Antidiabetic property of Aerva lanata (L.) Juss. ex Schult. is mediated by inhibition of alpha glucosidase, protein glycation and stimulation of adipogenesis (L.)

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1 This article is protected by copyright. All rights reserved. Antidiabetic property of Aerva lanata (L) Juss. ex Schult. is mediated by inhibition of alpha glucosidase, protein glycation and stimulation of adipogenesis 1 Mariam Philip RIYAa, Kalathookunnel Antony ANTUa, Savita PAL b, Karuvakandy Chandrasekharan CHANDRAKANTHa, Karunakaran Sasikala ANILKUMARc, Akhilesh Kumar TAMRAKARd, Arvind Kumar SRIVASTAVAb, Kozhiparambil Gopalan RAGHU a* aAgroprocessing and Natural Products Division, Council of Scientific and Industrial Research - National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Thiruvananthapuram, Kerala, India, Pin-695019. bDivision of Biochemistry, CSIR - Central Drug Research Institute (CDRI ), Lucknow, Uttar Pradesh, India, Pin-226001. cMedicinal Chemistry division, CSIR-CDRI, Lucknow, Uttar Pradesh, India, Pin-226001. dDivision of Pharmacology, CSIR-CDRI, Lucknow, Uttar Pradesh, India, Pin-226001. *For correspondence: Dr. K. G. Raghu, Agroprocessing and Natural Products Division, Council of Scientific and Industrial Research - National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Thiruvananthapuram, Kerala, India, 695019. Tel: +919495902522, Fax: +91-471-2491712/2491585. Email:, Running title: Basis of antidiabetic property of Aerva lanata This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1753-0407.12216 Accepted Article 2 This article is protected by copyright. All rights reserved. Abstract Background: Diabetes is the leading cause of morbidity and mortality, with a number currently diagnosed as high as 371 million. Plant-based therapy could be an ideal choice because of fewer side effects and wider acceptability. Hence, the antihyperglycemic potential of Aerva lanata, a herb prescribed for diabetes in Ayurveda was evaluated to elucidate its possible mechanism of action. Methods: HPLC analysis was employed for the characterization of 70% ethanolic (ALE) and ethyl acetate (AEA) extracts. Further, they were evaluated for their antioxidant, alpha glucosidase inhibition, protein glycation inhibition, dipeptidyl peptidase IV (DPP IV), protein tyrosine phosphatase 1B (PTP1B), glucose uptake and glitazone like property (adipogenic potential) using in vitro models. The promising alpha glucosidase inhibitory potential of ALE was further evaluated in normal and streptozotocin (STZ) diabetic rats. Results: ALE inhibited yeast (IC50- 81.76 g/mL) and rat intestinal alpha glucosidase (IC50- 108.7 g/mL), protein glycation, DPP IV enzyme (IC50- 118.62 g/mL) and PTP1B (IC50- 94.66 g/mL). ALE stimulated maximal adipogenesis at 50 g/mL and enhanced insulin mediated glucose uptake (3 fold of basal) at 100 g/mL in L6 myotubes. ALE (500mg/kg b.w) showed a significant antihyperglycemic activity in sucrose loaded STZ normal (15.57%) and diabetic (18.44%) rats. HPLC analysis of ALE revealed the presence of bioactives like alpha amyrin, betulin and beta sitosterol. Conclusions: Alpha glucosidase inhibition, antiglycation, and adipogenic potential significantly contribute to the antidiabetic property of Aerva lanata. In addition, insulin sensitization and antioxidant potential also enhance its therapeutic potential. Significant findings of the study: Accepted Article 3 This article is protected by copyright. All rights reserved. Antihyperglycemic potential of ALE in STZ diabetic rats via alpha glucosidase inhibition, enhanced glitazone like potential in 3T3-L1 and in vitro antiglycation potential. Promising glucose uptake in L6 myotubes, DPP IV and PTP1B inhibition. What this study adds: Alpha glucosidase inhibition, protein glycation and enhanced glitazone like potential are the key mechanisms responsible for the antidiabetic potential of Aerva lanata. In vitro studies have also shown the possible contribution of other associated mechanisms to its therapeutic efficacy. Key words: Aerva