INTRODUCTION:
Noise is an inevitable part of our day-to-day lives
and has become gradually increasing today’s major problem on the standard of
lives. People are daily exposed to various noise levels produced by a variety
of sources like commercial and industrial activities, transportation, social
events, household sources, etc1. There are several physiological
effects of noise exposure such as cardiovascular disease, irritation, sleep
disturbance and ischemic heart disease2. The impact of high level of
noise exposure to changes in the immune system and birth defects, in addition
to altered serum lipid, plasma viscosity, platelet count, blood glucose level
and reduced motor efficiency3.
In
fact, epidemiological studies had reported that, exposure to a noise even <60
dB intensity may trigger depressive symptoms4.
The
high level of noise exposure has shown to produce a number of physiological,
biochemical, and neurochemical rejoinder in both human and animals. Acute and
chronic exposure to loud noise induces oxidative stress that generates
excessive free radicals3. Excessive free radicals generation during
stress exposure induces the lipid peroxidation in many organs especially in the
brain5 and decreases the enzymatic levels in other organs in the
body. Many plant extracts and plant products have been shown to have
significant antioxidant activity6, which may be an important property
of medicinal plants associated with the treatment of several noise related
diseases7,8. With growing interest in indigenous herbal plants for
their therapeutic efficacy, various efforts are now concentrated in documenting
herbal plants as a potential source of modern medicine.
The
North American cranberry (Vaccinium macrocarpon Ait.) and its products
have been widely used as nutraceuticals due to their anti-microbial,
anti-mutagenic, anti-angiogenic, and anti-oxidant properties9. The
American cranberry is a conspicuous horticultural nourishment food produced in
Massachusetts, Wisconsin, Michigan, Canada, New Jersey, Oregon, and Washington.Cranberry extracts rich in phytochemicals
such as flavonoids (anthocyanins, flavonols and flavanols), tannins (proanthocyanidins,
ellagitannins and gallotannins), quercetin, phenolic acids, these compounds
reportedly inhibit oxidative process including oxidation of low-density
lipoprotein10, oxidative damage to neuron during
stimulated ischemia and oxidative and inflammatory damage to the vascular
endothelium11, urinatory tract infections12.
Cranberry ranks high among fruits in both antioxidant quality and quantity
because of its flavonoid content and phenolic acids. Many in vivo tests
have been conducted with cranberry extract for its hepatoprotective13,
hypolipidemic14, hypoglycaemic and antioxidant activity15 Cranberry consists of several bioactive compounds such
as fructose, vitamin C, flavonoids, anthocyanidins, catechins, triterpenoids
and phenolic compounds that are numerously used due to their healthcare
benefits and against different types of cancer16. This study is
aimed to investigate the effect of ethanolic extract of cranberry on the
alterations of liver functions enzymes and lipid profile parameters upon noise
exposure to rats.
MATERIALS AND METHODS:
Collection of plant material:
The healthy dried Cranberry (Delmonte dried Cranberries)
were collected from field Fresh Food Private Limited. Haryana, India. The
collected specimen was authenticated in National institute of Siddha, Tambaram,
Chennai. (Authentication No: NISMB3102017).
Preparation of the plant extract:
Preparation of the extracts was done
according to a combination of the methods used by Pizzale et al., (2002)
and Lu and Foo (2001). About 15g of dried fruit fine powder of cranberries were
extracted with 150 ml ethanol (75%) extract for 1 min using an Ultra Turax
mixer (13,000 rpm) and soaked overnight at room temperature. The sample was
then filtered through Whatman No.1 paper in a Buchner funnel. The filtered
solution was evaporated under vacuum in a rota-evaporator at 40°C to a constant
weight and then dissolved in respective solvents. The concentrated extracts
were stored in airtight container in refrigerator below 10ºC.
Animals:
Six-week-old male albino Wistar rats (mean
body weight, 180g) was procured from
Kings Institute, Chennai. They
are maintained at (25±2)°C and 45%-55% of humidity in 12 h light/12 h dark
cycle. Animals were given free access to standard laboratory food and water ad
libitum. The experiments were conducted according to the CPCSEA guidelines, the
Ethical Committee Approval No:
TMC/IAEC/01/004.
