiation of 3T3-L1 preadipocytes and obesity in high fat diet fed rats. 5 In obese or over-weight individuals, the increased fat mass can affect cell size. Adipose tissue hyperplasia is a result of increased adipogenesis, which include adipocyte differentiation. 6, 7 Preadipocyte cell lines are useful models for investigating the adipogenesis process. 3T3-L1 preadipocyte, which can be induced to differentiate into adipocyte cells, is one of the most studied preadipocyte cell line. 8, 9, 10 During adipocyte differentiation, transcriptional factors such as peroxisome proliferator-activated receptor (PPAR) γ and CCAAT element binding protein (C/EBPs) are involved in the sequential expression of adipocyte-specific proteins. In this regard, C/ EBP is expressed at the onset of preadipocyte differentiation 11, 12, 13, whereas expression of PPARγ and C/EBP, which trigger the expression of adipocyte-specific proteins, are induced during terminal differentiation of the adipocyte 14, 15 lineage. The cytotoxicity assay depends on the reduction of tetrazolium salt, MTT (Micro titre tetrazolium), by living cells to form a blue formazan product. This cytotoxicity assay is based on the assumption that dead cells or their products do not reduce tetrazolium. The assay depends both on the number of cells present and on the mitochondrial activity per cell. The principle involved is the cleavage of tetrazolium salt 3-(4, 5 dimethyl thiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) into a blue coloured product (formazan) by mitochondrial enzyme succinate dehydrogenase. The number of cells was found to be proportional to the extent of formazan production by the 16, 17 cells used.

2 Geetha Kodali et al. / Journal of Pharmacy Research 2014,8(1), In this study, we screened six crude extracts from four plants as potential anti-hyperlipidemic agents through an in-vitro study. Boswellia ovalifoliolata Bal.Henry (Burseraceae) is an endemic medicinal tree found on the Eastern Ghats of India. The leaves, stem bark and gum resin of the plant are highly medicated and are traditionally used in the treatment of ulcers, dysentery, inflammation, arthritis, obesity, and diabetes 18. Commiphora caudata Wight & Arn (Burseraceae) commonly known as hill mango, is a potential medicinal plant, distributed in India and Srilanka 19. The leaves and bark have the odour of mangoes, and the oleo-gum resin obtained from the tree is used as incense. The stem, bark and leaves are traditionally used in the treatment of rheumatism, ulcers, diarrhea and spasms. 20 Saccharum spontaneum (Poaceae) commonly known as Wild sugarcane, is widely found throughout India along the streams and moist places. Scientific information on their pharmacognosy, phytochemistry and pharmacology are very scant. It is a popular folk medicine and considered as valuable medicinal herb in traditional systems of medicine in India. The rural people in Vellore district of Tamilnadu and Andhra Pradesh use fresh juice of the stem of Saccharum spontaneum plant for the treatment of mental illness and mental disturbances. The whole plant is used to treat mental diseases, abdominal disorders, dyspnoea, anaemia, obesity, diuretic, lithotriptic, purgative, tonic, aphrodisiac, gynecological troubles, respiratory troubles etc. 21 Garcinia mangostana Linn (Clusiaceae), commonly known as Mangosteen and Mangustan has a long history of use as a medicinal plant mostly in south Asia. The fruit, fruit rind, leaves and bark of the plant are used medicinally. Mangosteen is a popular health food supplement. It is used as antimicrobial, anti- inflammatory, antiulcer, febrifuge and in the treatment of diarrhea and dysentery. 22 The above plants were also found to be used by the tribals in different parts of India for various disorders including obesity. 23 These are less explored plants but possess phytoconstituents such as phenols and flavonoids which are purported to exhibit wide pharmacological activities. Hence we elucidated anti-hyperlipidemic effects on lipid accumulation in cultured 3T3-L1 adipocytes by measuring Oil Red O staining as indicators of lipid accumulation. There are no previous reports of these plant extracts being screened for their lipid inhibitory activity. 