4. EXPERIMENTAL INVESTIGATIONS -...

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4. EXPERIMENTAL INVESTIGATIONS

4.1. ETHNOBOTANICAL SURVEY

An ethno-medicinal survey was conducted to find the traditional medicinal

plants and their uses by the tribe of Tripura. Ethnomedicinal information on folk

medicinal plants was collected from local peoples (generally more than 50 years old)

through interviewing them. Survey was conducted different hilly and remote villages

of West and South Tripura District of Tripura state. Semi-structured interview, group

discussion, and field observation were used to obtain the information on medicinal

plants. Information on local names of the plants, the parts used, method of preparation

and traditional medicinal uses was collected during the survey. Each area was visited

different seasons to determine the authenticity of information collected during field

work. Proper identification of plants were carried out with the help of forest guards,

traditional medical practitioner (locally known as auchai, kabiraj or vaidyas) and

knowledgeable or scientific persons.

4.2. SELECTION OF PLANTS

A number of ethanobotanical survey and few books published on medicinal

plants of Tripura were reviewed systemically to gather the information about the folk

medicinal plants of Tripura and their traditional uses. It was observed that more than

200 medicinal plants are used by the people of Tripura. The plants and/or plant

materials were selected on the basis of literature survey and information obtained

from local people. In first phase 12 plants were selected based on the folk medicinal

information, which is listed on Table 4.1.

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Table 4.1: List of preliminary selected plants

Scientific Name Family Local Name Traditional Uses

Clerodendrum

viscosum

Verbenaceae bait gach,

chayakkhui

bufang, ghetu

fever, skin infection,

inflammation, laxative, cough,

blood dysentery, anthelmintic

Leea asiatica Leeaceae banchalita against guinea worms, in

wounds, bone fracture,

gastrointestinal diseases

Meyna spinosa Rubiaceae monkata skin disease, peptic ulcer,

diabetes, hepatic disorder

Microcos

paniculata

Tiliaceae pitchla, asar

bufang

bone fracture, cold, typhoid

fever

Phyllanthus

fraternus

Euphorbiaceae bhuiamla,

samuk gach

menstruation cycle disorders,

diabetes, dysentery

Paederia foetida Rubiaceae gandha

bhadali,

dukupui

rheumatic pain, amoebiasis,

tooth ach, stomach pain, as

blood purifier,

Cuscuta reflexa Cuscutaceae sarnalata cough, viral infection, itchy

skin

Galinsoga

parviflora

Asteraceae gangaful,

garingburani

sam

fever, liver problem, wound,

inflammation, insect

bite, toothache

Ipomea aquatica Convolvulaceae kalmi skin disease, dysentery

Meriandra

benghalensis

Labiatae bangal sage high blood pressure, diabetes,

tonsillitis, skin disease

Marsilea minuta Marsileaceae susni sak respiratory troubles, sleeping

disorders, diabetes

Oxalis

corniculata

Oxalidaceae amlati,

amchukai

dysentery, stomach disorders,

rheumatism, toothache

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Scientific literatures on those plants were extensively reviewed. Reported

activities were matched with traditional knowledge. Initially in the view of relation

between oxidative stress and diseases, it was selected to screen antioxidant

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

and other activities were already been reported. Taking this as consideration 5 plants

(Meyna spinosa, Leea asiatica, Marsilea minuta, Meriandra benghalensis, Galinsoga

parviflora) were selected.

In next stage/phase information about freely available plants in Tripura among

the primarily selected plants were collected. It was observed that two indigenous

medicinal plants viz. Meyna spinosa and Leea asiatica has lot of traditional

importance, easily available in the state, widely used by the tribes of Tripura and

preliminary investigation of the leaves showed good DPPH radical scavenging effect.

But these plants were not scientifically investigated for their medicinal uses despite of

vast utility in folk medicine. Therefore I have concentrated on these two plants viz.

Meyna spinosa and Leea asiatica to bring their folk medicinal importance to a

scientific approach.

4.3. PLANT COLLECTION AND AUTHENTICATION

Fresh mature whole leaves of Meyna spinosa Roxb. were collected in October

2010 from Khowai subdivision of Tripura. While the leaves of Leea asiatica (L.)

Ridsdale were collected from Agartala subdivision of Tripura in April 2011. Both the

plants were identified by its vernacular name and later validated by Dr. B.K. Datta,

Department of Botany, Tripura University, Tripura, India. A voucher specimen of

herbarium of plants (TU/BOT/HEB/SS23072011a and TU/BOT/HEB/SS23072011c)

was deposited at the Plant Taxonomy & Biodiversity Laboratory, Department of

Botany, Tripura University.

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4.4. DRUGS AND CHEMICALS

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

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

agencies were tabulated in table 4.2.

Table 4.2: List of chemicals and name of their manufacturing agencies

Company Chemicals

Sigma Aldrich

(Bangalore,

India)

2, 2-diphenyl-picrylhydrazyl (DPPH), Alloxan, Sodium

nitroprusside (SNP), Phenazine methosulfate (PMS), Nitro blue

tetrazolium (NBT), Naphthyl ethylene diamine dihydrochloride

SD Fine Ltd.

Mumbai

Hydrogen peroxide (H2O2), 2-deoxy-ribose, Trichloro acetic acid

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

Petroleum ether, Ethanol, Chloroform, Quercetin, Aluminium

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

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

Sisco Research

Laboratories

Pvt. Ltd.,

Mumbai

Linoleic acid, Ammonium thiocyanate, Ascorbic acid, α-

tocopherol, Butylated hydroxyanisole (BHA), Epinephrine,

Ascorbic acid, Gallic acid, Sulphanilamide, Nicotinamide adenine

dinucleotide-reduced (NADH).

Loba Chemie

Pvt Ltd.,

Mumbai

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

Chlorosulfonic acid, Lead acetate, Phenazone,

Ethelenediaminetetraacetic 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,

Albumin, Glucose, High density lipids (HDL), Low density lipids

(LDL), and α–amylase.

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

analytical grade.

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4.5. INSTRUMENT USED

Following major instruments were used for the present study,

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

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)

4.6. PREPARATION OF EXTRACTS

Leaves of both the plants were cleaned to remove unwanted materials. The

leaves were dried in shade under room temperature, powdered mechanically and

sieved through No. 22 mesh sieve. The finely powdered leaves were kept separately

in an airtight container until the time of use. The leaf powder (1 kg) was extracted

with methanol (5 L) using Soxhlet apparatus. The procedure is repeated by taking

fresh leaves powder and extracted with ethyl acetate and petroleum ether separately.

Each filtrates were then concentrated under reduced pressure and evaporated

at 40ºC to obtain crude methanol extract of M. spinosa (MEMS), ethyl acetate extract

of M. spinosa (EEMS), petroleum ether extract of M. spinosa (PEMS), methanol

extract of L. asiatica (MELA), ethyl acetate extract of L. asiatica (EELA), and

petroleum ether extract of L. asiatica (PELA) leaves. All extracts were stored at 4ºC

till the time of use.

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4.7. PHYSICOCHEMICAL PROPERTIES OF EXTRACT

Physiochemical evaluation of extracts was carried out by determining yield,

colour, pH, density and specific gravity of crude extract.

4.7.1. Colour and Yield Determination

Colour of the extracts was observed in naked eye. The physical properties of

extracts were noted, including percentage yield.

4.7.2. Determination of Specific Gravity

An empty pycnometer was taken and the weight (Wp) of the same was

recorded. Sample (10g) was placed in the pycnometer and the weight (Wps) was

recorded. Pycnometer was filled half or ¾ with distilled water and soaked for10

minute. Partial vacuum was applied to the content for 10 minute to remove entrapped

air. The pycnometer was filled with distilled water up to the mark; the exterior surface

of the pycnometer was cleaned with clean and dry cloth and the weight (Wb) was

determined. Then the pycnometer was cleaned fully by removing the content and

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

was determined using the following formula [194],

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

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Figure 4.1: Extraction of plant materials through Soxhlet apparatus

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4.7.3. Determination of pH

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

pH was recorded by using Elico digital pH meter [195].

4.8. PRELIMINARY PHYTOCHEMICAL INVESTIGATION

Qualitative phytochemical investigation of extracts was carried out to analyse

phytochemicals present in the MEMS, EEMS, PEMS, MELA, EELA and PELA.