Noise stress Induction procedure:
Broad band (white) noise was produced by a
white noise generator and amplified by an amplifier (40w) which was connected
to a loud speaker located at 30cm above the animal cage. The intensity of the
sound was measured by a sound level meter and maintained at 100 dB intensity19
(Manikandan et al., 2013). The background noise level in the room
was recorded due to the ventilation system. Stress group animals were exposed
to noise (100dB 4h/day for 30 and 60 days) at 08:00 to 12:00 h daily and
control animals kept in noise chamber for the same period of time without
switching on the generator.
Experimental Design:
The study was conducted on 48 male albino
wistar rats which was divided in to eight groups,
Group I (Saline control) - Rats received
vehicle only
Group II (CBE control) - Rats received
500mg/kg b.wt cranberry extract (CBE) p.o for 60 day
Group III (30 DNS) - Rats exposed to noise
(100dB 4h/day for 30days) at 8:00 to12:00 A.M daily
Group IV (60 DNS) - Rats exposed to noise
(100dB 4h/day for 60 days) daily.
Group V (CBE250+30DNS) - Rats treated with
CBE (250mg/kg b.wt) orally for 30 days(Starting
from the day of noise stress exposure before the rats were placed in noise
chambers)
Group VI (CBE 500+30DNS) - Rats treated
with CBE (500mg/kg b.wt) orally for 30 days along with the noise exposure.
Group VII (CBE 250+60DNS) -Rats treated
with CBE (250mg/kg b.wt) orally cranberry extract for 60 days along with the
noise exposure.
Group VIII (CBE 500+60DNS) - Rats treated
with CBE (500mg/kg b.wt) orally cranberry extract for 60 days along with the
noise exposure.
After the completion of treatment period the animals were
euthanized by co2 inhalation and blood
serum was collected for estimation of liver enzymes (SGOT and SGPT) and lipid
profile (total cholesterol, HDL and LDL) and
liver tissues were collected for carrying out histology study.
Biochemical studies:
Blood samples (5mL) were collected immediately in
eppendoff tube and kept in centrifuge for 15 min at 3000 rpm. Approximately 3mL
of serum was collected and stored at −20∘C until assay. The collected serum stored at -20ºC until biochemical
determinations of total protein, aspartate aminotransferase (AST or SGOT),
alanine aminotransferase (ALT or SGPT), total cholesterol, HDL and LDL levels.
Determinations were done spectrophotometrically using Randox analytical kits
according to standard procedures of manufacturer’s protocols.
Histopathology
studies
After
the fixation of liver tissue in formal saline (10%) for 24 hours, the tissue were
subjected to dehydration using series of alcohols (methyl, ethyl and absolute ethyl
alcohol) followed by embedding in paraffin wax at 56ºC. After that, the wax tissue
blocks were sectioned by sliding microtome and then prepared for the histological
slide examination under the light electric microscope (Olympus Cx21 with attached
digital camera) after staining with hematoxylin and eosin stain.
Statistical Analysis
All
data were presented as the mean±S.E.M. for n=6. Statistical analysis was
performed using one-way ANOVA followed by Dunnett’s T3 multiple comparison
test. Comparisons between two groups were analyzed using the Student’s t-test.
A P-value of less than 0.05, 0.01, 0.001 was considered to be statistically
significant.
RESULTS:
Fig
1. Effect of CBE on aspartate transaminase and alanine transaminase levels in
30 days noise stress induced rats.
Results
represents the mean ± SEM, n=6. ***p<0.001, statistically significant as
compared with saline control rats; #p<0.05, statistically
significant as compared with 30DNS rats; @@@p<0.001,
statistically significant as compared with 30 DNS rats. DNS- Days Noise Stress;
CBE – Cranberry Extract
Fig
2. Effect of CBE on aspartate transaminase and alanine transaminase levels in
60 days noise stress induced rats.
Results
represents the mean ± SEM, n=6. ***p<0.001, statistically significant as
compared with saline control rats; ##p<0.01, ###p<0.001statistically
significant as compared with 60DNS rats; @@@p<0.001,
statistically significant as compared with 60 DNS rats.
Fig. 1& 2 depicts the SGOT and SGPT levels in both
30 and 60 DNS serum of experimental animals. The results showed that rats
treated with CBE alone caused no significant change in (SGOT and SGPT) when
compared to control. The noise exposure animals showed (p<0.001) significant
increase in the levels of SGOT and SGPT in both 30 and 60 DNS groups as
compared with saline control. The noise exposure group treated with CBE (250
and 500 mg/kg) showed (p<0.05) significant reduction in SGOT and SGPT levels
for 30 days, whereas (p<0.01) (p<0.001) significant decrease for 60 days.
