131

CHAPTER – 7

INVITRO AND INVIVO ANTI-OXIDANT ACTIVITY

Chapter No Contents Page No.

Invitro Anti-Oxidant Activity of Combined Extract of CissusQuadrangularis and Aegle Marmelos

7.1. Introduction 132

7.2. Materials and Methods 133

7.3. Results and Discussion 138

7.4. Conclusions 139

Invivo Antioxidant Activity of Combined Extract of Cissus

Quadrangularis and Aegle Marmelos

7.5. Introduction 139

7.6. Materials and Methods 141

7.7. Results and Discussion 147

7.8. Conclusions 151

132

INVITRO ANTI-OXIDANT ACTIVITY OF COMBINED EXTRACT OF

CISSUS QUADRANGULARIS AND AEGLE MARMELOS

7.1. Introduction

The antioxidants are radical scavengers which protect the human body against

free radicals1. The reactive oxygen species, with superoxide, hydroxyl radical and

hydrogen peroxide are produced in definite organelles in the cell under normal

physiological conditions. Excessive production of these ROS, beyond antioxidant

defense capacity of the body can cause oxidase stress2. The reactive oxygen species

(ROS) and free radicals mediated reactions are involved in various pathological

conditions such as anaemia, asthma, inflammation, neurodegeneration against ageing

process and perhaps dementia. Diet has a main role in the growth of chronic diseases,

such as, cancer, coronary heart disease, diabetes, hypertension and cataract3.

Researches have also indicates that utilization of food and beverages high in phenolic

content is correlated with reduced incidence of heart diseases, anaemia, asthma, arthritis,

inflammation, neurodegeneration. Recently, there has been considerable interest in finding

natural antioxidants from plant sources to replace synthetic ones. Natural antioxidant

substances are considered to be safe since they occur in plant foods and are desirable than

their synthetic counter parts. The scientific reports and experimental studies have shown that

plants contain a large variety of phytochemicals that have antioxidant property4. The most

familiar plant phenolic antioxidants consist of flavonoid compounds, cinnamic acid

derivatives, coumarins, tocopherols and poly functional organic acids. Studies on antioxidants

have been explored in various plants and plant products. In this antioxidant survey two

common plants like Sauropus androgynous and Alternanthera pungens were selected based

on their use as edible greens5. There is scanty information on the antioxidant property of these

plants and hence the present work was taken up to fill the gap.

133

Recent study with main bioactive substances in various plants and food substances

are of greater interest6. It has been exposed that free radicals could induce cellular damage

and may be involved in numerous human diseases such as cancer, arteriosclerosis, diabetic

mellitus, hypertension,AIDS and in aging processes of different types of natural antioxidants,

flavonoids and phenolic compounds have achieved more attention7. Polyphenolic compounds,

like flavonoids and phenolic acids, generally originate in plants have been reported to have

many biological effects, including antioxidant activity8.

A. lanata has been studied for flavonoid glycosides, β-sitosterol, α-amyrin,

compesterol, chrysin, four new alkaloids viz. aervine, methylaervine, aervoside and

aervolanine. Similarly, from A. persica alkaloids, leucoanthocyanidins, flavonoids,

triterpenoids, cardiac-glycosides, coumarins and saponins are reported to possess antioxidant

activity9.

7.2. MATERIALS AND METHODS

7.2.1. Materials

Combined ethyl acetate extract of stem bark of Cissus quadrangularis and fruit

pulp of Aegle marmelos (c-EACA) and combined ethanol extract of stem bark of Cissus

quadrangularis and fruit pulp of Aegle marmelos (c-ECA).

7.2.2. Experiment

7.2.2.1. DPPH Method

The free radical scavenging activity by different plant extracts was done according

to the method reported10. The plant extract of volume fifty micro liters is mixed with

methanol, provides 100 μg/ml, mix 450 μl of 50 mM Tris-HCl buffer (pH 7.4) and 1ml

of 0.1mM DPPH in methanol solution in every reaction. Methanol (50 μl) without any

substances was taken as control. The reduction of the DPPH free radical was measured

reading the absorbance at 517 nm after incubation at room temperature for 30 min. BHT

134

and L-Ascorbic acid were used as controls. The percent inhibition was determinedfrom the

following formulae:

