Journal of Ethnopharmacology 227 (2018) 69–81

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Hepatoprotective effects of green Capsicum annum against ethanol induced T

oxidative stress, inflammation and apoptosis in rats
Moumita Dasa, Subhashree Basub, Bhaswati Banerjeec, Anurupa Send, Kuladip Janac,
⁎
Gouriprosad Dattaa,
a
Department of Physiology, Rammohan College, 85A, Raja Rammohan Sarani, Kolkata 700009, West Bengal, India
b
Department of Physiology, Tamralipta Mahavidyalaya, Tamluk, Poorba Medinipur, India
c
Department of Molecular Medicine, Bose Institute, P-1/12 C.I.T. Scheme VIIM, Kolkata 700054, West Bengal, India
d
Department of Physiology, City College, Kolkata, India

A R T I C LE I N FO A B S T R A C T

Keywords: Ethnopharmacological relevance: Capsicum annum L. (CA) is used extensively as a spice and is a rich source of
Capsicum annum antioxidant vitamins. It has long been used in Indian, Native American, and Chinese traditional medicine as a
Antioxidant carminative and an appetizer that normalizes liver function. However, its hepato-protective activity has so far
Apoptosis not been studied.
Hepato-protective
Aim of the study: The present study was undertaken to evaluate the efficacy of aqueous extract of CA at two
Interleukin 6
different doses (125 mg/kg body weight and 250 mg/kg body weight), against ethanol induced oxidative stress
Tumour necrotic factor alpha
and apoptosis in liver tissue.
Materials and methods: Adult male Wistar rats, weighing 150–200 g, were randomly grouped (n = 6) and treated
with ethanol (2 g/kg bw, i.p.), CA125 (125 mg/kg bw, i.p.), CA250 (250 mg/kg bw, i.p.), ethanol with CA (similar
doses), and control (0.5 ml normal saline, i.p.) for 30 days. Lipid peroxidation (LPO) and reduced glutathione
content (GSH) in tissue homogenate, along with catalase (CAT), superoxide dismutase (Cu-Zn-SOD & Mn-SOD),
glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GST) and glucose-6-phos-
phate dehydrogenase (G-6-P-D) activity were evaluated. Serum levels of alanine transaminase (ALT), aspartate
transaminase (AST), alkaline phosphate (ALP), triglyceride (TG), total cholesterol (CHLS), high density lipo-
protein (HDL), low density lipoprotein (LDL) very low density lipoprotein (VLDL), tumour necrotic factor alpha
(TNF-α) and interleukin 6 (IL-6) were also measured using ELISA kits. Histopathological evaluation of the he-
patic tissue was performed by hematoxylin and eosin (H&E) and periodic acid-schiff (PAS) staining. TUNEL assay
was performed for apoptosis detection.
Results: Ethanol significantly (p < 0.001) increased ALT, AST, ALP, TNF-α, IL-6, LPO, Cu-Zn-SOD, GST, GPx,
TG, CHLS, LDL, VLDL levels, along with significant (p < 0.001) decrease in HDL, Mn-SOD, CAT, GSH, GR and
G6PD activity. Co-administration of CA along with ethanol alleviated changes in the above parameters
(p < 0.001) in a dose-dependent manner and also reduced the number of apoptotic death cells. Histo-patho-
logical and histo-chemical studies of liver sections also ascertained the outcomes of this study.
Conclusion: Thus, it can be concluded that the aqueous extract of green CA can exert a protective effect against
ethanol induced hepato-toxicity. The possible mechanism may be by acting as an antioxidant; preventing ethanol
induced apoptosis and reducing pro-inflammatory cytokine levels.

1. Introduction
Hepatotoxicity is one of the common complaints leading to several
metabolic disorders (Patel et al., 2008) and at times can even be fatal.
Ethanol being a xenobiotic is metabolized primarily in the liver and
excess consumption of ethanol results in acute hepatic toxicity.
Ethanol has long been consumed by most people of all socio-eco-
nomic strata in the form of alcohol. It is a commonly consumed re-
creational beverage of modern society and when in excess, is re-
sponsible for causing Alcoholic Liver Disease (ALD). Study of literature
suggests that the underlying mechanism of ethanol induced hepato-
toxicity is oxidative stress and endotoxin mediated activation of Kupffer

⁎
Corresponding author.
E-mail address: dattagp@yahoo.co.in (G. Datta).

