North South University Department of Pharmaceutical Sciences Bachelor of Pharmacy

North South University
Department of Pharmaceutical Sciences
Bachelor of Pharmacy (Project)
Approval
Phytochemical Screening, Analgesic and Antibacterial activities of ethanolic extract of Abroma augusta(Linn.) leaves.

………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………….

Is submitted by
Tamony Tanjum Mon
ID # 1320715046 for partial fulfilment of the requirements for the degree of Bachelor of Pharmacy.
…………………………………………………………………
Dr. Hasan Mahmud Reza
Professor & Chairman
Department of Pharmaceutical Sciences
…………………………………………………………………
Name of Supervisor
Designation
Department of Pharmaceutical Sciences
Declaration
I do hereby declare that the paper entitled “Ispaghulahusk- A holistick approach to relief constipation” presented to Department of Pharmaceutical Science, North South University, is the outcome of the study performed by me under the supervision of Dr………………………………………, Professor, Department of Pharmaceutical Science, North South University. I also declare that no part of this report has been or is being submitted elsewhere for the award of any degree.

Submitted by
Name
ID:

TABLE OF CONTENT
No.

Topic Name
Page No.

01 Abstract 02 Introduction 03 Materials & Method 04 Phytochemical Screening 05 Antimicrobial Activity Screening 06 Analgesic Activity Screening
Acetic Acid Induced Test
Tail Immersion Test 07 Conclusion
ABSTRACT
Objective: Abroma augusta Linn (Family-Malvaceae) commonly known as Ulatkambal traditionally used as dysmenorrhoea, ammenorrhoea, wound healing, sterlilty and other menstrual disorder. The purpose of this study was to evaluate the methanolic extract Abroma augusta Linn leaves for antimicrobial & analgesic activity & phytochemical screening.

Methodology: The antimicrobial properties have been characterized by disc diffusion method where the 200mg and 400mg doses of extract were compared with standard antibiotic, Ciprofloxacin. The analgesic effect of the extract was evaluated by performing acetic acid-induced mouse writhing test and tail immersion test on Swiss albino mice using 200mg/kg and 400mg/kg doses of the extract comparing to the standard drug, diclofenac Na. Phytochemical screening was carried out by performing various chemical tests on the extract to determine the presence of alkaloids, steroids, flavonoids, tannins, carbohydrate and glycosides, etc.
Results and Conclusion: The results obtained from the tests showed significant analgesic property but no antimicrobial effect. The phytochemical screening showed the presence of alkaloids,steroids, tannins, carbohydrates, glycosides & reducing sugar.

INTRODUCTION
Over the years, plants have generally proven to be veritable sources of drugs used in orthodox medicine. This has in recent times encouraged the search for newer more efficacious and better tolerated drugs from plants. A criterion that has been used over the years for the selection of plants for pharmacological investigations is reported use in traditional medicine.

leftcenter Abroma augusta (Malvaceae) is commonly known as Ulatkambal in Hindi, Devil’s cotton in English and Kepashantu in Bahasa Malayu. It is widely distributed in Asia, South and East Africa and Australia1. The species is of Indo-Malaysian origin and it is found in Perak, Pahang, Selangor, and Kelantan States of Peninsular Malaysia. A. augusta is a small shrub with a long history of medicinal use in Ayurveda system. In India, the mother tincture of A. augusta is widely used in homeopathic medicine to treat uterine disorders and diabetes mellitus. Its root-bark is highly useful as uterine tonic and to treat amenorrhoea, dysmenorrhea, abortifacient and antifertility. Leaves are used for diabetes, rheumatic pains and headache with sinusitis. Leaves and stem are used as demulcent. Cool water extract of fresh leaves and stems is very efficacious in gonorrhoea2-4. The different solvent fractions showed the presence of tannins, glycosides, steroids and alkaloids. The whole plant contains several alkaloids and secondary metabolites including steroids, triterpenes, flavonoids, megastigmanes, benzohydrofurans and their glycosides and phenylethanoid glycosides 3. The leaves of A. augusta contain octacosanol, taraxerol, b- sitosterol acetatelupeol, an aliphatic alcohol and mixture of long chain fatty diols.

