chapter 5 therapeutic impact of newly isolated bioactive...

CHAPTER 5

Therapeutic Impact of Newly Isolated

Bioactive Compound from Ficus virens Ait on

Cigarette Smoke Induced Oxidative Stress and

Hyperlipidemia

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Danish Iqbal, et al., pharmacognosy magazine, 2015 (communicated) 144

5.1 INTRODUCTION

Cardiovascular diseases (CVD) contribute around 17.3 million deaths

globally (WHO, 2015). Cigarette smoking is one of the major risk factor for

CVD and is the leading cause of preventable death and a major public health

concern (WHO, 2015). Consumption of tobacco causes killing of nearly 6

million people worldwide (WHO, 2014) and increasing number of untimely

deaths in India (Jha et al., 2008). Large household surveys have shown the

alarming increase of about 120 million smokers in India (Rani et al., 2003).

Smoking is responsible for about 1 in 20 deaths of women and 1 in 5 deaths

of men in India (Jha et al., 2008). It is estimated that smoking may cause 53

% of myocardial infarction among urban males in India (Rastogi et al., 2005).

According to nationally representative case–control study in neighbouring

India showed that in 2010 smoking caused around 20 % of all deaths among

males aged 30 to 69 years (Alam et al., 2013) and by the year 2030 smoking

will kill around 10 million people per year, among them 70 % will be in low-

and middle-income countries. There is a preponderance of evidence showing

a strong association between cigarette smoking and alarming increase in the

mortality rate from smoking-related diseases such as pulmonary diseases,

cardio and cerebrovascular diseases, cancers and several others (Jha et al.,

2013; US DHHS, 2014).

The specific ingredients of cigarette smoke (CS) responsible for its

cardiovascular effects are not fully recognized, but comprises of polycyclic

aromatic hydrocarbons, particulate matter and oxidizing agents (Ambrose and

Barua, 2004). Both active and passive cigarette smoking exposure predispose

to cardiovascular events (Ambrose and Barua, 2004). Majority of other

compounds in cigarette smoke such as nicotine and carbon monoxide (CO)

have been reported to further increase the risk of CVD in chronic smokers

(Papathanasiou et al., 2014). It has been known that nicotine alone acutely

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increases endothelial dysfunction by means of impaired vascular reactivity

(Neunteufl et al., 2002; Papathanasiou et al., 2014). Cigarette smoke contain

carbon monoxide that constitute constitutes 4 % of cigarette smoke and

directly leads to high levels of carboxyhemoglobin. Continuos exposure to

high levels of CO, chronic hypoxia ensues, leads to increased exercise-

induced ischemia, ventricular dysfunction and arrhythmias in patients with

coronary artery disease (CAD) (Allred et al., 1989).

Smoking can raise the cholesterol and free fatty acid concentrations in

the blood by increasing plasma total cholesterol (TC), triglycerides (TG) and

low density lipoprotein-cholesterol (LDL-C) including Apo-B and decreasing

the cholesterol and ApoA-1 level of high density lipoprotein (HDL) (Ambrose

and Barua, 2004). Significant generation of free radicals during smoking

induced oxidative stress causes lipid peroxidation, LDL oxidation and

decreased levels of antioxidants in the plasma of smokers (Mohod et al.,

2014) that ultimately results in CVD (Ambrose and Barus, 2004). Evidence of

oxidized LDL has been obtained from arterial walls of animal models of

atherosclerosis and of CHD patients (Yla¨-Herttuala et al., 1989;

Nordestgaard and Nielsen, 1994; Kapourchali et al., 2014). Cigarette smokers

encounter a sustained free radical load that can contribute to the oxidation of

LDL in-vitro (Church and Pryor, 1985; Yokode et al., 1988), although data

are conflicting (Chen and Loo, 1995). Moreover, erythrocytes from CS

induced-hypercholesterolemia or oxidative stress may result in accelerated

peroxidation reactions, cellular aberration and alterations in lipid and protein

structure (Achudume and Aina, 2012). Generation of free radicals due to CS

exposure could be responsible for alterations in the activities of Enzymatic

and non-enzymatic antioxidants viz., superoxide dismutase (SOD), catalase

(CAT), glutathione peroxidase (Gpx), glutathione reductase (Gred),

glutathione-s-transferase (GST) and non-enzymic antioxidants namely GSH,

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because of excessive utilization in inactivating the free radicals or insufficient

availability of GSH (Ramesh et al., 2007; Ramesh et al., 2010; Thirumalai et

al., 2010). Thus, natural compounds with antioxidant properties contribute the

protection of cell and tissue against deleterious effects caused by CS

generated reactive oxygen species (ROS). Several natural antioxidants have

been experimentally proved as protective agents against smoke induced-

oxidative stress and hyperlipidemia (Ramesh et al., 2007; Ramesh et al.,

2010; Anbarasi et al., 2006).

Ficus species due to their strong antioxidant and biological properties

are known previously to diffuse the toxic free radical and can be used as a

possible protective agent for treatment of oxidative stress related disorders

(Sirisha et al., 2010; Shridhar, 2013). As already described and documented

in chapter 2 and 4, that Ficus virens bark methanolic (FVBM) extract

contained large amount of antioxidant with significant hypolipidemic

property, the work presented in this chapter illustrate the protective role of

FVBM extract and it’s principal bioactive compound, n-Octadecanyl-O-α-D-

glucopyranosyl(6’→1’’)-O-α-D-glucopyranoside in one of the risk factor

induced CVD. Thus, the putative preventive effects of F. virens extract and its

bioactive compound were investigated on overall antioxidant capacity,

hypolipidemic property, hepatic enzyme and non-enzymatic defence system

as well as histopathological studies in CS-induced oxidative stress and

hyperlipidemia.

5.2 MATERIAL AND METHODS

5.2.1 Chemicals

Bradford dye (Sigma Aldrich, India), hydrogen peroxide (H2O2),

isopropeol, glacial acetic acid (Merck Pvt Ltd, India), hemoglobin assay kit,

total cholesterol (TC) and triglycerides (TG) kits was procured from Span

Diagnostics Ltd. (India). Rodent Chow (Ashirwad pellets), capston cigarette

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(Capston, India) sodium dodecyl sulphate (SDS), thiobarbituric acid (TBA),

trichloroacetic acid (TCA), hydrochloric acid (HCL) and all other chemicals

were procured from Himedia Laboratories, Mumbai, India. All other

chemicals and solvents used in this study were of analytical grade.

5.2.2 Animals

Male Sprague-Dawley (SD) rats weighed around 100-150 gm were

procured from Indian Institute of Toxicology Research Center, Lucknow. The

study protocol was approved by Institutional Animal Ethics Committee

(IAEC) (registration number: IU/Biotech/project/CPCSEA/13/11). The rats

have been housed 5 per cage for one week in the animal house for

acclimatization at a temperature of 21-22˚C with 12 hour light and dark cycle.

The rats were given standard diet and water ad libitum.

5.2.3 Dose preparation

Sequentially extracted FVBM extract, its bioactive fraction (F18) and

reference drug atorvastatin were dissolved in 10 % dimethyl sulfoxide

(DMSO) at different concentrations and were homogenized with saline. The

doses of the extracts were selected on the basis of previously published

reports (Jayashree et al., 2012; Hafeez et al., 2013).

5.2.4 Diet/exposure to cigarette smoke

FVBM extract, its bioactive fraction (F18) and atorvastatin suspension

was administrated through gastric intubation in two divided doses (morning

and evening) of 0.5 ml each/rat/day. Rats in smoking control group received

0.5 ml of saline containing 10 % DMSO (vehicle) twice daily while rats in

normal control group received 0.5 ml of saline containing 10 % DMSO twice

daily. The rats were divided randomly and equally (5 rats in each group) in

groups as illustrated in Table 5.1. Rats were exposed to cigarette smoke in

morning by keeping two rats in bottomless metallic container (10 x 11 x 16

inch), having two holes of 3 and 1.5 cm diameter, one on the either side. A

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burning cigarette was introduced through one hole (3 cm) and the other hole

(1.5 cm) was used for ventilation. Animals were exposed to CS for 30

minutes, daily for 4 weeks with interval of 10 min between each 10 min

exposure, using 3 cigarettes/day/2 rats in each group (Anbarasi et al., 2006).

Table 5.1. Protocol for the treatment of cigarette smoke induce

hyperlipidemia in rats

Group Treatment

N-C Normal control

S-C Cigarette smoking control + vehicle

FVT-1 Smoke-exposed + plant extract (FVBM) (50 mg/rat/day)

FVT-2 Smoke-exposed + plant extract (FVBM) (100 mg/ rat/day)

CT Smoke-exposed + bioactive compound (F18) (1 mg/ rat/day)

AT Smoke-exposed + standard (Atorvastatin) (1 mg/ rat/day)

5.2.5 Collection of blood, plasma and packed erythrocytes

At the end of the experiment, all the rats were anaesthetized and blood was

collected in heparinized tubes by cardiac puncture. Plasma was collected from

blood by centrifugation at 2,500 rpm for 30 min, aliquoted and stored at either

4 or −20˚C for future use.

Packed erythrocytes hemolysate was prepared as described by Lakshmi

and Rajagopal (1998). The packed erythrocytes obtained after the separation

of plasma and buffy coat, were washed thrice with normal saline and a portion

of washed erythrocytes was lysed in hypotonic (10 mM) sodium phosphate

buffer, pH 7.4. A portion of the washed packed erythrocytes was stored at 4˚C

for further use.

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5.2.6 Collection of different organs

In addition to the blood, liver and lung were excised. Liver and lung were

kept in ice-cold saline. A portion of liver and lung were immediately fixed in

10 % neutral formalin for histopathological studies.

5.2.7 Preparation of homogenate and post mitochondrial supernatant

At the end of the experiment, homogenate and post mitochondrial

supernatant (PMS) of liver and lung were prepared as illustrated in section

4.2.8.5.

5.2.8 Determination of hemoglobin (Hb) in blood

Hemoglobin level was estimated by cyanmethemoglobin method of

Drabkin and Austin (1932) according to the procedure described in the

instruction sheet enclosed with the reagent kit supplied by Span diagnostic.

The percent blood hemoglobin was determined by measuring the absorbance

of cyanomethemoglobin at 540 nm in eppendorf spectrophotometer using a

hemoglobin standard.

5.2.9 Determination of nicotine content in blood

Nicotine content in blood samples was determined by the method of

Varley et al. (1976). One ml of fresh blood was collected in heparinized

tubes; pH was adjusted to 9-10 by adding ammonium hydroxide (0.1 %) and

then extracted 3 times with 5 ml chloroform. The chloroform extract was

washed once with 5 ml water, extracted with 5 ml dilute sulphuric acid (0.5

M), pH was adjusted to 10 using ammonium hydroxide (0.1 %), then

extracted 3 times with 5 ml chloroform, washed once with 5 ml water and

dried by adding 1 gm anhydrous sodium sulphate. The solution was filtered

and the optimal density of the filtrate was read at 260 nm. Nicotine standard

was treated in the similar manner and used in the calculation of nicotine

present in the blood samples.

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5.2.10 Determination of carbon monoxide saturation in a mixture

containing hemoglobin and carboxy hemoglobin

Carbon monoxide Saturation (SCO) in blood samples was determined

by the method of Varley et al. (1976). Hundred microliter of heparinised

blood was mixed with 20 ml of 0.1 % ammonia and 20 mg of sodium

dithionate were added to convert oxyhaemoglobin (HbO2) to Hb. The optimal

density was measured at 538 nm and 578 nm (isobestic wavelength) L=1.0

cm, slit width 0.02 nm and the measurement was carried out within 10 min of

the addition of sodium dithionate. The carbon monoxide saturation was

calculated from the equation.

