Popular Ethnomedicinal Plant Alstonia scholaris Induces Neurotoxicity-Related Behavioural Changes in Swiss Albino Mice

Research Article

Austin Neurol. 2021; 4(1): 1016.

Popular Ethnomedicinal Plant Alstonia scholaris Induces Neurotoxicity-Related Behavioural Changes in Swiss Albino Mice

Laskar YB, Laskar IH, Gulzar ABM, Vandana UK, Bhattacharjee N, Mazumder PB* and Bawari M

Department of Biotechnology, Natural Product & Biomedicine Research Laboratory, Assam University, Silchar, India

*Corresponding author: Pranab Behari Mazumder, Department of Biotechnology, Assam University, Silchar, India

Received: April 30, 2021; Accepted: May 27, 2021; Published: June 03, 2021


Plants constituents are a reliable source of the remedial need of humanity for ages by being the basis of the traditional medicinal system and often serving as the prototype for designing modern medicine. Several plants are used in traditional medicine for ages without proper administration guidelines in terms of dosages. Several toxicological analyses revealed side-effects of such therapies beyond a specific dose. One such plant is Alstonia scholaris, widely used in numerous traditional medicines to treat diseases like ulcers, asthma, diabetes, etc. The present study investigated the neurotoxic effect of the plant extract through oxidative stress in Swiss albino mice. The treated mice showed anxiety, neophobic and depression-like properties compared to control mice. The biochemical parameters show an increase in Malondialdehyde (MDA) concentration while decreasing the total protein content in different brain regions of treated mice. The Glutathione Reductase (GR) activity shows an increase in treated mice compared to the control one. The study indicates that Alstonia scholaris may cause severe damage to the central nervous system when administered without a proper guideline.

Keywords: Alstonia scholaris; Neurotoxicity; In vivo; Malondialdehyde; Glutathione Reductase


Traditional medicines that primarily include plant-based preparations are the most commonly used source of inexpensive and accessible remedies in many developing countries [1]. In developed countries, they are used as alternative medicines either with current therapies for synergistic effect or as a substitute for expensive treatments [2]. The low therapeutic index of many modern drug formulations also aided traditional medicines’ popularization in treating chronic illnesses [3]. Several clinically successful plantbased compounds and their derivatives are administered directly as alternative medicine or in combinational therapies to treat lifethreatening diseases like Cancer, Diabetes, Alzheimer’s, etc [4,5]. These compounds include taxols (Larotaxel, Paclitaxel, Cabazitaxel) for cancer treatment [6,7], Arteether/Artemotil (an artemisinin derivative) as anti-malarial [8], Galantamine for Alzheimer’s-related dementia [9], Apomorphine (a morphine derivative) for Parkinson’s disease [10], and Tiotropium (an atropine derivative) for pulmonary diseases [11]. The toxicological limitations of such clinically administered plant-based drug formulations are well-documented and followed extensively. However, no specific toxicological guideline is followed for plant-based preparations in traditional therapies that might have serious consequence [12-14]. Some recent studies proposed that several extensively used herbal formulations showed nephrotoxicity, neurotoxicity, cardiotoxicity, hepatotoxicity and reproduction-related side effects when used without a proper guideline of administration [15-18].

Alstonia scholaris, or the devil tree, belonging to the plant family Apocynaceae is a tropical tree commonly found in tropical South- East Asia, including India, China, Bangladesh up to the African and Australian continent [19]. In Indian Traditional Medicinal Systems (Ayurveda, Siddha, and Unani), the plant’s parts are used extensively to treat fever, chronic diarrhoea, dysentery, gout, rheumatism, skin diseases, malaria, jaundice, cancer, etc [19,20]. In Chinese traditional medicine, the plant’s parts are used to treat respiratory diseases, such as cough, asthma, phlegm, and COPD [19]. Additionally, its preparations are widely used in several countries and among several ethnic groups as a cost-effective alternative treatment for antipyretics in malaria, rheumatic pains, diabetes, and placenta expel after childbirth [19]. Although the plant’s preparations are used widely in traditional therapies, some reports suggest that the plant’s extracts may have several acute and chronic toxicity, including oral toxicity [21] and developmental toxicity [22] when used beyond a specific limit. Besides, Baliga et al. (2004) reported that high doses of Alstonia scholaris cause severe damage to all major organs of experimental rodent models [23]. The present study preliminarily evaluates the neurotoxic effect of the methanolic extract of the plant and the corresponding behavioural changes in experimental animals. The specific findings might help consider the neurotoxic side-effects while using the plant’s preparations in traditional therapies and regularizing standard administration doses.


