Plumbagin exerts antiobesity effects through inhibition of pancreatic lipase and adipocyte differentiation

S.A. Pai1 | E.A.F. Martis2 | S.G. Joshi3 | R.P. Munshi3 | A.R. Juvekar1

1 Pharmacology Research Laboratory 1, Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Nathalal Parekh Marg, Mumbai, Maharashtra 400 019, India
2 Molecular Simulations Group, Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Kalina, Santa Cruz (E), Mumbai, Maharashtra 400 098, India
3 Department of Clinical Pharmacology, T. N. Medical College and BYL Nair Charitable Hospital, Dr. A. L Nair Road, Mumbai Central, Mumbai, Maharashtra 400 008, India
Correspondence
Prof. Archana Juvekar, A‐243, Pharmacology Research Laboratory‐1, Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga, Mumbai, Maharashtra 400019, India.
Email: [email protected]

1 | INTRODUCTION

The roots of Plumbago zeylanica Linn. (Chitraka) have been described in ancient Ayurvedic texts to possess antiobesity and hypolipidemic activities (Kumari, Pushpan, & Nishteswar, 2013). Furthermore, the aqueous extract of roots of P. zeylanica has shown promising antihyperlipidemic effect in rats (Pendurkar & Mengi, 2009) as well as in rabbits (Sharma, Gusain, & Dixit, 1991). In the light of these observations, we evaluated the antiobesity potential of plumbagin, a naphthoquinone and one of the active constituents found in the roots of P. zeylanica. Plumbagin has been reported to have anti‐ inflammatory, antimicrobial, anticancer, and antidiabetic activities (Panichayupakaranant & Ahmad, 2016).

Pancreatic lipase (PL) is involved in the absorption of dietary lipids from the gastrointestinal tract. Therefore, inhibiting PL is one of the important strategies for combating obesity (Chatzigeorgiou, Kandaraki, Papavassiliou, & Koutsilieris, 2014). Thus, the aim of this study was to determine the PL inhibitory potential of plumbagin. The preliminary binding affinity was computed using molecular docking simulation and validated by in vitro inhibition of PL. Furthermore, the effect of plumbagin on acute lipid absorption in rats, fed a high‐fat diet, was also studied. The effects of plumbagin on in vitro adipocyte differenti- ation were investigated using 3T3‐L1 cells.

2 | MATERIAL AND METHODS

3T3‐L1 fibroblast cell line was obtained from National Centre for Cell Science, Pune, India. 3‐Isobutyl‐methylxanthine, insulin (from bovine pancreas), dexamethasone, porcine pancreatic lipase (Type II), p‐nitro- phenyl palmitate (p‐NPP), thiazolyl blue tetrazolium bromide (MTT), and plumbagin were procured from Sigma Ltd., Bengaluru, India. Fetal bovine serum, Dulbecco’s modified Eagle medium, and trypsin were procured from Gibco™, Thermo Fisher Scientific. Orlistat was provided as a gift sample by Macleods Pharma Pvt. Ltd., Mumbai, India. Intralipid™ 10% (Fresenius‐Kabi) was purchased from a local drug store whereas triglycerides (TG) kit was purchased from Erba Mannheim GmbH. All other chemicals used in the study were of analytical grade.

2.1 | Molecular docking

The binding affinity of plumbagin against porcine PL was estimated by molecular docking approach using GLIDE (Friesner et al., 2004) mod- ule in the Schrödinger suite. The X‐ray crystal structures for human pancreatic lipase (PDB id: 1LPB; Egloff et al., 1995) and the porcine pancreatic lipase (PDB id: 1ETH) were imported from the protein data bank. The colipase chain and other heteroatoms, except the C11 alkyl phosphonate (MUP), were deleted. The X‐ray structures were corrected using the Protein Preparation Wizard in the Schrödinger suite. This step composes of adding hydrogen atoms, assigning bond orders, assigning atom types according to the OPLS‐2005 force field, and deleting water molecules beyond 5 Å from the ligand centre of mass. The protonation states for amino acid residues were generated using PROPKA at pH 7.4. Subsequently, the structures were mini- mized in vacuum using the restrained minimizer until the convergence criteria for heavy atom root‐mean‐squared deviation of 0.01 Å was achieved. The two X‐ray structures were aligned using Maestro‐GUI, and MUP coordinates from human PL were copied on the porcine PL. One more round of energy minimization was performed to clear any clashes that might have appeared during the coordinate transfer. Plumbagin structure was prepared for docking using the LigPrep mod- ule in the Schrödinger suite.