https://doi.org/10.1016/j.jep.2018.08.019
Received 15 August 2017; Received in revised form 19 July 2018; Accepted 13 August 2018
Available online 16 August 2018
0378-8741/ © 2018 Elsevier B.V. All rights reserved.
M. Das et al.
cells (Thurman et al., 1997) along with release of cytokines and che-
mokines causing increase in macrophage infiltration and inflammation
(Zhou et al., 2003).
In view of severe adverse effects of synthetic drugs that claim to
cure liver damage, there is a budding focus on systematic research
methodology and evaluation of scientific basis for traditional herbal
medicines, which claim to possess hepatoprotective activity
(Chatterjee, 2000). Plant derived natural products such as flavonoids,
terpenoids and steroids have received considerable attention in recent
years due to their varied pharmacological properties including anti-
oxidant and hepatoprotective activity (Banskota et al., 2000; DeFeudis
et al., 2003; Takeoka and Dao, 2003). The preventive action of herbs
and spices against hepatic damage may be attributed to their anti-
oxidant property and free radical scavenging potential.
Capsicum annum L., one of the popularly used spices in global cui-
sines is a rich source of antioxidants. It is a genus of the flowering plant
belonging to the nightshade family, Solanaceae. The fleshy fruit is
available in green, red, orange, yellow and purple colours and is a rich
source of vitamin A, C, beta carotene, capsaicin and its analogues
(Cantrill, 2008). Traditionally, crude extract form has been used as a
counter irritant in the treatment of rheumatism, arthritis, lumbago, sore
muscles, toothache, neuralgia, stomach ache, skin rashes, dog/snake
bite, poor appetite, and wounds (Meghvansi et al., 2010). Capsicum
annum, and/or its active component, capsaicin has been reported to
possess antioxidative (Deepa et al., 2007; Ou et al., 2002), analgesic
(Epstein and Marcoe, 1994; Lynn, 1990), hypolipidemic (Kawabata
et al., 2009; Lee et al., 2009), anti-diabetic and anti-hypertensive (Kwon
et al., 2007; Zhang et al., 2007), anti-inflammatory (Khabade et al.,
2012; Park et al., 2004) and anti-carcinogenic (Chou et al., 2009;
Thoennissen et al., 2010) activity.
Though the traditional use of Capsicum annum in folklore medicine
as an appetizer and carminative that improves liver function has been
reported (Trease and Evans, 2002; Barnes et al., 2007; Khan and
Abourashed, 2010; Pawar et al., 2011; Gupta et al., 2015), till date no
study has been conducted to testify the hepatoproctective activity of
Capsicum annum against ethanol induced toxicity. Moreover, Blanco-
Ríos et al. (2013) revealed that the phenolic content and antioxidant
activity of green capsicum is more compared to the red and yellow
variety. In addition, our previous study showed that the aqueous extract
of green capsicum has more antioxidant potency with better free radical
scavenging power compared to the ethanolic counterpart (Das et al.,
2017).
So, the present study was undertaken to evaluate the hepatopro-
tective activity of green Capsicum annum L. in a dose dependent
manner, against ethanol induced hepatotoxicity. As hyperlipidemia,
inflammation and oxidative stress is closely linked to the pathogenesis
of ALD, the anti-oxidative and anti-inflammatory property of Capsicum
annum is biochemically and histo-pathologically analysed to evaluate
the claimed hepatoprotective activity of Capsicum annum.
2. Materials and methods
2.1. Chemicals
Ethanol, Di-sodium hydrogen phosphate, Sodium di-hydrogen
phosphate, Trichloro-acetic acid (TCA), Thiobarbituric acid (TBA),
Hydrogen peroxide (H2O2), Ethylene diamine tetra acetic acid (EDTA),
Magnesium chloride (MgCl2), Per-iodic acid and Basic fuschin were
purchased from Merck, India. Tris buffer, Pyragallol, Di-thio bis nitro
benzoic acid (DTNB), 1-chloro, 2,4-dinitrobenzene (CDNB), Reduced
glutathione (GSH), Oxidised glutathione (GSSG), Glucose-6-phosphate
(G-6-P), Nicotinamide adenine dinucleotide phosphate (NADP),
Reduced nicotinamide adenine dinucleotide phosphate (NADPH),
Sodium azide, Hematoxylin, Eosin, were purchased from HiMedia
Laboratories Pvt. Ltd., Mumbai, India. Direct red and capsaicin (≥ 95%
purity) were purchased from Sigma-Aldrich, USA. Silymarin as
70
Journal of Ethnopharmacology 227 (2018) 69–81
standard hepatoprotective drug was procured from Micro Labs Ltd.,
India. All reagents used in the experiment were of analytical grade. All
Kits were procured from Span Diagnostics Pvt. Ltd. Antibodies for cy-
tokine assessment were purchased from Raybiotech, USA and for
TUNEL assay was procured from Roche, Germany.
2.2. Procurement and authentication of plant material
Green variety of Capsicum annum L. (CA) was procured from local
Kolkata Municipal Corporation approved vegetable market. The col-
lection period was from November to February. It was then authenti-
cated by the Botanical Survey of India (BSI), Howrah, West Bengal,
India. A herbarium of the specimen was maintained in the institute li-
brary bearing the number RMC/PHY/MD/01/14.
2.2.1. Preparation of aqueous extract and RP-HPLC standardization
After procurement, the vegetable was washed thoroughly under
running tap water, sliced into cubicles and shade dried. It was then
extracted using distilled water with the help of Soxhlet apparatus
continuously for 72 h. The mixture was then centrifuged at 4000 rpm
for 15 min. The supernatant, considered as aqueous extract of Capsicum
annum, was concentrated to dryness at 40 ± 5 °C by using temperature
controlled hot plate (temp ranging: 25–250 °C, M/s. B.C. Chatterjee,