2. METHODS: 2.1 Plant collection and authentication: Fresh plant materials were collected from the Hills of Tirumala region, Chittoor (dist), A.P, India and authenticated by Dr. Madhava chetty, Assistant Professor, Department of Botany, S.V. University, Tirupathi. A voucher specimen of all the plants was kept in our herbarium, Dept of Pharmacognosy and Phytochemistry, College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh. 2.2 Preparation of alcoholic extracts of the selected plants B. ovalifoliolata, C. caudata, S. spontaneum and G. mangostana: Freshly collected plant material was dried under shade and coarsely powdered in a willey mill. The coarsely powdered material (500 g) was extracted with petroleum ether to remove the fatty material and further extracted in a Soxhlet apparatus and subjected to continuous extraction with ethanol (95%). The liquid extract was collected and concentrated under reduced pressure until a waxy mass was obtained. The concentrate was thoroughly air dried to remove all traces of the solvent and the percentage yield was calculated.the extractive value of the extraction was obtained by using the relation, % Extraction = Weight of concentrate Weight of fresh material X In-vitro cytotoxicity of the selected plant extracts in 3T3-L1 cells For testing the in-vitro cytotoxicity of the selected plant extracts, an MTT assay was performed to assess the cell viability with concentrations of test extracts of the selected plants ranging from to 1000 µg/ml Chemicals: 3-(4,5 dimethyl thiazol 2 yl) 5 diphenyl tetrazolium bromide (MTT), Fetal Bovine serum (FBS), Phosphate Buffered Saline (PBS), Dulbecco s Modified Eagle s Medium (DMEM) and Trypsin were obtained from Sigma Aldrich Co, St Louis, USA. EDTA, Glucose and antibiotics from Hi-Media Laboratories Ltd., Mumbai. Dimethyl Sulfoxide (DMSO) and Propanol from E.Merck Ltd., Mumbai, India Cell lines and culture medium: 3T3L1 cell line was cultured in DMEM supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/mL), streptomycin (100 µg/ml) and amphotericin B (5 µg/ml) in an humidified atmosphere of 5% CO 2 at 37 C until confluent. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm 2 culture flasks and all experiments were carried out in 96 microtitre plates (Tarsons India Pvt. Ltd., Kolkata, India) Preparation of test solutions: For cytotoxicity studies, the test sample provided in liquid form (1.5 mg/ml) was made up with DMEM supplemented with 2 % inactivated FBS to obtain a stock solution of 750 mg/ml concentration and sterilized by filtration and centrifuged. Serial two fold dilutions were prepared from this for carrying out cytotoxic studies Procedure: The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 x 10 5 cells/ml using DMEM containing 10% FBS. To each well of the 96 well microtitre plate, 0.1 ml of the diluted cell suspension (approximately 10,000 cells) was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off and the monolayer was washed once with medium and 100 µl of different test concentrations of test drugs were added on to the partial monolayer in microtitre plates. The plates were then incubated at 37 o C for

3 Geetha Kodali et al. / Journal of Pharmacy Research 2014,8(1), 3 days in 5% CO 2 atmosphere, and microscopic examination was carried out and observations were noted every 24 h interval. After 72 h, the drug solutions in the wells were discarded and 50 µl of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 h at 37 o C in 5% CO 2 atmosphere. The supernatant was removed and 100 µl of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (CTC 50 ) is generated from the dose-response curves for each cell line. % Growth Inhibition = 100 Mean OD of individual test group Mean OD of control group x In-vitro anti-adipogenic studies of selected plant extracts in 3T3- L1 cells The test concentrations