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4. EXPERIMENTAL INVESTIGATIONS

4.1. SELECTION OF PLANTS

A number of ethnobotanical survey and books describing the ethno-medicinal

plants of North East India were reviewed systemically to gather the information about

the folk medicinal knowledge of tribes of the region and to find potential medicinal

plants used by them. It was observed that more than thousands of plants are used by

more than 450 ethnic groups live in this region. The ethnomedicinal information

obtained from local people was the primary basis for the selection of medicinal plants.

In first phase 15 plants were selected based on the folk medicinal information, which

were distributed throughout the entire region commonly used by ethnic peoples. List

of those plants with their traditional and reported pharmacological effect given in

Table 4.1.

Table 4.1: List of preliminary selected plants

Scientific

name

Family Traditional uses Investigated

pharmacological effect

Dillenia

indica

Dilleniaceae Gastrointestinal pain,

diarrhoea, respiratory

diseases, cough, fever,

dysmenorrhoea,

infection

Antioxidant effect,

maltase inhibitory

effect, anti-amylase,

anti-glucosidase,

antihistamine release,

anti-leukemic activity

Litsea

glutinosa

Lauraceae Treat muscular, non-

bleeding injuries,

gonorrhea,

constipation,

diarrhoea,

rheumatism, wound,

antispasmodic

Antibacterial,

antioxidant, anti-

inflammatory, wound

healing, anthelmintic,

free radical scavenging,

aphrodisiac activity

112

Oxalis

corniculata

Oxalidaceae Treat dysentery,

stomach disorders,

rheumatism,

toothache, cold, fever,

skin diseases,

diarrhoea,

dysmenorrhoea

Anti-inflammatory,

anxiolytic,

anticonvulsant,

antifungal, antiulcer,

antinociceptive,

anticancer, antidiabetic,

hepatoprotective,

hypolipedemic,

abortificient,

antimicrobial and

wound healing

properties

Phyllanthus

acidus

Phyllanthaceae Astringent, cathartic,

antihypertensive,

emetic, purgative,

laxative; to treat

respiratory disorders,

cough, bronchitis,

liver disorders,

psoriasis, diabetes,

pain, fever

Antidiabetic,

antimicrobial, cytotoxic

and selective

antioxidant, treatment of

cystic fibrosis,

hepatoprotective against

CCl4 induced

hepatotoxicity

Smilax

zeylanica

Smilacaceae Treat hepatitis, urinary

disease, skin disease,

veneral diseases, skin

disorders, sores,

swellings, rheumatism

and pain

Antidiabetic, pesticidal,

anthelmintic, analgesic,

antibacterial,

antipyretic,

anticonvulsant,

antiepileptic,

hepatoprotective,

antioxidant effect

Marsilea

minuta

Marsileaceae Treatment of cough,

bronchitis, insomnia,

headache, mental

problems,

hypertension,

gastrointestinal

disorders, renal

diseases

Hypocholesterolemic,

anxiolytic, antifertility,

anti stress,

antidepressant,

hepatoprotective against

CCl4 induced

hepatotoxicity, few in

vitro antioxidant study

113

Aphanamixis

polystachya

Meliaceae Treat stomach pain,

skin disease, tumours,

cancer, spleen

diseases, rheumatism,

liver disease

Hepatoprotective,

antimicrobial, antiviral

and

antibacterial activity,

radioprotective,

antioxidant, antipyretic,

antiulcer, hematinic,

diuretic

Asparagus

racemosus

Liliaceae Treatment of

diarrheoa, fever,

measles, stomach pain,

ulcerative disorders,

menstruation problem

and to increase milk

secretion during

lactation

Antiulcer, galactogogue,

immunomodulating,

antineoplastic,

hepatoprotective,

antioxidant, cardio

protection

Coccinia

grandis

Cucurbitaceae Treatment of diabetes,

scabies, skin infection,

jaundice, bronchitis,

skin eruptions, burns,

insect bites, fever,

indigestion, nausea,

eye infections, allergy,

syphilis, gonorrhoea

Antidiabetic,

antioxidant,

antinociceptive and

anti-inflammatory

activity, antipyretic,

hypolipedemic, anti-TB,

anthelmintic, antitussive

effect

Ipomea

aquatic

Convolvulaceae Treatment of nervous

and general debility,

piles, worm infections,

leucoderma, leprosy,

jaundice, liver

disorders, high blood

pressure, skin disease,

dysentery; used as

purgatives, diuretics

Nootropic,

antihyperglycemic,

antiulcer antimicrobial,

cytotoxic, diuretic,

antioxidant,

antiepileptic effect

114

Mimosa

pudica

Mimosaceae Treatment of leprosy,

burning sensation,

fever, alopecia, tumor,

diarrhoea, dysentery,

insomnia, and various

urogenital infections

Wound healing activity,

regeneration of sciatic

nerve, antidepressant,

anticonvulsant,

hyperglycemic, diuretic,

antifertility,

hepatoprotective,

antioxidant,

antimicrobial effect,

aphrodisiac property,

Paederia

foetida

Rubiaceae Treatment of

amoebiasis, tooth

ache, stomach pain,

asthma, bowel

complaints, diarrhea,

diabetes, seminal

weakness, and as

antiarthritic,

diaphoretic,

antispasmodic,

expectorant

Anti-diarrhoeal,

antioxidant,

hepatoprotective,

antitussive,

anthelmintic,

antispasmodic

Typhonium

trilobatum

Araceae Treatment of bowl

disease, constipation,

body pain,

rheumatism, traumatic

injury, tuberculosis,

bronchitis, vomiting,

cough, asthma,

headache, ulcer

Antimicrobial,

nematocidal, larvicidal

activity, analgesic, anti-

inflammatory and anti

diarrheal effect

Alternanther

a

philoxeroides

Amaranthaceae Treatment of stomach

pain, dysentery,

stomach disorders,

wound, ulcer

Analgesic,

antiinflammatory,

anthelmintic,

antioxidant, antipyretic,

antibacterial,

hepatoprotective,

antiulcer, diuretic effect

115

Centella

asiatica

Apiaceae Treatment of stomach

and liver disorder,

asthma, leprosy

Wound healing,

sedative and anxiolytic,

epatoprotective,

antioxidant,

antiepileptic, antiulcer,

antileprotic

cardioprotective,

antidepressant,

antinociceptive, anti-

inflammatory,

neuroprotective effect

Scientific literatures on these plants were extensively reviewed. Reported

activities were observed to be matched with traditional knowledge. In the view of

relation between oxidative stress and diseases, it was planned to screen antioxidant

potential of those plant first. It was observed that for some of the plants antioxidant,

hepatoprotective and other activities were already been reported. Taking this as a

consideration 4 plants viz. Marsilea minuta (whole plant), Oxalis corniculata (leaf),

Phyllanthus acidus (leaf), Dillenia indica (leaf) were selected for primary study.

In next stage a preliminary antioxidant study was conducted using DPPH and

nitric oxide radical scavenging assay method. This study was very initial study using

only two concentrations, to find more effective plant among these four. All methanol

extracts were taken to perform activity. It was observed that all the extract showed

antioxidant activity, but two plants viz, Marsilea minuta and Phyllanthus acidus

showed better effect as compared with other two. It was also observed that having a

lot of traditional medicinal importance of these plants was not scientifically

investigated despite of vast utility as folk medicine. Therefore I have concentrated on

these two plants (Marsilea minuta and Phyllanthus acidus) to focus their folk

medicinal importance by a scientific approach.

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4.2. PLANT COLLECTION AND AUTHENTICATION

Whole plant of Marsilea minuta L. was collected during the month of

March/April 2011 and the leaves of Phyllanthus acidus were collected in February

2011 from Tripura, India. The time of collection purposefully taken based on the

suggestion of botanis/phytochemist. The plant was identified vernacular name, later

validated and authenticated by Dr. BK Datta, Department of Botany, Tripura

University, Tripura, India. Voucher specimens (TU/BOT/HEB/SS23072011b and

TU/BOT/HEB/RC25092011b) were deposited at the herbarium of Plant Taxonomy &

Biodiversity Laboratory, Tripura University.

4.3. DRUGS AND CHEMICALS

Chemicals, reagents, biochemical diagnostic kit, drugs were purchased from

reputed companies. List of the important chemicals and name of their manufacturing

agencies were tabulated in Table 4.2.

Table 4.2: List of chemicals and name their manufacturing agencies

Company Chemicals

Sigma Aldrich

(Bangalore,

India)

DPPH, Sodium nitroprusside (SNP), Phenazine methosulfate

(PMS), Nitro blue tetrazolium (NBT), Naphthyl ethylene

diamine dihydrochloride

SD Fine Ltd.