lanata, antioxidant, diabetes, dipeptidyl peptidase IV, protein tyrosine phosphatase 1B Accepted Article 4 This article is protected by copyright. All rights reserved. Introduction Diabetes is a chronic metabolic disorder characterized by insulin resistance, impaired insulin secretion and hyperglycemia1. Elevated postprandial glycaemia (PPG) is an important indicator of pre-diabetes and hence is a valuable tool for its early diagnosis2. Persistent hyperglycemia is associated with irreversible damage to eyes, kidneys, nerves and blood vessels. So targeting PPG significantly aid in reducing the rapid onset of diabetic complications. Currently available drugs include inhibitors of alpha glucosidase3, DPP IV4, 5 and sensitizers like metformin6 and rosiglitazone7. PTP1B is an emerging drug target and its inhibition can improve insulin sensitivity8. But, the cost of the prescribed drugs and their undesirable side effects (due to their long-term use), emphasis the need for a traditional plant-based therapy. Aerva lanata, commonly known as bhadrika in Sanskrit, is a prostrate decumbent, sometimes erect herb, widely used in Ayurveda for the treatment of diabetes mellitus9, 10. It is also consumed as a vegetable in many of the Asian and African nations11, 12. Pharmacological studies have shown that Aerva lanata also possess diuretic, anti-lithiasis, anti-inflammatory, antimicrobial, antitumour and antioxidant_ENREF_13 properties13,14. The antidiabetic potential of aerial part and root have been previously reported13, 15. However, there has been no detailed study employing currently explored drug targets for diabetes. In the present study, we have evaluated Aerva lanata (whole plant) to unveil its possible mechanism of action using in vitro and in vivo models. Methods Plant material and preparation Aerva lanata (L) Juss. ex. Schult. was collected from Thiruvananthapuram and identified by Dr H. Biju, Taxonomist from the Jawaharlal Nehru Tropical Botanical Garden Research Institute (JNTBGRI), Palode, Thiruvananthapuram, Kerala. After identification, voucher specimen (No. Accepted Article 5 This article is protected by copyright. All rights reserved. 66499) was deposited at JNTBGRI herbarium, for future reference. One kilogram of fresh whole plants were air dried and sequentially extracted using different organic solvents (3.5-4 litres) in their increasing order of polarity i.e. hexane (AHE), dichloromethane (ADE), ethyl acetate (AEA) and 70% ethyl alcohol (ALE) until colourless. The resulting solvent fractions were concentrated in a rotary evaporator (Laborota4010; Heidolph, Schwabach, Germany) at 40-45 C. They were lyophilized and stored at 4 C until use. Cell-culture and treatment All cells were cultured at 37 C in a humidified 5% CO2, 95% air atmosphere. HepG2 (human hepatocellular carcinoma), L6 myotubes, 3T3-L1 murine preadipocytes were obtained from ATCC (Manassas, VA, USA). HepG2 was maintained in low glucose (5.5mM) Dulbeccos Modified Essential Media (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and 1% antibiotic-antimycotic solution (10,000 U/mL). L6 myotubes were maintained in alpha-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and 1% antibiotic-antimycotic solution. Experiments were performed in differentiated myotubes (DMEM with 2% FBS) 6-7 days after seeding. 3T3-L1 preadipocytes were cultured in DMEM (25 mM glucose) supplemented with 10% FBS and antibiotics. Differentiation into adipocytes was induced by switching to DMEM with 500 M 3-isobutyl-1-methylxanthine, 10 M dexamethasone and 500 nM insulin (Sigma, St Louis, MO, USA) for 48 h. Cells were then maintained in DMEM containing 10% FBS and 500 nM insulin for 8 days. Determination of cell viability Cells were seeded at a density of 4 104 cells/well in 24 well plates and incubated for 24 or 48 h. Cells were subsequently incubated with various concentrations (10, 50, 100 and 200 g/mL) of Accepted Article 6 This article is protected by copyright. All rights reserved. the extracts for 24 h or 48 h. Cell viability was evaluated using MTT assay kit (Cayman, USA). Further