Kolkata, India) and stored in air tight plastic vials at − 20 °C for further
use. The percentage yield was 19% (w/w).
The aqueous extract was subjected to standardization with respect
to the marker compound capsaicin, using High Performance Liquid
Chromatography (UFLC, Shimadzu, Japan), (Materska and Perucka,
2005; Butnariu et al., 2012; Juangsamoot et al., 2012), with slight
modifications. A C18 column was used for chromatographic separation
and isocratic elution was carried out using solvent mixture of methanol:
water (80:20). The flow rate was kept constant at 1.0 ml/min and the
injection volume was 10 µL. The UV-detector was set at 280 nm and the
column temperature was maintained at 40 °C. All solvents and diluents
used were of HPLC grade and filtered via 0.45-µm filters.
2.2.2. GC-MS analysis and identification of components
Qualitative analysis of the phyto-constituents present in aqueous
extract of CA was carried out using Gas Chromatography Mass
Spectrometry (GC-MS, Thermo scientific trace GC ultra) technique.
Flow rate of mobile phase (carrier gas: He) was set at 1.0 mL/min. In
the gas chromatography part, temperature programme (oven tem-
perature) was initially set at 50–250 °C with increasing rate of 5 °C/min
and holding time of about 2 min. Finally, the temperature was raised to
300 °C at a rate of 10 °C/min and injection volume was 1 µL. Sample
dissolved in methanol were run fully and interpretation on mass spec-
trum of GC-MS was done using the database of National Institute
Standard and Technology (NIST)/National Bureau of Standard (NBS)
and Wiley. The spectrum of the components was compared with the
spectrum of the known components stored in the inbuilt library.
2.3. Animal care
Male Albino wistar rats (150–200 g) used for the experiment were
kept in autoclavable, polyvinyl cages at temperature (23–25 °C) con-
trolled and well-ventilated rooms of institutional animal house. A
12:12 h light: dark cycle was maintained and all rats were allowed to
have a free access to a standard diet and water. Food and water were
supplied adlibitum. The animals were maintained according to the
guidelines recommended by Animal Welfare Board and approved by
our Institutional Animal Ethical Committee (IAEC) bearing the regis-
tration number 1795/PO/Ere/S14CPCSEA, constituted under the
guidelines of Committee for the Purpose of Control and Supervision on
Experimental Animal (CPCSEA), Ministry of Environment, Government
of India, New Delhi. The animals were acclimatized to laboratory
conditions for 7 days prior to the experimental protocol to minimize
M. Das et al.
any nonspecific stress.
2.4. Oral toxicity studies and dose standardization
Generally, capsicum tincture or oleoresin was used in folk medicine
and both were formerly official in The United States Pharmacopeia and
The National Formulary (USP–NF). The prescribed dosage for tincture
is 0.1 gm/ml or less than 5 g for capsule (Leung and Foster, 1996). The
repeated dose oral toxicity of the extract was determined on male Al-
bino wistar rats by fixed dose method of OECD guide line (TG 407,
adopted 3rd October 2008) (OECD, 2008) given by CPCSEA. Groups of
6 rats were administered test drug by intraperitoneal (i.p.) route in the
range of 100–2000 mg/kg and mortality was observed. The LD50 value
for the aqueous extract of CA was determined by modified Lorke's
method (Lorke, 1983), using the formula:
LD50 = √ (D0 × D100)
D0 = Highest dose that gave no mortality,
D100 = Lowest dose that produced mortality.
As per our previous study, the standardized dose of ethanol for in-
ducing hepatotoxicity was taken to be 40% v/v ethanol, at 2 g per kg
body weight (Datta et al., 2012). The extract was prepared by dissol-
ving 125 mg and 250 mg of the concentrated extract in 0.5 ml of 0.9%
normal saline. Silymarin was used as the standard drug.
In the present experiment, intraperitoneal route of drug adminis-
tration has been chosen to avoid first pass metabolism (Iwaniec and
Turner, 2013). Moreover, forced alcohol and extract administration can
initiate stress response, established by an enhanced release of gluco-
corticoids, which is immunosuppressive (Collier et al., 2000; El-Guindy
et al., 2007).
2.5. Experimental design
Male Albino wistar rats were randomly divided into six groups,
(n = 6) in each group. Treatment was carried out as per the schedule
mentioned below.
Group I: Control (Cont) rats; treated with normal saline (0.9 g/dL),
i.p., once every day for a period of 30 days.
Group II: Ethanol (EtOH) treated rats; dose, 40% v/v ethanol (2 g
per kg body weight), i.p., once every day for a period of 30 days.
Group III: Silymarin treated; dose, 50 mg/kg body weight, followed
by ethanol, (SILY + EtOH), i.p., once every day for a period of 30 days.
Group IV: Aqueous extract of CA treated; dose, 125 mg/kg body
weight, followed by ethanol, (CA125 + EtOH), i.p., once every day for a
period of 30 days.
Group V: Aqueous extract of CA treated; dose, 250 mg/kg body
weight, followed by ethanol, (CA250 + EtOH), i.p., once every day for a
period of 30 days.
Group VI: Only aqueous extract of CA (Only CA125) treated; dose,
125 mg/kg body weight, i.p., once every day for a period of 30 days.
Group VII Only aqueous extract of CA (Only CA250) treated; dose,
250 mg/kg body weight, i.p., once every day for a period of 30 days.
2.5.1. Animal sacrifice and collection of blood and tissue samples
At the end of the treatment period, the animals were kept fasting
overnight, and post-experimental body weight of all animals were re-