SCO % = (2.44 D538

/ D578

-2.68) 100

The constants 2.44 and 2.68 have been calculated from a series of 30

measurements of D538

/ D578

to 0 % COHb (100 % Hb) and 100 % COHb. The

isobestic point was established at = 578 0.5 nm (n=100).

5.2.11 Isolation of Plasma LDL and HDL

The precipitation method described by Wieland and Seidel (1983) was

used for the isolation of plasma LDL and the method of Patsch et al. (1989)

was used for the isolation of HDL. The detailed protocol was mentioned in

section 4.2.8.6.

5.2.12 Measurement of ex-vivo and in-vitro Cu++

-mediated susceptibility

of LDL to oxidation

The ex-vivo and in-vitro Cu++

-mediated susceptibility of isolated LDL

to oxidation was assessed by determining the lag phase of conjugated diene

(CD) formation using the method of Esterbauer et al. (1989; 1992). Prior to

oxidation studies LDL samples were dialyzed against 5 mM phosphate buffer

saline (PBS), pH 7.4, for 12 h. The incubation mixture contained 50 g of

LDL in 1.0 ml of 10 mM PBS, pH 7.0. At time zero, before the initiation of

Cu++

-mediated oxidation, 1.0 ml aliquot of each sample was added to a tube

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containing 0.5 M EDTA, pH 7.4, for the assessment of ex-vivo levels of CD.

Then, lipoprotein samples were mixed with CuSO4 to a final concentration of

2.5 M and incubated at 37˚C. At different time intervals of oxidation, 1.0 ml

aliquots from LDL incubation mixture were taken out, mixed with 0.5 mM

EDTA, pH 7.4, stored at 4˚C and used for the assessment of CD. The

oxidation for LDL was carried out for 12 h. The formation of CD in each

aliquot was measured by monitoring absorbance at 234 nm in a Beckman DU

640 spectrophotometer. Conjugated diene was calculated by using an

extinction coefficient of 2.52 x 104 M

-1 cm

-1 and expressed as nmole

malondialdehyde (MDA) equivalent per mg LDL protein.

The MDA content in LDL was assayed by the method of Niehaus and

Samuelsson (1968). Briefly, 0.3 ml of LDL mixture in triplicate was

thoroughly mixed with 2 ml of reagent containing 15 % TCA, 0.375 % TBA

and 0.25 N HCL and was heated for 15 min in a boiling water bath. After

cooling, the flocculent precipitate was removed by centrifugation at 1000 rpm

for 10 min. The absorbance of the samples was determined at 535 nm against

a blank that contained all the reagents except the LDL. The MDA

concentration of the samples was calculated by using an extinction coefficient

(1.56 x 105 M

-1cm

-1).

5.2.13 Measurement of plasma “total antioxidant power” (FRAP)

The method of Benzie and Strain (1996) was used for measuring the

ferric reducing ability of plasma (FRAP assay) which estimates the “total

antioxidant power”, with minor modification as described in section 4.2.8.9.

5.2.14 Measurement of MDA release from intact erythrocytes

The procedure of Cynamon et al. (1985) was employed for the

determination of MDA release from erythrocytes. Two aliquots of 0.22 ml of

washed packed erythrocytes in duplicate were taken in two separate tubes.

One series of aliquots were suspended in 4.18 ml of phosphate buffered

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saline, pH 7.4 and the second series of aliquots of erythrocytes were

suspended in 4.18 ml of phosphate buffered saline, pH 7.4, containing 4 mM

sodium azide. Both the suspensions were vortexed for 15 seconds. One ml of

erythrocytes suspension of series one was taken in triplicate and mixed with

1.0 ml of freshly prepared 3 % hydrogen peroxide, whereas, 1.0 ml of 0.75 %

hydrogen peroxide was added to the second series of erythrocytes suspension.

The samples were mixed for 10 seconds and incubated for 1 h at 37˚C. At the

end of incubation, 1.0 ml of 28 % TCA containing 100 mM sodium arsenite

was added to each tube and centrifuged at 3,000 rpm for 10 min. Two

millilitre of the supernatant from each tube was taken in triplicate and mixed

with 0.5 ml of 1 % TBA prepared in 50 mM NaOH. The mixture containing

sample was then boiled for 15 min at 95˚C, cooled to room temperature and

the absorbance was recorded at 535 nm against a reagent blank in a Beckman

DU 640 spectrophotometer. The MDA concentration of the samples was

calculated by using an extinction coefficient (1.56 x 105 M

-1cm

-1).

5.2.15 Determination of MDA content in erythrocytes

The determination of MDA in erythrocytes was carried out by adopting

standardized protocol of Stocks and Dormandy (1971). Briefly, 0.3 ml of

packed erythrocytes in triplicate was made up to 1.0 ml in phosphate buffered

saline, pH 7.4. To each tube, BHT in ethanol (0.75 mM) was added, followed

by the addition of 0.5 ml of 30 % TCA. Tubes were vortexed and allowed to

stand in ice-bath for 2 h. Tubes were then centrifuged at 2,000 rpm for 15 min

and 1.0 ml of the supernatant of each tube was transferred to another tube. To

each tube, 75 l of 100 mM EDTA and 250 l of 1 % TBA in 50 mM NaOH

was added, mixed and kept in a boiling water bath for 15 min. After cooling

the tubes to room temperature the absorbance of each sample was read against

a reagent blank at 532 nm in a Beckman DU 640 spectrophotometer. The

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MDA concentration of the samples was calculated by using an extinction

coefficient (1.56 x 105 M

-1cm

-1).

5.2.16 Determination of plasma triglycerides and very low density

lipoprotein-cholesterol

Plasma TG was determined by using enzymatic kit (Merck, India) based

on glycerol-3-phosphate oxidase peroxides (GPO-POD) method (Trinder,

1969). The very low density lipoprotein-cholesterol (VLDL-C) in plasma was

calculated by dividing plasma TG values (mg/dl) by a factor of 5 as described

by Friedewald et al. (1972).

5.2.17 Determination of total cholesterol in plasma and lipoprotein

Plasma TC, LDL-C and HDL-C were determined by using cholesterol

enzymatic kit (Merck, India) based on cholesterol oxidase phenol

aminophenazone (CHOD-PAP) method and results were expressed as mg/dl.

5.2.18 Determination of triglycerides and total cholesterol in liver and

lung homogenates

For the extraction of lipid contents from plasma and tissues, the method

of Folch et al. (1957) was employed. Briefly, 1 ml of plasma or tissue

homogenate was mixed with 5 ml of chloroform: methanol (2:1), followed by

centrifugation at 1,000 rpm for 5 min to separate the phases. Most of the

upper layer was removed and 3.0 ml of the lower chloroform layer was

recovered. The lower chloroform was placed in test tube this phase containing

lipids is evaporated at 45˚C till dryness. The lipid residues were dissolved in

1.0 ml of chloroform. One hundred microliter of samples (in triplicate) was

taken for the determination of triglyceride in liver and lung, by using

enzymatic kit. The method uses a modified Trinder colour reaction to produce

a fast, linear, end point reaction (Trinder, 1969). The intensity of the colour

produced after incubation is directly proportional to the concentration of the

triglyceride in the samples when measured at 500 nm in a Beckman DU 640

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spectrophotometer.

For the determination of total cholesterol in liver and lung

homogenates, fifty microliter of lipid residues in chloroform was mixed with

500 l of isopropanol, allowed to stand for 5 min and centrifuged at 3,000

rpm for 10 min. A suitable aliquot of isopropanol extract was used for

cholesterol determination by using cholesterol enzymatic kit (Merck, India)

based on CHOD-PAP method and results were expressed as mg/dl.

5.2.19 Estimation of lipid peroxides in plasma, liver and lung

homogenates

For the determination of lipid peroxidation products viz., CD, lipid

hydroperoxide (LOOH) and MDA content in plasma, liver and lung

homogenates different protocols were followed as illustrated in section

4.2.8.10.

5.2.20 Assay of HMG-CoA reductase activity in liver homogenate

HMG-CoA reductase enzyme activity in liver homogenate was

estimated indirectly by Rao and Ramakrishnan (1975) method as illustrated in

section 4.2.8.11.

5.2.21 Activities of antioxidant enzymes

5.2.21.1 Determination of catalase activity in liver and lung

The enzymatic activity of catalase in PMS of liver and lung was

measured by the procedure described by Sinha (1972). Briefly, the reaction

mixture (1.0 ml) contained 10 mM phosphate buffer, pH 7.0 and, 100 l of

liver (987-1234 g) or 100 l of lung (524-612 g) PMS protein. After a

preincubation of 10 min at 30˚C to this mixture, the reaction was initiated by

the addition of hydrogen peroxide resulting in a final concentration of 100

mM and the tubes were incubated for 5 min at 30˚C. At the end of incubation,

the reaction was stopped by the addition of 1.0 ml dichromate-acetic acid

reagent (5% potassium dichromate and glacial acid were mixed in 1:3 ratio).

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For zero time control, hydrogen peroxide was added after stopping the

reaction. The tubes were boiled for 15 min, cooled and the absorbance was

taken at 590 nm against a reagent blank in a Biospectrometer. Liver catalase

activity was calculated by using a standard hydrogen peroxide calibration

curve.

5.2.21.2 Determination of superoxide dismutase activity in liver and lung

Enzymatic activity of SOD in PMS fraction of liver and lung was

determined by the method as described by Kakkar et al. (1984) based on the

50 % inhibition of the formation of nicotinamide adenine dinucleotide

(NADH)-phenazine methosulphate-nitroblue tetrazolium formazan at 560 nm.

The assay mixture containing 52 mM sodium pyrophosphate buffer, pH 8.3,

300 M nitroblue tetrazolium, 186 M phenazine methosulphate and 100 l

of liver (987-1234 g) or 100 l of lung (524-612 g) PMS protein was

preincubated for 5 min at 30˚C. The reaction was initiated by the addition of

NADH (780 M), followed by the incubation of samples for 4 minute at

30˚C. The reaction was stopped by the addition of glacial acetic acid (0.5 ml).

For zero time control, to the above incubation mixture, 0.5 ml of glacial acetic

acid was added prior to the addition of NADH. After incubation, 2.0 ml of n-

butanol was added to each tube including reagent blank, this mixture was

rigorously extracted and centrifuged at 3,000 rpm for 5 min. The colour

intensity of the chromogen observed in the butanol extract was measured at

560 nm against a reagent blank in a Biospectrometer. Superoxide dismutase

activity was calculated in terms of an arbitrary unit, which illustrated as the

enzyme concentration of enzyme required to inhibit the chromogen formation

by 50 % in one min under the above assay conditions.

