Antidiabetic effect of mangiferin in combination with oral hypoglycemic agents metformin and gliclazide

Accepted Manuscript

Antidiabetic effect of mangiferin in combination with oral hypoglycemic agents metformin and gliclazide Vidhushini Sekar , Sugumar Mani , R. Malarvizhi , P. Nithya , Hannah R Vasanthi PII: DOI: Article Number: Reference:

S0944-7113(19)30071-6 https://doi.org/10.1016/j.phymed.2019.152901 152901 PHYMED 152901

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

7 November 2018 19 March 2019 21 March 2019

Please cite this article as: Vidhushini Sekar , Sugumar Mani , R. Malarvizhi , P. Nithya , Hannah R Vasanthi , Antidiabetic effect of mangiferin in combination with oral hypoglycemic agents metformin and gliclazide, Phytomedicine (2019), doi: https://doi.org/10.1016/j.phymed.2019.152901

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ACCEPTED MANUSCRIPT

Antidiabetic effect of mangiferin in combination with oral hypoglycemic agents metformin and gliclazide Vidhushini Sekara, Sugumar Mania, Malarvizhi. Ra, Nithya. Pa, Hannah R Vasanthia,* *, a

Department of Biotechnology, School of Life Sciences, Pondicherry University,

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Puducherry, India

Running Title: Synergistic effect of mangiferin with hypoglycaemic agents

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*Author correspondence A. Hannah Rachel Vasanthi Professor

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Department of Biotechnology

Pondicherry University Puducherry- 605014

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Tele: 0413-2655 745

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School of Life Sciences

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Tele Fax: 0413-2655 714

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eMail ID : [email protected]

ACCEPTED MANUSCRIPT Abstract Background Diabetes mellitus poses serious threat to the global population due to the alarming diabetic complications it leads to. The current therapeutic options available can be improved for better efficiency and maximum benefits. Combination therapy has been commonly used

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to improve the efficacy and to minimize the side effects of drugs in current clinical use. Purpose

The present study aims to assess the interaction between a natural molecule mangiferin with the commercially available oral hypoglycemic drugs metformin and

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gliclazide in diabetic rats. Methods

In this study, the in vitro cytotoxicity and glucose uptake studies were performed in HepG2 cells. Based on experimental data, the combination index of the hypoglycemic drugs like metformin and gliclazide in combination with different doses of mangiferin was

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determined using COMPUSYN software. Further, in vivo studies were performed in HFD + STZ induced diabetic male Sprague Dawley rats. Serum parameters, enzyme markers, hepatic

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oxidative stress markers, gene and protein expression studies and histopathological analyses

administration.

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Results

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were performed in rat liver to identify the mode of action of the combination drug

The in vitro studies on HepG2 cells suggest a positive interaction of mangiferin with

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both metformin and gliclazide at specific concentrations as evidenced by glucose uptake. The hepatic enzymes, oxidative stress markers, carbohydrate metabolizing enzymes, gene (AMPK, Akt, ACC β and Glut-2) and protein (PPARα, PPARγ) expression confirmed the results of the in vitro studies. Both the combinations of mangiferin with metformin and mangiferin with gliclazide exhibited potent antidiabetic effect. The combination of mangiferin with metformin was insulin dependent (Akt pathway) whereas the combination of mangiferin and gliclazide was insulin independent (AMPK pathway). Conclusion

ACCEPTED MANUSCRIPT The overall results suggest that combination of mangiferin with both metformin and gliclazide alleviates diabetic conditions potentially at specific doses and modulates the adverse effect of high dose of commonly used OHD’s. This combination therapy can be translated for its clinical use as a diabetes management strategy. Keywords: Combination therapy, type 2 diabetes mellitus, HepG2 cells, mangiferin,

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metformin, gliclazide List of abbreviations:

ACCβ - Acetyl-CoA carboxylase; ALP - Alkaline phosphatise; ALT – Alanine aminotransferase; AMPKα - 5' adenosine monophosphate-activated protein kinase; AST -

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Aspartate aminotransferase; CI – Combination Index; DMEM - Dulbecco's modified Eagle medium; DMSO - Dimethyl sulfoxide; Glut 2 - Glucose transporter 2; HFD – High fat diet; LDH - Lactate dehydrogenase; MTT-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide; OHD’s – Oral hypoglycemic drugs; PPAR - Peroxisome proliferator-activated receptors; STZ – Streptozotocin; TCA- Tricarboxylic acid; γGT - Gamma-glutamyl

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transferase.

ACCEPTED MANUSCRIPT Introduction Diabetes mellitus (DM) is a chronic metabolic disease affecting millions worldwide. The current estimate of diabetes across the globe is 422 million people which is nearly 8.8% of world adult population (Cho, et al. 2018) and this estimate is said to expand twice by the

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year 2045. Such significant afflicted population can drastically affect the social and economic growth of a country especially a developing country (Datta 2018). Hence, focusing on effective treatment modalities for managing diabetic conditions in a holistic manner is the need of the hour. Phytomedicines and their involvement in diabetes management are

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currently sought after as they are proving to be effective and possess relatively lower sideeffects (Khazaei, et al. 2018). In recent years, combination therapy of different hypoglycemic drugs offers an effective approach of glycemic control in diabetes management (Bell 2013). While combination therapy offers a better approach, involvement of phytochemicals in

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combination therapy offers an even more effective therapeutic intervention with significantly

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reduced side effects of the chemically synthesized OHDs prescribed (Prabhakar, et al. 2014; Prabhakar and Doble 2009).

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Mangiferin(1,3,6,7-Tetrahydroxy-2-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-

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(hydroxymethyl)oxan-2-yl]xanthen-9-one) is a glycosyl xanthone found in certain medicinal plants such as Salacia spp. and more abundantly in Mangifera indica L. (Anarcadiaceae)

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Some of the pharmacological properties of mangiferin include antioxidant, antimicrobial, anticancer, antiinflammatory and most notably antidiabetic activity (Fomenko and Chi 2016). Further, interaction studies of mangiferin with commercially prescribed hypoglycemic drugs such as sitagliptin, a dipeptidyl peptidase-4 inhibitor (Hou, et al. 2012) and combination of mangiferin with berberine improves glucose uptake in L6 myotubes (Xuejian, et al. 2008) . Hence, this study has investigated the antidiabetic properties of mangiferin in combination with commonly consumed oral hypoglycemic drugs (OHD’s) in HepG2 cells and

ACCEPTED MANUSCRIPT subsequently went on to confirm them in vivo in experimentally induced diabetic rats. In addition to this, the possible mode of action (insulin dependent or independent pathway) of the combinations was also evaluated by analyzing target genes and proteins. Materials and methods

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Chemicals Bovine serum albumin (BSA), DMEM high glucose, heat in-activated foetal bovine serum (FBS), L-glutamine, penicillin, streptomycin and non-essential amino acids were procured from Himedia chemicals Pvt. Ltd., (Mumbai, India), STZ (streptozotocin) was

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purchased from Santa Cruz Biotechnology, (Dallas, Tx, USA). Mangiferin was purchased from Nanjing Nutri Herb BioTech Co.,Ltd, (Nanjing, China) (purity 99.7% by HPLC (Area normalization method), equal to 95.6% by HPLC (external standard method) and

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comply with CP2015 standard). Metformin was purchased from MP Biomedicals, (Solon, Ohio, USA), gliclazide, thiobarbituric acid and TRIzol reagent were purchased from Sigma

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Aldrich, (St. Louis, MO, USA). All the primers were purchased from Eurofins India Pvt.Ltd,