% DPPH radical-

scavenging= 100)(

)(

controlofAbsorbance

sampletestofAbsorbancecontrolofAbsorbance

Table 7.1: Antioxidant activity by DPPH method

S.No. Sample Absorbance % of scavenging DPPH

1. c-EACA(100µg) 0.21 60.38

2. c-ECA(100µg) 0.23 56.60

3. Negative control (Water) 0.53 -

4. Methanol 0.51 -

5. Standard (Vit E) 0.05 90.57

Sample 1 Sample 2Control MethanolStandard0.000.050.100.150.200.250.300.350.400.450.500.55

Groups

Abso

rban

ce

Fig.7.1: Antioxidant activity by DPPH method

135

7.2.2.2. FTC Method

The standard method as described by Palombo et al.,11 was followed. A mixture

was prepared by mixing 4.0 mg plant extract in 4 ml absolute ethanol, 4.1 ml of 2.5%

linolenic acid in absolute ethanol, 8.0 ml of 0.05 M phosphate buffer (pH 7.0) and 3.9

ml of water was filled in a vial by means of a screw cap and then dried in an oven at 40 °C.

To 0.1 ml of this solution was mixed 9.7 ml of 75% ethanol and 0.1 ml of 30%

ammonium thiocyanate. Accurately 3 min after addition of 0.1 ml of 0.02 M ferrous

chloride in 3.5% HCl to the reaction mixture, the absorbance of red colour was calculated

at 500 nm each 24 hr til the day after absorbance of control attained maximum. α-

tocopherol was considered as positive controls, mixture without plant sample was

considered as the negative control.

Table 7.2: Antioxidant activity by FTC method

S.No Sample Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

1. c-EACA (100µg) 0.76 0.92 1.06 0.69 0.68 0.68

2. c-ECA (100µg) 0.77 0.91 1.05 0.69 0.69 0.69

3. -ve control 0.20 0.39 0.40 0.41 0.44 0.44

4. +ve control 0.20 0.23 1.04 1.06 1.06 1.07

136

Sample 1 Sample 2 Control Standard0.00.10.20.30.40.50.60.70.80.91.01.1

GroupsAb

sorb

ance

Figure 7. 2: Antioxidant activity by FTC method

7.2.2.3. Thiobarbituric acid (TBA) method

The method of Koleva et al., 12 was used. Two ml of 20% trichloroacetic acid and 2

ml of 0.67% 2-thiobarbituric acid was mixed with 1 ml of sample solution, as such in

FTC method. Then it is immersed in a boiling water bath and cooled, it was subjected to

centrifugation for 20 min at 3000 rpm. Absorbance of supernatant was calculated at 552

nm. Antioxidant activity depends on the absorbance on the final day.

Table 7.3: Antioxidant activity by TBA method

S.No. Sample Absorbance

1. c-EACA (100µg) 0.15

2. c-ECA (100µg) 0.36

3. -ve control 0.08

4. +ve control 0.12

137

Sample 1 Sample 2 Control Standard0.0

0.1

0.2

0.3

0.4

Groups

Abso

rban

ce

Fig.7.3: Antioxidant activity by TBA method

7.2.2.4. Qualitative analysis

For chromatographic separation, spot the ethanol extract on a TLC plate (100

μg/ml) using the mobile phase methanol: chloroform (95:5, v/v). The chromatogram is

made to develop for 30 minutes. After the chromatogram is completed the entire plate was

sprinkled with DPPH (0.15 % w/v) solution by means of an atomizer. In pinkish

background yellowish colour is developed on the TLC plate were noted which represents

the presence of antioxidant substances.

Fig.7.4.Chromatographic separation of the extract using TLC

138

7. 4. RESULTS AND DISCUSSION

It is familiar that free radicals are the one of the reason of numerous diseases,

Parkinson’s disease, coronary heart disease and cancer13. This study demonstrates that

ethanolic and ethyl acetate extracts of Aegle marmelos and Cissus quadrangularis, have

tremendous antioxidant activity. It is remarkable and valuable to scrutinize the latent

efficiency of combined extracts of Aegle marmelos and Cissus quadrangularis

The importance of natural antioxidants as of combined extracts of Aegle

marmelos and Cissus quadrangularis can be distinguished, For health conscious

consumer, the word “free radicals and antioxidants” has become very important.

Antioxidants assist the organisms to face oxidative stress. It is possible to

diminish the threat of chronic diseases and avoid their development by either rising the

body’s antioxidant defense or by enrichment with established nutritional antioxidants14.