Preparation of plant extract

The leaf samples were collected from different locations of the Cachar district of Assam, India. The samples were dried under shade for seven days. The plant materials were extracted by following the methods described by Bello et al. (2016) [24] with a few modifications. Briefly, the dried samples were ground to powder and loaded in a cellulose thimble for extraction using a soxhlet apparatus. The repetitive extraction was carried out at 40°C using methanol as a solvent until the colourless solvent was observed. The collected extract was filtered through Whatman filter paper (No. 1). Further, the filtrate was concentrated by removing the solvent using a vacuum rotary evaporator. The extracted materials were kept in a -20°C refrigerator for further experiments. Later, two doses of the extract (100 and 300 mg/ml) were prepared as a low and high dose by dissolving them in distilled water for oral administration.

Experimental animals

Twenty-one (21) Swiss albino mice, about 8-10 weeks old (weighing between 25 and 30g), were obtained from the College of Veterinary Sciences at Assam Agriculture University, Guwahati, India. The animals were acclimatized to standard laboratory conditions for seven days before commencing the experiments. They were housed in large, clean polypropylene cages in a temperaturecontrolled room with 12hrs light and dark cycle, free access to water ad libitum and fed with a standard diet. Approval was obtained from the Institutional Animals Ethics Committee of Assam University, and all the ethical guidelines were followed strictly. After acclimatization, the animals were divided into three groups (n=7): Group A (served as standard control, administered with distilled water for seven days.), Group B (administered with ASME low dose) and Group C (administered with ASME high dose). The two concentration of the extract dissolved in distilled water were administered via oral gavage. The experimental animals were kept under regular and individual observation for body weight changes, behavioural changes and toxicity signs for the next seven days. Upon completing the experiments, the mice were sacrificed, and the blood/tissue samples were collected for biochemical assays.

Behavioural studies

Elevated plus maze test: The Elevated Plus Maze (EPM) Method is a regularly used behavioural assay for rodents that helps to estimate the anti-anxiety effects of pharmacological agents and steroid hormones [25]. The method has been employed in several recent impactful reports [26-28] that reflects the efficiency of the technique in accessing neurotoxicity-induced anxiety-related behaviour in rodents. The experiment was performed following the protocol described by Casarrubea et al. (2013) [29]. Briefly, the animals were transferred from the animal house to the experiment room in homecages to minimize the transfer effect. They were acclimatized for another 30 minutes to overcome any possible visual and olfactory influences. The EPM apparatus consisted of two open arms (16cm × 5cm) and two covered arms (16×5×12 cm). The arms extended from a central platform (5cm × 5cm), and the maze was elevated to a height of 25cm from the floor. Each animal was placed in the central platform, facing an open arm, and allowed to explore for 5min freely. The EPM apparatus was cleaned with ethyl alcohol (10%) after each observation to remove scent cues left from the preceding animal. The time spent by animals in the open arms, in the central platform and in the closed arms was calculated minute-by-minute and recorded for further analysis.

