Vol. 05,Special Issue 01, (ICOSD-2020) January 2020, Available Online: www.ajeee.co.in/index.php/AJEEE ANTINOCICEPTIVE STUDY OF METHANOLIC EXTRACT OF ANNONA
SQUAMOSA ON MICE
Achla Vyasa1, Aayushi Nigama2, R. K. Nemaa, Kapil Vyasb3 and Aarti Nandwanaa4
1,2,3Lakshmi Narain Institute of Pharmacy (RCP), Revati, Sanwer Road, Indore, M.P., India, 453331.
4Symbiotec Pharmalab Pvt. Limited, SEZ, Pithampur, M.P., India.
Abstract:- Soreness produce discomfort, pain is the most common sign of any sickness or grievances. On the basis of pain, we diagnose a number of diseases that is very uncomfortable for the patient. To overcome this situation we used pain killers or antinociceptive drugs that are also known as an analgesic that are the molecules which produce relief from pain. Nowadays there are so many types of the antinociceptive present in the market but the aim of the study is to reboot the traditional system of medication because the organic molecules create dependence for our body & for this our body easily create tolerance for the particular drug. In our study, we used plant Annona squamosa to determine its antinociceptive properties. Activity involves, selection of plant, collection of leaves, methanolic extract, phytochemical & pharmacological study. Tail flick method is used for the examination of antinociceptive activity in mice.
Keywords:- Annona squamosa, antinociceptive, pharmacological study, tail flck method.
1. INTRODUCTION
Antinociceptives are very common nowadays we use mostly to reduce pain but we innovate more potency of the drug in less amount of drug with avoid side effects. Pain is the most common sign of any disease, injuries. On the basis of pain, we diagnose a number of diseases, that is very uncomfortable for the patient. We used so much amount of antinociceptive drugs are used with the combination of other drugs in many diseases.
Basically, that drug molecules are organic molecules that affect the body in a negative manner also like:- dose-dependent, tolerance, addiction are the most common symptoms.
In our traditional system, we used leaf juices & leaps to treat pain in the body or at the side of the injury. In Indian, Thai, American medicinal system they are used to treat dysentery & UTI. According to Indian traditional system, people’s are used Annona squamosa plant leaves crushed past on wound. That indicates about the plant antibacterial activity, Annona squamosa plant is very useful to plant and filled with lots of goodness.
This fruit is filled with immune modifier property, bark, leaves, seeds having many medicinal properties like analgesics, antioxidant, antibacterial, antifungal, antiulcer, anticancer, antidiabetic.
This is the herbal effective molecule we used in this activity and the most important thing there is no side effect of the herbal drug. We want to reuse that traditional culture with modification of science. Annona squamosa a small well-branched tree or shrub that bears edible fruitscalled sugar-apple, species of the genus Annona and member of the family Annonaceae1. The tree is native from the Caribbean islands to throughout India and has been cultivated since ancient times. It is mainly grown for its fruit which is sweet in taste and as a source for preparation of sugar apple wine. The whole plant of Annona squamosa possess medicinal values apart from its sweet taste.
The seeds of the plant are found to antidiabetic 2 anti thyroid3, antioxidant4, and anti-lice activity5. On the other hand, the whole plant extract and the leaves are found to possess anti lipidemic6 and vasorelaxant acitivity7. The leaves of the plant have been used in India by the local people as a medium to relieve the pain of medium to moderate intensity. The present work aims at scientifically judging the antinociceptive and anti- inflammatory activity of Annona squamosa leaves. Annona Squamosa is commonly known as sitafal plant. That is also called a sugar apple plant. It is a small branched tree or shrub.
Custard apple is a common name for a fruit.
The species is widely grown as a commercial fruit tree both within its native range and in tropical regions around the world. Pharmacological properties of plant Annona squamosa is proved earlier, this plant exibits Anti-inflammatory activity, Antifertility activity, Antidiabetic activity,Anticancer activity, anti-microbial activity, cytotoxic activity, anti-oxidant activity, anti-lipidomic activity, anti-ulcer activity etc.
