METHODOLOGY - AXIOLYTIC PROPERTY OF CAESALPINIA PULCHERRIMA LEAVES EXTRACTS IN EXPERIMENTAL ANIMALS
methodology
4.1. Materials:
4.1.1. Plant material:
The Caesalpinia pulcherrima leaves are found throughout
4.1.2. Drug:
Diazepam
I.P- Reliance Formulation Private Limited, Chaekosons Chemic,
4.1.3. Reagents:
1. Benedict's reagent.
2. Barfoed'ss reagent.
3. Millon’s reagent.
4. Dragendroff's reagent.
5. Hager’s reagent.
6. Mayer's reagent.
7. Wagner's reagent.
4.1.4. Chemicals:
All chemicals used were of analytical grade.
1. Petroleum ether-SDFCL.Fine-chem.Ltd; Mumbai.
2. Chloroform- SDFCL.Fine-chem.Ltd; Mumbai.
3.
Ethanol - Ghangshu Yangyuan
chemical;
4. Tween 80 - SDFCL.Fine-chem.Ltd; Mumbai.
5. Gum Acacia
4.1.5. Instrument:
- Elevated plus maze apparatus- INCO, Medicraft.
- Open field test apparatus.
- Light/dark test box.
- Hole board test apparatus
- Soxhlet apparatus-
- Double distillatory- Infustil India Pvt. Ltd.
- Electronic weighing balance- Citizen
- Oral feeding needle- Space Lab,
4.1.6. Animals:
Albino Wistar rats of either sex weighing between 150 to 200gm and Albino mice of either sex weighing between 20 to 30gms were procured from registered breeders (146/999 CPCSEA, Mahavir Enterprises, Hyderabad). The animals were housed under standard conditions of temperature (25± 2°C) and relative humididty (30-70%) with a 12:12 light-dark cycle. All the animals were acclimatized for seven days before the study. The animals were housed individually in sanitized polypropylene cages containing sterile paddy husk as bedding. They had free access to standard pellets as basal diet V.R.K Nutritional solutions, Pune and water ad libitum.
Animals were habituated to laboratory conditions for 48 hours prior to experimental protocol to minimize if any of non-specific stress. All the studies conducted were approved by the Institutional Animal Ethical Committee (IAEC) (REG.No. 346/CPCSEA; Annexure-III) according to prescribed guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.
4.2 Methodology:
4.2.1. Extraction: 119, 120, 121
The authenticated Caesalpinia pulcherrima leaves were collected from the surroundings
of Luqman College of Pharmacy. The leaves were shade dried at room temperature
and pulverized. The powder obtained was
subjected to successive soxhlet extraction with analytical grade solvents with
increasing order of polarity i.e Pet. Ether (60–80°), Chloroform (59.5–61.5°), Ethanol (64.5-65.5°) and Distilled water. In each case, the powder weighing approximately 200 gm was
extracted by adding 2000 ml of the solvents. The duration of extraction varies
with the solvent and was found to be between 08-12 h with all the solvents.
The extracts were concentrated under
reduced pressure and stored in a desicator until further use. The yields are
tabulated in Table 1.
4.2.2. Qualitative chemical test:
122, 123
Preliminary phytochemical
investigation of extract:
Qualitative
chemical tests were conducted for petroleum ether, chloroform, ethanolic and
aqueous extracts of leaves of Caesalpinia pulcherrima to identify the various
phytoconstituents. The various tests and reagents used are given below and
observations are recorded and tabulated in results (Table 2).
Tests for Carbohydrates:
Molisch's test (General test):
To
2-3 ml aqueous extract, few drops of α-naphthol solution in alcohol was added,
shaken and concentrated H2SO4 was added from the sides of the test tube. It was
observed for violet ring at the junction of two liquids.
For Reducing Sugars:
a) Fehling's
test:
1 ml Fehling's A and 1ml Fehling's B solutions was mixed and boiled for one min. Equal volume of test solution was added. Heated in boiling water bath for 5-10 min and observed for a yellow, then brick red precipitate.
b) Benedict's test:
Equal volume of Benedict's reagent and test solution in test tube were mixed. Heated in boiling water bath for 5 min. Solution may appear green, yellow or red depending on amount of reducing sugar present in test solution.
Test for Monosaccharide’s:
Barfoed's test:
Equal volumes of Barfoed's reagent and test solution were added. Heated for 1-2 min, in boiling water bath and cooled. Observe for red precipitate.
Test for Hexose Sugars:
Cobalt-chloride test: 3 ml of test solution was mixed with 2ml cobalt chloride, boiled and cooled, added few drops of FeCl3 and NaOH solution. Solution was observed for greenish blue (glucose), purplish (Fructose) or upper layer greenish blue and lower layer purplish (Mixture of glucose and fructose).