The noise exposure group treated with CBE showed promising hepato protective
potential by lowering the serum levels of SGOT and SGPT.
The
effect of different doses (250 and 500mg/kg) of CBE on the levels of LDL, HDL
and total cholesterol in the serum of all experimental animals were depicted in
Table 1. Our results indicated that the exposure of animals to noise stress
cause significant (p< 0.001) elevation in the LDL, TC levels and a decrease
in HDL levels in both 30 and 60 days. Whereas, the noise exposure groups
treated with CBE at different doses (250 and 500mg/kg) caused a significant
decrease (p< 0.01) in the levels of LDL, TC and increased in the levels of
HDL for 30 days and (p<0.001) significant alterations for 60 days.
The
histology photomicrograph of liver tissues was depicted in Figure (a-h). Saline
control rats and drug control rats showed normal well-developed hepatocytes,
sinusoidal spaces and Kupffer cells. The 30 days noise exposed liver tissue
group showed irregular architecture with
widening of sinusoidal space with infiltration and degenerative changes in
hepatocytes such as condensed and
pyknotic nuclei with granulated cytoplasm. We also observed that apoptosis and
well defined spotty necrosis in hepatocytes exposed to noise stress increased
after 60days. Dose of 250mg/kg/bw of CBE decreased the apoptotic cells and
cellular damage in hepatic tissue architecture upon 30 days and 60 days treatment
whereas the dose of 500mg/kg /bw of CBE showed more effects by increasing the
normal number of hepatocytes and total inhibition of necrosis and proves it
hepatoprotective effect.
DISCUSSION:
Noise
is recognized as one of the most harmful physical factors and its harmful
effects on living entities and biological systems are reported in systematic
form as well as inducing hearing loss and damage to other organs20.
Literature survey has revealed that noise can alter neurotransmitter levels,
cause metabolic, anatomical changes and altered antioxidant enzyme status have
often been regarded as the reason behind redox imbalance in the cell.
Deficiencies in the antioxidant defense system impair protection against
reactive oxygen/nitrogen species (ROS/RNS) leading to damage in vulnerable
targets such as unsaturated fatty acyl chains in the membrane, proteins and DNA21.
The
AST (aspartate aminotransferase) and ALT
(alanine aminotransferase), enzyme are a
sensitive marker of liver damage due to toxic drugs, alcohol and virus, The
increased activity of serum AST, ALT were explained by excess free radical
which reacts with polyunsaturated fatty acids of cell membrane leading to
impairment of mitochondrial and plasma membranes resulting in enzyme leakage22.
Results
of the present study showed that noise exposure group resulted in a significant
increase in SGPT, SGOT levels when compared to the saline control group. The
observed increase activity of serum SGPT and SGOT may be attributed to
excessive release of marker enzymes from the damaged liver cells into the blood
circulation23. Upon treatment with CBE of different doses (250mg/kg
and 500mg/kg b.wt.) shows significant decrease in the SGOT and SGPT levels
compared to the noise exposure group. Studies have reported that, cranberry
flavonoid effectively reduce the elevated level of AST and ALT, and also
prevent accumulation of lipid membrane droplets in rat’s liver24.
Plasma
lipid response to stress varies from stressor to other according to severity
and combination of more than stressor. So, stress may influence lipid concentration
and metabolic thought variety of physiological and behavioral mechanisms. All
type of plasma cholesterol levels increased in response to stress particularly
LDL-C which constitute the bad type of cholesterol25. The stress
induced an elevation in serum total cholesterol concentration which may persist
through the recovery period, increased triglyceride level, fatty acids, LDL and
HDL26. Based on the results of the present study, noise has also led
to the increase of LDL and TC level and decreased HDL levels in blood serum in
the noise exposure groups in both 30 and 60 DNS compared to saline control
group. The CBE alone treated group has no significant change when compared to
control groups, whereas noise exposed animals treated with CBE of different
doses at 250mg/kg and 500mg/kg b.wt. shows significant changes in the LDL, TC
and HDL levels compared to the noise exposure group.