corded. The animals were then euthanized via intravenous ketamine
injection, after being anaesthetized via intraperitoneal injection of a
combination of 100 mg/kg ketamine and 10 mg/kg xylazine (Mitra
et al., 2014). Blood was then carefully collected by cardiac puncture
after opening the chest cavity. The blood was allowed to clot at room
temperature for 30 min, and then centrifuged at 3000 rpm for 15 min
for obtaining the serum. The liver of the sacrificed animals were also
removed immediately, rinsed properly in ice-cold normal mammalian
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Journal of Ethnopharmacology 227 (2018) 69–81
saline, blotted dry and weighed, to note the organ weight. A part of the
hepatic tissue was then immediately immersed in appropriate fixative
for further histo-pathological study. The remaining part of the liver was
kept for biochemical assay.
2.5.2. Measurement of liver weight to body weight ratio
Body weight of all animals at the end of the treatment period, along
with their respective liver weight on the day of sacrifice was noted to
determine the liver weight to body weight ratio of animals in each
group.
2.6. Assessment of hepato-protective activity
2.6.1. Preparation of tissue homogenate
A 10% w/v liver tissue homogenate was prepared in ice cold
Phosphate buffer saline (PBS) containing 1 mM EDTA (pH 7.4), using
tissue homogenizer. The homogenate was then centrifuged at
10,000 rpm for 30 min, at 4 °C. The supernatant thus obtained was used
further, for the following biochemical assay of tissue oxidative stress
markers.
2.6.2. Preparation of mitochondrial fraction
For mitochondrial preparation, the liver homogenate was first
centrifuged at 2000 rpm for 5 min. The supernatant was collected and
centrifuged again at 10,000 rpm for 20 min. The supernatant was dis-
carded and the pellet was suspended in Tris-HCl buffer (pH 7.4).
2.6.3. Estimation of lipid peroxidation (LPO)
Lipid peroxidation was estimated by measuring thiobarbituric acid
reactive substance (TBARS), according to the method of Buege and Aust
(1978). 1 ml of cytosol was mixed with 10% TCA and 0.67% TBA re-
agent, and boiled for 20 min in water bath. The samples were then
cooled and centrifuged at 3000 rpm for 10 min at room temperature.
The optical density of the pink chromogen present in the clear super-
natant was measured at 532 nm using a UV–VIS spectrophotometer.
The values were calculated using molar extinction co-efficient, and
expressed as µmoles of malondialdehyde (MDA)/100 gm tissue.
2.6.4. Estimation of cytosolic superoxide dismutase (Cu-Zn SOD) and
mitochondrial superoxide dismutase (Mn-SOD)
Cu-Zn SOD activity was measured according to the method of
Marklund and Marklund (1974). 2 ml of Tris-buffer containing 0.1 mM
EDTA was mixed 10 µL of tissue cytosol and the reaction was initiated
by adding pyragallol (20 mM in Tris-HCl buffer, pH 6.6). The absor-
bance was then measured at 420 nm for 5 min, at an interval of every
1 min. For Mn-SOD, the cytosolic fraction was replaced with mi-
tochondrial suspension. The enzymatic activity was expressed as U/
min/mg protein of liver extract and 1 U of enzyme is defined as the
enzyme activity that inhibits auto-oxidation of pyragallol by 50%.
2.6.5. Estimation of catalase (CAT)
Catalase activity was determined by the decomposition of H2O2 at
240 nm, according to the method of Beers and Sizer (1952). 3 ml of
H2O2 solution (10 mmol/L in 50 mmol/L potassium phosphate buffer,
pH 7.0) was mixed with 1 µL of cytosol and the decrease of absorbance
in every 30 s was recorded over a period of 3 min. Change in the rate of
absorbance was converted µmoles of H2O2/min/mg protein.
2.6.6. Estimation of reduced glutathione (GSH)
Reduced glutathione content was estimated according to the
method of Sedlak and Lindsay (1968). 1 ml cytosol was deproteinized
using 10% TCA. It was then centrifuged and the supernatant obtained
was used for the assay. 0.5 ml of the clear supernatant was diluted with
phosphate buffer containing 1 mM EDTA (pH 8), and then DTNB was
added for colour development. After incubation for 30 min the absor-
bance was measured at 412 nm and the values were calculated using
M. Das et al.
molar extinction co-efficient, and expressed as mg/100 gm tissue.
2.6.7. Estimation of glutathione peroxidase (GPx)
Glutathione Peroxidase activity was measured according to the
method of Rotruck et al. (1973). 200 µL of cytosol was diluted with
0.4 M phosphate buffer (pH 7.0) and mixed with 100 µL of Sodium
azide (10 mM), 200 µL of Glutathione (4 mM) and finally, 100 µL of
(0.2 mM) H2O2 was added into it and incubated at 37 °C for 10 min. The
reaction was then stopped by adding 400 µL of 10% TCA. It was then
centrifuged at 3000 rpm for 20 min, and the clear supernatant obtained
was assayed for GSH content using DTNB. Glutathione Peroxidase ac-
tivity was expressed as µMoles of GSH consumed/min/mg protein.
2.6.8. Estimation of glutathione-S-transferase (GST)
Glutathione-S-Transferase was estimated according to the method of
Habig et al. (1974), by observing the conjugation of CDNB with GSH at
340 nm. 100 µL of cytosol was appropriately diluted and mixed with
0.1 M phosphate buffer (pH 6.5), 100 µL CDNB (1 mM in Ethanol) and
100 µL of GSH (1 mM). The absorbance was then recorded for 5 min, at
an interval of every 1 min, against blank containing all reagents except
the cytosol. The values were calculated using molar extinction co-effi-
cient, and the activity was expressed as µMoles of CDNB-GSH conjugate
formed/min/mg protein.
2.6.9. Estimation of glutathione reductase (GR)