Preliminary phytochemical screening will give the idea about the chemical

constituents present in the respective extracts.

Phytochemical test were carried out to find the presence of alkaloids,

carbohydrates, glycosides, protein, amino acids, tannins, flavonoids, triterpenoids and

steroids in the extracts. The screening was done as explained in the standard

literatures [196, 197].

Table 4.3: Procedure for preliminary phytochemical investigation

Test procedure Observation for

positive test

A. Detection of Alkaloids: A small quantity of solvent free extract was stirred

with little quantity of dilute hydrochloric acid (HCl) and filtered, which was used

to test to find the presence of alkaloids. The screening test for alkaloids were

performed using the following tests,

Mayer’s test: Few millilitres of filtrate were mixed with 2

drops Mayer’s reagent (potassium mercuric solution) along

the sides of tube.

White or creamy

precipitate.

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Wagner’s test: Few millilitres of filtrate were mixed with

few drops of Wagner’s reagent (iodine-potassium iodide

solution) along sides of tube.

Reddish brown or

orange precipitate.

Hager’s test: Few millilitres of filtrate were mixed with

2.0 ml of Hager’s reagent (saturated solution of picric

acid).

Prominent yellow or

crystalline yellow

precipitate.

Dragendroff’s test: Few millilitres of filtrate were mixed

with 2.0 ml of Dragendroff’s reagent (potassium bismuth

iodide solution).

Prominent reddish

brown or orange red

precipitate.

B. Detection of Carbohydrates: A small quantity of the extract was dissolved in

few millilitre of distilled water and filtered. The filtrate was subjected to the

following tests,

Molish’s test: Two drops of Molish reagent (alcoholic

solution of α-naphthol) was added to 2.0 ml of filtrate.

The mixture was shaken well and 1.0 ml concentrated

sulphuric acid was added slowly along the sides of the

tube, cool the tube in ice water and allowed to stand.

A violet colour ring

appears at the junction of

layers.

Fehling’s test: One millilitre of filtrate was mixed with

1.0 ml each of Fehling’s solutions A and B. The solution

was heated on water bath.

Brick red precipitate

indicates presence of

reducing sugar.

Barfoed’s test: One millilitre filtrate was added with 1.0

ml Barfoed’s reagent in a test tube and heated on water

bath for 2 minute.

Red precipitate indicates

presence of

monosaccharides.

Benedict’s test: Equal volume of filtrate and Benedict

reagent was taken in a test tube and heated on water bath

for 2 minute.

A characteristic colored

precipitate indicates

presence of reducing

sugar.

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C. Detection of Protein and Amino Acid: Small quantity of sample was

dissolved in few millilitre of distilled water and filtered through Whatman no.1

filter paper and the filtrate was subjected to tests for proteins and amino acids as

follows,

Millon’s test: Two millilitre of filtrate was mixed with few

drops of Millon’s reagent. (Test for amino acid)

White precipitate

indicates

Ninhydrin test: Two drops of ninhydrin solution was

added to 2.0 ml of filtrate and boiled.

Purple/blue/violet

colour for amino acid.

Biuret test: Two millilitre of filtrate was added to 2.0 ml

Biuret reagent.

Violet colour indicates

presence of protein

Xanthoprotein test: Five millilitre of test solution were

mixed with 1.0 ml concentrated HNO3 solution and boiled.

On cooling 40% NaOH solution was added.

Orange colour indicates

presence of protein.

D. Detection of Glycosides (General test for glycosides)

Test A: 200 mg of drug was extracted with 5.0 ml of dilute

H2SO4 by warming on a water bath. The solution was

filtered and extract was neutralised with 5% NaOH

solution. Fehling's solution A and B (each 0.1 ml) was

mixed until it becomes alkaline and heated on a water bath

for 2 minute. The quantity of red precipitate formed during

the test was measured.

Test B: About 200 mg of drug extracted with 5.0 ml of

water by warming on a water bath. Equal amount of water

was added as used for NaOH in the Test A. Fehling's

solution A and B (each 0.1 ml) was mixed until it becomes

alkaline and heat on water bath for 2 minutes. Quantity of

precipitate formed in Test B compared with that of forms

in Test A.

If the precipitate formed

in Test A is more than

in Test B then glycoside

may be present (Test A

represent free reducing

sugar plus those related

on acid hydrolysis of

any glycoside in the

crude drug, whereas

Test B represents the

amount of free reducing

sugar in the crude drug).

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E. Detection of Specific Glycosides: Tests for specific glycosides like

anthraquinone, cardiac glycosides, coumarin, saponin glycosides was also

performed using the following methods,

Borntrager's test: Test sample was boiled with 1.0 ml of

sulphuric acid in a test tube for 5 minute. The solution was

filtered while hot, cool the filtrate and shake with equal

volume of chloroform. Lower layer of chloroform was

separated and shaken with half of its volume of dilute

ammonia.

A rose pink to red

colour will be produce

in the ammoniacal layer

if anthraquinone

glycosides present.

Keller-killiani test: The sample was extracted with

chloroform, and evaporates to dryness. Glacial acetic acid

(0.4 ml) containing trace amount of ferric chloride was

added. Concentrated H2SO4 (0.5 ml) was added carefully

by the side of the test tube.

Acetic acid layer shows

blue colour indicates

presence of cardiac

glycosides.

Raymond’s test: The test solution was treated with hot

methanolic alkali.

Violet colour indicates

presence of cardiac

glycosides.

A small amount of sample in test tube was taken and

covers the test tube with a filter paper moistened, with

dilute NaOH solution. The test tube was placed on water

bath for several minutes. The paper was removed and

expose to UV light.

Paper shows green

fluorescence indicates

presence of coumarin

glycosides.

Haemolysis test: Two millilitre NaCl solution (1.8%) was

taken to the two test tubes. To one of these 2.0 ml of

distilled water and to other 2.0 ml of sample solution was

added. The concentration of NaCl in each tube now is

isotonic to blood. Blood was obtained by pricking the

thumb at the base of the nail, and 5 drops of blood was

added to each and mixed gently.

Haemolysis observed

under the microscope in

the tube containing the

sample, but no

haemolysis in control


saponin glycosides

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Froth formation test: Small amount of extract was

shaken in a test tube with a little quantity of water.

The foam produced will

persists for 10 minute


saponin glycosides.

F. Detection of Steroids and Triterpenoids: The test for flavonoids was

carried out by the following methods,

Libermann-Burchard’s test: The sample was treated

with few drops of acetic anhydride, heated to boiling, and

cooled. One millilitre of concentrated H2SO4 was added

along the side of test tube.

Brown ring at the junction

and upper layer turns to

green indicated presence

of presence of steroids.

Brown ring at the junction

and upper layer turns to

deep red indicates

presence of triterpenoids

Salkowski Test: The sample was treated with few drops

of concentrated H2SO4.

Red colour at lower layer

indicated presence of

presence of steroids.

Yellow colour at lower

layer indicates presence

of triterpenoids

Tschugajeu test: To the chloroform solution of sample

excess of acetyl chloride and a pinch of zinc chloride

were added. Test tube was kept aside for reaction and

then warm on water bath

Erosin red colour

produced indicates


Brieskorn and Brinar test: To the chloroform solution

of test sample a few drops of chrolosulfonic acid in

glacial acetic acid (7:3) was added.

Red colour in few

minutes indicates


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G. Detection of Tannins: The presence of tannin in crude extract was

determined by the following methods,

Ferric chloride test: A small quantity extract was boiled

in few millilitre of distilled water in a test tube and then

filtered. A few drops of 0.1% ferric chloride were added.

Brownish green or a

blue-black colour.

Phenazone test: To 5.0 ml of aqueous extracts sodium

acid phosphate (0.5 g) was added. The mixture was

warmed and filtered. Phenazone solution (2%) was added

in the filtrate.

Bulky precipitate which

often coloured

Gelatin test: A small quantity of sample was dissolved in

distilled water, and 2.0 ml of 1% gelatine solution

containing 10% NaCl was added.

White precipitate

H. Detection of Flavonoids: The presence of flavonoids was determined by the

following methods,

Lead acetate test: To the alcoholic solution of the extract/,

few drops of 10% lead acetate solution was mixed

Yellow precipitate.

Alkaline reagent test: To the few millilitre of test

solution, few drops of 10% NH4OH was added

Intense yellow colour

formed which turns to

colourless on addition

of few drops of dilute

acid.

