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. 2021 Apr 23;11(5):233. doi: 10.1007/s13205-021-02771-2

A bioactive fraction of Pterocarpus santalinus inhibits adipogenesis and inflammation in 3T3-L1 cells via modulation of PPAR-γ/SREBP-1c and TNF-α/IL-6

Karunakaran Reddy Sankaran 1, Muni Swamy Ganjayi 1, Lokanatha Oruganti 1, Appa Rao Chippada 1,✉, Balaji Meriga 1,✉
PMCID: PMC8065073  PMID: 33968577

Abstract

Pterocarpus santalinus has huge demand owing to its commercial and medicinal value. However, there are limited research studies on its therapeutic activity against obesity and obesity-induced inflammation and underlying mechanism of action. Therefore, in the present study, chloroform bioactive fraction of P. santalinus (CFP) was isolated and evaluated for its activity against adipogenesis and adipogenesis-induced inflammation in 3T3-L1 cell culture model. LC–MS/MS analysis of CFP was performed to identify the compounds present. CFP-treated 3T3-L1 cells (50, 100 and 200 μg/ml) have significantly (p < 0.01 or < 0.05) enhanced glycerol release and adiponectin level, but reduced lipid accumulation and leptin, and MTT assay demonstrated CFP was non-toxic till a dose of 300 µg/ml at 24 and 48 h. A considerable reduction in tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels was witnessed in lipopolysaccharide (LPS)-induced 3T3-L1 cells with CFP treatment in dose-dependent manner. Gene expression studies demonstrated down-regulation of mRNA expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), sterol regulatory element-binding protein-1c (SREBP-1c), leptin, TNF-α and IL-6 but up-regulation of adiponectin and uncoupling protein-1 (UCP-1) and the same trend was observed in protein expression also. In conclusion, it is suggested that CFP could be beneficial to treat obesity and associated inflammation.

Keywords: Adipogenesis, Adipokines, Inflammation, Lipolysis, Oil-red O stain, Proinflammatory cytokines

Introduction

Obesity is a complex multi-factorial disorder and a potential risk factor to develop hypertension, diabetes mellitus, dyslipidemia, cardiovascular diseases, chronic inflammation and certain types of cancers (Kyrou et al. 2018). Prolonged imbalance in energy homeostasis leads to overweight and obesity. An increase in body weight means an increase in white adipose tissue mass (MacLean et al. 2015; Romieu et al. 2017). Adipose tissue mass increases by two processes namely hypertrophy (increase in cell size) and hyperplasia (increase in cell number) (Jo et al. 2009). Adipose tissue has been recognized as a vital endocrine organ because it not only stores excess triglycerides but also produces a variety of adipokines (leptin, adiponectin, resistin, etc.) and triggers cytokines which influence the regulation of energy homeostasis, insulin resistance, inflammation, immunity and metabolic disorders (Coelho et al. 2013; Balistreri et al. 2010; Shi et al. 2019).

Adipogenesis is a process of fat accumulation in visceral, subcutaneous, peritoneal and abdominal regions leading to obesity (Silva and Baptista 2019). Adipose tissue contains several types of cells, including endothelial cells, blood cells, fibroblasts, pre-adipocytes, macrophages, other immune cells and mature adipocytes (Burhans et al. 2018). When adipocytes increase in large numbers due to excessive accumulation of triglycerides, they become the main cell type and then form adipose tissue (Armani et al. 2010).

The cellular process of adipogenesis includes three major steps namely commitment of mesenchymal stem cells (MSCs) to become adipocytes, mitotic clonal expansion and terminal differentiation (Moseti et al. 2016). This process of adipogenesis involves expression of key genes at transcriptional level such as CCAAT/enhancer-binding proteins (C/EBPs) and PPARs and induction of lipogenic genes such as SREBPs, acetyl Co-A carboxylase (ACC), and Fatty acid synthase (FAS) (Park et al. 2020). There are other signaling pathways also such as AKT, AMPK and MAPK that can regulate energy homeostasis and adiposity (Boucher et al. 2014; Lopez 2018; Song et al. 2019). Drugs/formulations that can regulate the expression of key genes/protein involved in adipogenesis and lipogenesis pathways can be worked on as target(s) to treat obesity ailments (Balaji et al. 2016).