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(Bengaluru, Karnataka, India), Rabbit polyclonal IgG PPAR-α (Cat no. sc-9000) and PPAR-γ antibodies (Cat no. sc-7196), mouse anti-rabbit IgG-HRP secondary antibodies (Cat no. sc-

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2357) were procured from Santa Cruz Biotechnology. HepG2 cell line was procured from National Centre for Cell Science (Pune, Maharashtra, India). Unless mentioned, all other

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chemicals and reagents were of analytical grade. Cell culture and treatment Human liver cancer HepG2 cells were cultured in Dulbecco’s modified Eagle Medium, supplemented with 10% (v/v) FBS, 2 mmol/l L-glutamine, 50 U/ml penicillin, 100

ACCEPTED MANUSCRIPT mg/l streptomycin and non-essential amino acids, and maintained at 37˚C in a humidified 5% CO2 atmosphere. Medium was changed every 48 h. Cell cytotoxicity assay HepG2 cells were seeded on 96-well plates at a density of 0.5 X 105 cells/well. The

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cultures were grown for 24 h followed by the addition of fresh medium containing different doses of mangiferin, metformin and gliclazide (5, 10, 20, 40, 60, 80, 125, 250, 500 µM). Cell viability was determined by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Rao, et al. 2009). After incubation for 24 h with each drug (mangiferin,

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metformin, gliclazide), 50 µl of MTT reagent (5 mg/ml MTT in phosphate-buffered saline) was added to each well and incubated in a CO2 incubator for 4 h. The medium was aspirated from each well and dissolved with the remaining MTT-formazan crystals by adding 200 µl of

Combination Index assessment

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DMSO to all the wells and the absorbance was measured in a micro plate reader at 570 nm.

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The combination of mangiferin with hypoglycemic drugs at different concentrations (mangiferin: 80, 40, 20, 10 µM, metformin: 20, 10, 5, 2.5 µM, gliclazide: 10, 8, 4, 2, 1 µM)

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and their effects were analysed. The combination index CI was determined by employing the

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COMPUSYN software (ComboSyn, Inc. 2005) using the formula CI = (Ca/ICa)+(Cb/ICb) where Ca and Cb are the concentrations of compound A and compound B used in

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combination to achieve a desirable effect herein, the glucose uptake. ICa and ICb are the concentration of the compound required individually to achieve the same effect. A CI < 1 indicates synergy, CI = 1, additivity; CI > 1 antagonism, between the compounds. Glucose uptake assay employing 2-deoxyglucose Cellular glucose uptake in the HepG2 cells was quantified by the 2-NBDG (2-(N-(7nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose) assay using a microplate reader

ACCEPTED MANUSCRIPT (Biotek, Winooski, VT, USA). Cells were plated in 24-well plates treated with mangiferin (80, 40, 20, 10 µM), metformin (20, 10, 5, 2.5 µM), gliclazide (10, 8, 4, 2, 1 µM) individually and in combinations of mangiferin + metformin (5.0+40.0, 10.0+20.0, 20.0+10.0, 40.0+5.0, 80.0+2.5 µM respectively) and mangiferin + gliclazide (80.0+2.0, 40.0+4.0, 20.0+ 8.0, 10.0+10.0 µM respectively). 2-NBDG was added at 50 µM concentration and incubated for

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30 min at 37°C. Cells were washed twice with PBS followed by addition of serum-free medium and then fluorescence intensity was immediately measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. After being taken up by the cells, 2-NBDG was converted to a non-fluorescent derivative (2-NBDG metabolite). A fair

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estimation of the overall glucose uptake was obtained by quantifying the fluorescence (Prabhakar and Doble 2009).

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Experimental animals

Adult male Sprague Dawley rats (160 - 180g) were procured from Invivo

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Biosciences, Bengaluru, India and were maintained at 25 ± 2оC in 12-h dark/12-h light cycles, with both standard pelleted diet and water ad libitum in accordance to the CPCSEA

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guidelines. After the initial acclimatization period, except for the normal control group, all

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other group animals received high fat diet (HFD) from VRK Nutrition Solutions, Pune, India. The composition of HFD was casein 30%, cholesterol 10%, ground nut oil 14%, corn starch

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41%, vitamin/mineral 05%, (protein 24%, carbohydrate 41% and fat 24%). The experiments were carried after necessary clearances from the Institutional Animal Ethics Committee of Pondicherry University, India. (Approval No:PU/SLS/AH/2016/02) and the experiments were carried out as per CPSCEA guidelines (Govt. of India). Diabetes induction and in vivo experimental design

ACCEPTED MANUSCRIPT Followed by 8 weeks of HFD, the experimental rats were fasted overnight and injected with STZ (35 mg/kg b.w) in citrate buffer (pH 4.5) intraperitoneally. After 72 h, blood was collected and animals with glucose level ≥ 250 mg/dl were considered diabetic. Followed by diabetes induction, the animals were administered with the test drugs and their combinations orally for a period of 28 days. The dose was determined from earlier literature

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sources (Sellamuthu, et al. 2009) and accordingly calculated based on human dose to animal dose conversion technique (Nair and Jacob 2016). Further, based on the results of the in vitro combination study, the doses for the combination drug treatment were fixed.

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Group I (Normal control: abbreviated as NC): rats received standard pellet diet with neither diabetes induction nor drug treatment and considered as vehicle control Group II (Diabetic control: abbreviated as DC): rats received HFD + STZ and no treatment

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mangiferin (40 mg/kg b.w)

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Group III (mangiferin: abbreviated as Mangi): rats received HFD + STZ and treated with

Group IV (metformin: abbreviated as Met): rats received HFD + STZ and treated with

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metformin (100 mg/kg b.w)

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Group V (mangiferin + metformin: abbreviated as Mangi + Met HD): rats received HFD + STZ and treated with the combination of mangiferin (40 mg/kg b.w) and metformin (100

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mg/kg b.w)

Group VI (mangiferin + metformin: abbreviated as Mangi + Met LD): rats received HFD + STZ and treated with the combination of mangiferin (40 mg/kg b.w) and metformin (50 mg/kg b.w) Group VII (gliclazide: abbreviated as Gli): rats received HFD + STZ and treated with gliclazide (10 mg/kg b.w)

ACCEPTED MANUSCRIPT Group VIII (mangiferin + gliclazide: abbreviated as Mangi + Gli HD): rats received HFD + STZ and treated with the combination of mangiferin (40 mg/kg b.w) and gliclazide (10 mg/kg b.w) Group IX (mangiferin + gliclazide: abbreviated as Mangi + Gli LD): rats received HFD +

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STZ and treated with the combination of mangiferin (40 mg/kg b.w) and gliclazide (5 mg/kg b.w)

Biochemical analysis of serum parameters and enzyme markers

At the end of the experimental period (28 days), approximately 2 ml blood was

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collected through retro-orbital puncture in a sterile micro centrifuge tube for serum separation and stored at -80º C until further analysis. The separated serum was used to check serum glucose, triglycerides and cholesterol levels using Accurex kits (Boisar, Maharashtra, India).

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Serum insulin, a specific marker of type II diabetes was quantified using an ELISA kit (Ray Biotech, GA, USA) as per manufacturer’s instruction and the OD was measured at 450 nm in

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a microplate reader (BIO- RAD). Accordingly, the insulin resistance was determined using

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the homeostasis model assessment formula. Insulin resistance = [Glucose (mM/l) X Insulin (mU/l)]/ 22.5. The serum enzyme (ALT, AST, ALP, LDH and γGT) levels were estimated

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using Spinreact kits (Girona, Catalonia, Spain).