Zinc Hydrochloride test: To the alcoholic test solution of

the extract a pinch of zinc dust and few drops of

concentrated HCl was mixed.

Red/Megneta colour

developed after few

minute.

Ferric chloride test: To the alcoholic solution of extract,

few drops of ferric chloride were added.

Green colour indicated

presence of flavonoid in

the sample.

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4.9. EXPERIMENTAL ANIMALS

Healthy Albino mice (20-30 g) and Wistar rats (250-250 g) were used for the

study. In acute toxicity study, nephroprotective activity mice were used, and rats were

used for ex vivo antioxidant, antidiabetic, hepatoprotective activity. Animals were

maintained under standard laboratory conditions at a temperature of 23±2ºC, with 12

h light-dark cycle, and relative humidity (5010%) for 1 week. All animals had free

access to pallet chow (Nutrivet Life Science, Pune) and water ad libitum until the day

before experiment, when only water was made available to them.

Indian adult earthworms (Pheretima posthuma) were used for anthelmintic

activity. The species Pheretima posthuma were supplied and authenticated by the

Yamuna Vermi Compost, Kurnool, Andhra Pradesh. The earthworms were collected

from moist soil and to remove all fecal matter washed with normal saline.

Earthworms with 3-5 cm length and 0.1-0.2 cm width were used for in vitro

anthelmintic activity.

All animal procedures have been approved by Institutional Ethical Committee

of Creative Educational Society’s College of Pharmacy, Kurnool, Andhra Pradesh

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

4.10. DETERMINATION OF ANTIOXIDANT COMPOUNDS

4.10.1. Total Phenolic Content Determination

The amount of total phenolics in the crude extracts was determined using

Folin-Ciocalteu reagent following the colorimetric method [92]. The ethanol solution

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of each extract (0.5 ml, l.0 mg/ml) were added into the test tubes containing 2.5 ml of

10% v/v Folin-Ciocalteu reagent and 2.0 ml of 2% w/v sodium carbonate. The tubes

were shaken thoroughly and incubated at 45°C for 15 minute with intermittent

shaking. Absorbance was observed at 765 nm using ELICO SL 164 UV–Vis

spectrophotometer. Gallic acid (0 to 800 µg/ml) was used as standard to get a

calibration curve, 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.

4.10.2. Total Flavonoid Content Determination

Total flavonoid content of extracts was estimated by aluminium nitrate-

potassium acetate reagent method. Briefly, 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. The mixture was mixed thoroughly and allowed to stand for 40

minute at room temperature. The absorbance of the supernatant was measured at 415

nm [94]. The estimation was carried out in triplicate and calculations were based on a

calibration curve obtained with quercetin (0 to 16 µg/ml). The results were expressed

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

4.11. IN VITRO ANTIOXIDANT ACTIVITY

4.11.1. DPPH Radical Scavenging Assay

The hydrogen atom or electron-donation ability of the corresponding sample

was measured from the bleaching of a purple-coloured ethanol solution of DPPH. The

DPPH radical is a stable radical with a maximum absorption at 517 mm that can

108

readily undergo reduction by an antioxidant. Free radical scavenging capacity of

extracts/fractions was measured in terms of hydrogen-donating or radical scavenging

ability, using the stable DPPH radical [98].

Briefly, 1.0 ml of 0.1 mM DPPH methanolic solution was mixed with 3.0 ml

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

room temperature in the dark for 30 min and the absorbance was recorded at 517 nm

[98]. The assay was carried out in triplicate for each sample. One millilitre methanol

with 3.0 ml extracts solution was used as a blank, DPPH solution (1.0 ml, 0.1 mM)

with ethanol (3.0 ml) was served as negative control. The radical scavenging activity

of ascorbic acid was also determined, which served as positive control. The decrease

in absorbance on addition of test samples was used to calculate the antiradical

activity, as 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.

4.11.2. Superoxide Anion Radical Scavenging Activity

The scavenging activity of the sample towards superoxide anion radicals was

measured by the method of Nagulendran et al. [93]. Superoxide anions were formed

in a non-enzymatic PMS-NADH system through the reaction of PMS, NADH, and

oxygen, which was assayed by the reduction of NBT. Superoxide anion reduces the

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yellow dye NBT2+

to produce the blue formazan which was determined

spectrophotometrically at 560 nm.

Briefly, 3.0 ml of Tris-HCl buffer (100 mM, pH 7.4) containing 0.75 ml of

NBT (300 μM) solution, 0.75 ml of NADH (936 μM) solution were mixed with 0.3

ml of extract at different concentration. The reaction was started by mixing 0.75 ml

PMS (120 μM) to the mixture. After 5 minute of incubation at room temperature, the

absorbance of mixture was recorded at 560 nm. BHA was used as standard, all the

tests were carried out in triplicate. Scavenging activity was calculated according to the

equation 1.

4.11.3. Hydroxyl Radical Scavenging Activity

The hydroxyl radical scavenging activity of sample was assayed by using the

2-deoxyribose oxidation method. The 2-deoxyribose is oxidized by hydroxyl radical

that is generated by the Fenton reaction and degraded to malondialdehyde [100].

Therefore decomposing effect of sample on hydroxyl radicals was estimated by the

assay of malondialdehyde chromogen formation due to 2-deoxy 2-ribose degradation.

Briefly, 0.2 ml KH2PO4–KOH (100 mM), 0.2 ml deoxyribose (15 mM), 0.2 ml

FeCl3 (500 mM), 0.1 ml EDTA (1 mM), 0.1 ml ascorbic acid (1 mM), and 0.1 ml

H2O2 (10 mM) were mixed with 0.1 ml sample. The mixture was incubated at 37ºC

for 1 h. After incubation time, 1.0 ml of TBA (1% w/v) was added to mixture

followed by the addition of 1.0 ml of TCA (2.8% w/v). The resultant mixture was

heated on a water bath at 80ºC for 20 minute, so that pink coloured developed. The

absorbance of the solution was measured at 532 nm [106]. Quercetin was used as the

positive control. The scavenging activity of extracts or standard (I %) was calculated

using the equation (1).

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4.11.4. Nitric Oxide Radical Scavenging Activity

Nitric oxide impulsively produced from an aqueous solution of sodium

nitroprusside at physiological pH, and interacts with oxygen to produce nitrite ions,

which may be quantified by the Griess Illosvoy reaction. The nitrite ions thus

produced react with Griess reagent that leads to a chromophore formation. The

concentration of this chromophore is proportional to that of the generated nitrite ions.

Antioxidant compounds compete with oxygen leading to reduced production of nitric

oxide [92, 98].

Four millilitres of sample solution at different concentrations were mixed with

1.0 ml of 25 mM sodium nitroprusside solution in a test tube, and incubated for 2 h at

37ºC. An aliquot (2.0 ml) of the incubated solution was taken and mixed with 1.2 ml

Griess reagent (1% sulfanilamide in 5% H3PO4 and 0.1% naphthyl ethylene diamine

dihydrochloride). The chromophore formed by diazotization of the nitrite with

sulfanilamide and subsequent coupling with naphthylethylenediamine

dihydrochloride. The absorbance was measured immediately at 570 nm [95]. Control

experiment was also carried out in similar manner, using distilled water in the place of

sample solution. The experiment was performed in triplicate, ascorbic acid was used

as positive control and I% was calculated using the equation (1).

4.11.5. Metal Chelating Ability

The capacity of antioxidants to form insoluble metal complexes with ferrous

ion or to generate steric hindrance which avert the interaction between metal and lipid

can be investigated using the iron chelating ability assay method. Ferrozine can make

111

red colored complexes with ferrous ions, which was interrupted by chelating agents.

Thus, the activity of antioxidants is determined by monitoring the decrease in

absorbance of the red Fe2+

-ferrozine complex as antioxidants compete with ferrozine

in chelating ferrous ion [93, 101].

Extracts at different concentration (0.4 ml) was mixed with 2 mM FeCl2

solution (0.05 ml). The reaction was started by the mixing of 0.2 ml of ferrozine (5

mM), and total volume of the solution was adjusted to 4.0 ml using ethanol. The

mixture was shaken vigorously and left standing for 10 minute at room temperature.

The absorbance of the solution was measured spectrophotometrically at 562 nm [104].