Glutathione reductase was estimated according to the method of
Racker (1955), based on amount of NADPH utilized to convert GSSG to
GSH. 100 µL of cytosol diluted with 0.01 M phosphate buffer (pH 7.0)
was taken in a cuvette, and mixed with an equal volume of EDTA
(1 mM) and NADPH (2 mM). The reaction was then initiated by adding
GSSG (1 mM). The decrease in absorbance was then recorded for 5 min,
at an interval of 1 min, against blank containing all reagents except the
homogenate. The values were calculated using Molar extinction co-ef-
ficient, and the activity was expressed as µMoles of NADPH utilized/
min/mg protein.
2.6.10. Estimation of Glucose-6-Phosphate Dehydrogenase (G-6-P-D)
Glucose-6-Phosphate Dehydrogenase was estimated according to
the method of Balinsky and Bernstein (1963). Appropriately diluted
cytosol was mixed with 100 µL each of 0.1 M Tris-HCl (pH 8.2), 0.2 mM
NADP and 0.1 M MgCl2. The reaction was then initiated by adding
6 mM Glucose-6-phosphate, and the change in absorbance was recorded
at 340 nm for 3 min. The activity was expressed as U/mg protein where,
0.01 change in OD/min corresponded to 1 U.
2.7. Serum analysis
2.7.1. Liver function enzymes
Liver function was assessed by estimating serum levels of liver
marker enzymes, such as Alanine Transaminase (ALT), Aspartate
Transaminase (AST) by the method of Reitman and Frankel (1957), and
Alkaline Phosphatase (ALP) by the method of Kind and King (1954),
using commercially available kits. The results were expressed in U/L.
2.7.2. Assessment of lipid profile
Lipid profiling was done by using commercially available kits. Total
Cholesterol (CHLS), was estimated by cholesterol oxidase- peroxidase
(CHOD-PAP) enzymatic end point assay method (Allain et al., 1974).
Triglyceride (TG), was estimated by glycerol phosphate oxidase (GPO-
PAP) method (Bucolo and David, 1973). Then the High density lipo-
protein (HDL), Low density lipoprotein (LDL) and Very low density li-
poprotein (VLDL) contents were calculated from CHLS and TG levels,
using equations mentioned in manufacturer's manual.
2.7.3. Cytokine assessment
Assessment of pro-inflammatory cytokines, viz., tumour necrotic
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Journal of Ethnopharmacology 227 (2018) 69–81
factor alpha (TNF-α) and interleukin 6 (IL-6) was also carried out using
commercially available ELISA kit and expressed in picogram per mil-
lilitre (pg/mL).
2.8. Estimation of serum and tissue protein
Serum and tissue protein were estimated according to the method of
Lowry et al. (1951) using BSA as standard.
All absorbance at different wavelengths were carried out using
UV–VIS spectrophotometer (Systronics 118) and ELISA micro-plate
reader (Alere, AM 2100).
2.9. Histological and histochemical studies
A part of the rat liver was immediately fixed in 10% formalin buffer
and embedded in paraffin after routine clearing and dehydration. 5 µm
thick sections was then prepared from the tissue blocks using rotary
microtome. The sections were then stained with hematoxylin-eosin (H-
E) stain, periodic-schiff (PAS) stain, feulgen and Sirius red (SR) stain.
Percentage of hepatic lesion area was estimated as [inflammatory area/
total area] × 100 (Singhal et al., 2012).
The tissue sections were viewed under microscope (Magnus, MLXi)
and images were captured using a digital camera (Olympus) attached to
it at (200×) magnification. For feulgen stained sections, images were
captured at (400×) magnification.
2.9.1. Fibrosis quantification by confocal microscopy
Tissue sections stained with Sirius red according to the method of
Roy et al. (2009) was viewed under laser scanning confocal microscope
(Leica SP8, Germany) and the stacked images through multiple slices
were captured (200× magnification). The images were then analysed
using image analysis system (imageJ, NIH Software, Bethesda, MI) and
the total collagen area fraction of each image was measured and ex-
pressed as % collagen content.
2.9.2. Apoptosis detection by TUNEL assay
For detection of apoptosis deoxynucleotidyl transferase-mediated
dUTP nick end labeling (TUNEL) assay was performed on liver tissue
sections using TUNEL assay kit (Roche, Germany). The nuclei were
counterstained using 4,6-diamidino-2-phenylindole (DAPI). The TUNEL
stained sections were viewed under laser scanning confocal microscope
(Leica SP8, Germany) at two different wavelengths for fluorescein
isothiocyanate (FITC) and DAPI respectively, and images of five ran-
domly selected fields were captured at 400× magnification for each
slide. The apoptotic index (AI) was then calculated as the percentage of
TUNEL positive cells, as per the equation: AI = (Number of TUNEL
positive cells/ Total number of cells) × 100% (Kitamura et al., 2004).
2.10. Statistical analysis
Each experiment was repeated at least three times. Data are pre-
sented as means ± S.E. Significance of mean values of different para-
meters between the treated groups were analysed using one way post
hoc tests (Tukey's HSD test) of analysis of variances (ANOVA) after
ascertaining the homogeneity of variances between the treatment
groups. Statistical tests were performed using SPSS software version
20.0. A value of p < 0.05 was considered statistically significant.
3. Results
3.1. Standardization of aqueous extract of CA
The HPLC chromatogram of standard capsaicin and aqueous extract
of CA is shown in Fig. 1. The active component in CA has been iden-
tified by comparing its retention time and absorption spectrum profile
with that of the standard pure capsaicin.
 MS.
M. Das et al.