The minimized complex was used to prepare the necessary files for molecular docking using GLIDE. Then MUP centre of mass was used to define the centre of the grid for docking. The van der Waals radii for ligand and protein atoms were scaled down by a factor of 0.8 to allow sufficient cushioning effect during molecular docking. The hydroxy groups in serine, threonine, and tyrosine residues in the binding site were free to rotate to maximise the chance of forming hydrogen bonds between the ligand and receptor atoms. The docking protocol was validated by redocking MUP (cocrystallised ligand) starting from various random conformations generated using ConfGen module in the Schrödinger suite. The docking conformations were scored using the GLIDE standard scoring function, and the best con- formation was selected on the basis of the GLIDE Emodel score.

2.2 | In vitro PL inhibition assay

In vitro PL inhibition assay was carried out using p‐NPP as the substrate (Slanc et al., 2009). Briefly, 16 μl of inhibitor (different concentrations of plumbagin), 12 μl of porcine PL, and 162 μl of 75 mM Tris–HCl buffer (pH = 8.5) were added to a 96‐well microplate and incubated at room temperature for 30 min. Then 10 μl of p‐NPP was added, and the micro- plate was incubated for another 30 min. A series of blank solutions were made as described above but without the enzyme. The actual absorbance (Atestactual) of the test solutions were calculated as the dif- ference between the observed absorbance of the test solution and the absorbance of the corresponding blank solution. The absorbances of the solutions were determined at 405 nm. The control solution consisted of p‐NPP, enzyme, and buffer. The percentage inhibition of the test solutions was calculated using Equation 1 given below
Acontrol−Atestactual.

2.3 | Kinetics of PL inhibition by plumbagin

To determine the kinetics of inhibition of PL by plumbagin, the enzyme concentration was held constant (1 mg/ml) whereas the sub- strate (p‐NPP) concentration was varied (0, 250, 500, 1,250, 2,500, 5,000, and 10,000 μM). Three concentrations of plumbagin (50, 100, and 200 μM) were incubated, each with the different concen- trations of p‐NPP as described for the determination of IC50. A Lineweaver–Burk plot was used to determine the type of inhibition (Wang et al., 2014).

2.4 | Oral fat tolerance test

The protocol for the animal study was approved by the Institutional Animal Ethics Committee of T. N. Medical College and BYL Nair Hos- pital, Mumbai, India (Protocol number: IAEC/2016/8A). Male Wistar rats (180–200 g) were procured from the Central Animal House of Bombay Veterinary College, Parel, Mumbai, and acclimatized to the conditions of the animal house for 1 week. The rats were placed in polypropylene cages and had access to food (Amrut rat and mice food manufactured by Pranav Agro Ltd., Sangli, Maharashtra, India) and water ad libitum. For the oral fat tolerance test (OFTT), the rats were randomised into four groups, each containing eight rats and received treatments as follows:

Group 1: Vehicle Control (0.5% carboxymethylcellulose)
Group 2: Orlistat (45 mg/kg p.o.)
Group 3: Plumbagin (0.5 mg/kg p.o.)
Group 4: Plumbagin (1 mg/kg p.o.)

The rats were fasted overnight, and the next morning, blood was withdrawn (0 hr) from the retro‐orbital plexus under light ether anaes- thesia. The rats then received the different treatments as described above. Thirty minutes later, the rats were fed Intralipid™ orally and then blood was withdrawn after 1, 2, 3, 4, and 6 hr, serum was sepa- rated and used for the estimation of TG and the area under the curve of serum TG from 0 to 6 hr (Tamaru et al., 2013).

2.5 | Cell culture and cell viability studies

3T3‐L1 preadipocytes were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum in a carbon diox- ide (5%) incubator maintained at 37 °C. The cells were subcultured when they reached about 80–90% confluency.Cell viability was measured using (3‐(4,5‐dimethylthiazol‐2‐yl)‐ 2,5‐diphenyltetrazolium bromide; MTT) assay. The stock solution of plumbagin (1 mg/ml equivalent to 5,319.14 μM) was prepared in dimethyl sulfoxide (DMSO), and the concentrations used for the cell viability studies were diluted using 0.9% sodium chloride. The con- centration of DMSO in the final dilutions was not more than 0.1%. The cells were seeded in a 96‐well plate (1 * 105 cells/well) and treated with 0.05, 0.1, 0.26, 0.53, 1.06, and 2.65 μM of plumbagin and isopropanol was added to each well to solubilize the formazan crystals. The absorbance of the solution was measured at 570 nm, and cell viability was expressed as a percentage of cell control (Mangal et al., 2017).