Cancer chemoprevention is by means of antioxidant advancements has been

recommended to propose a good possibility in providing betterment to health and is

accepted by many researchers for inhibiting, hindering, or reverse the course of

carcinogenesis15,16 .

DPPH is a constant radical which is used to estimate the antioxidant activity of

plant17. FTC test measures the peroxide compound which is produced at the preliminary

stage of oxidation while TBA test measures the minor products such as aldehyde and

ketone18.

139

7. 5. CONCLUSION

Both extracts exhibited significant antioxidant activity in all the four methods.

In DPPH assay method Both Sample 1(c-ECA) and Sample 2 (c-EACA) have

better antioxidant activity. Sample 2 have more antioxidant activity when compared to

Sample 1.

In FTC method, Both Sample 1(c-ECA) and Sample 2 (c-EACA) have better

antioxidant activity. Sample 1 has more antioxidant activity when compared to Sample 2.

In TBA method, Both Sample 1(c-ECA) and Sample 2 (c-EACA) have better

antioxidant activity. Sample 1 has more antioxidant activity when compared to Sample 2.

In the qualitative analysis by TLC plate method. Both Sample 1(c-ECA) and

Sample 2 (c-EACA) have better antioxidant activity.

INVIVO ANTIOXIDANT ACTIVITY OF COMBINED EXTRACT

OF CISSUS QUADRANGULARIS AND AEGLE MARMELOS

7.5. Introduction

Phagocytic cells generate poisonous reactive oxygen intermediates (ROI) along with

injury and swelling of tissues as a method to destroy attack microbes19, 20.

Oxidative stress has been exposed to produe lesser harm through late cellular death

and swelling consequently, reducing oxidative stress can stop cellular death, reduces swelling

and avoid morbidity and mortality21.

Oxidative stress is related with numerous neonatal diseases for instance retinopathy

of prematurity, periventricular leucomalacia, necrotizing enterocolitis and bronchopulmonary

dysplasia22.

140

Heavy metal pollution of soils expected substantial awareness as a value of the

amplified ecological pollution from industrialized and agricultural source23, 24.

Antioxidants play a main role in the body defense system against reactive oxygen

species (ROS), which are dangerous by yields which are produced during ordinary cell

aerobic respiration25. Antioxidants are radical scavengers, which protect the human from

diseases like ischemia, anaemia, asthma, arthritis, inflammation, neuro-degeneration and

ageing process26.

Free radicals attack the unsaturated fatty acids in the cell biomembranes leads to

membrane lipid peroxidation, decline in membrane fluidity, enzyme loss and receptor tend to

activates and membrane proteins damage causing cell inactivation27. If the in vivo activity of

scavengers is very low to slow down these radicals, produce diseases like arteriosclerosis,

liver disease, diabetes, inflammation, renal failure or aging 28.

Lipid peroxidation is linked with aging and carcinogenesis29. Though, living

systems are guarded from active oxygen species by enzymes like superoxide

dismutase, glutathione peroxidase, glutathione reductase and catalase30.

Therefore, to justify the traditional claims the present study was undertaken to

find out if combined ethyl acetate extract of stem bark of Cissus quadrangularis and

fruit pulp of Aegle marmelos (c-EACA) and combined ethanol extract of stem bark of

Cissus quadrangularis and fruit pulp of Aegle marmelos (c-ECA) demonstrates the

antioxidant activity against CCl4 induced rats models of erythrocyte damage by

estimation of anti-oxidant enzymes such as and the antioxidants superoxide dismutase

(SOD), catalase, glutathione peroxidase, glutathione reductase and lipid peroxidation

as biomarkers. Hence, the current research was considered to confirm the state of the

native practitioners.

141

7.6. MATERIALS AND METHODS

7.6.1. Materials

Combined ethyl acetate extract of stem bark of Cissus quadrangularis and

fruit pulp of Aegle marmelos (c-EACA) and combined ethanol extract of stem bark of

Cissus quadrangularis and fruit pulp of Aegle marmelos (c-ECA), carbon

tetrachloride, Vehicle -1% tween 80.

7.6.2. Experimental Animals

Wistar male albino rats bearing the weight of 150-220 gm were used. They

maintained in Santhiram College of Pharmacy, Nandyal, Andhra Pradesh, India. The animals

were kept in a well-ventilated room with at 12:12 hr light, dark cycle in polypropylene cages.

Institutional Animal Ethical Committee (IAEC) clearance was done with reference no

1519/PO/a/11/CPCSEA).