5.2.21.3 Determination of glutathione peroxidase activity in liver and lung

Glutathione peroxidase activity in liver and lung homogenate was

assayed by a modification of Mill's procedure 2 (1959) as reported by

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Hafeman et al. (1974). The reaction mixture contained 80 mM sodiumz

phosphate buffer, 80 M EDTA, pH 7.0, 1 mM sodium azide, 400 M GSH

and 100 l of liver (987-1234 g) or 100 l of lung (524-612 g) homogenate

protein in a total volume of 0.5 ml. After a 5 min preincubation at 37˚C, the

H2O2 (250 M) was added to start the reaction and the tubes were incubated

for 6 mins at 37˚C. After indicated time, the reaction was terminated by the

addition of 0.5 ml of 10 % TCA. After that, tubes were centrifuged at 3,000

rpm for 10 min and 0.5 ml supernatant from each tube was added to tubes

containing 2.0 ml of 0.4 M sodium phosphate buffer, 0.4 mM EDTA, pH 7.0

and 0.5 ml of 1 mM 5, 5’-dithiobis 2-nitrobezoic acid (DTNB). Absorbance

of sample mixture was recorded at 412 nm against a reagent blank within 2

min of DTNB addition in a Biospectrometer. The enzyme activity was

calculated after deducting the absorbance of samples from the zero time

control values by utilizing a standard glutathione.

5.2.21.4 Assay of glutathione reductase activity in liver and lung

The enzymatic activity of Gred was determined according to the

method of Carlberg and Mannervik (1975). The incubation mixture of 82.5

mM sodium phosphate buffer containing 25 M EDTA, pH 7.6, 25 M

oxidized glutathione and 100 l of liver (987-1234 g) or 100 l of lung

(524-612 g) PMS protein was preincubation at 25˚C for 5 min. The tubes

were incubated for 4 min at 25˚C and NADPH (5 M) was added to start the

reaction. The reaction was terminated at one minute time interval by

incubating the samples in an ice-bath. Zero time control tubes were prepared

as above except NADPH (5 M) was added after stopping the reaction. The

optical density of each sample was recorded at 340 nm against a reagent blank

in a Biospectrometer. By using the zero time control value the enzyme

activity was calculated on the basis of a molar extinction coefficient of

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NADPH (6.22x103M

-1cm

-1). .

5.2.21.5 Determination of glutathione-s-transferase activity in liver and

lung

Method of Habig et al. (1974) was used to measure the GST activity in

PMS fraction of liver or lung. The assay mixture (1.0 ml) containing 100 mM

potassium phosphate buffer, pH 6.5, 1 mM GSH and 50 l of liver (494-617

g) or 50 l of lung (246-319 g) PMS protein. The samples were

preincubated for 10 min at 30˚C and 1-chloro 2, 4-dinitrobenzene (CDNB),

prepared in absolute ethanol (1 mM), was added to these samples to start the

reaction followed by incubation for 4 min at 30˚C. At the end of incubation,

reaction has been stopped by placing the tubes in an ice-bath for 15 min and

the absorbance was recorded at 340 nm against a reagent blank in a

Biospectrometer. The enzyme activity was calculated using molar extinction

coefficient of CDNB (9.6 x 103 M

-1cm

-1) after an appropriate deduction of

zero time control values.

5.2.21.6 Determination of GSH content of glutathione in liver and lung

For the determination of GSH content in liver or lung homogenate, the

method of Ellman (1959) as modified by Sedlack and Lindsay (1968) was

followed. In order to assay GSH content, 0.5 ml of 10 % homogenate was

added to 0.5 ml of 4 % sulphosalicylic acid to precipitate the homogenate

(Jollow et al., 1974). The samples were incubated for 60 minute at 4˚C

followed by centrifugation at 6,000 rpm for 5 min and 0.5 ml aliquots of the

supernatant were used for the assay of GSH content. The reaction mixture

includes 267 mM tris buffer, 13 mM EDTA, pH 8.9 and 0.33 mM DTNB

(prepared in absolute methanol). The optical density of the samples was

recorded against a reagent blank at 412 nm within 2 minutes of DTNB

addition. Glutathione content in the samples were calculated using a standard

calibration curve of reduced glutathione.

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5.2.22 Histopathological studies of liver and lung

For histopathological study, a portion of liver and lung were used. For

microscopic preparation of the above tissues, method of Disbrey and Rach

(1970) was used. Two formalin fixed samples from each tissue were

embedded in paraffin and sectioned after block preparation. The paraffin

block was sectioned in 4~5 μm thickness by using a microtome to make a

slide and then it was dyed with hematoxylin-eosin (H&E) and observed with a

light microscope and photographs were taken (Kiernan, 2001).

5.2.23 Protein estimation

The protein concentration of plasma, LDL, liver or lung homogenate

and PMS was analysed by the method of Bradford (1976), using bovine

serum albumin as standard. Aliquots were first precipitated with 10 % TCA

followed by centrifugation at 1500 rpm for 10 min. The pellets containing

protein were dissolved in 0.5 N NaOH and suitable aliquots were used for

protein determination.

5.2.24 Data analysis

For all assays, samples were analyzed in triplicate and the results were

expressed as mean ± SD and the results were evaluated using one-way

analysis of variance (ANOVA) and two tailed Students t-test. Statistical

significance were expressed as *p<0.05, **p<0.01 and ***P<0.001.

5.3 RESULTS

5.3.1 Average body weight of rats in each group before and after 4 weeks

of treatment

As shown in Table 5.2, there was significant decrease in body weight

of smoke-exposed rats from 130.85 gm ± 5.56 in N-C to 80.56 gm ± 3.14 gm

in S-C rats after 4 weeks of exposure to CS. Whereas, the average body

weight of smoke-exposed rats treated with different doses of F. virens

methanolic extract (FVBM-50 & FVBM-100), bioactive compound (F18) and

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Table 5.2. Average body weight of rats in each group before and after 4

weeks of FVBM extract, F18 bioactive compound and atorvastatin treatment

Group* Before treatment After treatment

N-C 125.14±2.67 130.85±5.63

S-C 135.85±3.93 80.56±3.14 a

FVT-1 125.28±7.86 90.28±3.14 c

FVT-2 133.46±6.07 115.57±4.45 a

C-T 122.24±7.86 125.28±5.14 a

A-T 126.78±6.07 120.57±5.45 a

*Values are mean (grams)SD from 5 rats in each group.

N-C, normal control; S-C, smoke-exposed control; FVT-1, fed 50 mg FVBM

extract/rat/day; FVT-2, fed 100 mg FVBM extract/rat/day; C-T, fed 1 mg F18

bioactive compound/rat/day and A-T, given 1 mg atorvastatin/rat/day for 4

weeks.

Significantly different from N-C at ap<0.001.

Significantly different from S-C at ap<0.001.

Significantly different from S-C at cp<0.05.

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atorvastatin was 90.28±3.14, 115.57±4.45, 125.28±5.14 and 120.57±5.45

(gm), respectively, which indicates a significant regain of average body

weight when compared to S-C rats.

5.3.2 Impact of FVBM extract, F18 bioactive compound and

atorvastatin on hemoglobin (Hb), blood carbon monoxide saturation and

blood nicotine in smoke-exposed rats treated for 4 weeks

Results presented in Table 5.3, indicated the Hb, blood carbon

monoxide saturation (carboxyhemoglobin) and blood nicotine level in N-C, S-

C and plant extract bioactive compound treated rats. Hemoglobin level was

significantly reduced by 22 % in smoke-exposed (S-C) rats, when compared

to N-C value. However, treatment with higher dose of plant extract (100

mg/rat), purified compound (F18) and atorvastatin showed a highly

significant increase in Hb concentration of 26 %, 27 % and 21 % respectively,

when compared to S-C rats. Both FVBM extract and F18 bioactive compound

administration to smoke-exposed rats restored the Hb levels close to normal

value. Furthermore, in S-C rats blood carbon monoxide saturation and blood

nicotine levels were increased from 3.26 (SCO %) and 0.998 µg/ml in N-C to

5.78 (77 %) and 2.16 µg/ml (116 %) respectively. After 4 weeks of treatment

blood carbon monoxide saturation and blood nicotine levels showed reduction

of 11 % & 2 % in FVT-1; 30 % & 32 % in FVT-2, 18 % & 26 % in C-T and

31 % & 8 % in A-T rats, respectively, in comparison to values in S-C rats.

5.3.3 Effect of FVBM extract, F18 bioactive compound and

atorvastatin on plasma, liver and lung lipids; plasma lipoprotein profile

and hepatic HMG-CoA reductase activity in smoke-exposed rats treated

for 4 weeks

The potential of FVBM extract (50 & 100 mg/rat/day), F18

(1mg/rat/day) and atorvastatin (1mg/rat/day) in ameliorating the increase in

plasma, liver and lung lipids as well as plasma lipoprotein profile was

CHAPTER 5


Table 5.3. Impact of FVBM extract, F18 bioactive compound and atorvastatin

on blood haemoglobin, carbon monoxide saturation and nicotine in cigarette

smoke-exposed rats after 4 weeks of treatment

Group* Haemoglobin

(g/dl)

Carbon monoxide

saturation (SCO %)

Nicotine

(µg/ml)

N-C 14.860.764 3.260.089 0.9980.012

S-C 11.600.515

(-21.9%)b

5.780.095

(+77.3%)a

2.160.033

(+116.4%)a

FVT-1 12.350.617

(+6.4%)c

5.160.080

(-10.7%)a

2.110.024

(-2.3%)d

FVT-2 14.620.721

(+26.1%)b

4.070.091

(-29.6%)a

1.480.042

(-31.5%)a

C-T 14.740.732

(+27.1%)b

4.760.078

(-17.6%)a

1.590.035

(-26.4%)a

A-T 13.990.677

(+20.6%)b

3.970.043

(-31.3%)a

1.980.022

(-8.3%)b

*Values are mean SD from blood of 5 rats in each group.

Significantly different from N-C at bp<0.01.


Significantly different from S-C at bp<0.01.


Non-significantly different from S-C at dp>0.05.

CHAPTER 5


investigated in cigarette smoke-exposed rats, after 4 weeks of administration

and the results are illustrated in sections as given below:

5.3.3.1 Effect on plasma lipids

The results illustrated in Table 5.4 showed that, all the plasma lipids

parameters, TC, TG and non-HDL-C were significantly increased from 85.42,

64.05 and 55.53 mg/dl in N-C rats to 136.87, 171.05 and 125.93 mg/dl,

respectively, in S-C rats. After 4 weeks of treatment with FVBM extract (50

& 100 mg/rat/day), levels of TC, TG and non-HDL-C were significantly

decreased by 20 %, 30 %, 29 % and 30 %, 50 %, 47 % respectively, when

compared to corresponding S-C values. Whereas, marked reduction of 33 %,

55 %, 56 % and 36 %, 61 %, 54 % was observed in TC, TG and non-HDL-C

level of F18 and atorvastatin treated rats, when compared to corresponding

values in S-C group. These results demonstrate that 4-week treatment to

cigarette smoke-exposed rats mediated a significant diminution in above lipid

parameters.

5.3.3.2 Effect on plasma lipoprotein lipids

Results shown in Table 5.5 depicted that plasma LDL-C and VLDL-C

levels were significantly increased from 42.7 and 12.8 mg/dl in N-C to 91.7

mg/dl (115 %) and 34.2 mg/dl (167 %) respectively, in S-C rats. After 4

weeks of treatment with FVBM extract (at higher dose), both LDL-C and

VLDL-C levels showed a significant reduction of 46 % and 50 %,

respectively, whereas, FVT-1 group exhibited much less reduction.

Furthermore, LDL-C and VLDL-C level in C-T group were significantly

reduced by 50 % and 55 % respectively, in comparison to corresponding

values in S-C rats which was almost equivalent to the reduction observed in

atorvastatin treated rats. Plasma HDL-C level were decreased from 30 mg/dl

in N-C to 11 mg/dl (63 %), in S-C values which was subsequently attenuated

after the treatment with FVBM extract, bioactive compound and standard.

