Effect of Nelumbo Nucifera Petal Extracts on Lipase, Adipogenesis, Adipolysis, and Central Receptors of Obesity
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Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 145925, 7 pages http://dx.doi.org/10.1155/2013/145925 Research Article Effect of Nelumbo nucifera Petal Extracts on Lipase, Adipogenesis, Adipolysis, and Central Receptors of Obesity Chandrasekaran Chinampudur Velusami,1,2 Amit Agarwal,1 and Vijayalakshmi Mookambeswaran2 1 R&D Centre, Natural Remedies Pvt. Ltd., Plot No. 5B, Veerasandra Industrial Area, 19th K.M. Stone, Hosur Road, Bangalore, Karnataka 560100, India 2 CenterforBioseparationTechnology,VITUniversity,Vellore,TamilNadu632014,India Correspondence should be addressed to Chandrasekaran Chinampudur Velusami; [email protected] Received 22 July 2013; Revised 30 September 2013; Accepted 2 October 2013 AcademicEditor:C.S.Cho Copyright © 2013 Chandrasekaran Chinampudur Velusami et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. N. nucifera is one among the important medicinal plants assessed for its antiobesity action in various preclinical models. The present study was aimed at investigating the antiobesity effect of methanol and successive water extracts of petals of N. nucifera by studying its effect on adipogenesis, adipolysis, lipase, serotonin (5-HT2C), cannabinoid (CNR2), melanocyte concentrating hormone (MCHR1), and melanocortin (MC4R) receptors. Both methanol and successive water extracts of N. nucifera petals had an effect on inhibition of lipid storage in adipocytes and on increasing lipolysis. N. nucifera petal methanol extract exhibited the concentration- dependent inhibitory effect on lipase activity with an IC50 value of 47 g/mL. N. nucifera petal extracts showed evident agonist and antagonist activity towards 5-HT2C and CNR2 receptors, respectively, while it showed no effect towards MCHR1 and MC4R receptors. Overall, methanol extract of N. nucifera petals showed better activity than successive water extract. 1. Introduction roemerine, nuciferine, nornuciferine, nelumboside, anon- aine, 5-methoxy-6-hydroxyaporphine, liensinine, and asim- Several herbs have been indicated for weight management ilobine [15]. Bisbenzylisoquinoline alkaloids from N. nucifera [1]. One such plant used for weight management is Nelumbo were shown to be bioavailable after oral administration to rats nucifera Gaertn.N.nucifera,knownbyanumberofnames at a dose of 20 mg/kg [16]. N. nucifera alkaloid was shown including Indian lotus, sacred lotus, bean of India, or simply to inhibit 3T3-L1 preadipocyte differentiation and improve lotus, is one of two species of aquatic plant in the family high-fat diet-induced obesity and body fat accumulation in Nelumbonaceae. Almost all parts of N. nucifera are edible, rats [17]. and in many Asian countries it was found in the recipe of Several flavonoids and nonflavonoids from flowers of N. food [2]. Extracts of N. nucifera flowers, seeds, rhizomes, nucifera reported by several authors were consolidated in a and leaves have been reported to have varied therapeutic review by Mukherjee et al. [14]. Flavonoids include myricetin- potential including antistress [3], antiobesity [4], antioxi- 3-O-b-D-glucopyranoside, quercetin-3-O-b-D-glucuronide, dant [5], hepatoprotective [6], antidiabetic activity [7], anti- astragalin, quercetin, 3,4-dihydroxybenzoic, kaempferol, p- inflammatory8 [ , 9], antipyretic [10], antibacterial [11], and hydroxybenzoic acid, and b-sitosterol which were isolated immunomodulatory [12, 13]activities. from ethanol extract of the petals of N. nucifera [18]. Several bioactive phytocompounds derived from these Nonflavonoid compounds, including adenine, myo-inositol, plant parts were belonging to different chemical groups, arbutin, and sitosterol glucopyranoside, were identified in including alkaloids, flavonoids, glycosides, triterpenoid, and flower extract [14]. Wuet al. [19] demonstrated the antiobesity vitamins [14]. Leaves, root, and the embryonic stage of N. effect of a flavonoid-enriched extract from N. nucifera leaf nucifera have been reported to contain alkaloids such as (NLFE) in high-fat diet (HFD) fed C57BL/6 mice and 2 Evidence-Based Complementary and Alternative Medicine concluded its action via lipid-regulated enzymes, thereby in cell plate reagent in 96-well tissue culture plates for 48 h attenuating body lipid accumulation and preventing obesity. and 24 h, respectively. AntiobesityactionofleavesandseedsofN. nucifera was extensively studied in in vitro and in vivo models by many 2.4. Cell Viability Determination. Initial experiments using researchers [4, 20–22]. The present study was designed to 3T3-L1, CHO-K1, and U2OS cells were conducted to assess