cell line studies were based on cytotoxicity experiment. Animals Male albino Sprague Dawley (SD) rats (16020 g, 7-8 week old) bred at animal facility of Central Drug Research Institute of India, Lucknow were utilized for this study. All the procedures complied with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) formed by the Government of India in 1964 (Ethics committee reference number IAEC/2008/63/Renewal 04 dated 16.05.2012). Rats were housed in polypropylene cages under an ambient temperature of 23 2 C; 50-60% relative humidity; light 300 lux at floor level with regular 12 h light/dark cycle. Animals were maintained on a standard pellet diet and water was provided ad libitum. Chemical composition Analysis of fibre and mineral content Crude fibre was estimated by adopting the official method of analysis by Association of Analytical Chemists (AOAC)16. The mineral constituents were determined by digesting 1 g of the sample in a mixture of nitric acid and hydrochloric acid (1:3)16 and quantified by atomic absorption spectrophotometer (AAS; Perkin Elmer A Analyst 100; Perkin Elmer, MA, US). Results are expressed as mg/g of sample. Quantification of total phenolic, flavonoid and tannin content Total phenolics17, flavonoid18 and tannin content19 were determined spectrophotometrically. Results were expressed as gallic acid equivalents/g extract (mg GAE/g) for total phenolics, quercetin equivalents/g extract (mg QE/g) for total flavonoid and tannic acid equivalents/g extract (TAE/g) for total tannin content. Accepted Article 7 This article is protected by copyright. All rights reserved. High-performance liquid chromatography (HPLC) Betulin, beta sitosterol and alpha amyrin (Sigma, St Louis, MO, USA) were identified and quantified using HPLC. Chromatographic separation was performed using Agilent HPLC 1100 series equipped with C-18 column (150 x 4.6, 5 m). Acetonitrile: water in the ratio of (30:70) v/v was used as the mobile phase at a constant flow rate of 1.0 mL/min. Peaks were detected at 220 nm with UV Detector. Determination of antioxidant potential The antioxidant activity was measured by DPPH20 and ABTS21 method. Percentage of radical scavenging activity obtained was plotted against the corresponding concentration of the extract to obtain IC50 value. Gallic acid for DPPH and Trolox for ABTS served as standards. Measurement of hyperglycemia induced intracellular reactive oxygen species (ROS) Cells were maintained in low glucose medium for 24 h, and then switched to high glucose (25 mM) medium with or without the extracts or quercetin for another 24 h. Treated cells were loaded with H2DCFDA (10 M)22 in serum free medium for 30 min at 37 C. Subsequently the cells were rinsed with prewarmed PBS and fluorescence intensity was measured (ex 490 nm; em 525 nm). In addition, fluorescent images were also captured using BD 855 bio-imaging system (USA). Alpha glucosidase inhibition Alpha glucosidase (EC inhibitory activity was assessed using yeast alpha glucosidase and rat intestinal enzyme as reported elsewhere23. Percentage inhibition was plotted against the corresponding concentration to obtain the IC50 value. Acarbose served as the reference standard. In vivo study Induction of diabetes Accepted Article 8 This article is protected by copyright. All rights reserved. Diabetes was induced by a single intraperitoneal injection of STZ at 60 mg/kg b.w (Sigma, St Louis, MO, USA) dissolved in 0.1 M citrate buffer, pH 4.5. After 72 h, induction of diabetes was confirmed and animals with blood glucose >270mg/dL were included in the study24,25. Oral sucrose tolerance test (OSTT) in normoglycemic and STZ diabetic rats Both normal and diabetic rats fasted overnight were divided into 5 groups (n = 6 per group). Animals of control and experimental groups were administered suspension of the vehicle or ALE (100, 250 & 500 mg/kg b.w in 1.0% gum acacia)/acarbose (100 mg/kg b.w) orally by a gavage needle (18-gauge, 38 mm long curved, with a 21/4 mm ball end). Sucrose load of 10 g/kg b.w for normoglycemic and 2.5 g/kg b.w for diabetic rats