The aim of this study is to evaluate the phytochemical screening, analgesic and antimicrobial activities of the methanolic extract of Abroma augusta (linn.) leaves.
MATERIALS ; METHOD
CHEMICALS
The following drugs and chemicals were used in the current study: Diclofenac NA (Square Pharmaceuticals Ltd), Ciprofloxacin antibiotic disc, Acetic acid, 0.9% Saline, Methanol ; distilled water.

COLLECTION OF PLANT MATERIAL
The leaves of Abroma augusta was collected from the botany department of Chittagong University, Chittagong, Bangladesh. The leaves were then air dried under shade for 12 days. Then they were broken into smaller sizes by hand and oven dried for 8 hours.

38296856087745The dried leaves were then grinded into powder using Toshiba single phase electric machine, which has the speed of 1400 rpm. After grinding, we got about 580g of coarse powder, which was stored in air tight container.

PREPARATION OF EXTRACT
Among the 580g powder of A. augusta Linn, about 100g of powder was weighed and soaked in 900ml of methanol in a conical flask and in a rotary shaking machine at North south university, which has rpm of 140.

After 7 days the powder were uniformly dissolved in the solvent (methanol). Then, the extract was filtered using a normal white cotton cloth followed by whatman filter paper.

Finally, the extracts were evaporated using Rotary evaporator, which has rpm of 100. In evaporation process, at first water was pumped to heat the evaporator machine (In water bath, water was heated at 600C according to the solvent).

When we were done with the evaporation, we got liquid crude extracts which we collected in a petri dish and then dried under the fan at room temperature. After a while finally we got gummy concentrates of the crude extracts of A. augusta Linn.10 The petri dish containing our extract was tightly taped and labelled after which it was preserved in the refrigerator at 40C temperature.

ANIMALS
16 swiss albino mice, all males of about 35 to 45kg, were collected from the animal house of the Department of Pharmaceutical Sciences in North South University. They were kept at the animal house of North South University where they were maintained under standard environmental conditions. The animals were fasted overnight before the experiments.

DRUGS ; TREATMENT
For antimicrobial property evaluation, 200mg and 400mg doses of extract were compared with standard antibiotic disc of Ciprofloxacin. For analgesic activity test, Diclofenac Sodium (5mg/kg) was used as standard drug in both writhing and tail immersion test. The drug was administered intraperitonially to the mice. Saline water (0.1ml/mouse) was administered to the mice in control group. Methanolic extract of the plant was administered orally to the test groups at 200mg/kg and 400mg/kg doses.
PHYTOCHEMICAL SCREENING
The methanol extract of A. augusta (Linn.) leaves was screened for the presence of phytochemical constituents such as reducing sugars, tannins, alkaloids, steroids, glycosides, carbohydrates, saponins and flavonoids following the standard procedures. The standard methods that were used to perform the tests are as follows:
TEST FOR ALKALOIDS
2ml extract solution and 0.2ml dilute HCL mixed together with 0.1ml of iodine solution (wagner’s reagent). Reddish brown precipitated was obtained to confirm the presence of alkaloids.
2ml extract solution and 0.2ml diluted HCL mixed together with 0.1ml of picric acid (Hager’s reagent)21. Yellowish precipitate was observed which confirms the presence of alkaloids.

TEST FOR STEROIDS
10mg of extract was dissolved in 1ml of chloroform. Then 1ml sulfuric acid was added. It was observed that chloroform layer acquired reddish brown color and acid layer showed green fluorescence thus confirming the presence of steroids.

TEST FOR FLAVONOIDS
10ml of the extract solution was hydrolyzed with 10% H2SO4 acid. This was extracted with ether and divided into 3 portions:
To 1st potion, 1ml dilute sodium carbonate solution was added and pale yellow color were seen which confirms the presence of flavonoids.