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Measurement of hepatic markers of oxidative stress A portion of liver was taken from all the groups and 10% w/v homogenate was

prepared in 10 mmol potassium phosphate buffer (pH 7.4) for the determination of lipid peroxidation (LPO) by thiobarbituric acid reactive substances (TBARS) (Ohkawa, et al. 1979) and reduced glutathione (GSH)(Moron, et al. 1979). The remaining portion of liver was taken from all the groups and 10% w/v homogenate was prepared in 0.05 M phosphate buffer (pH 7.4). The homogenate was subjected to cold centrifugation at 4◦C for 20 min and

ACCEPTED MANUSCRIPT used for the estimation of superoxide dismutase (SOD) (Marklund and Marklund 1974), catalase (CAT) (Sinha 1972), Glutathione Peroxidase (GPx) (Rotruck, et al. 1973) Glutathione S Transferase (GST) (Habig, et al. 1974) and protein content (Lowry, et al. 1951).

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Measurement of hepatic carbohydrate metabolizing enzymes The activity of the hepatic carbohydrate metabolizing enzymes such as Hexokinase D (Brandstrup, et al. 1957), 1,6-Bisphosphatase (Gancedo and Gancedo 1971), Glucose 6phosphatase (Hikaru and Toshitsugu 1959) and Glucose 6-phosphate dehydrogenase (Ells

Relative quantification of gene expression

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and Kirkman 1961) were estimated in the liver homogenate using standard protocols.

Quantitative real time PCR (qPCR) amplifications of Glut 2, AMPK, Akt, ACCβ and

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GAPDH were performed in triplicates using a Roche master cycler. The total RNA was extracted from the liver tissue using TRIzol Reagent (Sigma-Aldrich). After homogenization,

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the tubes were incubated for 10 min and centrifuged at 12,000 g for 5 min and the RNA was

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precipitated after processing with suitable reagnts and the pellets were resuspended in RNase free water and stored at −80°C until use. The isolated RNAs were subjected to reverse

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transcription and polymerization reactions to obtain cDNA using the PCR master cycler gradient (Agilent Technologies Sure Cycler 8800). Reaction mixture contains 1 µl of

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template (cDNA), 300 nmol each of forward and reverse primers and 1x SYBR green PCR master mix (Applied Biosystems, Vilnius, Baltic, Lithuania,). The pre-denaturation condition for RT-PCR was kept at 95˚C for 30s, denaturation at 95°C for 5s and Annealing for 55°C for 10s and amplified for 40 cycles. The changes in fluorescence of SYBR green dye in every cycle were monitored, and threshold cycle (ct) above background for each reaction was calculated. The primer sequences used are as follows, rat AMPK α (Forward 3’ TTC GGG

ACCEPTED MANUSCRIPT AAA GTG AAG GTG GG 5’; reverse 3’ GGT TCT GGA TCT CTC TGC GG 5’), rat Glut 2 (Forward 3’ CAC ATC CTA CTT GGC CTA TCT G; reverse 3’ CTT TGC CCT GAC TTC CTC TT), rat Akt (Forward 3’ GCT GGA GGA CAA CGA CTA TG 5’; reverse 3’ CTT CTC ATG GTC CTG GTT GTA G 5’), rat ACCβ (Forward 3’ CTT GGG GTG ATG CTC CCA TT 5’; reverse 3’ GCT GGG CTT AAA CCC CTC AT 5’), rat GAPDH (Forward 3’

Protein expression analysis by immunoblotting

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AGA CAG CCG CAT CTT CTT GT 5’; reverse 3’ CTT GCC GTG GGT AGA GTC AT 5’).

The liver homogenates prepared using RIPA (Radioimmunoprecipitation assay)

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buffer were centrifuged at 12000 g at 4°C for 15 min and the concentration of the total protein was determined from the supernatant by Lowry’s method. Samples containing 40 g protein were separated in SDS-PAGE (30 mA) and transferred to a nitrocellulose membrane

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(30 mA for 120 min). Membranes were blocked with Tris buffered saline (TBS) containing 5% non-fat dry milk for 2 h to avoid non-specific binding sites and incubated with 1:1000

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dilution of primary antibodies PPAR α, PPAR γ, β actin overnight, and then washed with TBS containing 0.1% Tween-20 and incubated with HRP-conjugated secondary antibody at

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1:2500 dilution for 90 minutes at room temperature (Towbin, et al. 1979). The transferred

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proteins were visualized on X-ray photographic film and quantified by Image J software.

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Histopathological analysis The liver tissue from each group was examined for histopathological changes. Tissues

were fixed in 10% formalin solution and fixed overnight in cassettes. The paraffin-embedded tissues were sectioned on a microtome to a thickness of 5-7 mm, stained with hematoxylin and eosin (H&E), and observed under a light microscope for histo architectural variations and documented by taking photomicrographs. Statistical analysis

ACCEPTED MANUSCRIPT All the data were expressed in mean  standard error mean (SEM). Mean difference between the groups were analysed using one-way ANOVA followed by Tukey’s multiple comparison test as post-hoc using Graph Pad Prism 5.0. A p-value <0.05 was considered to be statistically significant.

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Results Interaction of mangiferin with OHD’s

The cell viability assay was performed to check the concentrations at which the test

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compounds are toxic to the cells. Based on the OD values, IC50 concentrations were calculated and they are as follows: mangiferin (Mangi) 160.1 µM, metformin (Met) 62.21 µM, gliclazide (Gli) 14.82 µM. Based on the cell viability assay, the corresponding doses below the IC50 concentrations for each compound were selected for combination cytotoxicity.

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Among the combinations studied, Mangi exhibited synergistic interaction with both Met and Gli. The combinations of Mangi (10 µM) and Met (20 µM); Mangi (20 µM) and Met (10

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µM); Mangi (40 µM) and Met (5 µM); Mangi (80 µM) and Met (2.5 µM) showed a CI < 1

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and thus showing synergistic interaction. Similarly, the combinations of Mangi (80 µM) with Gli (2 µM); Mangi (40 µM) with Gli (4 µM) and Mangi (20 µM) with Gli (8 µM), displayed

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synergistic action as the CI < 1. The natural compound Mangi exhibited synergistic interaction with both Met and Gli at the above mentioned concentrations and combinations.

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The values showing synergistic action were further chosen for glucose uptake study (Fig. 1a and 1b).

Influence of combined drug therapy on glucose uptake in HepG2 cells The glucose uptake efficiency was measured by employing 2 NBDG glucose uptake assay. The individual drug’s glucose uptake at different doses are tabulated in Table 1.The

ACCEPTED MANUSCRIPT maximum fold change in glucose uptake was seen in Met (40 µM), Gli (8 µM) and Mangi (80 µM). Among the individual glucose uptake, Met (40 µM) produced the highest 2.09-fold change in glucose uptake compared to Mangi and Gli. The combination effect over glucose uptake in HepG2 cells were estimated and are

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tabulated in Table 2 and 3. The high dose combination of Mangi (80 µM) with Met (40 µM) exhibited the maximum glucose uptake which is 1.7-fold and 1.4-fold higher than Mangi and Met individual glucose uptake. Similarly, in the combination of Mangi with Gli the highest fold change of 2.82 was observed in the concentration of Mangi (80 µM) and Gli (8 µM).

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These combination concentrations [Mangi (80µM) + Met (40 µM) and Mangi (80µM) + Gli (8 µM)] exhibited better glucose uptake efficiency in terms of fold change than the individual test drugs.