Mumbai

H2O2, 2-deoxy-ribose, Trichloro acetic acid (TCA),

Thiobarbituric acid (TBA), Methanol, Ethyl acetate, Petroleum

ether, Ethanol, Chloroform, Quercetin, Aluminium chloride

(AlCl3), Sodium nitrite (NaNO2), Sodium hydroxide (NaOH),

Folin-Ciocalteu reagent, Tris-HCl buffer, Phosphoric acid,

Carragennan, Formalin

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Sisco Research

Laboratories

Pvt. Ltd.,

Mumbai

Linoleic acid, Ammonium thiocyanate, Ascorbic acid, α-

tocopherol, Folin–Ciocalteau reagent, BHA, Epinephrine,

Ascorbic acid, Gallic acid, Sulphanilamide, NADH

Loba Chemie

Pvt Ltd.,

Mumbai

Ferric chloride (FeCl3), Ferrous chloride (FeCl2), Zinc chloride,

Chlorosulfonic acid, Lead acetate, Phenazone, Ethelene diamine

tetra acetic acid (EDTA), Potassium acetate, Aluminium nitrate

Agapee

Diagnostic Ltd.,

Kerala

Diagnostic kit for - Serum glutamic oxaloacetate transaminase

(SGOT), Serum glutamate pyruvate transaminase (SGPT),

Alkaline phosphatase (ALP), Bilirubin, Cholesterol,

Triglycerides, Blood urea nitrogen (BUN), Creatinine, Uric acid,

Total protein, and Albumin.

All other chemicals used in the study were obtained commercially and were of

analytical grade.

4.4. INSTRUMENTS USED

Following major instruments were used for the study,

Double beam UV-Vis Spectrophotometer (Elico SL 164).

Plethysmometer (AARSON Scientific Works, Haryana)

Digital pH Meter (Elico LI 127).

Research Centrifuge (Remi R-24).

Semi Auto Analyser (Mispa neo).

Tissue Homogenizer (Remi RQ-127A/D).

Ultrasonic Baths (Edutek Instrumentation, Ambala).

Vacuum Pump (Advanced Technocracy Inc., Ambala).

Melting point Apparatus (Advanced Technocracy Inc., Ambala).

Digital Balance (Contech CA-123)

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4.5. PREPARATION OF EXTRACTS

Collected whole plant/leaves were washed in running water to remove

unwanted materials, and dried under shade. The air dried materials were powdered

mechanically by using a grinder and sieved through sieve 22 mesh to obtain fine

powder and stored in air tight container. Dried powdered plant material was extracted

using Soxhlet apparatus with several solvents like methanol, ethyl acetate, petroleum

ether separately. About 2 kg of Marsilea minuta and 1kg of Phyllanthus acidus leaves

were extracted by using 10 L and 5 L of each solvent respectively. The extracts were

concentrated under reduced pressure by using rotary vaccume evaporator to obtain

methanol extracts of Marsilea minuta (MEM) and Phyllanthus acidus (MEP), ethyl

acetate extract of Marsilea minuta (EEM) and Phyllanthus acidus (EEP), petroleum

ether extract Marsilea minuta (PEM) and Phyllanthus acidus (PEP) which were used

for further study.

Figure 4.1: Extraction of plant materials

119

4.6. PHYSICOCHEMICAL PROPERTIES OF EXTRACT

Physiochemical properties of the extracts were evaluated. The yield, colour,

pH, density and specific gravity of crude extracts were determined by following

standard procedure.

4.6.1. Colour and Yield Determination

Colour of the extracts was observed in bare eye. The physical properties i.e.

colour, percentage yield of the extracts were also observed and reported.

4.6.2. Determination of pH

The individual extract solution was filtered by using Whatman filter paper and

the pH was recorded by using Elico digital pH meter [199].

4.6.3. Determination of Specific Gravity

The weight of an empty pycnometer (Wp) was recorded. About 10 g of sample

was placed in the pycnometer and weight (Wps) was taken. Half or ¾ of pycnometer

was filled with distilled water and soaked for10 min. Partial vacuum was applied to

remove entrapped air. And then pycnometer was filled with distilled water up to the

mark. The exterior surface of the pycnometer was cleaned properly and the weight

(Wb) was determined. After removing the content, the pycnometer was cleaned, filled

with distilled water, and the weight (Wa) was measured again. Specific gravity was

determined using the following formula [200],

120

Specific Gravity (Gs) = W0

W0 - (Wa - Wb)

W0 = Weight of the sample (Wps - Wp)

Wa = Weight of pycnometer filled with water

Wb = Weight pycnometer filled with water and sample

4.7. PRELIMINARY PHYTOCHEMICAL INVESTIGATION

The presence of Alkaloids, carbohydrates, glycosides, protein, amino acids,

tannins, flavonoids, triterpenoids and steroids were carried out by quantitative

phytochemical investigation using the standard chemical methods [201, 202].

4.7.1. Detection of Alkaloids

Small quantity of dried extract/fractions was stirred with dilute hydrochoric

acid and filtered. Filtrate was tested for alkaloids with various reagents.

Mayer’s test: Reddish brown or orange precipitate obtains when few millilitres of

filtrate were mixed with 2 drops of Mayers reagent (potassium mercuric solution)

along with the side of the test tube.

Wagner’s test: Few drops of Wagner’s reagent (iodine-potassium iodide solution)

were mixed with few millilitres of filtrate along sides of the tube to obtain precipitate.

Hager’s test: Prominant yellow or crystalline yellow precipitate was the positive test

when few millilitre of filtrate mixed with 2.0 mL of Hager’s reagent (saturated

solution of picric acid).

121

Dragendroff’s test: Prominent reddish brown or orange red precipitate can be

observed, when few millilitres of filtrate were mixed with 2.0 mL of Dragendroff’s

reagent (potassium bismuth iodide solution).

4.7.2. Detection of Carbohydrates

A solution was prepared by taking a small quantity of the extract/fraction in

few mL of distilled water, filtered and was subjected for the following tests.

Molish’s test: Around 2 mL filtrate was taken in a test tube, two drops of Molish

reagent (alcoholic solution of α-naphthol) was added. Solution was mixed well and

1.0 mL concentrated sulphuric acid added slowly along the sides of the tube. Cooled

the tube in ice water and allowed to stand. Appearance of violet colour ring at the

junction of layers was the conformation for carbohydrate.

Fehling’s test: The test used to identify reducing sugar, and was performed by taking

few millilitre of filtrate mixing with 1.0 mL each of Fehling’s solutions A and B. The

solution was heated on water bath. Formation of brick red precipitate was the positive

test.

Benedict’s test: Few mL of filtrate and few mL of Benedict reagent was taken in a

test tube and heated on water bath for 2 min. reducing sugar confirmed by formation

of colored precipitate.

Barfoed’s test: One mL Barfoed’s reagent with few milliliter of filtrate was added in

a test tube and heated on water bath for 2 min. Monosaccharides confirmed when red

precipitate forms.

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4.7.3. Detection of Protein and Amino Acid

A solution was prepared by taking small quantity of sample in few mL of

distilled water and filtered through Whatman filter paper no 1. The filtrate was

subjected for the following tests.

Millon’s test: Formation of white precipitate, when the filtrate was mixed with few

drops of Millon’s reagent.

Ninhydrin test: Two drops of ninhydrin solution was added to 2.0 mL of filtrate and

boiled in a water bath. Purple/blue/violet colour could be observed for positive test.

Biuret test: Formation of violet colour was the conformation, when equal volume of

filtrate and Biuret reagent mixed together.

Xanthoprotein test: A solution was prepared by taking 5 mL of filtrate with 1mL

concentrated HNO3 and boiled in water bath, 40% NaOH was added after cooling.

Orange colour indicates the presence of protein.

4.7.4. Detection of Glycosides

4.7.4.1. General test for glycosides

Test A: About 200 mg of drug and 5.0 mL of dilute sulphuric acid warmed on a water

bath. The filtrate was neutralised with 5% NaOH solution and mixed with 0.1 mL of

Fehling's solution A and B until it becomes alkaline (tested with pH paper) and heated

on a water bath for 2 min. The quantity of red precipitate formed during the test was

measured.

Test B: Warmed about 200 mg of drug extract with 5.0 mL of water on a water bath.

Equal amount of water as used for sodium hydroxide in the Test A was added.

Fehling's solution A and B was added untill the solution became alkaline (tested with

123

pH paper) and the mixture heated on water bath for 2 minutes. Quantity of red

precipitate formed was noted and compared with Test A.

Presence of glycoside is confirm if the precipitate formed in Test A is more

than in Test B (Test A correspond to free reducing sugar plus those associated on acid

hydrolysis of any glycoside in the crude drug, whereas Test B stands for the amount

of free reducing sugar present in the crude drug).

