Synthesis & Characterization of AV-AgNPs
Silver nanoparticles produced by the method of Chandran et al. (2006) using aqueous aloe vera leaves extract and synthesized silver nanoparticles (AV-AgNPs) were characterized using analytical techniques via Shimadzu UV-visible spectrophotometer (UV-1900, Japan) and scanning electron microscopy (JEOL-Japan-JSM 6380A) described in detail in our previously published report (Begum and Mahboob 2019).
Reagents and chemicals used were analytical grade and procured from the local suppliers (Pakistan) although silver nitrate was acquired from Sigma-Aldrich (USA) and streptozotocin was obtained from Calbiochem (Germany).
Healthy, male adult Wistar albino rats (n=50) of the identical age group with a bodyweight of 200 (±20 g) procured by the International center for chemical and biological sciences (ICCBS), University of Karachi, accustomed to laboratory environments one-week before experimentation and caged in a moderate temperature maintained room at 23 ± 4 ºC and 12 hours light-dark cycles. Rats were fed on rodent pellet food and water ad libitum with free access. The experimental procedures were designed by following the world-wide accredited health research extension act (1985).
Induction of diabetes
To make diabetes model, rats were injected (once) IP, freshly prepared STZ (35 mg/ml/kg in citrate buffer; pH= 4.5) to overnight-fasted rats (Gayathri and Kannabiran 2008) then, 2% glucose solution (once) was given to the rats via orally to overcome the hypoglycemic shock and confirmed diabetes later 72 hours of STZ injection (IP) via glucose levels >250 mg/dl and proceed for the further experimental protocol.
Experimental animals segregated into five groups comprises (n=10) and received the bellow mentioned treatment.
Group-I (C): Control group continue untreated, received a normal rat diet.
Group-II (DC): Diabetic Control group treated with Streptozotocin (STZ) freshly dissolved in chilled citrate buﬀer at pH:4.5, given i. p (once) 35 mg/ml/kg body weight (Gayathri and Kannabiran 2008).
Group-III (D+AV-AgNPs): Diabetic group received freshly prepared green synthesized silver nanoparticles (AV-AgNPs) orally 10 mg/ml/kg body weight dissolved in aqueous media for 28 days daily after induction of diabetes.
Group-IV (D+AVLE): Diabetic group received freshly prepared AV-aliquot orally 100 mg/ml/kg body weight for 28 days daily after induction of diabetes.
Group-V (D+GLB): Diabetic group received freshly prepared Glibenclamide orally 600 µg/kg body weight dissolved in aqueous media for 28 days daily after the induction of diabetes (Prabhu et al. 2018).
During the experiment, animals were weighed, monitored individually, and regularly from all experimental groups while % Change in body weight was calculated via formula (Tabassum and Mahboob 2018) of each rat.
Samples of blood and liver were collected later 24 hours of the last dose of administration by decapitating the animals from the neck wound. Plasma separated via centrifugation (2000 rpm) for 20 minutes while after excised liver, trimmed then rinsed with ice-chilled saline, dried, weighed, and a piece of tissue for histological examination immersed in 10% formalin. Whereas, the remaining part kept at -80 °C for biochemical analysis.
Liver homogenate preparation
Liver (1:10 w/v) homogenized with sodium phosphate buffer (10% of 0.1 M at pH-7.4) and centrifuged (1000 rpm) for 10 min (4◦C) to get supernatant and subsequently added 10 µl BHT (0.5 M in acetonitrile) in a fraction to avert further oxidation whereas other part recentrifuged (12000 rpm for 20 min at 4◦C) for further estimations (Khan et al. 2011).
Estimation of blood glucose
Blood glucose determined on alternate seven days for 28 days. Rats fasted for 12 hours, blood specimens attained from the tail vein, and measured levels of glucose using a Glucometer (On-Call EZ II ACON Laboratories, Inc., USA). Further, oral glucose tolerance tests (OGTT) executed on day 28. Briefly, animals fasted overnight and, then the concentration of glucose was determined, followed by OGTT via gavage 2g/kg glucose to rats and after the 30-minute interval for 2 hours concentration of glucose assessed by using a glucometer.
Biochemical indices of liver function such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) were analyzed using a commercially prepared kit (LABKIT, Chemelex, S.A., Canovelles-Barcelona, Spain) as described and resulting values expressed as U/L.
The Malondialdehyde (MDA), is the degree of lipid peroxidation determined by the scheme of Okhawa et al. (1979) based on thiobarbituric acid (TBA) reacting substance interaction with MDA thus produced pink color which was determined by Shimadzu Spectrophotometer (UV-1900, Japan) at 530 nm absorbance and resulting values were expressed as nmol/gm of tissue.
In the liver homogenate, Superoxide dismutase (SOD) activity was assessed by the scheme of Kono (1978) in the cell-free supernatant. the rate of NBT reduction in terms of inhibition (%) in the reaction mixture was noted per minute at 560 nm absorbance on Shimadzu Spectrophotometer (UV-1900, Japan) and resulting values expressed as U/gm of tissue.
The activity of Catalase (CAT) in liver homogenate was assessed by the scheme of Sinha et al. (1972). In brief, the sample was added to Hydrogen peroxide and phosphate buffer (pH;7) at 100℃ and subsequently added dichromate acetic acid reagent into a fraction of reaction mixture then allowed to boil for 10 minutes, cooled, note the absorbance on Shimadzu Spectrophotometer (UV-1900, Japan) at 570 nm and resulting values expressed as nmol/gm of tissue.
In the liver homogenate, Glutathione reductase (GSH) activity was measured by the scheme of Carlberg and Mannervik (1985). Briefly, the sample was mixed with BSA, β-NADPH, potassium phosphate buffer, and oxidized-Glutathione. Note the absorbance of each reaction mixture for 5 min at 25◦C by Kinetic-Spectrophotometer (PRIM-500, Germany) at 340 nm and resulting values expressed as U/gm of tissue.
Rats liver removed quickly, washed with phosphate-buffered saline to eradicate debris, and a piece of tissue immersed in 10% formalin. About 5 μm tissue slices implanted in molten paraffin wax, stained with hematoxylin and eosin (H&E), and tissue morphology was analyzed by light microscopy at 10x magnification.
Data exhibited as mean ± SD (n=10) considered signiﬁcant when p < 0.05 using ANOVA (One-way) followed by post hoc test (Tukey’s) using IBM-SPSS version 22 (IBM, Anmork, NY, USA).