was given 30 min after administration of the vehicle/ALE/acarbose. Blood was collected from the tail vein and glucose levels were determined using glucometer (Accu-check, Roche Diagnostics, USA) at 30 min interval for 2 h after the sucrose load in normoglycemic24 and at 0, 30, 60, 90, 120, 180, 240 and 300 min respectively in diabetic rats24, 26. Percentage antihyperglycemic activity was calculated by comparing the area under curve (AUC) of experimental and control groups. Determination of antiglycation activity BSA derived advanced glycation end products (AGEs) were measured as previously reported 27 with slight modifications. BSA (10 mg/mL) in the presence of ribose (500 mM) in phosphate buffered saline (PBS) was used as control. Quercetin (100 M) served as the reference standard. AGE fluorescence (ex370 nm; em 440 nm) was measured (Biotek, USA) after 24 h. Samples were analysed for changes in the complexity of glycated product on EVO 18 special edition model of Carl Zeiss (Carl Zeiss, Munich, Germany) scanning electron microscope28, 29. All images of samples were captured at 16K x magnification for comparison. DPP IV and PTP 1B inhibitory potential Accepted Article 9 This article is protected by copyright. All rights reserved. DPP IV (EC was measured using commercially available kit from Cayman (Ann Arbor, Michigan, USA). PTP1B (EC was measured using commercially available kit from Calbiochem, Darmstadt, Germany). Diprotin A for DPP IV and suramin for PTP1B served as standards. IC50 value was calculated as mentioned above. 2-deoxy glucose (2-DG) uptake 2-DG uptake in L6 myotubes was performed as reported elsewhere30, 31 with slight modification_ENREF_25. Briefly, the myotubes were incubated with extracts or standards for 16 h. Cells were serum deprived for 3 h before the experiment. For assessment of insulin stimulated glucose uptake, cells were stimulated with 100 nM insulin for 20 min. Basal glucose uptake was determined without insulin stimulation. Glucose uptake was assessed after incubation in HEPES-buffered saline containing 10 M 2-DG (0.5 CimL-1 2-[3H] DG) at room temperature for 5 min. Subsequently, cells were rinsed with ice-cold solution of 0.9% NaCl and 20 mM D-glucose and lysed in 0.05 N NaOH. Radioactivity in the lysate was determined using scintillation fluid in a counter (Perkin Elmer, USA). Cytochalasin B (50 M) was used to determine non-specific glucose uptake. Results were expressed as fold induction with respect to unstimulated cells after normalization to the total protein content. Metformin (10 mM) served as positive control. Adipocyte differentiation assay Oil Red O staining and quantitation The adipogenic potential of the extracts was tested for triglyceride content in differentiating 3T3-L1 preadipocytes over an 8 day period32. Cells were stained with Oil Red O and triglyceride content was measured as previously reported33. Rosiglitazone (10 M) was used as positive control. Accepted Article 10 This article is protected by copyright. All rights reserved. Determination of glycerol-3-phosphate dehydrogenase (GPDH) activity, diacylglycerol O-acyltransferase 1 (DGAT1) and adiponectin On day 8, adiponectin level in the media of different treatment groups was determined using kit from Cayman (Ann Arbor, Michigan, USA). Cells were washed twice with PBS and harvested into 25 mM Tris buffer (pH 7.5) containing 1 mM EDTA and 1 mM DTT. Cells were disrupted by sonication and then centrifuged at 12,000g for 20 min at 4 C. The supernatants were assayed for GPDH activity according to the method of Wise and Green 34. DGAT1 level was determined using kit from (San Diego, California, USA). Results were normalized to protein content using BCA method. Statistical analysis Results are expressed as mean standard error of mean (SEM). IC50 value was interpolated from three independent experiments with triplicates using non-linear regression. Quantitative glucose tolerance was calculated by AUC using GNU PSPP ( AUC of the control and the experimental groups were compared for percentage antihyperglycemic activity. Results