4.7.4.2. Tests for specific glycosides

Tests for specific glycosides also performed using the following methods,

Borntrager's test: Crude extract was boiled with 1.0 mL of sulphuric acid in a test

tube for 5 min and filtered while hot. Cooled the filtrate and shaked with equal

volume of chloroform. Lower layer of chloroform was separated and mixed with half

of its volume of dilute ammonia and shaked well. Anthraquinone glycoside confirms

if a rose pink to red colour produces in the ammoniacal layer.

Keller-killiani test: Extracted the crude extract with chloroform, and evaporated to

dryness. Add 0.4 mL glacial acetic acid containing trace amount of ferric chloride. A

volume of 0.5 mL concentrated H2SO4 was added carefully by the side of the test

tube. Blue colour in acetic acid layer indicates the presence of cardiac glycosides.

Raymond’s test: If a violet colour develops when the test solution treated with hot

methanolic alkali, confirms cardiac glycosides.

A small amount of sample in test tube was taken and heated for several minutes on a

water bath by covering the test tube with a filter paper moistened, with dilute sodium

hydroxide solution. On exposure to UV light the paper shows green fluorescence

indicates presence of coumarin glycosides.

124

Froth formation test: Small amount of extract/fraction was shaken in a test tube

with a little quantity of water. If it results in formation of foam persists for 10 min

indicates presence of saponin glycosides.

Haemolysis test: A volume of 2 mL 1.8% NaCl solution was taken to the two test

tubes, made the solution isotonic to blood after adding one of these test tube 2.0 mL

of distilled water and to other 2.0 mL of sample solution. Each of these test tubes was

gently mixed with 5 drops of blood which was obtained by pricking the thumb at the

base of the nail. Haemolysis observed under the microscope in the tube containing the

sample, but no haemolysis in control indicates presence of saponin glycosides.

4.7.5. Detection of Tannins

Tannin in crude extract or fraction was determined by the following methods,

Ferric chloride test: A small quantity extract was boiled in few mL of distilled water

in a test tube and then filtered. Few drops of 0.1% ferric chloride was added to the

filtrate. A brownish green or a blue-black colour develops after represents the

presence of tannin.

Phenazone test: In a test tube 5 mL of aqueous extract was mixed with 0.5 g of

sodium acid phosphate, warmed the mixture and filtered. A solution of 2% phenazone

was added to the filtrate. Formation of bulky precipitate (which often coloured)

represents the positive test for tannin.

Gelatin test: Small quantity of sample dissolved in distilled water and mixed with a

solution of 1% gelatin (2.0 mL) containing 10% NaCl. Formation white precipitate

indicates the presence of tannin.

125

4.7.6. Detection of Flavonoids

Flavonoids in crude extract or fraction was determined by the following

methods,

Lead acetate test: Alcoholic solution of the extract/fraction mixed with few drops of

10 % lead acetate solution. Yellow precipitate indicates the presence of flavonoid.

Alkaline reagent test: Few mL of test solution, was mixed with few drops of 10%

NH4OH. Intense yellow colour develops and turns to colourless on addition of few

drops of dilute acid confirms the presence of flavonoid.

Zinc Hydrochloride test: Red colour/Megneta colour cis conformation when

alcoholic test solution of the extract/fraction mixed with a pinch of zinc dust and few

drops of concentrated HCl.

Ferric chloride test: To the alcoholic solution of extract, few drops of ferric chloride

were added. If it gives green colour, flavonoid is confirmed.

4.7.7. Detection of Steroids and Triterpenoids

Flavonoids in crude extract or fraction was determined by the following

methods,

Libermann-Burchard’s test: The sample and few drops of acetic anhydride, heated

to boil, and cooled. Along with the side of the test tube, 1 mL of concentrated H2SO4

was added. If it results in formations of brown ring at the junction and upper layer

turns to green indicates presence of steroids and brown ring at the junction and upper

layer turns to deep red indicates presence of triterpenoids.

126

Salkowski Test: The sample was mixed with few drops of concentrated sulphuric

acid. Red colour at lower layer indicates presence of steroids where as yellow colour

at lower layer indicates the presence of triterpenoids.

Tschugajeu test: Chloroform solution of sample mixed with excess of acetyl chloride

and a pinch of zinc chloride in a test tube and was kept aside for few minute. It was

warmed on a water bath. Formation of erosin red colour indicates the presence of

triterpenoids.

Brieskorn and Brinar test: Chloroform solution of test sample was added to a

mixture of chrolosulfonic acid in glacial acetic acid (7:3). Development of red colour

in few minutes indicates presence of triterpenoids.

4.8. EXPERIMENTAL ANIMALS

Healthy Wistar rats (150–200 g) and albino mice (20–30 g) between 2 and 3

months of age were used. Animals were housed under standard environmental

conditions (24±1°C) with 12 h light – 12 h dark cycles. The animals had free access to

water and food. The animal studies were carried out in CES College of pharmacy,

Kurnool, Andhra Pradesh. All animal procedure have been approved by institutional

animal ethical committee of CES college of Pharmacy (Reg. no:

1305/ac/09/CPCSEA) in accordance with animal experimentation and care.

4.9. DETERMINATION OF ANTIOXIDANT COMPOUNDS

4.9.1. Total Phenolic Content Determination

The total phenolic content in the crude extracts was determined by using

Folin-Ciocalteu reagent by colorimetric method [76]. A solution of 0.5 mL ethanol

solution of extract/fraction (l.0 mg/mL) was mixed in a test tube with 2.5 mL of 10%

127

v/v Folin-Ciocalteu reagent and 2.0 mL of 2% w/v sodium carbonate. The tubes were

shaken thoroughly and incubated for 15 min at 45°C with intermittent shaking.

Absorbance was taken at 765 nm using ELICO SL 164 UV–Vis spectrophotometer. A

calibration curve was plotted by taking gallic acid as standard (0 to 800 µg/mL), and

results were expressed as gallic acid equivalents in milligram per gram (mg GAE/g)

of dried extract. The estimation was carried out in triplicate.

4.9.2. Total Flavonoid Content Determination

Aluminium nitrate-potassium acetate reagent method was used to determine

total flavonoid content of extracts. A volume of 0.5 mL of sample solution (1.0

mg/mL) was mixed with 0.1 mL of 10% aluminium nitrate, 0.1 mL 1 M potassium

acetate and 4.3 mL of 80% ethanol was mixed thoroughly and allowed to stand for 40

min at room temperature. Absorbance of the supernatant solution was measured at

415 nm [86]. A calibration curve was plotted by taking quercetin (0 to 16 µg/mL) as

standard. The estimation was carried out in triplicate. The results were expressed as

quercetin equivalents in milligram per gram (mg QE/g) of dried extract.

4.10. IN VITRO ANTIOXIDANT ACTIVITY OF EXTRACTS

4.10.1. DPPH Radical Scavenging Assay

Ability of the sample to donate hydrogen atom or electron was measured from

the bleaching of a purple-coloured methanol solution of DPPH. Antioxidants can

readily reduce DPPH a stable radical with an absorption maximum at 517 mm. Free

radical scavenging capacity of extracts/fractions was measured using the stable DPPH

radical [87].

128

Methanolic solution of DPPH (1.0 mL, 0.1 mM) was mixed with 3.0 mL of

sample solution of different concentrations. The reaction mixture was incubated in

dark at room temperature for 30 min and the absorbance was recorded at 517 nm [87].

The assay was carried out in triplicate for each sample. Methanol (1 mL) with 3.0 mL

extracts/fraction solution was used as a blank and DPPH solution (1.0 mL, 0.1 mM)

with methanol (2.5 mL) served as negative control. The radical scavenging activity of

ascorbic acid was also determined as positive control. The decrease in colour intensity

on addition of test samples was used to calculate the antiradical activity, measured by

taking absorbance and compared with negative control. The activity was expressed by

the inhibition percentage (I %) of DPPH radical, following the equation (1)

I % = [(Ac-As)/Ac] x 100

Where, Ac and As are the absorbance of the control and of the test/standard sample

respectively. From a plot of concentration against I%, a linear regression analysis was

performed to determine the IC50 (extract concentration resulting in a 50% inhibition)

value for each sample.

4.10.2. Superoxide Anion Radical Scavenging Activity

The scavenging activity of the sample towards O2•−

was measured by the

method described by Nagulendran et al. [77]. Superoxide anions can be formed in a

non-enzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS-

NADH) system. PMS, NADH, and oxygen reacts to give O2•−

which was assayed by

the reduction of nitroblue tetrazolium (NBT).

