Antidiabetic and antioxidant potential of Alnus nitida leaves in alloxan induced diabetic rats

Journal Pre-proof Antidiabetic and antioxidant potential of Alnus nitida leaves in alloxan induced diabetic rats Moniba Sajid, Muhammad Rashid Khan, Hammad Ismail, Sara Latif, Amna Abdul Rahim, Ramsha Mehboob, Sayed Afzal Shah PII:

S0378-8741(19)33810-3

DOI:

https://doi.org/10.1016/j.jep.2020.112544

Reference:

JEP 112544

To appear in:

Journal of Ethnopharmacology

Received Date: 25 September 2019 Revised Date:

4 December 2019

Accepted Date: 1 January 2020

Please cite this article as: Sajid, M., Khan, M.R., Ismail, H., Latif, S., Rahim, A.A., Mehboob, R., Shah, S.A., Antidiabetic and antioxidant potential of Alnus nitida leaves in alloxan induced diabetic rats, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/j.jep.2020.112544. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Antidiabetic and antioxidant potential of Alnus nitida leaves in alloxan induced diabetic rats 1

1*

Moniba Sajid , Muhammad Rashid Khan , Hammad Ismail2, Sara Latif1, Amna Abdul Rahim1, Ramsha Mehboob1, Sayed Afzal Shah3 1

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University,

Islamabad, 45320, Pakistan 2

Department of Biochemistry and Molecular Biology, University of Gujrat, 50700 Gujrat,

Pakistan 3

Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University,

Islamabad, 45320, Pakistan *

Corresponding author.

E-mail addresses: [email protected](M. Sajid) [email protected] (M. Rashid Khan) [email protected] (H. Ismail) [email protected] (S. Latif) [email protected] (A. A. Raheem) [email protected] (R. Mehboob) [email protected] (S. Afzal Shah).

Abbreviations: ANME: A. nitida leaves methanol extract; ANHE: n-hexane fraction of ANME; ANCE: Chloroform fraction of ANME; ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME. ALT; alanine transaminase, AST: aspartate transaminase, BUN; blood urea nitrogen, CAT; catalase, POD; peroxidase, SOD; superoxide dismutase, GSH; reduced glutathione, TBARS; thiobarbituric acid reactive substances, AST; aspartate transaminase, ALT; alanine transaminase, BUN; blood urea nitrogen, HDL; heavy density lipoprotein, LDL; low density lipoprotein.

Abstract Ethnopharmacological relevance: Leaves of Alnus nitida are used by local communities for the management of diabetes and in inflammatory disorders. Methods: Powder of shade dried leaves of A. nitida was extracted with methanol (ANME) and fractionated in escalating polarity i.e n-hexane (ANHE), chloroform (ANCE), ethyl acetate (ANEE) and soluble residual aqueous fraction (ANAE). The extract /fractions were evaluated for antidiabetic in vitro assays; α-amylase, α-glucosidase and dipeptidyl peptidase-4 (DPP-4). The in vivo investigations were carried out on ANEE and ANAE (100 mg/kg; 200 mg/kg, p.o.) in alloxan (125 mg/kg i.p.) induced hyperglycemic rats. Serum analysis was performed on liver, pancreas and kidney function markers. Analysis of antioxidant enzymes and genotoxic studies were carried out on pancreas, liver and kidneys tissues. GC-MS analysis was performed on ANME whereas HPLC analysis was carried out on ANME, ANEE and ANAE. Results: Preliminary in vitro assays indicated appreciable antidiabetic activity of ANEE and ANAE against α-amylase, α-glucosidase and DPP-4 assay. Furthermore, in vivo antidiabetic effect of ANEE and ANAE was inveterate by anti-hyperglycemic action in normal glucose loaded and diabetic glucose loaded animals. Single dose of alloxan (125 mg/kg) decreased the level of insulin and high density lipoprotein while raised the level of amylase and lipase, ALT, AST, total lipids, triglycerides, cholesterol, creatinine, BUN, CPK, CK-Mb in serum. Concentration of H2O2, lipid peroxidation (TBARS) and nitrite was increased (P < 0.05) whereas level of tissue protein, glutathione content (GSH) and antioxidant enzymes decreased in pancreas, liver and kidneys as compared to control group. Administration of ANEE and ANAE for 14 days after induction of diabetes decreased the hyperglycemia and restored the level of these parameters. Histopathological and genotoxic studies also endorsed the defensive strategies of ANEE and ANAE. GC-MS analysis of ANME demonstrated the presence of antidiabetic constituents i.e. linalool, Vitamin E and phytol. Conclusion: Results obtained in this study suggests antidiabetic and antioxidant abilities and provides the scientific proof of the folklore medicine. Keywords: Alnus nitida; antidiabetic; antioxidant; hyperglycemia; lipid peroxidation. 1. Introduction Medicinal plants have extensively been practiced for ethno medical management of diabetes or hyperglycemia in local medicine systems and are presently recognized as a substitute for

managing diabetes (Shah and Khan, 2014; Rashid et al., 2019). Diabetes mellitus is an ailment with homeostasis of lipid, carbohydrate metabolism is inadequately controlled by insulin causing advancement of fasting and postprandial blood glucose concentrations. As a result of metabolic variations, no less than 50% individuals with diabetes encompass prominent microvascular (neuropathy, retinopathy and nephropathy) and macrovascular (heart failure, myocardial infarction and brain stroke) impediments (Stadler et al., 2015). These impediments are the common attributes of type I and type II diabetes. Injection of alloxan and streptozotocin are used in rodents as diabetogenic agents. Metabolism of alloxan yields free radicals which have been proved as cytotoxic towards pancreatic-β cells (Rohilla and Ali, 2012). This process leads to hyperglycemia in rodents similar to diabetic patients. The most important aspect of chronic hyperglycemia is the glycation of proteins a source of free radical generation. Perpetual generation of free radicals in diabetic patients direct the complications of hyperglycemia in various organs on account of cellular impairment and injuries. The same process is also linked with pancreatic-β cell impairment and resistance to insulin. The oxidative stress can cause endoplasmic reticulum swelling and in turn activation of inflammatory pathways (Batool et al., 2018). Secondary metabolites of plants have shown antidiabetic and antioxidant effects in many experimental settings (Shah and Khan, 2014; Rashid et al., 2019). Alnus nitida (Spach) Endl. (Betulaceae) locally called as Sharol and Seril is native of western Himalayas (Stewart,1972). The leaves of A. nitida are used by the local communities in the form of infusion or decoction for the management of diabetes. For this purpose crushed leaves are either soaked in water overnight or boiled in water and the extract obtained is used in the morning (Yaseen et al., 2015). The stem bark of A. nitida is used for injuries, swelling, aching and bone fractures (Rokaya et al., 2010). The antioxidant potential of stem bark of A. nitida has been reported during in vitro antioxidant assays and hepatoprotective abilities against CCl4 induced liver damage in rat (Sajid et al., 2016). Anti-inflammatory and analgesic activities of A. nitida have also been reported (Sajid et al., 2017). The plant A. nitida exhibited potential anticancer activities against lung cancer during cell culture and in nude mice (Sajid et al., 2019). Despite the use of A. nitida leaves as a local treatment of diabetes, no scientific investigations have been documented. The recent investigation was executed to evaluate the antidiabetic potential of A. nitida leaves and hyperglycemia prompted oxidative stress in rat. Further we have

