ANXIOLYTIC EFFECT OF ETHANOLIC EXTRACT OF FLOWERS OF CHRYSANTHEM INDICUM IN ALBINO MICE

ANXIOLYTIC EFFECT OF ETHANOLIC EXTRACT OF FLOWERS OF CHRYSANTHEMUM INDICUM IN ALBINO MICE

Sheela ^*1, V. Srinivasan ¹ and E. Sukumar ²

Department of Pharmacology ¹, Department of Research and Development ², Saveetha Medical College and Hospital, Saveetha Nagar, Thandalam, Chennai - 602105, Tamilnadu, India

ABSTRACT: Mental disorders are commonly occurring and often seriously impairing in many countries throughout the world. The total number of people suffering from anxiety or depression amount to 615 million, Close to 10% of the world’s population is affected. Chrysanthemum indicum is an ancient herbal medicine used as an anti-arthritic, anti-inflammatory and anti-anxiety in many Asian countries. The elevated plus-maze (EPM) and open field test has been most familiar tool in the investigation of the neurochemical transmission basis of anxiety, for testing anxiolytic drugs. The anxiolytic activities of Ethanolic and Chloroform extracts of Chrysanthemum indicum Linn flowers were evaluated using the elevated plus-maze (EPM) and Open field. The ethanolic extract  results are  suggestive of anxiolytic activity in dosages around 250 and 500 mg/kg, as expressed by elevation of the time spent on the open arms in the plus-maze; a decrease of freezing and increase of deambulation in the open field test.

Chrysanthemum indicum,

Anti-anxiolytic effects, Elevated plus maze, Open field, Ethanolic extract

INTRODUCTION: Anxiety generally is defined as a state of diffuse, vague, very unpleasant feeling of fear or apprehension ¹. Anxiety is one of the common psychiatric disorders affecting all the age group people around the world. In most of the countries, the life time prevalence of panic disorder is around 7-9% and in India it is been reported to be about 1%. According to WHO, the prevalence of generalised anxiety disorder in common population is about 8.5% and in worldwide population, it is about 16.6% ². Compounds varying in selectivity as 5-HT1A receptor antagonists having benzo diazepine-like activity are recent targets to assess antianxiety effects in mice ³.

A common factor responsible for affecting both physiological and psychological state in human is stress. Anxiety and other mental health disorder require a life time treatment ⁴. An Indian study has reported the incidence of childhood psychiatric disorder was about 18 per 1000 per year ⁵. Anxiety, if it is been untreated it may lead to depression and also increases the risk of cardiovascular morbidity and mortality ⁶. The various other mental disorders like generalised anxiety, social phobias, agoraphobia, claustrophobia, panic disorder, obsessive compulsive disorder also require a chronic treatment. Anxiety is  been treated by using some of the common drugs like selective serotonin reuptake inhibitor and benzodiazepines which are  known for its side effect such as suicidal intention, decreased alertness, sexual dysfunction and dependency ⁷.

With the above known side effects, the researcher aims in treating the generalised anxiety with the safe and effective drug by using the alternate traditional treatment. Herbs have been used for centuries by the folk in the traditional treatment of anxiety. Some of the herbs and other nutrients used by the folk as traditional medicine in calming the mind and elevating the mood are Passion flower, lavender, lemon balm, black cohosh, Ginkgo biloba, Kava, St. John wort (SJW), Valerian root and others like amino acid lysine, S-adenosyl –Methonine, B-Vitamin, inositol, choline, omega -3 fatty acid, gamma amino butyric acid (GABA), tryptophan,5- hydroxyl tryptophan and other cation magnesium ^{8, 9}. The beneficiary use of the herbs and other alternative nutrients in treating anxiety and depression was reviewed and published over few decades ¹⁰.

Chrysanthemum indicum, an ancient herb used in the traditional medicine shows various biological effect which has been documented in a number of studies ¹¹. They are the traditional Chinese medicine reported to possess anti-arthritic, anti-inflammatory, immunomodulatory property. The plant extract was used as analgesic, antipyretic and also in reducing the blood pressure, respiratory disorders, deterioration of bone and muscles ¹². The aerial parts of the plant used are Stem, flower and leaves specifically the tea prepared from the flowers of Chrysanthemum indicum is been used in Korea to relieve anxiety and to enhance the mood, antioxidant and DNA damage preventive activity was found in the flower extract ¹³.

There are studies which show the aqueous extract of the chrysanthemum indicum to be used in relieving anxiety ¹⁴. The present study aims in the ethanolic extract of Chrysanthemum indicum and also to find the effective dose of the extract in anxiety.

