CHAPTER III

METHODOLOGY

This chapter consists and discusses the different methods that

will be followed and employed to come up with the desired results. This

will also include the preparation of materials and subjects, research

design, data gathering tools and statistical treatment of data to be used.

Research Design

The researchers will be utilizing the randomized post-test only

control group experimental method of research in evaluating the

antinociceptive and anti-inflammatory activities of Paragis (Eleusine

indica).

Young Swiss mice aged about 4-5 weeks with average weight of

25-35 grams and adult Albino rats of either sex having average weight

100-130 grams will be used in assessing the antinociceptive and anti-

inflammatory activities of Paragis (Eleusine indica) ethanolic leaf extract.

The methods that will be used for the pharmacological assay of

Paragis will be employed to evaluate the antinociceptive and anti-

inflammatory activities. Hot plate test and acetic acid-induced writhing

method in mice will be used for the antinociceptive. Anti-inflammatory

activity will be tested using carrageenan-induced paw edema method in

Albino rats.

The extract that will be obtained after rotary evaporation will be

subjected to different test which include phytochemical screening using

the standard procedure based on the Guidebook to Plant Screening:

Phytochemical and Biological (Guevara, 2005) to determine the

presence of alkaloids, flavonoids, anthraquinones, saponins,

carbohydrates, tannins, cardiac glycosides, fixed oils, and volatile oils.

Locale of the Study

The extraction of the active constituents present in Paragis

leaves, phytochemical screening of the extract and pharmacological

assay will be conducted at the laboratory of Pharmacy Department,

College of Health Sciences, Mariano Marcos State University, Batac

City.

Data Gathering Procedures

Collection, Preparation and Authentication of Plant Sample

Fresh Paragis (Eleusine Indica) leaves will be collected from

Brgy. 15, Valdez, Banna, Ilocos Norte. Authentication of the plant

samples will be carried out at the National Museum where the specimens

will be deposited. After which, the plant samples will be washed with

clean water to wipe off dirt and contaminants. The plant specimens will

be air dried at room temperature to make sure that moisture is properly

evaporated off and confined the important constituents of the plant

samples.
Extraction of Plant Material

Maceration

The fresh leaves (2 kg) of the plant will be dried on laboratory

table for 2 weeks and will be reduced to powder. The powder that will be

obtained will be macerated in 95% ethanol (300ml) for 72 hours.

Rotary evaporation

The liquid filtrate obtained will be concentrated in vacuum at

40°C. The percentage yield was 4.83% w/w. The extract will be stored

in a refrigerator at 4°C using rotary evaporator until used for experiment.

The final weight and volume of the extract will be recorded and

calculated.

Selection, Care and Use of Animals

Young Swiss mice aged about 4-5 weeks with average weight of

25-35 grams and adult Albino rats of either sex having average weight

100-130 grams will be used in the study of antinociceptive and anti-

inflammatory effect. The animals will be housed in separate cages

depending on the group they will belong, under environment conditions

of room temperature at 24±10C and 55-65% relative humidity with 12

hour dark light cycle. They will be supplied with adequate food and water

and will be acclimatized for one week for them to be comfortable during

the evaluation of antinociceptive and anti-inflammatory activities of

Eleusine indica leaf extract.

Phytochemical Screening

Phytochemical Screening of the plant extracts will be carried out

as per the methods described by Guevarra (2005) (as cited by Amoroso,

et. al, 2014).

A. Test for Alkaloids

An equivalent of two (2) ml of Paragis Leaf Extract will be placed

in an evaporating dish and will be evaporated to a syrupy consistency

over a steam bath. Five (5) ml of 2M HCl will be added to the

concentrated extract. It will be heated with constant stirring for about five

(5) minutes and then cooled at room temperature. Five (5) drops of 10%

NaCl solution will be added to it while heated for another two (2) minutes.

Enough 2M HCl that will be freshly prepared will be added to

wash. The solution will be filtered and the filtrate will be bring to a final

volume of five (5) ml. To one (1) ml of the filtrate, a few drops of Mayer’s

reagent will be. The formation of white precipitate will indicate the

presence of alkaloids. To another one (1) ml of the filtrate, few drops of

Dragendorff’s reagent will be added and formation of orange precipitate

will indicate the presence of alkaloids.

B. Test for Saponin Glycosides

 Froth Test

A volume of the Paragis Leaf Exract equivalent to two (2) milliliters

will be taken. For control, two (2) ml of 10% “gogo” extract (1 gram of

gogo bark with 10 ml of ethyl alcohol) will be used in separate test tubes.
Ten (10) ml of distilled water will be to each test tube and shaken for 30

(thirty) seconds. It will be allowed to stand and then observed in 10 (ten)

minutes. The presence of honeycomb froth greater than two (2) cm in

height from the surface of the liquid will yield a positive result.