3.1.1. Identification of components

sulfanyl] ethanol); flavonol (2-[4-(Dimethylamino)]-3-hydroxy-4H-

(4-ethoxyphenyl)-1-hydroxy 2- propanone); furanonaphthoquinone

strated in Fig. 2. The large compound fragments into small compounds

homomonocyclic compound (3-Phenyl propionitrile). Table 1 below

presence of several hydro-carbons along with glycoaldehyde (2,3-Di-

derivative (2,6-Dimethylnapthol [1,2-b] furan-4,5-dione) and aromatic

hydroxy 1,4-dioxane); ester derivative (2-[{(Methylsulfonyl) methyl}
MS library. GC-MS analysis of aqueous extract of CA revealed the
library. The peaks in the chromatogram were integrated and compared

Fig. 2. Total ion chromatogram of aqueous extract of Capsicum annum by GC-

chromen-4-one); fatty acyl (5,9-Tetradecadienedioic acid); ketone (1-
with the database of spectrum of known components stored in the GC-
with crude aqueous extract of CA. The GC-MS spectrum confirmed the

are fingerprint of that compound which can be identified from the data
identify the nature and structure of the compounds. These mass spectra
giving rise to appearance of peaks at different m/z ratios. The mass
presence of various components with different retention times as illu-
Gas chromatography mass spectroscopy analysis was carried out
Fig. 1. HPLC chromatogram of Capsaicin standard and aqueous extract of CA.

spectrometer analyzes the compounds eluted at different times to

73
Table 1
Components present in aqueous extract of CA.
Sl no. Retention time Compound Formula Pharmacological/biological activity