were analysed by one-way analysis of variance with Dunnett post hoc test. P 11 This article is protected by copyright. All rights reserved. ALE has highest amount of phenolics including flavonoids and tannins followed by AEA and ADE (Table1). HPLC fingerprint of ALE clearly shows the presence of alpha amyrin (8.74 mg/g), betulin (5.4 mg/g) and beta sitosterol (18.3 mg/g extract) (Supplementary Fig 1). ALE and AEA were taken further in this study. Determination of in vitro antioxidant potential Antioxidant potential was assessed using DPPH (IC50 value for ALE and AEA-124.24 g/mL and 268.28 g/mL respectively, IC50 for gallic acid - 6.5 g/mL, Table 2) and ABTS (IC50 value for ALE and AEA -167.91 g/mL and 189.54 g/mL respectively, IC50 for trolox-3.8 g/mL, Table 2) assay. The extracts were found to be safe up to 200 g/mL. ALE and AEA (10, 50 and 100 g/mL) were further evaluated in cell based system (HepG2) to check their potential to prevent hyperglycemia induced ROS generation. Hyperglycemia induced 155% increase in ROS compared to vehicle control (Fig 1A & 1B a, b). ALE and AEA (100 g/mL) demonstrated a significant decrease in oxidative stress by 41% and 37% respectively compared to hyperglycemia control. 52% decrease in ROS was observed after treatment with positive control (Fig 1A & 1B b, c; P 12 This article is protected by copyright. All rights reserved. Treatment with ALE at 100, 250 & 500 mg/kg b.w exhibited 10.09%, 13.57% and 15.57% reduction in plasma glucose when compared to vehicle control (Fig 3A & B). Acarbose at 100 mg/kg b.w decreased blood glucose by 20.4% when equated to vehicle control. ALE treatment (500 mg/kg b.w) prevented a sharp rise in postprandial blood glucose (18.44%) after 5 h of treatment compared to STZ control (Fig 3C & 3D). Results were comparable with acarbose treated group which showed 21.7% improvement in blood glucose profile compared to STZ control. Antiglycation property Antiglycation potential was evaluated for their ability to inhibit the formation of AGEs using fluorescence assay and scanning electron microscopy. ALE and AEA exhibited promising antiglycation potential (IC50- 160.82 g/mL and 268.99 g/mL; Fig 4A). Control group showed extremely granular agglomeration with uneven pores and complex microstructure (Fig 4B a). ALE and AEA showed a noticeable decrease in complexity of the microstructure (Fig. 4B c-f). Quercetin (IC50- 30.1 g/mL) showed a significant decrease in fluorescence and complexity of microstructure (Fig 4A & B). DPP IV and PTP1B inhibitory potential ALE showed promising inhibition against DPP IV enzyme in vitro. IC50 value of ALE and AEA was 118.62 & 304.62 g/mL respectively, whereas Diprotin A showed an IC50 value of 1.54 mg/mL (Fig. 5A). Extracts also showed promising PTP1B inhibition (IC50 value of ALE and AEA was 94.66 & 150.73 g/mL) respectively. IC50 value of Suramin was 14.01 g/mL (Fig 5B). Glucose uptake in L6 myocytes ALE and AEA increased glucose uptake at 100 g/mL (1.6 and 1.4 fold increase in basal glucose uptake, Fig 6, P 13 This article is protected by copyright. All rights reserved. basal glucose uptake (Fig 6, P 14 This article is protected by copyright. All rights reserved. Alpha glucosidase inhibitors are regarded as the first line of treatment for preventing the early-onset of diabetes complications by suppressing postprandial hyperglycemia37, 38. They inhibit the action of carbohydrate digestive enzymes in the small intestine and thereby slow down the liberation of glucose from oligosaccharides and disaccharides3, 39. Cinnamon40, kotalanol41 from Salacia reticulata, triterpenes42 from Syzygium aromaticum are some of the promising alpha glucosidase inhibitors from plants. ALE showed significant reduction in enzyme activity both in vitro (Fig 2A & B) and in vivo (Fig 3A & B). ALE (500 mg/kg b.w) suppressed the postprandial blood glucose AUC0-300 min in STZ induced diabetic rats, whereas control animals showed elevated blood glucose even after 5 h of sucrose load (Fig 3A & B) which was comparable with acarbose. Thus, ALE could be beneficial in reducing