CHAPTER 5


Table 5.4. Effect of FVBM extract, F18 bioactive compound and atorvastatin

on plasma triglycerides, total cholesterol and non-HDL-cholesterol in

cigarette smoke-exposed rats after 4 weeks of treatment

Group* Total Cholesterol Triglycerides Non-HDL-chloesterol

†

N-C 85.42±3.22 64.05±2.31 55.53±2.34

S-C 136.87±8.42

(+60.2%)a

171.05±6.64

(+167.1%)a

125.93±5.94

(+126.8%)a

FVT-1 109.49±7.42

(-20.0%)a

120.5±4.32

(-29.6%)a

89.78±3.45

(-28.7%)a

FVT-2 95.67±5.44

(-30.1%)a

86.1±3.22

(-49.7%)a

66.96±2.35

(-46.8%)a

C-T 91.43±3.48

(-33.2%)a

76.28±2.38

(-55.4%)a

55.84±2.06

(-55.7%)a

A-T 87.04±4.62

(-36.4%)a

67.45±2.39

(-60.6%)a

58.52±2.74

(-53.5%)a

†For the calculation of non-HDL-C, data is taken from Table 5.4 and 5.5.

*Values are mean (mg/dl)SD from plasma of 5 rats in each group.



CHAPTER 5


Table 5.5. Effect of FVBM extract, F18 bioactive compound and atorvastatin

on plasma LDL-C, HDL-C and VLDL-C in cigarette smoke-exposed rats

after 4 weeks of treatment

Group* LDL-C HDL-C VLDL-C

N-C 42.72±1.42 29.89±1.34 12.81±0.54

S-C 91.72±4.45

(+114.7%)a

10.94±0.54

(-63.4%)a

34.21±1.36

(+167.1%)a

FVT-1 65.68±2.24

(-28.4%)a

19.71±1.07

(+80.2%)a

24.1±1.17

(-29.6%)a

FVT-2 49.74±1.87

(-45.8%)a

28.71±1.24

(+162.4%)a

17.22±1.04

(-49.7%)a

C-T 46.12±1.36

(-49.7%)

30.06±1.35

(+174.8%)a

15.25±0.57

(-55.4%)a

A-T 45.03±1.29

(-50.9%)a

28.72±1.36

(+162.5%)a

13.49±0.52

(-60.6%)a

Values are mean (mg/dl)SD from LDL-C, HDL-C and VLDL-C, isolated

from plasma of 5 rats in each group.



CHAPTER 5


5.3.3.3 Impact on the ratios of LDL-C/HDL-C, HDL-C/TC, HDL-

C/LDL-C and TC/HDL-C

The LDL-C/HDL-C, HDL-C/TC, HDL-C/LDL-C and TC/HDL-C

ratios calculated from the data presented in Table 5.4 and 5.5 are shown in

Table 5.6. In contrast to normal ratio’s, significant decrease of 4.4 and 5.8

fold in HDL-C/TC and HDL-C/LDL-C ratios was observed; whereas a

marked increase of 4.4 fold and 5.9 fold in TC/HDL-C and LDL-C/HDL-C

ratios was observed in S-C group. Furthermore, atorvastatin and bioactive

compound treated rats exhibited marked increase of 4.1, 5.3 and 4.4, 5.8 fold

in HDL-C/TC and HDL-C/LDL-C ratio. An opposite pattern was observed in

TC/HDL-C and LDL-C/HDL-C ratios. These results indicated a significant

restoration of these ratios close to the normal values, which in turn indicated

the normalization of lipoprotein associated cholesterol level in CS-exposed

rats treated with FVBM extract, F18 and atorvastatin.

5.3.3.4 Lipid lowering effect on liver and lung TG and TC

The result presented in Table 5.7 showed that hepatic levels of TC and

TG were significantly increased in cigarette smoke-exposed rats by 91 % and

92 %, respectively, when compared to corresponding values in N-C,

Similarly, in lung, TC and TG levels were significantly increased by 55 %

and 27 %, respectively. In comparison to corresponding values in S-C,

administration of FVBM extract, F18 and atorvastatin to smoke-exposed rats

for 4 weeks were associated with a significant decline in liver TC level by 20

%, 39 %, 47 % and 33 %, respectively, whereas, TG level was reduced by 18

%, 32 %, 48 % and 40 % respectively. Parallel, to hepatic TC and TG levels,

lung TC level was reduced by 15 %, 29 %, 35 % and 33 %, whereas, 6 %, 16

%, 21 % and 16 % of reduction were observed in TG level of FVT-1, FVT-2,

F18 and atorvastatin, respectively. These results demonstrate that similar to

CHAPTER 5


Table 5.6. Effect of FVBM Extract, F18 bioactive compound and atorvastatin

on the ratios of plasma HDL-C/TC, HDL-C/LDL-C, TC/HDL-C and LDL-

C/HDL-C in cigarette smoke-exposed rats after 4 weeks of treatment

†For the calculation of ratios, data is taken from Table 5.4 and 5.5.

*Values are mean (ratio)SD from plasma of 5 rats in each group.



Group*/Ratio

† HDL-C/TC HDL-C/LDL-C

TC/HDL-C

LDL-C/ HDL-C

N-C 0.35±.015 0.70±0.031 2.85±0.105 1.43±0.057

S-C 0.08±0.003

(-4.4 f)a

0.12±0.006

(-5.8f)a

12.51 ±0.46

(+4.4 f)a

8.38±0.35

(+5.9 f)a

FVT-1 0.18±0.008

(+2.3 f)a

0.30±0.014

(+2.1 f)a

5.55±0.24

(-2.3 f)a

3.33±0.186

(-2.5 f)a

FVT-2 0.30±0.013

(+3.8 f)a

0.58±0.026

(+4.8 f)a

3.33±0.135

(-3.8 f)a

1.73±0.079

(-4.8 f)a

C-T 0.33±0.016

(+4.4 f)a

0.65±0.029

(+5.8 f)a

3.04±0.091

(-4.4 f)a

1.53±0.061

(-5.9 f)a

A-T 0.33±0.015

(+4.1 f)a

0.64±0.028

(+5.3 f)a

3.03±0.129

(-4.1 f)a

1.56±0.064

(-5.4 f)a

CHAPTER 5


Table 5.7. Impact of FVBM extract, F18 bioactive compound and atorvastatin

on liver and lung TC and TG level in cigarette smoke-exposed rats after 4

weeks of treatment

Liver Lung

Group* Total

cholesterol

Triglycerides Total

cholesterol

Triglycerides

N-C 7.4±0.385 4.25±0.215 5.93±0.304 3.72±0.102

S-C 14.1±0.883

(+90.5%)a

8.2±0.416

(+92.3%)a

9.23±0.452

(+55.2%)a

4.75±0.109

(+27.7%)a

FVT-1 11.3±0.576

(-19.8%)b

6.7±0.324

(-18.29%)b

7.86±0.357

(-14.8%)c

4.46±0.104

(-6.1%)c

FVT-2 8.67±0.405

(-38.5%)a

5.56±0.236

(-32.1%)a

6.52±0.294

(-29.3%)a

4.01±0.082

(-15.6%)c

C-T 7.5±0.346

(-46.8%)a

4.3±0.217

(-47.6%)a

5.98±0.283

(-35.2%)a

3.75±0.107

(-21.1%)b

A-T 9.39±0.372

(-33.4%)a

4.89±0.238

(-40.3%)a

6.18±0.321

(-33.1%)a

3.97±0.106

(-16.3%)b

*Values are mean (mg/gm protein) SD from homogenate of liver or lung of

5 rats in each group.





CHAPTER 5


plasma, TC and TG levels in liver and lung were significantly increased in

smoke-exposed rats, which was subsequently attenuated after feeding of

FVBM extract, F18 and atorvastatin to smoke-exposed rats.

5.3.3.5 Regulation of enzymatic activity of hepatic HMG-CoA

reductase

Enzymatic activity of hepatic HMG-CoA reductase in cigarette smoke-

exposed rats was associated with significant amplification of 2.31 fold when

compared to N-C value. Administration of plant extract in two different doses

(FVBM 50 and 100 mg/rat) resulted in a significant decrease in HMG-CoA

reductase activity by 1.53 and 1.99 fold, respectively, when compared to S-C

value. It is noteworthy to mention that isolated compound (F18) exerts better

and significant reduction (2.21 fold) than the atorvastatin treated (A-T) group

(1.96 fold) (Table 5.8). These results demonstrate that FVBM extract and F18

bioactive compound mediated decrease in lipid and lipoprotein levels of

cigarette smoke-exposed rats is obviously due to reduction in the enzymatic

activity of hepatic HMG-CoA reductase, the rate limiting enzyme in the

biosynthetic pathway of cholesterol.

5.3.4 Impact of FVBM extract, F18 bioactive compound and

atorvastatin on lipid peroxidation status in plasma, erythrocytes, liver

and lung of smoke-exposed rats treated for 4 weeks

5.3.4.1 Impact on plasma total antioxidants and lipid peroxidation

products

Data in Table 5.9 demonstrate the antioxidant efficacy of FVBM

extract, F18 bioactive compound and atorvastatin on ex-vivo plasma

concentrations of total antioxidants, CD, LOOH and MDA in CS-exposed

rats. Table 5.9 illustrated that cigarette smoke causes substantial decrease in

plasma total antioxidants level and was reduced by 48 % while CD, LOOH

and MDA levels were increased by 163 %, 182 % and 116 % respectively.

CHAPTER 5


Table 5.8. In-vivo regulation of hepatic HMG-CoA reductase activity in

cigarette smoke-exposed rats treated with FVBM extract, F18 bioactive

compound and atorvastatin for 4 weeks of treatment

Group* HMG-CoA reductase activity†

N-C 4.34±0.25

S-C 1.87±0.11

(+2.31 f)a

FVT-1 2.87±0.14

(-1.53 f) a

FVT-2 3.73±0.17

(-1.99 f) a

C-T 4.14±0.21

(-2.21 f) a

A-T 3.67±0.18

(-1.96 f) a

†Expressed as ratio of HMG-CoA to Mevalonate; lower the ratio higher the

enzyme activity.

*Values are mean SD from liver homogenate of 5 rats in each group.



CHAPTER 5


Table 5.9. Antioxidant impact of FVBM, F18 bioactive compound and

atorvastatin on plasma total antioxidants, CD, LOOH and MDA contents in

cigarette smoke-exposed rats after 4 weeks of treatment

Group* CD LOOH MDA Total

antioxidants

N-C 2.86±0.141 1.02±0.022 1.24±0.015 85.47±3.46

S-C 7.52±0.325

(+162.9%)a

2.88±0.104

(+182.4%)a

2.68±0.125

(+116.1%)a

44.21±2.04

(-48.27%)a

FVT-1 5.28±0.216

(-29.8%)a

1.96±0.083

(-31.9%)a

1.95±0.098

(-27.2%)b

56.42±2.79

(+27.6%)a

FVT-2 3.21±0.110

(-57.3%)a

1.29±0.034

(-55.2%)a

1.46±0.062

(-45.5%) a

81.53±3.16

(+84.4%) a

C-T 2.91±0.108

(-61.3%)a

1.08±0.028

(-62.5%)a

1.28±0.042

(-52.2%) a

79.18±3.73

(+79.1%) a

A-T 5.74±0.209

(-23.7%)b

2.28±0.026

(-20.8%)a

2.14±0.044

(-20.1%) c

51.79±2.92

(+17.1%) a

*Values are mean (µmole/dl) ± SD from plasma of 5 rats in each group.