Another important protein to reduce obesity is UCP-1, present in inner-mitochondrial membrane which uncouples cellular respiration from the generation of ATP and leads to heat generation. This UCP-1 is well expressed in hibernating animals providing them required heat to continue basal metabolism and it is also expressed in brown adipose tissue (BAT) usually present in infants (Laursen et al. 2015; Demine et al. 2019). In adults, BAT % is very low while that of WAT % is high. If UCP-1 can be activated in WAT, it mimics like BAT and dissipates heat. Therefore, targeting UCP-1 is an important therapeutic option which promotes thermogenesis and contributes to attenuation of adiposity (Vergnes et al. 2020).

Several infectious conditions increase pro-inflammatory cytokines such as IL-6 and TNF-α which in turn activate other cytokines and causes inflammation (Kany et al. 2019). Obesity also induces chronic low-grade inflammation resulting in altered secretion and expression of inflammatory cytokines, adipokines, eicosanoids, etc., which disturb energy homeostasis process and causes obesity-related co-morbidities (Ritter et al. 2020). Therefore, therapies that can modulate the inflammatory state of adipose tissue and inhibit adipogenesis are being much considered for the treatment of obesity. Since, natural product-based therapies are considered as safe with no or minimum side effects than synthetic drugs, there is growing interest among researchers and the general public for drugs/formulations from plants and herbs.

Pterocarpus santalinus belongs to Fabaceae family and is a native to South India. It has enormous demand in India and overseas for its commercial and medicinal value. The heartwood of this plant is reported to possess antioxidant, anti-diabetic, anti-cancer, anti-inflammatory and anti-pyretic activities (Kondeti et al. 2010; Bulle et al. 2016). Due to restrictions from the forest department for its collection and usage, not much research work has been done in recent years. Some of the major phyto-constituents present in it include santalin A, B and Y, isoflavone glucoside, 4-hydroxybenzoic acid, vanillic acid, α-resorcyclic acid, gentisic acid, kinotannic acid, lupeol, β-sitosterol, homopterocarpin, pterocarpin, epicatechin gallate, savinin, calocedrin, pterolinus K and L, and pterostilbenes (Abouelela et al. 2019). In view of the inadequate studies on anti-adipogenic activity of P. santalinus and underlying therapeutic mechanism of action, the present study was carried out to evaluate its anti-adipogenic and anti-inflammatory activity in 3T3-L1 cells, the most widely tested cell line model for adipogenesis studies.

Materials and methods

Chemicals and reagents

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), 3­isobutyl­1­methyl xanthine (IBMX), isopropanol, insulin, dexamethasone (DEX), LPS, penicillin and streptomycin were procured from Thermo Fisher Scientific. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and Oil-red O (ORO) stain were procured from Sigma Aldrich. Other chemicals, solvents and reagents used were of Analytical Grade.

Isolation of bioactive fraction and LC–MS/MS analysis

The heartwood pieces of P. santalinus were obtained with the prior permission of the Principal Chief Conservator of forests, Govt. of A.P. and authenticated by a botanist (Voucher No. 1708) in Department of Botany, Sri Venkateswara University, Tirupati. Heartwood pieces were pulverized to a coarse powder and extracted with methanol following cold extraction method (Padmaja et al. 2014). The methanol crude extract was then fractionated using hexane, ethyl acetate, chloroform and ethanol based on their polarity in a column chromatography using silica gel as the column material. The collected fractions were concentrated under reduced pressure in Heidolph rotary-evaporator. LC–MS/MS analyses of P. santalinus fractions were carried out on 6520 Accurate Q-TOF (Agilent Santa Clara, CA) mass spectrometer at Indian Institute of Chemical Technology (IICT), Hyderabad, India. Among these, CFP revealed the presence of about 19 major phyto-compounds including santalin, savinin, pterolinus, vanillic acid and pterostilbene. Based on preliminary studies and phytochemical analysis, CFP was used for further experiments.

Induction of adipogenesis and inflammation

The 3T3­L1 pre-adipocytes of American Type Culture Collection (ATCC) cells were maintained and cultured in DMEM supplemented with 10% FBS at 37 °C in a humidified atmosphere with 5% CO2. The 3T3-L1 cells were cultured as five different groups into culture plates: DMI-induced/differentiated cells (control), DMI + DMSO-treated cells (0.01%) (vehicle control), and DMI + CFP-treated cells (50, 100 and 200 µg/ml). For adipogenesis studies, 3T3-L1 pre-adipocytes were grown to confluence, then switched to differentiation medium of induction (DMI) consisting of DMEM, 10% FBS, 0.5 mM IBMX, 1 μM DEX and insulin (10 μg/ml) for 2 days followed by treating cells with differentiation medium (DM) (DMEM with 10% FBS and insulin, 10 μg/ml) for 8–10 days in the presence or absence of CFP (50, 100 and 200 µg/ml). All the media that we used contained 100 IU/ml penicillin and 100 mg/ml streptomycin (Martino et al. 2016). For anti-inflammatory studies, the differentiated adipocytes on day 8 were induced with LPS (0.1 µg/ml) for 3 h in the same medium along with 25 mM HEPES buffer, followed by treatment with CFP (50, 100 and 200 µg/ml) for 24 h in 24 well plate. Then, the cultured medium was collected to evaluate secreted levels of TNF-α and IL-6 (Fabian et al. 2013).