postprandial hyperglycemia via digestive enzyme inhibition. AGEs represent a heterogeneous group of entities resulting from non-enzymatic reaction between reducing sugars and proteins, lipids and nucleic acids. Numerous studies have shown that build-up of cross linked AGEs on long lived proteins accelerate secondary complications in diabetic patients 36,43. Aminoguanidine44 has demonstrated powerful ability to inhibit or break AGEs, but has not been successful due to concerns regarding their toxicity in patients45. Numerous studies suggest that compounds with combined antiglycation and antioxidant properties could be more beneficial. Carnosine46, tannins in green tea47, garcinol from Garcinia indica fruit rind48 and S-allyl cysteine from aged garlic extract49 have been found to possess promising antiglycation and antioxidant activity47. Likewise, the present study shows that ALE possesses both the antiglycation (Fig 4A & B) and antioxidant activity (Table 1, Fig 1A & B). DPP IV inhibitors decrease hyperglycemia and improve glucose metabolism by glucose dependent insulin secretion from pancreas50. They prolong the action of endogenous incretin and Accepted Article 15 This article is protected by copyright. All rights reserved. hence beneficial for diabetic patients51. Results from similar studies 52 show that DPP IV inhibition is an important target for diabetes. ALE inhibited the DPP IV enzyme significantly in a dose dependent manner compared to AEA (Fig 5A). PTP1B inhibitors increase insulin sensitivity by blocking the PTP1B mediated negative insulin signalling pathway53. In the present study, ALE exhibited significant inhibition of the enzyme over AEA (Fig 5B). Reduced uptake of glucose in insulin sensitive tissues like skeletal muscle54 also contributes to postprandial hyperglycemia. We have screened ALE and AEA for their ability to stimulate glucose uptake in L6 under both basal and insulin stimulated conditions. ALE and AEA enhanced basal glucose uptake (1.6 fold and 1.4 fold respectively at 100 g/mL; Fig 6) compared to 10 mM metformin (1.7 fold increase; Fig 6). Insulin stimulated control exhibited 1.9 fold increase of glucose uptake. Pretreatment with ALE and AEA (100 g/mL) for 16 h prior to insulin stimulation, enhanced glucose uptake (3 fold of basal and 2.6 fold of basal; Fig 6). Results were compared to metformin (3.8 fold of basal; Fig 6). Lipid storage in adipocytes improve glucose homeostasis and helps prevent peripheral lipotoxicity55. Activators of PPAR like thiazolidinediones (TZDs) commonly referred as glitazones improve insulin sensitivity by stimulating lipid accumulation in adipose leading to a decrease in lipid content in liver and muscle56. TZDs promote maturation and redistribution of adipose and improve adiponectin profile. They also negates the action of TNF alpha and IL-6. ALE showed a positive adipogenic potential (Fig 7A-E) with an increase of TG57, GPDH34, 57, DGAT157 and adiponectin58, the hall marks of adipogenesis. Siveen and Kuttan 201159 have shown that ethanolic extract of Aerva lanata stimulate bone marrow cells, which in turn can reduce the incidence of bone fracture observed with TZDs therapy60, 61. Previous studies have shown that Aerva lanata contains a number of pharmacologically active compounds such as alpha Accepted Article 16 This article is protected by copyright. All rights reserved. amyrin24, 62-64, betulin64, 65_ENREF_43, beta sitosterol13, 63, 64, 66-69 and alkaloids like canthine-6-one and carboline15, 59, 70. We have identified and quantified alpha amyrin, betulin and beta sitosterol in ALE by HPLC (Supplementary Fig 1). We have also shown the presence of significant amount of fibre besides minerals like potassium, zinc_ENREF_5071 and magnesium72, 73 which also possibly contribute to its antidiabetic potential. Therefore, we believe that significant alpha glucosidase inhibition, antiglycation and adipogenic potential play an important role in the antidiabetic property of Aerva lanata. In addition, moderate antioxidant, DPP IV and PTP1B inhibition along with glucose