CHAPTER 5


Administration of FVBM extract (50 & 100 mg/rat/day), F18 and atorvastatin

to smoke-exposed rats significantly increased the total antioxidants levels by

28 %, 84 %, 79 % and 17 %, respectively, when compared to S-C value. On

the other hand, CD, LOOH and MDA levels were significantly decreased by

30 %, 32 % and 27 % in FVT-1; 57 %, 55 % and 45 % in FVT-2; 61 %, 63 %

and 52 % in C-T and 24 %, 21 % and 17 % in A-T rats, when compared to

corresponding values in smoke-exposed rats. In four treated groups, plasma

antioxidant and lipid peroxidation product levels were significantly

ameliorated close to corresponding normal values, indicating a potent in-vivo

antioxidative effect of above fractions/compounds in the order C-T>FVT-

2>FVT-1>A-T.

5.3.4.2 Effect on membrane lipid peroxidation in erythrocytes

As seen in Table 5.10, erythrocytes from smoke-exposed rats (S-C)

group showed a greater susceptibility to hydrogen peroxide-induced lipid

peroxidation than those from N-C group. The MDA level was substantially

increases by 125 % in S-C rats, when compared to N-C value. Formation of

MDA was markedly decreased by 23 %, 46 %, 54 % and 25 %, after the

administration of FVBM extract, F18 and atorvastatin, respectively, when

compared to the corresponding values in S-C. Similarly, H2O2-mediated

release of MDA erythrocytes was increased from 22.45 in N-C to 46.6

nmol/gHb (108 %) in S-C rats. A highly significant decrease of 27 %, 36 %,

50 % and 21 % in MDA release was seen in smoke-exposed rats treated with

FVBM extract, F18 and atorvastatin, respectively, when compared to

corresponding values in S-C rats. These results demonstrate that exposure of

cigarette smoke to rats for 4 weeks was associated with a significant increase

in both ex-vivo and in-vivo erythrocytes membrane lipid peroxidation product

and MDA, which was significantly prevented by the administration of FVBM

extract, F18 and atorvastatin.

CHAPTER 5


Table 5.10. Basal MDA contents and its H2O2-induced MDA release in intact

erythrocytes of cigarette smoke-exposed rats after 4 weeks of FVBM extract,

F18 bioactive compound and atorvastatin treatment

Group* MDA

content

(nmole/g Hb)

MDA release

(percent)

N-C 5.67±0.24 22.45±0.92

S-C 12.74±0.64

(+124.7%)a

46.6±2.13

(+107.6%)a

FVT-1 9.78±0.46

(-23.2%)a

34.26±1.92

(-26.5%)a

FVT-2 6.82±0.31

(-46.4%)a

29.87±1.12

(-35.9%)a

C-T 5.82±0.23

(-54.3%)a

23.68±1.02

(-49.2%)a

A-T 9.54±0.32

(-25.1%)a

36.81±1.24

(-21.0%)a

*Values are mean SD from packed erythrocytes of 5 rats in each group.



CHAPTER 5


5.3.4.3 Impact on liver and lung lipid peroxidation products

In all four treated groups, the CS-mediated increase in CD, LOOH and

MDA was significantly blocked, with a maximum decrease of 31 %, 40 %

and 23 % in FVT-2 and 26 %, 47 % and 34 % in C-T rats, respectively, when

compared to corresponding values in S-C group (Table 5.11). Similar

prototype was shown in lung, CD, LOOH and MDA levels which were

significantly reduced after treatment with FVBM, F18 and atorvastatin with a

maximum decrease of 39 %, 39 % and 30 % and 46 %, 48 % and 37 % in

FVT-2 and C-T group, respectively. These results demonstrate that increased

levels of CD, LOOH and MDA in liver and lung of smoke-exposed rats were

significantly reduced after 4 weeks of treatment, with a maximum effect

depicted by FVT-2 and F-18 group treated with FVBM (100 mg/rat/day) and

F18 compound, respectively.

5.3.5 Ex-vivo and in-vitro Cu++

-mediated oxidation of LDL in smoke-

exposed rats treated with FVBM extract, F18 bioactive compound and

atorvastatin

5.3.5.1 Antioxidant effect on basal and maximal level of CD formation and

MDA content in LDL

As depicted in Table 5.12, the ex-vivo basal CD level of LDL in smoke-

exposed rats was increased by 48 %, in comparison to the corresponding N-C

values. Administration of FVBM-50, FVBM-100, F18 and atorvastatin to

these smoke-exposed stressed rats partially blocked their in-vivo LDL

oxidation and reduced their basal CD levels by 11 %, 27 %, 29 % and 8 %,

respectively, in comparison to the corresponding S-C values. The maximal

CD value of LDL in N-C was substantially increased by 430 % in comparison

to corresponding basal CD value in N-C. When compared to corresponding

maximal CD value in N-C rats, LDL associated CD formation of S-C rats was

increased by 43 %.

CHAPTER 5


Table 5.11. Effect of FVBM extract, F18 bioactive compound and atorvastatin on liver and lung CD, LOOH and MDA

contents in cigarette smoke-exposed rats after 4 weeks of treatment

Liver Lung

Group* CD LOOH MDA CD LOOH MDA

N-C 5.69±0.211 0.72±0.041 2.86±0.085 1.88±0.111 0.92±0.041 2.16±0.085

S-C 9.81±0.417

(+87.8%)a

1.22±0.086

(+97.4%)a

4.42±0.115

(+56.1%)a

3.61±0.217

(+92.0%)a

1.81±0.086

(+96.7%)a

4.12±0.115

(+90.7%)a

FVT-1 8.71±0.325

(-11.2%)c

1.05±0.054

(-19.0%)c

4.05±0.168

(-8.2%)c

3.04±0.225

(-15.8%)c

1.25±0.054

(-30.9%)a

3.28±0.168

(-20.4%)b

FVT-2 6.81±0.221

(-30.5%)a

0.79±0.051

(-39.5%)a

3.38±0.124

(-23.4%)a

2.20±0.121

(-39.1%)a

1.10±0.051

(-38.9%)a

2.89±0.124

(-29.8%)a

C-T 7.22±0.210

(-26.4%)a

0.76±0.048

(-46.5%)a

2.96±0.112

(-33.8%)a

1.96±0.110

(-45.7%)a

0.94±0.048

(-48.1%)a

2.59±0.112

(-37.1%)a

A-T 8.35±0.240

(-15.3%)b

1.08±0.062

(-11.5%)d

3.97±0.142

(-10.1%)c

3.12±0.140

(-13.6%)c

1.42±0.062

(-21.5%)c

3.56±0.142

(-13.6%)b

*Values are mean (nmole/mg protein) SD from homogenate of liver or lung of 5 rats in each group.

Significantly different from N-C at ap<0.001. Significantly different from S-C at

ap<0.001. Significantly different from S-

C at bp<0.01. Significantly different from S-C at

cp<0.05. Non-Significantly different from S-C at

dp>0.05

CHAPTER 5


Table 5.12. Ex-vivo and Cu++

-catalyzed in-vitro oxidation of LDL, from

plasma of cigarette smoke-exposed rats after 4 weeks of FVBM extract, F18

bioactive compound and atorvastatin treatment

Group LDL oxidation+

Conjugated diene

formation* MDA content

#

Basal Maximal Lag

Phase**

Basal Maximal¥

N-C 178.49 945.86

(+430%)

88 5.43±0.212 15.26

S-C 264.36

(+48.3%)†

1348.56

(+42.6%)€

68

(-22.7%)¶

8.64±0.364a

(+53.2%)†

23.65

(+54.9%)€

FVT-1 234.72

(-11.4%)††

1264.35

(-6.2%)α

75

(+10.3%)§

7.43±0.202 b

(-14.0%)††

20.26

(-14.3%) ♯

FVT-2 192.41

(-27.3%)††

1048.95

(-22.2%) α

85

(+25.0%)§

6.24±0.244 a

(-27.8%)††

18.45

(-21.9%) ♯

C-T 187.32

(-29.2%)††

989.35

(-26.6%) α

87

(+27.9%)§

5.87±0.231 a

(-32.1%)††

16.6

(-29.8%) ♯

A-T 244.54

(-7.5%)††

1294.28

(-4.0%) α

73

(+7.4%)§

7.56±0.292 c

(-12.5%)††

20.43

(-13.6%) ♯

*The CD values are expressed as nmole MDA equivalents/mg protein. Basal

conjugated diene represent the in-vivo status of oxidized LDL. **

The lag phase is defined as the interval between the intercept of the tangent

of the slope of the curve with the time expressed in minutes. ¥The maximum

in-vitro oxidation of LDL was achieved after 12hrs of incubation with CuSo4

in each group, †Percent increase with respect to basal value in N-C,

††Percent

decrease with respect to basal value in S-C, ¶Percent decrease with respect to

lag phase value in N-C, §Percent increase with respect to lag phase value in S-

C, €Percent increase with respect to maximal value in N-C,

♯Percent decrease

with respect to maximal value in S-C, Significantly different from N-C at aP<0.001.

+Values are obtained from LDL subpopulation, isolated from

plasma of 5 rats in each group.

Significantly different from N-C at ap<0.001, significantly different from S-C

at ap<0.001, significantly different from S-C at

bp<0.01, significantly different

from S-C at cp<0.05.

CHAPTER 5


Being a potent antioxidant, FVBM-100 and F18 significantly blocked the

maximal CD concentration and reduced them by 22 % & 27 %, respectively,

in comparison to corresponding maximal values in S-C rats. As expected, the

lag phase time of LDL oxidation was reduced from 88 min in N-C to 68 min

in S-C. Treatment of smoke-exposed rats with FVBM-50, FVBM-100, F18

and ATR, restored the lag phase time of LDL oxidation to 75 min, 85 min, 87

min and 73 min, respectively. Similar to ex-vivo basal and in-vitro Cu++

catalyzed maximal CD values, the ex-vivo basal MDA content in LDL was

significantly increased by 53 % in S-C rats, when compared to corresponding

values in N-C rats. FVBM-50, FVBM-100, F18 and atorvastatin treatment to

smoke-exposed rats significantly blocked the ex-vivo increase in LDL MDA

formation in S-C rats and reduced their levels by 14 %, 28 %, 32 % and 13 %

respectively. An almost similar pattern was observed in maximal MDA

content of LDL. These result demonstrate that feeding of FVBM extract (at

higher dose) and F18 bioactive compound to smoke-exposed rats mediated an

antioxidant effect by preferentially and significantly blocking the in-vivo and

in-vitro formation of CD and MDA content of atherogenic LDL as well as

increasing the lag phase time of atherogenic LDL.

5.3.6 Regulatory effect of FVBM extract, F18 bioactive compound and

atorvastatin on antioxidant defence system in smoke-exposed rats after 4

weeks of treatment

Production of free radicals is associated with cigarette smoke which in

turn generates oxidative stress that links with atherosclerosis. Since,

protection against oxidative stress/ROS is provided by enzymatic and non-

enzymatic antioxidants, therefore, the status of antioxidant enzymes, such as

CAT, SOD, Gpx, GST and Gred including GSH concentrations in liver and

lung of experimental smoke-exposed rats are important.