evaluated the inhibition of α-amylase, α-glucosidase and DPP-4 activity through in vitro assays. Serum analysis for various markers and antioxidant behavior of A. nitida in liver, pancreas and kidneys was also determined in alloxan induced diabetic rats. Furthermore, genotoxicity evoked by alloxan and defensive strategy of A. nitida was also studied. 2. Materials and methods 2.1. Reagents and chemicals Reduced glutathione (GSH), bovine serum albumin (BSA), oxidized glutathione (GSSG), 1,2 dithiobis nitrobenzoic acid (DTNB), nicotinamide adenine dinucleotide phosphate (reduced NADPH), glucose 6 phosphate, 2,8 dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium hydroxide, sodium tungstate, trichloro acetic acid (TCA) and alloxan were bought from the Sigma Chemicals Co., USA. 2.2. Plant collection Leaves of A. nitida were collected from the Charbagh town (Swat, Pakistan) in April, 2015. Area of assemblage was at 34.842727º north latitude and 73.431089º east longitude at an altitude of 1000 meter. Documentation of plant was done by Flora of Pakistan (Nasir et al., 1972) and then identified by Dr. Sumaira Sahreen, Associate Curator, Pakistan Museum of Natural History, Pakistan. The authentic sample with voucher number 1279633 was apprehended at the Pakistan Museum of Natural History, Islamabad. 2.3. Extraction and fractionation The leaves of A. nitida were air dried in shade at room temperature for 15 days and powdered by using Willy Mill to 60-mesh size. Leaves powder weighing 5 kg was soaked in 15 liters of commercial methanol and the soaking process was repeated twice. The extract obtained was strained with Whatman No. 1 filter paper and dried in rotary evaporator under vacuum to get the viscous material (ANME). A part of ANME was swayed in distilled water and solvents including, n-hexane (ANHE), chloroform (ANCE) and ethyl acetate (ANEE) were employed in escalation direction of polarity. Remaining soluble fraction was used as an aqueous fraction (ANAE). Solvents of all the fractions were evaporated by rotary evaporator and afterwards maintained at 4°C. 2.4. High performance liquid chromatography (HPLC-DAD) analysis HPLC-DAD (Agilent 1200, Germany) was utilized for the analysis of ANME, ANEE and ANAE. The equipment was aided with Zorbex RXC8 analytical column having 25 ml capacity

and 5 µm particle size. Mobile phase comprised of eluent A (acetonitrile : methanol : water : acetic acid; 7: 10: 80: 1) and eluent B (acetonitrile : methanol : acetic acid; 30: 60:1). The gradient (A: B) used was the following: 0–30 min (0 to 50 % B), 21–25 min (50 to 100 % B), 26–30 min (100 % B) and 31-40 (100 to 0 % B) at the flow rate of 1 ml/min. Sample and standards were dissolved in HPLC grade methanol at 1 mg/ml concentration. The solutions were filtered using 0.45 µm membrane and 20 µl was injected in the system. The standards were examined at different wavelengths (rutin at 257 nm, catechin and gallic acid at 279 nm, caffeic acid and apigenin at 325 nm however quercetin, myricetin and kaempferol at 368 nm). The experiment was run in triplicate and the equipment was renovated 10 minutes after each run. The quantification was done by utilizing standard external method. 2.5. Gas chromatography-Mass spectrometry (GC-MS) analysis ANME was scrutinized for the occurrence of active constituents on “Thermo GC-Trace Ultra Ver; 5.0” gas chromatograph attached with a “Thermo MS DSQ II” in order to verify mass. Chemicals was detached on a “ZB 5-MS Capillary Standard Non-polar Column” with 50 m length having 255 µm thick film. During experiment, the temperature was elevated from 70 to 260 °C at the rate of 8 °C/min. flow-rate of carrier gas; helium was 1 ml/min. The volume of sample used was 1 µl for GC-MS analysis. Retention times and mass spectra are compared for chemical characterization of constituents and was based on those gained from authentic specimen and/or NIST/NBS and Wiley libraries spectra (Cha et al., 2005). 2.6. In vitro antidiabetic activity 2.6.1. α- Amylase inhibition assay In vitro inhibition of α-amylase activity was carried out by the method of Sumathy (2013). The reaction mixture was prepared by the addition of 5 µl of extract/fraction (200, 100 and 50 µg/ml) with 40 µl of starch (0.05%) and 30 µl of potassium phosphate buffer (pH 6.8) in 96-well micro titer plate. A volume of 10 µl of α-amylase (0.2 U/well) was added to the reaction mixture and incubated at 50 °C for 30 min. Then a volume of 20 µl HCl (1M) was added to stop the reaction and was followed by addition of 100 µl iodine reagent (5 mM KI and 5 mM I2). The absorbance was recorded at 540 nm with microplate reader (BioTek, Elx800). Acarbose and DMSO were the positive and negative controls respectively. 2.6.2. α- Glucosidase inhibition assay

For estimation of α-glucosidase inhibition activity reaction mixture was prepared by adding 300 µl of carbohydrate (30 mg/ml) and 150 µl either of PBS (pH 7.2 as control), acarbose (30 mg/ml, as a positive control) or extract (500 mg/ml) and incubated at 37 °C for 10 minute. Rat intestinal acetone extract was added to the reaction mixture to start the reaction. Concentration of glucose was measured at 0, 30, 60 and 90 min after incubation time by using an enzymatic assay kit (Analox Ltd. London, UK) and detected on a PGM7 Micro-Stat Analyser (Analox Instruments Ltd; London, UK) according to method of Panwar et al. (2014). 2.6.3. DPP-4 inhibition assay DPP-4 inhibition was determined by employing the scheme of Fujiwara and Tsuru (1978) in which Gly-pro-AMC, DPP-4 substrate enlightened the amount of free 7 amino 6 methyl coumarin (AMC). The activities were performed in triplicate in 96-well micro titer plate using fluorescence and quantified at Em430 nm resulting excitation at Ex351 nm employing a Tecan Safire fluorometer (Reading, England, UK). The samples were primed in 50 mM HEPES buffer at pH 9.6. Every well comprised of 25 µl of DPP-4 (1 U/ml), 25 µl of test sample and 28 µl of 1 mMGly-pro-AMC substrate. Incubation was done at 37°C with mild agitation for 1 h and then 200 µl of 3 mM acetic acid was poured to inhibit reaction. Berberine (13 mM) was employed as positive control. 2.7. Animals Sprague Dawley male rats (150-200 g) were acquired from the primate facility situated at the Quaid-i-Azam University, Islamabad, Pakistan. The rats were housed separately in normal steel cages under standard conditions (12 h light and 12 h-dark cycle; 25±30 °C). The rats were served with usual diet (rodent chow and water ad libitum). The experimental protocol (Bch=0275) was sanctioned by ethical committee of the university. 2.8. Glucose tolerance test in normal animals Glucose tolerance test was accomplished in overnight (15 h) fasted Sprague Dawley male rats with free access to water ad libitum. The animals were distributed in six groups containing 7 rats in each group. Group I used as normal control and was administered with 5% of DMSO at a dose of 1 ml/kg to each rat. Other groups were administered orally with glibenclamide (10 mg/kg), ANEE (100 mg/kg), ANEE (200 mg/kg), ANAE (100 mg/kg) and ANAE (200 mg/kg). The animals were treated with 5% dextrose (10 g/kg) after 30 min of extract administration. The amount of glucose in blood was recorded by glucometer by puncturing tail tip at 0 min and after

30 min, 60 min and 120 min of glucose administration. The values of glucose were recorded in mg/dl of blood. 2.8. Glucose tolerance test in diabetic animals For this study Sprague Dawley male rats were treated intraperitoneally with alloxan monohydrate (125 mg/kg) in 0.9% saline in 15 h fasted animals. The normal control animals received 0.9% saline intraperitoneally. The level of glucose in blood was recorded after 48 h of treatment in 12 h fasted rats. The animals showing blood glucose >230 mg/dl were used for further studies. There rats were distributed in to 7 groups with 7 in each group. The rats of Group I was normal control and have received only 5% of DMSO at a dose of 1 ml/kg to each rat. Group II was diabetic control and was treated with 5% of DMSO. The rats of Group III were treated with glibenclamide (10 mg/kg) whereas rats of other groups were treated with ANEE (100 mg/kg), ANEE (200 mg/kg), ANAE (100 mg/kg) and ANAE (200 mg/kg). The animals were treated with 5% dextrose (10 g/kg) after 30 min of extract administration. Level of glucose in blood was monitored with glucometer at a time period of 0 min, 30 min, 60 min and 120 min from tail vein after glucose administration to rat. 2.9. Induction of Type II diabetes Sprague-Dawley male rats were used to evaluate the utility of ANEE and ANAE fractions in alloxan induced diabetic rats. Diabetes was induced in 15 h fasted rats by intraperitoneal injection of alloxan monohydrate (125 mg/kg) prepared in 0.9% saline. Normal control group received the 0.9% saline intraperitoneally. To prevent excessive hypoglycemia alloxan treated rats received 5% glucose for 24 h instead of simple water. After 72 h of alloxan injection in 12 h fasted animals level of glucose was monitored in each and rats showing glucose concentration above 230 mg/dl were included in the diabetic experiment. Diabetic animals were distributed in 6 groups (7 rats in each). The rats were served with usual diet (rodent chow and water ad libitum) in this experiment. The ANEE and ANAE were dissolved in 5% DMSO and animals were treated with The schematic treatment is as follows.

