2.1 Chemicals and instruments
High performance thin layer chromatography (HPTLC) profiling was done on pre-coated silica gel 60F254 TLC plate (Merck, India). The mobile phase was standardized as toluene:ethyl acetate:methanol in the ratio 8:2:1. The chromatogram was developed in a saturated chromatographic chamber (Camag, Switzerland), and the developed plate was visualized under 366 nm. LC/MS analysis was conducted on Agilent 6520 accurate mass Q-TOF LC/MS coupled with Agilent LC 1200 equipped with Extend-C18 column of 1.8 μm, 2.1 × 50 mm. The APCI source was operated with following settings in positive mode: drying gas (nitrogen) flow 8 L/min; nebulizer pressure 40 psig; drying gas temperature 325 °C; capillary voltage + 3000 V; fragmentor volt 125 V; Oct Rf Vpp 750 V. Gradient elution was performed with water/0.05% formic acid (solvent A) and acetonitrile (solvent B) at a constant flow rate of 0.9 ml/min. Column temperature was maintained at 30 °C.
2.2 Plant material
Mature tubers of I. mauritiana were collected from Herb garden, Kottakkal, Kerala, India, and authenticated by Dr. K M Prabhukumar, Plant Systematics and Genetic Resources Division, Centre for Medicinal Plants Research (CMPR), Kerala, India, and a voucher specimen was deposited in CMPR herbarium.
2.3 Extraction of coumarins
Coumarins were separated by specific method. Briefly, 50 g of the material was extracted with methanol by soxhlet extraction method for 48 h. After evaporating the extract under reduced pressure, the residue was refluxed with 5% HCl for 2 h. After filtration two fractions were obtained, residue and aqueous—acid phase. The residue was extracted with methanol and evaporated (Fr 1) and aqueous—acid phase was extracted with diethyl ether and was evaporated (Fr 2). The fractions (Fr 1 and Fr 2) were kept under refrigerator until further analysis.
2.4 Acute oral toxicity study
2.4.1 Plant extracts
The pharmacological evaluation was done in aqueous extract. Two hundred fifty grams of the tubers of IM was extracted with water using soxhlet extraction method for 72 h. The final extract was concentrated to dryness under reduced pressure using rotary evaporator (Heidolph, Germany).
2.4.2 Target animals
The experiment was conducted on Wistar rats (females) weighing 147 to 204 g and aged 8 to 9 weeks obtained from the Animal House, J.S.S. College of Pharmacy, Ootacamund-Tamil Nadu. The rats were distributed into 5 groups with 6 animals in each group. The experimental procedures relating to the animals were authorized by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Approval No: JSSCP/IAEC/OT/Ph.D/Ph.Cology/06/2017-18) before starting the study and were conducted under the internationally accepted principles for laboratory animal use and care.
The extracts were prepared from a plant material having a high safety margin, and hence, it was decided to use 2000 mg/kg (limit test) for this study. The test item was prepared immediately prior to administration on respective treatment days. A quantity of 2 g of the test item was dissolved in distilled water, and the volume made up to 10 ml to get a test item concentration of 200 mg/ml. Homogeneity of the test item in the vehicle was maintained during treatment by constant stirring and mixing. The test substance was administered soon after preparation. The prepared test item solutions were administered once orally as gavage to the fasted (16–18 h) rats at the dose volume of 10 ml/kg b.wt. to deliver a dose of 2000 mg/kg b.wt. Food was offered about 3–4 h after dosing. Water was not withheld.
The treated rats were observed five times during day 1 (day of administration), i.e., at 30 min and four times at hourly (post-administration) intervals and once daily and, thereafter, for a total of 14 days. The clinical signs were recorded on all working days. The body weights of rats were recorded on test day 1 (pre-administration), day 8 (7 days post-administration), and day 15 (14 days post-administration). The rats were euthanized by using diethyl ether anesthesia and necropsied.
2.4.3 Evaluation of anti-amnesic activity
The test item was prepared immediately prior to administration on respective treatment days. The extract was prepared as solution in distilled water at concentration equal to 1/10th of the dose and administered at a dose volume of 10 ml/kg, b.wt. Animals were randomly divided into seven groups containing 6 each.
Group treatment
1Normal (vehicle 10 ml/kg, p.o.)
2Control (vehicle 10 ml/kg, p.o.)
3Rivastigmine (1.5 mg/kg., p.o.)
4IM (100 mg/kg., p.o.)
5IM (200 mg/kg., p.o.)
The above treatment was given for a period of 14 days, and a day before the memory test, all the animals were given appropriate training. On 14th day, 1 h after the respective treatments, scopolamine (3 mg/kg., i.p.) was administered to all the groups, except group 1, normal. Step down and step through latency were evaluated 45 min after the scopolamine administration to assess the memory, and the radial arm maze memory test was carried out on day
Step through latency was evaluated by the standard procedure. Each rat was individually placed in the bright part of a two-chambered apparatus for training. The door was closed once rat enters the dark chamber to prevent it from escaping. Later a foot shock (1 mA, 1 s) was applied through the grid floor, and the rat was then returned to the home cage.
Twenty-four hours later, testing was repeated by placing the animal again in the bright chamber. The latency period to enter the second darker chamber was measured. A prolonged latency indicates that the animal remembers that it has been punished and, therefore, does avoid the darker chamber.
Step down latency was assessed using a rectangular box (50 × 50 cm) with electrifiable grid floor and 35 cm fits over the block. The grid floor is connected to a shock device which delivers scrambled foot shocks. The experiment was conducted in three phases: (1) familiarization—the animal was placed on the platform, released after raising the cylinder, and the latency to descend is measured. After 10 s of exploration, it was returned to the home cage. (2.) Learning—immediately after the animal has descended from the platform, an unavoidable foot shock was applied (foot-shock 50 Hz; 1.5 mA; 1 s) and the animal is returned to the home cage, (3) retention test—24 h after the learning trial the animal was again placed on the platform, and the step-down latency was measured. The test was finished when the animal steps down or remains on the platform (cutoff time 60 s).
Radial arm maze test was performed in a eight arm radial maze. Animals were placed in wooden elevated eight-arm radial maze with the arms extending from a central platform 26 cm in diameter. Each arm is 56 cm long and 5 cm wide with 2 cm high rails along the length of the arm. The maze was well illuminated and numerous cues are present. Food pellets (reward) will be placed at the end of the arms. During the test, rats were well fasted to motivate them to run the maze. Animals were trained on a daily basis in the maze to collect the food pellets. The session was terminated after 8 choices, and the rat has to obtain the maximum number of rewards with a minimum number of errors.
The number of errors (entries to non-baited arms) will be counted during the session: reference memory error: Visit to unbaited arm more than once; working memory error: Visit to baited arm more than once.
2.5 Statistical analysis
For determination of significant intergroup differences of each parameter one-way analysis of variance (ANOVA) was carried out. Dunnet’s test was used for individual comparisons after significant ANOVA results. The differences with p < 0.05 were considered statistically significant. Graph pad Prism-6 software (Graph pad software, Inc., USA) was used for the statistical analysis.