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In vivo serum biochemical parameters and enzymes of diabetic animals Serum biochemical parameters depict the test drugs’s activity and the physiologic

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outcomes. Elevated blood glucose and depleted insulin levels leading to chronic

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hypoglycaemia was noted in high fat diet and STZ induced diabetic rats as compared to the normal control rats. Interestingly the increased serum glucose levels of drug treated groups

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reduced to 3-fold. However, better reduction was noted in the combination groups, more prominently in the Mangi + Gli HD (82. 29 mg/dl) combination comparable to the NC group

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(84.89 mg/dl) (Fig. 2a). Insulin resistance also contributes to the pathophysiology of diabetes mellitus. In this

study, insulin resistance exhibited by the experimental animals were compared before (0th day) and after (28th day) drug treatment. The reduced insulin levels and insulin resistance was observed in the pre-treatment conditions across all treated groups (Fig. 3). Post-treatment, the resistance to insulin was increased 3-fold in the DC as compared to the normal control (p <

ACCEPTED MANUSCRIPT 0.001). However, it was significantly reduced in all the treated groups (p < 0.01). The most notable activity against insulin resistance was observed in the combination of Mangi + Gli HD (p<0.001). The analysis of serum biochemical parameters, serum triglyceride and serum

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cholesterol levels were also monitored throughout the study. Nearly 2-fold increase in serum triglycerides level was noted in the DC group (168.82 mg/dl) as compared to the NC group (93.46 mg/dl). The combination of Mangi + Met HD and Mangi + Gli HD reduced the triglyceride content in the serum, slightly better than individually treated Mangi (127.1

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mg/dl), Met (121.43 mg/dl) and Gli (127.13 mg/dl) animals (Fig. 2b). However, no remarkable changes were seen in the serum cholesterol levels throughout the study period. Serum hepatic enzymes are markers of hepatocellular injury. The high levels of ALT

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(54.23 U/l), ALP (684.04 U/l), AST (81.41 U/l) in the DC groups are indicative of possible hepatic injury (Table 4). Post-treatment, the combination of Mangi+ Met HD decreased the

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ALT values comparable to NC group (37.10 U/l) and the action of Mangi (43.69 U/l), Met (39.93 U/l) and Gli (38.09 U/l) were in close range to NC group. Likewise, the γGT levels

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were elevated in the DC groups (7.93 U/l). In the drug treated groups however, a marked

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reduced γGT levels were observed especially in the Mangi + Met HD (2.82 U/l) group. From the results, it is clear that individually treated drugs were more effective in bringing down the

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hepatic ALP, AST and LDH enzyme levels better than the combination group. Combined drug therapy on hepatic stress markers In the case of chronic hyperglycemia, the cells are more susceptible to oxidative damage. The extent of oxidative damage was assessed by depleted levels of catalase, SOD, GPx, GST and GSH and high malonaldiehyde (MDA) in the hepatic tissue of diabetic rats (Table 5). Among the treatment groups, the individually treated natural compound Mangi,

ACCEPTED MANUSCRIPT exhibited highest anti-oxidative property followed by the combination groups Mangi+ Met HD and Mangi + Gli HD. The lipid peroxidation levels indicated by high levels of MDA were restored by Mangi (4.73 µM/g tissue) and Met (4.25 µM/g tissue) and also in the combination groups Mangi + Met LD (4.26 µM/g tissue). Similarly the combination of Mangi + Met LD best restored the Gpx activity. Likewise, the depleted levels of GSH were

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also regained in the groups of Mangi + Gli HD (1.35 nM/mg protein). Both the standard drugs Met and Gli equally improved the SOD levels (49.56 U/mt/ mg protein) and (49.28 U/mt/ mg protein) this activity was closely followed by both the combination of Mangi + Met

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HD (48.95 U/mt/ mg protein) and Mangi + Gli HD (48.24 U/mt/ mg protein). Combined drug therapy on carbohydrate metabolizing enzymes

Post-treatment with test drugs restored the elevated levels of gluconeogenic enzymes

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as observed in the DC groups and elevated the enzymes involved in glycolysis (Table 6). The Mangi combination with Met and Gli showed both the gluconeogenic and glycolytic

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activities across the groups. The elevated levels of glucose-6 phosphatase DC (3.29 µm/min/mg protein) and fructose-1,6-bisphosphatase (7.37 µM/min/mg protein) in the

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diabetic group were restored to near normal levels in the treated groups. However, the

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combination of Mangi with Met and Gli was better at lower doses. Similarly, the combination groups Mangi + Met HD (0.400 µM/min/mg protein) and Mangi + Gli HD (0.402

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µM/min/mg protein) restored the depleted levels of hexokinase to NC (0.411 µM/min/mg protein). Likewise, the glucose-6 phosphate dehydrogenase activity in DC (0.072 units/mg protein) were brought to near normal levels of NC (0.90 units/mg protein) by all the groups, particularly in Mangi + Gli HD (0.094 units/mg protein). The molecular mechanisms leading to better glucose metabolizing efficiency was further evaluated by gene and protein expression studies.

ACCEPTED MANUSCRIPT Effect of combination drugs on gene and protein expression markers of glucose metabolism In the present study, the oral hypoglycemic drugs used are of two different classes: a biguanide and a sulfonylurea which act in different but vital pathways. Increased relative expression of AMPKα and Akt influences the increased translocation of Glut 2 for better glucose transport and also suppresses the expression of ACCβ. The combination of Mangi

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with Met significantly upregulated the AMPKα levels (p<0.01). Similarly, the combination of Mangi with Gli significantly increased the Akt levels (p<0.01). The results reveal a better increase in Glut 2 and downregulate ACCβ expression levels in the combination group than

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the individually treated group (p<0.01) as seen in (Fig.4).

The results also showed a downregulation of PPAR α in the DC group. Notably 3-fold increase in the expression of PPAR α in the Mangi + Gli HD combination group followed by

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Mangi + Met HD group was observed. Interestingly, the downregulation of PPAR α in the metformin treated group was comparable to the NC group. In the combination group Mangi +

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Met LD, PPAR α activation could be a possible influence of Mangi (Fig. 5A). However, the combination of Mangi + Gli HD was better in the downregulation of PPAR γ closely

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followed by Mangi + Met HD combination (Fig. 5B). These results can be interpreted as that

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combination drugs have a significant effect over the PPARs and thereby play a possible role both in the glucose and lipid metabolism as seen earlier.

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Effect of combination drugs on histological changes in the hepatic tissue The present study, critically analysed the histological changes in the hepatic tissue.

The drug combination Mangi + Met HD showed better hepatoprotective effect by visibly reducing the necrosis, bile duct hyperplasia and the sinusoidal congestion than the changes observed in individually treated (Mang, Met, Gli) groups as seen in (Fig. 6). Based on the severity of lesions, scoring of hepatic tissue reveals a significant reduction (p <0.001) in

ACCEPTED MANUSCRIPT lesions in the combination groups (Mangi + Met HD and Mangi + Gli HD) than the individually treated or reduced dose combination groups. Discussion As the primary goal was to reduce the side effects of the OHD’s by supplementing

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with higher concentration of natural compound, the combination doses were chosen accordingly (Fig. 1 and 2). Mangi exhibited synergistic interaction with Met at a dose of Mangi 80 µM and Met 2.5 µM. Comparatively, better than the glucose uptake by the individual drugs, the combination of Mangi with OHD’s exhibiting synergistic interactions

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showed an improved glucose uptake. This is in harmony with the earlier work on the similar premise (Prabhakar and Doble 2009; Prabhakar and Doble 2011). Improvement in the glucose uptake efficiency is a forerunner in understanding the drug’s role in the pathology of

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the disease. It is worth to be noted that Mangi and Met improves glucose uptake by the activation of insulin independent Activated Protein Kinase (AMPK) pathway (Niu, et al.