CHAPTER 5


5.3.6.1 Impact on the regulation of hepatic and lung catalase, superoxide

dismutase, glutathione peroxidase, glutathione reductase, glutathione-s-

transferase activities and reduced glutathione content

The enzymatic activities of hepatic CAT, SOD, Gred and GST in S-C rats

were significantly decreased from N-C values of 62.28, 7.65, 18.29 and 132.8

U/mg protein to 32.45, 4.8, 11.28 and 68.12 U/mg protein, respectively, while

Gpx activity in S-C rats was increased from N-C values of 44.2 U/mg protein to

57.28 U/mg protein (Figure 5.1). The restoration in above enzymatic activities of

hepatic CAT, SOD, Gred and GST in FVT-2 treated rat was 24 %, 32 %, 48 %

and 36 % of respective normal control values. The values of these enzymes in C-

T group, in comparison to S-C values, were significantly increased by 35 %, 53

%, 60 % and 43 % respectively. In contrast the hepatic Gpx activity was

significantly decreased by 14 %, 18 %, 20 % and 10 % in FVT-1, FVT-2, C-T

and A-T rats. As depicted in Figure 5.3, the hepatic GSH content were reduced

by 43 % when compared to value of N-C group. Administration of FVBM

extract, F18 bioactive compound and atorvastatin to CS-exposed rats resulted in

a significant increase in GSH content by 22 %, 32 %, 45 % and 18 %,

respectively.

Moreover, similar pattern was observed in lung enzymatic and non-

enzymatic antioxidant defence system. As depicted in Figure 5.2 the enzymatic

activities of lung CAT, SOD, Gred and GST in S-C rats were significantly

decreased from N-C value of 32.5, 14.7, 24.3 and 127.7 U/mg protein to 21.86,

9.56, 11.78 and 102.47 U/mg protein. Whereas, lung Gpx activity in S-C rats

was significantly increased from N-C values of 59.68 U/mg protein to 82.73

U/mg protein. The restoration in above enzymatic activities of lung CAT, SOD,

Gred and GST in FVT-2 treated rat was 34 %, 40 %, 41 % and 20 % of

respective N-C values. The values of these enzymes in C-T group, in

comparison to N-C values, were significantly increased by 42 %, 50 %, 47 %

and 24 % respectively. In contrast the lung Gpx activity was significantly

CHAPTER 5


Figure 5.1. Impact of FVBM extract, F18 bioactive compound and

atorvastatin on Liver CAT, SOD, Gpx, Gred and GST activities in cigarette

smoke-exposed rats after 4 weeks of treatment.

0

50

100

150

Gpx Gred GST CAT SOD

Liver Antioxidant Enzymes

Units/

mg P

rote

in

N-C S-C FVT-1 FVT-2 C-T A-T

aa

a a a a a

aa

aa

a a

b b

b b

b b

b b

a

cc

c

One unit (U/ mg protein) of Gpx activity is defined as nmole oxidized

glutathione formed/min/mg homogenate protein. One unit (U/ mg protein) of

Gred activity is defined as nmole NADPH oxidized/min/mg PMS protein.

One unit (U/ mg protein) of GST activity is defined as the nmole of 1-chloro

2,4-dinitrobenzene (CDNB) conjugate formed/min/mg PMS protein. One unit

(U/mg protein) of CAT activity is defined as the µmoles of H2O2

decomposed/min/mg protein. One unit (U/mg protein) of SOD activity is

defined as the amount of enzyme required to inhibit O.D. at 560 nm of

chromogen production by 50 % in one minute.

Values are mean SD from homogenate/PMS fraction of liver of 5 rats in

each group.

Significantly different from N-C at ap<0.001, significantly different from S-C

at ap<0.001, significantly different from S-C at

bp<0.01, significantly different

from S-C at cp<0.05.

CHAPTER 5


Figure 5.2. Impact of FVBM extract, F18 bioactive compound and

atorvastatin on lung CAT, SOD, Gpx, Gred and GST activities in cigarette

smoke-exposed rats after 4 weeks of treatment.

0

20

40

60

80

100

120

140

Gpx Gred GST CAT SOD

Lung Antioxidant Enzymes

Units/

mg P

rote

in

N-C S-C FVT-1 FVT-2 C-T A-T

a

a

aa a

a

a

ba

a a a

b b

b b

b

bbb b

c

c

c

b

One unit (U/ mg protein) of Gpx activity is defined as nmole oxidized

glutathione formed/min/mg homogenate protein. One unit (U/ mg protein) of

Gred activity is defined as nmole NADPH oxidized/min/mg PMS protein.

One unit (U/ mg protein) of GST activity is defined as the nmole of 1-chloro

2,4-dinitrobenzene (CDNB) conjugate formed/min/mg PMS protein. One unit

(U/mg protein) of CAT activity is defined as the µmoles of H2O2

decomposed/min/mg protein. One unit (U/mg protein) of SOD activity is

defined as the amount of enzyme required to inhibit O.D. at 560 nm of

chromogen production by 50 % in one minute.

Values are mean SD from homogenate/PMS fraction of Lung of 5 rats in

each group.

Significantly different from N-C at ap<0.001. Significantly different from N-C

at bp<0.01. Significantly different from S-C at

ap<0.001. Significantly

different from S-C at bp<0.01. Significantly different from S-C at

cp<0.05.

CHAPTER 5


Figure 5.3. Effect of FVBM extract, F18 bioactive compound and atorvastatin

on liver and lung GSH content in cigarette smoke-exposed rats after 4 weeks

of treatment.

Values are mean (nmole SH group/mg protein) SD from homogenate of

liver or lung of 5 rats in each group.





CHAPTER 5


decreased by 11 %, 17 %, 26 % and 16 % in FVT-1, FVT-2, C-T and A-T

treated rats. Similarly, lung GSH content which was significantly reduced by

33 % in S-C rats was significantly increased by 20 %, 37 %, 46 % and 28 %

after administration of FVBM extract, F18 bioactive compound and

atorvastatin. Here it is interesting to mention that atorvastatin also exhibited

significant amelioration in all the enzymatic and non-enzymatic activities but

is consider to be less effective than higher dose of FVBM extract and F18

bioactive compound,

In summary, hepatic and lung CAT, SOD, Gred, GST enzymes and

non-enzymatic GSH, which constitute a mutually supportive team of defence

against ROS, are significantly ameliorated after feeding of FVBM extract and

F18 bioactive compound as well as substantially quenches the free radicals,

thus positively normalizing the above enzyme levels close to normal values.

5.3.7 Histopathological studies of liver and lung

As shown in Figure 5.4A, histopathology of the liver of the control rats

showed normal looking uniform hepatocytes with small vesicular nuclei and

abundant eosinophilic granular cytoplasm with distinct cell boundaries.

Architecture of liver is well maintained. Interstitial cells normal with normal

vascularity. CS-exposed rats showed smaller hepatocytes with small vesicular

or pyknotic nuclei and reduced granularity of cytoplasm. Cytoplasmic

boundaries are relatively indistinct. Architecture not well maintained.

Interstitial cells increased and vascularity is reduced (Figure 5.4B).

Administration of FVBM extract at lower dose, shows smaller hepatocytes

with smaller vesicular nuclei and eosinophilic cytoplasm with indistinct cell

boundaries showing proliferation. Architecture not well maintained but

vascularity has relatively increased (Figure 5.4C). Moreover, liver section of

FVT-2 rats (higher dose) (Figure 5.4D) shows proliferated normal hepatocytes

with normal vesicular nuclei and abundant eosinophilic cytoplasm with

CHAPTER 5


Figure 5.4A. Microphotograph of liver section from normal control rats.

Figure 5.4B. Microphotograph of liver section from cigarette smoke-exposed

rats after 4 weeks of cigarette smoke exposure.

CHAPTER 5


Figure 5.4C. Microphotograph of liver section from cigarette smoke-exposed

rats after 4 weeks of FVBM-50 treatment.

Figure 5.4D. Microphotograph of liver section from cigarette smoke-exposed


CHAPTER 5


distinct cell boundaries. Architecture well maintained with normal interstitial cell

and vascularity. Similarly, liver section of C-T rats shows proliferated

hepatocytes with normal morphology as well as normal architecture is well

maintained. Interstitial cells normal with normal vascularity, also no toxic effects

noted (Figure 5.4E). Furthermore, liver section of A-T rats shows normal

looking hepatocytes with normal vesicular nuclei and abundant eosinophilic

cytoplasm. Architecture well maintained and normal vascularity is seen (Figure

5.4F).

Histopathology of lung in N-C group shows normal lung stroma and

characteristic spongy appearance of the lung with normal looking numerous

alveolar spaces, had normal blood vessels and bronchi. The branchoalveolar unit

parenchyma in the normal lung was within the limits (Figure 5.5A). The lung

section of CS-exposed rats shows increased volume of stroma with presence of

few collections of stomal cells with reduced alveolar spaces, reduced vascularity

and constricted bronchi (Figure 5.5B). There was obliteration of most alveoli and

subsequently compensatory dilatation and expansion of the contiguous ones with

destruction of alveolar wall. On treatment with lower dose of FVBM extract,

section of lung shows reduced stroma, increased alveolar spaces, increased

vascularity with patent blood vessels and dilated bronchi (Figure 5.5C). While,

on treatment with higher dose, section of lung shows above mentioned changes

but bronchi are relatively constricted (Figure 5.5D). In addition, in lung section

of C-T rat observed that stroma is further reduced with presence of large alveolar

spaces. Blood vessels are normal but smaller bronchi are further constricted

(Figure 5.5E). Furthermore, lung slice of atorvastatin treated group shows

alveolar spaces comparatively less than normal with corresponding increase in

stromal elements. Blood vessels and bronchi are normal with presence of few

constricted smaller bronchi (Figure 5.5F).

CHAPTER 5


Figure 5.4E. Microphotograph of liver section from cigarette smoke-exposed

rats after 4 weeks of F18 bioactive compound treatment.

Figure 5.4F. Microphotograph of liver section from cigarette smoke-exposed

rats after 4 weeks of atorvastatin treatment.

CHAPTER 5


Figure 5.5A. Microphotograph of lung section from normal control rats.

Figure 5.5B. Microphotograph of lung section from cigarette smoke-exposed

rats after 4 weeks of cigarette smoke exposure.

CHAPTER 5


Figure 5.5C. Microphotograph of lung section from cigarette smoke-exposed


Figure 5.5D. Microphotograph of lung section from cigarette smoke-exposed


CHAPTER 5


Figure 5.5E. Microphotograph of lung section from cigarette smoke-exposed

rats after 4 weeks of F18 bioactive compound treatment.

Figure 5.5F. Microphotograph of lung section from cigarette smoke-exposed

rats after 4 weeks of atorvastatin treatment.

CHAPTER 5


5.4 DISCUSSION

Cigarette smoking (CS) is the foremost cause of morbidity and mortality

worldwide (Jha et al., 2008; Alam et al., 2013) and is considered to be the

preventable risk factor for CVD (Ambrose and Barua, 2004). Cigarette

smoking contribute to oxidative stress responsible for enhanced lipid

peroxidation, protein oxidation, DNA damage and endothelial damage in

several organs of animals results in various diseases such as CVD, stroke,

different types of cancer and diabetes (Willi et al., 2007; Valavanidis et al.,

2009; Messner and Bernhard, 2014; Mohod et al., 2014).