1. 2.14 2,3-Dihydroxy 1,4-dioxane C4H8O4 Antidote, Testosterone hydroxylase inducer, 17 beta-hydroxysteroid dehydrogenase inhibitor, Vasodilator, Diaphoretic, appetizer, diuretic,
Anti-asthmatic, Hepatoprotective
2. 2.59 2-[{(Methylsulfonyl) methyl} sulfanyl] ethanol C4H10O3S2 Ethanol absorption inhibitor, Ethanolytic, Alcohol dehydrogenase inhibitor, Catechol-O-methyl transferase inhibitor, Methyl-Guanidine
inhibitor, Anti-cancer
3. 3.06 Benzyl 2-(acetyl sulfanyl) propionate C12H14O3S Anti-inflammatory, Acetyl CoA Carboxylase Inhibitor, Osteoblast stimulant
4. 3.67 2-Hydroxy-2-methyl malonic acid C4H6O5 Anti-inflammatory, Methyl-Guanidine inhibitor, Inhibits uric acid production, Urinary Acidulant
5. 6.60 4-[5-(Methylsulfinyl)-1,3,4 thiadiazole-2-yl] C8H7N3OS2 NO-scavenger and inhibitor, Catechol-O-methyl transferase inhibitor, Methyl-Guanidine inhibitor, Hepatoprotective
pyridine
6. 14.88 2-Chloronicotinotrile C6H3ClN2 NA
7. 17.84 Acetin monoacetate C5H10O4 NA
8. 20.94 2-[4-(Dimethylamino)]-3-hydroxy-4H-chromen-4- C17H15NO3 Anti-inflammatory,
one
9. 23.88 6,7-Dimethyl [1,2,4] triazolo[4,3-b][1,2,4] triazine C6H7N5 Anti-microbial, Anti-viral (anti-AIDS), anti-cancer, Neuroprotective, HMG-CoA Inhibitor, Immuno-modulator
10. 24.81 2-[N-phenyl formimidoyl] C12H10N2 NA
11. 26.24 5,9-Tetradecadienedioic acid C20H34O4 Anti-inflammatory, Inhibits uric acid production, Urinary Acidulant
12. 28.31 1-(4-ethoxyphenyl)-1-hydroxy 2- propanone C11H14O3 Anti-oxidant, Anti-inflammatory, Testosteron hydroxylase inducer, 17 beta-hydroxysteroid dehydrogenase inhibitor (anti-cancer)
13. 31.55 2,6-Dimethylnapthol [1,2-b] furan-4,5-dione C14H10O3 Anti-bacterial, antioxidant, Anti-neoplastic
14. 32.19 p-(Isopropylidene cyclo-propyl) C13H16 Anti-asthmatic, Anti-arthritis, Anti-bacterial, Catechol-O-methyl transferase inhibitor, Methyl-Guanidine inhibitor
15. 33.19 (1,1-Dimethyl-2-butynyl) benzene C14H14 Anti-asthmatic, Anti-arthritis, Anti-bacterial, Anti-inflammatory, Anti-depressant, Immunomodulatory
16. 35.41 3-Phenyl propionitrile C8H8N Anti-asthmatic, Anti-arthritis, Bronchodilator, Nephro-protective, Anxiolytic, Neuro-protective

* Source of Biological Activity: Dr. Duke's Phytochemical and Ethnobotanical databases online [Jim Duke, 1998: Dr. Jim Duke of the Agricultural Research Service/USDA].
Journal of Ethnopharmacology 227 (2018) 69–81
M. Das et al. Journal of Ethnopharmacology 227 (2018) 69–81