α-tocopherol was used as standard and percentage of inhibition of ferrozine–Fe2+

complex formation (I%) was calculated by using equation (1).

4.11.6. Reducing Power Ability

The potassium ferricyanide reduction method was used to evaluate the ability

of phenolic compounds to quench radicals through electron donation. Antioxidant

capacity of the sample was monitored by measuring the absorbance of Perl’s Prussian

blue complex at 700 nm, the absorbance was increases as antioxidants reduce the

ferric ion/ferricyanide complex to the ferrous form [101].

Briefly, different concentration of sample solutions (1.0 ml) were mixed with

0.2 M phosphate buffer (2.5 ml, pH 6.6) and 1% (w/v) K3Fe(CN)6 (2.5 ml). The

resulting mixture was incubated at 50ºC for 20 minute, and then 10% TCA (2.5 ml)

was added. The solution was centrifuged at 12000 rpm for 10 minute, and 2.5 ml of

supernatant solution was pipette out to mix with 2.5 ml of distilled water, 0.5 ml of

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0.1% (w/v) FeCl3 solution. The absorbance of solution was determined at 700 nm

using ELICO SL 164 UV–Vis spectrophotometer [105]. The reducing power ability

of ascorbic acid (25-400 µg/ml) was also determined. The test was run in triplicate

and averaged.

4.11.7. Hydrogen peroxide Scavenging Activity

The hydrogen peroxide-scavenging ability of extracts was determined

according to the method of used by Bozin et al. [96]. Briefly, a solution of 40 mM

H2O2 and crude extracts/standard in different concentrations were prepared in

phosphate buffer (pH 7.4). An aliquot (3.4 ml) sample solution was added to 0.6 ml

of H2O2 solution. The absorbance of resulting solutions was taken at 230 nm. Gallic

acid was used as positive control. The percentage of H2O2 scavenging (I%) of

examined extracts was calculated by equation (1).

4.11.8. Ferric Thiocyanate Method (FTC)

The FTC method was employed to determine the peroxide level during the

initial stage of lipid oxidation. Linoleic acid oxidation cause the generation of

peroxides, which react with Fe2+

to form Fe3+

. The ferric ion form a red colored

complex with SCN– and this complex has a maximum absorbance at 500 nm [103].

Sample (200 µg) in 4.0 ml ethanol was mixed with 2.5% linoleic acid in

ethanol (4.0 ml), phosphate buffer (8.0 ml, 0.05 M, pH 7.0) and distilled water (4.0

ml). The mixture was placed in screw cap tube, mixed vigorously and incubated in the

113

dark conditions at 40ºC. One millilitre solution was drawn from the incubated reaction

mixture and mixed with 9.7 ml ethanol (75%) and 0.1 ml ammonium thiocyanate

(30%). Then, 0.1 ml 20 mM ferrous chloride in 3.5% HCl was added to the reaction

mixture, and exactly 3 minute later the absorbance of the red colour solution was

measured at 500 nm. The procedure was repeated every 24 h until the absorbance of

the control reached a maximum [103]. The control and standard were prepared as the

sample. Linoleic acid mixture without the addition of sample was used as the control;

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

4.12. EX VIVO ANTIOXIDANT ACTIVITY

4.12.1. Lipid Peroxidation Assay

A healthy Wister rat was fasted for 16 h, and sacrificed by decapitation. The

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

homogenate was prepared in phosphate buffer saline. One millilitre liver homogenate

was mixed with 100 μl of sample solution and incubated for 2 h at 37°C. After

incubation time, 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 minute and

cooled. The final volume of the solution was made up to 5.0 ml with deionized water

and then centrifuged for 10 minute at 2800 rpm. The supernatant solution was

removed carefully and absorbance supernatant was recorded at 532 nm [99].

Control was prepared without extracts. Rutin was used as positive control. The

inhibition of lipid peroxidation was calculated according to equation (1), where,

114

Asample is the absorbance of an extract/standard in presence of liver homogenate and

Acontrol is the absorbance of the solution containing all reagents except the

test/standard sample.

4.12.2. Oxidative Haemolysis Assay

The assay method of Su et al. (2009) was performed with slight modifications

as described by Coulibaly et al. (2011) [97, 102]. Blood sample was collected from rat

eyepit under mild anaesthesia and centrifuged at 1500 × g for 10 minute at 4ºC. The

erythrocytes were isolated from the plasma and buffy coat, and suspended in 10.0 ml

phosphate buffer saline (PBS, 10 mM, pH 7.4). The erythrocyte in PBS was

centrifuged again at 1500 × g for 5 minute to remove buffy coats. The process was

repeated three times; the supernatant and buffy coats of white cells were carefully

removed after each wash. Washed erythrocytes made to 0.5% erythrocyte suspension

in PBS (10 mM, pH 7.4) for the assay.

Briefly, the reaction mixture was consisted of 0.5 ml of erythrocyte

suspension, 0.5 ml extract/standard drug solution at different concentration and 0.05

ml of 100 mM H2O2. Mixture was kept for incubation at 37ºC for 60 minute, and after

incubation period 4.2 ml of distilled water was added. The solution was centrifuged

for 10 minute at 1000 rpm, and absorbance of the supernatant was measured at 415

nm.

Control contains supernatant without extract. 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).

115

4.13. CHROMATOGRAPHIC FRACTIONATION OF EXTRACT

In vitro and ex vivo antioxidant study revealed that methanol extract of both

plant possess superior antioxidant activity. Therefore, most effective solvent extract

viz. methanol extract of leaves of M. spinosa and methanol extract of leaves of L.

asiatica was chromatographed in silica gel column (borosilicate column, 40 mm ×

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

was mixed with 200 ml of petroleum ether, and poured slowly to pack the column to

get a hight of 20 cm. The procedure was carefully performed to avoid deposition of

any air bubble. A quantity of 80 g of extract was dissolved in little quantity of

methanol and digested with 120 g of silica gel and dried under a stream of cold air,

then spread over the column packing and eluted successively with petroleum ether,

ethyl acetate and methanol. A volume of 2.5 l petroleum ether was eluted first

followed by 2 l of ethyl acetate and 2 l of methanol to obtain petroleum ether fraction

(PFMS), ethyl acetate fraction (EFMS), methanol fraction (MFMS) of methanol

extract of Meyna spinosa, and petroleum ether fraction (PFLA), ethyl acetate fraction

(EFLA), methanol fraction (MFLA) of methanol extract of Leea asiatica.

4.14. THIN LAYER CHROMATOGRAPHY (TLC) STUDY

Thin layer chromatography (TLC) was performed on pre-coated aluminium

sheets with silica gel 60 (20 × 20 cm, layer thickness 0.2 mm) procured from SD

Fine-Chen Limited, Mumbai. The plates were cut in smaller pieces to get 5 × 10 cm

size of each smaller plate.

116

Figure 4.2: Diagram of column chromatography

117

The 2-5 μl of the extract/fraction filtered solution (usually 1%) was spotted

using a fine bore capillary tube. The spot was placed (1 cm above the base) at

equidistance from each other. Chromatogram were developed using different solvent

systems in a room temperature (28°C and at an angle of 75°). The solvent system was

allowed to run to a distance of 8 cm from the application point of the extract/fraction

in the plates. The time required for the completion of run varied from 20-30 minute,

and then the plates were removed from the chamber and allowed to dry in air. These

plates were placed in iodine chamber and the presence of the spots was identified

[197]. Retardation factor (Rf) is the ratio of distance travelled by the solute (extract/

fraction) and the distance travelled by the respective solvent front. Rf values were

calculated using the following formula,

Distance traveled by the solute

Distance traveled by the solvent Rf value =

4.15. ACUTE TOXICITY STUDY

The acute toxicity for samples (potent extract and its fractions) was

determined in Albino mice. The animals were fasted overnight prior to the

experiment. Fixed dose [Organisation for Economic Co-operation and Development

(OECD) Guideline no. 423, Annexure 2d] method of Committee for the Purpose of

Control and Supervision of Experiments on Animals (CPCSEA) was adopted for

toxicity studies. The tested extracts suspended in 0.5% w/v sodium carboxy methyl

cellulose (CMC) and were administrated orally (1 ml/100 g) in 3 animals. The

presence or absence of any signs of toxicity or mortality was monitored at 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 recorded within the 7 days of

observation [198].