Group-1: Normal control group treated with 1 ml/kg of vehicle (5% DMSO) Group-2: Diabetic control group treated with 1 ml/kg of vehicle (5% DMSO) Group 3: Diabetic positive control group received reference drug glibenclamide (10 mg/kg) Group 4:-Diabetic animals received ANEE (100 mg/kg) dissolved in 5% DMSO Group 5:-Diabetic animals received ANEE (200 mg/kg) dissolved in 5% DMSO

Group 6: Diabetic animals received ANAE (100 mg/kg) dissolved in 5% DMSO Group 7: Diabetic animals received ANAE (200 mg/kg) dissolved in 5% DMSO 2.9.1. Assessment of antidiabetic activity Rats were fed with usual diet (rodent chow and water ad libitum) and received their respective treatments for 14 days. In fasted animals (12 h) level of glucose in blood of each animal was measured after 1st, 7th and 14th day from tail vein with glucometer. After 14th day, the animals were euthanized under mild ether anesthesia. The blood was collected by the cardiac puncture in plane tubes and stored at 4 °C. Kidneys, liver and pancreas was removed and rinsed in cold saline. A portion of the pancreas was processed for histology while a portion of pancreas and other organs was placed in liquid nitrogen for antioxidant enzymatic and other biochemical assays. 2.9.2. Biochemical studies of serum Quantitative measurement of insulin level in serum was done by using the ELISA kit (Ultrasensitive Rat Insulin ELISA from Mercodia) according to manufacturer’s directions. Level of biochemicals in serum i.e. amylase, lipase, alanine transaminase (ALT), aspartate transaminase (AST), low density lipoproteins (LDL), high density lipoprotein (HDL), cholesterol, triglyceride, creatinine, blood urea nitrogen (BUN) was assessed by employing standard diagnostic kits purchased from Stattogger Strasse 31b 8045 Graze, Austria. 2.10. Homogenate preparation of tissues Tissues of the organs i.e. kidneys, liver and pancreas were homogenized and 10X volume was prepared by addition of 100 mM potassium phosphate buffer prepared in 1.2 mM EDTA (pH 6.4). Homogenate was centrifuged at 1500 g at 4 °C for 25 min. The supernatant obtained was used for the estimation of activity level of antioxidant enzymes. 2.10.1. Catalase (CAT) activity For the assessment of catalase activity the method of Chance and Maehly (1955) was used. The reaction mixture consisted of 630 µl of 40 mM potassium phosphate buffer (pH 8.5), 150 µl of 5.6 mM H2O2 and 25 µl of the supernatant. After 1 min of mixing the reaction contents, variations in the absorbance at 230 nm were recorded. One unit of the catalase activity was specified as change in absorbance of 0.01 and was expressed as units/min. 2.10.2. Peroxidase (POD) activity

POD activity was assessed by the method of Chance and Maehly (1955). Reaction mixture was prepared by the addition of 70 µl of 45 mM hydrogen peroxide, 20 µl of 25 mM guaiacol in 620 µl of 55 mM potassium phosphate buffer (pH 6.6). The reaction was commenced by the addition of 2 µl of supernatant. After one minute, variation in absorbance was noted at 460 nm. One unit of POD activity was considered as absorbance change of 0.01 and results were stated as units/min. 2.10.3. Superoxide dismutase (SOD) activity Activity level of SOD was assessed by the method of Kakkar et al. (1984). The homogenate was centrifuged at 15000 g for 15 min and then at 10,00 g for 10 min and 100 µl of supernatant was mixed with 800 µl of 0.05 mM of sodium pyrophosphate buffer (pH 6.5) and 60 µl of 180 mM of phenazine methosulphate. To start the reaction 200 µl of 980 µM NADH was added. After 2 min, 600 µl of glacial acetic acid was added to end the reaction. Absorbance of the reaction mixture was recorded at 540 nm. Results were stated in units/mg protein. 2.10.4. Protein assessment The method of Lowry et al. (1951) was used to assess the total soluble protein in different organs. For this purpose 200 mg of organ was taken and homogenized in potassium phosphate buffer. After homogenization, centrifugation was done at 4°C at 10,000 g for 10-15 min to get supernatant. An aliquot of 1 ml of the alkaline solution was added in 1.2 ml of supernatant and combined by vortex machine and was incubated for 30 min. Later the variation in absorbance was recorded at 630 nm using the micro plate reader. Bovine serum albumin (BSA) curve was employed as standard to evaluate the amount of serum proteins in given sample. 2.10.5. Glutathione (GSH) assessment Assessment of glutathione was done according to the method of Jollow et al. (1974). An aliquot of 600 µl of 4% sulfosalicylic acid was mixed with 500 µl of tissue homogenate and incubated for 1 h at 4 °C. After centrifugation for 20 min at 1200 g, 30 µl of supernatant was mixed with 910 µl of 0.2 M potassium phosphate buffer (pH 9.6) and 60 µl of 200 mM dithio-bis nitro benzoic acid (DTNB). Absorbance of reaction mixture was recorded at 405 nm. The GSH activity was stated as µM GSH/g tissue. 2.10.6. Lipid peroxidation assay For the assessment of lipid peroxidation, the method of Iqbal et al. (1996) was used. The reaction mixture was prepared by addition of 280 µl of 0.11 M phosphate buffer (pH 9.4), 20 µl of 150

mM ferric chloride, 150 µl of 100 mM ascorbic acid and 200 µl of the homogenate. Reaction mixture was incubated at 37 °C for 1 h in the shaking water bath followed by the addition of 400 µl of 10% trichloroacetic acid to stop the reaction. Then 550 µl of 1.68% thiobarbituric acid was added and tubes were retained in the water bath again for 20 min. The tubes were removed from the water bath and placed in crushed ice for 10 min and then centrifuged at 25000 g for 10-20 min. Absorbance of the supernatant was noted at 540 nm against a reagent blank. By employing molar extinction coefficient of 1.59×105/M/cm, results were expressed as nM of thiobarbituric acid (TBARS) produced per min per mg tissue at 37 °C. 2.10.7. Nitrite assay The assessment of nitrite content in the samples was made by using the Griess reagent according to the method of Green et al. (1982). Samples were deproteinized by addition of 200 µl each of 2% ZnSO4 and also 0.5 M NaOH. After centrifugation at 6400 g for 12-15 min, 10 µl of supernatant was mixed with 1.5 ml of the Griess reagent in cuvette. The absorbance of the reaction mixture was noted at 570 nm by using Griess reagent as a blank. Sodium nitrite standard curve was used for estimation of the nitrite concentration in the samples. 2.10.8. Hydrogen peroxide assay Assessment of H2O2 content in the samples was made by the method of Pick and Keisari (1981). The reaction mixture was prepared by addition of 500 µl of 0.02 M phosphate buffer (pH 6.7), 200 µl of supernatant, 200 µl of 0.20 nM solution of phenol red, 200 µl of 6.5 nM of dextrose and 8.5 units of the horse radish peroxidase and was left at room temperature for 60 min. Then 200 µl of 1 N NaOH was added to stop the reaction. Afterwards, it was centrifuged for 10-20 min at 600 g and absorbance of supernatant was recorded at 559 nm against blank. Standard curve of H2O2 oxidized by phenol red was used to assess the formation of H2O2 as nM H2O2/min/mg tissue. 2.11. Histopathological examination A part of pancreas was fixed in a fixative (absolute alcohol : formaldehyde : acetic acid ; 6: 3: 1). The tissues sections of 4 µm thin were stained with hematoxylin/eosin and studied under light microscope (DIALUX 20 EB) at 40X magnification. Slides of all groups were studied and photographed. 2.12. Comet assay