To 2nd portion, 1ml dilute sodium hydroxide solution was added and yellow color was observed, which means that flavonoid is present.

To 3rd portion, 1ml dilute ammonia solution was added and greenish yellow color was obtained that showed flavonoid is present.

TEST FOR REDUCING SUGARS
3923665866140To 5ml of extract solution 5ml of Fehling A and Fehling B solutions were added. The mixture is boiled for 5 minutes in a water bath. No Brick red color precipitate was observed thus confirming the presence of reducing sugars.

To 5ml of extract solution 5ml of Benedict’s reagent was added. The mixture is boiled for 5 minutes in boiling water bath. No Brick red color precipitate was observed thus confirming the presence of reducing sugars.
TEST FOR TANNINS
5ml extract solution was mixed with 1ml of 10% lead acetate solution. Yellow precipitate was obtained, which confirms the presence of tannins.

To 5ml extract solution, 1ml of 10% potassium dichromate solution was added. No yellowish brown precipitate was obtained which confirms the presence of tannins.

TEST FOR GLYCOSIDES
45053256680200To a small amount of extract, 1ml of water and few drops of Sodium hydroxide (NaOH) solution were added. Yellow color was observed, which confirms the presence of glycosides.
4229100-114300TEST FOR CARBOHYDRATE
2ml of extract sample was taken and 2ml conc. Sulfuric acid was added to it. Reddish yellow ring confirms the presence of carbohydrate.
TEST FOR SAPONINS ( FOAM TEST)
1ml of extract solution was taken and diluted to 20ml with distilled water. It is then shaken for 15 minutes. No layer of foam was formed which suggest that saponins is absent.

RESULT & DISCUSSION
The preliminary phytochemical screening of the plant extract was done to ascertain the presence or absence of bioactive components present in the leaves of Abroma Augusta Linn. The presence of Tannins, Flavonoids, Steroids, Alkaloids, Carbohydrates, Glycosides and Reducing sugars was confirmed whereas, Saponins were absent.
In the following table, (+) sign means the presence of chemical active groups and (-) sign means the absent of chemical groups in the methanolic extract of leaves of Abroma Augusta Linn.

CHEMICAL GROUPS RESULTS
Alkaloids +
Steroids +
Flavonoids +
Reducing sugars +
Carbohydrate +
Tannins +
Glycosides +
Saponins-
Table 1: Results of different chemical group tests performed on the methanolic leaves extract of Abroma Augusta Linn.

The preliminary phytochemical screening tests may be beneficial in the detection of the bioactive components and may lead to the drug formulation and great importance in the field of drug research or drug discovery field. These tests facilitate their qualitative separation and quantitative estimation of pharmacologically active chemical compounds 25 26. The results of the phytochemical screening of methanolic extract of A. Augusta leaves has revealed the presence of Tannins, Flavonoids, Glycosides, Carbohydrates, Reducing sugars and Alkaloids which could be responsible for the versatile medicinal properties or pharmacological actions of this plant part, like treating uterine tonic, dysmenorrhoea, amenorrhoea, strerility and other menstrual diseases, uterine disorders, wound healing, rheumatic pain of joints, headache with sinusitis and diabetes mellitus, etc. Moreover, the bioactivities that were shown by the extractives of A. Augusta Linn support the traditional uses of these plants against various diseases.

ANTIMICROBIAL ACTIVITY SCREENING
The susceptibility of various fungi and bacteria to any agent can be ascertained by antimicrobial screening which is the first stage of antimicrobial drug research. The ability of each test sample to inhibit the in vitro fungal and bacterial growth can be measured by this test. This ability may be estimated by any of the following three methods (Ayafor et al., 1972):-
Disc diffusion method
Serial dilution method
Bioautographic method
But there is no standardized method for expressing the results of antimicrobial screening (Ayafor et al., 1972). The diameter of zone of inhibition and/or the minimum weight of extract to inhibit the growth of microorganisms are used by some investigators. However, the results can be influenced by a great number of factors viz., the extraction methods, inoculums volume, culture medium composition (Bayer et al., 1966), pH, and incubation temperature, etc. Among the above mentioned techniques the disc diffusion (Bayer et al., 1966) is a widely accepted in vitro investigation for preliminary screening of test agents which may possess antimicrobial activity. We followed the disc diffusion method to determine the resistance of E. coli against the extract of the leaves of A. Augusta at North south university.1112 The sensitivity or resistance of the microorganisms to the test materials is indicated by this quantitative or qualitative test. However, this method cannot distinct between bacteriostatic and bactericidal activity (Roland, 1982).