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2012; Zang, et al. 2004) whereas, Gli employs the insulin dependent Protein Kinase B (Akt) pathway for the glucose uptake (Salani, et al. 2005). This would be the possible reason for the

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better glucose uptake of Mangi with Met combination than of Mangi with Gli combination,

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as the former has the combined advantage of acting on the same signaling pathway. Elevated body weight, serum glucose and triglyceride levels correspond to insulin

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resistance mimicking type 2 diabetic conditions. The best reduction in the serum parameters was observed in the combination of Mangi with Gli. Gli achieve anti-hyperglycemic effect by sensitizing the insulin secreting cells which produce more insulin. Almost similar results were observed in the serum hepatic enzyme markers such as ALT, AST, ALP, γGT and LDH. Elevated levels of these enzymatic markers are indicative of liver or tissue injury. Compared to the DC group, the treatment groups showed considerable decreased levels of the

ACCEPTED MANUSCRIPT enzymes in all the treatment groups indicating better compliance. These biochemical analysis are in agreement with earlier works on the combination of metformin and glibenclamide with honey which further improves the glycemic control by the regulation of serum parameters (Erejuwa, et al. 2011) and the synergistic interaction of a phytocompound with OHD’s (Prabhakar, et al. 2013) in a similar fashion. Thus the combination of a natural compound

OHD’s dose to abridge the side effects on prolonged usage.

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with OHDs can effectively maintain the serum metabolites and can be extended to reduce the

The extent of oxidative stress in the hepatic tissues by the studied parameters (SOD,

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GPx, GST, GSH, LPO and catalase) were altered in the DC group. The depleted GSH and GST levels which are imperative for the cellular protection were restored to near normal levels in all the treatment groups and a significant result was found in the Mangi combination groups (Table 5). Mangi is known for their antioxidative properties, since the catechol moiety

M

stabilizes with its ferrous complexes and prevents the formation of Fenton type reactions

ED

leading to better radical scavenging and also helps in the maintenance of the inbuilt antioxidant enzymes (Leiro, et al. 2003).

PT

The carbohydrate metabolizing enzymes are quintessential to referee the health status

CE

of the liver under stressful conditions. Hexokinase and glucose-6-phosphate dehydrogenase are the rate limiting enzymes in glycolysis and pentose phosphate pathway respectively. The

AC

depleted levels of these two enzymes were restored in the treated groups. Likewise, glucose6-phosphate dehydrogenase and fructose-1-6-bisphosphatase elevated levels signify the condition of gluconeogenesis and increased hepatic endogenous glucose production in the diabetic conditions. These enzyme levels were substantially reduced in the treated groups. The results are in compliance with earlier studies on investigating the role of a natural compound ursolic acid and the combined effect with rosiglitazone (Sundaresan, et al. 2012).

ACCEPTED MANUSCRIPT The signaling pathways are excellent therapeutic targets for pharmacological agents for a suitable standard drug design and delivery. Met is the standard drug for the improvement of AMPK pathway and insulin independent pathway (Zhou, et al. 2001). Interestingly, recent scientific reports claim that Mangi and its metabolite improves glucose metabolism via AMPK pathway (Niu, et al. 2012; Wang, et al. 2014). Conversely, Gli brings

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about a tight glycemic control by improving the insulin secretion by the β cells of the pancreas by activating the insulin dependent PI3k/Akt pathway (Bösenberg and van Zyl 2008). Herein, a significant activated increase in the AMPKα expression levels were observed in the Mangi combination with Met group. Similarly, significant action was

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observed in the expression of Akt in the Mangi with Gli combination group. In the case of combination of two compounds with varied mechanistic pathways, the final glucose uptake efficiency remains unaffected as evidenced by significantly increased levels of Glut 2 and

M

ACCβ in both the combination groups of Mangi with Met and Mangi with Gli.

ED

Similar to glucose metabolism, lipid metabolism also plays a significant role in the overall glucose homeostasis (Saltiel and Kahn 2001). Peroxisome proliferator activated

PT

receptors (PPARs) are a family of nuclear receptors which plays an influential role in lipid metabolism in a wide range of biological processes. In our study, based on the protein

CE

expression results (Fig. 5) we strongly suspect that the improvement in the carbohydrate

AC

metabolizing enzymes and the changes in the serum triglyceride levels could be rendered by the corresponding activation of the PPARs by the respective action of the Mangi combination groups.

Histopathological data are visual manifestations of the drugs effect on the physiologic functioning of the system. In the diabetic conditions, the liver histoarchitecture is affected by noticeable sinusoidal congestion, mild necrosis and vacuolar degeneration (Fig. 6A and 6B). Research suggests that these pathological changes could be the result of fatty liver conditions

ACCEPTED MANUSCRIPT and oxidative damage in hyperglycemia and could be reversed in treatment with natural compounds (Ijaz, et al. 2003; Murugan and Pari 2006). In this study, the drug combination Mangi and Met established a better hepatoprotective effect by visibly reducing the necrosis and the sinusoidal congestion and bile duct hyperplasia than with individual treatment alone.

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Conclusion The results of the present study showed that Mangi with Met and Gli strongly ameliorates the hyperglycemic conditions in both in vitro and in vivo diabetic models in a synergistic manner dose dependently. In conclusion, both the combinations of Mangi with

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Met and Gli offers better glycemic control than when treated individually in both in vitro and in vivo models. The synergistic interaction of Mangi with therapeutic doses of antidiabetic agent’s Met (100 mg/kg b.w.) and Gli (10 mg/kg b.w.) improved the overall diabetic

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conditions and can be translated in the clinical practice also. Acknowledgement

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The histopathological and microphotographs prepared at Central Inter-Disciplinary

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Research Facility (CIDRF) of Mahatma Gandhi Medical College and Research Institute (MGMCRI) Pondicherry, are acknowledged with special mention. The authors also express

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their gratitude to UGC (SAP) and DST (FIST) grants for infrastructural facilities in the Department of Biotechnology, Pondicherry University and Dr. P. Shonima, Veterinarian for

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helping in animal experimentation. Conflict of interest The authors declare no conflict of interest.

References Bell, DSH

ACCEPTED MANUSCRIPT 2013 Combine and conquer: advantages and disadvantages of fixed‐dose combination therapy. Diabetes, Obesity and Metabolism 15(4):291-300. Bösenberg, Liesel Hedwig, and Danie Gerhadus van Zyl 2008 The mechanism of action of oral antidiabetic drugs: a review of recent literature. JEMDSA. 13 (3)(3):80-88. Brandstrup, N, JE Kirk, and C Bruni

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1957 The hexokinase and phosphoglucoisomerase activities of aortic and pulmonary artery tissue in individuals of various ages. J. Gerontol. 12 (2)(2):166-171. Cho, NH, et al. 2018 IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes research and clinical practice 138:271-281. Datta, Partha Sarathi 2018 Economic Development By Reducing The Burden Of Non-Communicable Diseases In India. Economic Development of India 1(1):1-8. Ells, H Ar, and HN Kirkman

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2011 Glibenclamide or metformin combined with honey improves glycemic control in streptozotocin-induced diabetic rats. Int. J. Biol. Sci. 7 (2)(2):244. Fomenko, Ekaterina Vladimirovna, and Yuling Chi

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2016 Mangiferin modulation of metabolism and metabolic syndrome. BioFactors 42 (5)(5):492-503. Gancedo, Juana M, and Carlos Gancedo

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1971 Fructose-1, 6-diphosphatase, phosphofructokinase and glucose-6-phosphate dehydrogenase from fermenting and non fermenting yeasts. Arch. Microbiol. 76 (2)(2):132138. Habig, William H, Michael J Pabst, and William B Jakoby

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1974 Glutathione S-transferases the first enzymatic step in mercapturic acid formation. JBC. 249 (22)(22):7130-7139. Hikaru, Koide, and Oda Toshitsugu

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1959 Pathological occurrence of glucose-6-phosphatase in serum in liver diseases. Clin. Chim. Acta. 4 (4)(4):554-561. Hou, Jun, et al. 2012 Combination of mangiferin and dipeptidyl peptidase-4 inhibitor sitagliptin improves impaired glucose tolerance in streptozotocin-diabetic rats. Pharmacology 90(3-4):177-182. Ijaz, Samia, et al. 2003 Impairment of hepatic microcirculation in fatty liver. Microcirculation. 10 (6)(6):447456. Khazaei, Mohammad Rasool, et al. 2018 An overview of effective herbal and antioxidant compounds on diabetes. Journal of Contemporary Medical Sciences 4(3).