for anti-adipogenic studies of the selected plant extracts were determined based on the results obtained in cytotoxicity studies. The anti-adipogenic studies were carried out on 3T3- L1 cells and the effect of plant extracts on inhibition of fat droplet formation was determined by quantification of Oil Red O staining method Chemicals: Fetal Bovine serum (FBS) was obtained from Sigma Aldrich Co, St Louis, USA. EDTA, glucose, insulin, dexamethasone, oil O red stain and antibiotics from Hi-Media Laboratories Ltd., Mumbai Equipments: Automated Microplate reader ( Biotek, USA), CO 2 Incubator (Nuaire, USA), Inverted Tissue culture Microscope (Lyzer, India) were used for the Anti-adipogenic studies Cell lines and culture medium: 3T3-L1 cell line was cultured in DMEM supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (100 IU/mL), streptomycin (100 µg/ml) and amphotericin B (5 µg/ml) in a humidified atmosphere of 5% CO 2 at 37 C until confluent. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm 2 culture flasks and all experiments were carried out in 30 mm gelatin coated petri plates (Tarsons India Pvt. Ltd., Kolkata, India) Preparation of test solutions: For adipogenesis studies, the test sample extracts were weighed and made up with DMEM supplemented with 2% inactivated FBS to obtain a stock solution of 1000 mcg/ml concentration and sterilized by filtration and centrifuged Culture and differentiation: Day 0: 3T3-L1 preadipocytes (NCCS, India) were cultured in 4.5 g/l glucose- DMEM with 10% calf serum, antibiotic solution in 30 mm plastic Petri dishes until they reached 100% confluence. Day 1: For differentiation, 2-day post-confluent cells were incubated for 48 h in DMEM with 10% FBS, antibiotics, and a differentiation cocktail termed MDI, which contained 0.5mM isobutylmethylxanthine, 1µM dexamethasone, and 100 nm insulin along with extracts. Day 3: After 48 hours, cells were maintained with DMEM with 10% FBS, Insulin, antibiotics, and the test samples for 6 days. Day 9: After 6 days, the cells were maintained with DMEM with 10% FBS along with test samples for 48 hours Oil Red O Staining: 3T3-L1 adipocytes were washed with phosphate-buffered saline (PBS) and fixed with 10% formalin for 30 min. After washing twice with distilled water, cells were stained for at least 1 h at room temperature in freshly diluted Oil Red O containing 0.5% Oil Red O in isopropanol. Finally, the dye retained in the 3T3-L1 cells was eluted with isopropanol and quantified by measuring the optical absorbance at 500 nm 24, RESULTS: 3.1Preparation of alcoholic extracts of the selected plants B. ovalifoliolata, C. caudata, S. spontaneum and G. mangostana: The alcoholic extracts were obtained as intractable gum and possessed characteristic odour. The percentage yield of the extracts obtained was found to be 13.5 (EBOL), 7.5 (EBOG), 8 (ECCL), 19.5 (ECCB), 4.2 (ESSW) and 18.5 (EGMP). 3.2 In-vitro cytotoxicity of the selected plant extracts in 3T3-L1 cells The results of in-vitro cytotoxicity were presented in table 1 and Figure 1 shows the Cytotoxic effect of selected plant extracts in 3T3- L1 cells. 3.3 In-vitro anti-adipogenic studies of selected plant extracts in 3T3- L1 cells Effect of plant extracts on Oil Red O staining in cultured 3T3-L1 cells was shown in figure 3 and the percentage inhibition was presented in table 2.

4 Geetha Kodali et al. / Journal of Pharmacy Research 2014,8(1), Table 1: Cytotoxic properties of selected plant extracts on 3T3-L1 cells 3T3L-1 S.No. Extract Test Conc. % Cytotoxicity CTC 50 (µg/ml) ( µg/ml) 1 EBOG ± ± ± ± ± ± ± ECCB ± ± ± ± ± ± ±0.45 Cell control EBOG 3 EBOL ± ± ± ± ± ± ± ECCL ± ± ± ± ± ± ±0.49 ECCB 5 ESSW ± ± ± ± ± ± ± EGMP ± ± ± ± ± ± ± 0.34 Table 2: Anti-adipogenesis activity of selected plant extracts in 3T3L1 cells S. No Name of the Extract Test Concn. % Inhibition ( µg/ml) EBOL ECCL 1 EBOG ± ECCB ± EBOL ± ECCL ± ESSW ± EGMP ±0.014 EBOG: Ethanolic extract of B.ovalifoliolata gum ECCB: Ethanolic extract of C.caudata bark EBOL: Ethanolic extract of B.ovalifoliolata leaf ECCL: Ethanolic extract of C.caudata leaf, ESSW: Ethanolic extract of S.spontaneum whole plant EGMP: Ethanolic extract of G.mangostana pericarp ESSW EGMP EBOG: Ethanolic extract of B.ovalifoliolata gum ECCB: Ethanolic extract of C.caudata bark EBOL: Ethanolic extract of B.ovalifoliolata leaf ECCL: Ethanolic extract of C.caudata leaf, ESSW: Ethanolic extract of S.spontaneum whole plant EGMP: Ethanolic extract of G.mangostana pericarp Figure 1: Cytotoxic effect of selected plant extracts in 3T3-L1 cells