Studies
have reported that antioxidants phyto chemical such as flavonoid, saponin and
phenolic acids, reduced the LDL, TC and increased the HDL level in hyper
lipidimic rats27,28,29. It is well known that cranberry is having
more antioxidant phytoconstituents; this phytochemicals might be responsible
for its hypo lipidimic property.
The
histological alterations observed might be an indication of hepatocyte injury
due to the oxidative stress induced by noise. The hepatic structural
disturbances increased in rats based upon the time exposure to noise. The
cytoplasmic degeneration and destruction of nuclei in hepatocytes suggests that
noise stress leads to reactive oxygen species which, in turn, may induce stress
in hepatocytes, promoting apoptosis, and necrosis. The dose and time dependent
protection was noted in CBE treated rats due to the hepato protective property
of cranberry. Studies have shown that several plants have anti-hepatotoxic
property30,31,32, the antioxidants compounds present in the plants ameliorates
the oxidative stress induced hepatotoxicity. Previous studies have found
cranberry bio actives improve lipid and cholesterol profiles in animals and
humans, reduce blood markers of oxidative stress in humans, and reduce
inflammations33. The cranberry powder diet has been reported to
alleviate inflammatory response and lipid oxidation, which is useful to
individuals with the metabolic syndrome34. Daily intake of cranberry
extract has recently been reported to decrease high fat diet-induced weight
gain, hepatic triglyceride accumulation, and oxidative stress in high fat/high
sucrose-fed mice35. Elevated serum biomarker enzymes SGOT, SGPT and
cholesterol associated with hepatocellular necrosis indicating the liver damage36.
Accumulating evidences such a biochemical and histological studies proved that
CBE has the potential ability to promote a healthy metabolic organ by reducing
the hepatotoxicity caused by noise exposure.
CONCLUSION:
In
the present, cranberry ethanolic extract restored the increased serum enzyme
levels, lipid profile and prevented the morphological changes induced by
chronic noise exposure, and exerted effective hepatoprotective and
hypolipidemic property. The presence of several bioactive antioxidants
components in cranberry, particularly flavonoids and phenolic compounds, might
be responsible for its hepatoprotective property. This is the first report to provide
a direct evidence for the hepatoprotective effect of cranberry extract in noise
stress animal models. Further studies are needed to elucidate the exact
mechanism of action of cranberry and its active principle as hepatoprotective
and hypolipidemic agent.
ACKNOWLEDGEMENTS:
We
are thankful to the Principal and Management Tagore Dental College and
Hospital, Chennai for their encouragement to carry out this study.
CONFLICTS
OF INTEREST:
The
authors have no conflict of interest.
REFERENCES:
1. Gregory A. Flamme, Mark R. Stephenson,
Kristy Deiters, Amanda Tatro, Devon Van Gessel, Kyle Geda, Krista Wyllys, and
Kara McGregor. Typical noise exposure in daily life. Int J
Audiol.2012;52(01)S3-11.
2. Arabmoazzen S, Sarkaki A, Saki Gh,
Mirshekar MA. Antidiabetic effect of honey feeding in noise induced
hyperglycemic rat: involvement of oxidative stress. Iran J Basic Med Sci. 2015;
18(8):745‐751.
3. Ravindran R, Rathinasamy SD, Samson J,
Senthilvelan M. Noise stress induced brain neurotransmitter changes and the
effect of Ocmium Sanctum (Linn) treatment in albino rats. J
Pharm Sci. 2005; 98: 354-360.
4. Haider S, Naqvi F, Batool Z, Tabassum S,
Perveen T, Saleem S, Darakhshan JH. Decreased Hippocampal 5-HT and DA levels
following sub-chronic exposure to noise stress: impairment in both spatial and
recognition memory in male rats. Scientia Pharmaceutica. 2012; 80:1001-1011.
5. Vinay Kumar, Ahmed Abdullah Khan, Anu
Tripathi, Praveen K. Dixit, U.K. Baja. Role of oxidative stress in various
diseases: Relevance of dietary antioxidants The Journal of Phyto pharmacology.
2015; 4(2): 126-132.
6. Deepak M. Kasote, Surendra S. Katyare,
Mahabaleshwar V. Hegde, Hanhong Bae. Significance of Antioxidant Potential of
Plants and its Relevance to Therapeutic Applications. Int J Biol Sci. 2015;
11(8): 982–991.
