Collection and identification of the plant material
The mature fruits of Morus alba L. were collected from the easternmost region of (27° 47″ latitude and 94° 91″ longitude), Assam, India, in the month of March 2018. The plant was identified and authenticated by the Eastern Regional Centre of the Botanical Survey of India (No.: BSI/ERC/2018/Tech/468, dated 22 October 2018).
Preparation of the extract
The fruits were washed thoroughly and dried under shaded condition. The dried material was subjected to pulverization and then passed through sieve no. 30 to get a coarse powder of the crude drug. The dried powder was extracted with 70% methanol by cold maceration at room temperature for 48 h. The extract was filtered through Whatman no.1 filter paper. This was repeated for multiple times, and similar extracts were pooled together, concentrated and vacuum evaporated using rotary evaporator at a temperature of 45 °C, and the solvent traces were removed by lyophilization. The freeze-dried extract thus obtained was stored at low temperature (− 8 °C) in air tight containers.
High-performance thin layer chromatography analysis
High-performance thin layer chromatography (HPTLC) analysis was performed using the markers quercetin, rutin and resveratrol. The reported contents of quercetin, rutin and resveratrol in mulberry fruits are 0.038, 0.433 and 0.03 mg/g of fruit extract respectively . The standards (1 mg/ml) were prepared by dissolving 10 mg of each of the standard (quercetin, rutin and resveratrol) in 10 ml of methanol in separate volumetric flasks and were sonicated (Ultrasonic cleaner STD Rivotek, India).The extract was prepared by dissolving 100 mg of 70% methanol extract of M. alba fruits in 1 mL methanol, sonicated followed by centrifugation (RM-03 Plus, REMI, India), and the supernatant was used for the analysis.
HPTLC was performed on silica gel 60F254 pre-coated plates (Merck, Mumbai, India). The applications were made as bands 8.0-mm wide, 11.5-mm apart and 10.0-mm from the bottom edge of the chromatographic plate by the use of a Camag (Muttenz, Switzerland) Linomat V sample applicator equipped with a 100-μL Hamilton (USA) syringe. Ascending development to a distance of 70 mm was performed at room temperature (28 ± 2 °C), with a suitable mobile phase, in a Camag glass twin-trough chamber previously saturated with mobile phase vapour for 20 min. The mobile phases used as follows—for quercetin (toluene: ethyl acetate: formic acid: 10:8:0.4), for resveratrol (hexane: ethyl acetate: glacial acetic acid: 8:12:0.2) and for rutin (ethyl acetate: formic acid: acetic acid: water: 15:1:1:1.5). After development, the plates were dried and then scanned at specified wavelength with a Camag TLC Scanner. The compounds were identified by superimposing the UV spectra of the standards and the samples within the same Rf range.
Animals and housing conditions
The animal studies were performed in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and was approved by the Institutional Animal Ethics Committee (IAEC), vide approval no. IAEC/DU/166, dtd. 10.12.2018. Female mice of Swiss strain (20–25 g) and male and female rats of Wistar strain (150 ± 20 g, 5–6 weeks old), obtained from breeding house, were used for in vivo neurotoxicity study. The animal house was well ventilated, and the temperature was maintained at 22 °C (± 3 °C). Lighting was provided according to 12 h light/dark cycle. The animals were housed in large spacious cages in small groups of same sex to allow to acclimatize to laboratory conditions 5–7 days before the commencement of the experiment. For feeding, conventional laboratory diets were used with an unlimited supply of drinking water.
Acute toxicity study
The acute oral toxicity study was based on ‘Up-and-Down’ procedure as described by Dixon according to OECD 425 guidelines . Healthy adult female Swiss albino mice selected for the study were fasted overnight prior to dosing. MA extract was dissolved in 10% carboxymethyl cellulose (CMC) and administered orally at a single dose of 2000 mg/kg of body weight (b.w) to all the female mice (n = 6). Following the dosing, the animals were observed individually once, periodically during the first 24 h (with special attention given during the first 4 h) and daily thereafter, for a total of 14 days for any delayed toxic effects. The behavioural changes and death were the main parameters observed during the study period.
The neurological safety profile of MA was evaluated by performing various neurobehavioural tests as per OECD guideline 424 . Based on acute toxicity study result, three doses were selected for further studies (1000, 300 and 100 mg/kg). The control group animals received a definite volume of distilled water. Accordingly, the animals were randomly divided into 4 groups (comprising n = 12 animals; 6 males and 6 females in each group). Repeated daily dose of design regimen was administered orally for a period of 28 days.
Neurotoxicity of the plant was determined using open field test on actophotometer for motor coordination and ataxia, sensory response analysis using hot plate test and stereotyped behaviour (rearing, grooming, latency, level of mobility, urination/defecation, tremor/convulsions, vocalization, arousal, posture, pilo-erection, gait abnormalities, stereotypy and/or unusual behaviours) using light-dark box test.
Assessment of motor dysfunction
Spontaneous locomotor activity
The locomotor activity can be an index of wakefulness (alertness) of mental activity. The spontaneous locomotor activity was performed in actophotometer (Orchid Scientific & Innovative India Pvt. Ltd., Nashik, India). Each animal was allowed for a period of 5 min in a square closed field arena (30 × 30 × 30 cm) equipped with 6 photocells in the outer wall for spontaneous locomotion. Interruption of photocell beams (locomotor activity) was recorded by means of a 6 digit resettable counter. Increase in count was regarded as central nervous stimulation, decrease in count as central nervous depressant activity .
Assessment of behavioural impairment
Light-dark box test
The light-dark box (LDB) (Orchid Scientific & Innovative India Pvt. Ltd., Nashik, India) setup consisted of two compartments: one lit compartment (40 × 50 cm, 350 lux; light box) and one dark compartment (40 × 30 cm, 70 lux) . The floors in each compartment were divided into squares (10 × 10 cm), and the compartments were connected via a small opening (7.5 × 7.5 cm) enabling transition between the compartments . Rats were placed in the dark compartment and rearing and grooming, number of entries and time spent in light compartment, and latency to first light compartment entry during the 5 min test were assessed by all maze video tracking software (Orchid Scientific & Innovative India Pvt. Ltd., Nashik, India).
Assessment of sensory dysfunction
Hot plate reaction time in rats
Rats were screened by placing them on a hot plate maintained at 55 ± 1 °C and recording the reaction time in seconds for forepaw licking or jumping. Only animals which reacted within 15 s and which did not show large variation when tested on four separate occasions, each 15 min apart, were taken for the test. The time for forepaw licking or jumping on the heated plate of the analgesiometer maintained at 55 °C was taken as the reaction time .
Body weight and food consumption
All animals were weighed once a week. The food intake was also recorded weekly.
Biochemical and haematological analysis
At 28th day of the treatment, blood samples were collected from the animals in two batches. From one batch of blood sample, serum was separated by centrifuging at 6000 rpm for 10 min and was used for biochemical investigations. The other batch was used for haematological study.
Necroscopy and histopathology
After euthanasia, the internal organs (liver, spleen, lung, heart, kidneys, testis and ovaries) were harvested, and gross findings as well as organ weights were recorded. The liver, spleen, lung, heart, kidney and brain were fixed with neutral formalin for 48 h, dehydrated with alcohol, embedded in paraffin, cut into thin sections and stained with haematoxylin-eosin dye for photomicroscopic examinations.
Data of all experiments were expressed as mean ± Standard deviation (SD) of six animals in each group. Differences amongst different treatment groups were determined by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls comparison test unless stated otherwise. GraphPad Prism 5 was used for statistical analysis.