MATERIALS AND METHOD:

Authentication of plant material: Flowers were procured from Tirunelveli district, Tamil Nadu. The flower was identified and authenticated by a renowned Botanist.

Drugs and chemicals: Diazepam was obtained from GlaxoSmithKline Pvt. Ltd. and petroleum ether, methanol (LR grade) were procured from H. Chandanmal & Co, Chennai.

Preparation of extract:

Alcoholic extract of Chrysanthemum indicum: The shade dried flowers of the Chrysanthemum indicum weighing about 10 gm were subjected to the 90 % ethanolic extraction using the Soxhlet apparatus and then filtered. The filtrate was concentrated using a rotary vacuum evaporator. The alcoholic extract of the chrysanthemum indicum was about approximately 2 % in the end.

Chloroform extract of Chrysanthemum indicum: About 10 gm shade dried flowers were extracted three times using lab grade chloroform using the Soxhlet apparatus and were filtered. The filtrate was concentrated using a rotary vacuum evaporator. The chloroform extract yielded about 1% only.

Animals: Female Swiss albino mice (weighing 30-40 gm) bred and maintained in the animal house facility of Saveetha Medical College were procured for the study. Animals were randomly allotted to five groups with six animals in each group. Animals were housed in polypropylene cage with three animals per cage and the dry husk was used as the bedding material. Animals were fed with water and food ad libitum and maintained at controlled room temperature of about (18 + 2 °C) with a 12:12h light and dark cycle. The animals selected for the study was brought to the department of pharmacology for acclimation the environment. The guidelines of the “committee for the purpose of control and supervision of experiments on animals (CPCSEA, India) was strictly followed for the maintenance of animals in this study. The study was carried out after the prior approval from the animal ethics committee [SU/CLAR/RD/009/2016].

Oral administration of extract: The alcoholic extract and the chloroform extract of the Chrysanthemum indicum was freshly prepared using hydroxyl methyl cellulose as the vehicle. About 250 mg/kg and 500 mg/kg of both the extract were given orally by gavage and the diazepam was maintained as a standard drug which was injected intraperitoneally. The extract was given at a volume of 1ml/kg of the body weight of the animal and the test was carried out only after 30 minutes.

Different treatments:

The following treatments were employed in the study:

Group 1- vehicle (1% HMC in distilled water)

Group 2- Standard drug (Diazepam 2.5 mg/kg p.o. suspended in HMC vehicle)

Group 3-5- Test groups (EE and CE extracts of Chrysanthemum indicum at two different doses- 250, 500 mg/kg p.o. suspended in HMC vehicle)

The effects of the drugs were estimated 30 min after the administration of the dose. In each experiment, apparatus was cleaned using 5% ethanol before introducing the next animal to preclude the possible cueing effects of odours left by previous subjects.

Animal Model for Anxiety:

Elevated plus maze: Anxiolytic effect of the extract was evaluated using elevated plus maze. The standard elevated plus maze consists of two open arms (25x5x0.5 cm) and two closed arms (25x5x16 cm) with the wall connected to the central zone (5x5x0.5cm) at a height of about 50 cm from the floor. The open arms wall is too small to decrease the number of falls, whereas the closed arms had a high (16 cm) wall to enclose the arm. Each animal was placed in the central zone facing the open arm ¹⁵. During the 5 min experiment, behaviour of the mice was noted as a) number of entries into the open arm & closed arm b) average time spent by the animal in open arm. The antianxiety activity was recorded as average time spent by the animals in the open arms of the EPM.

Open field apparatus: The open field apparatus was used to conduct the open field test in open aired Plexiglass cube with the dimension of 40 cmx40cmx30cm. The each animal was individually placed in the same left corner of the arena and was allowed to explore for 5 mins. The floor of the field was subdivided into 16 squares of equal size (4 center, 12 peripheral). A camera was mounted on the apparatus and was used to record the movement of the animal which would be used to evaluate the anxiety behaviour ¹⁶. The open field apparatus should be cleaned before introducing the other animal with 70 % ethyl alcohol and allow it to dry in order to avoid the bias during the experiment.

The parameters used to evaluate the exploratory behaviour and anxiety in this instrument is

1. Number of squares crossing with all four paws
2. Number of center square entries
3. Duration of stay in the central square
4. Rearing frequency
5. Grooming frequency
7. Statistical analysis: All the result values were expressed in Mean + SEM calculated using SPSS statistical software package. One Way Analysis of Variance (ANOVA) was performed, followed by Post hoc test to compare the inter-group difference. A P value of <0.05 was considered to be statistically significant.

