Animals
Adult male Wistar Albino rats of uniform weight (180–250 g) were used in this study, which were commercially purchased from the enlisted supplier of the University (M/S Chakraborty Enterprise, Kolkata, West Bengal, India). The animals were free to access food and potable water as they underwent acclimatization under laboratory conditions for 2 weeks prior to the experiment. All experiments were conducted during the day time (between 0900 and 1600 h), after an overnight fasting period. During the entire experimental period, the animals were maintained as per the guidelines of the Guide for the Care and Use of Laboratory Animals, National Institutes of Health (NIH). The rats were hygienically maintained in polypropylene cages in an air-conditioned environment of 22–25 °C, 40–70% humidity with 12 h light-dark cycles, and ventilation of 15–21 air changes/h. The entire study was permitted by the Institutional Animal Ethics Committee (IAEC), Assam, and conducted by following the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA); with prior approval number IAEC/DU/60 dated 24/09/2013.
Plant material and extraction
The collection of the bark of Garcinia lanceifolia was done from the University campus and neighboring areas of Dibrugarh, Assam India in accordance to the Good Agricultural and Collection Practice (GACP) guidelines. Images of the plant and collected fresh bark are shown in Fig. 1. The plant herbarium was authenticated by Dr. A. A. Mao, Botanical Survey of India, Eastern Regional Centre, Shillong (Letter no.: BSI/ERC/2014/Plant identification/882. A herbarium specimen of the plant was submitted at the Pharmacognosy Research Lab of the Institute for further references. After proper cleaning of the collected bark, it was cut into smaller pieces and dried to constant weight. Thereafter, the dried bark were pulverized in a mechanical grinder and stored in hermetically sealed containers until extraction process commenced.
The hydroalcoholic extract of the stem bark of Garcinia lanceifolia (HAEGL) was prepared by using cold maceration process with 1000 mL of water:ethanol (80:20) mixture. The extracts were concentrated by distillation, followed by vacuum drying using a rotary evaporator. Preliminary phytochemical tests were carried out with all the extracts in order to evaluate for the presence of different phytochemical constituents.
Chemicals
All chemicals used in this assay were of analytical grade and obtained from reputed suppliers like SRL Sisco Research Laboratories Pvt. Ltd., Mumbai, India; Roche (Products) Pvt. Ltd., Bayer Diagnostics, Mumbai, India; Himedia Laboratory, Mumbai, India; Loba Chemie, Mumbai, India; Rankem Chemicals, Faridabad, India; Otto Chemie, Mumbai, India; SRL Sisco Research Laboratories, Mumbai, India; Spectrochem, Mumbai, India; and Beacon Diagnostics Pvt. Ltd; Gujarat, India.
Acute toxicity and LD50 calculation of HAEGL
To process the toxicological studies of HAEGL, a series of doses, viz; 175 mg/kg, 550 mg/kg, 1750 mg/kg, 5000 mg/kg body weight were used.
To obtain the LD50 of HAEGL, the experiments were planned according to the methods approved by the Organization for Economic Cooperation and Development (OECD). After acclimatization of the environment, rats were administered HAEGL with doses in the sequence listed previously by a single oral gavage. The highest dose limit of 5000 mg/kg for HAEGL was determined by subjecting to a limit test at 5000 mg/kg, as per OECD guideline 425. Each dose was given to a single animal only. If the animal survived the current dose, the second animal received a consecutive higher dose. If the animal does not survive, the second animal received a lower dose. Since HAEGL did not show any toxicity reactions below the regulatory limit doses (i.e., 5000 mg/kg); hence, it was evaluated by a limit test of 5000 mg/kg. The LD50 and 95% profile likelihood (PL) of HAEGL were obtained by analyzing the experimental data using the AOT425 program (OECD guideline 425) [21,22,23].
Antidiabetic assay
Oral glucose tolerance test
Initial screening of the extract for the hypoglycemic activity was done in healthy rats by conducting oral glucose tolerance test (OGTT). The OGTT was performed for two different doses of HAEGL (250 and 500 mg/kg of bodyweight per orally) and blood glucose level was measured by one touch glucometer (ACCU-CHEK®, Roche India Pvt. Ltd., Mumbai, India). The blood glucose levels were estimated at the intervals of 0th, 30th, 60th, 90th, and 120th min after the administration of extract [24].