Briefly, different concentration of 0.3 mL of extract was mixed with 3.0 mL of Tris-

HCl buffer (100 mM, pH 7.4) containing 0.75 mL of NBT (300 μM) solution, 0.75

129

mL of NADH (936 μM) solution. The reaction was started by mixing 0.75 mL of

PMS (120 μM) to the mixture and after 5 min of incubation at room temperature, the

absorbance of mixture was recorded at 560 nm. For comparison, BHA was used as

standard; all the tests were carried out in triplicate. The super oxide anion scavenging

activity was calculated by the equation 1.

4.10.3. Hydroxyl Radical Scavenging Activity

Hydroxyl radical was generated by oxidising 2-deoxyribose (Fenton reaction)

and degraded to malondialdehyde [85]. Therefore decomposing effect of sample on

hydroxyl radicals was estimated by the assay of formed malondialdehyde chromogen

formation.

Briefly, 0.2 mL of 100mM KH2PO4–KOH, 0.2 mL of 15mM deoxyribose, 0.2 mL

of 500mM FeCl3, 0.1 mL of 1mM ethylene diamine tetra acetic acid (EDTA), 0.1 mL

of 1mM ascorbic acid, and 0.1 mL of 10mM H2O2 were mixed with 0.1 mL sample

with different concentration. The mixture was incubated for 1 h at 37ºC. After

incubation, 1.0 mL of 1% w/v TBA and followed by 1.0 mL of 2.8% w/v TCA was

added to the mixture. The resultant mixture was heated for 20 min at 80ºC on a water

bath results in development of pink colour which was measured at 532nm [89].

Quercetin was used as the positive control. The scavenging activity (I %) was

calculated using the equation (1).

4.10.4. Nitric Oxide Radical Scavenging Activity

Aqueous solution of sodium nitroprusside at physiological pH produces nitric

oxide, which interact with oxygen to produce nitrite ions, which was quantified by the

130

Griess Illosvoy reaction. The nitrite ions thus produced react with Griess reagent that

leads to formation of a chromophore which can be determined

spectrophotometrically. The concentration of chromophore is proportional to that of

the generated nitrite ions. Antioxidant compounds present in the sample compete with

oxygen leading to reduced production of nitric oxide [76, 87].

An aliquot of extract solution (4 mL) at different concentrations were mixed

with 1.0 mL of 25 mM sodium nitroprusside (SNP) solution in a test tube, and

incubated for 2 h at 37ºC. Incubated solution (2 mL) was mixed with 1.2 mL Griess

reagent (1% sulfanilamide in 5% H3PO4 and 0.1% naphthylethylenediamine

dihydrochloride) which results in diazotization of the nitrite with sulfanilamide and

subsequent coupling with naphthylethylenediamine dihydrochloride to form a

chromophore. The absorbance of chromophore was measured immediately at 570 nm

[78]. Control experiment was also carried out in similar manner taking same volume

of distilled water in the place of sample solution. The experiment was performed in

triplicate, ascorbic acid was used as positive control and percentage scavenging

activity was calculated using the equation (1).

4.10.5. Hydrogen Peroxide Scavenging Activity

A solution of 40 mM H2O2 and crude extracts/standard in different

concentration were prepared in phosphate buffer (pH 7.4). An aliquot (3.4 mL)

sample solution was added to 0.6 mL of H2O2 solution and the absorbance of resulting

solutions was measured at 230 nm. Gallic acid was used as standard. The percentage

of H2O2 scavenging (I %) of extracts was calculated by equation (1) [79].

131

4.10.6. Reducing Power Ability

The ability of phenolic compounds to quench radicals by electron donation

was determined by potassium ferricyanide reduction method. The absorbance of

Perl’s Prussian blue complex was at 700nm. Increase in absorbance may be due to

reduction of ferric ion/ferricyanide complex by antioxidants to ferrous form [88].

Briefly, 1 mL of sample solutions with different concentration were mixed

with 0.2 M phosphate buffer (2.5 mL, pH 6.6) and 1% (w/v) K3Fe(CN)6 (2.5 mL) and

the mixture was incubated at 50ºC for 20 min, and then mixed with 2.5mL, 10%

TCA. The solution was centrifuged at 12000 rpm for 10 min. A volume of 2.5 mL

supernatant solution was mixed with 2.5 mL of distilled water, 0.5 mL of 0.1% (w/v)

FeCl3 solution. The solution was mixed properly and the absorbance of solution was

measured at 700 nm using ELICO SL 164 UV–Vis spectrophotometer [80]. Ascorbic

acid (25-400 µg/mL) was used as standard. The test was run in triplicate and average

was calculated.

4.10.7. Metal Chelating Ability

Ferrozine with ferrous ion makes red colored complex, which was interrupted

by chelating agents. Thus, the activity of antioxidants was determined by monitoring

the decrease in absorbance of the red Fe2+

/ferrozine complex as antioxidants present

in the test sample compete with ferrozine to chelate ferrous ion [77, 88].

A volume of 0.4 mL different concentration extracts was mixed with 0.05 mL,

2 mM FeCl2 solution. The reaction was started by the mixing 0.2 mL, 5mM ferrozine.

132

Total volume of the final solution was adjusted to 4.0 mL using ethanol. The mixture

was mixed by shaking vigorously and incubated for 10 min at room temperature. The

absorbance of the solution was measured at 562nm spectrophotometrically [81]. The

procedure was also performed using α- tocopherol as standard and percentage of

inhibition of ferrozine–Fe2+

complex formation (I%) was calculated by using equation

(1).

4.10.8. Ferric thiocyanate method (FTC)

Oxidation of Linoleic acid causes the generation of peroxides, which react

with Fe2+

to form Fe3+

. The ferric ion forms red colored complex with SCN– and this

complex has a maximum absorbance at 500 nm. The method was employed to

determine the peroxide level during the initial stage of lipid oxidation [15].

A quantity of 200 µg of sample in 4.0 mL ethanol was mixed with 4 mL,

linoleic acid (2.5 % in ethanol), 8.0 mL phosphate buffer (0.05 M, pH 7.0) and 4 mL

distilled water. The mixture was mixed vigorously in a screw cap tube and incubated

in dark at 40ºC. One millilitre of above solution was mixed with 9.7 mL ethanol

(75%) and 0.1 mL ammonium thiocyanate (30%) followed by 0.1 mL 20 mM ferrous

chloride in 3.5% hydrochloric acid and exactly 3 min later the absorbance of the red

colour solution was measured at 500 nm. The sample was withdrawn after every 24hr

and the procedure was repeated until the absorbance of the control reached a

maximum [15]. Linoleic acid mixture without the addition of sample was used as the

control and α-tocopherol was used as the positive control at the same concentration.

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4.11. EX VIVO ANTIOXIDNT ACTIVITY OF EXTRACTS

4.11.1. Lipid Peroxidation Assay

A healthy Wister rat was sacrificed by decapitation after fasten for 16 h. The

liver was dissected out carefully and washed with normal saline and 5.0 %w/v liver

homogenate was prepared in phosphate buffer saline. From the above mixture 1 mL

was mixed with 100 μL of sample solution and incubated for 2 h at 37°C. A volume

of 1.0 mL of 15% w/v TCA and 1.0 mL of 0.67% w/v TBA were added to the

mixture. This solution was heated on boiling water bath for 15 min and kept aside

until it comes to room temperature. Deionised water was used to make the final

volume 5.0 mL. The mixture was then centrifuged for 10 min at 2800 rpm. The

supernatant solution was removed carefully and absorbance was recorded at 532 nm

[82]. Control was prepared by following the same procedure without extracts. Rutin

served as positive control. The inhibition of lipid peroxidation was calculated using

the equation (1), where, Asample was the absorbance of an extract/standard in presence

of liver homogenate and Acontrol was the absorbance of the solution containing all

reagents except the test/standard sample.

4.11.2. Oxidative Haemolysis Assay

Oxidative haemolysis assay method was followed as described by Su et al.

(2009) with slight modification as represent by Coulibaly et al. [83]. Blood sample

was collected from rat eyepit under mild anaesthesia and followed by centrifuged at

1500 × g for 10 min at 4ºC. The erythrocytes were separated from the plasma and

buffy coat. The erythrocytes were suspended in 10 mL phosphate buffer saline (PBS,

134

10 mM, pH 7.4). The erythrocyte in PBS centrifuged again at 1500 × g for 5 min to

take out buffy coats. The process was repeated three times and washed erythrocytes

made to 0.5% erythrocyte suspension in PBS (10 mM, pH 7.4) for the assay.

