1-Introduction: Anxiety affects one-eighth of the total population worldwide and has become an important area of research in psychopharmacology during this decade

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Anxiety affects one-eighth of the total population worldwide and has become an important area of research in psychopharmacology during this decade. Benzodiazepines (BZDs) are the major class of compounds used in anxiety and they remain the most commonly prescribed treatment for anxiety. However, the realization that BZDs have a narrow safety margin has prompted many researchers to evaluate new compounds in the hope of identifying other anxiolytic drugs with fewer unwanted side effects1. Anxiety is a normal emotional behaviour. it is a bodily response to a perceived threat or danger. It is triggered by a combination of biochemical changes in the body, the patient's personal history and memory, and the social situation. It is important to distinguish between anxiety as a feeling or experience and an anxiety disorder as a psychiatric diagnosis. A person may feel anxious without having an anxiety disorder. In addition, anxiety frequently occurs as a symptom in other categories of psychiatric disturbance. Whereas most people will be fearful in physically dangerous situations, and can agree that fear is an appropriate response in the presence of danger, anxiety is often triggered by objects or events that are unique and specific to an individual. An individual might be anxious because of a unique meaning or memory being stimulated by present circumstances, not because of some immediate danger2.

In clinical usage, "fear", "anxiety" and "phobia" have distinct meanings, though the words are often used interchangeably in casual discourse to describe ubiquitous emotions3. The exact cause of anxiety disorders is unknown; but anxiety disorders - like other forms of mental illness-are not the result of personal weakness, a character flaw or poor upbringing. anxiety disorders may be caused by chemical imbalances in the body. Studies have shown that severe or long-lasting stress can change the balance of chemicals in the brain that control mood. Other studies have shown that people with certain anxiety disorders have changes in certain brain structures that control memory or mood. In addition, studies have shown that anxiety disorders run in families, which means that they can be inherited from one or both parents, like hair or eye color. Moreover, certain environmental factors-such as a trauma or significant event-may trigger an anxiety disorder in people who have an inherited susceptibility to developing the disorder4.

Besides a number of allopathic medications available, there is considerable evidence of an increase in demand for medicinal plants, as these plants have no side effects rather it is beneficial to provide sustainability to the body. Herbal medicines are the gift of “mother nature to its children”, to the man kind. Two hundred and fifty years ago there were few or no synthetic drugs and the species of plants were the main source of medicines to the world. Today many drugs we use based on the folk remedies and subsequent Ethno Pharmacological studies. A number of indigenous plant products are listed in Ayurvedic Pharmacopoeia for the treatment of anxiety. The indigenous tropical plant products have the potential of drug development, which may make the health care cheap and more acceptable; therefore there is a need to evaluate the psychopharmacological properties of the indigenous plant products. Hence in the present study, we are interested in screening of Nymphaea Alba for it antianxiety activity5.

2. Need for the study:

Anxiety disorders have a high impact on daily life and cause a great deal of suffering for the individual patient. They also have a substantial impact economically and incur a great deal of expenditure by society as a whole6. Not all patients with anxiety require treatment, but for more severe cases, treatment is recommended. The important thing to remember is that anxiety disorders do respond to treatment and therefore everyone can be helped.

There are several reasons why it is important for patients with severe anxiety symptoms to get help. Anxiety doesn't always go away by itself! It often progresses and if left untreated, anxiety disorders may eventually lead to a diagnosis of major depression, or interfere with the patient's education or ability to keep a job.

In addition, many anxious patients develop addictions to drugs or alcohol when they try to medicate their symptoms. Moreover, since children learn ways of coping with anxiety from their parents, adults who get help for anxiety disorders are in a better position to help their families cope with factors that lead to anxiety than those who remain untreated.

Because anxiety often has more than one cause and is experienced in highly individual ways, its treatment usually requires more than one type of therapy. In addition, there is no way to tell in advance how patients will respond to a specific medication, remedy or therapy.

Sometimes health care professionals will need to try different methods of treatment before finding the best combination for the particular patient. It usually takes about six to eight weeks to evaluate the effectiveness of a treatment regimen7.