2.6 | Cell differentiation

The confluent cells (1 * 104 cells/well) were seeded in a 24‐well plate. To induce differentiation, on day three, 1 ml of a cocktail of insulin, IBMX, and dexamethasone was added to the wells along with different concentrations of plumbagin (0.05, 0.1, 0.26, 0.53, and 1.06 μM). After 48 hr, insulin was added. Pioglitazone (0.003 μM) was used as a positive control as it stimulates the dif- ferentiation of preadipocytes to adipocytes.
Undifferentiated cells (cell control, CC) served as negative control. The media was replaced every 48 hr. The experiments were carried out in repli- cates (n = 6; Mangal et al., 2017).

2.7 | Oil red O staining and TG estimation

On the 10th day after differentiation of adipocytes, the cells were fixed with 10% formalin followed by staining with oil red O (ORO). The plate was incubated for 30 min. The ORO was aspirated, and the stained cells were washed with isopropanol. The absorbance of the solution was read at 500 nm. For the estimation of TG, the cells were trypsinised, centrifuged, and the cell pellet was treated with lysis buffer (50 mmol/L Tris, 0.15 mol/L sodium chloride, 10 mmol/L ethyl- ene diamine tetraacetic acid, 0.1% Tween‐80, and adjusted to pH 7.5). The TG contents of the cell lysate were determined using a commer- cially available kit. The TG content of the adipocytes was expressed as milligram per milligram protein (Mangal et al., 2017).

2.8 | Statistical analysis

Data are represented as mean ± SEM. Statistical analysis was carried out using GraphPad Prism version 5, CA, USA using two‐way analysis of variance (ANOVA) followed by Bonferonni post hoc test for OFTT and one‐way ANOVA followed by post‐hoc Tukey’s multiple compar- ison test for cell viability, ORO staining, and estimation of TG contents in adipocytes. Differences were considered to be statistically significant if p < .05.

FIGURE 1 Plumbagin inhibits pancreatic lipase enzyme. (a) Binding conformation of plumbagin predicted by molecular docking. (b) Lineweaver– Burk plot of reaction rate versus substrate concentration in the presence of different concentrations of plumbagin (n = 3). (c) Effect of plumbagin on oral fat tolerance test. Data are represented as mean ± SEM of eight rats. *p < .05; **p < .01 when compared with vehicle control group (two‐ way analysis of variance followed by Bonferroni's test), n = 8. (d) Area under the curve for serum triglycerides from 0 to 6 hr. Data are represented as mean ± SEM of eight rats. *p < .05; **p < .01 when compared with vehicle control group one‐way analysis of variance followed by Tukey's multiple comparison test. VC = vehicle control; Orli = orlistat 45 mg/kg p.o; PL‐0.5 = plumbagin 0.5 mg/kg p.o.; PL‐1 = plumbagin 1 mg/kg p.o. (n = 8) [Colour figure can be viewed at wileyonlinelibrary.com]

3 | RESULTS

3.1 | Molecular docking

Molecular docking revealed that the naphthoquinone carbonyl group overlaps with the phosphonate group from the MUP. However, the major difference in their binding is that MUP is covalently bound to PL through Ser153 whereas plumbagin binds noncovalently with a strong coulombic interaction between the naphthoquinone carbonyl group and the Ser153 side chain that is a part of the catalytic triad. This indicates that plumbagin contains necessary chemical features to strongly inhibit PL. Moreover, the protein–ligand interactions between plumbagin and PL are dominated by van der Waals interactions (Figure 1a).

3.2 | Plumbagin inhibits PL in vitro through mixed type of inhibition

The IC50 for plumbagin for porcine PL was found to be 82.08 ± 9.47 μM. The inhibition was found to be of mixed type as seen from the Lineweaver–Burk plot (Figure 1b).The km in the absence of plumbagin, 50, 100, and 200 μM of plumbagin were found to be 613.3 ± 77.35; 652.48 ± 92.54; 692.93 ± 86.05; and 735.53 ± 82.21 μM, respectively.The Vmax in the absence of plumbagin, 50, 100, and 200 μM of plumbagin were found to be 504.54 ± 7.31; 387.79 ± 49.82; 302.54 ± 50.77; and 291.29 ± 27.12 μM/min, respectively.

3.3 | Plumbagin inhibits lipid absorption in vivo after OFTT

Serum TG increased until 2 hr after Intralipid™ administration in all the groups. Plumbagin 1 mg/kg significantly reduced serum TG at 2, 3, 4, and 6 hr (p < .05) when compared with vehicle control. Treatment with orlistat significantly reduced serum TG compared with vehicle‐treated group at 1, 2, 3, 4 (p < .01), and 6 hr (p < .05; Figure 1c). The integrated area under the curve of serum TG from 0 to 6 hr for orlistat (p < .01) and PL‐1 (p < .05) groups were also significantly reduced compared with vehicle‐treated group (Figure 1d).

