Evaluation of Anti-anxiety Activity of Ethanolic Extract of Doronicum Hookeri Rhizome in Swiss Albino mice

 

Ruchi Singhal, Anant Kumar Patel, Pawan Kumar Dubey*

Swami Vivekanand College of Pharmacy, Indore, MP.

 

ABSTRACT:

The Aim of this study is the “Evaluation of anti-anxiety activity of Ethanolic Extract of plant Doronicum Hookeri Rhizome in Swiss Albino Mice.” Methods: Twenty Swiss Albino Mice are of either sex selected with weight 20-30gm. Elevated Plus Maze and Light and Dark Exploration test was performed on them with full safety and hygiene. This consists of a 10cm x 10cm central platform, elevated 50cm off the ground, connected to two open arms 50x10cm and two closed arms 50cm x 40cm x 10cm. Swiss albino rats weighing 20-40g were treated with OS extract, diazepam, and gum arabic for 30 min and then individually placed in the middle of an increasing number of arm diameters. Closing time is 5 minutes for both arms to open and close. Elapsed time is in seconds. The number of entries into the open and closed arms was counted during the test. Entry means that all four paws are on the arms, and in Light and Dark Exploration test: the installation consisted of two square boxes, each measuring 50cm x 50cm x 50cm, separated by wooden walls. One of the boxes is dark and the other is illuminated by a 7W/12V lamp. There is an opening (6cm x 6cm) in the middle of the wooden wall that can be opened and closed using a transparent Plexiglas sliding door through which the animals can move from both sides. Mice were placed individually in the center of the light box and monitored for the next 5 min Time spent in both boxes in seconds. The number of passes in the box is also indicated. Mice were treated with OS extract, diazepam, for 30 min being placed in the light box. Result: Ethanolic extract of Doronicum Hookeri is found satisfactory, results show the increase in time spent in Open Maze and decreased in closed maze. Similarly time spent in Open Arena is increased as in closed arena. The value is calculated as Controlled, Standard, Low Dose and High Dose. Conclusion: The ethanolic extract showed significant anti-anxiety effect (p<0.04) when compared to control group.

 

 

 

INTRODUCTION:

Neuropharmacology is the study of how drugs affects function in the nervous system, and also the neural mechanism due to this it influence behaviour. Neuropharmacology is wide topic to study that why it is divided into two main branches: behavioural and molecular.

·       Behavioural neuropharmacology: This branch focuses on the study of how drug affect’s the human behaviour (also known as neuropsychopharmacology), including the study of how drug became addicted and drug dependency affect the Brain of Human.¹

·       Molecular neuropharmacology: this study of neurons and their neuro-chemical interactions, with the overall goal of developing drugs that have beneficial effects on neurological function.¹

 

Figure 1.1: Neuron¹

 

Both the fields are closely connected, both are concerned with the interactions of neurotransmitters, neurohormones, neuropeptides, neuromodulators, enzymes, co-transporters, ion channels, second messengers and receptor proteins in the central and peripheral nervous systems. Study of the interactions, researchers are trying to developing drugs to treat many of the different neurological disorders, including pain, neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, addiction, psychological disorders, and many others diseases related to neurology.¹   

 

2.     Nervous System:

The nervous system includes spinal cord, the brain, and a complex network of nerves. This system transfer messages back and forth between the brain and the body. The brain is the organ controls all the body's functions. The spinal cord runs from the brain down through the neck side back.

 

The central nervous system (consist of the brain and spinal cord) is usually considered to have seven basic parts: the spinal cord, the medulla, the cerebellum, the midbrain, the pons, the diencephalon, and the cerebral hemispheres.^2,3

 

3.     Human Nervous System:

Neurons are excitable cells, due to on its surface membrane there are an abundance of proteins known as ion-channels that help small charged particles to pass in and out of the cell. The neuron’s, the starting that allows chemical information to be received by its dendrites, propagated through the parikaryon (which is known as cell body) and down to cell body axon is last, and passing on to other neurons through its axon terminal. These voltage-gated ion channels responsible for rapid depolarization throughout the cell. This depolarization, if it reaches a certain threshold, due to this an action potential is caused. Once the action potential reaches the axon terminal, it helps in influx of calcium ions into the cell.^2,3,4