There is a tremendous increase in the use of herbs for the treatment of anxiety. The most commonly used herbs for the treatment of anxiety are Bramhi, Rauwolfia, passion flower etc. They are widely used because of their less toxic in nature when compared to Allopathic drugs. More over the chances of drug dependence with herbal drugs is very less, which is most common with the allopathic drugs acting on CNS. All these facts evoked a deep interest to work on the topic of evaluating anxiolytic activity of Nymphaea alba.

3. Review of literature:

Plant profile8:

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Nymphaeales

Family : Nymphaeaceae

Genus : Nymphaea

Species : N. alba

Binomial name: Nymphaea alba

Common names: Water lily, Pond-lily, White pond-lily, Water nymph, Water cabbage.

Plant part used: Stalks, roots and flowers.

Chemical Composition: Nymphaea alba is rich in

Mucilages (polysaccharides)

Flavonoids: quercetin and derivatives, kaempferol and derivatives, apigenin...

Tannins: ellagic and gallic acids

Phenolic acids: parahydroxybenzoic acid

Alkaloids: Nupharine and nymphaeine

Ascorbic acid (vitamin C)9.
Medicinal Uses: Medicinal properties of White Lotus include astringent, antiseptic, anesthetic, anaphrodisiac and sedative.  Medicinally, Lotus (its roots, stems and leaves) found uses both as a culinary delight and starchy food staple as well as being used internally as a treatment for gastrointestinal disorders and jaundice. The Lotus was also used in medicine for its narcotic properties, most commonly applied as an anesthetic. Soaking the Lotus flower petals or leaves in wine and then ingesting the extract also lent itself well as an aid in meditation and relaxation. The rhizome and the flowers are used in herbal preparations10 . They are also used in leucorrhea, ulcers of mouth, constipation, stomachic and gonorrhea11.
Reported biological activity of the plant:

  1. Inhibition of potassium bromate induced renal oxidative stress and hyperproliferative response by Nymphaea alba in Wistar rats12.

  2. Anti carcinogenic effect of Nymphaea alba against oxidative damage, hyperproliferarive response and renal carcinogenesis in wistar rats13.

4. Objective of the study: Investigation of Anti-anxiety activity of alcoholic extract of Nymphaea

Alba in experimental animals.

5. Materials & Methods:

Male Swiss albino mice, weighing 25-35 g, will be procured from the Central Animal House, , Sree Siddaganga College of Pharmacy, Tumkur, The animals will be maintained at the Central Animal House and fed on a standard balanced with water ad libitum. All studies will be conducted in accordance with the CPCSEA guideline.

Diazepam obtained commercially, manufactured by Ranbaxy laboratories Ltd., will be used as standard drug.

Plant material and preparation of extracts:

The plant material will be collected, dried in shadow and coarsely powdered. The powdered plant material will be extracted using alcohol and Soxhlet apparatus.The suspension of extract will be used for oral administration.


100 and 200 mg/kg will be used orally.12

Grouping of animals

Each group contains 6 animals

  • GROUP I – serves as control (Normal Saline,Orally)

  • GROUP II – Diazepam (1mg/kg) Orally .

  • GROUP III – 100 mg/kg, of test extract, Orally.

  • GROUP IV – 200 mg/kg of test extract, Orally.

Test parameters

Following test parameters will be carried out for the assessment of anxiolytic activity.

  • Foot shock induced aggression (FSIA)

  • Elevated plus maze (EPM)

  • Open field test (OFT)

  • Light and dark box arena

  • Rotarod test


  • Foot shock induced aggression (FSIA):

Five groups (n=6) will placed in a box with a grid floor consisting of steel rods with distance of 6 mm. A constant current of 0.6 or 0.8 mA will be supplied to the grid floor by a LVE constant current shocker with an associated scrambler. A 60-Hz current will be delivered for 5 s, intermission for 3 min. Each pair of mice will be dosed and tested without previous exposure. The total number of fights is recorded for each pair during the 3 min. period. The fighting behavior consists of vocalization, leaping, running, rearing and facing each other with some attempt to attack by hitting, biting or boxing.It will be observed and noted14.

  • Elevated plus maze (EPM): EPM consists of two open arms 50x10x40 cm. And two enclosed arms, 50x10x40 cm. with an open roof, arranged so that the two open arms will be opposite to each other. The maze will be elevated to a height of 50 cm. One hour after the administration of the test drug or the standard, the rat will be placed in the center of maze, facing one of the enclosed arms, during a 5 min test period. The following measures will be taken the number of entries into and time spent in the open and enclosed arms; the total number of arm entries. The procedure will be conducted preferably in a sound attenuated room1.