7.6.3. Acute Toxicity Study

As per the OECD guideline no. 423 (Acute Toxic Method) the acute toxicity of c-

EACA and c-ECA was estimated. The test extract was found to be safe even at 2000 mg/kg

dose. Hence, 1/8th (250 mg/kg) and 1/4th (500 mg/kg) of this dose were preferred for further

study31.

7.6.4. Experimental Design:

Body weight of animals was noted and they were separated into 6 groups each

group is comprised of 6 rats each.

1% Tween 80 was used as a vehicle of extracts in addition to carbon

tetrachloride (1 ml/kg body weight), given intraperitoneally in alternate days for 14

days.

The following groups were used:

142

Group I - Vehicle 1% Tween 80 (5 ml/kg, p.o) [Normal Control]

Group II - CCl4 (1 ml/kg of body weight), i.p[Negative Control]

Group III - Combined ethyl acetate extract of stem bark of Cissus quadrangularis and fruit

pulp of Aegle marmelos (c-EACA) 250 mg/kg, p.o + CCl4 (1 ml/kg of body weight), i.p

Group IV - Combined ethyl acetate extract of stem bark of Cissus quadrangularis and fruit

pulp of Aegle marmelos (c-EACA) 500 mg/kg, p.o + CCl4 (1 ml/kg of body weight), i.p

Group V - Combined ethanol extract of stem bark of Cissus quadrangularis and fruit pulp of

Aegle marmelos (c-ECA) 250 mg, p.o + CCl4 (1 ml/kg of body weight), i.p

Group VI - Combined ethanol extract of stem bark of Cissus quadrangularis and fruit pulp

of Aegle marmelos (c-ECA) 500 mg/kg, p.o + CCl4 (1 ml/kg of body weight), i.p

Vehicle and extract treatments were given once daily for 14 days, but CCl4 (1 ml/kg

of body weight, i.p), was given in alternate days for 14 days.

On the 15th day all group of animals were fasted for 16 hours then it was

sacrificed by cervical dislocation. The blood was collected from the jugular vein into

tubes containing heparin (anticoagulant), the buffy coat obtained was removed by

using centrifugation at 3000 rpm for 15 min. The packed cells were washed with

physiological saline (0.9% NaCl) for three times, by suspending them in cold distilled

water It is lysed, and again centrifuged at 7000 rpm for 30 min. The pellet was formed

along with the erythrocyte membrane and the supernatant denotes the haemolysate.

7.6.4.1. Biochemical estimation

As a result of centrifugation, plasma was formed that measures the lipid

peroxidation by the method of Gutteridge and Wilkins32 Superoxide dismutase was

estimated by haemolysate 33 and catalase34 activities. Lipids were extracted from the

143

erythrocyte membrane by means of method of Folch et al35. The concentration of

cholesterol and phospholipids were evaluated by established methods36, 37. The

cholesterol/phospholipid ratio was determined.

7.6.4.2. Estimation of Lipid Peroxidation

It was determined in the plasma sample by quantifying the amount of

malondialdehyde (Gutteridge and Wilkins, 1982)32.

Reagents

1. 20% Acetic acid

2. 8.1% Sodium dodecyl sulphate (SDS)

3. 0.8 % Thiobarbituric acid (TBA)

4. N-Butanol-Pyridine mixture (15:1v/v)

Procedure

0.2 ml of plasma sample was added to 1.5 ml of 20% acetic acid, 0.2 ml of sodium

dodecyl sulphate and 1.5 ml of thiobarbituric acid, then it was made up to the volume to 4.0

ml with distilled water. It was heated at 95oC for 60 min in a water bath and it was incubated

and cooled to room temperature and made up to the final volume of 5.0 ml in all tubes. Then

5.0 ml n-butanol pyridine (15: 1) mixture was added and mixed carefully by vortexing for 2

minutes. Centrifugation was done at 3000 rpm for 10 min, the organic upper layer was taken

and its optical density was read at 532 nm against a suitable blank.

The levels of lipid peroxides were represented by n moles of malondialdehyde

(MDA)/min/mg protein in plasma sample.

7.6.4.3. Estimation of Superoxide Dismutase

Superoxide dismutase was assayed by Marklund and Marklund, 1974. Method33.