PRINCIPLE OF DISC-DIFFUSION METHOD
In this classical method, on nutrient agar medium uniformly seeded with the test microorganisms, dried and sterilized filter paper discs (6 mm diameter) containing the test samples of known amounts are placed. Antibiotics diffuse from a confined source through the nutrient agar gel and create a concentration gradient. Standard antibiotic (Ciprofloxacin) discs and blank space (Methanol) are used as positive and negative control.
To allow maximum diffusion of the test materials to surrounding media these plates are kept at low temperature (4 °C) for 16 to 24 hours (Barry, 1976). For optimum growth of the organisms the plates are then inverted and incubated at 37 °C for 24 hours. The test materials having antimicrobial property inhibit microbial growth in the media surrounding the discs and thereby yield a clear, distinct area defined as zone of inhibition. The diameter of zone of inhibition expressed in millimeter is then measured to determine antimicrobial activity of the test agent (Barry, 1976; Bayer et al., 1966). In our study, the crude extracts were tested for antimicrobial activity by disc diffusion method. The experiment was carried out more than once and the mean of the readings was required (Bayer et al., 1966).

DESCRIPTION OF THE EXPERIMENT
Antimicrobial screening by disc diffusion method was performed under sterile condition where laminar air flow hood was used to prevent any means of bacterial contamination. The apparatus, reagents and instruments used are listed in the table below.

Filter paper disc Sterile cotton
Autoclave Petri dishes
Mueller-Hinton agar media Inoculating loop
Nutrient broth media Sterile forceps
Laminar air flow Spirit burner
Hot water bath Incubator
Micropipette Refrigerator
Screw cap test tubes Nose and hand gloves
Table 2: List of apparatus, reagents & instruments used.

Test micro-organism:
Gram negative Bacteria (Escherichia Coli) were collected from the microbiology lab of North south university for this experiment as pure culture. The organism is inoculated from a pure culture and incubated overnight prior experiment.

Preparation of the test materials:
Methanolic extract of A. Augusta Linn were collected from the refrigerator after it was stored as gummy extracts. We prepared four doses of the extracts (200,250, 400 and 500 microgram per disc). 200mg, 250, 400mg and 500mg of the extract is measured and each of them is dissolved in 1ml methanol separately to prepare four test solutions. Then 10 microlitre of the solutions is placed onto the discs.

Preparation of the medium:
Mueller-Hinton agar media and Nutrient broth were collected from the Store house of North south university. Then, 2.28g of Mueller-Hinton agar media was dissolved into 60ml of distilled water to make the volume up to 30ml for each petri dish (38g should be dissolved into 1000ml distilled water) and 0.125g of nutrient broth were dissolved into required amount of distilled water to make the volume up to 5ml for each test tube. After that, both of the solutions were placed into autoclave along with test tube, measuring cylinder, beaker, test tubes, forceps and petri dishes. All the materials are sterilized by autoclave at 1210c temperature for about 50 minutes.
60ml of Mueller-Hinton agar media solution were placed uniformly into two petri dishes (30ml each) with measuring cylinder under laminar air flow to avoid any kind of contamination. To assure homogenous distribution of the solutions, the petri dishes were rotated several times. Then, petri dishes were kept in refrigerator at 40c upside-down for about 24 hours.