RESULTS:

TABLE 1: ANXIOLYTIC EFFECT OF ETHANOL AND CHLOROFORM EXTRACT OF FLOWERS OF CHRYSTHAMUM INDICUM AND DIAZEPAM ON ELEVATED PLUS MAZE. DATA ARE EXPRESSED AS MEAN + SEM

*P < 0.05 (ANOVA, Post hoc) compared with multiple groups

Control= 1% HMC, STD=Diazepam (2.5 mg/kg p.o.). EE= Ethanolic extract (250, 500 mg/Kg p.o), CE= Chloroform extract (250, 500 mg/Kg p.o), Results are expressed as mean±SEM (n=5); P<0.05 was considered statistically significant.

TABLE 2: ANXIOLYTIC EFFECT OF ETHANOL AND CHLOROFORM EXTRACT OF FLOWERS OF CHRYSTHAMUM INDICUM AND DIAZEPAM ON OPEN FIELD. DATA ARE EXPRESSED AS MEAN + SEM

*P < 0.05 (ANOVA, Post hoc) compared with multiple groups

Control= 1% HMC, STD=Diazepam (2.5 mg/kg p.o.). EE= Ethanolic extract (250, 500 mg/Kg p.o), CE= Chloroform extract (250, 500 mg/Kg p.o), Results are expressed as mean±SEM (n=5); P<0.05 was considered statistically significant.

DISCUSSION: Psychiatric disorders are now considered as a universal problem where there is a need for safety treatment. Benzodiazepines have been used for more than 40 years for the treatment of several types of anxiety ¹⁷. The alternation of neurotransmitters, GABA, serotonin, adrenaline and dopamine play the important role in the development of psychiatric problems ¹⁸. The current drugs which are used for the treatment of anxiety have failed to reduce the untoward effects produced by them. Anxiety disorders require a chronic treatment and the above mentioned reason have forced the researchers for the alternate traditional method to overcome the current scenario. The Indian history shows the custom of using the medicinal herbs for various neurological conditions ¹⁹ and even the present drug is being used in using in china for treating Parkinson's disease (PD) ²⁰. However, to date there have been fewer scientific observations of this flower extract which has led to the current study to demonstrate the anxiolytic effect of this flower extract using elevated plus maze and open field apparatus.

When the lab animals are removed from the home cage and produced to the new environment, the alteration in the behaviour of the animals is termed as anxiety. The most common lab method used in the animals to evaluate the anxiolytic effect was elevated plus maze ^{21, 22}. The normal behaviour of aversion towards the open area and the greater preference towards the protective area was used to demonstrate the effectiveness of the anxiolytic effect. The ethanolic and chloroformic extract of the flowers of the Chrysanthemum indicum increased the time spent and the number of entries in the open arm. There was a significant increase in the entries and the time spent in the open arm of ethanolic extract than the chloroformic extract of the flowers (Table 1). Open field test used the emotional state of the animal to evaluate the anxiolytic effect of the animal ^{23, 24}. The oral administration of the ethanolic extract of Chrysthamum indicum has produced a marked increase in the time spent in the centre and significant reduction in the grooming time. The extract produced anxiolytic effect in a dose dependant manner (Table 2).

CONCLUSION: The present study has revealed the anxiolytic activity of the ethanolic extract and chloroformic extract of Chrysthamum indicum at 250, 500 mg/kg dose. The ethanolic extract has marked anxiolytic effect than the chloroformic extract. The presence of the phytochemicals like flavinoids, phenols, and terpinoids may influence the anxiolytic effect of the herbal plants. Further research may be needed to isolate and quantify the phytochemicals which may be responsible for the anxiolytic effect ²⁵.

ACKNOWLEDGMENT: Authors are thankful to Dr. C.B. Tharani, Prof & HOD, Dept of Pharmacology, Dr. R. Vijayaraghavan, Director of Research and Dr. S Porchelvan, Prof of Statistics, Dept of Social & Preventive Medicine to help conduct.

DECLARATION OF INTEREST: The authors declare that there is no conflict of interest.

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    How to cite this article:

    Sheela D, Srinivasan V and Sukumar E: Anxiolytic effect of ethanolic extract of flowers of Chrysanthem indicum in albino mice. Int J Pharm Sci Res 2017; 8(6): 2573-77.doi: 10.13040/IJPSR.0975-8232.8(6).2573-77.

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