118

4.16. HEPATOPROTECTIVE AND IN VIVO ANTIOXIDANT

ACTIVITY

Hepatoprotective and in vivo antioxidant effect of MEMS, MELA and

fractions of MEMS, MELA were evaluated against paracetamol induced

hepatotoxicity [111, 112].

4.16.1. Paracetamol Induced Hepatotoxicity

Experiment was performed two stages. In first stage hepatoprotective activity

of extract was performed and in second stage effect of fractions on paracetamol

induced hepatotoxicity was evaluated.

In first stage, animals were divided into seven groups each containing six,

Group I served as control, Group II served as negative control, Group III treated with

standard drug silymarin, Group IV-V treated with MEMS (150 and 300 mg/kg, b.w.),

Group VI-VII treated with MELA (150 and 300 mg/kg, b.w.).

To determine hepatoprotective effect of fractions animals are divided into 15

groups (n=6) in following manner,

Group I (Control) : Vehicle (water, 1 ml/kg)

Group II (Negative control) : Vehicle (water, 1 ml/kg)

Group III (standard) : Silymarin (25mg/kg)

Group IV-IX : MFMS, EFMS, PEMS at 75 and 150 mg/kg

Group X-XV : MFLA, EFLA, PELA at 75 and 150 mg/kg

119

Animals of the test groups were given the respective drug treatment one daily

for consecutive three days. All the extracts/fractions, silymarin, and paracetamol were

administered orally by suspending in 0.5% sodium carboxy methyl cellulose (CMC).

Animals of all groups except normal control group received paracetamol (3 gm/kg) as

single dose on 3rd

day, thirty minutes after the administration of test samples or

vehicle respectively. After 48 h of paracetamol oral administration, the blood was

collected by direct cardiac puncture under light ether anaesthesia and serum was

separated by centrifugation of blood at 4000 × g, which was used for the biochemical

estimations.

4.16.2. Biochemical Determinations

Biochemical parameters like SGOT, SGPT, ALP, total bilirubin, direct

bilirubin, TC, TG were determined by using commercial biochemical kit obtained

from Agapee Diagnistic Ltd., Kerala

4.16.2.1. SGOT determination

Quantitative determination of SGOT in serum was performed according to the

manufacturer’s procedure (SGOT Kit Batch no: 4501, Manufacturing date: 9/11,

Expiry date: 11/13), which is based on the following reactions,

Aspartate + alpha Ketoglutarate Oxaloacetate + L-Glutamate

Aspartate aminotransferase

Oxaloacetate + NADH + H+

Malatedehydrogenase

L - Malate + NAD+

120

Laboratory procedure: Serum sample (0.1 ml) was mixed with 1.0 ml of working

reagent and incubated for 1 minute at 37°C. The absorption of the resulting was taken

at semi auto analyser. Following parameter were feed in auto analyser before the

determination of absorbance, 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. Results was

expressed as U/l.

4.16.2.2. SGPT determination

Quantitative determination of SGPT in serum was performed according to the

procedure described by the manufacturer (SGPT Kit Batch no: 4063, Manufacturing

date: 9/11, Expiry date: 11/13), which is based on the following reactions,

Alanine + alpha Ketoglutarate Pyruvate + L-Glutamate

Aspartate aminotransferase

Pyruvate + NADH + H+

Lactatedehydrogenase

L - Lactate + NAD+

Laboratory procedure: Serum sample (0.1 ml) was mixed with 1.0 ml of working

reagent and incubated for 1 minute at 37°C. The absorption of the resulting was taken

at semi auto analyser. Following parameter were feed in auto analyser before the


decreasing, blank – distilled water, wavelength – 340 nm, factor – 1745 , linearity –


expressed as U/l.

121

4.16.2.3. ALP determination

The level of ALP in serum was determined according to the procedure

described by the manufacturer (ALP Kit Batch no: 8721, Manufacturing date: 8/11,

Expiry date: 8/13), which is based on the following reactions,

p-Nitrophenylphosphate + H2

O

Alkaline

phosphatasep-Nitrophenol +

Inorganic phosphate

Laboratory procedure: Working reagent (1.0 ml) and test serum (0.02 ml) was mixed

and incubated again 1 minute at 37°C. The absorption of the resulting was taken at

semi auto analyser. Following parameter were feed in auto analyser before the


increasing, blank – distilled water, wavelength – 405 nm, factor – 2750 , linearity –


expressed as U/l.

4.16.2.4. Bilirubin determination

Total and direct bilirubin in serum was performed according to the

procedure described by the manufacturer (Bilirubin Kit Batch no: 670, Manufacturing

date: 06/11, Expiry date: 06/13). The assay is based on the principle that, salfanilic

acid (present in direct bilirubin reagent) reacts with sodium nitrate to produce in the

presence of diazotied sulfanilic acid to form azobilirubin. In the absence of dimethyl

sulfoxide only direct bilirubin reacts to give azobilirubin.

122

Laboratory procedure: Test and blank solution were prepared in the following

manner,

Reagents/ sample Total bilirubin Direct bilirubin

Sample blank Test Sample blank Test

Total bilirubin reagent 1.0 ml 1.0 ml - -

Direct bilirubin reagent - - 1.0 ml 1.0 ml

Serum 0.05 ml 0.05 ml 0.05 ml 0.05 ml

All samples were mixed well and incubated at room temperature for 5 minute.

The absorption of the resulting was taken at 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. Result was expressed

as mg/dl.

4.16.2.5. Total cholesterol determination

Total cholesterol (TC) was estimated by CHOD-PAP method as per

manufacturer procedure (Total cholesterol Kit Batch no: CH-0612, Manufacturing

date: 1/12, Expiry date: 1/13). In this assay, cholesterol esters are dissociated into

cholesterol and fatty acids in presence of cholesterol esterase. In the subsequent

enzymatic oxidation by cholesterol oxidase, H2O2 and cholestenone was formed. In

presence of peroxidase, H2O2 reacts with p-aminoantipyrine and phenol to form a red

quinoeimine dye.

123

Chloesterol ester + H2OCholesterol esterase

Cholesterol + Fatty acids

Cholesterol + O2Cholesterol oxidase

4 Cholesten-3-one + H2O2

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

Procedure: One millilitre of working reagent mixed with 0.01 ml of serum sample or

standard and mixed well. Solution was and incubated for 37°C for 5 minute. The

absorbance of sample and standard was measured against the blank (working reagent).

The absorption of the resulting was taken at 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 – 505 nm, linearity – 600 mg/dl,

standard concentration – 200 mg/dl. Result was expressed as mg/dl.

4.16.2.6. Total triglycerides determination

Total triglyceride (TG) in serum was estimated by GPO-PAP method using

the manufacturer procedure (Triglyceride Kit Batch no: 650, Manufacturing date: 5/11,

Expiry date: 5/13). The assay is based on following principle,

Triglycerides + H2O

Lipoprotein lipaseGlycerol + Fatty acid

Glycerol + ATP Glycerol kinase

Glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O2

Glycerol-3

Dihydroxyacetone phosphate + H2O

2

H2O

2 + 4 Aminoantipyrine + p-chlorophenol

PeroxidaseRed quinoneimine

-phosphate oxidase

124

Procedure: One millilitre of working reagent was mixed with 0.01 ml of serum

sample or standard and mixed well. Solution was incubated for 37°C for 5 minute.

The absorbance of sample and standard was measured against the blank (working

reagent). The absorption of the resulting was taken at 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 – 505 nm, linearity –

1000 mg/dl, standard concentration – 200 mg/dl. Result was expressed as mg/dl.

4.16.3. In vivo Antioxidant Activity

In vivo antioxidant activity was evaluated by determining the level of SOD,

CAT, GPx, GSH in liver tissue, and blood plasma GSH level in drug treated and

hepatotoxic animals.

4.16.3.1. Preparation of liver homogenate

The liver was dissected out from animals and was perfused with cooled 0.15

M KCl. The 10% homogenate in 0.15 M KCl-10 mM potassium phosphate buffer, pH

7.4 was centrifuged at 3000 rpm for 10 minute [199].