Reagents

144

1. 0.1 M, Tris – HCl buffer, pH 8.2

2. 0.5 M, Tris – HCl buffer, pH 7.4

3. 2 mM Pyrogallol solution

4. Absolute alcohol

5. Chloroform

Procedure

1 ml of the sample was added with 0.25 ml of absolute ethanol and 0.15 ml of

chloroform. It was shaken for 15 min by means of a mechanical shaker centrifuge the

suspension and the supernatant attained comprises the enzyme extract. The mixture for auto-

oxidation consists of 2 ml of buffer, 0.5 ml of 2 mM pyrogallol and 1.5 ml of water. Auto-

oxidation rate of pyrogallol was recorded at an interval of 1 min for 3 min. The enzyme

mixture have 2 ml of 0.1 M Tris – HCl buffer, 0.5 ml of pyrogallol, aliquots of the enzyme

preparation and made up to 4 ml with water. The inhibition rate of pyrogallol auto-oxidation

after adding the enzyme was recorded. The superoxide dismutase activity was estimated by

the inhibition of pyrogallol auto-oxidation at 420 nm for 10 min. To bring about 50%

inhibition of auto-oxidation by pyrogallol the amount of one unit of superoxide dismutase

enzyme is essential. The enzyme activity was represented in terms of units/min/mg protein.

7.6.4.4. Estimation of Catalase

Catalase of tissue homogenate was estimated according to the method of Beers RF

and Sizer IW (1952)34.

Reagents

1. Phosphate buffer (M/15, pH 7.0)

2. Hydrogen peroxide – phosphate buffer, pH 7

145

Procedure

Plasma sample (haemolysate) was centrifuged with M/15 phosphate buffer at 1 to 4oC

and. The deposit is stirred with cold phosphate buffer and allowed to stand in the cooled

environment with occasional shaking.This was repeated once or twice; the resulting

supernatants were collected together and used for assay. A cuvette was filled with 3 ml of

H2O2 phosphate buffer and 0.01 – 0.04 ml sample and compared against a control cuvette with

enzyme solution without H2O2 phosphate buffer at 240 nm. It was observed for a decrease in

the optical density from 0.450 to 0.400. The calculations were made by using these values.

7.6.4.5. Estimation of Glutathione Reductase

Glutathione reductase activity of sample was measured by the method of Dobler and

Anderson (1981)35.

Reagents

1. 50 mM Sodium Phosphate buffer pH 7.5

2. 10 mM Ethylene Diamine Tetra Acetic Acid (EDTA)

3. 0.9 mM Glutathione oxidized (GSSG)

4. Nicotinamide Adenine Dinucleotide Phosphate reduced (NADPH)

Procedure

The reaction mixture containing 50 mM sodium Phoshpate buffer pH 7.5, 10 mM

EDTA, 0.67 mM glutathione oxidized and 0.1 mM NADPH was made up to 3ml with water.

The Change in the optical density was monitored after adding 0.1 ml of sample at 340 nm for

3 mins at 30 second interval. The enzyme activity is represented in n moles of GSSG

utilized/min/mg protein in plasma sample.

7.6.4.6. Estimation of Glutathione Peroxidase (GSH-Px)

Glutathione peroxidase of sample was evaluated by the method of Lawrence and

Burk (1976)36.

146

Reagents

1. 75 mM Phosphate buffer, pH 7.0

2. 60 mM Glutathione

3. 30 units/ml Glutathione reductase

4. 15 mM EDTA

5. 7.5 mM H2O2

6. 3 mM NADPH

Procedure

The assay mixture consists of 2 ml of 75mM Phosphate buffer (pH 7.0), 60 mM

Glutathione, 0.1 ml of 30 units/ml Glutathione reductase, 0.1 ml of 15 mM EDTA, 0.1 ml of 3

mM NADPH and supernatant of plasma sample and made upto final volume of 3.0 ml. The

reaction starts by adding 0.1ml of 7.5 mM H2O2.

The change in the absorbance rate during the conversion of NADPH to NADP+ was

measured spectrophotometrically at 340 nm for 3 mins. The activity of glutathione peroxidase

was reprented in moles of NADPH oxidized to NADP+ / min/ mg protein in plasma sample.

7.6.4.7. Statistical Analysis

The data were recorded as mean ± standard error mean (S.E.M). The importance of

variations between the groups was evaluated by means of one way and multiple way analysis

of variance (ANOVA). The test followed by Dunnett’s test p values less than 0.05 were noted

as significance.

