Chemicals
Propionic acid and neurotransmitters estimation kits (Glutamate Assay Colorimetric kit, Serotonin ELISA kit) were purchased from Sigma-Aldrich–Merck, Bengaluru, India, and supplied by Southern India Scientific Corporation, Kandanchavadi, Chennai, India, whereas all other chemicals and reagent were of analytical grade.
Experimental animals
Healthy Wistar male rats between 4 and 8 weeks of age and weighing about 150–180 gm were used for the study. Animals were purchased from the animal house of Tamil Nadu Veterinary and Animal Sciences University, Chennai. They were housed in polypropylene cages at a temperature of 25–30˚C and relative humidity 35–45%, light and dark cycles of 12 and 12 h, respectively, for one week before and during the experiments. Animals were provided with free access to a standard rodent pellet diet and water ad libitum. All animal studies were performed after getting the approval from the ‘Institutional animal ethical committee with an approved reference number'; also possible measures were taken to minimize the suffering of the animals.
Collection and Preparation of Clitoria ternatea L. root extract
Fresh roots of Clitoria ternatea L were collected, authentified, shade-dried at room temperature to remove moisture, and then coarsely powdered by using an electric grinder. The powdered materials were stored in an air-tight container and used for further extraction.
Extraction procedure
Extraction of roots of Clitoria ternatea L. was carried out using ethanol by hot continuous extraction method with Soxhlet apparatus. The extract obtained was collected and concentrated by gentle heating followed by using rotarat vacuum evaporator. The concentrated extract was then weighed, calculated the percentage yield, and stored. The extract was subjected to various preliminary phytochemicals tests [10].
Experimental design
A total of twenty-four rats were utilized for the study. Before starting the study procedure, all the rats were undergone a surgical procedure for cannula implantation. After 14 days of the surgery, the rats were stratified in to four separate groups containing six Wistar rats each.
Group I: Received vehicle alone per oral (p.o) (1% Tween-80 solution).
Group II: Received vehicle alone p.o (1% Tween-80 solution).
Group III: Received ethanolic extract of Clitoria ternatea L.(EECT) [11] at dose of 250 mg/kg -suspended in 1% Tween-80 solution p.o.,
Group IV: Received ethanolic extract of Clitoria ternatea L.(EECT) [11] at dose of 500 mg/kg-suspended in 1% Tween-80 solution p.o.,
The vehicles and the extract were administered orally using an intragastric tube daily for 28 days. After 21 days of extract administration, each group of animals except Group I received intracerebroventricular (ICV) infusions of propionic acid (PPA) (4.0 µl of a 0.26 M solution PPA was dissolved in phosphate-buffered saline (PBS) vehicle) daily for 7 consecutive days (between 22nd and 28th days). Group I rats received ICV infusions PBS (4.0 µl of 0.1 M PBS) between 22nd and 28th days.
A schematic representation of the experimental design is provided in Fig. 1.
Induction of ASD
Surgical procedures for cannula implantation
Surgical procedures were completed under aseptic conditions. All rats were implanted with a 23gauge (ga) guide cannula using standard stereotaxic techniques. Before surgery, each rat was treated with atropine methyl nitrate (0.1 ml Subcutaneous injection). Before placing the stereotaxic apparatus, rats were anesthetized using inhaled isoflurane and 3% oxygen. During surgery, body temperature was maintained normal using a heating pad.
The tip of the guide cannula was placed in such a way that it will penetrate into the brain lateral ventricle just below the border of corpus callosum. For proper infusion into lateral ventricle below the tip of the guide cannula, another 30ga injection cannula is placed so that the tip of injection cannula was further protruded 0.5 mm deepar. Each cannula was provided with an obturator closing, which can be removed before each intracerebroventricular (ICV) infusion. A Sage syringe pump with sterile PE10 tubing was attached with 30ga injection cannula for the purpose of infusion. Small screws were placed in the top of the skull, and the cannula was affixed to the skull with dental acrylic. Oral extract/vehicle administration began approximately 14 days after surgery [4, 12].
Intracerebroventricular (ICV) infusion procedure
Prior to the ICV infusion of propionic acid (PPA), it was dissolved in appropriate vehicle. Phosphate-buffered saline (PBS) was used as vehicle which was buffered to pH 7.5 by means of concentrated hydrochloric acid and sodium hydroxide. All the animals except Group I received ICV infusions of PPA (4.0 µl of a 0.26 M solution) and the Group I rats received ICV infusions PBS (4.0 µl of 0.1 M PBS). The infusions were given for 7 consecutive days (between 22nd and 28th days) in twice daily manner. The infusion duration was about 60 s, but an additional 60 s was allowed for the infusion cannula to remain in the position before being removed. Assessment of habituation behavior and memory assessments were done on each specific day (day 22 to day 28) following the infusion of PPA. Habituation behavior assessment was screened by performing a ‘novel object recognition test,’ and memory evaluation was done using ‘elevated plus maze’ [4, 12]. The effects of PPA infusion and the effectiveness of the extract to prevent those effects were evaluated by performing a comparative statistical analysis with the results obtained for each group by carrying out both in vivo test—novel object recognition test and elevated plus maze test.
Novel object recognition (NOR) test
This experiment took placed in a square open enclosure of 50 × 50cm2, surrounded by four walls of 50cm2 elevation. At the beginning of the test, each rat was allowed to liberally explore the device for 5 min in the absence of any object. Twenty-four hours later, the animals have explored two matching objects (A1 and A2) positioned at the two adjacent corners of the device at 10 cm from the walls, for 5 min; this was the ‘acquisition’ session. Exploration was only called when the rat had a nose directed fewer than one cm to the object while nibbling and marking were not measured exploration. After a postponement of 24 h, the rats were again positioned in the device, but this time, one of the two identical objects was replaced by another entirely different (B). The animal is permitted to explore the two different objects for 5 min [13, 14].
The state of memory was calculated by the following equation:
$${\text{Recognition}}\;{\text{index}} = {\text{time}}\;{\text{spent}}\;{\text{in}}\;{\text{discovering}}\;{\text{the}}\;{\text{object}}\;{\text{B}}/{\text{Sigma}}\;{\text{time}}\;{\text{spent}}\;{\text{exploring}}\;{\text{objects}}\;{\text{A}}\;{\text{and}}\;{\text{B}} \;[{13}].$$
Elevated plus maze
The memory power of the rodents can be investigated efficiently by using an elevated plus maze. Rat elevated plus maze is specified with a central platform (10 cm × 10 cm) associated with two open arms (50cmx10cm) and two enclosed arms (50 cm × 40 cm × 10 cm) [15]. An elevation of 50 cm height from the floor was provided while positioning the maze. On the sixth day of propionic acid administration, the experiments were carried out to record the ‘transfer latency (TL)’ in seconds [15]. This was considered as a training session for each rat; before returning to its home cage rats were permitted to explore the maze for an extra 2 min. After 24 h (i.e., the seventh day of PPA infusion/28th day of the extract administration), each of the rats was examined for their retention memory [15, 16].
On the 8th day (29th day of the study), animals were killed by ethically approved euthanasia method—cervical dislocation, and brain tissues extracted and homogenized using a tissue homogenizer, and it was preserved /stored at −80˚C for further analysis of neurotransmitters like serotonin [17] and glutamate [18].
Statistical analysis
Statistical validation of the data was done with the help of computer software GraphPad Prism version 8; statistical test utilized was ANOVA, followed by Dunnett's multiple comparison test.
All the values in the tabular column represent the Mean ± SD (Standard deviation), n = 6 (6 rats in each group).