Tests for Non-Reducing Sugars:
a) Test solution does not give response to Fehling's and Benedict's test.
b) Tannic acid test for starch: With 20% tannic acid, test solution was observed for precipitate.
Tests for Proteins:
a) Biuret
test:
(General test): To 3 ml test solution added 4% NaOH and few drops of 1% CuSO4 solution and observed for violet or pink color.
b)
Millon's test: (for proteins):
Mixed 3 ml test solution with 5 ml Millon’s reagent, whiteprecipitate obtained. Precipitate warmed turns brick red or precipitate dissolvesgiving red color.
c)
Xanthoprotein test: (For protein containing
tyrosine or tryptophan):
Mixed 3ml test solution with 1 ml concentrated H2SO4, observed for white precipitate.
d) Test
for protein containing sulphur:
Mixed 5
ml test solution with 2 ml 40% NaOH and 2 drops 10% lead acetate solution.
Solution was boiled, turns black or brownish due to lead sulfide formation.
Tests for Steroids:
a) Salkowski Reaction:
To 2 ml of extract, 2 ml chloroform and 2 ml concentrated H2SO4 was added. Shook well, whether chloroform layer appeared red and acid layer showed greenish yellow fluorescence was observed.
b) Liebermann-Burchard Reaction:
Mixed 2ml extract with chloroform. Added 1-2ml acetic anhydride and 2 drops concentrated H2SO4 from the side of test tube, observed for first red, then blue and finally green color.
c) Liebermann’s reaction:
Mixed 3 ml extract with 3 ml acetic anhydride. Heated and cooled. Added few drops concentrated H2SO4, observed for blue color.
Tests for Amino Acids:
a) Ninhydrin test (General test):
3 ml test solution and 3 drops 5% Ninhydrin solution were heated in boiling water bath for 10 min and observed for purple or bluish color.
b) Test for Tyrosine:
Heat 3 ml test solution and 3 drops Millon’s reagent. Solution was observed for dark red color.
c) Test for Tryptophan:
To 3 ml test solution added few drops glycoxalic acid and concentrated H2SO4 observed for reddish violet ring at junction of the two layers.
Tests for Flavonoids:
a) Shinoda test: To dried powder or extract, added 5 ml 95% ethanol, few drops concentrated HCl and 0.5 g magnesium turnings. Pink color was observed.
b) To small quantity of residue, added lead acetate solution observed for Yellow colored precipitate.
c) Addition of increasing amount of sodium hydroxide to the residue was observed as to whether it showed yellow coloration, which was decolorized after addition of acid.
d) Ferric chloride test: To test solution, added few drops of ferric chloride solution observed for intense green color.
Tests for Alkaloids:
a) Dragendroff's test:
To 2-3 ml filtrate added few drops Dragendroff's reagent and was observed for orange brown precipitate.
b) Mayer's test:
2-3 ml filtrate with few drops Mayer's reagent was observed for precipitate.
d) Wagner's test:
2-3 ml filtrate with few drops of Wagner's reagent was observed for reddish brown precipitate.
Tests for Tannins and Phenolic
Compounds:
To 2-3 ml test solution, added few drops of following solutions and was looked for respective coloration or precipitate:
- 5% Ferric chloride solution: - Deep blue-black colored.
- Lead acetate solution: - White precipitate.
- Bromine water: - Decoloration of bromine water.
- Acetic acid solution: - Red color solution.
- Potassium dichromate: - Red precipitate.
- Dilute iodine solution: - Transient red color.
- Dilute Nitric acid: - Reddish to yellow color.
Tests for Glycosides:
General test for Glycosides:
Part A:
To 2-3 ml of extract dil. H2SO4 was added and heated on a water bath for 1- 2 min. Neutralize with 10% NaOH, check with litmus paper and to resulting solution add Fehling's A and B. Increased red precipitate in this case shows glycosides are present.
Part B:
To 2-3 ml of extract, water was added and heated. According to need, NaOH was added for neutralization and also added equal quantity of water. To the resulting solution added Fehling's A and B. Increased red precipitate in this case showed glycosides are absent. Compare Part A and B.