The method of Dhawan and Anderson (2009) was used to evaluate DNA injury. Slides were sterilized and immersed in 1% NMA i.e. normal melting agarose then permitted to solidify at the room temperature. Small section of the tissue was put in 1.5 ml of chilled lysing solution, crushed into tiny portions and assorted with 70 µl of agarose solution. This solution was covered on already prepared cover slip and slides were gradually positioned on it. Slide was put on ice packs for around 10-15 min. The cover slip was detached and again the low melting point agarose was poured and put on ice packs for solidification. Afterwards covered with the LMA i.e. low melting point agarose and placed again in lysing solution for about 8-10 min and kept in refrigerator for 3 h. After electrophoresis CASP 1.2.3.b was used to analyze images for assessment of the magnitude of DNA damage. For every sample 50-100 cells were scrutinized for comet size, tail length, head length, tail moment and DNA content. 2.13. Statistical analysis The values were stated as mean ± standard deviation. For in vivo studies, the variations among different treatments were assessed by one way analysis of variance by using Statistix 8.1. Noteworthy variation among groups was calculated by Tukey’s multiple comparison tests at Pvalue ≤ 0.05. 3. Results 3.1. HPLC-DAD analysis The presence of known potent antioxidant compounds was scrutinized by the help of reverse phase HPLC technique in ANME, ANEE and ANAE. In this analysis the standards used were gallic acid, rutin, caffeic acid, catechins, apigenins, myricetin, kaempferol and quercetin The results are summarized in Table 1 and the demonstrative chromatogram of ANME showing the peaks for different compounds is shown in Figure 1S. From HPLC profiling of ANME, ANEE and ANAE, it was revealed that the ANME consisted of gallic acid (788 µg/g), rutin (729 µg/g), catechin (1021 µg/g), caffeic acid (2053 µg/g) and myricetin (285 µg/g). ANEE was constituted of gallic acid (2270 µg/g) and quercetin (870 µg/g) whereas ANAE have gallic acid (1350 µg/g) and quercetin (3650 µg/g). 3.2. GC-MS analysis The ANME was used for GC-MS analysis to dig out the presence of chemical classes and compounds. GC-MS analysis specified that ANME contained 30 chemical components eluted between 6.30 and 40.05 min (Fig. 2S). The documentation of these chemical components was

established on the basis of evaluation of their respective retention times and mass spectra to those attained from authentic samples or the NST/NBS and Wiley libraries standard spectra (Table 2). In ANME, out of these 30 chemical constituents, there were 2 siloxanes (0.21% on the basis of peak area), 2 alcohols (7.25%), 2 alkenes (1.2%), 1 amine (0.07%), 1 silicate (0.06%), 1 silane (0.75), 4 terpene alcohols (2.59%), 1 arene (0.33%), 6 esters (68.07%), 2 ketones (2.05%), 1 acid amide (0.10%), 1 phosphol (15.23%), 4 alkanes (0.67), 1 terpenoid (1.68%) and 1 carotenoid (0.11%). 3.3. In vitro antidiabetic assays 3.3.1. α- Amylase inhibitory studies The inhibitory activity of extract/fractions and their IC50 values against α-amylase are shown in Table 3. The IC50 values determined for the ANEE and ANAE were 199.2 µg/ml and 176.4 µg/ml as compared with the IC50 value of standard drug acarbose (IC50=194.3 µg/ml). The IC50 values obtained for ANME, ANHE and ANCE were 206.5 µg/ml, 904.2 µg/ml and 218.2 µg/ml. 3.3.2. α- Glucosidase inhibitory studies The IC50 values displayed for the ANEE after 30 min, 60 min and 90 min were 223.9 µg/ml, 215.6 µg/ml and 208.8 µg/ml as compared to the standard drug acarbose IC50 values of 92.11 µg/ml, 88.72 µg/ml and 83.83 µg/ml at the respective times. The ANAE exhibited IC50 values of 125.8 µg/ml, 120.9 µg/ml and 116.3 µg/ml after 30 min, 60 min and 90 min, respectively. Other extract/fractions used in this experiment had less inhibitory activities (Table 3). 3.3.3. DPP-4 inhibitory studies The IC50 values determined for various extract/fractions and for the standard drug are shown in Table 3. ANEE and ANAE exhibited remarkable low IC50 values of 110.8 µg/ml and 107.8 µg/ml as compared with the standard berberine (IC50 = 94.82 µg/ml). 3.4. In vivo antidiabetic studies 3.4.1. Hypoglycemic potential of A. nitida leaves in glucose loaded normal animals The effects of ANEE and ANAE on glucose tolerance in rat are displayed in Table 4. The blood glucose level was assessed at various time intervals i.e. 0, 30, 60 and 120 min after the administration of glucose to ANEE and ANAE at 100 mg/kg and 200 mg/kg treated rats. An elevation in the blood glucose concentration was detected till 30 min and then a decline in blood glucose was recorded in all the groups (Table 4). ANAE at its both concentrations i.e. 100 mg/kg and 200 mg/kg elicited remarkable hypoglycemic effect (P < 0.05) after 120 min as compared

with the normal control. ANEE also exhibited significant hypoglycemic effects but were less as compared to the ANAE treated groups. The ANAE (200 mg/kg) exhibited less area under curve as against the other treatments. However ANAE (100 mg/kg) and ANEE (200 mg/kg) showed nearly similar area under curve in the normal glucose loaded rats (Table 4). 3.4.2. Anti-hyperglycemic potential of A. nitida leaves in glucose loaded diabetic animals There was an appreciable elevation in blood glucose level of rats in alloxan-inducted diabetic rats. Treatment of diabetic rats with ANEE and ANAE declined the blood sugar level in a dose dependent manner at various time periods as compared to diabetic control group. Treatment of ANAE at 100 mg/kg and 200 mg/kg showed remarkable decrease (P < 0.05) in blood glucose concentration as compared to diabetic control after 0, 30, 60 and 120 min of extract treatment (Table 5). In diabetic glucose loaded rats administration of ANAE at both dosages (100 mg/kg and 200 mg/kg) showed less area under curve as compared to the glibenclamide treated rats. However, administration of ANEE exhibited higher area under curve as against the glibenclamide treated rats. 3.4.3. Hypoglycemic activity in alloxan induced diabetic animals Hypoglycemic potential observed for different treatments is shown in Table 6. ANAE at 200 mg/kg demonstrated significantly higher (P < 0.05) hypoglycemic activity at 7th day whereas after 14th day the hypoglycemic activity was not different (P > 0.05) as compared to glibenclamide treated rats. The other treatments showed lower hypoglycemic activity after 7th and 14th day as compared to glibenclamide treated rats. The values of area under curve for different groups are shown in Table 6. The results indicated less area under curve by the treatment of ANAE (200 mg/kg) as compared to glibenclamide treated rats. The administration of ANEE at both dosages and ANAE at 100 mg/kg indicated higher area under curve as compared to glibenclamide treated rats. 3.5. Effect of A. nitida leaves on biomarkers of serum In this study the serum profile of various biomarkers is given in Table 7. In alloxan induced diabetic rats the level of amylase, lipase, BUN, creatinine, ALT and AST in serum increased significantly (P < 0.05) as compared to normal control rats. However, the level of total protein in serum decreased significantly (P < 0.05) as compared to normal control rats. Administration of glibenclamide to diabetic rats restored the concentration of above parameters towards the normal control rats. Administration of ANAE and ANEE at both dosages restored the level of above