4.16.3.2. Estimation of superoxide dismutase

SOD activity was determined by the inhibition of autocatalyzed adrenochrome

formation at 480 nm in the presence of tissue homogenate. The reaction mixture

contained 150 µl of homogenate, 1.8 ml of 30 mM carbonate buffer (pH, 10.2), and

0.7 ml of distilled water and 400 µl of epinephrine (45 mM). Auto oxidation of

125

epinephrine to adrenochrome was performed in a control tube without the

homogenate [200]. The enzymatic activity was calculated as nM of epinephrine

protected from oxidation/min/mg protein using a molar extinction coefficient of 4.02

× 103 M-1

cm-1

.

4.16.3.3. Estimation of catalase

The catalysis of H2O2 to H2O in an incubation mixture adjusted to pH 7.0 was

recorded at 254 nm. The reaction mixture contained 1.9 ml of 50 mM potassium

phosphate buffer pH 7.0 and 0.1 ml of supernatant and the reaction was started by the

addition of freshly prepared 0.1 ml of 30 mM H2O2. The rate of H2O2 decomposition

was measured spectrophotometrically at 240 nm [201]. One unit of catalase activity

was defined as the amount of enzymes causing the decomposition of µM H2O2/mg

protein/min at pH, 7.0 at 25ºC using a molar extinction coefficient of 43.6 M-1

cm-1

.

4.16.3.4. Estimation of glutathione peroxidase

The reduction of GSSG is coupled to the oxidation of NADPH through

glutathione reductase. The reaction mixture contained 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. After 5 minute the reaction was started by the addition of 100 µl

2.6 mM H2O2 and the absorbance change at 340 nm in 3 min was recorded at 37ºC

[202]. Activity of GPx was determined using the molar extinction coefficient of

NADPH 6220 M−1

cm−1

and expressed as µM NADPH oxidized/min/mg protein at

37ºC.

126

4.16.3.5. Estimation of blood GSH

Blood glutathione was estimated by the method of Khynriam and Prasad

(2001). Briefly, fresh blood (0.1 ml) was mixed with 0.9 ml of water and 1.5 ml of

precipitating solution (1.67 g glacial metaphosphoric acid, 0.2 g Sodium EDTA, 30.0

g NaCl in 100 ml water) were added immediately, mixed well and incubated at room

temperature for 5 minute. After incubation, the mixture was incubated centrifuged at

3000 × g at 4°C for 15 minute. Clear supernatant (0.5 ml) was taken out and mixed

with 2.0 ml of 0.3 mol/l phosphate solution and 0.25 ml 0.2% DTNB in 1% sodium

citrate solution were added and mixed thoroughly. Blank solutions contain 1.0 ml 0.3

mol/l phosphate solution, 1.0 ml water, 0.5 ml precipitating solution and 0.25 ml

DTNB solution. Absorbance of both blank and sample reaction mixtures were taken

against water at 412 nm. The gluthathione concentration was calculated by following

formula taking extinction coefficient 13.6 and molecular weight of glutathione 307

[203].

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) = Absorbance of sample - Absorbance of blank]

4.16.3.6. Estimation of GSH in liver tissue

Briefly, 125 μl of 25% TCA was mixed with 0.5 ml of tissue homogenate in a

test tube to precipitate the protein. The test tubes were cooled in ice for 5 minute and

0.6 ml of 5% TCA again mixed with the previous mixture and centrifuged at 1500

rpm for 10 minute. After centrifugation 0.3 ml of supernatant was collected and

mixed with 0.7 ml of phosphate buffer (0.2 M, ph 8). Freshly prepared 2.0 ml DTNB

127

solution (0.6 mM in 0.2 M sodium phosphate buffer, pH 8) was mixed with above

mixture. The absorbance of mixture was measured after 10 minute 412 nm [204]. The

GSH content was calculated using a standard curve varying from 5-100 nm in 5%

TCA for assay, and results were expressed as nmol/mg protein.

4.17. NEPHROPROTECTIVE ACTIVITY

Nephroprotective effect of MEMS, MELA and fractions of MEMS (i.e.

MFMS, EFMS, PFMS), MELA (i.e. MFLA, EFLA, PFLA were evaluated against

cisplatin induced nephrotoxicity [126].

4.17.1. Experimental Design

Experiment was performed two stages. In first stage nephroprotective activity

of extract was performed and in second stage effect of fractions on cisplatin induced

nephrotoxicity was evaluated.

In first stage, animals were divided into seven groups each containing six,

Group I served as control, Group II served as negative control, Group III treated

with standard drug rutin, Group IV-V treated with MEMS (150 and 300 mg/kg,

b.w.), Group VI-VII treated with MELA (150 and 300 mg/kg, b.w.).

All the test drugs were administered for 6 days once daily. On fourth day

nephrotoxicity was induced in animals of group II-VII by a single i.p. injection of

cisplatin (20 mg/kg). Animals were monitored carefully and sacrificed at 72 h after

nephrotoxicity induced.

128

To determine nephroprotective effect of fractions animals were divided into 15

groups (n=6) in following manner,

Group I (Control) : Vehicle (Normal saline, 0.5 ml/animal/day)

Group II (Negative control) : Vehicle (Normal saline, 0.5 ml/animal/day)

Group III (standard) : Rutin (20 mg/kg)

Group IV-IX : MFMS, EFMS, PEMS at 75 and 150 mg/kg

Group X-XV : MFLA, EFLA, PELA at 75 and 150 mg/kg

All the test drugs, standard and vehicle were administered for six consecutive days

once daily by oral route. On fourth day nephrotoxicity was induced in animals of

group II-XVI by a single i.p. injection of cisplatin (20 mg/kg). Animals were

monitored carefully and sacrificed at 72 h after nephrotoxicity induced.

4.17.2. Sampling and Biochemical Analyses

The mice were sacrificed 72 h following cisplatin administration. Blood

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

which were used for the estimation of blood urea nitrogen (BUN), serum creatinine,

uric acid, total protein, and albumin.

All these estimation was carried out by using commercial biochemical kit

obtained from Agapee Diagnistic Ltd., Kerala. Kidney tissue was taken to determine

level of tissue MDA.

129

4.17.2.1. Estimation of blood urea nitrogen (BUN)

Quantitative determination of BUN in serum was determined by following

manufacturer procedure (Blood urea nitrogen Kit 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 were 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) =(T

1 - T

2) sample

(T1 - T

2) standard

X 23.4

4.17.2.2. Estimation of serum creatinine

Modified Jaffe’s method was used to determine the amount of creatinine in

serum as per procedure described by manufacturer (Serum creatinine Kit Batch no:

769, Manufacturing date: 1/12, Expiry date: 1/13). In this assay, creatinine reacts with

picric acid to form creatinine alkaline picrate, a coloured compound. The change in

absorbance is proportional to creatinine concentration.

Standard sample was prepared by mixing of 1.0 ml of working reagent with

0.1 ml of standard given with commercial kit. Sample solution was prepared by

mixing 1.0 ml of working reagent with 0.1 ml of serum sample. The solutions were

mixed well and optical density (T1) was determined after 60 sec. Second reading (T2)

was taken exactly 60 sec after first reading at 492 nm. Distilled water used as blank.

130

The concentration of creatinine was calculated using the following formula,

Creatinine concentration (mg/dl) =(T

1 - T

2) sample

(T1 - T

2) standard

X 2

4.17.2.3. Estimation of serum uric acid

Serum uric acid level was determined using the uricase methodology as

described by manufacturer (Uric acid Kit Batch no: 871, Manufacturing date: 6/11,

Expiry date: 6/13). The assay is based on the following reaction,

Uric acid + O2

+ 2H2O

UricaseAllantoine + CO

2 + H

2O

2H2

O2 + 4 -AAP + EHSPT

PeroxidaseRed quinone

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

4 -AAP = Amino-4-antipyrine



mixing 1.0 ml of working reagent with 0.025 ml of serum sample. The solutions are

mixed and incubated at 37°C for 5 minute. the absorption was taken at 546 nm.

Working reagent was used as blank. The concentration of uric acid was calculated

using the following formula,

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

Absorption of standardX 8

131

4.17.2.4. Estimation of serum total protein

Direct biuret method was employed to determine total protein concentration as

per manufacturer procedure (Total protein Kit Batch no: 298, Manufacturing date:

2/12, Expiry date: 2/12). In this colorimetric assay, protein in serum sample forms a

blue coloured complex when treated with cupric ions in alkaline solution (working

reagent). The intensity of the blue colour is proportional to the protein concentration.




mixed and incubated at 37°C for 10 min. The absorption was measured at 546 nm

against reagent blank. The concentration of uric acid was calculated using the

following formula,

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


4.17.2.5. Estimation of serum albumin

Bromocresol green method was employed to determine albumin concentration

as per manufacturer procedure (Albumin Kit Batch no: 888, Manufacturing date:

10/11, Expiry date: 10/13). In this colorimetric assay, albumin from serum sample

reacts with a dye bromocresol-green (working reagent) to produce a colour change

that is proportional to the protein concentration.