MTT assay

The cytotoxic effect of CFP on 3T3­L1 cells and their proliferation was determined using MTT assay (Jo et al. 2015). Cells were plated at a density of 1.5 × 104 cells/ml in a 96-well plate and incubated for 24 h. Then, cells were treated with CFP (50–300 μg/ml) for 24 h or 48 h followed by incubation with MTT solution for 3 h at 37 °C. The supernatants were aspirated, DMSO was added to each well, and the plates were agitated to dissolve the crystal product. Absorbance was measured after 24 h and 48 h at 570 nm using a microplate reader (Bio-Rad). The cytotoxicity was calculated based on the absorbance value of untreated (control) cells as the baseline.

Oil-red O staining

In differentiated adipocytes, lipid contents were visualized as well as measured using ORO staining (Kraus et al. 2016). After completion of 8 days, adipocytes were washed with phosphate-buffered saline (PBS, pH 7.2) and fixed with 10% buffered formalin for 1 h at room temperature. Then cells were stained with 0.5% ORO-staining solution for 30 min followed by 3 washes using wash buffer and visualized under bright field microscope. The lipid contents were quantified by extracting the stain from adipocytes using 60% isopropanol (to extract intracellular ORO stain). The absorbance was measured at 540 nm by microplate reader.

Lipolysis assay

The magnitude of lipolysis was assessed by measuring glycerol levels released into the medium, using commercial kit (Lipolysis assay kit, ab185433, Abcam, Shanghai) following manufacturer’s instructions. Briefly, the differentiated 3T3-L1 adipocytes were treated with CFP (50, 100 and 200 μg/ml) for 24 h. Fifty microliters of the medium was incubated with 50 μl of reaction mixture for 30 min at room temperature. The glycerol content was quantified by measuring absorbance at 570 nm using microplate reader. Measurements were performed in triplicates and the results were presented as nmol/well.

Assessment of adipokines and inflammatory cytokines by ELISA

Secreted levels of leptin and adiponectin were measured in 3T3-L1 adipocytes containing media in the presence and absence of CFP using an enzyme-linked immunosorbent assay kit (ElabScience, USA) according to the manufacturer’s instructions. Similarly, the secreted levels of TNF-α and IL-6 were measured in LPS-induced 3T3-L1 adipocytes containing media as per the manufacturers protocol as per manufacturers protocol (ElabScience, USA). In brief, the culture media was collected and centrifuged to remove impurities and the absorbance was measured at 450 nm. The experiments were performed in three replicates and the results were presented as pg/ml.

RT-PCR analysis—mRNA expression

Total RNA was isolated from 3T3-L1 cells using tri-reagent (Sigma Aldrich, USA) according to manufacturer’s protocol and reverse transcribed to obtain cDNA using cDNA synthesis kit (Applied Bio Systems, Foster City, USA). Two nanograms of cDNA was used for RT-PCR. The PCR amplification was performed with transcript specific primers. The primer sequences used for PCR analysis are shown in Table 1.

Table 1.

Primers used in this study

Gene Accession number Primer sequence bp length
UCP-1 NM_009463.3 F: 5´-CACGGGGACCTACAATGCTT-3´ 20
R: 5´-ACAGTAAATGGCAGGGGACG-3´
PPAR-γ NM_001308354.1 F: 5´-GGCTGCAGCGCTAAATTCTT-3′ 20
R: 5´-GGCATTGTGAGACATCCCCA-3´
SREBP-1c NM_011480.4 F: 5´-CGGCTCTGGAACAGACACT-3´ 19
R: 5´-CAGCAGTGAGTCTGCCTTGA-3´
Leptin NM_008493.3 F: 5´-TCTGAAAGATCCCACGTGCC-3´ 20
R: 5´-AGGTTCTCTGAGCGAAAGGC-3´
Adiponectin NM_028320.4 F: 5´-CAGGAATGCCTGGATCGGA-3´ 19
R: 5´-GTCTCTGTGTGGATGCGGAA-3´
TNF-α NM_001278601.1 F: 5´-CCCTCACACTCACAAACCAC-3´ 20
R: 5´-GGGGCTCTGAGGAGTAGACA-3´
IL-6 NM_001314054.1 F: 5´-GCCTTCTTGGGACTGATGCT-3´ 20
R: 5´-TGTGACTCCAGCTTATCTCTTGG-3´
β-Actin NM_007393.5 F: 5´-TAGGCGGACTGTTACTGAGC-3′ 20
R: 5´-CTCAGACCTGGGCCATTCAG-3′
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Immunoblotting—protein expression