ACCEPTED MANUSCRIPT Leiro, José Manuel, et al. 2003 In vitro effects of mangiferin on superoxide concentrations and expression of the inducible nitric oxide synthase, tumour necrosis factor-α and transforming growth factor-β genes. Biochem. Pharmacology 65 (8)(8):1361-1371. Lowry, Oliver H, et al. 1951 Protein measurement with the Folin phenol reagent. JBC. 193 (1)(1):265-275. Marklund, Stefan, and Gudrun Marklund

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1974 Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. FEBS J. 47 (3)(3):469-474. Moron, Maria S, Joseph W Depierre, and Bengt Mannervik 1979 Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. BBA- Gen. Subjects. 582 (1)(1):67-78. Murugan, Pidaran, and Leelavinothan Pari

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2006 Antioxidant effect of tetrahydrocurcumin in streptozotocin–nicotinamide induced diabetic rats. Life Sci. 79 (18)(18):1720-1728. Nair, Anroop B, and Shery Jacob 2016 A simple practice guide for dose conversion between animals and human. J. Basic Clin. Pharm. 7 (2)(2):27. Niu, Yucun, et al.

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2012 Mangiferin decreases plasma free fatty acids through promoting its catabolism in liver by activation of AMPK. PloS one. 7 (1)(1):e30782. Ohkawa, Hiroshi, Nobuko Ohishi, and Kunio Yagi

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1979 Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Chem. 95 (2)(2):351-358. Prabhakar, PK, Anil Kumar, and Mukesh Doble

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2014 Combination therapy: a new strategy to manage diabetes and its complications. Phytomedicine 21(2):123-130. Prabhakar, Pranav Kumar, and Mukesh Doble

—

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2009 Synergistic effect of phytochemicals in combination with hypoglycemic drugs on glucose uptake in myotubes. Phytomedicine. 16 (12)(12):1119-1126.

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2011 Effect of natural products on commercial oral antidiabetic drugs in enhancing 2deoxyglucose uptake by 3T3-L1 adipocytes. Ther. Adv. Endocrinol. Metab. 2 (3)(3):103-114. Prabhakar, Pranav Kumar, et al. 2013 Synergistic interaction of ferulic acid with commercial hypoglycemic drugs in streptozotocin induced diabetic rats. Phytomedicine. 20 (6)(6):488-494. Rao, BS Satish, MV Sreedevi, and B Nageshwar Rao 2009 Cytoprotective and antigenotoxic potential of Mangiferin, a glucosylxanthone against cadmium chloride induced toxicity in HepG2 cells. Food Chem. Toxicol. 47 (3)(3):592600. Rotruck, JT, et al.

ACCEPTED MANUSCRIPT 1973 Selenium: biochemical role as a component of glutathione peroxidase. Science. 179 (4073)(4073):588-590. Salani, Barbara, et al. 2005 Glimepiride activates eNOS with a mechanism Akt but not caveolin-1 dependent. Biochem. Biophys. Res. Commun. 335 (3)(3):832-835. Saltiel, Alan R, and C Ronald Kahn

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2001 Insulin signalling and the regulation of glucose and lipid metabolism. Nature. 414 (6865)(6865):799. Sellamuthu, Periyar Selvam, et al. 2009 Antihyperglycemic effect of mangiferin in streptozotocin induced diabetic rats. J. Health Sci.55 (2)(2):206-214. Sinha, Asru K 1972 Colorimetric assay of catalase. Anal. Biochem. 47 (2)(2):389-394. Sundaresan, Arjunan, Ranganathan Harini, and Kodukkur Viswanathan Pugalendi

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2012 Ursolic acid and rosiglitazone combination alleviates metabolic syndrome in high fat diet fed C57BL/6J mice. Gen. Physiol. Biophys. 31 (3)(3):323. Towbin, Harry, Theophil Staehelin, and Julian Gordon 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. 76 (9)(9):4350-4354. Wang, Fang, et al.

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2014 Mangiferin and its aglycone, norathyriol, improve glucose metabolism by activation of AMP-activated protein kinase. Pharm. Biol. 52 (1)(1):68-73. Xuejian, Li, et al.

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2008 Experimental Study on Hypoglycemic Effect of the Mixture of Mangiferin and Berberine [J]. Modern Chinese Medicine 12. Zang, Mengwei, et al.

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2004 AMP-activated protein kinase is required for the lipid-lowering effect of metformin in insulin-resistant human HepG2 cells. J. Biol. Chem. 279 (46)(46):47898-47905. Zhou, Gaochao, et al.

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2001 Role of AMP-activated protein kinase in mechanism of metformin action. J. Clin. Invest. 108 (8)(8):1167-1174.

Fig. 1a Combination Effect of Mangiferin with Metformin in HepG2 Cells Normalized Isobologram for Combo: MM (Mangi+Met)

(A)

Ca/ICa

(B)

Combination Index Plot

ACCEPTED MANUSCRIPT

Combination Effect

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Cb/ICb

CI Data for Non-Constant Combo: MM (Mangi+Met) Dose Mangi (μm)

Effect

CI

40.0 20.0 10.0 5.0 2.5

0.88 0.84 0.78 0.75 0.7

1.16186 0.61517 0.42131 0.49463 0.68136

M

5.0 10.0 20.0 40.0 80.0

Dose Met (μm)

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(C)

AC

CE

PT

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Combination effect of mangiferin with metformin in HepG2 cells. The combined effects were assessed by median effect analysis. (A) Normalized Isobologram profile of combined dose treatment (B) Combination Index Plot. (C) Effect of combination at different concentration. Data points below, above or on the line respectively indicate synergistic, antagonistic or additive nature of the combination.

ACCEPTED MANUSCRIPT Fig. 1b Combination Effect of Mangiferin with Gliclazide in HepG2 Cells

Normalized Isobologram for Combo: MG (Mangi+Gli)

Combination Index Plot

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(B)

(A)

Combination Effect

Ca/ICa

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Cb/ICb

Function Effect level (Fa)

Dose Gli (μm)

Effect

CI

80.0

2.0

0.71

0.88257

40.0

4.0

0.62

0.47373

20.0

8.0

0.7

0.93630

10.0

10.0

0.7

1.04934

CE

ED

Dose Mangi (μm)

PT

(C)

M

CI Data for Non-Constant Combo: MG (Mangi+Gli)

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Combination effect of mangiferin with Gliclazide in HepG2 cells. The combined effects were assessed by median effect analysis. (A) Normalized Isobologram profile of combined dose treatment (B) Combination Index Plot. (C) Effect of combination at different concentration. Data points below, above or on the line respectively indicate synergistic, antagonistic or additive nature of the combination.