147

7.7. RESULTS AND DISCUSSION

7.7.1. Effects of c-EACA and c-ECAon lipid peroxidation and primary

antioxidant enzymes of the erythrocytes of carbon tetrachloride -intoxicated rats

Table-7.4 shows the effect of c-EACA and c-ECA on oxidative stress induced

by carbon tetrachloride. The extracts considerably (P <0.01) prohibited the

accumulation of lipid peroxidation substances in the plasma. The rats were intoxicated

by carbon tetrachloride led to considerable increase in superoxide dismutase, catalase,

glutathione reductase and glutathione peroxidase activities, at the same time

instantaneous administration of carbon tetrachloride with the c-EACA and c-ECA

significantly (P <0.01) decreased these activities than control group. c-ECA treated

group showed more significant effect than c-EACA treated group (Fig 7.1 to7. 4).

7.7.2. Effects of c-EACA and c-ECAon cholesterol and phospholipids

Carbon tetrachloride intoxication produces a raise in membrane cholesterol, a

reduction in membrane phospholipid and a consequent enhancement in the cholesterol

to phospholipid ratio. Both extract of c-EACA and c-ECA at the doses of 250 & 500

mg/kg considerably (P<0.01) reduces the cholesterol and phospholipids. Both extracts

showed dose dependent activity and significantly decreased the

cholesterol/phospholipid ratio. (Table 7.2; Fig 7.5

148

Table 7.4: Effects of c-EACA and c-ECAon lipid peroxidation and primary antioxidant enzymes of the

erythrocytes of carbon tetrachloride -intoxicated rats

Values arerepresented asmean ± SEMof sixobservations.Statisticalsignificant testfor comparisonwas made byANOVA,followed byDunnet’stest.a -Comparisonbetween GroupI Vs Group II.b- Comparisonbetween GroupII Vs GroupIII, IV, V &VI. *p<0.05**;p<0.01

Group

Design oftreatments

Enzyme activities

Lipidperoxidation

x 10 -6 (units)

SOD

Units/mgprotein

CatalaseUnits/mgprotein

GlutathioneReductase

GSSGutilized/min/mg protein

Glutathione PeroxidaseNADP+ / min/ mg protein

I Vehicle 1%Tween 80 (5ml/kg, p.o)

27.17±0.7491 192.67±0.8028

1.73±0.076 25.33±0.7601 35.5±0.7638

II CCl4 (1 ml/kg ofbody weight), i.p

49.17±0.9458**a

261±3.777**a 4.53±0.0989**a

15.17±0.4773**a

22±0.5774**a

III c-EACA 250mg/kg +CCl4

38.17±0.4014**b

234.50±1.408**b

3.53±0.0494**b

21±0.5164**b 24.33±0.5578*b

IV c-EACA 500mg/kg +CCl4

34.67±0.6667**b

216.33±1.382**b

2.57±0.0421**b

23.83±0.4773**b

26.67±0.4944**b

V c-ECA250 mg +CCl4

35.17±0.7923**b

229±1.155**b 3.22±0.0477**b

23.67±0.8028**b

25.83±0.4773**b

VI c-ECA 500mg/kg, +CCl4

32.67±0.7149**b

208±1.653**b 2.43±0.0558**b

26.67±0.7491**b

28.17±0.3073**b

149

I II III IV V VI05

10152025303540455055

IIIIIIIVVVI

Groups

Lipi

d pe

roxid

atio

n in

Uni

ts

Fig. 7.5: Effects of c-EACA and c-ECAon lipid peroxidation

I II III IV V VI0

100

200

300IIIIIIIVVVI

Groups

SOD

Units

/mg

prot

ein

Fig. 7.6: Effects of c-EACA and c-ECAon SOD

I II III IV V VI0

1

2

3

4

5IIIIIIIVVVI

Groups

Units

/mg

prot

ein

Fig. 7.7: Effects of c-EACA and c-ECAon Catalase

150

Glut Red Glut Per0

10

20

30

40IIIIIIIVVVI

Groups

Fig. 7.8: Effects of c-EACA and c-ECAon Glutathione Reductase&Glutathione Peroxidase

Table 7.5: Effect of c-EACA and c-ECA on erythrocyte membrane lipids and

cholesterol/phospholipid ratio of carbon tetrachloride - intoxicated rats

Group

Design of treatments Cholesterol

(mg/100μl)

Phospholipid

(mg/100μl)