132


mixed and incubated for 1 minute. The absorption was measured at 630 nm against

reagent blank. The concentration of uric acid was calculated using the following

formula,

Albumin concentration (mg/dl) =Absorbtion of sample


4.17.2.6. Estimation of MDA

Lipid peroxidation results oxidative degradation of polyunsaturated fatty acids

to MDA, which is usually determined by the chromogenic thiobarbituric acid reaction

and expressed as total TBARS. Animals were sacrificed by cervical dislocation; the

kidneys were isolated quickly and washed with saline, kidney was dried using filter

paper and weighed. A 10% (w/v) kidney tissue homogenate was prepared in

phosphate buffer (pH 7.4) using a homogenizer. Unbroken cells, cell debris and nuclei

were sedimented by centrifugation at 2000 × g force for 10 minute, and the

supernatant was pipetted into plastic tubes. This kidney homogenate thus obtained

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

Briefly, 2.5 ml of 10% (w/v) TCA solution was mixed with 0.5 ml tissue

homogenate, and placed on a boiling water bath for 15 minute and then cooled in tap

water. The mixture was centrifuged at 1000 × g force for 10 minute. After

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

solution in a test tube and the tube was placed again in a boiling water bath for 15

minute. The solution was then cooled in tap water and its absorbance of solution was

taken at 532 nm. The concentration of MDA was calculated by using absorbance

coefficient = 1.56×105 cm

−1M

−1) and the results were expressed as nM/min/mg tissue

protein [133].

133

4.18. ANTI-DIABETIC ACTIVITY

The effect of fractions of methanol extract of Meyna spinosa leaves was

investigated. MEMS showed better antioxidant activity therefore, MEMS and its three

fractions of methanol extracts viz, PFMS, EFMS and MFMS were selected for

antidiabetic activity.

4.18.1. Induction of Type 2 Diabetes

Type 2 diabetes was induced experimentally induced by intraperitoneal

injection of alloxan after administration of high fat diet for 10 days as described by

Shu et al. (2009) [140]. Healthy rats were treated orally with high fat diet (fat 20%,

cholesterol 5%, glucose 5%, fructose 5%, glutamine 5% and methylthiouracil 1%)

continuously for 10 days. On 11th

day alloxan was prepared in a concentration of 100

mg/ml in physiological saline solution and injected into the caudal vein of overnight

fasted rats, at a dose of 120 mg/kg body weight. Fifteen minute later 0.4 U insulin

was injected intraperitoneally; 2.5 and 5.0 hour later, 25% glucose (10 ml/kg)

administered orally to decrease animal mortality rate resulted from hypoglycemia and

hyperglycemia in rats with injection of alloxan. Twenty four hour after first alloxan

injection, freshly prepared alloxan solution (100 mg/kg body weight) was injected

again into the caudal vein of overnight fasted rats. Insulin (0.4 U) was injected (i.p.)

15 minute later to rats, then 25% glucose (10 ml/kg) administered orally 2.5 and 5.0

hour later respectively. The blood glucose was tested and animals with fasting blood

glucose more than 200 mg/dl were used for the experiments.

134

4.18.2. Antidiabetic Activity

The experiment was carried out in two stages, at first stage antidiabetic effect

of extracts were evaluated. Animals were divided into 6 groups (n=6).

Group I (normal control) : received vehicle (0.5% sodium CMC solution)

Group II (diabetic control) : received vehicle (0.5% sodium CMC solution)

Group III (standard drug) : diabetic rat treated with glibenclamide (5 mg/kg)

Group IV-VI : treated with MEMS at 100, 200, 300 mg/kg b.w. respectively.

Treatment was continued for 21 days body weight and serum glucose level

were estimated at 0, 7, 14, and 21 of treatment.

In second stage antidiabetic effect of fractions were evaluated. Healthy and

diabetic animals were grouped into nine groups of six rats each in following manner,

Groups I: Normal control received vehicle (0.5% sodium CMC solution)

Group II: Diabetic control received vehicle (0.5% sodium CMC solution)

Group III: Diabetic rat treated with 5 mg/kg/day glibenclamide

Group IV & V: Diabetic rat treated with the MFMS (75 and 150 mg/kg/day)

Group VI & VII: Diabetic rat treated with the EFMS (75 and 150 mg/kg/day)

Group VIII & IX: Diabetic rat treated with the PFMS (75 and 150 mg/kg/day)

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The volume of administration was 5 ml/kg, and the treatment was lasted for 21

days. The animals were carefully monitored every day and weighed and serum

glucose level was estimated every week. Initial and final serum lipid profile, α–

amylase level were also evaluated in this stage.

4.18.3. Estimation of Biochemical Parameters

The blood samples were withdrawn through the retro orbital plexus from

overnight fasted rats under light ether anaesthesia using a glass capillary. Blood

samples were allowed to clot and serum separated by centrifugation at 3500 rpm for

10 minute, which was used to estimate different biochemical parameters.

Serum glucose level was determined in weekly basis. Initial (0 day) and final

(after 21 day treatment) serum lipid profile like total cholesterol (TC), triglyceride

(TG), high density lipids (HDL), low density lipids (LDL), very low density lipids

(VLDL), and α–amylase level in serum were estimated using a commercially

available standard kit in a semi-autoanalyzer. All diagnostic kits were stored at cool

place (2-8°C) till the time of use.

4.18.3.1. Serum glucose estimation

Serum glucose level was estimated using the manufacturer procedure using

GOP-PAP methodology (Glucose Kit Batch no: 9741, Manufacturing date: 3/12,

Expiry date: 3/13). The principle of this enzymatic colorimetric determination of

glucose was based on the following reaction,

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Glucose + O2 + H2OGlucose oxidase

Gluconic acid + H2O2

2H2O2 + Phenol + 4-Aminoantipyrine Quinonimine + 4 H 2OPeroxidase

(Red Colour)

Procedure: To the sample tube, 0.01 ml of serum and 1.0 ml of working reagent were

added. In another tube, 0.01 ml of standard and 1.0 ml of working reagent were

mixed. The working reagent alone served as the blank. All solutions were mixed

properly and incubated for 10 minute at 37°C. The absorbance of the sample and

standard against the blank was measured at 505 nm. The concentration of glucose was

determined by the following formula,

Glucose concentration (mg/dl) = Absorbance of sample

Absorbance of standardX 100

4.18.3.2. Estimation of total cholesterol and triglycerides

Serum level of TC and TG in normal, diabetic, standard and fractions treated

animals were determined as per the commercial assay kit procedure, which is

described in ‘hepatoprotective activity’ section (section, 4.14.2.5 and 4.14.2.6).

4.18.3.3. Estimation of high density lipids (HDL)

Determination of HDL cholesterol was performed by manufacturer procedure

(HDL Kit Batch no: 822, Manufacturing date: 9/11, Expiry date: 11/12). In this assay

anti human β-lipoprotein antibody present in reagent 1 bind to lipoproteins like LDL,

VLDL, and chylomicrons. The antigen-antibody complexes formed block enzyme

reaction after addition of reagent 2. Cholesterol esterase and cholesterol oxidase of

regent 2 react only with HDL-C. Enzyme reaction with HDL-C yields a blue color

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complex upon oxidative condensation with F-DAOS [N-ethyl-N-(2-hydroxy-3-

sulfopropyl)-3,5-dimethoxy-4-fluroaniline, sodium salt] and 4-aminoantipyrine in

presence of peroxidase. HDL-C concentration can be calculated when compared with

absorbance of the HDL-C calibrator. Blank, sample and HDL calibrator were

prepared by following way,

Reagents/Samples Blank Calibrator Sample

Reagent 1 0.45 ml 0.45 ml 0.45 ml

HDL-C direct Calibrator - 0.005 ml -

Sample - - 0.005 ml

Mix the samples and incubate for 5 minute at 37°C

Reagent 2 0.15 ml 0.15 ml 0.15 ml

After addition of R2 all samples were mixed well and incubated for 5 min at

37°C. The absorbance of calibrator and sample was measured against reagent blank.