Prepared 0.5 mL of erythrocyte suspension, 0.5 mL extract/standard drug

solution at different concentration and 0.05 mL of 100 mM H2O2 was mixed together

and kept for incubation at 37ºC for 60 min. After incubation period 4.2 mL of distilled

water was added and the solution was centrifuged for 10 min at 1000 rpm. The

absorbance of the supernatant was measured at 415 nm. Control was prepared by

taking supernatant without extract and ascorbic acid was used as positive control. The

protective effect of extract was calculated as inhibition percentage of erythrocyte

haemolysis (I%) using the following equation (1).

4.12. ACUTE TOXICITY STUDY

Albino mice maintained under standard condition used for the acute toxicity

study for the samples (potent extract and its fractions). The overnight fasted animals

were taken and fixed dose (OCED Guideline no. 423, Annexure 2d) procedure of

CPCSEA was adopted for toxicity studies. Solvent free dried extract or fraction was

orally administered in acute toxicity and in screening of other biological activity to

experimental animal. In acute toxicity study, the test material suspended in 0.5% w/v

sodium carboxy methyl cellulose (CMC) and was orally administrated (1 mL/100 g)

in 3 animals. The mortality was observed at a dose of 2000 mg/kg in the all cases.

Common side effects such as, mild diarrhea, lose of weight and depression of treated

groups of animals were observed for 7 days.

135

4.13. ANIMAL GROUPING

The experiments were performed in two stages. At first stage activity of crude

extracts were evaluated, and in second phase the activity of fractions of more potent

extract were determined. For each experiment animals were divided into eight groups

in following manner.

Group I : Normal control (treated with only vehicle)

Group II : Negative control (diseased group)

Group III : Standard

Group IV : Lower dose of methanol extract or methanol fraction

Group V : Higher dose of methanol extract or methanol fraction

Group VI : Lower dose of ethyl acetate extract or ethyl acetate fraction

Group VII : Higher dose of ethyl acetate extract or ethyl acetate fraction

Group VIII : Lower dose of Pet ether extract or pet ether fraction

Group VIII : Higher dose of Pet ether extract or pet ether fraction

Lower dose of extracts indicates 250 mg/kg b.w. higher dose indicates 500

mg/kg b.w. For fractions lower dose indicates 75 mg/kg b.w. and higher dose

indicates 150 mg/kg b.w. Hepatoprotective and nephroprotective activity of both the

plants were evaluated therefore Group I, II. III were common for both the plants.

136

4.14. ANALGESIC AND ANTI-INFLAMMATORY ACTIVITY OF

P. ACIDUS

4.14.1. Nociceptive Tests

4.14.1.1. Writhing reflex induced by acetic acid in mice

The extracts/fraction, indomethacin, and vehicle were administered orally.

Indomethacin at a dose of 5mg/kg was used as standard. After 1 h each mouse was

tested with acetic acid (0.6%, v/v, 10 mL/kg), and the intensity of nociceptive

behaviour was quantified by counting the total number of writhes over a period of 25

min [102]. The percentage analgesic activity was calculated by using the following

equation:

Percentage analgesic activity = [(Nc − Nt)/ Nc] × 100%

Where Nc was the average number of stretches of the control group, and Nt was the

average number of stretches of the test drug group.

4.14.1.2. Tail immersion test

In hot water (temperature was maintained at 55±0.5°C) extreme 3 cm of the

Albino mouse tail immerged in that water. Within a time period each mouse was

reacted by withdrawing the tail, and the reaction time was recorded with a stopwatch.

The drugs were given orally to the respective groups as described above. The

experiment was repeated at 0, 0.5, 1, 2, 3, 5 h after administration of extracts/fraction

and standard drug [104]. Morphine was used as standard at a dose of 10mg/kg.

137

4.14.1.3. Formalin-induced licking response in mice

One hour after oral administration of vehicle, extract/fraction and diclofenac

sodium (10 mg/kg), 25 μL of 1% formalin in saline was injected subcutaneously in

subplantar area of right hind paw. The mice were instantly placed in a cleaned jar.

The time spent on licking the injected paw was recorded. The first period was

recorded at 0-5 min considered as early phase response and the second period was

recorded at 10-35 min considered as late phase response [114].

4.14.2. Anti-inflammatory Activity

4.14.2.1. Carrageenan-induced paw oedema in rats

A solution of 1%, 0.1 mL carrageenan was injected subcutaneously into the

plantar surface of left hind paw to induced paw inflammation in rats after respective

drug treatment to each group. Indomethacin (5mg/kg) used as standard. The volume

of the rat paws was measured with a plethismometer before and after 1, 2, 3, 4, and 5

h [109].

14.4.2.2. Granuloma formation induced by cotton pellet in rats

A 30 mg sterilized cotton pellet was put subcutaneously into the interscapular

area of anaesthetized rats using 25 mg/kg of pentobarbitone sodium under sterilized

condition. The extract/fraction solution, indomethacin (5 mg/kg, p.o.), and vehicle

water were administered 5 consecutive days once in a day. On the 5th

day, animals

were killed via cervical dislocation after 1h of drug treatment and the cotton pellets

with granuloma tissue around them were dissected out carefully. The weights of these

pellets (wet and dry) were measured and the anti-proliferative effects of

extracts/fraction and indomethacin were determined by comparing with control group

[103].

138

14.4.2.3. Membrane stabilizing activity

The membrane stabilizing activity was performed by the method used by

Shinde et al. [101].

Preparation of erythrocyte suspension

Under anaesthesia whole blood was collected in a heparinized tube from mice.

Blood sample was washed with 0.9% saline for three times. Isotonic buffer solution

(pH 7.4) was used to make 40% (v/v) erythrocyte suspension. Buffer solution was

prepared by following formula, NaH2PO4. 2H2O – 0.26 g, NaHPO4 – 1.15 g, NaCl – 9

g (10 mM sodium phosphate buffer) in 1 L distilled water.

Heat induced haemolysis

Isotonic buffer containing different concentration of extract/fraction were

taken into 2 duplicate sets of centrifuge tubes. Vehicle was prepared by adding same

amount of water to another centrifuge tube. To each tube 30 µL erythrocyte

suspension was added and mixed gently by inversion. A pair of tubes was kept in

incubator for 20 min at 54ºC, another pair of test tube kept in an ice bath at 0–5ºC.

After incubation, reaction mixture was centrifuged at 1300 × g for 3 min. The

absorbance (A) was read at 540 nm using UV-Vis spectrophotometer. Acetyl salicylic

acid used as standard.

Percent (%) activity = [1 – (A2-A1)]/ [A3-A1] × 100

A1 = test sample unheated; A2 = test sample heated; A3 = control sample heated

139

Hypotonic solution induced haemolysis

The isotonic buffer solution was prepared by taking 154 mM NaCl in 10 mM

sodium phosphate buffer (pH 7.4). The test was performed in duplicate pair of test

tubes. Hypotonic solution containing different concentration of extract/fraction was

taken in test tube and mixed with 30 µL of stock erythrocyte suspension, while

control sample was prepared with drug free solution. The test tubes were incubated

for 10 min in room temperature, and then centrifuged at 1300 × g for 3 min. The

absorbance (A) was measured at 540 nm. Acetyl salicylic acid used as standard.

Percent (%) activity = [1 – (A2-A1)]/ [A3-A1] × 100

A1 = test sample in isotonic solution; A2 = test sample in hypotonic solution; A3 =

control sample in hypotonic solution.

4.15. EXPECTORANT AND ANTITUSSIVE ACTIVITY OF M.

MINUTA

4.15.1. Antitussive Activity

4.15.1.1. Ammonium liquor induced cough

Briefly, extract/fraction, vehicle, standard was orally administered 1h prior to

each mouse, was placed in a glass chamber exposed to 0.3 mL 25% NH4OH produced

by a nebulizer for 45 s. Animals were monitored during ammonia exposure and the

cough frequency and latent period of cough were recorded for 6 min. Codeine

phosphate (30mg/kg) used as standard [99].

140

4.15.1.2. Sulphur dioxide (SO2) induced cough

Mice were treated with extract/fraction, standard, vehicle continuously for 5

days. NaHSO3 solution was prepared in water (500 mg/kg, 2.0 mL) was placed at the

base of special deigned desiccator and 0.2 mL of sulphuric acid was added using a

pipette; results in formation of sulphur dioxide gas. After 15 seconds, each mouse was

placed in desiccator and exposed to SO2 for 45 s. The mice were then removed and

placed in a clear glass chamber for counting of bouts of cough for next five minutes.

Codeine phosphate (30mg/kg) used as standard [92].

4.15.2. Expectorant Activity

After 1 week of acclimatization, mice were divided into different groups as

described earlier and 0.5%, 1.0 g/kg ammonium chloride was used as positive control.