Cholesterol

/Phospholipid

I Vehicle 1% Tween 80 (5ml/kg,

p.o)

0.66±0.0183 1.11±0.008 0.59

II CCl4 (1 ml/kg of body weight), i.p 0.92±0.0095**a 0.85±0.0109**a 1.08

III c-EACA 250mg/kg + CCl4 0.86±0.0213*b 0.90±0.0119*b 0.96

IV c-EACA 500mg/kg + CCl4 0.73±0.0076**b 0.98±0.011**b 0.74

V c-ECA 250mg + CCl4 0.79±0.0083**b 0.95±0.0128**b 0.83

VI c-ECA 500mg/kg, + CCl4 0.69±0.0159**b 1.01±0.0101**b 0.68

Values are represented as mean ± SEM of six observations.Statistical significant test for comparison was made by ANOVA, followed by Dunnet’s test.a - Comparison between Group I Vs Group II.b - Comparison between Group II Vs Group III, IV, V & VI. *p<0.05**; p<0.01.

151

Cholesterol Phospholipid0.00.10.20.30.40.50.60.70.80.91.01.11.2

IIIIIIIVVVI

Groups

Fig. 7.9: Effect of c-EACA and c-ECA on erythrocyte membrane cholesterol &

phospholipid of Carbon tetrachloride –intoxicated rats

7.8. CONCLUSION

The reports of the current study undoubtedly specified that the rigidity of the membranes

after administration of both extract of c-EACA and c-ECA at the doses of 250 & 500 mg/kg.

Administration of c-EACA and c-ECA prohibited alteration in membrane phospholipids and in

membrane fluidity. Many of the researchers proved free radicals are importantly implicated in

different pathological conditions like cancer, arthritis, inflammation and liver diseases37.

Carbon tetrachloride intoxication breaks the erythrocytes was proved by the elevation of

lipid peroxidation, superoxide dismutase and catalase activities and inversely reduces the

glutathione reductase & glutathione peroxidase in erythrocyte membrane fluidity. The enhanced

superoxide dismutase activity leads to the accumulation of hydrogen peroxide, which enhances

the catalase activity.

152

Experimental animals were pre-treated with the c-EACA and c-ECA reveals a better free

radical scavenging leads toreduced actions of superoxide dismutase and catalase, the

concentration of lipid peroxidation products tends to be regular.

Previous studies documented that changes in glutathione peroxidase activity in

erythrocytes were inversely correlated with intensity of lipid peroxidation. It may be supposed

that decrease in glutathione peroxidase and glutathione reductase activity causes failure of H2O2

detoxification.

H2O2 accumulated in erythrocyte cells ions present may undergo Fenton’s reaction in

which hydroxy radicals are produced. These reactive oxygen species participate in lipid

peroxidation processes38-40. Increases in lipid peroxidation in the present study were dependent

on decrease in glutathione peroxidase & glutathione reductase activity, suggested that oxidative

stress and lipid peroxidation rise might occur after CCl4 administration.

In present study results showed that c-EACA and c-ECA significantly decreased lipid

peroxidation and increased glutathione peroxidase & glutathione reductase. Participation of

oxygen free radicals and oxidative stress in carbon tetrachloride (CCl4) induced erythrocyte

damage may indirectly be confirmed by antioxidant activity of c-EACA and c-ECA extracts.

The cumulative effect of carbon tetrachloride intoxication resulted that micro-viscosity of

a membrane enhances with enhancement in cholesterol to phospholipid ratio results in cellular

rigidity41. Experimental animals were intoxicated with carbon tetrachloride changes the

membrane structure and its role as exposed by the enhancement in cholesterol and consequent

reduction in phospholipid concentrations, therefore enhanced cholesterol to phospholipid ratio.

Cooper et al., 42 states that modification of bio-membrane lipid profile agitates its fluidity,

153

permeability, activity of associated enzymes and transport system. Thus c-EACA and c-ECA

extracts plays a key role in peroxidation by hindering the free radical attack on bio-membranes.

The reports of phytochemical test of the c-EACA and c-ECA extractsrevealed that presence of

flavonoids. The presence of flavonoids provides information to guard lipids, blood and body fluids

against the attack of reactive oxygen species such as superoxide, peroxide and hydroxyl radicals43-46. The

presence of flavonoids in c-EACA and c-ECA extractsmay be dependable for their antioxidant activity.

Ever since reactive oxygen species and free radicals were involved in oxidative stress.