 

The calcium ions then cause vesicles, small packets are filled with neurotransmitters, to bind to the cell membrane and are release the contents into the synapse, cell is known as the pre-synaptic neuron, and the cell which interacts with the neurotransmitters are released in the synapse space that is known as the post-synaptic neuron. The neurotransmitter are released into the synapse, it can either bind to receptors, present on the post-synaptic cell, and the pre-synaptic cell can re-uptake that and save it for further transmission, either it can be broken down by enzymes in the synapse. Above, 3 different activities are major areas which shows how drug action can affect communication between neurons.^3,4

 

The brain is that part, which serves as the center of the nervous system in all vertebrate and invertebrate animals. The brain is the largest bunch of neurons is located in the parts such as head, and organs for senses such as vision, hearing and olfaction (eyes). It is the organ which consume most energy of the body, and also the most specialized, and responsible for endocrine regulation, sensory perception, motor control, and the development of intelligence.^3,4

 

While invertebrate brains are paired segmental ganglia (each of which is only responsible for the respective of their function of the body segment) of the ventral nerve cord, vertebrate brains grows axially from the midline of the dorsal nerve cord as a vesicular enlargement at the rostral end of the neural tube, which is centralized control over all body segments.^3,4

 

4.     Human Brain:

The cerebral cortex contains approximately around 14–16 billion neurons, and the estimated no. of neurons in the cerebellum is 55–75 billion. Each neuron are connected to each other by synapses, typically communicating with one another via root-like bulge called dendrites and long fiber-like county agent called axons, which are usually may be myelinated and carry trains of rapid micro-electric signal called action potential  which target specific recipient cells in other areas of the brain or different parts of the body. The pre-frontal cortex, which controls cognitive control, is peculiarly well developed in humans.^4,5

 

The shape and size of the brain varies between species, and identifying common features is often difficult to detect. There are a number of principles of the brain designed to apply across a wide range of species. Some of the aspects of brain structure are common to almost the entire range of animals; others distinguish "advanced" brains from most primitive ones, or severalize vertebrates from invertebrates.^4,5

 

5.     Anxiety Activity:

Anxiety is an emotion that arises from an unpleasant internal state of anxiety and fear about upcoming events. Anxiety is not fear, defined as an emotional response to a current threat; Anxiety is the anticipation of the next threat. This is often followed by neurological behaviors such as flashbacks, somatic complaints, and rumination.^5,6

 

Anxiety is a feeling of disquietude and worrying, commonly generalised and unfocussed as an over-reaction to a state of affairs that is may be immanent seen as threat. It is sometimes accompanied by muscular tenseness, restlessness, tiredness, inability to gimmick one's breath, stringency in the abdominal part, nausea, and issues in the concentration. Anxiety is related to fear, the re-action to a existent or sensed quick menace (i.e. fight or flight reactions); anxiety is involve in the outlook of a future menace include horrible. People are facing anxiety that may drawback from situations which have aggravated anxiety in the past.^5,6

 

The emotion of anxiety can base beyond the evolutionary to a particular time periods of action towards specific events, and may be changed to the one to various anxiety diseases (e.g. generalised anxiety disorders, affright disorder). The difference between anxiousness (i.e. mental disease) and anxiety (i.e. normal emotion problem), is people with this disorder experiences anxiousness most of the times in days during 6 months appox, or may be for shorter time periods in childrens. Anxiety is a disease which is the most horrible mental problem which some times last decades. Anxiety is observed in other mental disorders, such as - post-traumatic stress disorder, obsessive-compulsive disorder.^5,6

 

Symptoms:

Depression can cause long term, persistent daily symptoms that reduce quality of life, called chronic (or generalized) depression, as well as shortterm, vomiting, stress anxiety, called severe pain over time. The no., intensity and frequency of anxiety causes vary from people to people. But most people do not suffer from chronic stress. Other effects may include changes in sleep patterns, behavioral changes, increased or decreased activity, and increased activity (such as foot tapping). Feelings of anxiety may include "feelings of worry or fear, depression, anxiety, or stress, thinking the worst, restlessness, feeling uncomfortable, looking for (and waiting for) signs (and events) of danger, and feeling like one is empty." Nightmares/nightmares, brooding thoughts thinking about experiences in your head, thinking that everything is dangerous will involve mystery and a feeling of dissatisfaction.^6,8,9

 

The physiological symptoms of anxiety may include:

·       Neurological, as headache, presyncope, vertigo, paresthesias, or fasciculations.