Modified hole board test: The Modified Hole Board Method (mHB) is another powerful method widely accepted to measure multiple dimensions of unconditioned behaviour in rodents [30]. Recently, many researchers used this method to validate their findings on the neurotoxic effect of different plant infusions that alters the locomotion and exploratory behaviour of experimental animals [31-33]. The grey wooden mHB box (60×60×25 cm) with sixteen equidistant holes (3cm in diameter) on the floor was raised 28 cm above the ground on a stand. Each equidistant hole was 28cm from the nearest wall of the box. The box was divided into squares of (20cm × 20cm). This experiment was performed by following the protocols described by Pyrzanowska et al. (2021) [33] with few modifications. Briefly, the experimental animal was placed in the corner facing the wall and allowed to explore for 5 minutes freely. The total number of heads-dips recorded. A mouse was considered to make a head-dip if both eyes disappeared into the hole. The box was cleaned with 10% alcohol after each observation to avoid any bias based on olfactory cues.

Forced swim test: The Forced Swim Test (FST) is a rodent behavioural test used to evaluate the depression-like symptoms and stress-coping behaviour in experimental animals when treated with new compounds/drugs or neurotoxic agents [34-36]. The FST was performed according to the method described by Tanaka et al. (2020) [37] recently. Briefly, the individual mice were placed in a cylindrical glass container of 12 cm in diameter and 30 cm in height and left there for 5min. The cylinder was filled with water (25±1 °C) to a height of 20cm. The duration of swimming and immobility was recorded. Freshwater was used for each mice. A mouse was judged to be immobile when it ceased struggling and remained floating motionless in the water, making only those movements necessary to keep its head above water [38].

Biochemical assays

Following the behavioural studies, the mice were sacrificed immediately, and the brain tissues (cerebral cortex) were isolated by dissection. The tissues were washed with ice-cold saline, blot dry with filter paper, weighed and homogenized in a cool environment. The homogenates were used for the biochemical analyses.

Lipid peroxidation (LPO) assay: Lipid peroxidation by Reactive Oxygen Species (ROS) is a well-established detrimental mechanism for several acute and chronic brain disorders [39]. The Thiobarbituric Acid assay (TBA test) developed by Ohkawa et al. (1979) [40] is still used as a robust biochemical assay for detecting lipid peroxides in animal tissues. Thiobarbituric Acid (TBA) reacted with Malondialdehyde (MDA), which resulted in a colour compound. Briefly, to 0.2ml of the test sample, 0.2ml of SDS, 1.5ml of acetic acid and 1.5ml TBA was added. The mixture was made up to 4ml with water and then heated in a water bath at 95°C for 60 minutes. After cooling, 1ml of water and 5ml of n-butanol/pyridine mixture was added and shaken vigorously. After centrifuging at 4000 rpm for 10 minutes, the organic layer was formed, and its absorbance was measured spectrophotometrically at 532nm. The level of lipid peroxidation was expressed as nmoles of MDA released per gram of wet tissue. The concentration of MDA was expressed using the formula: Absorbance at 532nm × D /L × €, where L= light path (1cm), D=Dilution factor and, €= extinction coefficient (1.56×105M-1cm-1).

Total protein estimation: The total protein content of the samples was assessed using Folin-Lowry’s method of protein estimation [41]. The absorbance of standards was measured at 550nm against the blank, and a standard curve was generated. From this standard curve, the protein concentration for samples was calculated.

Glutathione reductase (GR) activity assay: The GR activity in the experimental tissue samples was determined spectrophotometrically at 340nm by following Carlberg and Mannervik (1975) method [42]. Briefly, the GR activity was determined by measuring the oxidation of NADPH at 340nm in a reaction mixture containing 1.8ml phosphate buffer, 300μl each of EDTA, NADPH, oxidized glutathione (GSSG) and enzyme extract. The unit of GR activity was expressed as the amount that catalyzes the consumption of 1μmol of substrate per minute, using a molar extinction coefficient of 6.2mM-1cm-1 for NADH/NADPH.


Change in body weights

Body weights of both the control and treated animals were measured on an alternative day until the experiments. The body weights of LD-treated and HD-treated animals showed a 2.58% and 0.23% increased in weight, respectively. In contrast, control animals showed a 5.18% weight gain on the 7th-day than the initial body weight (Figure 1).