Vol. 05,Special Issue 01, (ICOSD-2020) January 2020, Available Online: www.ajeee.co.in/index.php/AJEEE 1.1 Scientific classification
Kingdom : Plantae
Subkingdom : Tracheobionta
Super division : Spermatophyta
Division : Magnoliophyta
Class : Magnoliopsida
Sub class : Magnoliidae
Order : Magnoliales
Family : Annonaceae
Genus : Annona L.
Species : Annona squamosa
1.2 Synonames
Annona asiatica L Annona cinerea
Dunal Guanabanus squamosus (L.) M.Gómez
Sitaphal
Sitafalam
Annona forskahlii DC.
2. MATERIAL AND METHODS 2.1 Collection of plant material
The leaves of Annona squamosa collected from the medicinal garden of Lakshmi Narain College of Pharmacy (RCP) Indore. The mature leaves of Annona squamosa were collected in the month of September. The leaves are authenticated by botonist. The collected plant material (leaves) was washed thoroughly with water to remove the adhering soil, mud, and debris. All insect damage or fungus infected leaves were removed. The plant material was dried in the shade at room temperature to a constant mass. The plant material was coarsely powdered using blender. The powder was stored in an air tight container and protected from light.
2.2 Extraction
Collected dried leaves of Annona squamosa were converted into small pices then then dried under the shade at room temperature. Powder of dried leaves about 45 g were prepared using a mechanical mixer. Soxhlet apparatus is used for extraction process for this extraction process 45 g powdered drug take in filter paper bag and kept this bag on soxhlet, then after we added solvent (methanol) in small quantity up to 450 ml. we extracted for 24 hrs at constant temperature 40°C.
After that, we collect the residue and evaporated this at low temperature till we get solid mass. After the complete evaporation process, we get a dark green colour sticky product was found. Obtained extract stored in refrigerator -10°C until further use. We perform TLC of obtained extract. Perform Phytochemical screening of plant extract, the extract was subjected to detect the presence of glycoside, alkaloids, amino acids, flavonoids, carbohydrates, saponins, phenols, steroids, tannins by standard methods.
Vol. 05,Special Issue 01, (ICOSD-2020) January 2020, Available Online: www.ajeee.co.in/index.php/AJEEE 3. PHYTOCHEMICAL SCREENING
Alkaloids (Meyer’s test): A quantity of 0.5 g of the dried powdered sample was boiled in 20 ml of water and filtered. To a few drops of the filtrate, a drop of Meyer’s reagent was added by t1 side of the test tube. crimish precipitate indicates the test is positive.
Glycosides: About 0.5 g of the extract was dissolved in 2 ml of glacial acetic acid containing 1 drop of 1% FeCl3. This was under laid with concentrate H2SO4. A brown ring obtained at the interface indicated the presence of deoxy sugar, characteristic of cardiac glycosides. No violet/greenish ring appear.
Flavonoids: A portion of the powdered sample was heated with 10 ml of Ethyl acetate over a steam bath for 3 minutes and then the mixture was filtered. 4ml of the filtrate was shaken with diluted Ammonia. The presence of flavonoid is supported by Light yellow colouration.
Steroids: The crude plant extract (1 mg) was taken in a test tube and dissolved with 10 ml of chloroform, then added an equal volume of concentrated sulphuric Acid to test tube by sides. The upper layer was in the test tube does not turn to red and sulphuric acid layer showed yellowish-green colour. Test shows absence of Steroids.
Phenolic compounds (Ferric chloride test): Three hundred mg of extract was diluted to 5 ml of distilled water and filtered. To the filtrate, 5 % of ferric chloride was added. Brownish green colour indicates the absence of Phenol compounds.
Tannins: A quantity of 0.5 g of the dried powdered sample was boiled in 20 ml of water and filtered. A few drops of 0.1% Ferric chloride was added and observed for brownish or bluish-black colour.