The current chapter basically illustrate the use of FVBM extract and

F18 compound in prevention and protection of CS-induced oxidative stress,

hypercholesterolemia and subsequent atherosclerosis. This study recognised

that CS-exposed rats showed significant loss of weight and this loss was

restored by treatment with FVBM and F18 bioactive compound. Rats exposed

to cigarette smoke showed loss of appetite, moderate irritability and breathing

difficulties which were in agreement with previously published reports that

showed acute effects of cigarette smoking in human and animals leads to

breathing difficulties due to marked airway inflammation (Vaart et al., 2004;

Ghobashy et al., 2010). The weight after CS-exposed rats was markedly less

than that of controls, perhaps due to reduced food intake or gastrointestinal

irritation. Other reports also stated that smoker's weight on average is about 4

kg less than non-smokers, mainly because of reduced food intake (Rang et al.,

2001).

The gas phase and tar of cigarettes contain free radicals, responsible for

oxidative stress, has been hypothesized to be involved in the pathogenesis of

smoking-related atherosclerosis (Yokode et al., 1995). Moreover, cigarette

smoking generates many toxic and carcinogenic compounds harmful to the

health, such as nicotine, nitrogen oxides, carbon monoxide (CO), hydrogen

CHAPTER 5


cyanide and free radicals (Neunteufl et al., 2002; Valavanidis et al., 2009;

Papathanasiou et al., 2014). It has been known that nicotine, a component of

the tar phase, is an addictive substance of cigarette smoke and acutely

increases endothelial dysfunction by means of impaired vascular reactivity

(Neunteufl et al., 2002; Powell, 1998). Similarly, CO directly leads to high

levels of carboxyhemoglobin and continual exposure of high levels of CO,

leads to increased exercise-induced ischemia, ventricular dysfunction and

arrhythmias in patients with CAD (Allred et al., 1989). However, more recent

data suggest that CO from cigarette smoke was an unlikely cause for

atherosclerosis or thrombus (Rochette et al., 2013; Papathanasiou et al., 2014;

Chung et al., 2015). Similar to these reports, our results showed significant

increase of blood carbon monoxide saturation (carboxyhemoglobin) and

nicotine level in CS-exposed rats. Further, significant restoration of 30 %, 32

% & 18 %, 26 % in blood carbon monoxide saturation and nicotine level was

observed after 4 weeks of treatment with higher dose of FVBM extract and

F18 compound. These results specify a strong protective effect of FVBM

extract and F18 compound, which may help in lowering the menace of

myocardial infarction in smokers.

Numerous smoking consequences have been described as being

atherogenic, like direct vascular actions, oxidative stress, thrombogenic

factors and secondary dyslipidemia (Powell, 1998; Mohod et al., 2014; Chung

et al., 2015). In this context, our data recognized that exposure of cigarette

smoke to rats results in elevated plasma TC, TG and LDL-C levels. The

increase in cholesterol and TG level of CS-exposed rats is in consensus with

previously published reports (Chitra et al., 2000; Ambrose and Barua, 2004;

Li et al., 2013). In addition, Kavitharaj and Vijayammal (1999) reported that

chronic administration of nicotine was found to produce enhanced synthesis

of triglycerides which is an independent risk factor for coronary heart disease.

CHAPTER 5


The dyslipidemic profile in smokers showed significantly higher TC, TG and

LDL but HDL is lower than in non-smokers (Craig et al., 1989). Among

apolipoproteins, apoB is known to measure the total number of atherogenic

particles and has been identified in VLDL and LDL (Boekholdt et al., 2012).

Hence, apoB is a more accurate measure than LDL-C with regard to

predicting the CAD. Though both cholesterol and apoB concentrations of

LDL particles were significantly increased in mild, moderate and heavy

smokers, apoB increase was much stronger. Moreover, elevated LDL-C in

CS-exposed rats is highly susceptible to oxidation and oxidized LDL-C plays

an important role in the development of atherosclerotic process and

endothelial dysfunction (Messner and Bernhard, 2014).

The increase in plasma TC level in S-C rats is apparently due to

increased cholesterol synthesis (Craig et al., 1989; Chitra et al., 2000; Li et

al., 2013), through the induction of hepatic HMG-CoA reductase activity-the

rate limiting enzyme in the biosynthetic pathway of cholesterol. The TG and

HDL abnormalities may be linked to insulin resistance which is a potential

key link between CS and CVD (Reaven et al., 2003). Enhance generation of

free radical load in smokers might stimulate VLDL production by increasing

adipose tissue lipolysis, increasing hepatic de novo fatty acid synthesis and

decreasing hepatic fatty acid oxidation, all of which provide fatty acid

substrate for esterification into TG and assembly into VLDL particles in the

liver as well as increase in plasma free fatty acid. Chen and Loo (1995) earlier

reported that the increase in the cholesterol content of TG rich VLDL might

be due to CS-induced reduction of lipoprotein lipase, which is responsible for

the removal of TG from VLDL particles. Thus, the increased level of VLDL-

C in S-C rats is also responsible for high concentrations of atherogenic

cholesterol rich circulating LDL. The observed high level of VLDL-C in CS

rats may responsible for reduced level of antiatherogenic HDL-C because of

CHAPTER 5


decreased availability of phospholipid remnants needed for the configuration

of HDL from VLDL and a concomitant decline in lecithin cholesterol:

acyltransferase (LCAT) activity.

Administration of FVBM extract at higher dose (100 mg/rat) and F18

bioactive compound to CS-exposed rats effectively blocked the increase in

TG, TC, LDL-C and non-HDL-C level and reversed them to a level close to

their normal control values, which is almost comparable to amelioration

exhibited by standard drug atorvastation in cigarette smoke-exposed rats.

Moreover, the present study observed diminished level of HDL-C in cigarette

smoke-exposed rats when compared to normal rats which were in agreement

with earlier reports (Craig et al., 1989; Vella et al., 1994). Simultaneous

administration of FVBM extract and F18 compound to CS-exposed rats

significantly increases the HDL-C level by 80 %, 162 % and 175 %, which

might be due to its profound antioxidant activity which in turn offer better

protection to LDL as well as HDL from oxidative stress through its associated

antioxidant enzyme paraoxonase (PON) (Watson et al., 1995). Consistent

with earlier reports (Lemieux et al., 2001; Millán et al., 2009) that established

lipoprotein ratios, LDL-C to HDL-C and HDL-C to TC, are good forecaster

for the existence and severity of CAD, our results depicted a significant

decrease in HDL-C/LDL-C and HDL-C/TC ratio and a increase in TC/HDL-

C and LDL-C/HDL-C ratios in CS-exposed hyperlipidemic rats. All the ratios

were significantly attenuated in CS-exposed rats treated with higher dose of

FVBM extract (100 mg/rat) or bioactive compound (F18). These results

which represent an initial demonstration indicate strong hypocholesterolemic

property of FVBM extract and F18 compound.

The significant amelioration in plasma and lipoprotein lipid level

exerted by these fractions was due to the marked suppression in the hepatic

HMG-CoA reductase activity. Among all the treated groups FVT-2 and C-T

CHAPTER 5


exhibited marked decline of 1.99 and 2.21 fold in HMG-CoA reductase

activity, respectively, which was better or equivalent to the decline observed

in atorvastatin treated rats. These data suggest that FVBM extract/compound

may exert their cholesterol lowering effect in CS-exposed hyperlipidemic rats

via suppression of hepatic HMG-CoA reductase m-RNA expression which in

turn cause inhibition of cholesterol synthesis (Maurya and Srivastava, 2011).

These results are in agreement with previous published reports that revealed

hypolipidemic property of natural bioactive compound might be due to

decrease in the efficiency of HMG-CoA reductase m-RNA translation and

increasing the degradation of reductase protein post translationally (Elson et

al., 1999; Parker et al., 1993; Pakpour, 2013). These results represent an

initial demonstration and exhibit strong rationale in support of the use of

FVBM extract/bioactive compound, preferably F18, as a functional food in

the prevention and treatment of tobacco induced dyslipidemia/hyperlipidemia

and atherosclerosis.

Free radicals are highly reactive molecules occurs in normal consequence of a

variety of biochemical reactions (Velavan, 2011). Overload of free radicals

from exogenous sources like, smoking, alcohol abuse, UV radiations and air

pollution added to the endogenous production of free radicals that results in

oxidative stress and oxidative damage to the tissues (Wu and Cederbaum,

2003; Ghobashy et al., 2010; Mohod et al., 2014). Oxidative stress cause

extensive damage to DNA, proteins and membrane lipids (Wu and

Cederbaum, 2003; Achudume and Aina, 2012). It is well known that CS is

associated with substantial increase in oxidative stress which is mainly due to

increased lipid peroxidation or reduced antioxidants (Ambrose and Barua,

2004; Mohod et al., 2014).

Throughout the course of CS-induced oxidative stress, oxygen derived

free radicals like superoxide (O¨), hydrogen peroxide (H2O2), hydroxyl (•HO),

CHAPTER 5


peroxyl (ROO), alkoxy (RO) and nitric oxide (NO) are known to be generated

in the cell, which can lead to oxidative modification of lipids and cause

membrane damage, resulting in cell death (Kalra et al., 1991; Pryor and Stone

et al., 1993; Yokode et al., 1995; Messner and Bernhard, 2014; Mohod et al.,

2014). The process of lipid peroxidation is initiated by interaction of hydroxyl

radical or peroxynitrite with polyunsaturated fatty acid, that are particularly

susceptible to attack by these free radicals from CS, leading the generation of

various by-products of lipid peroxidations viz., CD, LOOH and MDA (Pryor

and Stone et al., 1993; Lykkesfeldt, 2007; Thirumalai et al., 2010). The cells

of the artery wall, including macrophages, endothelial cells and smooth

muscle cells secrete oxidative products that can initiate lipid oxidation and

peroxidation products have been found in atheroma (Chisolm and Steinberg,

2000; Messner and Bernhard, 2014).

Cigarette smoke is known to contain reactive peroxyl radicals and

acetaldehyde which can increase lipid peroxidation (Pryor and Stone et al.,

1993; Thirumalai et al., 2010). Higher level of lipid peroxidation products and

oxidised LDL (OxLDL) are reported in smokers and passive smokers

(Yamaguchi et al., 2005; Mohod et al., 2014). It was previously found that

serum MDA concentrations were higher in smokers and rats exposed to CS

(Lykkesfeldt, 2007; Mohod et al., 2014). The excessive generation of free

radicals in young novice smokers exhibited greater degree of lipid

peroxidation and lower plasma antioxidant capacity compare to non-smokers

(Bloomer, 2007). A similar finding was observed by Mohod et al. (2014) that

depicted cigarette smoking imposes oxidative stress, promote lipid

peroxidation and consequently perturb the antioxidant defence systems in

blood and tissues of smokers. Similar to plasma, erythrocytes are also

susceptible to damage by CS-induced oxidative stress. Erythrocytes contain

haemoglobin molecules and PUFAs which are extremely prone to oxidative

CHAPTER 5


damage induced by ROS because they are constantly exposed to both

extracellular and intracellular sources of ROS and cause profound alteration

in the structure and function of cell membrane that lead to cell death

(Achudume and Aina, 2012). Cigarette smoke is highly responsible for

permanent inflammation and leads to imbalance in the profile of lipid

peroxidation products (Vaart et al., 2004), which can cause number of

membrane changes including lipid peroxidation in CS-exposed erythrocytes

of rats (Achudume and Aina, 2012). The studies also showed that

occupationally exposed human subjects indicate enhance lipid peroxidation

and alter antioxidant systems in erythrocytes (Durak et al., 2002).