20ml solutions of nutrient broth were placed uniformly into 2 test tubes (5ml each) with measuring cylinder under laminar air flow to avoid any kind of contamination. To assure homogenous distribution of the solutions, the test tubes were rotated several times. After that, bacterial samples (E.coli) were transferred into the test tubes by using inoculating loop and shake it for homogenous distribution. Finally, test tubes containing the broth solutions are placed into beaker along with some empty test tubes filled with normal water. All the test tubes were capped and placed into hot water bath in North south university at 370c temperature for continuous shaking for about 12 hours.

Finally, into the petri dish of Mueller-Hinton agar media solutions, broth solutions are dispersed and rotated several times for uniform distribution in the petri dishes.

Preparation of the disc:
Sterilized Whatman filter paper discs, 6 mm in diameter, were taken in a blank petri dish under the laminar hood. These discs are capable of absorbing 10 L. They are soaked with solutions of test samples and dried.

Standard Ciprofloxacin (10 g/disc) discs were used as positive control to ensure the activity of standard antibiotic against the test organisms as well as for comparison of the response produced by the known antimicrobial agent with that of produced by the test sample. In order to ensure that the residual solvents (left over the discs even after air-drying) and the filter paper were not active themselves, blank space (methanol) were used as negative controls.

Diffusion and Incubation:
The sample discs, the standard antibiotic discs and the control discs were placed gently on the previously marked zones in the agar plates pre-inoculated with test bacteria. The plates were then inverted and kept in an incubator at 37 0C for 24 hours to determine the zone of inhibition.
Determination of Zone of Inhibition:
If the test agents in discs possess antimicrobial potency, they would prevent the growth of the microorganisms surrounding the discs which gives clear zone of inhibition. The antimicrobial activities of the test materials were determined by measuring the diameter of the zones of inhibition in millimeter with a transparent scale after incubation.

Result and Discussion:
The methanolic extracts of the leaves of Abroma Augusta Linn were stored as gummy extracts in the refrigerator. The methanolic leaves extract of the plant was subjected to go under antimicrobial screening with a concentration of 200g/disc, 250g/disc, 400g/disc and 500g/disc.

Standard disc (Ciprofloxacin) used as positive control showed zone of inhibition (3cm and 3.5cm) in both petri dishes. However the test sample doesn’t show any zone of inhibition.

We can conclude that, the methanolic leaves extract of Abroma Augusta Linn has no antibacterial property and further deep studies are required to find out other antimicrobial properties.

ANALGESIC ACTIVITY SCREENING
Acetic-Acid Induced Writhing Test
INTRODUCTION
Pain has been officially defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage. Pain acts as a warning signal against disturbances of the body and has a proactive function 27.

Peripheral analgesic activity can be evaluated by acetic acid induced writhing method 28. In this method, acetic acid is administered intraperitoneal to the experimental animals to create pain sensation. As a result, the animals squirm their body at regular interval out of pain. This squirm or contraction of the body is termed as “writhing”. As long as the animals feel pain, they continue to give writhing. Each writhing is counted and taken as an indication of pain sensation. Any substance that has got peripheral analgesic activity is supposed to lessen the number of writhing of animals within in a given time frame and with respect to the control group. The writhing inhibition of positive control is taken as standard and compared with test samples. As positive control, any standard NSAID drug can be used. In this study, diclofenac Na was used as a standard drug 29. According to this principle the crude methanolic extract of leaves of A. Augusta Linn was subjected to analgesic testing at two different doses (Dose: 400 mg/kg and 200 mg/kg of body weight).

43243506943090EXPERIMENTAL ANIMAL
Swiss albino mice of male sex, aged 8-10 weeks, obtained from the animal house of North South University were used for the experiment. They were housed in standard polypropylene cages and kept under controlled room temperature (24 ± 2º C; relative humidity 60-70 %) in a 12 h light-dark cycle and fed formulated rodent food and normal water. As these animals were very sensitive to environmental changes, they were kept before the test for at least 3-4 days in the environment where the experiment took place.