3.4 | Effect of plumbagin on adipocyte viability and differentiation

Plumbagin at concentrations from 0.05–1.06 μM did not cause cyto- toxicity when compared with untreated cells (cell control); however, it was found to be cytotoxic at 2.65 μM (Figure 2a). Hence, we evalu- ated the concentrations of plumbagin up to 1.06 μM for ORO staining and TG estimation. Upon treatment with differentiation medium, the preadipocytes differentiated into adipocytes with a resultant signifi- cant increase in ORO staining when compared with undifferentiated cells. Pioglitazone treatment further augmented the differentiation of adipocytes. Treatment with plumbagin at 0.05, 0.1, 0.26, 0.53, and 1.06 μM resulted in a dose‐dependent decrease in ORO staining (Figure 2b). However, only cells treated with 1.06 μM showed a significant decrease in ORO staining and TG content when compared with differentiated control. The effect of PL on TG accumulation in adipocytes was in agreement with that of ORO staining with1.06 μM showing a significant decrease in TG content when compared with differentiation control (Figure 2c).

FIGURE 2 Effect of plumbagin on cell viability and differentiation of adipocytes. (a) Plumbagin at concentrations up to 1.06 μM did not adversely affect the viability of 3T3‐L1 cells when compared with vehicle control. (b) Plumbagin reduced oil red O staining of adipocytes when compared with DC. (c) Plumbagin reduced triglyceride content of adipocytes when compared with DC. *p < .05 when compared with CC and **p < .05 when compared with DC (n = 6). Abbreviations: CC = cell control; DC = differentiation control; Pio = pioglitazone; VC = vehicle control; PL‐0.05, PL‐0.1, PL‐0.27, PL‐0.53, PL‐1.06, and PL‐2.65 = plumbagin 0.05, 0.1, 0.27, 0.53 and 1.06 and 2.65 μM, respectively.

4 | DISCUSSION

The current work was embarked upon to investigate the antiobesity potential of plumbagin and to elucidate its mechanism(s) of action(s). Inhibition of PL is known to be one of the relatively well‐tolerated approaches, until date, for the treatment of obesity as demonstrated by the long‐standing use of orlistat, vis‐à‐vis other antiobesity drugs that were withdrawn from the market owing to safety issues (Chatzigeorgiou et al., 2014). Hence, we first determined the PL inhib- itory potential of plumbagin. Preliminary clues were obtained from molecular docking studies, and it was seen that plumbagin possessed the necessary chemical groups to be identified as a binder of PL. How- ever, there is a large scope to modify plumbagin to explore the large binding cavity for optimal interactions and tight binding. Nonetheless, we believe that plumbagin has shown promising results in the in silico studies that is validated experimentally in this current work. The Vmax decreased in a dose‐dependent manner in the presence of plumbagin. On the contrary, the km increased in a dose‐dependent manner in the presence of plumbagin. We further investigated the mechanism of inhibition, and it was found that plumbagin shows mixed type of inhi- bition as exemplified by the change in km and Vmax of the enzyme in presence of the inhibitor (Cox & Nelson, 2008). The PL inhibitory potential of plumbagin was corroborated in the in vivo studies in rats fed with Intralipid™. Thus, we provide compelling evidence that inhibi- tion of PL is one of the mechanisms responsible for the antiobesity effect of plumbagin.

Hypertrophy of adipocytes is a hallmark of obesity, and molecules that can inhibit the differentiation of adipocytes are believed to act as potential antiobesity drugs (Virtue & Vidal‐Puig, 2008). Therefore, we investigated the effects of plumbagin on the differentiation of adipo- cytes in vitro using 3T3‐L1 cells. Cell viability studies revealed that plumbagin was not cytotoxic up to concentration of 1.06 μM. Further- more, plumbagin also reduced ORO staining and TG accumulation within the adipocytes, suggesting that it possesses antiadipogenic activity.

Therefore, we conclude that plumbagin exhibits potential antiobe- sity effects by inhibiting PL as well as by inhibiting the differentiation of adipocytes. The effects of plumbagin on markers of adipocyte differentiation, fatty acid synthesis, lipolysis, and apoptosis warrant further investigation.

ACKNOWLEDGEMENTS

This work was supported by a grant from University Grants Commis- sion, New Delhi, India (Letter no. F.25‐1/2014‐15 (BSR)/No. F.5‐63/ 2007 (BSR) dated on 16 Feb 2015) awarded to the first author, Ms. Sarayu Pai. The authors wish to thank Dr. Ashish Mungantiwar, Senior Vice President, Macleods Pharmaceuticals Ltd. for providing a gift sample of orlistat and Ms. Chaitali Deshpande for her assistance in carrying out in vitro adipocyte differentiation studies.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

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