Plant material and extraction
The mature fruits from Litsea cubeba (Lour) Pers. (Lauraceae) were collected from Gangtok, East Sikkim, India in the month of August 2018. The plant specimen was authenticated at the Central National Herbarium, Botanical Survey of India, Shibpur, Howrah, West Bengal, India and a voucher specimen has been kept in the Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India.
The air-dried fruits of Litsea cubeba were ground mechanically, weighed (500 g) and then it was immersed in petroleum ether and kept it in a mechanical shaker for 48 h, after that the solvent was evaporated in air and the air-dried powder was again extracted with methanol for 48 h using cold maceration process. The solvent was evaporated under reduced pressure by rotary vacuum evaporator and the dry extract (MELCF) was obtained. Then it was stored in a refrigerator at 40 °C for further use. To determine the chemical constituents present in the sample of MELCF, the phytochemical screening was performed by following the qualitative methods (Bhattacharya and Zaman 2009).
Drugs and chemicals
The chemicals and drugs used in the experiments were of analytical grade and procured from Hi-Media, Mumbai, India. All the test kits were purchased from Arkray Health Care Private Limited (Autospan), Gujarat, India.
In vitro assessments
Alpha amylase inhibitory activity
Alpha amylase inhibitory assay in vitro was performed by the 3,5-dinirosalicylic acid (DNSA) method (Patra et al. 2020). MELCF was dissolved in 10% dimethyl sulphoxide (DMSO) and again dissolved in phosphate buffered saline (PBS) of pH 6.9. Various concentrations (10 to 500 μg/ml) of extract was prepared. α-amylase solution 200 μl (2U/ml) was mixed with it and incubated at 30 °C for 10 min. Afterwards, 200 μl (1% w/v) starch solution was added to it again incubate for 3 min. Reaction was stopped by addition of 200 μl DNSA (3,5-dinitro salicylic acid) reagent and boiled it for 10 min in hot water bath. Afterwards, it was cooled in running tap water and absorbance was taken at 540 nm using a UV–visible spectrophotometer. In a same manner, a positive control test was performed by using acarbose. The alpha amylase inhibitory activity was expressed by using percentage of inhibition which is calculated by using the following equation:
$${\text{Percentage}}\;{\text{of}}\;{\text{inhibition}} = \left( {{\text{Absorbance}}\,{\text{of}}\;{\text{control}} - {\text{Absorbance}}\;{\text{of}}\;{\text{sample}}} \right)/{\text{Absorbance}}\;{\text{of}}\;{\text{control}}) \times {1}00$$
Alpha glucosidase inhibitory activity
Alpha-glucosidase inhibitory activity of MELCF in vitro was carried out according to the standard method with minor modifications (Kumari et al. 2021). 50 μl of phosphate buffer taken (100 mM, pH = 6. 8) in a 96-well plate, then 10 μl of alpha-glucosidase (1 U/ml), 20 μl of different concentrations of MELCF (0.1 to 0.5 mg/ml) was added to it and it incubated at 37 °C for 15 min. Afterwards, as a substrate, 20 μl P-NPG (4-nitrophenyl-β-D-glucopyranoside) (5 mM) added and incubated further at 37 °C for 20 min. The reaction was stopped by adding 50 μl sodium carbonate (Na2CO3) (0.1 M). The released p-nitro phenol’s absorbance was measured at 405 nm by spectrophotometer (Spectramax). As a standard various concentrations of acarbose (0.1–0.5 mg/ml) taken. There was a parallel set up without any test substance and the experiment was performed in triplicates. The results are expressed as percentage inhibition and the calculation is done by using the following formula:
$${\text{Percentage}}\;{\text{of}}\;{\text{inhibition}} = \left( {{\text{Absorbance}}\;{\text{of}}\;{\text{ control}} - {\text{Absorbance}}\;{\text{of}}\;{\text{sample}}} \right)/{\text{Absorbance}}\;{\text{of}}\,{\text{control}}) \times {1}00.$$
DPPH radical scavenging assay
The MELCF at different concentrations (40, 80, 160, 200, 400, 600, 800 µg/ml) was mixed with 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl). In the dark place, the mixture was kept at room temperature for 30 min and against blank the absorbance was measured at 517 nm. Ascorbic acid was employed as standard (Bhattacharya et al. 2010).
$${\text{Percentage}}\;{\text{of}}\;{\text{inhibition = }}\left( {{\text{Absorbance}}\;{\text{of}}\;{\text{control}} - {\text{Absorbance}}\;{\text{of}}\;{\text{sample}}} \right)/{\text{Absorbance}}\;{\text{of}}\;{\text{control}}) \times {1}00.$$
Nitric oxide scavenging assay
Through Griess Illosvoy reaction, nitric oxide scavenging activity was determined (Bhattacharya and Haldar 2020). The MELCF of different concentrations (40, 80, 160, 200, 400, 600, 800 µg/ml) were mixed with 2 ml of 10 mM sodium nitroprusside in 0.5 ml phosphate buffer (0.5 M, pH 7.4) the final volume is 3 ml. It was incubated at 37 °C for 60 min and then Griess reagent i.e., N-(1-Naphthyl) ethylenediamine (0.1%) and sulphanilic acid (1%) in H3PO4 (5%) was added. Pink chromophore was generated and finally measured spectrophotometrically at 540 nm. As a standard, ascorbic acid was used. The result was calculated according to the formula:
$${\text{Percentage}}\;{\text{ of}}\;{\text{inhibition}} = \left( {{\text{Absorbance}}\;{\text{of}}\;{\text{control}} - {\text{Absorbance}}\;{\text{of}}\;{\text{sample}}} \right)/{\text{Absorbance}}\;{\text{of}}\;{\text{control}}) \times {1}00.$$
Animals
The study was conducted on adult male Swiss albino mice and animals were purchased from the M/s Chakraborty Enterprise (CPCSCA registered). The age of the animals was around five to six weeks, weighing 35 ± 5 g were housed in a ambient temperature 25 ± 2 °C, relative humidity was 45–55% and 12 h in light and 12 h dark cycle was maintained. The animals were kept for two weeks in the laboratory to acclimatize with the environment. The animals were fed normal pellet diet purchased from Hindustan Lever Ltd., India and water ad libitum. As per the Institutional Animal Ethics Committee, Jadavpur University, Kolkata, India all the animal experiments were carried out (Ref. No. AEC/PHARM/1502/09/2015).