EXPERIMENTAL DESIGN
Ten experimental animals were randomly selected and divided into five groups denoted as group-I, group-II, group- III, group-IV A and group-IV B, consisting of two mice in each group. Each group received a particular treatment. Prior to any treatment, each mouse was weighed properly and the doses of the test samples and control materials were adjusted accordingly. As it was difficult to observe the biologic response of three mice at a time receiving same treatment, it was necessary to identify individual animal of a group during the treatment. The animals were individualized and marked.

PREPARATION OF TEST MATERIALS
In order to administer the extract at doses of 400 mg/kg of body weight and 200 mg/kg of body weight of mice, the exactly weighed extracts were measured respectively according to the weight of mice. Normal saline (0.9% Nacl) was administered through the route of IP into the control group also termed as negative control (Group-I). Disease Group (Group-II) was administered 1% acetic acid in the Intra-peritoneal route at the concentration of 10mg/kg of body weight of mice. For the preparation of standard also termed as positive control (Diclofenac), 5 mg of diclofenac was taken and a suspension of dose using distilled water and raw diclofenac sodium powder of 5 mg/kg of body weight was made (Group-III). Two different doses of extract 200mg/kg and 400mg/kg were administered into the mice based on their body weight (Group-IV A and Group-IV B). If the sample possesses analgesic activity, the animal that received the sample, will give lower number of writhing than the control, i.e. the sample having analgesic activity will inhibit writhing 30.

PROCEDURE
2581275546735At zero hour test samples, control and diclofenac sodium were administered orally by means of a long needle with a ball-shaped end.

26479507499351ml of 1 % acetic acid (1 ml acetic acid diluted to 100 ml distilled water) was injected after 30 minutes of administration of diclofenac Na and test samples to each of the animals of all the groups.

Responses were measured for next 10 minutes after the first 5 minutes of acetic acid injection.

44862757715250
COUNT OF WRITHING
Each mouse of all groups were observed individually for counting the number of writhing they made in 10 minutes commencing just 5 minutes after the intraperitoneal administration of acetic acid solution. Full writhing was not always accomplished by the animal, because sometimes the animals started to give writhing but they did not complete it. This incomplete writhing was considered as half writhing.
2716530286385Acetic acid (1 %) I.P
2714625236220Inflammation in situ
2714625242570Localized biogenesis of prostaglandin
2714625300355Prostacyclin (PGI2) and (PGE2)
2714625253365Stimulation unmyelinated C fibers and myelinated A-delta fiber
2714625295275Release of substance P and other nociceptive neurokinins2710815248920Nociception or pain
2781300258445Biological response
Writhing
Figure 1: Mechanism of action of pain induction and writhing.

RESULTS AND DISCUSSION
The different test samples were subjected to screening for peripheral analgesic activity by acetic acid test. The test was performed by taking samples at doses 400 and 200 mg/kg of body weight. The result was statistically evaluated % writhing and % inhibition values were determined. Both the test materials exhibited statistically significant peripheral analgesic activity but none showed as strong effect as the standard drug. However, we recommend further higher studies to investigate the plant’s analgesic properties.

Animal Group Writhing count Average of writhing (Mean) % Writhing % Inhibition
M1 M2 Control (0.9% Nacl) 0 0 0 0 0
Disease – 1% acetic acid, 10mg/kg
41 45 43 100 0
Disease + control (Std- diclofenac sodium)
19 16 17.5 40.7 59.3
Disease + control (extract- 200mg/kg)
25 27 28 65.1 34.8
Disease + control (extract- 400mg/kg) 23 19 21 48.8 51.1
Table 3: Peripheral analgesic activity of methanolic crude extract of A.augusta (Linn.) leaves.