uptake in skeletal myotubes also possibly contribute to its efficacy. Presence of bioactives like alpha amyrin, betulin and beta sitosterol adds to its therapeutic advantage. So, Aerva lanata deserves special attention as a potential therapeutic/adjunct for diabetes. A detailed investigation is in process to understand its efficacy in ameliorating diabetes induced secondary complications. Acknowledgement Riya MP and Antu KA acknowledge CSIR and Indian Council of Medical Research (ICMR) and UNDO CSIR (BSC103) 12th Five Year Plan Project for financial assistance. We thank Director, CSIR-NIIST and CSIR-CDRI for providing necessary laboratory facilities. We also thank Dr.H.Biju, JNTBGRI for identification of the plant material. Disclosure Authors declare that there is no conflict of interest. Accepted Article 17 This article is protected by copyright. All rights reserved. References 1. Lin Y, Sun Z. Current views on type 2 diabetes. J Endocrinol. 2010;204:1-11. 2. American Diabetes Association. Postprandial Blood Glucose. Diabetes Care. 2001;24:775-8. 3. Standl E, Schnell O. Alpha-glucosidase inhibitors 2012 - cardiovascular considerations and trial evaluation. Diab Vasc Dis Res. 2012;9:163-9. 4. Hocher B, Reichetzeder C, Alter ML. 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Accepted Article 21 This article is protected by copyright. All rights reserved. Accepted Article 22 This article is protected by copyright. All rights reserved. Figure legend Figure 1 A & B ALE and AEA attenuated high glucose-induced intracellular ROS levels in HepG2 cells. (A) The quantitative analysis of fluorescence from 3 doses of ALE and AEA (10, 50 & 100 g/mL) (B) representative microscopic scans a-e (VC - Vehicle control, HG - High glucose, Q quercetin, HG + Q (10g/mL), HG + ALE (10g/mL & 100g/mL), HG + AEA (10g & 100g/mL). Values are means with SEM of 3 independent experiments, each performed in triplicate. * denote P 23 This article is protected by copyright. All rights reserved. independent experiment, each performed in triplicate. *p < 0.05 relative to MDI-ve, #P 24 This article is protected by copyright. All rights reserved. Table 1 Total phenolics, flavonoid and tannin content in different extracts of Aerva lanata (ALE - ethanolic extract, AHE - hexane extract, ADE - dichloromethane extract, AEA - ethyl acetate extract. Results for total phenolics were expressed as gallic acid equivalents/g extract (mg GAE/g) or quercetin equivalents/g extract (mg QE/g) or tannic acid equivalents/g extract (TAE/g). Table 2 DPPH & ABTS radical scavenging activity of various extracts obtained by sequential extraction from Aerva lanata Extracts DPPH radical scavenging activity IC50 (g/mL) ABTS radical scavenging activity IC50 (g/mL) ALE 124.24 167.91 AHE 150.81 178.8 ADE 224.56 237.6 AEA 268.28 189.54 Gallic acid 6.5 -- Trolox -- 3.8 (ALE-ethanolic fraction, AHE-hexane fraction, ADE- dichloromethane fraction, AEA- ethyl acetate fraction. IC50 value for DPPH and ABTS radical scavenging assay is expressed as g/mL which indicates its radical scavenging potential). Fractions Total phenolics (mg GAE/g) Total flavonoid (mg QE/g extract) Total tannin (mg TAE/g extract) ALE 55.46 44.6 19.91 AHE 21.55 15.8 -- ADE 34.82 26.0 -- AEA 51.54 39.9 12.85 Accepted Article 25 This article is protected by copyright. All rights reserved. JDB_12216_F1A JDB_12216_F1B JDB_12216_F2A Accepted Article 26 This article is protected by copyright. All rights reserved. JDB_12216_F2B JDB_12216_F3A JDB_12216_F3B Accepted Article 27 This article is protected by copyright. All rights reserved. JDB_12216_F3C JDB_12216_F3D JDB_12216_F4A Accepted Article 28 This article is protected by copyright. All rights reserved. JDB_12216_F4B JDB_12216_F5A Accepted Article 29 This article is protected by copyright. All rights reserved. JDB_12216_F5B JDB_12216_F6 Accepted Article 30 This article is protected by copyright. All rights reserved. JDB_12216_F7A JDB_12216_F7B Accepted Article 31 This article is protected by copyright. All rights reserved. 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