Acute toxicity
MELCF was administered orally to the Swiss albino mice for evaluation of the acute toxicity according to the method of Organization for Economic Co-operation and Development (OECD) guideline no. 425 (Anonymous 2008).
Induction of diabetes
The mice were acclimatized for 2 weeks in the laboratory. Afterwards, the animals were fasted overnight. STZ was dissolved in citrate buffer (0.1 M sodium citrate and 0.1 M citric acid, pH 4.5). The diabetes was induced by giving STZ (60 mg/kg) through intraperitoneal injection for 3 consecutive days (Biswas et al. 2011). The blood glucose was measured by taking a drop of blood from the tail vain with the help of a single touch glucometer (Accu-check, Roche Diagnostic, USA). The mice with blood glucose more than 250 mg/dl were selected for the experiment.
Oral glucose tolerance test (OGTT)
The OGTT was performed in the overnight (12 h) fasted mice. Animals were divided into six groups (n = 6). Group 1 was treated as normal control which was given water 5 ml/kg. Groups 2, 3, 4 animals were orally given MELCF 100, 200 and 300 mg/kg respectively. Group 5 was orally given metformin 200 mg/kg. After 30 min of administration of extract and standard drug, glucose solution (2.5 g/kg) was administered orally. After administration of glucose solution 0, 30, 60 and 120 min, blood glucose level was measured with the help of single touch glucometer (Accu-check, Roche Diagnostic, USA) (Kumar et al. 2011).
Experimental design
The MELCF was evaluated for the antidiabetic activity and the selected animals were divided into six groups (n = 6) as follows (Das et al. 2011).
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Group 1: Normal control (untreated).
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Group 2: Diabetic/disease control (streptozotocin 60 mg/kg body eight).
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Group 3: Diabetic mice treated with MELCF 100 mg/kg body weight.
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Group 4: Diabetic mice treated with MELCF 200 mg/kg body weight.
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Group 5: Diabetic mice treated with MELCF 300 mg/kg body weight.
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Group 6: Diabetic mice treated with metformin 200 mg/kg body weight.
All the animals were treated orally for 28 consecutive days. Fasting blood glucose levels of the mice were monitored in the overnight (12 h) fasted mice on 0, 7, 14, 21 and 28 days of the treatments along with corresponding body weights.
Determination of biochemical parameters
After 24 h of last dosing on the 28th day, the blood samples were collected from the overnight fasted mice in each group by cardiac puncture for determination of glycosylaed haemoglobin (HbA1c). For liver function tests aspartate aminotransferase (AST)/serum glutamic oxaloacetic transaminase (SGOT), alanine aminotransferase (ALT)/serum glutamic pyruvic transaminase (SGPT) were performed. For kidney function tests urea, creatinine tests were done. For lipid profile, total cholesterol (TC), triglyceride (TG) were determined by using the reagent kits as mentioned under ‘drugs and chemicals’ sub-section.
Liver and kidney antioxidant status
On the 29th day, animals of each group were scarified by cervical dislocation the liver and the kidney of the scarified animals were taken out and homogenized in 10 ml 20 mM phosphate buffer (pH: 7.4). Then it was centrifuged in 12,000 rpm for 10 min in 4 °C the supernatant was collected and used for the estimation of the lipid peroxidation assay (LPO/MDA), reduced glutathione (GSH) and superoxide dismutase (SOD) as per the procedures stated in the respective kits.
Histopathological studies of pancreas, liver and kidney
After 28 days treatment, all animals were sacrificed on the 29th day by cervical dislocation, afterwards pancreas, liver and kidney tissues were isolated from the animals of each group and cut in a small piece measuring about 1 cm and kept it in 10% formaldehyde solution at 4 °C and histology was done in the laboratory, then it was dehydrated gradually by increasing the concentration of ethanol (50–100%), it was cleared with xylene and embedded in paraffin and cut by using a semi-automated rotary microtome (Leica Microsystem, RM-2245, Wetzlar, Germany). Each section was about 5 μm thickness and it was stained with haematoxylin and eosin. The microscopic slides of pancreas, liver and kidney tissues were photographed under 100 × magnification by binocular microscope (Olympus, India).
Statistical analysis
The determinations were carried out in triplicates for in vitro studies and the values are expressed as mean ± standard error of mean (SEM). For in vivo study, statistical significance was analyzed by one way analysis of variance, followed by Dunnett’s test. The name of the software used is GraphPad Prism 5.0. (Graph Pad Software Inc, La Jolla, USA). P < 0.05 considered as statistically significant.