Tests for Cardiac Glycosides:
a) Baljet's test:
The
test solution was observed for yellow to orange color withsodium picrate.
b) Legal's test (For cardenoloids):
To aqueous or alcoholic test solution, added 1 ml pyridine and 1 ml sodium nitroprusside, observed for pink to red color.
c) Test for deoxysugars (Keller
Killani test):
To 2 ml extract added glacial acetic acid, one drop of 5% FeCl3 and concentrated H2SO4, observed for reddish brown color at junction of the two liquid and upper layers bluish green.
d) Liebermann’s test (For bufadenolids):
Mixed 3 ml extract with 3 ml acetic anhydride. Heated and cooled. Added few drops concentrated H2SO4 observed for blue color.
Tests for Saponin Glycosides:
a) Foam test:
The drug extract or dry powder was shaken vigorously with water. Persistent foam was observed.
Test for Monoterpenoids: 124
Lieberman-Burchard test:
Dissolve a few mg of the residue in about 1 ml of chloroform, add a few drops acetic anhydride. Add concentrated sulphuric acid by the side of the test tube. Production of purple coloration indicates presence of triterpenoids and a blue-green colour indicates presence of sterols.
4.3 Methods:
4.3.1. Acute oral toxicity study: 125
Acute
oral toxicity study for the formulation was carried out using OECD guideline
425 (Adopted 3rd October 2008). The test procedure minimizes the
number of animals required to estimate the oral acute toxicity of a chemical
and in addition estimation of LD50, confidence intervals. The test also allows
the observation of signs of toxicity and can also be used to identify chemicals
that are likely to have low toxicity.
Principle of the FDP:
The fixed dose procedure is method for assessing acute oral toxicity that involve the identification of a dose level that cause evidence of non-lethal toxicity (termed evident toxicity) rather than a dose level that cause lethality. Evident toxicity is terms describing clear signs of toxicity following administration of test substance, such that an increase to the next highest fixed dose would result in the development of sever toxic signs and probably mortality.
Procedure:
As suggested, after acclimatization of animals for 4-5 days, study was carried out as follows:
- Healthy, young adult Albino Swiss female mice (20-30gm), nulliporous and non pregnant were used for this study Food, but not water was with held for 3-4 hours and further 1-2 hours post administration of sample under study.
- Fixed dose level of 5, 50, 500 mg/kg were initially chosen as dose level that would be expected to allow the identification of dose producing evident toxicity.
- During the validation procedure, a fixed dose of 2000mg/kg was added to provide more information on substance of low acute toxicity.
- Dosed one animal at the test dose by oral route.
- Since, this first test animal survived, four other animals were dosed (orally) as subsequent days, so that a total of five animals were tested.
Observation:
After the administration of pet.ether and ethanolic extracts, animals were observed individually during the first 30 min and periodically during 24 hours with special attention during the first four hours and daily thereafter for a period of 14 days. After 21 days study it was concluded that both the extracts were safe upto 2000 mg/kg body weight with fewer side effects like grooming and hair loss and no adverse effects.
4.3.2. Elevated Plus-Maze Test in mice: 98,126,127,128
Elevated
plus maze (EPM) one of the commonly used animal model (exteroceptive aversion
stimulus model) for testing anti-anxiety drugs was employed to assess
anxiolytic activity of sample under study. EPM is based on the apparent natural
aversion of rodents to open and high spaces animals have tendency to spend more
time in enclosed arms than in the open arms.
Grouping:
Six groups of six mice (20-30 gm) in each were used in this
model.
Group I : Animals (-veControl) were administered with 3% Tween 80.
Group II : Animals were administered with Diazepam 2mg/kg; p.o suspended in 2% gum acacia
Group III : Animals were administered with Pet. ether extract of CP in 3% tween 80 at 400 mg/kg; p.o
Group IV : Animals were administered with Pet. ether extract of CP in 3% tween 80 at 800 mg/kg; p.o in 3%
Group V : Animals were administered with ethanolic extract of CP at
400 mg/kg; p.o
Group VI : Animals were administered with ethanolic extract of CP at
800 mg/kg; p.o
Procedure:
Albino mice of either sex weighing between 20-30 gm were dividing into four experimental groups of six animals each.
Standard drug (Diazepam) was administered 45 min prior to testing and extracts were administered p.o 45 min prior to testing. Anxiolytic activity was measured using the elevated plus maze test. The maze consisted of two open (28 cm x 5 cm) and two closed (28 cm x 5 cm x 14 cm) arms, extending from the central platform (5 cm x 5 cm) and elevated up to the height of 25 cm above the floor. The entire maze was made of clear Plexiglass. Mice were individually placed on the centre of the maze facing an open arm, and the number of entries and the time spend in closed and open arm were recorded during a 5 min observation period. Arm entries were defined as entry of all four paws in to the arm.
The following parameters were calculated for each animal.