% OF WRITHING
SAMPLES

Figure 2: Comparison of % of writhing among different samples

Tail Immersion Test
INTRODUCTION
Analgesics are agents which selectively relieve pain by acting in the CNS and peripheral pain mediators without changing consciousness. Analgesics may be narcotic or non-narcotic. Analgesics drugs are used to treat or reduce pain and the classical analgesic drugs notably opiates and non-steroidal anti-inflammatory drugs have their origin in natural products but many synthetic compounds that act by the same mechanism have been developed and are associated with serious adverse effects such as ulceration, gastrointestinal bleeding, additive potential, respiratory distress, drowsiness, nausea etc . Based on these therefore, there is the need for the search for bioactive compounds from natural products especially from medicinal plants for use as alternative analgesics with little or no side effects. Tail emersion method has been developed to be selective for morphine-like compounds. The procedure is based on the observation that morphine-like drugs are selectively capable of prolonging the reaction time of the typical tail-withdrawal reflex in rats induced by immersing the end of the tail in warm water of 55°C.

EXPERIMENTAL ANIMAL
34290005943600Swiss albino mice of male sex, aged 8-10 weeks, obtained from the animal house of North South University were used for the experiment. They were housed in standard polypropylene cages and kept under controlled room temperature (24 ± 2º C; relative humidity 60-70 %) in a 12 h light-dark cycle and fed formulated rodent food and normal water. As these animals were very sensitive to environmental changes, they were kept before the test for at least 3-4 days in the environment where the experiment took place.

EXPERIMENTAL DESIGN
Ten experimental animals were randomly selected and divided into five groups denoted as group-I, group-II, group- III, group-IV A and group-IV B, consisting of two mice in each group. Each group received a particular treatment. Prior to any treatment, each mouse was weighed properly and the doses of the test samples and control materials were adjusted accordingly. As it was difficult to observe the biologic response of three mice at a time receiving same treatment, it was necessary to identify individual animal of a group during the treatment. The animals were individualized and marked.

PREPARATION OF TEST MATERIALS
In order to administer the extract at doses of 400 mg/kg of body weight and 200 mg/kg of body weight of mice, the exactly weighed extracts were measured respectively according to the weight of mice. Normal distilled water 0.1ml was given to control group (Group-1). For the preparation of standard also termed as positive control (Diclofenac), 5 mg of diclofenac was taken and a suspension of dose using distilled water and raw diclofenac sodium powder of 5 mg/kg of body weight was made and administered to standard group (Group-2). Two different doses of extract 200mg/kg and 400mg/kg were administered into the mice based on their body weight (Group- 3 and Group-4).

PROCEDURE
Young male swiss albino mice (30-45 g body weight) are used. They are placed into individual restraining cages leaving the tail hanging out freely. The animals are allowed to adapt to the cages for 30 min before testing. The lower 2 cm portion of the tail is marked and this part of the tail is immersed in a cup of freshly filled water of exactly 55°C. Within a few seconds the mice reacts by withdrawing the tail. The reaction time was the time taken by the mice to deflect their tails. The latent period of the tail-flick response was taken as the index of antinociception and was determined at 0, 30, 60, 90 and 120 min after the administration of all the samples and standard drug 33. If the plant has analgesic activity then reflex time will increase after increasing the dose.