The treatment was last for 5 days; after 30 min of last administration mice were

injected with 5% phenolsulfonphthalein and after 40 min animals were dissected to

take out trachea and bronchia parts. NaHCO3 solution (5 %) was used to flush inside

of these parts three times with 0.5 mL for each time, which were collected and

centrifuged to measured the absorbance of supernatant at 546 nm using UV–Vis

spectrophotometer [93].

4.16. CHROMATOGRAPHIC SEPARATION OF EXTRACTS

Most potent bioactive extract from each plant was identified by in vitro and ex

vivo antioxidant activity, analgesic and antiinflammatory activity, expectorant and

antitussive activity. Identified potent extract (methanol extract of both plants) was

fractionated using column chromatography technique by taking borosilicate column

(40 mm × 600 mm). Silica gel (60-80 mesh) served as stationary phase. About 120 g

141

of silica gel was mixed with 200 mL of petroleum ether, and poured slowly to pack

the column up to a hight of 20 cm. The procedure was carefully performed to avoid

deposition of any air bubble during packing. A quantity of 30 g of extract was

digested with 30 g of silica gel and dried under a stream of cold air, then spread over

the column packing. Fractions were eluted successively with petroleum ether, ethyl

acetate and methanol. A volume of 2.5 L of each solvent was used to elute

successively with petroleum ether followed by EtOAc and MeOH to obtain petroleum

ether fraction (PFM), ethyl acetate fraction (EFM), methanol fraction (MFM) of

methanol extract of Marsilea minuta, and petroleum ether fraction (PFP), ethyl

acetate fraction (EFP), methanol fraction (MFP) of methanol extract of Phyllanthus

acidus. All the eluents were dried by using rotary vaccume evaporator.

4.17. DETERMINATION OF RETARDATION (Rf) VALUE

TLC was carried out using pre-coated aluminium sheets with silica gel 60 (20

× 20 cm, layer thickness 0.2 mm) (SD Fine-Chen Limited, Mumbai). The plates were

cut into smaller pieces (5 × 10 cm size). The 1% extract/fraction filtered solution (2-5

μl) was spotted using a fine bore capillary tube 1 cm above the base at same distance

from each other. Chromatograms were developed in a room temperature (28°C) using

different solvent systems at an angle of 75°. The solvent system was allowed to run to

a distance of 9 cm from the base of the plates. The time required for the completion

chromatogram varied from 20-30 min, and then the plates were taken out from the

chamber and allowed to dry in air. The plates were placed in iodine chamber to

identify the presence of the spots [202]. Rf (Retardation factor) values were calculated

using the following formula,

Distance traveled by the solute

Distance traveled by the solvent R

f value =

142

4.18. IN VITRO AND EX VIVO ANTIOXIDANT ACTIVITY OF

FRACTIONS

DPPH radical scavenging activity, nitric oxide radical scavenging activity, total

antioxidant activity, lipid peroxidation activity was performed for the fractions using

the same method discussed earlier.

4.19. HEPATOPROTECTIVE AND IN VIVO ANTIOXIDANT

ACTIVITY OF FRACTIONS

Hepatoprotective and in vivo antioxidant effect of MFP, EFP, PFP, MFM,

EFM, and PFM were evaluated against paracetamol induced hepatotoxicity as

described by Singh and Handa (1995) and Manokaran et al. (2008) [128, 129].

4.19.1. Paracetamol Induced Hepatotoxicity

Fractions of different concentrations, paracetamol, and silymarin were

administered orally and the dose was prepared by suspending in 0.5% sodium CMC.

Respective drug treatment was continued once in a day for consecutive three days. A

single dose paracetamol on 3rd

day at a dose of 3g/kg was administered orally to the

animals of all groups except normal control, after 30 min the test sample in vehicle

was administered. Blood sample was collected by direct cardiac puncture under light

ether anaesthesia after 48 h of paracetamol oral administration, and serum was

separated for the biochemical estimations.

143

4.19.2. Biochemical Determinations

Biochemical parameters related to hepatotoxicity like SGOT, SGPT, ALP,

total bilirubin, direct bilirubin, total cholesterol, total triglycerides were assayed by

using commercial diagnostic kits.

4.19.2.1. SGOT determination

Quantitative determination of SGOT in serum was performed by using Agapee

Diagnostic kit (Batch no: 4501, Manufacturing date: 9/11, Expiry date: 11/13) and the

principle mechanism of estimation is based on the following reactions,

Aspartate + Alpha Ketoglutarate Oxaloacetate + L-Glutamate

Aspartate aminotransferase

Oxaloacetate + NADH + H+

Malatedehydrogenase

L - Malate + NAD+

Laboratory procedure: the estimation was performed by Mispa semi auto analyser

and the following parameter was imputed in the system before analysing the samples.

Mode of reaction – kinetic, Slope of reaction – decreasing, Blank – distilled water,

Wavelength – 340 nm, Factor – 1745, Linearity – 1000 U/L, Delay time – 60 sec, no

of readings – 3, Interval – 20 sec.

Sample was prepared by mixing 0.1 mL of serum sample with 1.0 mL of working

reagent, incubated for 1 min at 37°C and the sample was analysed. Results were

expressed as U/L.

144

4.19.2.2. SGPT determination

Quantitative determination of SGPT in serum was performed by using Agapee

Diagnostic kit (Batch no: 4063, Manufacturing date: 9/11, Expiry date: 11/13) and the

principle mechanism of estimation is based on the following reactions,

Alanine + Alpha Ketoglutarate Pyruvate + L-Glutamate

Aspartate aminotransferase

Pyruvate + NADH + H+

Lactatedehydrogenase

L - Lactate + NAD+

Laboratory procedure: the estimation was performed by Mispa semi auto analyser

and the following parameter was imputed in the system before analysing the samples.

Mode of reaction – kinetic, Slope of reaction – decreasing, Blank – distilled water,

Wavelength – 340 nm, Factor – 1745, Linearity – 1000 U/L, Delay time – 60 sec, no.

of readings – 3, Interval – 20 sec.

Sample was prepared by mixing 0.1 mL of serum sample with 1.0 mL of working

reagent, incubated for 1 min at 37°C and the sample was analysed. Results were

expressed as U/L.

4.19.2.3. ALP determination

Quantitative determination of ALP in serum was performed PNPP kinetic

method by using Agapee Diagnostic kit (Batch no: 4781, Manufacturing date: 8/11,

Expiry date: 8/13) and the principle mechanism of estimation is based on the

following reactions,

p-Nitrophenylphosphate + H2O

Alkalinephosphatase

p-Nitrophenol + Phosphate

145

Laboratory procedure: the estimation was performed by Mispa semi auto analyser

and the following parameter was imputed in the system before analysing the samples.

Mode of reaction – kinetic, slope of reaction – increasing, blank – distilled water,

wavelength – 405 nm, factor – 2754 , linearity – 1000 U/L, delay time – 60 sec, no. of

readings – 4, interval – 60 sec.

Sample was prepared by mixing 0.1 mL of serum sample with 1.0 mL of working

reagent (mixed after incubating 37°C for 1 min), incubated for 1 min at 37°C and the

sample was analysed. Results were expressed as U/L.

4.19.2.4. Bilirubin determination

Quantitative determination of total and direct bilirubin in serum was

performed by using Agapee Diagnostic kit (Batch no: 670, Manufacturing date: 6/11,

Expiry date: 6/13) and the principle mechanism of estimation is based on the reaction

between sulfanilic acid (present in direct bilirubin reacgent) with sodium nitrate in

presence of diazotied sulfanilic acid to form azobilirubin. Direct bilirubin only reacts

in absence of methyl sulfoxide to give azobilirubin.

Laboratory procedure: The test and blank solution was prepared in the following

manner,

Reagents/ sample Total bilirubin Direct bilirubin

Sample blank Test Sample blank Test

Total bilirubin reagent 1000 mL 1000 mL - -

Direct bilirubin reagent - - 1000 mL 1000 mL

Serum 50 mL 50 mL 50 mL 50 mL

146

All solutions were mixed well, incubated for 5 min at room temperature and the

absorption of the resulting solutions was taken using semi auto analyser. Following

parameter were feed in auto analyser before the determination of absorbance.

Mode of reaction – end point, slope of reaction – increasing, wavelength – 546 nm,

linearity – 20 mg/dL, factor for total bilirubin – 20.5, factor for total bilirubin – 16.5.

Results were expressed as mg/dL.

4.19.2.5. Total cholesterol determination

Quantitative determination of total cholesterol in serum was performed

CHOD-PAP method by using Agapee Diagnostic kit (Batch no: CH-0612,

Manufacturing date: 1/11, Expiry date: 1/13) and the mechanism of estimation is

based on the principle that, cholesterol esters are dissociated into cholesterol and fatty

acids in presence of cholesterol esterase. Subsequently cholestenone forms by

enzymatic oxidation with cholesterol oxidase, hydrogen peroxide. Red quinoeimine

dye forms when hydrogen peroxide reacts with p-aminoantipyrine and phenol in

presence of peroxidase.