Control and treated 3T3-L1 adipocytes were treated with lysis buffer, and total proteins were extracted and quantified using Bradford method. Equal amount of protein was resolved on 10% SDS-PAGE gel and transferred on to a PVDF membrane (Millipore, Billerica, MA, USA) (Reckziegel et al. 2017). To block non-specific binding sites, blots were incubated at room temperature with 5% skimmed milk (v/v) for 1 h followed by overnight incubation with primary antibodies of rabbit anti-UCP-1, anti-PPAR-γ, anti-SREBP-1c, anti-TNF-α, anti-IL-6 and mouse anti-β actin (Abclonal, USA) at 4 ºC (1:1,000 dilution). The immune reactive antigen was then recognized by incubation with HRP-conjugated secondary antibody (1:1,000, Abclonal). After washing with TBST, the membrane was treated with chemiluminescent ECL detection reagent (Bio-Rad) followed by exposure to chemiluminescence detection system.

Statistical analysis

The data are expressed as mean ± standard deviation (SD) of triplicate, and comparison was made using one-way ANOVA programme (SPSS version 17.0, SPSS Inc., Chicago, IL, USA), followed by Tukey’s post hoc tests to study the significance level.

Results

LC–MS/MS analysis of active fraction

From methanolic crude extract of P. santalinus heartwood, solvents, such as hexane, ethyl acetate, chloroform and water, based on polarity, were sequentially used to collect respective fractions. Based on preliminary phytochemical tests, TLC and LC–MS/MS analysis, chloroform fraction was selected. Based on retention time and molecular mass, major phytochemical groups identified in CFP were pyrans, benzoates, lignans, chalcones and stilbenoids and some of the identified compounds included savinin, santalin, pterolinus and vanillic acid (Fig. 1).

Fig. 1.

Fig. 1

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LC–MS/MS analysis of CFP phyto-constituents. a LC–MS/MS peaks represent retention time of different phyto-compounds. b The presence of savinin, santalin, vanillic acid and pterolinus K in CFP based on molecular mass

Effect of CFP on adipocytes’ viability

The effect of CFP on viability of 3T3-L1 cells and its cytotoxicity was analyzed at 24 and 48 h using MTT assay. At a dose range of 50–200 µg/ml, CFP did not show any toxic effects on 3T3-L1 cells, however, a small decrease (but not significant) in cell viability was observed at 300 µg/ml (Fig. 2a). Figure 2b–g represents the structure, morphological changes and percent viability of 3T3-L1 adipocytes.

Fig. 2.

Fig. 2

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Effect of CFP on viability of 3T3-L1 cells by MTT assay. a Percentage of cell viability in control and treated groups at 24 and 48 h. b–g Images representing the adipocytes of control and CFP-treated groups at 48 h. Arrows indicate healthy adipocytes in images ‘b, c’, shrunken adipocytes in images ‘d–f’ and some dead cells in image ‘g’

Effect of CFP on cellular lipid content

When compared to untreated cells, CFP-treated 3T3-L1 adipocytes showed significant (p < 0.01) decrease in intracellular lipid content and adipogenesis in a dose-dependent manner as observed by ORO-stained images (Fig. 3b). We also quantified the lipid content by extracting the ORO stain (using 60% isopropanol) released into the media from 3T3-L1 adipocytes and the results showed decreased lipid content in CFP-treated adipocytes when compared to untreated adipocytes (Fig. 3a).

Fig. 3.