C. Test for Steroids

 Leibermann-Burchard Test

An equivalent of ten (10) grams of the Paragis Leaf Extract will be

evaporated using a steam bath. It will be cooled to room temperature.

Ten (10) ml of hexane will be added to the cool residue will be stirred for

a few minutes. It will be allowed to settle and decant off supernatant

liquid. The procedure will be repeated until the hexane extract that will

be removed most of the color then hexane extract will be discarded. Ten

(10) ml of chloroform will be added to the residue and stirred for minutes.

It will be decanted into a test tube containing about 100 mg of anhydrous

sodium sulphate. It will be shaken and passed through a filter paper. The

filtrate will be divided into two (2) clean and dry test tubes. The one

portion will be as a reference. Three (3) drops of acetic anhydride will be

added to the other portion and mixed gently. One (1) drop of sulphuric

acid will be added and then mixed gently and will be observed for any

color changes.

D. Test for Flavonoids

After ten (10) grams of the plant extract will be evaporated to

incipient dryness cooled at room temperature, it will be defatted with nine

(9) ml hexane and water at 2:1 ratio, the defatted residue will be diluted
to a ten (10) ml of 80% ethyl alcohol. The mixture will be filtered and the

filtrate will be treated with 0.5 ml of 12M HCl. The mixture will be warmed

for fifteen (15) minutes in a water bath and color change will be

observed. An expected result of a strong violet or red color will indicate

the presence of leucoanthocyanins.

E. Test for Tannins and Polyphenolic Compounds

The plant extract equivalent to ten (10) grams of plant extract will

be taken and evaporated to incipient dryness over a water bath then

cooled. The residue will be extracted with twenty (20) ml of hot distilled

water then cooled. Five (5) drops of 10% of NaCl solution will be added

to salt out undesirable constituents then filtered. The filtrate will be

divided into 3 test tubes labeled A, B, C. Test tube A will be reserved as

blank.

 Ferric Chloride Test

Three (3) drops of FeCl3 solution will be added to test tube C. Any

color changes will be observed. A blue-black color will indicate the

presence of hydrolysable tannins, while a brownish-green color will

indicate condensed tannins if the gelatin test is positive. Polyphenolic

compounds will give a negative gelatin test.

F. Test for Anthraquinones

 Borntrager’s Test

An equivalent of one (1) gram of the plant extract will be

processed into incipient dryness using a water bath. The residue will be
taken up in ten (10) of distilled water, and then it will be filtered, the filtrate

will be extracted with five (5) ml of benzene twice. The combined

benzene extracts will be divided into two (2) portions, one portion

reserved as blank and the second portion will be added to five (5) ml of

ammonia solution. Any alkaline layer and formation of red color will

indicate the presence of anthraquinone compounds.

G. Test for Carbohydrates

 Molisch’s Test

An equivalent of two (2) ml of the filtrate will be treated with two

(2) drops of alcoholic a-naphthol solution in a test tube. Preparation of

Molisch’s test: weighed 15 grams of a-naphthol in 100 ml of alcohol or

chloroform. The formation of a violet ring at the junction of the test tube

will indicate the presence of carbohydrates.

H. Test for Proteins

An equivalent of two (2) grams of the plant material will be

evaporated over a water bath. The residue will be taken with ten (10) ml

of water.

 Xanthoproteic Test

One (1) ml of the ethanolic extract and ten (10) drops of HNO3

will be added and will be observed for the formation of white precipitate.
  Millon’s Test

One (1) ml of the ethanolic extract and ten (10) drops of Millon’s

reagent will be added. It will be placed in a boiling water bath and the

color will be observed.

I. Test for Fixed Oils, Fats and Volatile Oils

Equivalent of two (2) grams of the plant extract and ten (10) ml of

petroleum ether or hexane will be boiled. A piece of white paper will be

taken and will be placed on its surface two (2) drops of the petroleum

ether extract. Any stain that will be produced will indicate the presence

of fixed oils (Guevarra 2005).

Pharmacological Assay

Antinociceptive Activity

The antinociceptive activity of the ethanolic extract from the

leaves of Paragis (Eleusine indica) will be investigated using the

following models: (1) Acetic acid-induced writhing based on chemical

induced algesia and (2) Hot plate method based on pain sensation at

55 °C.