Induction of non-insulin dependent diabetes mellitus
Following overnight fasting, type II diabetes was stimulated in the experimental animals by an intraperitoneal (i.p.) inoculation of streptozotocin (Spectrochem, Mumbai, India) dissolved in 0.1 M cold citrate buffer (pH 4.5), at a dose of 65 mg/kg of bodyweight; followed 110 mg/kg of nicotinamide via the i.p. route (Spectrochem, Mumbai, India). The control rats were administered with the vehicle alone. The induced rats were tested for the presence of elevated blood glucose levels after 7 days and the rats with moderate hyperglycemia (blood glucose range of above 250 mg/dl) were utilized for the experiment [25].
Experimental design
The rats were segregated into five groups and for each group five animals were taken as follows:
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Group 1: Normal control rats administered with 0.5% carboxymethylcellulose (CMC) vehicle 5 mL/kg bodyweight per orally.
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Group 2: Streptozotocin (STZ)-induced diabetic control rats.
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Group 3: Diabetic control rats treated with HAEGL (250 mg/kg body weight/day) dissolved in aqueous solution of 0.5% CMC per orally for 15 days.
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Group 4: Diabetic control rats administered orally once a day for 15 days with HAEGL (500 mg/kg body weight/day) dissolved in aqueous solution of 0.5% CMC. Group 3 and 4 were served as test drug treated groups.
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Group 5: Diabetic control rats treated orally once a day for 15 days with the standard drug metformin (Yarrow Chem Products, Mumbai, India) at a dose 10 mg/kg of bodyweight which served as standard control.
Blood was sampled from the tail vein of the overnight (12–15 h) fasted rats and fasting blood glucose levels along with the average body weights were closely monitored on 0th, 5th, 10th and 15th day. On the 15th day, all the animals underwent euthanasia via cervical dislocation under moderate anesthesia and evaluated for various biochemical parameters, histopathology and in vivo anti-oxidant status [26].
Biochemical investigation
Total cholesterol (TC), triglycerides (TG) in EDTA plasma, and aspartate transaminase (SGOT), alanine transaminase (SGPT), alkaline phosphatase (ALP) levels in serum were measured colorimetrically using specific kits (Beacon Diagnostics Pvt. Ltd; Gujarat, India) as per the manufacturers’ instructions.
Histological studies
After euthanizing the animals, the pancreas of the experimental animals were collected and analyzed for histological changes. The skin samples were fixed in a fixative mixture of fixative (picric acid, formaldehyde 40%, and glacial acetic acid) for 24 h and thereafter embedded in paraffin with a melting point of 55–57 °C. Sections of 6 μm were obtained and stained with hematoxylin and eosin (H&E) stain to assess any structural alterations in the pancreas. All stained specimens were observed using optical light microscope and photographed using a camera.
In vivo antioxidant assay
About 400 mg of liver tissue was samples from each experimental animal subject, rinsed in normal saline, and blotted with filter paper. The tissues were then homogenized in 1.15% potassium chloride (KCl) and centrifuged at 1200 rpm at 40 °C for 10 min. The supernatant was collected which were again centrifuged at 10,000 rpm at 40 °C for 10 min. Again the supernatant were collected and centrifuged at 14,000 rpm for 60 min at 40 °C. The microsomal fraction were taken, suspended in KCl, and stored at − 20 °C.
Thereafter, the processed samples were analyzed separately to determine the levels of lipid peroxidation (LPO), reduced glutathione (GSH), and catalase activity (CAT) as per methods described earlier, with trivial modifications [27,28,29].
Antiulcer assay
Absolute alcohol induced ulcer model
The study was carried out as per the methods described by Umamaheswari et al. 2007 and Deore et al. 2011 [12, 30]. The rats were divided into five groups and for each group five animals were taken as follows:
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Group 1: Normal control rats administered with 0.5% carboxymethylcellulose (CMC) vehicle 5 mL/kg bodyweight per orally.