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Fig. 2a Influence of combined drug therapy on serum glucose Normal Control Diabetic control

500

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Mangiferin (40mg/kg) Metformin (100mg/kg)

##

Mangi (40mg/kg)+Met (100mg/kg)

##

Mangi (40mg/kg)+Met (50mg/kg)

300

Gliclazide (10mg/kg)

Mangi (40mg/kg)+Gli (10mg/kg) Mangi (40 mg/kg)+Gli (5mg/kg)

200

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28

M

0

14

th

th

D

ay

D ay

0

ay

****** **** ** **

100

th

Glucose (mg/dl)

400

AC

CE

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Fig. 2b Influence of combined drug therapy on serum triglycerides

ACCEPTED MANUSCRIPT

Normal Control Diabetic control Mangiferin (40mg/kg) Metformin (100mg/kg)

300

Mangi (40mg/kg)+Met (50mg/kg) Gliclazide (10mg/kg)

##

200

Mangi (40mg/kg)+Gli (10mg/kg) Mangi (40 mg/kg)+Gli (5mg/kg) *

100

ay

28

th

D

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0

14

th

th

D

ay

D ay

0

* *

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Triglycerides (mg/dl)

Mangi (40mg/kg)+Met (100mg/kg)

M

Values are expressed in mean±SEM, n=7 animals/group, Statistical analysis was performed using one way ANOVA followed by Tukey’s multiple comparison test, # and ## indicates p value< 0.05, 0.01 vs Normal Control; *, ** and *** indicates p value < 0.05, 0.01, 0.001 vs Diabetic Control group.

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Fig. 3 Effect of combined drug therapy on insulin resistance

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Diabetic control Mangiferin (40mg/kg)

###

Metformin (100mg/kg) Mangi (40mg/kg)+Met (100mg/kg) Mangi (40mg/kg)+Met (50mg/kg)

CE

400

AC

Gliclazide (10mg/kg) Mangi (40mg/kg)+Gli (10mg/kg)

**

Mangi (40 mg/kg)+Gli (5mg/kg)

**

200

** **

th

th

0

28

Da y

0 Da y

Insulin resistance

600

Normal Control

** ** **

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

AN US

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Values are expressed in mean±SEM, n=7 animals/group, Statistical analysis was performed using one way ANOVA followed by Tukey’s multiple comparison test, ### indicates p value< 0.001 vs Normal Control; **,*** indicates p value < 0.01 and 0.001 vs Diabetic Control group.

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ACCEPTED MANUSCRIPT

Fig. 4 Influence of combined drug therapy on gene expression

(A) AMPK

Normal Control

##

1.5

* **

*

*

**

**

*

Diabetic control Mangiferin (40mg/kg) Metformin (100mg/kg)

1.0

Mangi (40mg/kg)+Met (100mg/kg) Mangi (40mg/kg)+Met (50 mg/kg)

0.5

Gliclazide (10mg/kg) Mangi (40mg/kg)+Gli (10mg/kg) Mangi (40mg/kg)+Gli (5mg/kg)

(C) Glut 2 1.5

*

**

*

*

**

**

1.0

0.5

*

**

*

Mangiferin (40mg/kg)

Metformin (100mg/kg)

##

Mangi (40mg/kg)+Met (100mg/kg) Mangi (40mg/kg)+Met (50 mg/kg) Gliclazide (10mg/kg)

0.5

Mangi (40mg/kg)+Gli (10mg/kg) Mangi (40mg/kg)+Gli (5mg/kg)

CE

0.0

Diabetic control Mangiferin (40mg/kg) Metformin (100mg/kg) Mangi (40mg/kg)+Met (100mg/kg) Mangi (40mg/kg)+Met (50 mg/kg) Gliclazide (10mg/kg) Mangi (40mg/kg)+Gli (10mg/kg) Mangi (40mg/kg)+Gli (5mg/kg)

1.5 Normal Control

Akt Relative expression

1.0

ED

*

Diabetic control

**

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Glut 2 Relative expression

**

*

(D) Akt

Normal Control **

Normal Control

##

1.5

0.0

M

0.0

ACC Relative expressions

ACC Relative expressions

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(B) ACC 2.0

2.0

*

1.0

##

*

**

Diabetic control **

Mangiferin (40mg/kg) Metformin (100mg/kg) Mangi (40mg/kg)+Met (100mg/kg) Mangi (40mg/kg)+Met (50 mg/kg) Gliclazide (10mg/kg)

0.5

Mangi (40mg/kg)+Gli (10mg/kg) Mangi (40mg/kg)+Gli (5mg/kg)

0.0

AC

Values are expressed in mean ± SEM, n=5 animals/group, Statistical analysis was performed using one way ANOVA followed by Tukey’s multiple comparison test, ## indicates p value< 0.01 vs Normal Control; *, ** indicates p value < 0.05 and 0.01 vs Diabetic Control group.

ACCEPTED MANUSCRIPT

PPAR  Relative expression

3

2.0

Diabetic control

***

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Metformin (100mg/kg)

*

**

**

**

Mangi (40mg/kg)+Met (100mg/kg) Mangi (40mg/kg)+Met (50 mg/kg)

1 ###

Gliclazide (10mg/kg)

Mangi (40mg/kg)+Gli (10 mg/kg)

1.0 0.5

0.0 PPAR   actin

M ED *

Mangi (40mg/kg)+Gli (5 mg/kg)

Normal Control Diabetic control

#

PT

1.5

CE AC

Mangiferin (40mg/kg)

***

2

0 PPAR   actin

(B)

Normal Control

***

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(A)

PPAR  Relative Expression

Fig. 5 Influence of combined drug therapy on protein expression

Mangiferin (40mg/kg) **

**

*

Metformin (100mg/kg) Mangi (40mg/kg)+Met (100mg/kg) Mangi (40mg/kg)+Met (50 mg/kg) Gliclazide (10mg/kg) Mangi (40mg/kg)+Gli (10 mg/kg) Mangi (40mg/kg)+Gli (5 mg/kg)

Relative protein expression levels of (A) PPAR a and PPAR g (B) proteins in hepatic tissue. Densitometric analysis showed that the significant exression of both proteins in hepatic tissue. Statistical analysis was performed using one way ANOVA followed by Tukey's multiple comparison test.#, ### p values < 0.05, 0.001 respectively vs normal control. *, ** and *** p value < 0.05, 0.01 and 0.001 vs diabetic control.

ACCEPTED MANUSCRIPT

AC

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PT

ED

M

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Fig. 6 Histopathological changes in the hepatic tissue

(A) Liver tissue stained with haematoxylin and eosin (Magnification= 20X, Scale bar = 50µm). (B) Scoring based on severity of lesions in liver tissue (0- None, 1- Minimal, 2- Mild, 3- Moderate and 4-Marked). Values are expressed in mean ± SEM, n=5 animals/group, Statistical analysis was performed using one way ANOVA followed by Tukey's multiple comparison test, ### indicates p value < 0.001 vs Normal Control; *, ** and *** indicates p < 0.05, 0.01 and 0.001 respectively vs Diabetic Control group.