7. Loganathan Sundareswaran, Sakthivel
Srinivasan, Wankupar Wankhar, Rathinasamy Sheeladevi. Effect of Scoparia dulcis
on noise stress induced adaptive immunity and cytokine response in immunized
Wistar rats. Journal of Ayurveda and Integrative Medicine. 2017; 8(1): 13-19.
8. Isaac OA, Joseph OA, Victor OS. Mitigative
effects of antioxidants in Noise Stress. J Clin Nutr Diet. 2017;3:21.
9. Pappas E, Schaich KM. Phytochemicals of
cranberries and cranberry products: characterization, potential health effects,
and processing stability. Crit Rev Food Sci Nutr.2009; 49(9): 741-781.
10. Wilson T, Porcari JP, Harbin D. Cranberry
extract inhibits low density lipoprotein oxidation. Life Sci.
1998;62(24):381-6.
11. Blumberg JB, Camesano TA, Cassidy A,
Kris-Etherton P, Howell A,Manach C et al. Cranberries and Their Bioactive
Constituents in Human Health. Adv in Nutr. 2013;4:618-32.
12. Abdul-Lateef Lamees A, Hassan Emad, Taha
Abd-Alla Bactash Enas. Effect of Cranberry on biofilm formation by P. mirabilis
isolated from Patients Suffering from Urinary tract Infections. Research
Journal of Pharmacy and Technology. 2018;11(3):1097-1100.
13. Hussain F, Malik A, Ayyaz U, Shafique H, Rana Z, Hussain Z. Efficient hepatoprotective activity of
cranberry extract againt ccl4- induced hepatotoxicity in wistar albino rat
model: Down-regulation of liver enzyme and strong
antioxidant activity. Asian Pac J Trop Med. 2017;10:1054-8.
14. Kim MJ, Kim JH, Kwak HK. Effects of
cranberry powder on biomarkers of oxidative stress and glucose control in db/db
mice. Nutr Res Pract . 2013;7:430-8.
15. Kim MJ, Kim JH, Kwak HK. Antioxidant
effects of cranberry powder in lipopolysaccharide treated hypercholesterolemic
rats. Prev Nutr Food Sci. 2014;19:75-81.
16. Boshra SA, Mohammed A. Cranberry extract as
a supplemented food in treatment of oxidative stress and breast cancer induced
by N-methyl-N-nitrosourea in female virgin Rats. Intl J Phytomed. 2016; 8:217-227
17. Pizzale L, Bortolomeazzi R, Vichi S, Conte
LS. Antioxidant activity of sage and oregano extracts related to their phenolic
compound content. J Sci Food Agr. 2002;82:1645-51.
18. Lu Y and Foo Y. Antioxidant activities of
polyphenols from sage (Salvia officinalis). Food Chem. 2001;75:197-202.
19. Sundaramahalingam M, Ramasundaram S,
Rathinasamy SD, Natarajan RP, Somasundaram T. Role of acorus calamus and
α-asarone on Hippocampal Dependent Memory in Noise Stress Exposed Rats.
Pak J Biol Sci. 2013;16:770-8.
20. Basner M, Babisch W, Davis A, Brink M,
Clark C, Janssen S et al., Auditory and non-auditory effects of noise on
health. Lancet. 2014;383:1325-32.
21. Pallavi Sharma, Ambuj Bhushan Jha, Rama
Shanker Dubey, and Mohammad Pessarakli. Reactive Oxygen Species, Oxidative
Damage, and Antioxidative Defense Mechanism in Plants under Stressful
Conditions. Journal of Botany.2012; http://dx.doi.org/10.1155/2012/217037
22. Saher Mahmood Jwad, Bushra Abbas, Haider S.
Jaffat. Study of The Protective Effect of Vitamin C plus E on
Lincomycin-Induced Hepatotoxicity and Nephrotoxicity. Research Journal of
Pharmacy and Technology. 2015; 8(2):177-184.
23. Ajay Kshirsagar, Deepa Ingawale, Purnima
Ashok, Vrushali Thorve, Tanmay Dodal, Anurag Dodal, Mahesh Kahane, Bharat Zope.
Hepatoprotective and Antioxidant Potential of Calotropis gigantea in
Cyclosporine–An Induced Hepatotoxicity. Research J. Pharmacology and
Pharmacodynamics. 2010; 2(5): 343-347.