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Group 2: Rats with ethanol-induced ulcers (1 mL absolute alcohol per orally).
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Group 3: Ulcer-induced rats administered orally once a day for 5 days with HAEGL (250 mg/kg body weight /day) dissolved in aqueous solution of 0.5% CMC.
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Group 4: Ulcer-induced rats administered orally once a day for 5 days with HAEGL (500 mg/kg body weight/day) dissolved in aqueous solution of 0.5% CMC. Group 3 and 4 were served as test drug treated groups.
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Group 5: Ulcer-induced rats treated orally once a day for 5 days with the standard drug ranitidine (Yarrow Chem Products, Mumbai, India) at a dose 50 mg/kg of bodyweight which served as standard control.
On the 6th day, the animals were sacrificed by cervical dislocation under mild anesthesia and stomach was incised along the greater curvature and examined for ulcers. The ulcer index was counted, by taking into account the product of length and width of the ulcers observed in the glandular section of the stomach (square millimeters per rat). The summation of the lengths (mm) of all lesions for each stomach sample salvaged was termed as the ulcer index (UI)
$$ \mathrm{UI}=\left(n\ \mathrm{lesion}\ I\right)+\left(n\ \mathrm{lesion}\ II\right)+\left(n\ \mathrm{lesion}\ II I\right) $$
where UI = ulcer index; I = presence single, submucosal, punctiform hemorrhages along with of edema and hyperemia; II = presence of hemorrhagic submucosal lesions with minor erosions; III = evidence of profound ulcers with invasive lesions and erosions.
The percentage inhibition was estimated by utilizing the following formula;
$$ \%\mathrm{inhibition}=\left(\frac{UI_{\mathrm{control}}-{UI}_{\mathrm{treated}}}{UI_{\mathrm{control}}}\right)\times 100 $$
Acetic acid induced ulcer model
The rats were segregated into five groups and for each group five animals were taken as follows:
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Group 1: Normal control rats administered with 0.5% carboxymethylcellulose (CMC) vehicle 5 mL/kg bodyweight per orally.
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Group 2: Rats with acetic acid-induced ulcers (by injection of 0.05 ml of 10% acetic acid into the subserosal layer present in the glandular region of the anterior barrage of the stomach via a midline gastric incision performed under light anesthesia).
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Group 3: Ulcer-induced rats administered orally once a day for 20 days with HAEGL (250 mg/kg body weight /day) dissolved in aqueous solution of 0.5% CMC.
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Group 4: Ulcer-induced rats administered orally once a day for 20 days with HAEGL (500 mg/kg body weight/day) dissolved in aqueous solution of 0.5% CMC. Groups 3 and 4 were served as test drug-treated groups.
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Group 5: Ulcer-induced rats treated orally once a day for 20 days with the standard drug famotidine (Yarrow Chem Products, Mumbai, India) at a dose 20 mg/kg of bodyweight which served as standard control.
All treatments were administered orally 1 day post-surgery for 20 days, and on the 21st day all the animals underwent euthanasia by cervical dislocation under moderate anesthesia, stomachs were salvaged, and the healing progression of the ulcers were evaluated. Gastric lesions were assessed by investigating the inner gastric plane using a dissecting-binocular microscope. Thereafter, the ulcer area (mm2) and curative rate (%) were determined [30]. Thereafter, the stomach were immersed in 10% formalin and processed for histopathological evaluation. If presence of ulcerated tissue was detected, the center part of the damaged tissue was taken and dissected in half along the long diameter. In case of undamaged tissue, the sections from the basal part were taken.
Statistical analysis
All statistical analysis was performed by utilizing the GraphPad Prism software version 5.0 (GraphPad Software, La Jolla, CA, US). One-way ANOVA followed by various post-hoc tests was used to analyze the difference among multiple dosage groups. All values are expressed as mean ± SEM. A p < 0.05 value, evaluated at 95% level of confidence, was considered as statistically significant; unless otherwise indicated in the results.