·       Digestive, i.e. abdomen pain, nausea, diarrhoea, dyspepsia, xerostomia, or bolus. Stress hormones released in an anxious province have an impact on intestine mapping and can evident physical symptoms that may contribute to or aggravate IBS.

·       Respiratory, as breathlessness or decrease in breathing.

·       Cardiac symptoms, as trembling, abnormal fast heartbeats, or pain in chest.

·       Muscular, as tiredness, or tremor.

·       Cutaneous, as sweating, or fidgety skin.

·       Uro-genital, as short time urination, dyspareunia, urinary urgency, or impotence, chronic pelvic pain syndrome.

 

Types:

Anxiety is a critical part of various differ disease. These involve:

·       Panic disorder. It means facing of revenant attacks at unexpected times.

·       Phobia. It is an Visible fear of a specific Things, situations, and activity’s.

·       Social anxiety disorder. It is about highly feared of being judged by others in social networks.

·       Obsessive-compulsive disorder.  It means you have revenant irrational thoughts that lead to perform specific or repeated behaviours is observed.

·       Separation anxiety disorder. It means having a fearing to be away from home or with loved ones.

·       Illness anxiety disorder. It is anxiety about the health (once called as hypochondria).

 

Add-on, there are number of mental wellness condition and medical conditions can feature anxiety as a causes. These include:

·       Post-traumatic stress disorder (PTSD). It is a type of anxiety which is based on a traumatic event.

·       Major depressive disorders. A very strong relationship is present between depression and anxiety.

·       Chronic disease. Managing the disease - chronic obstructive pulmonary disease (COPD) and diabetes which may result in anxiety symptoms.

·       Inflammatory conditions.  Anxiety leads to be a chronic inflammation and the diseases may be arthritis.

·       Substance use disorders: Most of the people with anxiety try’s to Treat them self’s, to manage their symptoms.

·       Chronic pain. Anxiety is present in those with chronic pain diseases.

 

The anxiety is a disease which is frequently occurring mental disorders, and is common. It covers a group of conditions that share utmost or pathological anxiety as the principal perturbation of humor and emotional tone. Anxiety may be understand as the pathological vis-a-vis of normal fearfulness, is evident by disturbances in mood, thinking power, behavior symptoms, and physiological activity. The most common anxiety disorder involve panic disorder (include and non-includable account of agoraphobia), agoraphobia (include and non-includable account of panic disease), anxiety re-action, specified phobia, obsessive-compulsive disease, acute stress disease, post-traumatic stress disease and social phobia fear. ^6,8,9

 

MATERIAL AND METHOD:

Plant Material:

The Rhizome of Doronicum hookeri was collected from Local Area. The plant material was shade dried for 10 days and stored in glass bottle.

 

Preparation of Extract:

The Ethanol (EDH) and aqueous (ADH) extracts of powdered rhizome of Doronicum hookeri, was prepared by using Soxhlet’s apparatus.

 

Selection of Plant:

Selection of plant was based on traditional claim of medicine. Also the presence of phytochemicals in the ethanolic extract of D. Hookeri was suggestive of the anti-anxiety activity which encouraged us to select D. Hookeri rhizomes for the proposed activity,

 

Collection and Identification:

The plant material was collected from Local market in Indore and authenticated by botanist at Janata PG College, APS University, Rewa (M.P)

 

Preparation of Extract:

The Doronicum Hookeri rhizomes were washed under tap water and after that with distilled water to remove all physical impurities. The cleaned rhizomes were dried under shade and chopped into small pieces with a knife, pulverized mechanically with mortar pistle. The crushed rhizomes were converted into coarsely fine powder using electric grinder. The course powder was passed through sieve no. #40 and stored in air tight container for further investigation.