Fig. 3

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Effect of CFP on adipogenesis and lipid content in 3T3-L1 cells. a Lipid levels in control and treated groups. b ORO-stained adipocytes in control and treated groups (magnification ×40). c Glycerol release in to medium from control and treated 3T3-L1 cells. Data are presented as mean ± SD of triplicate (n = 3). * and ** indicate significant difference between control and CFP-treated adipocytes at p < 0.05 and p < 0.01, respectively

Effect of CFP on free glycerol release

Further, we demonstrated the lipolytic ability of CFP by quantifying the glycerol content released from adipocytes into the medium. Maximum glycerol release was observed in CFP (200 µg/ml) treated adipocytes which is significantly higher (p < 0.01) when compared to untreated and other treated cells confirming its lipolytic activity (Fig. 3c).

Effect of CFP on adipokine and cytokine levels

The secreted levels of leptin and adiponectin play significant role in adipogenesis and energy homeostasis. The levels of TNF-α and IL-6 alter during inflammatory state triggered by either obese condition or other ailments. Our data show significant (p < 0.01) and dose-dependent decrease in leptin, TNF-α and IL-6 levels and increase in adiponectin level in CFP-treated adipocytes than control cells (Fig. 4).

Fig. 4.

Fig. 4

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Effect of CFP on adipokines and pro-inflammatory cytokines in control and treated adipocytes. Data are presented as mean ± SD (n = 3). * and ** indicate significant difference between control and CFP-treated adipocytes at p < 0.05 and p < 0.01, respectively

Effect of CFP on mRNA expression of key genes

The mRNA expression levels of adipogenic (PPAR-γ, UCP-1 and SREBP-1c) and pro-inflammatory (TNF-α and IL-6) markers were measured in control and CFP-treated cells by RT-PCR using specific primers. Treatment with 200 µg/ml of CFP substantially reduced the mRNA expression levels of PPAR-γ, SREBP-1c, TNF-α, IL-6 and leptin but enhanced the expression levels of adiponectin and UCP-1 when compared to untreated adipocytes as depicted in Fig. 5.

Fig. 5.

Fig. 5

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Effect of CFP on mRNA expression levels of specific genes in control and treated adipocytes. a UCP-1, b PPAR-γ, c SREBP-1c, d leptin, e adiponectin, f TNF-α and g IL-6. Data are presented as mean ± SD (n = 3). * and ** indicate significant difference between untreated and CFP-treated adipocytes at p < 0.05 and p < 0.01, respectively

Effect of CFP on protein expression

To confirm the protein expression levels of key genes associated with adipogenesis and inflammation, we performed immunoblotting analysis in adipocytes. A dose-dependent down-regulation of PPAR-γ, SREBP-1c, TNF-α and IL-6 levels but up-regulation of UCP-1 was observed with CFP with profound effect being exhibited at 200 µg/ml when compared to control cells (Fig. 6).

Fig. 6.

Fig. 6

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Effect of CFP on protein expression levels in untreated and treated adipocytes. a Expression of UCP-1, PPAR-γ, SREBP-1c, TNF-α, IL-6 and β-actin. b Quantification of the protein expression levels. Data are presented as mean ± SD (n = 3). * and ** indicate significant difference between untreated and CFP-treated adipocytes at p < 0.05 and p < 0.01, respectively

Discussion

Lipid accumulation and adipocyte differentiation are the two major contributing factors for development of obesity and associated ailments. Therefore, discovering drugs/formulations that can target to reducing lipid digestion, absorption and accumulation or that can enhance lipolysis of already accumulated lipids is an effective strategy to regulate obesity. Another approach is to develop agents that can inhibit adipogenesis by interfering at transcriptional level or by influencing the genes either up-stream or down-stream of the master regulators of adipogenesis. Discovering molecules that can promote thermogenesis is another means to contain obesity. Therefore, it is obvious that a multi-target approach can be more effective in curbing obesity and co-morbidities and the present study was focused on these themes (Balaji et al. 2016).

In the present study, CFP was isolated from its heartwood and evaluated for its anti-adipogenic and anti-inflammatory activity in 3T3-L1 adipocytes. The CFP did not show any cytotoxic activity till a dose of 200 μg/ml either at 24 h or 48 h (Fig. 2). Reduced lipid accumulation and adipogenesis and increased release of glycerol in to the medium by CFP-treated 3T3-L1 adipocytes indicate its lipolytic and anti-adipogenic effects (Fig. 3). Adipocyte differentiation and adipogenesis occur due to coordinated regulation of genes (C/EBPs and PPAR-γ) at transcriptional level in a sequential manner (Lee et al. 2019). Similarly, activation of SREBP-1c and down-stream genes like FAS and ACC are responsible for triglyceride accumulation in adipocytes (Moseti et al. 2016). In our studies, the reduced adipogenesis and lipid accumulation in CFP-treated adipocytes could be due to PPAR-γ and SREBP-1c mediated down-regulation at transcriptional level and this is also further evident from protein expression studies (Figs. 5 and 6).