altered parameters towards the normal control rats. However the restoring effects were more pronounced with ANAE (200 mg/kg) as compared to ANEE at the respective dose. 3.6. Defensive effect of A. nitida leaves on lipid profile in serum Level of LDL, HDL, total cholesterol, triglycerides and insulin after 14 days in serum of various groups was recorded and presented in Table 8. There was a significant (P < 0.05) elevation in the level of LDL, cholesterol and triglyceride in alloxan induced diabetic animals as compared to normal control rats. Conversely, the level of HDL and insulin decreased (P < 0.05) after 14 days in diabetic control rats. Treatment of ANEE and ANAE at both dosages to diabetic rats revert the level of above serum markers towards the normal control rats in a dose dependent fashion. Though, administration of ANAE at both doses (100 mg/kg and 200 mg/kg) more effectively (P < 0.05) restored the alteration in above biomarkers as compared to respective doses of ANEE. 3.7. Defensive effect of A. nitida leaves on antioxidant status of pancreas, liver and kidney In alloxan induced diabetic animals the activity level of CAT, POD, SOD, tissue soluble protein and glutathione decreased while the level of TBARS, nitrite and H2O2 increased in pancreas, liver and kidneys of rat as compared to normal control group. Administration of ANEE and ANAE at both dosages (100 mg/kg and 200 mg/kg) restored the level of above biochemical towards the normal control rats in a dose dependent manner. The administration of ANAE at both doses was more effective in restoring the level of above biomolecules as compared to the ANEE respective treatment (Table 9). 3.9. Defensive effect of ANEE and ANAE on histopathology of pancreas Figure 1 depicted the histo-architecture of pancreas where normal control group displayed compact islets of Langerhans surrounded by normal intact acinar cells with prominent nuclei. A completely intact pancreatic duct was also observed in normal control with no inflammation or degeneration. In diabetic control rats the islets of Langerhans showed acute distortion and rupture of acinar cells and acinar cell steatosis. Administration of standard drug glibenclamide to diabetic rats expressively restored the normal structure of pancreas with only mild acinar cell disintegration. Administration of ANEE and ANAE at dose of 100 mg/kg and 200 mg/kg to diabetic rats reversed the pathological alterations towards normal control rats. The protective effects on the histopathology of pancreas were more pronounced with ANAE 200 mg/kg treatment to diabetic rat. 3.10. Defensive effect of ANEE and ANAE on genotoxicity in pancreas

Comet assay was used to assess the DNA damage and protective capacity of ANEE and ANAE in alloxan induced diabetic rats. In normal control rats pancreatic cells exhibited normal comet attributes; comet length, head length, tail length, % DNA in head, % DNA in tail and tail moment. However, in alloxan induced diabetic rats pancreatic cells displayed extensive DNA damages and resulted in enlarged comet length, comet tail and tail moment while a sharp decrease in % DNA in head as compared with normal control rats. Administration of ANEE and ANAE to diabetic rats notably decreased the alterations of comet parameters relative to diabetic control rats (Fig. 2). 4. Discussion The objective for management of diabetes is to sustain near average heights of glucose in both fasting as well as post prandial conditions. The restrictions of presently accessible pharmacological mediators for blood glucose control have encouraged development of new antidiabetic agents with diverse mode of action (Onitilo et al., 2014). Numerous natural assets have been explored pertaining to decrease the glucose assembly metabolized from carbohydrates in gut or in intestinal (Matsui et al., 2007). Dietary polysaccharides are metabolized by the action of various gastric enzymes especially α- amylase and α-glucosidase. The recent study revealed that ANME and its derived fractions have the potential to inhibit action of α-amylase and αglucosidase during in vitro conditions. Among the extract/fractions ANEE and ANAE remarkably obstructed the action of α-amylase and α-glucosidase and have exhibited low levels of IC50 values for inhibition of α-amylase and α-glucosidase. HPLC-DAD analysis of the ANME, ANEE and ANAE indicated the presence of myricetin, rutin and quercetin. In other studies these compounds have been implicated to prevent α-glucosidase and α-amylase activity (Ong and Khoo, 1997; Kamalakkannan and Prince, 2006). This study suggested that the use of ANAE and ANEE in the non-insulin dependent diabetes patients might decrease the postprandial hyperglycemia (Hamden et al., 2011b). Similar results have been reported in other studies where plant extract decrease the activity of digestive enzymes during in vitro conditions (Rashid et al., 2019). In recent years, DPP-4 has emerged as a novel target for diabetes treatment and has directed the researchers for the development of DPP-4 blockers with worthy protection and secure profile. Two incretin hormones; glucagon like peptide-1 (GLP-1) and glucose dependent insulinotropic polypeptide (GIP) are secreted from intestine. GLP-1 regulate the blood glucose by increasing

the synthesis of insulin, increase β-cell mass and inhibit the release of glucagon. But GLP-1 has sharp half-life and is degraded by the action of DPP-4. In this study ANEE and ANAE displayed appreciable inhibition of DPP-4 and have demonstrated low levels of IC50 values for inhibition of DPP-4 activity. Fractionation of ANME with solvents in escalating polarity efficiently concentrated the antidiabetic bioactive constituents in ANEE and ANAE. Presence of polyphenols in higher concentration in ANEE and ANAE might be the factors responsible for DPP-4 inhibitory action (Bansal et al., 2012; Parmar et al., 2012). Further, presence of terpenoids in ANME as indicated by the GC-MS analysis might also contributed towards the inhibition of DPP-4 activities and consequently causes hypoglycemia (Marques et al., 2010). Glucose prompted hyperglycemic model was chosen to evaluate the hypoglycemic action of ANEE and ANAE in normal rats. In the glucose loaded hyperglycemias model, ANEE and ANAE displayed significant anti-hyperglycemic activity at various intervals and the glucose levels have not surpassed those of normal control group, giving an implication with respect to the strong hypoglycemic activity of ANEE and ANAE. Further, ANEE and ANAE also showed antihyperglycemic effect in glucose loaded diabetic rats. The glucose tolerance might be credited to upgrade action of β-cells of pancreas for greater release of insulin. So system behind the ANAE and ANEE anti-hyperglycemic activity includes sensitivity of β-cells to glucose and thus elevated the secretion of insulin (Latha et al., 2004). Various plants with similar hypoglycemic activity have been reported (Shah and Khan, 2014; Rashid et al., 2019). The profound antihypergylcemic effects might be attributed by the synergic activity of diverse bioactive constituents in ANEE and ANAE. The additive effects of Maqui (Aristotelia chilensis) and lemon (Citrus x limon) juice also inhibited the postprandial increase of blood glucose upon intake of high glycemic index meals in healthy men (Avila et al., 2019). Prolonged hyperglycemia is linked with a range of metabolic ailments in animal and human diabetics triggering oxidative stress and reducing the action of antioxidant shield system (Hamden et al., 2009; Dahech et al., 2011). The oxidative stress is thought to be the source of cell damage in pancreas, kidney and liver (Hamden et al., 2011a). An augmented serum levels of amylase, lipase (indices of pancreas) of ALT, AST, LDL, HDL, cholesterol and triglyceride in serum (keys of liver dysfunction) and of creatinine, total serum protein and BUN (indices of kidney impairment) are recorded in diabetic rats of this study. As concerns the recent study,