Chloesterol ester + H2OCholesterol esterase

Cholesterol + Fatty acids

Cholesterol + O2Cholesterol oxidase

4 Cholesten-3-one + H2O2

2H2O2 + Phenol + 4 Aminoantipyrine Red quinone + 4 H2OPeroxidase

Procedure: Working reagent (1 mL) mixed with 0.01 mL of serum sample or standard

and mixed well. Solution was incubated for 5min at 37°C. The absorbance of sample

147

and standard was measured by taking working reagent as blank. Following parameter

were feed in auto analyser before the determination of absorbance.

Mode of reaction – end point, slope of reaction – increasing, wavelength – 505 nm,

linearity – 600 mg/dL, standard concentration – 200 mg/dL. Result was expressed as

mg/dL.

4.19.2.6. Total triglycerides determination

Quantitative determination of triglyceride in serum was performed CHOD-

PAP method by using Agapee Diagnostic kit (Batch no: 650, Manufacturing date: 5/11,

Expiry date: 5/13), Hyderabad and the mechanism of estimation is based on the

following principle,

Triglycerides Lipoprotein lipase

Glycerol + Free fatty acids

Glycerol + ATP

Glycerol kinaseGlycerol 3 phosphate + ADP

Glycerol 3 phosphate + O2 Glycerol 3 PO

Dihydroxyacetone phosphate + H2O2

H2O2 + 4 Aminoantipyrine + PhenolPeroxidase

Red quinoneimine dye + H2O

Procedure: Working reagent (1 mL) was mixed with 0.01 mL of serum sample or

standard. Solution was and incubated for 37°C for 10 min and the absorbance of

sample and standard was measured using working reagent as blank in semi auto

analyser. Following parameter were feed in auto analyser before the determination of

absorbance.

148

Mode of reaction – end point, slope of reaction – increasing, wavelength – 546 nm,

linearity – 1000 mg/dL, standard concentration – 200 mg/dL. Result was expressed

as mg/dL.

4.19.3. In vivo Antioxidant Activity

The level of SOD, CAT, GPx, GSH in liver tissue, and blood plasma GSH

level in drug treated and hepatotoxic animals was evaluated to determine in vitro

antioxidant activity.

4.19.3.1. Preparation of liver homogenate

Animal was anesthetised and the liver was dissected out and was perfused

with cooled 0.15 M KCl. The 10% homogenate was prepared in 0.15 M KCl-10 mM

potassium phosphate buffer, pH 7.4 and was centrifuged at 3000 rpm for 10 min

[203].

4.19.3.2. Estimation of superoxide dismutase

Inhibition of autocatalyzed adrenochrome formation in the presence of tissue

homogenate was determined at 480 nm to estimate SOD activity. The reaction

mixture was prepared by taking 150 µl of homogenate, 1.8 mL of 30 mM carbonate

buffer (pH, 10.2), 0.7 mL of distilled water and 400 µl of epinephrine (45 mM).

Control was prepared by performing auto oxidation of epinephrine to adrenochrome

without the homogenate [204]. The enzymatic activity was represented as nmol of

epinephrine protected from oxidation/min/mg protein using a molar extinction

coefficient of 4.02 × 103 M-1

cm-1

.

149

4.19.3.3. Estimation of catalase

The catalysis activity was determined by the conversion of H2O2 to H2O in an

incubation mixture adjusted at pH 7.0 and was recorded at 254 nm. The reaction

mixture was prepared by mixing 1.9 mL of 50 mM potassium phosphate buffer pH

7.0 and 0.1 mL supernatant of tissue homogenate and the reaction was started with the

addition of 0.1 mL of 30 mM H2O2. Absorbance was taken at 240 nm [205].One unit

of catalase activity was represents the amount of enzymes causing the breakdown of

µM H2O2/mg protein/min at pH, 7.0 at 25ºC using a molar extinction coefficient of

43.6 M-1

cm-1

.

4.19.3.4. Estimation of glutathione peroxidase

The reduction of oxidised glutathione occurs simultaneously with oxidation of

NADPH catalysed by of glutathione reductase. The reaction mixture was prepared by

taking 100 µl tissue homogenate solution and 800 µl 100 mM/l potassium phosphate

buffer (pH 7.4), containing 1 mM/l EDTA, 1 mM/l sodium azide, 0.2 mM/l NADPH,

1 U/mL glutathione reductase and 1 mM/l GSH. The reaction was started after 5 min

with the addition of 100 µl 2.6 mM H2O2 and the absorbance change was measured at

340 nm in 3 min was recorded at 37ºC [206]. Activity of GPx was determined using

the molar extinction coefficient of NADPH 6220 M−1

cm−1

and represented as µM

NADPH oxidized /min /mg protein at 37ºC

150

4.19.3.5. Estimation of GSH in liver tissue

To precipitate protein 0.125 mL of 25% TCA was mixed with 0.5 mL of tissue

homogenate in a test tube and cooled in ice for 5 min. Again 0.6 mL of 5% TCA was

mixed with the previous mixture and centrifuged at 1500 rpm for 10 min. After

centrifugation 0.3 mL of supernatant was transferred in another test tube and mixed

with 0.7 mL of phosphate buffer (0.2 M, ph 8), freshly prepared 2.0 mL DTNB [5'-5'

Dithiobis (2- nitro benzoic acid)] solution (0.6 mM in 0.2 M sodium phosphate buffer,

pH 8). The absorbance of mixture was measured after 10 min at 412 nm [207]. The

GSH was calculated using a standard curve plotted by taking different concentration

of GSH (5-100 nM) in 5% TCA, and results were expressed as nmol/mg protein.

4.19.3.6. Estimation of blood GSH

Blood glutathione was estimated by mixing fresh blood (0.1 mL) with 0.9 mL

of water and 1.5 mL of precipitating solution (1.67 g glacial metaphosphoric acid, 0.2

g Sodium EDTA, 30 g NaCl in 100 mL water). The solutions mixed well and

incubated at room temperature for 5 min and followed by centrifuged at 3000 × g at

4°C for 15 min. Clear supernatant (0.5 mL) was taken out and mixed thoroughly with

2.0 mL of 0.3 mol/L phosphate solution, 0.25 mL 0.2% DTNB in 1% sodium citrate

solution. A solution was prepared by taking 1.0 mL 0.3 mol/L phosphate solution, 1.0

mL water, 0.5 mL precipitating solution and 0.25 mL DTNB solution which served as

blank. Absorbance of both blank and sample reaction mixtures were taken against

water at 412 nm [208]. The gluthathione concentration was calculated by following

formula taking extinction coefficient 13.6 and molecular weight of glutathione 307.

151

Concentration of GSH (mg GSH/ 100 mL blood) = A X 2.75 X 2.75 X 307 X 100

13.6 X 0.1 X 0.5 X 1000

= A X 341.42

Absorbance (A) = Asample - Ablank

4.20. NEPHROPROTECTIVE ACTIVITY OF FRACTIONS

Nephroprotective effect of MFP, EFP, PFP, MFM, EFM, PFM were evaluated

against cisplatin induced nephrotoxicity [131].

Fractions, standard drug and vehicle were administered orally for six

consecutive days once daily. On fourth day cisplatin (20 mg/kg) injected

intraperitoneally to induce nephrotoxicity to all animals expect normal control group.

All animals were sacrificed 72 h after nephrotoxicity induced.

4.20.1. Sampling and Biochemical Analyses

Cisplatin was administered in mice and were sacrificed after 72 h. Blood

samples were collected and centrifuged at 5000 rpm for 10 min to obtain clear sera

which were used for the estimation of different biochemical parameters like BUN,

serum creatinine, uric acid, total protein, and albumin. Commercial biochemical kit

was used to estimate all biochemical parameters obtained from Agapee Diagnistic

Ltd., Kerala. Kidney tissue was used to determine level of tissue MDA.

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4.20.1.1. Estimation of BUN

Quantitative determination of BUN in serum was determined by following

manufacturer procedure (Batch no: 489, Manufacturing date: 1/12, Expiry date: 1/13).

Standard sample was prepared by mixing of 1.0 mL of working reagent with 0.01 mL

of standard given with commercial kit. Sample solution was prepared by mixing 1.0

mL of working reagent with 0.01 mL of serum sample. The solutions are mixed well

and optical density (T1) was determined after 30 sec at 340 nm. Second reading (T2)

was taken exactly 60 sec after first reading. Distilled water used as blank. The

concentration of BUN was calculated using the following formula,

BUN concentration (mg/dL) =(T1 - T2) sample

(T1 - T2) sandardX 23.4

4.20.1.2. Estimation of serum creatinine

Serum creatinine was determined by Modified Jaffe’s method as per procedure

described by manufacturer (Batch no: 769, Manufacturing date: 1/12, Expiry date:

1/13). Creatinine reacts with picric acid to form creatinine alkaline picrate, a coloured

compound. Observed change in absorbance is proportional to creatinine

concentration.