And quantity of HDL-C was determined by following formula,

HDL-C concentration (mg/dl) =Absorbance of sample

Absorbance of standardx Calibrator concentration

4.18.3.4. Estimation of low density lipids (LDL)

Determination of LDL cholesterol was performed by manufacturer

procedure (LDL Kit Batch no: 866, Manufacturing date: 5/11, Expiry date: 5/13). In

this assay serum was mixed with reagent 1 (R1). An amphoteric surfactant present in

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R1 protects LDL from enzyme reaction. Cholesterol esterase and cholesterol oxidase

react with non-LDL cholesterol and decomposed it to water by catalase. Reagent 2

(R2) enables conversion of LDL cholesterol to H2O2, which upon oxidative

condensation with HDAOS and 4-aminoantipyrine produces a blue colour complex,

which can be calculated when compared with the absorbance of LDL cholesterol

calibrator. Blank, sample and LDL cholesterol calibrator were prepared by following

way,

Reagents/Samples Blank Calibrator Sample

Reagent 1 0.45 ml 0.45 ml 0.45 ml

LDL-C direct Calibrator - 0.005 ml -

Sample - - 0.005 ml

Mix the samples and incubate for 5 minute at 37°C

Reagent 2 0.15 ml 0.15 ml 0.15 ml

After addition of R2 all samples were mixed well and incubated for 5 minute

at 37°C. The absorbance of calibrator and sample was measured against reagent

blank. And quantity of LDL-C was determined by following formula,

LDL-C concentration (mg/dl) =Absorbance of sample

Absorbance of standardx Calibrator concentration

4.18.3.5. Estimation of very low density lipids (VLDL)

VLDL level was calculated by the following formula [205]

VLDL (mg/dl) =Triglycerides

5

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4.18.3.6. Estimation of α-amylase

Quantitative estimation of α-amylase level in serum was carried by using

CNPG3 methodology using as described by manufacturer.

The principal of α-amylase estimation was based on following formula,

5 CNPG3

Amylase3 CNP + 2 CNPG2 + 3 Maltotriose + 2 Glucose

CNPG3 = 2-Chloro-4-Nitrophenyl-alpha-Maltotrioside

CNP = 2-Choloro-4nitrophenol

CNP-G2 = 2-Chloro-4-nitrophenyl-a-maltoside

Procedure: Briefly, 0.025 ml of serum was mixed with 1.0 ml of working reagent.

The reaction mixture was incubated at 37°C for 1 minute. Change in the absorbance

per minute (▲ OD/min) was measured during 3 minute using semi Autoanalyzer.

Following parameter were feed in auto analyser before the determination of

absorbance, mode of reaction – kinetic, slope of reaction – increasing, wavelength –

405 nm, linearity – 2000 U/l, factor – 3178, temperature – 37°C, blank – distilled

water, no. of readings – 3, interval – 60 sec. The α-amylase activity was expressed in

U/l.

4.18.4. Normoglycaemic Plus Glucose-Hyperglycaemic Model (NG-OGTT)

To find the effect of fractions on normal as well as glucose loaded animal a

combined normoglycaemic plus glucose-hyperglycaemic model (NG-OGTT) was

used in this present study.

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NG-OGTT in rats was performed according to the method used by Orhan et al.

(2005). This combined method was used in order to avoid use of excess animals.

Healthy rats are divided into eight groups each containing 6 animals.

After overnight fasting blood samples were collected from all the animals to

determine glucose level, and drugs were given immediately according to following

manner,

Groups I : received vehicle and served as control

Group II : served as standard and received glibenclimide (5 mg/kg)

Group III : treated with MFMS at a dose of 75 mg/kg, p.o.

Group IV: treated with MFMS at a dose of 150 mg/kg, p.o.

Group V: treated with EFMS at a dose of 75 mg/kg, p.o.

Group VI: treated with EFMS at a dose of 150 mg/kg, p.o.

Group VII: treated with PFMS at a dose of 75 mg/kg, p.o.

Group VIII: treated with PFMS at a dose of 150 mg/kg, p.o.

Blood glucose level was determined after 30 minute, 1 h, 2 h after drug

administration to assess the effect of the fraction on normoglycaemic animals.

After last measurement (at 2 h), 2 g/kg glucose was orally administered to each

animal and the blood was withdrawn in the following time pattern: 2.5, 3, 4 and 6 h to

access the effects of fractions on glucose-hyperglycaemic rats.

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4.19. ANTHELMINTIC ACTIVITY

Anatomical and physiological characteristic of Indian earth worm resemblance

with the intestinal round worm parasite of human being, therefore Pheretima

posthuma have taken in this study to assess anthelmintic activity of fractions of

MELA.

Indian earth worms are divided into twelve groups each containing six

earthworms approximately of equal size in following manner,

Group I : Control (3% Tween 80 in normal saline)

Group II - IV : MFLA (10, 20 and 50 mg/ml)

Group V - VII : EFLA (10, 20 and 50 mg/ml)

Group VIII - X : PFLA (10, 20 and 50 mg/ml)

Group XI-XII : Reference standard piperazine citrate (10 and 20 mg/ml)

Fifty millilitres of respective drug solutions were taken in a petri dishes and

the earthworms were released in to the solution. Earth worms were monitored

carefully and observations were made for the time taken to paralyze and death of

individual worms. Time taken to till paralysis was recorded when no movement could

be observed except when the worms were shaken vigorously. Times taken for death of

worms were noted after ascertaining that the worms lost their motility completely

with fading of their body colour. To confirm, the death worms were shaken

vigorously or dipped in warm water at 50°C but no movement was observed [151].

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Figure 4.3: Study of anthelmintic

activity

A: Control

B: Standard

C: MFLA (50 mg/ml)

D: EFLA (50 mg/ml)

E: FFLA (50 mg/ml)

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4.20. ISOLATION OF CHEMICAL CONSTITUENTS

Present pharmacological investigation showed that methanol fraction of

MEMS and ethyl acetate fraction of MELA showed superior activity then other

fractions (detail observation given in result section). Therefore most effective

fractions viz. MFMS, EFLA were further fractionated to isolate chemical constituents.

MFMS and EFLA was fractionated by using column chromatography by

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

sieve) was used as stationary phase.

To fractionate MFMS chloroform was used as solvent, while benzene was

used to fractionate EFLA. Silica gel (400 g) was stirred with 500 ml of respective

solvent to make slurry and the column was packed at height of 30 cm and a diameter

of 4 cm carefully to avoid the deposition of air bubbles in between compact filling.

Solvent level was kept few centimetres above of the silica gel layer. About 20 g of

MFMS and EFLA was dissolved in minimum quantity of methanol and ethyl acetate

respectively, and then mixed with 30 g of silica gel. The mixture was air dried and

spreaded slowly on the top of the column, which was covered with silica gel slurry (in

respective solvent) followed by with a piece of cotton wool. Chloroform and benzene

were used as solvent system to elude the components from MFMS and EFLA

respectively.

A total of 11 fractions were eluted form MFMS (named as MM-1 to MM-11)

and 7 fractions (named as LA-1 to LA-7) eluted form EFLA. All the eluants were

dried under reduced pressure.

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4.21. ANTIOXIDANT ACTIVITY AND TLC OF SELECTED

SUBFRACTIONS

Antioxidant activities of all sub-fractions obtained from MFMS and EFLA (MS-1

to MS-11 & LA-1 to LA-7) were screened by taking 2 fixed concentrations of

fractions (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 L. asiatica and 4 ‘more potent’

sub-fractions from M. spinosa were selected for further analysis.

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

discussed earlier, benzene was used as solvent (mobile phase). TLC was performed to

check purity of compound. One fraction of L. asiatica and 3 fractions of M. spinosa

were selected for spectral analysis as these compounds showed single spot in TLC.

4.22. SPECTRAL ANALYSIS

The compounds (one fraction form L. asiatica and one fractions of M. spinosa)

were subjected to IR spectroscopy in KBr (PwrkinElmer Spectrum Version 10.03.06),

1H and C13 NMR (Bruker DRX-300) and mass spectroscopy (ESI-HRMS, Agilent

6520 Q-Tof).

4.23. 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.

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