 

Preparation of doses:

The ethanolic extract of Doronicum hookeri and the standard drug was administered in à constant volume over range of doses to be tested by varying the concentration of dosing preparation. The test animal’s size determined the maximum amount of liquid that could be given at once. The volume in rats was typically not more than 1 millilitre per 100g of body weight. The dilutions of the ethanolic extract and standard drug was prepared in normal saline acting as a vehicle. The freshly prepared doses were administered to the rats to avoid stability issues.

 

Preparation of stock solution of test extract and reference drug for experimental animals.

According to OECD Guidelines, the test solution in low dose and high dose was constituted in appropriate vehicle in normal saline and the stock solution was prepared. The low dose of the ethanolic extract of D. Hookeri was selected as 200mg/kg and the high dose of the ethanolic extract was selected as 400mg/kg. The dose was calculated based on body weight of the animal. The low dose of ethanolic extract stock solution was prepared by dissolving 400mg of the ethanolic extract in 20ml of the normal saline. The standard stock solution of diclofenac sodium tablet 500mg was prepared by dissolving it in 100ml normal saline. The standard stock solution of Indomethacin tablet 25mg was prepared by dissolving it in 25ml of normal saline.

 

Acute oral toxicity:

According to literature ethanolic extract of rhizomes of Doronicum hookeri was found safe at 2g/kg body weight, no mortality was observed at dose and therefore, LD₅₀ of ethanolic extract of plant was reported 2000 mg/kg body weight.⁷

 

Evaluation Method for Anti-Anxiety Activity:

Elevated plus maze test:

These consists of a 10cm x 10cm central platform, elevated 50cm off the ground, connected to two open arms 50x10cm and two closed arms 50cm x 40cm x 10cm. Swiss albino rats weighing 20-40g were treatedwith OS extract, diazepam, and gum acasia for 30 min and then individually placed in the middle of an increasing number of arm diameters. Closing time is 5 minutes for both arms to open and close. Elapsed time is in seconds. The number of entries into the open and closed arms was counted during the test. Entry means that all four paws are on the arms.

 

Light and dark exploration test:

The installation consisted of two square boxes, each measuring 50cm x 50cm x 50cm, separated by wooden walls. One of the boxes is dark and the other is illuminated by 7W/12V lamp. There is an opening (6cm x 6cm) in the middle of the wooden wall that can be opened and closed using a transparent Plexiglas sliding door through which the animals can move from both sides. Mice were placed individually in the center of the light box and monitored for the next 5min. Time spent in both boxes in seconds. The number of passes in the box is also indicated. Mice were treated with OS extract, diazepam, and gum arabic for 30min before being placed in the light box.

 

OBSERVATIONS AND RESULTS:

Elevated plus maze test:

 

 

Light and dark exploration test

 

DISCUSSION:

In this Experiment, the recording is done on the basis of mice behaviour. This study shows about the Anti-Anxiety Activity which is study on Swiss Albino Mice. The Recording are in table format and also in graph form which is differentiated as four groups: controlled group, standard Group (Diazepam), low dose (100gm), and high dose (200gm).

 

According to this study, the result show the that the anti-anxiety activity on Doronicum hookeri is found satisfactory result as compared to controlled and standard groups. The experiment is done under guidance and research review of many journals. The ethanolic extract of D. hookeri rhizomes extract showed Anti-Anxiety property. The ethanolic extract showed significant anti-anxiety effect (p<0.05) when compared to control group. However, further studies are needed to isolate and characterize anxiety chemical constituents present in ethanolic extract of the rhizome.

 

CONCLUSION:

The present experimental protocol showed that the rhizomes of D. hookeri elicited a significant anti-anxiety activity is performed using Elevated Plus Maze Method and Light and Dark Exploration test. The ethanolic extract of D. hookeri rhizomes extract showed Anti-Anxiety property. The ethanolic extract showed significant anti-anxiety effect (p<0.05) when compared to control group. However, further studies are needed to isolate and characterize anxiety chemical constituents present in ethanolic extract of the rhizome.

 

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Received on 11.11.2024      Revised on 29.01.2025 Accepted on 01.03.2025      Published on 18.04.2025 Available online from April 22, 2025 Asian J. Res. Pharm. Sci. 2025; 15(2):123-128. DOI: 10.52711/2231-5659.2025.00019 ©Asian Pharma Press All Right Reserved
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