COUNT OF TAIL WITHDRAW-REFLEX TIME
Group-1 was treated with water, Group-2 was treated with standard drug (Diclofenac), Group-3 and Group-4 was treated with the plant extract at concentration of 200mg/kg and 400mg/kg of their body weight. When, they immersed in hot water bath they withdraw their tail after few seconds. Time taken to withdraw the tail is listed in the table.
Weight of mice Dose 0min 30min 60min 90min 120min
36.7g 0.1ml of water 2.10s 2.09s 2.05s 2.05s 2.01s
36.6g 0.1ml of water 1.08s 1.08s 1.10s 1.05s 1.04s
42.3g 0.1ml of water 2.05s 2.03s 2.01s 2.02s 2.02s
43.7g 0.1ml of water 2.09s 2.07s 2.09s 2.05s 2.04s
Table 4: Group 1 (Control – Water)
Weight of mice Dose (5mg/kg) 0min 30min 60min 90min 120min
36.9g 0.74ml 2.03s 5.10s 5.25s 5.30s 5.28s
37.0g 0.74ml 2.98s 5.58s 6.03s 6.05s 5.98s
43.2g 0.86ml 2.80s 5.76s 5.97s 6.02s 6.08s
38.4g 0.77ml 2.46s 5.21s 6.03s 6.03s 5.98s
Table 5: Group- 2 (Diclofenac Sodium)
Weight of mice Dose (200mg/kg) 0min 30min 60min 90min 120min
35.2g 0.7ml 2.15s 3.01s 3.93s 3.90s 3.81s
36.5g 0.73ml 1.91s 3.12s 3.83s 3.56s 3.45s
42.4g 0.85ml 1.95s 3.96s 4.10s 3.95s 3.94s
39.3g 0.79ml 2.34s 3.98s 4.05s 3.98s 3.95s
Table 6: Group-3 (Extract 200g)
Weight of mice Dose (400mg/kg) 0min 30min 60min 90min 120min
35.4g 0.71ml 1.97s 3.86s 4.05s 4.15s 4.13s
41.3g 0.83ml 2.06s 4.09s 4.04s 4.35s 4.30s
37.8g 0.76ml 2.12s 3.23s 3.96s 4.14s 4.15s
35.0g 0.7ml 1.93s 3.99s 4.20s 4.18s 4.17s
Table 7: Group-4 (Extract 400g)
RESULTS AND DISCUSSION
The different test samples were subjected to screening for analgesic activity by tail-emersion test. The test was performed by taking samples at doses 400 and 200 mg/kg of body weight. The tail-withdrawal reflex time of the mice to the hot water induced pain significantly increased after administration of methanolic extract of A. Augusta Linn (Table 8). The maximum effect of the extract was recorded at 60 and 90 mins. The response time of tail withdrawal of our plant extract at both doses seems closer to standard drug.

Group Dose Mean reaction time (s)
0min 30min 60min 90min 120min
Control 0.1ml water 1.82 1.81 1.81 1.79 1.77
Diclofenac Na 5mg/kg 2.56 5.41 5.82 5.85 5.83
Ext-200mg 200mg/kg 2.08 3.51 3.97 3.84 3.78
Ext- 400mg 400mg/kg 2.02 3.79 4.06 4.20 4.18
Table 8: Effect of A. Augusta on tail withdrawal reflex in mice
Reaction Time (s)
Time Interval (Min)

Figure 3: Comparison of reaction time of different samples

CONCLUSION
From the thousands of years, nature is giving us medicinal gift which act as natural source of modern drugs. Herbal plants and medicinal plants have been associated with the human health care system from time immemorial, particularly among tribal 34. Medicinal plants and herbal plants have assumed massive importance in present days due to the tremendous developments in the field of allopathic medicines during 20th century 35. Herbal plants plays important role in healthcare system of a large number of world’s population, plants all over the world which are being used traditionally in the prevention and treatment of many diseases and illness. One of the gift is Abroma augusta contains several pharmacological activity. To examine the pharmacological activity, a number of experiments were done. Based on the result of this present study, it can be concluded that the crude plant extracts of A. augusta possesses remarkable analgesic potential. Various phytochemical constituents like glycoside, alkaloids, tannins, reducing sugars, carbohydrates and steroids are present in this plant, as evident from phytochemical analysis. The plant extract showed moderate analgesic properties compared to respective standard drugs in both tail-emersion and acetic acid induced writhing test. At higher dose, notable analgesic activity was observed from acetic acid induced writhing test and tail immersion test. So it is clear that our experimental plant A. augusta Linn. on which we worked is a helpful plant and our work was only preliminary effort which will require further comprehensive exploration as well as depiction of active compounds and necessitates preformulation studies for the expansion of a potential dosage form. We also suggest finding out other properties this plant possesses like anti-diabetic and against dysmenorrhoea etc. We can conclude with that the leaves of A. augusta Linn might not have antimicrobial property but posseses potent analgesic properties as observed in both tail-emersion and acetic acid writhing test.