Consistent with these reports, our results demonstrated a significant

increase in lipid peroxidation products viz., CD, LOOH and MDA in plasma

and erythrocytes of subchronic cigarette smoke-exposed rats. The increase in

plasma lipid peroxidation product is well correlated with a significant decline

in plasma total antioxidant content which in turn is consistent with the

prooxidant effect of CS in rats. These results are in consensus with previous

finding (Thirumalai et al., 2010), where an increase correlation between

plasma MDA level and antioxidants has been reported in CS-exposed

animals. Moreover, the CS-exposed rats erythrocytes observed increased

tendency to H2O2-induced MDA release when compared to MDA level of

normal control rats. A similar increase in H2O2-induced MDA release of

intact erythrocytes from CS-exposed rats has been reported by Khan et al.

(2012). However, protective role of lipid lowering agent with potent

antioxidant property, such as FVBM extract and F18 compound, on the

formation of lipid peroxidation products in plasma and erythrocytes of CS-

exposed rats has not been reported. Our results showed that supplementation

of FVBM extract and F18 bioactive compound to CS-exposed rats caused a

significant decrease in plasma and erythrocytes lipid peroxidation products

CHAPTER 5


with a concomitant and significant increase in plasma total antioxidants and

restored their levels close to corresponding control values in N-C. These

results are in concordant with our above discussed in-vitro data that

demonstrated the profound antioxidant property of FVBM extract and F18

bioactive compound (page no: 146). Here it is interesting to mention that F18

compound, at a dose of 1 mg showed significant amelioration in reducing CS-

induced oxidative stress parameters which were almost comparable to the

FVBM extract at higher dose. The above data indicate that FVBM extract at

higher dose and F18 compound strongly inhibit the lipid peroxidation process

initiated by free radicals, thus preventing the membrane damage. Several lines

of research have established that lipoproteins play a pivotal role in

atherogenesis (Nordestgaard and Nielsen, 1994; Boekholdt et al., 2012).

Exposure of CS produces enhanced free radical that leads to the oxidative

modification of lipoprotein (Yokode et al., 1995). During oxidative

modification native LDL is converted to Ox-LDL which is a key mediator of

atherosclerosis and plasma concentrations has been found to be elevated in a

range of cardiovascular diseases (Craig et al., 1989; Yokode et al., 1995;

Yamaguchi et al., 2001). The uptake of Ox-LDL by macrophages results in

the formation of foam cells which accumulated in blood vessel walls and

promotes the development of the characteristic fatty streaks found in

atherosclerotic lesions (Yokode et al., 1995; Yamaguchi et al., 2001). The

filtration rate of LDL particles into the arterial intima is inversely related to

particle size. Lipoprotein profiles that are relatively rich in sd-LDL particles

are associated with up to a 3-fold greater risk of myocardial infarction (MI)

than those mainly consist of lb-LDL particles (Hirano et al., 2004). Small

dense LDL particles therefore may penetrate more easily into the

subendothelial space of artery wall from the circulation (Nordestgaard and

Nielsen, 1994; Chapman et al., 1998).

CHAPTER 5


Previous studies showed that elevated level of Ox-LDL concentrations

in plasma have been found in smoke-exposed rats (Ejaz, 2013). The ex-vivo

BDC levels and MDA have been suggested to reflect the in-vivo oxidation of

LDL (Planski et al., 1989). The result demonstrated that LDL in the CS-

exposed dyslipidemic rats had an increased susceptibility to oxidation when

compared to LDL of normal rats. Moreover, our result showed that the ex-

vivo BDC and MDA levels of LDL in CS-exposed hyperlipidemic rats was

increased by 48 % and 53 % respectively, in comparison to the corresponding

N-C values. This increase susceptibility may result from increased oxidative

stress, decrease total antioxidant and increase LDL cholesterol content. To the

best of our knowledge no one to date has demonstrated the potent protective

effect of F. virens extract or the isolated bioactive compound on the ex-vivo

and in-vitro Cu++

-catalyzed oxisability of plasma LDL of CS-exposed

dyslipideamic rats. Simultaneous supplementation of FVBM extract and

bioactive compound to CS-exposed rats blocked the in-vivo and in-vitro

oxidation of LDL as seen by a significant decrease in ex-vivo basal (27 % and

29 %) and maximal (22 % and 27 %) CD levels and increase in the lag time.

As expected, the lag phase time of LDL oxidation was reduced from 88 min

in N-C to 68 min in S-C. Treatment of CS-induced hyperlipidemic rats with

higher dose of FVBM and F18 compound restored the lag phase time of LDL

oxidation to 85 min and 87 min, respectively. Similarly, ex-vivo basal and

Cu++

induced maximal formation of MDA in LDL was significantly decreased

in CS-exposed rats treated with plant extract and the bioactive compound. The

data summarises that FVBM extract at a lower dose was least effective as an

antioxidant and was only able to partial restoration of the above lipid

peroxidation parameters. The potent hypolipidemic property of FVBM extract

(at higher dose) and the bioactive compound, as discussed above, is consistent

with the excellent antioxidant property of these fractions.

CHAPTER 5


Cigarette smoke is a complex situation possessing an array of free

radicals and ROS (Pryor and Stone, 1993). Modulation of antioxidant status

by increased lipid peroxidation throughout exposure to cigarette smoke has

been reported in various organs (Anbarasi et al., 2006; Luchese et al., 2009;

Thirumalai et al., 2010). Under an oxidative stress, the antioxidant enzyme

levels are ameliorated, in order to cope with the tremendous increase in the

production of ROS (Mccord, 1993; Anbarasi et al., 2006; Mohod et al.,

2014). However, after prolonged exposure, the toxic effects of cigarette

smoke emerge to override the adaptive mechanism of the body tissues, as

indicated by a decrement in the levels of these enzymes (Hulea et al., 1995).

As evident by above discussion CS-induces paramount oxidative stress and is

consistent with our results that demonstrate significant decrease in liver and

lung enzymatic activity of SOD, CAT, Gred, GST including the GSH level,

while Gpx activity was significantly increased in CS-exposed rats.

In antioxidant defence system SOD is known to be the first enzyme

which is responsible for scavenging the superoxide radicals to form H2O2

(Andersen et al., 1997). Further, this H2O2 is scavenged by catalase/GSH

(glutathione peroxidase) or it facilitate in the development of highly reactive

oxygen species (Inoue et al., 1994; Mohod et al., 2014). The tar phase of

cigarette smoke contains quinone–semiquinone radicals which are capable of

reducing molecular oxygen to superoxide radicals whose extreme generation

inactivates this enzyme (Church and Pryor, 1985; Durak et al., 2002). The

proposed decrease in SOD activity in CS-exposed rats could have resulted

from its inactivation by tar phase oxidants. The presence and production of

the free radicals from smoke lower enzymatic activity of CAT, leading to

accumulation of H2O2 and lipid hydroperoxides which further worsening the

tissue damage (Pryor and Stone, 1993). Inhibition of CAT activity in rat liver

CHAPTER 5


and lung by cigarette smoke has been previously reported (Ramesh et al.,

2007; Ramesh et al., 2010; Luchese et al., 2009).

However, a decrease in the activity of CAT in the present study

suggests the in-vivo decrease in antioxidant level which in turn unable to

defend the oxidative stress generated via cigarette smoke. This decrease in

SOD and CAT activity is well in consensus with our lipid peroxidation data

and plays an important role in scavenging the toxic intermediate product of

lipid peroxidation. Treatment with FVBM extract and F18 bioactive

compound to smoke-exposed rats resulted in a significant increase in both

liver and lung SOD and CAT activity which further strengthen the potent free

radical scavenging property of plant extract and bioactive compound.

Reduced glutathione play an important role in glutathione-dependent

antioxidant system that most probably act as free radicals scavenger or a

substrate for Gpx and GST during the detoxification of H2O2 (Masella et al.,

2005). Glutathione is maintained in body from its oxidized form by the

enzyme Gred, which requires NADPH as a cofactor (Carmel-Harel and Storz

2000). Previously it has been reported that GSH was depleted during CS-

exposure in various tissues. Meanwhile, the decrease in tissue GSH levels in

CS-exposed rats may be because of declined Gred activity and probably

reduced NADPH supply (Masella et al., 2005). GST is involved in the

detoxification of ROS and toxic compounds from cigarette smoke, by

conjugating them to GSH. This conjugation reaction results in depletion of the

intracellular GSH that may further enhances oxidant injury probably due to

non-availability of GSH for antioxidant enzymes such as Gpx. Recent

experimental data support the assumption that CS exposure increases

oxidative stress and act as a potential mechanism for initiating cardiovascular

dysfunction (Ambrose and Barua, 2004).

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Our data is well in agreement with above discussed reports and observed the

significant decline in GSH, Gred and GST level in contrast the Gpx activity in

liver and lung of CS-exposed rats was significantly increased which is due to

inability of CAT to cope with the oxidative stress. Helen and Vijayammal

(1997) also observed a similar decrease in the activity of SOD, CAT, Gred

and an increase in Gpx in rats exposed to cigarette smoke. Gpx is also a

scavenging enzyme, but an increase in its activity in tissues of CS-exposed

rats may further reduce the GSH content. In addition, an increased Gpx

activity represents a compensatory mechanism to degrade H2O2. Simultaneous

administration of FVBM extract (50 mg/rat and 100 mg/rat) and F18

bioactive compound (1 mg/rat) significantly increases CAT, SOD, Gred and

GST activity as well GSH concentration coupled with decrease in Gpx level

in CS-exposed stress rats. Thus, it has been concluded that administration of

FVBM extract at higher dose and F18 bioactive compound to CS-exposed rats

restore the enzymatic (SOD, CAT, Gred, GST and Gpx) and non-enzymatic

(GSH) antioxidant defence system and thus protect liver and lung cells

against oxidative stress-induced damage by directly counteracting ROS/free

radicals and by activating the overall antioxidant defence systems.

Histological observations noticed the pulmonary congestion and

thickening of interalveolar septa in rats exposed to cigarette smoke. The

present study showed devastation of some alveolar walls and foci of collapsed

alveoli with subsequent dilation of the adjoining alveolar spaces and

development of large irregular spaces (emphysematous changes). Cigarette

smoking causes endothelial dysfunction due to increased oxidative stress

(Messner and Bernhard, 2014). Our results coincided with those who

interrelated the thickening of the alveolar septa to modification in the vascular

bed resulting in inflammatory infiltration and oedema (Hora et al., 2003).

Chronic cigarette smoke exposure causes lung tissue destruction might be due

CHAPTER 5


to increased production of metalloproteinases (MMP) proteolytic enzymes by

macrophages (Barnes et al., 2003; Taraseviciene-Stewart and Voelkel, 2008).

The liver showed congestion in central vein, portal inflammation and necrosis

in CS-exposed rats. These morphological changes were significantly reversed

after treatment with FVBM extract and F18 compound. Thus biochemical and

histopathological studies suggested that, FVBM extract and F18 showed its

protective nature against CS-exposed rats.

In conclusion our combined results clearly demonstrated the protective

role of FVBM extract and F18 compound in risk factor induced

cardiovascular disease. Moreover, the test fractions/pure compound exhibited

significant protection against CS-induced severe oxidative stress and

hyperlipidemia. The results are well supported by histopathological

observations and indicates that F18/FVBM extract are highly promising

natural antioxidant as well as can be used as an antiperoxidative,

hypolipidemic and antiatherogenic agent. However, further large scale clinical

trials in hyperlipidemic smokers with and without coronary heart disease are

required to substantiate their antioxidative, hypolipidemic and

atheroprotective properties.

chapter 5 therapeutic impact of newly isolated bioactive...

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