24. Cheshchevik VT, Lapshina EA, Dremza IK,
Zabrodskaya SV, Reiter RJ, Prokopchik NI, Zavodnik IB. Rat liver mitochondrial
damage under acute or chronic carbon tetrachloride-induced intoxication:
protection by melatonin and cranberry flavonoids. Toxicol Applied Pharm.
2012;261(3): 271-279.
25. Seyedeh Negar Assadi. What are the effects
of psychological stress and physical work on blood lipid profiles?. Medicine
(Baltimore). 2017; 96(18): e6816.
26. Yanardag R, Bolkent S, Ozsoy-Sacan O,
Karabulut-Bulan O. The effects of chard (Beta vulgaris L. var. cicla) extract
on the kidney, tissue, serum urea and creatinine levels of diabetic rats. Phyto
ther Res. 2002;16(8):758–61.
27. Srikanth Jeyabalan and Muralidharan
Palayan. Antihyperlipidemic activity of Sapindus
emarginatus in Triton WR-1339 induced albino rats.. Research Journal of
Pharmacy and Technology. 2009;2(2):319-23.
28. Kanimozhi P and Karthikeyan J. Modulation
of Antioxidant Potential in Liver of Albino rats by seeds of Nelumbo nucifera
Gaertn. in 1,4 Dichlorobenzene induced hepato toxicity. Research Journal of
Pharmacy and Technology. 2012;4(5):538-40.
29. U. S. Mahadeva Rao, R. Babujanarthanam, B.
Arirudran. Clinical Evaluation to Assess the Efficacy of Ethanolic Extract of
Avocado Fruit on Diabetic Dyslipidemia Studied in STZ- Induced Experimental
Albino Rats. Asian Journal of Research in Chemistry. 2011;7(4):1131-6.
30. Jitendra Patel, Venkateshwar Reddy and G.S.
Kumar. Evaluation of hepatoprotective activity of ethanolic extract of
Diospyros melanoxylon (Roxb) leaves against CCl4 induced hepatotoxicity in
albino rats. Research Journal of Pharmacy and Technology. 2015;8(5):571-4.
31. Gauri Karwani, Siddhraj S. Sisodia.
Hepatoprotective activity of Chenopodiumalbum Linn. in Ethanol induced
Hepatotoxicity in Rats. Research Journal of Pharmacy and Technology.
2015;8(6):669-73.
32. Aparna Satapathy, Mandava V. Rao.
Protective effect of Curcumin on 2, 4- Dichlorophenoxy acetic acid exerted
Hepatotoxicity in Mice. Research Journal of Pharmacy and Technology.
2018;11(2):637-42.
33. Shannon L. Glisan, Caroline Ryan, Andrew P.
Neilson, and Joshua D. Lambert. Cranberry extract attenuates hepatic
inflammation in high fat-fed obese mice. J Nutr Biochem. 2016; 37: 60–66.
34. Nardi GM, Farias Januario AG, Freire CG,
Megiolaro F, Schneider K, Perazzoli MR et al., Anti-inflammatory activity of
berry fruits in mice model of inflammation is based on oxidative stress
modulation. Pharmacog Res. 2016; 8(1): 42-49.
35. Bajerska J, Chmurzynska A,
Mildner-Szkudlarz S, Drzymała-Czyz S, Gornas P. Effects of unextruded and
extruded cranberry pomace on selected metabolic parameters in high-fat diet fed
rats. Acta Sci Pol Tech Aliment. 2018;17(1):91-100.
36. T.S. Dhanaraj, R. Gowthami, S. Rajlakshmi, K.
Murugaiah. Antihepatotoxicity of Hygrophila auriculata on CCl4 Induced
Hepatotoxicity in Rats. Asian Journal of Research in Pharmaceutical Sciences.
2012;2(4):140-142.
(a)Saline control (b) CBE control (a,b)showing normal
architecture of liver with hepatocytes and sinusoidal spaces (c) 30DNS showed Widening of sinusoidal space with infiltration(
elongated arrow) degenerative changes in hepatocytes (small arrow) (d)60DNS showed Spotty
necrosis of hepatocytes (e) CBE (250mg)+30 DNS showed less apoptotic changes (f) CBE (500 mg)+30 DNS (g)
CBE(250 mg)+ 60 DNS showed less degenerative hepatocyes (h) CBE(500 mg)+60 DNS
showed normal liver architecture no apoptotic changes