administration of ANEE and ANAE to diabetic rats exhibited pronounced protective activities and effectively declined the impairment indices of pancreas, liver and kidneys. The present information expresses that alloxan treatment provoked high oxidative stress as proved by a decline in activity of antioxidant enzymes (CAT, POD, SOD), total protein and GSH levels and a higher concentration of TBARS, nitrite and H2O2 in pancreas, liver and kidneys tissues. The decrease of SOD action could be traced to the hyperglycemia that impelled oxidative stress in numerous tissues because of lack of enzyme cofactors; zinc and copper (Sharpe et al., 2013). Glutathione is vital in regulation of cell redox state and a decrease at cellular level in diabetes has been attributed due to oxidative stress (McLennan et al., 1991). Administration of ANEE and ANAE scavenge free radicals in diabetic rats, restore the level of antioxidant enzymes and consequently decrease the level of TBARS, H2O2 and nitrite in pancreas, liver and kidneys tissues. GC-MS and HPLC analysis indicated the presence of antioxidant constituents; rhodopin, phytol, vitamin E and polyphenols in ANME which might contribute toward the antioxidant effects of ANEE and ANAE against alloxan induced hyperglycemia in rat. Histopathological assessment of diabetic rats depicted destruction of islets of Langerhans in pancreas. However, restoration of number of islet of Langerhans towards the normal control rats with ANEE and ANAE in diabetic rats might indicate regenerative capacity of plant extract. In the present study, our aim was to assess free radical induced genotoxic effects of alloxan and ability of free radical scavengers present in ANEE and ANAE to ameliorate DNA damaging effects in pancreas. The DNA damages are detected by increase in comet length, % DNA in comet tail and tail moment in pancreatic cells; inducing pancreatic β-cells impairment and consequently to diabetes mellitus. However alloxan induced DNA damages were ameliorated by antioxidants present in ANEE and ANAE fraction. The anti-diabetic and antioxidant attributes of A. nitida may be related to the compounds; linalool, vitamin E, phytol and polyphenols as revealed by GC-MS and HPLC, which were previously reported as antidiabetic agent (Kulkarni et al., 2015). Linalool decreases plasma glucose level by enhancing level of insulin and encourages glucose consumption by the cells since linalool stimulates uptake of glucose in diaphragm. Linalool also decreases oxidative injury by decreasing the level of TBARS, TNF-α and TGF-β1 in diabetic rats (King, 2008; Ohga et al., 2007). Vitamin E and phytol act as an antidiabetic by decreasing lipid peroxidation, protein

glycosylation and insulin resistance in diabetic rat (Guimarães et al., 2010; Lima and Cardoso, 2013). 5. Conclusion Our studies indicate that administration of A. nitida leaves extract/fractions produce antihyperglycemic and antioxidant effects in diabetic rats. The administration of ANEE and ANAE maintains the antioxidant enzyme status in pancreas, liver and kidneys, accelerated the free radical scavenging activity and diminished the cellular damages induced by alloxan in diabetic rat. Competing interest Authors declare no conflict of interest. Funding The project was funded by the Department of Biochemistry Quaid-i-Azam University Islamabad Pakistan. Authors’ contributions: Moniba Sajid made significant contribution to experimentation, acquisition and drafting of the manuscript. Hammad Ismail made significant contribution for in vitro antidiabetic assays. Sara Latif, Amna Abdul Rahim, Sayed Afzal Shah and Ramsha Mehboob made significant contribution to experimentation and acquisition of data. Muhammad Rashid Khan edited the manuscript and has made substantial contribution to designing of experiment. The authors assign Muhammad Rashid Khan for correspondence and publication. All authors read and approved the final manuscript. Acknowledgements MRK is intensely acknowledged for his kind supervision, expert guidance and substantial facilitations of all necessary materials and equipment. References Ávila, F., Jiménez-Aspee, F., Cruz, N., Gómez, C., González, M. A. and Ravello, N., Additive effect of Maqui (Aristotelia chilensis) and lemon (Citrus x limon) juice in the postprandial glycemic responses after the intake of high glycemic index meals in healthy men, NFS Journal. 17, 2019, 8-16. Bansal, P., Paul, P., Mudgal, J., Nayak, P. G., Pannakal, S. T., Priyadarsini, K. and Unnikrishnan, M., Antidiabetic, antihyperlipidemic and antioxidant effects of the

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Figure 1: Protective outcome of A. nitida on pancreatic tissue. (A) Normal control, (B) Diabetic control, (C) Db + glibenclamide (10 mg/kg), (D) Db + ANEE (100 mg/kg), (E) Db + ANEE (200 mg/kg), (F) Db + ANAE (100 mg/kg), (G) Db + ANAE (200 mg/kg). IL: islets of Langerhans, AC: Acinar cells, MLD: mild Langerhans disruption, ALD: acute Langerhans disruption, LD: Langerhans disruption, AD: acinar disintegration, ACS: acinar cell steaosic, IC: inflammatory cells, PD pancreatic duct. ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME.

Figure 2: Fluorescence photomicrograph of pancreatic cells and protective outcome of A. nitida on genotoxicity. (A) Normal control, (B) Diabetic control, (C) Db + glibenclamide (10 mg/kg), (D) Db + ANEE (100 mg/kg), (E) Db + ANEE (200 mg/kg), (F) Db + ANAE (100 mg/kg), (G) Db + ANAE (200 mg/kg). ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME.

Supplementary files Figure S1: HPLC-DAD chromatogram of (A) ANME, (B) ANEE and (C) ANAE at different wavelengths Signal 1: 257λ, Signal 2:279λ, Signal 3: 325λ, Signal 4; 368λ. Conditions: Mobile phase A-ACN: MEOH: H2O: AA:: 5:10:85:1, Mobile phase B-ACN: MEOH: AA:: 40:60:1, Injection volume 20 µl, Flow rate 1 ml/min, Agilent RP-C8. ANME: A. nitida leaves methanol extract; ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME.

Figure S2: GC-MS analysis of A. nitida leaves crude extract

Table 1: HPLC-DAD profile of A. nitida leaves Extract ANME ANEE ANAE

Polyphenolics (µg/g of extract) Rutin Gallic acid Catechin Caffeic acid Apigenin Kaempferol Quercetin Myricetin 729 788 1021 2053 285 2270 870 3650 -

ANME: A. nitida leaves methanol extract; ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME. -: not detected. Table 2: GC-MS analysis of A. nitida leaves crude extract

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

Compound Cyclohexasiloxane, DodecamethylLinalool trans-4,7-Dimethyl-6,7-dihydro-5Hcyclopenta[c]pyridin-5-ol 3Z-3'S-gamma.-IRONE Cycloheptasiloxane, tetradecamethyl2-tert-Butyl-4-trifluoromethyl-1-methylimidazole SILICATE ANION TETRAMER Silane, (bromomethyl)Neophytadiene 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 3,7,11,15-Tetramethyl-2-hexadecen-1-ol Phenanthrene, 4-Bromobutanoic acid, tridec-2-ynyl ester Ethanone Phytol 6-Amino-1-[2-(3,4-dimethoxy-phenyl)-ethyl]-1Hpyrimidine-2,4-dione 1,2-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester (CAS) 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester (R)-5-Phenyl-4-trimethylsilyldibenzophosphole 15-Isobutyl-(13àH)-isocopalane Cyclohexane, 1,3,5-trimethyl-2-octadecyl- (CAS) Terephthalic acid, dodecyl 2-ethylhexyl ester Thiopheno[b,b']dicamphore 1,1-dioxide Vitamin E Cyclohexane, 1,1',1'',1'''-(1,6-hexanediylidene)tetrakis(CAS) Lucenin 2 bicyclo[3.2.0]hept-2,6-diene-1,2,3,4,4,5-d(6) Rhodopin Hexadecanoic acid, 2-phenyl-1,3-dioxan-5-yl ester, cis-(CAS) 2-(Phenylsulfanyl)cyclohexanol