- % of open arm time
- % of close arm time
- % open arm entry
- % closed arm entry
4.3.3 Hole-board test in rats: 129
Grouping:
Six groups
of six mice (20-30 gm) in each were used in this model.
Group I : Animals (-veControl) were administered with 3% Tween 80.
Group II : Animals were administered with Diazepam 2mg/kg; p.o
suspended in 2% gum acacia
Group III : Animals were administered with Pet. ether extract of CP in 3% tween 80 at 400 mg/kg; p.o
Group IV : Animals were administered with Pet. ether extract of CP in 3% tween 80 at 800 mg/kg; p.o
Group V : Animals were administered with ethanolic extract of CP at
400 mg/kg; p.o
Group VI : Animals were administered with ethanolic extract of CP at
800 mg/kg; p.o
Procedure:
Standard drug (Diazepam) was administered 30 min prior to testing and extracts were administered p.o 30 min prior to testing.
The hole-board apparatus was an open-field arena with 16 equally spaced holes of 3 cm diameter in the floor, similar to the box used in social interaction test. Rats were placed singly in the centre of the hole-board, and during a 5-min trial.
The following parameters were recorded.
1. Total time spend with the head dips
2. Number of head dips in the holes
3. Total locomotors activity (numbers of squares crossed)
4. Number of assisted rearing
A head dip was
scored if both eyes disappeared into the hole. The arenas were wiped with a
damp cloth after each trial and any faeces removed.
4.3.4. Light-dark transition test in mice: 130,131,132,133
The light/dark transition test is based on the innate aversion of rodents to brightly illuminated areas and on the spontaneous exploratory behavior of rodent in response to mild stressors, that is, novel environment and light. A natural conflict situation occurs when an animal is exposed to an unfamiliar environment or novel objects. The conflict is between the tendency to explore and the initial tendency to avoid the unfamiliar (neophobia). The exploratory activity reflects the combined result of these tendencies in novel situations. Thus, in the light/dark test, drug induced increase in behavior in the white part of a two compartment box, in which a large white compartment is illuminated and a small black compartment is darkened, is suggested as an index of anxiolytic activity.
Grouping:
Six groups
of six mice (20-30 gm) in each were used in this model.
Group I : Animals (-veControl) were administered with 3% Tween 80.
Group II : Animals were administered with Diazepam 2mg/kg; p.o
suspended in 2% gum acacia
Group III : Animals were administered with Pet. ether extract of CP in 3% tween 80 at 400 mg/kg; p.o
Group IV : Animals were administered with Pet. ether extract of CP in 3% tween 80 at 800 mg/kg; p.o
Group V : Animals were administered with ethanolic extract of CP at
400 mg/kg; p.o
Group VI : Animals were administered with ethanolic extract of CP at
800 mg/kg; p.o
Procedure:
Standard drug (Diazepam) was administered 30 min prior totesting and extracts were administered p.o 45 min prior to testing.
The apparatus for light/dark transition test consist of two compartments: one light area (27 L x 27 W x 27 H cm), illuminated by 100 W desk lamp was painted white, and the other dark area (18 L x 27 W x 27 H cm) was painted black. The two compartments were separated by a partition with a tunnel (7.5 x 7.5 cm) to allow passage from one compartment to the other. The experiments were performed between 09:00 and 14:00. Animal was placed in the centre of the light area with its back to the opening.
The following parameters were recorded during 5 min:
1. Total time spen in the light area.
2. Total time spen in the dark area.
4.3.5 Open field test: 134,135
Grouping:
Six groups
of six mice (20-30 gm) in each were used in this model.
Group I : Animals (-veControl) were administered with 3% Tween 80.
Group II : Animals were administered with Diazepam 2mg/kg; p.o
suspended in 2% gum acacia
Group III : Animals were administered with Pet. ether extract of CP in 3% tween 80 at 400 mg/kg; p.o
Group IV : Animals were administered with Pet. ether extract of CP in 3% tween 80 at 800 mg/kg; p.o
Group V : Animals were administered with ethanolic extract of CP at
400 mg/kg; p.o
Group VI : Animals were administered with ethanolic extract of CP at
800 mg/kg; p.o
Procedure:
Standard drug (Diazepam) was administered 30 min prior to testing and extracts were administered p.o 30 min prior to testing.
The apparatus consists of a wooden box (60X60X30cm). The apparatus is illuminated with 40-W lamp suspended 100cm above it. Mice will be fed orally with extract(s), vehicle (normalsaline) or diazepam (1mg/kg;p.o).
After 30 minutes following parameters are observedand recorded during the test session of 5 minutes.
1. Number of entry in central square.
2. Time
spent in
3. Number of squares crossed.
4. Number of assisted rearing.
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