5 Geetha Kodali et al. / Journal of Pharmacy Research 2014,8(1), % Fat inhibition over control ESSW 0 EBOG ECCB EBOL ECCL ESS W EGM P Figure 2: Anti adipogenesis activity of selected plant extracts in 3T3-L1 cells EGMP ECCL: Ethanolic extract of C.caudata leaf, EBOG: Ethanolic extract of B.ovalifoliolata gum ECCB: Ethanolic extract of C.caudata bark EBOL: Ethanolic extract of B.ovalifoliolata leaf ESSW: Ethanolic extract of S.spontaneum whole plant EGMP: Ethanolic extract of G.mangostana pericarp Figure 3: Effect of selected plant extracts on Oil Red O staining in 3T3-L1 adipocytes Cell control ECCL ECCB Adipogenesis control EBOG EBOL 4. DISCUSSION Hyperlipidemia is closely associated with several disorders like coronary heart disease, hypertension, type 2 diabetes mellitus, respiratory complications, and osteoarthritis. The results obtained in the present study demonstrate for the first time, the anti-adipogenesis activity of the selected plant extracts in 3T3-L1 preadipocytes without eliciting cytotoxicity of the cells. To examine the effect of plant extracts on cell viability of 3T3-L1 preadipocytes, an MTT assay was performed, which assess cell viability by measuring mitochondrial activity in 3T3-L1 cells, treated with different concentrations of test extracts of the selected plants B. ovalifoliolata, C. caudata, S. spontaneum and G. mangostana. The alcoholic extracts of the selected plants were tested at concentrations of to 1000 µg/ml. The extracts showed cytotoxicity with CTC50 ranging from 100±0.31 to 720±0.18 µg/ml against 3T3L1 cell line. The CTC50 values of the selected plant extracts were found to be in the order EBOG (100) > ECCB (120) > EBOL (190) > ESSW (246) > EGMP (287) > ECCL (720). The control sample methanol showed poor toxicity at tested concentrations. Of all the extracts tested, ECCL was found to be less toxic and EBOG was more toxic than the other extracts tested. One of the screening methods used in the discovery of anti-hyperlipidemic drugs is by measuring Oil Red O staining as indicators of lipid accumulation. The 3T3-L1 adipocytes were cultured and differentiated in a Dulbecco s Modified Eagle s Medium containing 10% fetal bovine serum for 6 to 8 days in the absence and presence of plant extracts. The effects of plant extracts on fat droplet formation in 3T3-L1 cells, and inhibition through the quantification of Oil Red O staining were presented in Fig. 3. Adipogenesis was substantially inhibited by the test extracts and among the six extracts screened for

6 Geetha Kodali et al. / Journal of Pharmacy Research 2014,8(1), anti adipogenic activity, test extracts ECCL, EBOG, ESSW and EGMP significantly reduced the lipid accumulation with 51.5, 46.1, 26 and 56.3 (%) over control at 100, 700, 250 and 250µg/mL respectively through the quantification method of Oil Red O staining. Other two extracts namely ECCB (4.8) and EBOL (8.3)) did not show significant inhibition of fat accumulation at the tested concentration. These observations demonstrate that among the six extracts tested for anti-adipogenesis activity in-vitro, ethanolic extracts of C.caudata leaf, B.ovalifoliolata gum, S.spontaneum whole plant and G.mangostana pericarp proved to be beneficial in the management of hyperlipidemia. Further investigations are in progress to screen the selected plant extracts for anti-hyperlipidemic and anti-atherosclerotic activities in-vivo. Conflict of interest: None to declare. REFERENCES: 1. Bhathena, S.J. and M.T. Velasquez. 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