Area % 0.15 1.07 0.09

Class Siloxane Terpene alcohol Alcohol

Retention time 9.15 9.88 10.30

0.31 0.06 0.07 0.06 0.75 0.89 0.16 0.26 0.33 0.06 0.10 1.10 0.10

Alkene Siloxane Amine Silicates Silane Alkene Terpene Alcohol Terpene Alcohol Arene Ester Ketone Diterpene alcohol Acid Amide

10.80 11.97 12.96 15.45 19.10 19.89 20.40 20.77 21.73 22.25 23.56 25.17 28.98

0.18

Ester

30.79

65.38

Ester

31.38

15.23 0.32 0.11 1.14 1.68 7.16 0.08

Phosphol Alkane Alkane Ester Terpenoid Alcohol Alkane

32.02 33.09 33.58 34.57 35.62 36.05 36.57

1.95 0.16

37.18 37.81

0.11 0.17

Ketone Deuterated cyclic Alkane Carotenoid Ester

0.76

Alcohol

39.64

38.59 39.08

Table 3: In vitro DPP-4, α-amylase and α-glucosidase inhibitory activity of A. nitida leaves IC50 (µg/ml) Time (min)

ANME

ANHE

ANCE

ANEE

ANAE

Standard

DPP-4

-

212.5±4.30c

900.8±5.37a

289.1±5.36b

110.8±1.98d

107.8±1.45de

94.82±3.33e

α-amylase

-

206.5±1.73bc 904.2±10.05a

218.2±3.56b

199.2±4.55c

176.4±1.78d

194.3±3.40c

α-glucosidase

30

263.2±2.98c

911.3±5.91a

356.8±4.32b

223.9±2.66d

125.8±4.31e

92.11±2.82f

α-glucosidase

60

255.3±3.21c

897.8±6.32a

343.2±3.99b

215.6±2.91d

120.9±5.21e

88.72±3.79f

α-glucosidase

90

249.5±6.86c

876.9±10.56a

339.9±7.96b

208.8±4.06d

116.3±4.14e

83.83±3.19f

. Values are presented as mean±SD (n=3). Means with different superscript (a-f) letters in column are significantly (P < 0.05) different from each other. ANME: A. nitida leaves methanol extract; ANHE: n-hexane

fraction of ANME; ANCE: Chloroform fraction of ANME; ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME. Standrads: DPP-4 – berberine, α-amylase – acarbose, α-glucosidase – acarbose

Table 4: Anti-hyperglycemic activity in glucose loaded normal animals Treatment

Blood glucose concentration (mg/dl)

Area under curve

0 min

30 min

60 min

120 min

Arbitrary units

Control

84.57±1.39

111.2±2.62a

101.2±1.97a

93.2±1.38a

11886

Glibenclamide (10 mg/kg)

86.14±1.95

92.4±2.22d

78.5±4.79e

72.1±1.76e

9696

ANEE (100 mg/kg)

83.21±2.64

102±3.41b

93.25±3.12b

88.54±2.74b

11019

ANEE (200 mg/kg)

85.28±1.60

95.8±2.03c

86.7±2.56c

79.85±2.85c

10352

ANAE (100 mg/kg)

85.42±1.13

97.42±2.50c

85.2±1.97c

81.14±1.67c

10309

ANAE (200 mg/kg)

84.85±1.67

91.85±1.67d

82.28±2.56d

76.57±2.22d

9943

Values expressed as mean ± SD (n = 7). Mean± SD with different superscript letter (a-e) within the column indicates significant difference (P < 0.05). ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME.

Table 5: Blood glucose concentration in glucose loaded alloxan induced diabetic rats Treatment

Blood glucose concentration (mg/dl)

Area curve

under

0 min

30 min

60 min

120 min

Arbitrary units

Normal control

85.71±1.25

110.28±2.25g

102.32±2.52e

92.14±2.62e

11889

Diabetic control

245.8±5.67

263.8±7.62a

289.5±3.20a

279.8±6.47a

33094

244.1±6.03

161.2±5.62d

106.7±5.56d

95.5±4.71e

16138

Db+ANEE (100 mg/kg)

246.5±4.07

193.0±6.08b

139.1±4.57b

147.2±5.87b

20254

Db+ANEE (200 mg/kg)

241.5±5.93

184.1±4.22c

129.2±3.14c

139.0±4.28c

19114

Db+ANAE (100 mg/kg)

248.0±5.52

151.0±4.70e

99.28±3.79e

103.2±3.38d

15814

Db+ANAE (200 mg/kg)

244.5±4.45

139.1±3.89f

86.85±3.34g

91.42±2.07e

14417

Db+Glibenclamide mg/kg)

(10

Values expressed as mean ± SD (n = 7). Mean± SD with different superscript letter (a-f) within the column indicates significant difference (P < 0.05). ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME.

Table 6: Hypoglycemic activity of A.nitida in diabetic animals Blood glucose concentration (mg/dl)

Area under curve

Treatment

1st day

7th day

14th day

Arbitrary units

Normal control

84.20±3.15

82.28±2.88f

83.2±3.63f

1079

Diabetic control

242.8±3.27

255.1±6.95a

257.4±7.87a

3327

Db+Glibenclamide (10 mg/kg)

241.1±2.03

182.28±6.56c

92.1±5.02e

2219

Db+ANEE (100 mg/kg)

232.5±3.07

206.4±5.50b

136.5±6.59b

2362

Db+ANEE (200 mg/kg)

242.3±5.93

181.8±6.03c

111.7±5.19d

2309

Db+ANAE (100 mg/kg)

251.2±4.52

172.7±6.11d

129.2±5.13c

2335

Db+ANAE (200 mg/kg)

241.5±3.45

152.57±5.61e

89.8±5.42ef

2041

Values expressed as mean ± SD (n = 7). Mean± SD with different superscript letter (a-f) within the column indicates significant difference (P < 0.05). ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME.

Table 7: Protective outcome of A. nitida on serum profile of pancreas, kidney and liver Treatment

Amylase (U/l)

Lipase (U/l)

Creatinine (mg/dl)

BUN (mg/dl)

147.2±3.60e

Total serum protein (mg/dl) 8.27±0.32a

Normal control

207.2±3.63f

1.12±0.19d

34.68±2.51e

25.14±2.96ef

34.85±2.41f

Diabetic control

397.0±7.87a

205.1±5.85a

4.85±0.24e

6.62±0.14a

76.68±3.96a

351.5±6.69a

321.1±6.57a

224.1±5.02d

157.2±4.49d

7.95±0.53b

2.00±0.18c

46.29±2.61c

31.14±2.96d

40.28±2.49e

Db+ANEE (100 mg/kg)

284.5±6.59b

175.1±4.47b

5.42±0.16d

3.03±0.37b

52.28±3.03b

53.00±3.41b

66.14±3.57b

Db+ANEE (200 mg/kg)

236.7±4.19c

161.0±5.91cd

6.67±0.26c

2.05±0.25c

41.71±2.13cd

27.42±1.51e

48.00±2.94d

Db+ANAE (100 mg/kg)

248.2±5.13c

163.8±6.54c

7.28±0.29b

1.18±0.18d

40.42±2.43d

39.57±5.34c

54.85±3.57c

Db+ANAE (200 mg/kg)

219.8±4.62e

145.5±4.37e

8.13±0.20a

0.84±0.11e

27.71±2.42f

29.42±1.13de

42.42±2.81e

Db+Glibenclamide mg/kg)

(10

ALT (U/l)

Values expressed as mean ± SD (n = 7). Mean± SD with different superscript letter (a-f) within the column indicates significant difference (P < 0.05). ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME.