  • Open field test (OFT): In the open field test each animal will be placed in one of the peripheral corner squares of the box and the number of peripheral and central squares crossed, the time spent in central squares and the number of rears will be observed for a five-minute period. The apparatus consisted of a large rectangular box (100 x 80 cm) with 60 cm high walls. The floor is divided into twenty-five squares (outer 16 and centre)14 .

  • Light and dark box: The apparatus consisted of an open top wooden box. Two distinct chambers, a black chamber (20 x 30 x 35 cm) painted black and a bright chamber (30 x 30 x 35 cm) painted white. The two chambers will be connected through a small open doorway (7.5 x 5 cm) situated on the floor level at the centre of the partition.-The testing apparatus consist of a light and dark chamber. Each animal will be tested in bright and dark arena paradigm in a single setting. The number of entries into and the time spent in the bright arena, the number of rears in the bright and dark arenas and the duration of immobility will be noted15 .

  • Rotarod test: Motor coordination and balance will be tested using accelerating rotarod. Mice will be placed on a horizontal metal coated rod with rubber (3 cm diameter) rotating at an initial speed of 10 rpm/min. Terminal speed of the rod will be 20 rpm in accelerated studies and rotational velocity of the rod will be linearly increased from 10 to 20 rpm within 20 s. The time each animal will be able to maintain its balance walking on top of the rod will be measured. Mice will be given two trials with a maximum time of 300 s and a 30 to 60 min intertrial rest interval. Before the beginning of all experiments, the riding ability of the animals in the rotarod will be checked. Thus, the mice will be initially put on a rotating rod, and mice that immediately dropped off (within 30 s) will be removed from the experiment16 .

6. Statistical analysis: It will be done by student ‘t’ test./ ANOVA
7. Experimental design:

Dose dependent studies:

Animal Swiss Albino Mice

Sl No



Route of


No. of Animals in each Group




Normal saline



1-Foot shock induced


2-Elevated plus maze


3-Open field test


4-Light and dark box


5-Rotarod test16.




1 mg/kg





100 mg/kg,

of Extract





200 mg/kg

Of Extract




  1. Yadav AV, Kawale LA, Nade VS. Effect of Morus Alba (Mulberry) leaves on Anxiety of Mice. IJP. 2008; 40: 32-35.

  2. Anxiety. - www.medicinenet.com

  3. Anxiety disorder - Wikipedia, the free encyclopedia.htm

  4. http://www.hhs.gov/faq/healthprograms/mentalhealth/1705.html

  5. http://www.helpguide.org/mental/anxiety_types_symptoms_treatment.htm

  6. http://www.webmd.com/anxiety-panic/guide/mental-health-anxiety-disorders?page=2

  7. http://psychiatry.healthse.com/psy/more/epidemiology_of_anxiety_disorders_introduction/

  8. http://www.nativeremedies.com/ailment/anxiety-disorders-info.html

  9. http://revadainter.com/upload/Prod/718/Nympheline_FTA.pdf

  10. http://www.iamshaman.com/lotus/whitelotus.htm.

  11. http://www.henriettesherbal.com/eclectic/kings/nymphaea.html.

  12. Khan N, Sultana S. Inhibition of potassium bromate induced renal oxidative stress and hyperproliferative response by Nymphaea alba in Wistar rats. J Enzyme inhib Med chem. 2005; 20: 275-283.

  13. Khan N, Sultana S. Anti carcinogenic effect of Nymphaea alba Against Oxidative damage, hyperproliferarive response and renal carcinogenisis in wistar rats. Mol Cell Biochem 2005; 271: 1-11.

  14. Gerhard Vogel H. Drug Discovery and Evaluation, Pharmacological Assays, ed 2nd Springer-Verlag Berlin Heidelberg New York 2002. P: 425,434.

  15. Gopala Krishna HN, Sangha RB, Misra N, Pai. MRSM. Antianxiety Activity of NR-ANX-C, a polyherbal preparation in rats. IJP. 2006: 38: 330-335.

  16. Hossein Hosseinzadeh Marjan Nassiri As Anticonvulsant, Sedative and Muscle Relaxant Effects of Carbenoxolone in Mice BMC Pharmacol. 2003; 3: 3.

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