Table 1 Glucose uptake level of gliclazide, metformin and mangiferin in HepG2 cells

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Gliclazide 1.80±0.02 1.85±0.24 1.91±0.10 1.92±0.06 1.81±0.03

Concentration (µM) 2.5 5 10 20 40

Metformin 1.53±0.07 1.69±0.20 1.79±0.24 1.88±0.05 2.09±0.23

Concentration (µM) 10 20 40 80 160

Mangiferin 1.51±0.20 1.62±0.11 1.67±0.13 1.70±0.23 1.78±0.09

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Concentration (µM) 1 2 4 8 10

Values are expressed in mean ± SD, µM; micromolar

80 µM

40 µM 20 µM 10 µM 5 µM

20 µM

3.02±0.27

2.81±0.06

2.80±0.15

2.79±0.02

2.37±0.22

2.48±0.15

2.32±0.13

2.23±0.05

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2.5 µM

40 µM

1.83±0.07

1.38±0.09

10 µM

2.82±0.12

2.40±0.11

2.65±0.29

2.24±0.11

2.23±0.26

1.79±0.20

1.33±0.14

1.36±0.13

1.12±0.32

0.89±0.07

M

Mangi Met

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Table 2 Glucose uptake level of mangiferin and metformin combination

CE

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Values are expressed in mean ± SD, µM; micromolar; Mangi: Mangiferin; Met: Metformin

Mangi

AC

Gli

80 µM

40 µM

20 µM

10 µM

8 µM

2.82±0.05

2.25±0.14

2.48±0.11

2.38±0.22

4 µM

2.55±0.12

2.26±0.13

2.37±0.31

2.04±0.03

2 µM

2.14±0.05

2.00±0.05

2.02±0.12

1.68±0.07

1µM

1.68±0.06

1.64±0.13

1.60±0.27

1.24±0.04

Table 3 Glucose uptake level of mangiferin and gliclazide combination

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Values are expressed in mean ± SD, µM; micromolar; Gli: Gliclazide; Mangi: Mangiferin

Table 4 Influence of combination drug therapy on hepatic enzymes Groups

γGT(U/l)

ALP(U/l)

AST(U/l)

ormal Control

37.10±1.31

51.28±3.17

49.72±9.63

4.49±0.18

233.89±26.70

abetic control

54.23±5.80

684.04±47.88###

81.41±4.57##

7.93±0.28#

987.86±34.78###

angiferin (40mg/kg)

43.69±3.44

508.55±19.24

45.54±4.64

3.74±0.79**

338.86±73.87***

etformin (100mg/kg)

39.93±4.52

190.33±27.98***

52.76±8.87

3.40±0.73***

190.45±34.28***

angi (40mg/kg)+Met (100mg/kg)

37.44±5.07

444.53±55.95*

64.56±4.69

2.82±0.87***

714.83±72.14*

angi (40mg/kg)+Met (50 mg/kg)

50.12±4.68

456.28±38.82

64.47±6.94

5.41±0.68

861.68±27.01

iclazide (10mg/kg)

38.09±5.11

338.98±59.92***

48.10±7.48

4.44±0.34*

314.28±47.14***

angi (40mg/kg)+Gli (10 mg/kg)

46.13±6.77

424.20±62.75*

50.45±6.62

3.17±0.73***

648.55±28.59***

angi (40mg/kg)+Met (5 mg/kg)

44.70±1.92

500.43±91.85

64.52±9.79

6.67±0.88

576.93±75.64***

LDH(U/l)

M

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ALT (U/L)

AC

CE

PT

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Values are expressed in mean±SEM, n=7 animals/group, Statistical analysis was performed using one way ANOVA followed by Tukey’s multiple comparison test, #, ##and### indicates p value< 0.05, 0.01 and 0.001 vs Normal Control; *,** and *** indicates p value <0.05, 0.01 and 0.001 respectively vs Diabetic Control group, Mangi+Met: Mangiferin+Metformin; Mangi+Gli: Mangiferin+Gliclazide; mg: milligram; kg: kilogram; U/l: unit per litre; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; AST: Aspartate Aminotransferase; γGT: Gamma Glutamyltransferase; LDH: Lactate dehydrogenase.

Table 5 Influence of combination drug therapy on hepatic oxidative stress markers

Treatment

SOD (U/min/mg protein)

GPx (nM/min/mg protein)

GST (nM/min/mg protein)

GSH (nM/g tissue)

LPO (µM/g tissue)

Catal (μM/m prote

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58.51±1.63

1.33±0.01

13.24±1.79

1.26±0.05

3.38±0.02

54.03±

trol

21.04±4.65##

0.77±0.02##

4.91±0.18##

0.94±0.01##

7.39±0.35##

34.09±

0mg/kg)

42.55±1.75

1.64±0.03**

8.91±0.30**

1.15±0.03*

4.73±0.48

41.17±

00mg/kg)

49.56±4.16**

1.46±0.02**

4.96±0.31**

1.14±0.02*

4.25±0.56*

38.96±

/kg) + Met (100mg/kg)

48.95±7.35**

1.68±0.02**

7.64±0.27**

1.25±0.05*

3.50±0.88**

38.42±

/kg) + Met (50 mg/kg)

44.86±6.68*

1.70±0.04**

5.96±0.52**

1.22±0.04*

4.26±0.81*

41.89±

0mg/kg)

49.28±3.45**

1.32±0.07**

7.63±0.55**

1.07±0.03

3.95±0.37**

47.35±

/kg) + Gli (10 mg/kg)

48.24±2.93**

1.54±0.14**

6.48±0.54**

1.35±0.05**

3.78±0.55**

43.96±

/kg) + Gli (5 mg/kg)

43.22±6.60*

1.59±0.02**

7.66±0.65**

1.28±0.05**

4.04±0.73**

48.82±

AN US

CR IP T

rol

PT

ED

M

Values are expressed in mean±SEM, n=7 animals/group, Statistical analysis was performed using one way ANOVA followed by Tukey’s multiple comparison test, # and ## indicates p value< 0.05 and 0.01 vs Normal Control; *,** indicates p value <0.05 and 0.01 respectively vs Diabetic Control group. Mangi+Met: Mangiferin+Metformin; Mangi+Gli: Mangiferin+Gliclazide; g: gram; mg: milligram; kg: kilogram; U: Unit; min: minute; nM: nanomolar; µM: micromolar; SOD: Superoxide Dismutase; GPx: Glutathione peroxidase; GST: Glutathione S- transferase; GSH: Reduced glutathione; LPO: Lipid peroxidation.

AC

CE

Table 6 Influence of combination drug therapy on hepatic carbohydrate metabolizing enzymes

34

ACCEPTED MANUSCRIPT

Values are expressed in mean±SEM, n=7 animals/group, Statistical analysis was performed using one way

Hexokinase

(µm/min/mg protein) Normal Control

0.411 ± 0.005

Diabetic control

0.317 ± 0.016

Mangiferin (40mg/kg)

0.375 ± 0.016

Metformin (100mg/kg)

0.433±0.016

Mangi (40mg/kg)+Met (100mg/kg)

0.400±0.010

Mangi (40mg/kg)+Met (50 mg/kg)

0.358±0.019

Gliclazide (10mg/kg)

0.358 ± 0.012

Mangi (40mg/kg)+Gli (10 mg/kg)

0.402 ± 0.019

Mangi (40mg/kg)+Gli(5 mg/kg)

0.361 ± 0.030

#

**

Fructose-1, 6-Bis

dehydrogenase

Phosphatase

(units/mg protein)

(µm/min/mg protein

0.090±0.006

2.93 ± 0.39

0.072±0.001

7.37 ± 0.66

0.083±0.005

3.74 ± 0.43

0.082±0.002

3.45±0.71

0.084±0.003

3.86±0.83

0.080±0.001

5.04±0.60

AN US

**

Glucose-6 phosphate

CR IP T

Treatment

*

0.084±0.004

4.26 ± 0.93

0.094±0.006

3.73 ± 0.74

0.080±0.003

4.31 ± 0.43

AC

CE

PT

ED

M

ANOVA followed by Tukey’s multiple comparison test, # and ## indicates p value< 0.05 and 0.01 vs Normal Control; *,** indicates p value <0.05 and 0.01 respectively vs Diabetic Control group. Mangi+Met: Mangiferin+Metformin; Mangi+Gli: Mangiferin+Gliclazide; mg: milligram; kg: kilogram; min: minute; µM: micromolar.

35

## **

* ** *