Table 8: Protective outcome of A. nitida on serum profile of lipids LDL

HDL

Cholesterol

Triglycerides

(mg/dl)

(mg/dl)

(mg/dl)

(mg/dl)

Normal control

30.42±1.90e

64.14±2.67a

114.8±2.95d

92.57±2.90f

15.57±1.13a

Diabetic control

208.2±6.79a

26.42±2.29f

435.2±7.30a

330.7±7.92a

5.14±0.69d

Db+Glibenclamide (10 mg/kg)

42.28±1.49cd

57.85±2.26b

122.0±2.16c

104.5±2.90de

13.42±1.51ab

Db+ANEE (100 mg/kg)

54.14±2.60b

35.85±2.19e

149.4±3.43b

127.5±3.81b

10.14±1.57c

Db+ANEE (200 mg/kg)

38.57±2.43d

50.14±1.77c

126.0±2.82c

109.0±2.76d

13.00±1.29ab

Db+ANAE (100 mg/kg)

43.85±2.03c

43.28±2.69d

124.7±3.54c

120.4±2.71c

12.71±1.38bc

Db+ANAE (200 mg/kg)

25.42±1.90f

61.28±2.98ab

102.0±2.82e

100.4±2.43e

14.57±1.51ab

Treatment

Insulin (U/l)

Values expressed as mean ± SD (n = 7). Mean± SD with different superscript letter (a-f) within the column indicates significant difference (P < 0.05). ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME.

AST (U/l)

Table 9: Protective outcome of A. nitida on antioxidant enzymes, tissue protein, GSH, TBARS, nitrite and H2O2 in various organs Tissue protein (µg/mg tissue) Pancreas

GSH (µM/g of tissue)

TBARS Nitrite of (nM/min/mg (µM/ml) of protein)

H2 O2 (µM/ml)

Treatment

CAT

POD

SOD

Normal control

8.31±0.24a

6.51±0.19a

6.82±0.22a

9.62±0.42ab

36.52±1.81a

2.17±0.49b

64.48±3.08c

5.42±0.22d

Diabetic control

1.62±0.18f

2.14±0.13e

1.92±0.24e

1.99±0.42c

13.52±1.18d

10.57±0.28a

104.5±3.26a

12.51±0.27a

c Db+Glibenclamid 6.24±0.26 e (10 mg/kg)

5.78±0.42b

5.98±0.53bc

8.93±0.63ab

34.22±1.98ab

2.76±0.38b

69.21±2.59bc

5.92±0.28cd

Db+ANEE mg/kg)

e (100 3.31±0.31

3.31±0.21d

4.94±0.19d

8.66±0.63b

25.38±1.46c

3.16±0.79b

73.54±2.76b

7.14±0.83b

Db+ANEE mg/kg)

c (200 5.80±0.53

4.91±0.20c

5.88±0.63bc

9.66±0.23ab

32.52±2.38b

2.99±0.68b

68.24±2.52bc

5.92±0.34cd

Db+ANAE mg/kg)

d (100 4.28±0.30

4.44±0.36c

5.44±0.42cd

8.78±0.75ab

28.82±1.67c

2.87±0.84b

73.57±2.68b

6.51±0.77bc

Db+ANAE mg/kg)

b (200 7.41±0.67

6.05±0.28ab

6.32±0.45ab

9.71±0.24a

35.1±1.99b

2.26±0.59b

65.45±3.40c

5.64±0.23cd

Normal control

16.28±0.24a

8.71±0.38a

11.18±0.62a

Liver 12.85±0.17a

51.85±1.40a

2.34±0.35d

63.30±2.78c

6.21±0.18d

Diabetic control

4.32±0.22f

3.05±0.26f

4.24±0.56d

7.02±0.16c

20.07±1.13d

12.11±0.40a

103.3±3.20a

13.14±0.26a

7.97±0.35b

10.15±0.88abc 12.72±0.11a

49.85±1.86ab

2.70±0.32bcd

69.7±3.68b

6.72±0.41cd

c Db+Glibenclamid 12.41±0.26 e (10 mg/kg)

Db+ANEE mg/kg)

e (100 7.22±0.35

5.34±0.22e

8.91±0.44c

10.86±0.92b

39.91±0.97c

3.20±0.28b

70.52±2.15b

8.12±0.25b

Db+ANEE mg/kg)

c (200 11.90±0.54

7.14±0.22c

9.77±0.83abc

12.94±0.57a

47.50±2.13b

3.03±0.26bc

67.87±1.69bc

6.81±0.48cd

Db+ANAE mg/kg)

d (100 8.38±0.34

6.22±0.17d

9.08±0.37bc

10.88±0.94b

42.04±3.48c

2.94±0.19bcd

70.07±2.28b

7.08±0.65c

Db+ANAE mg/kg)

b (200 13.8±0.47

8.14±0.28b

10.40±1.11ab

12.86±0.48a

50.27±2.00ab

2.46±0.40cd

64.34±2.02c

6.45±0.47cd

Normal control

13.45±0.28a

7.18±0.26a

7.61±0.24a

Kidney 12.00±0.18a

34.12±1.58a

1.97±0.22c

57.07±1.96c

4.42±0.26d

Diabetic control

3.55±0.19f

2.04±0.17f

2.40±0.23d

2.01±0.54c

12.55±0.90f

10.66±0.26a

99.72±2.73a

11.58±0.30a

Db+Glibenclami de (10 mg/kg)

10.32±0.29c

6.15±0.25b

6.94±0.29ab

11.79±0.64a

30.64±1.78bc

2.09±0.58c

61.65±1.34c

4.64±0.30cd

Db+ANEE (100 mg/kg)

6.27±0.21e

3.50±0.25e

5.93±0.49c

9.94±0.36b

21.38±0.93e

3.14±0.69b

66.65±2.29b

7.15±0.92b

Db+ANEE (200 mg/kg)

10.10±0.51c

5.45±0.24c

6.88±0.19b

11.95±0.31a

28.47±1.41cd

2.21±0.29c

60.91±0.80c

5.05±0.36cd

Db+ANAE (100 mg/kg)

8.24±0.25d

4.60±0.23d

6.48±0.59bc

10.05±0.48b

25.41±2.57d

2.21±0.41c

68.05±2.60b

5.61±0.62c

Db+ANAE (200 mg/kg)

12.0±0.35b

6.5±0.30b

7.10±0.15ab

12.10±0.37a

32.24±1.84ab

1.97±0.25c

58.21±3.51c

4.57±0.26d

Values expressed as mean ± SD (n = 7). Mean± SD with different superscript letter (a-f) within the column indicates significant difference (P < 0.01). ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME.

Table 10: Protective aptitude of A. nitida against alloxan induced genotoxicity in pancreas Treatment

Normal control

Comet length (µm) 46.01±2.64f

Head length (µm)

Tail length (µm)

% DNA head

8.22±1.43e

37.8±0.31de

97.9±1.62a

in % DNA in Tail tail moment (µm) 2.07±0.42f 23.00±1.01ef

Diabetic control

184.2±2.14a 29.11±1.31d

154.8±3.11a

18.3±1.03e

81.71±1.25a

92.10±2.11a

Db+Glibenclamide (10 mg/kg)

54.72±2.11e 12.52±1.6a

42.2±2.42d

90.11±1.44a

9.11±1.23e

27.36±1.61e

Db+ANEE mg/kg)

(100 180.1±2.45a

48.73±2.11c

131.4±2.11b

22.45±1.23d

77.55±3.11b

90.05±3.05a

Db+ANEE Db+ANAE mg/kg)

(200 168.4±2.21b 45.25±1.52c (100 161.2±2.32bc 49.22±1.24b

123.2±2.31bc 111.9±2.62c

24.21±1.33cd 45.70±2.81c

75.79±2.65b 54.32±2.3cc

84.20±2.02b 80.60±3.03c

Db+ANAE mg/kg)

(200 62.10±1.26e

16.3±1.11f

91.21±3.43a

8.79±1.11e

31.05±2.01d

45.81±1.22bc

Comet parameters are expressed as mean± SD (n= 7). Mean± SD with different superscript letter (a-d) within the column indicate significant difference (P < 0.01). ANEE: Ethyl acetate fraction of ANME; ANAE: Aqueous fraction of ANME.