investigate the effect of N. nucifera petal extracts on lipase, the cytotoxic concentrations of both methanol and successive adipogenesis, adipolysis, and central receptors in vitro. water extracts of N. nucifera. Cell viability was determined by a colorimetric MTT assay as described by Mosmann [24]. In brief, cells were cultured in 96-well plates at a seeding 2. Materials and Methods 3 density of 5 × 10 cells/well. After 24 h of seeding, the cells 2.1. Chemicals. Dexamethasone, isobutylmethylxanthine were treated with and without N. nucifera extracts up to a (IBMX), oil red O, porcine lipase enzyme, thiazolyl blue concentration of 100 g/mL. Thereafter, the cells were rinsed tetrazolium bromide (MTT), 4-methyl umbelliferyl oleate, and further incubated with MTT for 1 h. After 1 h, MTT GW 803430, melanocyte concentrating hormone (MCH), crystals were dissolved in 200 L DMSO. Optical density melanotan II, DL-isoproterenol hydrochloride, and orlistat was read at 570 nm, and, consequently, the noncytotoxic were procured from Sigma-Aldrich. AM630, CP 55,940, concentrations were chosen for conducting in vitro studies. AL34662, cell plating reagent-3, cell assay buffer, substrate reagent 1, and substrate reagent 2 were obtained from 2.5. Adipogenesis Assay. Effect of N. nucifera petal extracts DiscoveRx (USA). Insulin was procured from Biocon. Fetal on adipogenesis was evaluated by examining their ability bovine serum (FBS) and bovine calf serum (BCS) were to inhibit the differentiation of preadipocytes to adipocytes purchased from Hyclone. Enzychrom glycerol detection kit using 3T3-L1 cells as a test system. On day 0, mouse 3T3- 4 was procured from BioAssay systems. DMEM was obtained L1 fibroblasts were seeded at a density of 3 × 10 cells/well from Gibco Life Technologies. in a 48-well plate containing DMEM supplemented with 10% BCS.Onday1,cellswerechangedtoDMEMmediumsupple- 2.2. Plant Material. The petals of N. nucifera (100 g each) were mented with FBS (5%), IBMX (0.5 mM), insulin (10 g/mL), procured from a local commercial supplier and were authen- and dexamethasone (1 M) with and without N. nucifera ticated at National Institute of Science Communication and extracts. Guggulsterone was used as a reference control. On Information Resources (NISCAIR), New Delhi. A voucher day 3, cells were changed to DMEM supplemented with FBS specimen (no. 811) was deposited in our herbarium. Dried (5%) and insulin (5 g/mL) with and without N. nucifera petal petals were extracted with methanol (∼400 mL) by refluxing extracts. On days 5 and 7, the cells were changed to DMEM ∘ at 70 Cfor1hour.Extractsolutionwasfiltered,andthe supplemented with 5% FBS. On day 8, 3T3-L1 adipocytes remaining raw material was subjected to methanol extraction were rinsed with PBS and fixed using 10% formalin for 30 min by repeating the above steps twice. The liquid filtrates were followed by another rinse with 60% isopropanol solution and combined and concentrated by distillation under vacuum to a allowed to dry. The cells were stained with oil red O solution thick paste, followed by drying under vacuum at temperature (0.5% oil red O in isopropanol, diluted in proportion of 3 ∘ 70 C. The dried extract was named as methanol extract parts of oil red O stock and two parts of distilled water) for and utilized to perform in vitro experiments. Phytochemical 15 min at room temperature. Dye retained in adipocytes was investigation of methanol extract of N. nucifera was carried extracted with isopropanol and quantified by measuring the out by subjecting methanol extract to HPLC analysis to absorbance at 500 nm. identify the flavonoids as per the method described by Xingfeng et al. [23]. 2.6. Adipolysis Assay. Adipolysis assay was performed to Methanol extraction of the raw material was carried out evaluate the possible lipolytic activity of N. nucifera petal as mentioned above. The leftover raw material after methanol ∘ extracts by examining their ability to release glycerol from extraction was further boiled with water at 85–90 C(3 differentiated 3T3-L1 cells. On day 0, 3T3-L1 cells were seeded 4 times each with 500–600 mL water for 1 h) and filtered each at a density of 3 × 10 cells/well in a 48-well plate containing time. The liquid filtrates were combined and concentrated by DMEM medium supplemented with 10% BCS. On day 1, cells distillation under vacuum to a thick paste, followed by drying ∘ were changed to DMEM supplemented with 5% FBS, IBMX under vacuum at temperature 80 C. The dried extract was (0.5 mM), insulin (10 g/mL), and dexamethasone (1 M). named as successive water extract and used to perform in Onday2,cellswerechangedtoDMEM,supplemented vitro experiments. with FBS (5%) and insulin (5 g/mL), and left undisturbed for 2 days. On