Acetic acid-induced writhing

The antinociceptive activity of the plant samples will be evaluated

using acetic acid-induced writhing method following the method of

Koster et. al, 1959 and Vacher et al, 1964 (as cited in Florentino et al,

2013). In this method, acetic acid will be administered intraperitoneally

to the experimental animals to create pain sensation. Forty five Swiss

mice of both sexes will be randomly divided into five groups (A-E) of nine

mice per group. They will be fasted for 12 hours and later will be treated

as follows: Group A animals will be receiving distilled water (negative

control group), Group B will be treated with Indomethacin® at 10 mg/kg

dose (positive control group), while animals of Group C-E will be treated

with 250, 500, and 1000 mg/kg of Paragis (Eleusin indica) ethanolic leaf

extract respectively after an overnight fast. The test samples and vehicle

will be administered orally 30 minutes prior to intraperitoneally

administration of 1.2% acetic acid solution (10mL/kg). The number of

writhing that will be produced in each group for the following 30 min will

be counted and the results will be expressed as mean ± standard error

of mean (SEM) in percentage of control group.

Hot plate test

The plant samples will be evaluated using the hot plate test

following the method of Woolfe and MacDonald et. al, 1994. The hot

plate method will test the latency (in seconds) to reaction of the mice,

expressed as licking, shaking or lifting the hind paws, on hot plate at 55.5

+ 0.5 °C. The hot plate assay method will be employed for the purpose

of preferential assessment of possible centrally mediated antinociceptive

effects of Paragis (Eleusine indica) ethanolic leaf extract. Morphine® will

be used as the standard drug. In this experiment, forty five Swiss mice

of both sexes will be randomly divided into five groups (A-E) of five mice

per group. The animals will be placed on a hot plate maintained at room

temperature for 15 minutes. Food will be withdrawn on the preceding

night of the experiment. Group A will be receiving distilled water orally

(normal control group), Group B will be treated orally with the standard

drug, Morphine®, 10 mg/kg subcutaneously (positive control group),

Group C-E will be receiving ethanolic extract of Eleusine indica (250, 500

and 1000 mg/kg p.o respectively). Each animal will be then individually

placed gently on hot plate at 550C. The latency to pain reaction will be

measured at -60, -30, 0, 30, 60, 90, and 120 min of the treatment (as

cited by Florentino et al, 2013).

Anti-inflammatory Activity

The anti-inflammatory activity of the ethanolic extract of Paragis

(Eleusine indica) will be investigated using the following model.

Carrageenan-induced paw edema

Carrageenan-induced paw edema will be followed as described

by Passos et. al, 2007 (as cited by Florentino et. al, 2013). Forty-five

Albino rats of both sexes will be randomly divided into five groups (A-E)

of five rats per group. 0.9 gm/100 ml of sodium chloride will be prepared.

At zero hour; distilled water (Group A), Indomethacin® at 10ml/kg

(Group B) and the test samples (Group C, D, E) will be administered

orally by a feeding needle. One hour after administration of these agents,

edema will be induced by injection of 0.1 ml carrageenan (1% w/v in

saline) into the sub-plantar region of the right hind paw. Then, the edema

will be measured by the difference in the volume between the paws using

a plethysmometer (Ugo Bsile Co. – Italy) at several time-points (1, 2, 3

and 4 hr) after injection of phlogostic agent. The inhibitions of edema

formation among the five groups will be compared. Percentages of

inhibition will be obtained using the following ratio:

[(Vt-Vo) control - (Vt-Vo) treated / (Vt-Vo) control] X 100

Vt is the average volume for each group after treatment, and

Vo is the average volume for each group before any treatment.

 Collection of Paragis
(Eleusine indica) leaves

Extraction by Maceration

Rotary Evaporation for

Concentration

Phytochemical Screening Pharmacological Assay

*AntinociceptiveActivity
* Alkaloids
*Anti-inflammatory Activity
*Flavonnoids
* Tannins
*Anthraquinones
*Steroids
*Cardenolides and Bufadienolides
*Carbohydrates
*Proteins
*Saponins
*Fats, Fixed Oils, and Volatile Oils

Flowchart of Methodology
 Statistical Analysis

The data that will be obtained for the antinociceptive and anti-

inflammatory activities will be analysed using one way analysis of

variance (ANOVA) followed by Student’s t test when there will be

differences between two means. Differences between more than two

means will be detected using one-ways analysis of variance (ANOVA)

followed by Student-Newman-Keul’s test. Results were expressed as

means ± S.E.M. The results will be considered significant when p<0.05

(Sokal and Rohlf, 1981).