Saponins: 2 g of the powdered sample was boiled in 20 ml of distilled water in a water bath. 10ml of the filterable was mixed with 5 ml of distilled water shaken vigorously for a stable persistent froth. The following was mixed with 3 drops of Olive oil and shaken vigorously. Then observed for the formation of an emulsion.
Test for Carbohydrates: (Benedict test and Iodine test): Few drops of Benedict solution was added in the plant extract if it shows the brick read colour Confirms the presence of glucose and few drops of Iodine was added in another extract the dark blue colour confirms the presence of starch.
Test for Amino Acid: Ninhydrin Test: The plant extract is treated with 0.25% v/v ninhydrin reagent and boiled for a few minutes. Formation of blue colour indicates the presence of amino acids.
Table: 1 Phytochemical screening results are tabulated below:.
Where, (+) indicate present, (-) indicate absent 3.1 Experimental
3.1.1 Animals
Wistar Albino mice weighing select for the activity. The animals had free access to food and water and they were housed under a natural (12 h each) light-dark cycle with access to standard pellet chow and water ad libitum. The animals were acclimatized for 5 days to the laboratory conditions before performing the experiments.
Vol. 05,Special Issue 01, (ICOSD-2020) January 2020, Available Online: www.ajeee.co.in/index.php/AJEEE 4. METHODS
4.1 Tail Flick method
Pain is very difficult for everyone to overcome this situation we used antinociceptives. In this activity, we use a tail-flick method to measure analgesic response. In tail flick method we adjust the tail of mice over the heat source and note the time of tail withdrawal from the radiant heat source. To perform the task mice are divided into three groups, first group is control group, to this group distil water orally given by feeder needle, stander drug paracetamol (100mg/kg) given to the second group is, the third group is given Annona squamosa leaves to extract a three different dose(100 , 200, 500mg/kg).
4.2 Acid induced Writhing test
Painful reaction in animals may be produced by chemicals also. Acetic acid produced pain reaction that characterized as a writhing response like abdominal constriction, turning of the trunk, stretching of legs response of animals due to pain by chemically induced. In this study we are using three groups of animals first is a control that we administered acetic acid 1% (1ml/100g) according to the bodyweight of the animals.
Place them to a glass jar and note the response of abdominal constriction, trunk twist response and starching of legs response for 10 min. the second group we used standard drug indomethacin (20 mg/kg) after 15 min of acetic acid administration. Note the onset of writes. The third group we used Annona squamosa leaves extract after 15 min of acetic acid administration. Note the onset of writes. Calculate the mean writhing response of control, standard, and test group. An antinociceptive activity can also be expressed as maximal possible effects. (% MPE)
5. RESULT
Tail flick method is used for determination of antinociceptive activity in mice result, obtained against the three groups are tabulated below:
Table: 2 Results obtained from tail flick method
5.1 Results from Acid induced Writhing test
Vol. 05,Special Issue 01, (ICOSD-2020) January 2020, Available Online: www.ajeee.co.in/index.php/AJEEE Table: 3 Results obtained from acid induced Writhing test are tabulated below:
6. DISCUSSION
Pain is a subjective experience, which is difficult to define exactly even though everyone experiences it. Screening of centrally and peripherally acting analgesic activity to intraperitoneal injection of noxious chemicals is performed by the tail flick method and the writhing response of the animals. Acetic acid causes algesia by liberating endogenous substances that excite the pain nerve endings.
From the results it is apparent that the ethanolic extract of Annona squamosa showed a significant antinociceptive effect in the tail flick method and writhing response which is comparable to the standard. There are reports on the role of tannins and flavanoids in antinociceptive activities. The extract of Annona squamosa leaves was found to possess significant analgesic activity which is quite comparable to paracetamole.
Furthermore a detailed investigation on the extract is underway to determine the phytoconstituents that are responsible for these activities as well as to define the exact mechanism of action of the herbal drug.
6.1 Acknowledgement
The authors are thankful to the Director and Management of Lakshmi Narain Institute of Pharmacy (RCP), Raveti, Sanwer Road, Indore for providing the necessary facilities for performing the work.
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