Leptin and adiponectin are key adipokines of adipose tissue and their secreted levels vary with severity of obese condition. They have regulatory role on energy homeostasis, insulin resistance, inflammation and adipogenesis (Stern et al. 2016). In the present context, the results of ELISA depict that CFP-treated adipocytes showed reduced leptin but enhanced adiponectin secretion. This is in accordance with our RT-PCR results where decreased mRNA expression of leptin but increased adiponectin levels was observed in CFP-treated cells when compared to untreated cells (Fig. 5). Decreased leptin and elevated adiponectin secretion by CFP could be attributed to attenuation of adipogenesis brought about by CFP at molecular level (Fig. 4a).

Thermogenesis is a key process to reduce obesity through fat burning by activating UCP-1 (Liu J et al. 2019). In the present study, enhanced mRNA and protein expression of UCP-1 was observed in CFP-treated adipocytes (Figs. 5a and 6). Higher UCP-I levels favor heat generation which could enhance cellular energy catabolism and energy expenditure. Previous studies using β3-adrenergic receptor (β3-AR) activators confirmed that the sympathetic nervous system was the main trigger of UCP-1 activation, but the mechanism is not clear (Szentirmai and Kapas 2017).

A chronic low-grade inflammatory state has been attributed to adipose tissue enlargement and its dysfunction. This process may lead to activation of pro-inflammatory cytokines, TNF-α, IL-6 and monocyte chemoattractant protein-1 (MCP-1) and others (Park et al. 2014; Ellulu et al. 2017). This study demonstrates that CFP-treated LPS-induced adipocytes showed reduced secretion of TNF-α and IL-6 and also their down-regulation at mRNA and protein expression level demonstrating CFP’s anti-inflammatory activity (Fig. 4b). The reduced cytokines levels can be attributed to decreased adipogenesis by phytochemicals present in CFP.

Conclusion

Our results demonstrate that CFP at 200 μg/ml had substantially reduced lipid accumulation, adipogenesis and inflammation in 3T3-L1 cells through down-regulation of PPAR-γ, SREBP-1c, leptin, TNF-α, IL-6 and up-regulation of adiponectin and UCP-1 expressions. Hence, it is suggested that the heartwood of P. santalinus could be beneficial to treat obesity and inflammation.

Acknowledgements

The authors thank and acknowledge Indian Council of Medical Research, Govt. of India, New Delhi [Grant number: 59/25/2011/BMS/TRM, dated: 23-03-2015] for their support. The authors thank and acknowledge Dr. K. Suresh Babu, Principal Scientist, Indian Institute of Chemical Technology, Hyderabad, for supporting isolation and purification of phyto-constituents.

Abbreviations

CFP

Chloroform fraction of P. santalinus

PPAR-γ

Peroxisome proliferator-activated receptor-γ

SREBP-1c

Sterol regulatory element-binding protein-1c

UCP-1

Uncoupling protein-1

TNF-α

Tumor necrosis factor-α

IL-6

Interleukin-6

DMEM

Dulbecco’s Modified Eagle’s medium

FBS

Fetal bovine serum

MDI

Differentiation medium of induction

IBMX

3­Isobutyl­1­methyl­xanthine

MTT

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

Author contributions

CAR and M. Balaji conceived and developed the idea. RSK have done extraction and fractionation of P. santalinus. RSK, OL, and GMS conducted in vitro experiments (cell culture studies). RSK, GMS, OL and MB collected the data and performed statistical analysis. RSK, CAR, and MB analyzed data and/or interpreted the results. RSK and MB wrote the manuscript.

Availability of data and material

The analyzed and/or used datasets presented herein are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors have no conflict of interest to declare.

Consent for publication

All the authors here by give their consent for publication of the revised manuscript.

Contributor Information

Karunakaran Reddy Sankaran, Email: karunakaranrs.1986@gmail.com.

Muni Swamy Ganjayi, Email: muni.gbch@gmail.com.

Lokanatha Oruganti, Email: oruganti.loka@gmail.com.

Appa Rao Chippada, Email: chippadar@yahoo.com.

Balaji Meriga, Email: balaji.meriga@gmail.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The analyzed and/or used datasets presented herein are available from the corresponding author upon reasonable request.

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