Briefly, standard and sample solution was prepared by mixing of 1.0 mL of

working reagent with 0.1 mL of standard given with commercial kit and 0.1 mL of

serum sample respectively. The solutions were mixed well and optical density (T1)

was determined after 60 sec and second reading (T2) was taken immediately after 60

sec after first reading at 492 nm using distilled water as blank. The concentration of

creatinine was calculated using the following formula,

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Creatinine concentration (mg/dL) =(T1 - T2) sample

(T1 - T2) sandardX 2

4.20.1.3. Estimation of serum uric acid

Uricase methodology was used to determine serum uric acid level as described

by manufacturer. The principle of the assay is based on the following reaction,

Uric acid + O2 + 2H2O Uricase

Allantoine + CO2 + H2O

2H2O2 + 4 -AAP + EHSPTPeroxidase

Red quinone

EHSPT = N-ethyl N-(2-hydroxy-3-sulfopropyl) n-toluidine

4 -AAP = Amino-4-antipyrine

Standard and sample solution was prepared by mixing of 1.0 mL of working

reagent with 0.025 mL of standard given with commercial kit and 0.025 mL of serum

sample respectively. The solutions are mixed and incubated for 5min at 37°C and the

absorption was taken at 546 nm using working reagent as blank. The concentration of

uric acid was calculated using the following formula,

Uric acid concentration (mg/dL) =Absorbtion of sample

Absorption of satndardX 8

4.20.1.4. Estimation of serum total protein

Protein concentration was determined by direct biuret method as per

manufacturer procedure (Batch no: 298, Manufacturing date: 2/12, Expiry date: 2/13).

Briefly, cupric ion present in working reagent forms blue colour complex with protein

in serum sample. The intensity of the blue colour determined by calorimetric assay is

proportional to the protein concentration.

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Standard and sample solution was prepared by mixing of 1.0 mL of working

reagent with 0.02 mL of standard given with commercial kit and 0.02 mL of serum

sample respectively. The solutions are mixed and incubated for 10min at 37°C. The

absorption was measured at 546 nm taking reagent blank. The concentration of uric

acid was calculated using the following formula,

Total protein concentration (mg/dL) =Absorbtion of sample

Absorption of satndardX 6

4.20.1.5. Estimation of serum albumin

Albumin concentration was determined using bromocresol green method as

per manufacturer procedure (Batch no: 888, Manufacturing date: 10/11, Expiry date:

10/13). Briefly, albumin from serum sample reacts with a dye bromocresol-green

(working reagent) cause colour change which is proportional to protein concentration.

Standard and sample solution was prepared by mixing of 1.0 mL of working

reagent with 0.01 mL of standard given with commercial kit and 0.01 mL of serum

sample respectively. The solutions were mixed and incubated for 1 min and the

absorption was measured at 630 nm using reagent blank. The concentration of uric

acid was calculated using the following formula,

Albumin concentration (mg/dL) =Absorbtion of sample

Absorption of satndardX 3

4.20.1.6. Estimation of MDA

Lipid peroxidation responsible for oxidative degradation of polyunsaturated

fatty acids to MDA, and this formation of MDA is usually estimated by the

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chromogenic thiobarbituric acid reaction and expressed as total TBARS. After

physical euthanasia the kidneys were isolated quickly form each animal and washed

with saline. Kidney was dried using filter paper, weighed, and homogenised using

phosphate buffer (pH 7.4) 10% (w/v). Unbroken cells, cell debris and nuclei were

sedimented by centrifugation for 10 min at 2000 × g, and the supernatant was taken

out which was used for evaluate lipid peroxides (MDA production) in the kidney.

A volume of 0.5 mL tissue homogenate was mixed with 2.5 mL of 10% (w/v)

trichloro acetic acid in a tube. Tubes were placed in a boiling water bath for 15 min

and then cooled in tap water followed by centrifugation at 1000 × g for 10 min. After

centrifugation 2.0 mL of the supernatant was mixed to 1.0 mL of 0.67% (w/v) TBA

solution in a test tube and placed again in a boiling water bath for 15 min. The

solution was then cooled in tap water and its optical density was measured at 532 nm.

The concentration of MDA was calculated by using absorbance coefficient 1.56×105

cm−1

M−1

) and the results were represented as nM/min/mg tissue protein [144].

4.21. ANTI TUBERCULAR ACTIVITY OF FRACTIONS

The anti-TB activity of PFM, EFM, MFM, MFP, EFP, PFP were assessed

against M. tuberculosis using MABA as described by Lourenco et al. (21). MABA is

a non-toxic method, uses a thermally stable reagent and shows outstanding correlation

with propotional and BACTEC radiometric assay. Briefly, sterile 96 wells plate were

taken and sterile deionzed water (200 µl) was added to all outer perimeter wells of

sterile wells plate to reduce the evaporation of medium in the test wells throughout

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incubation. Middlebrook 7H9 broth (100 µl) was mixed in all wells plate and serial

dilution of test sample was made directly on plate. The final fraction concentrations

tested were 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50, and 100µg/mL. Each plate was

enclosed and sealed with parafilm and incubated at 37ºC for five days. Exactly 25 µl

of freshly prepared mixture of Almar Blue reagent was mixed after incubation period

and 10% tween 80 (1:1) was mixed to the plate and then incubated for 24 hrs. A blue

color in the well represents no bacterial growth, while pink colour interpreted as

bacterial growth. The MIC was determined as minimal sample concentration which

prevented the colour change from blue to pink.

4.22. ISOLATION AND IDENTIFICATION OF CHEMICAL

CONSTITUENTS

Pharmacological investigations reveal that methanol fraction from M. minuta

and P. acidus leaves methanol extract showed superior activity. To investigate and to

identify the possible active ingredients/chemical constituent, both the fractions were

further fractionated by using preparative column chromatography.

MFM and MFP were fractionated by using column chromatography by taking

borosilicate column with a dimension of 40 mm × 600 mm. Silica gel (60-120 sieve)

used as stationary phase. Chloroform was used as mobile phase. A weight of 400 g of

silica gel was stirred with 500 mL of chloroform to make slurry and the column was

packed at height of 30 cm and a diameter of 4 cm carefully to form a compact

packing, special care has been taken to avoid the deposition of air bubbles. Solvent

level was maintained few centimeters above of the silica gel layer. About 20 g of

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fraction was mixed with minimum volume of methanol and mixed with 30 g of silica

gel. The mixture was air dried under stream of cold air and spreaded slowly on the top

of the column. The top layer (sample-silica gel mixture) was covered with silica gel

slurry (in chloroform) followed by a piece of cotton wool.

A total of 13 fractions were eluted form MFM (named as MM-1 to MM-13)

and 6 fractions (named as PA-1 to PA-6) eluted form MFP. All the eluants were dried

under reduced pressure.

Figure 4.2: Fractionation of extract

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4.23. ANTIOXIDANT ACTIVITY AND SELECTION OF POTENT

SUB-FRACTION

Fractions PA 1-6 and MM 1-13 were screened for antioxidant activity by

taking 2 fixed concentrations ((20 and 40 μg/mL)) using DPPH● and NO

● scavenging

activity method. Procedures to perform the same were discussed in section 4.11.

Based on the antioxidant study 3 ‘more potent’ sub-fractions from P. acidus

and 3 ‘more potent’ sub-fractions from M. minuta were selected for further analysis

(details are given in result section).

4.24. TLC AND SPECTRAL ANALYSIS

Selected sub-fractions were subjected for TLC using the same procedure and same

solvent system as discussed earlier.

One fraction from P. acidus and one fraction from M. minuta were selected for

spectral analysis as they showed single spot in TLC. Spectra study like IR (in KBr),

1HNMR,

13CNMR were performed to find the possible structure of constituents.

Melting point was determined for isolated compounds by open capillary method

and was uncorrected.

4.25. STATISTICAL ANALYSIS

The data were subjected to analysis of variance (ANOVA) and expressed as

mean ± SEM (n=3 for in vitro and ex vivo model; n = 6 for in vivo tests). Statistical

analysis was carried out by analysis of variance followed by Tukey tests. A level of p

< 0.05 was used as the criterion for statistical significance. For in vitro and ex vivo

antioxidant activity IC50 value was the concentration which cause 50% scavenging

effect and determined graphically.