Table 2 Mn-SOD activity (p < 0.001). In case of catalase, the higher dose of CA
Effect of aqueous extract of CA against ethanol induced changes in liver weight was found to be more effective compared to the lower dose. The lower
to body weight ratio. dose of CA showed no significant difference in catalase activity
Groups Post- Liver weight (g) Liver weight: body (p > 0.05) when compared to EtOH treated group. The higher dose of
experimental weight ratio (%) CA however reverted back the catalase activity to near normal values.
body weight (g) Additionally, Table 3(b) depicts the changes in the cellular anti-
oxidant component, i.e., reduced glutathione and the related xenobiotic
Cont 172 ± 0.38 5.12 ± 0.16 3.05 ± 0.2
EtOH 173 ± 0.44 7.06 ± 0.23* 4.08 ± 0.16* detoxifying enzymes, i.e., GST, GPx and GR, along with G6PD. EtOH
SILY + EtOH 172 ± 0.21 5.27 ± 0.11**,*** 3.06 ± 0.17**,*** treated group show significant depletion (p < 0.001) of GSH, GR and
CA125 + EtOH 171 ± 0.32 5.93 ± 0.28**,*** 3.47 ± 0.12**,*** G6PD activity by 68%, 51% and 71% respectively. On the other hand,
CA250 + EtOH 166 ± 0.26 5.52 ± 0.09**,*** 3.33 ± 0.18**,***
GST and GPx activity was found to be significantly (p < 0.001) ele-
Only CA125 175 ± 0.35 5.79 ± 0.22**,*** 3.31 ± 0.21**,***
Only CA250 170 ± 0.18 5.36 ± 0.08**,*** 3.15 ± 0.06**,***
vated by 47% and 73% respectively, compared to control group. In case
of animals co-treated with CA at 125 mg/kg body weight and 250 mg/
The values are expressed as Mean ± S.E. kg body weight dose, the levels of GSH, GR and G6PD increased sig-
* p ≤ 0.001 vs control. nificantly (p < 0.001). Co-treatment with CA also caused significant
** p ≤ 0.001 vs EtOH. decrease in GST and GPx activity (p < 0.001). A dose dependent re-
*** p > 0.05 vs control. sponse was observed in case of GSH, GPx, GR and G6PD. Though the
lower dose of CA arrested the changes that occurred in EtOH treated
represents various components present in aqueous extract of CA as group, the higher dose showed more promising results. No significant
detected by GC-MS analysis, along with their pharmacological/biolo- changes were observed among the only extract and silymarin treated
gical activity. groups.

3.2. Oral toxicity study 3.4. Changes in liver function marker enzymes

After 72 h of extract administration, animals were found to tolerate
the administered dose at 1000 mg/kg body weight and there were no
significant changes in behaviour such as alertness, breathing, rest-
lessness, diarrhoea, convulsions, coma and appearance of the animals.
There was no mortality within 28 days of observations.
LD50 for aqueous extract of CA = √ (1000 × 1250) = 1118.03 mg/kg.
As per our observation and calculation, the LD50 for aqueous extract
of CA after intraperitoneal administration is found to be 1118.03 mg/
kg. Thus, according to Clarke and Clarke (1997) classification, aqueous
extract of CA can be considered as a non-toxic. For the present study, 1/
8th and 1/4th concentration of the maximum safe dose i.e., 1000 mg/
kg body weight were selected.
3.2.1. Changes in liver weight to body weight ratio
Table 2 represents the changes in liver weight to body weight ratio
of all animals belonging to different groups. A significant increase
(p < 0.001) in the liver weight and liver weight to body weight ratio
was observed in EtOH treated group compared to that of Cont group.
Conversely, co-treatment with aqueous extract of CA at both the higher
and lower dose, as well as with silymarin significantly reduced
(p < 0.001) the ratio near to control values. However, no dose de-
pendent response was observed among the two doses (125 mg/Kg body
weight and 250 mg/kg body weight) of CA. Moreover, no significant
difference (p > 0.05) in values was observed among the only extracted
treated groups when compared to control group.
3.3. Changes in antioxidant enzyme status in liver tissue
To assess whether the extracts of CA could prevent against EtOH
induced oxidative stress, level of lipid peroxidation along with activities
of antioxidant enzymes viz, cytosolic Cu-Zn-SOD, mitochondrial Mn-
SOD, and catalase were estimated. It is evident that treatment with
EtOH significantly increased lipid peroxidation by 48% (p < 0.001)
and Cu-Zn-SOD activity by 63% (p < 0.001). Treatment with EtOH
also significantly decreased Mn-SOD by 86% (p < 0.001) and catalase
by 60% (p < 0.001) activity. However, co-treatment with aqueous
extract of CA at both 125 mg/Kg body weight and 250 mg/Kg body
weight prevented the above changes. The results were comparable to
silymarin treated group. Table 3(a) below reveals that co-treatment
with CA significantly reduced lipid peroxidation (p < 0.001) and cy-
tosolic Cu-Zn-SOD activity (p < 0.001) and elevated mitochondrial
Table 4 indicates the alteration in the activities of liver marker
enzymes. Administration of EtOH significantly increased (p < 0.001)
ALT, AST and ALP levels by 69%, 62% and 45% respectively, along
with significant decrease (p < 0.001) in serum protein by 35%, when
compared to Cont group. Eventually, co-treatment with CA at both the
doses significantly reduced (p < 0.001) the elevated levels of ALT, AST
and ALP and likewise, elevated the serum protein content (p < 0.001),
as comparable to silymarin treated group. However, no significant
difference was observed among the two doses of CA.
3.5. Changes in lipid profile
It is relevant from Fig. 3 that treatment with ethanol also disrupts
the lipid profile homeostasis of the body, leading to an increase in
CHLS, TG, LDL and VLDL levels by 61%, 30%, 78% and 39% respec-
tively, along with a simultaneous decrease in HDL level (p < 0.001) by
75%. Co-treatment with aqueous extract of CA significantly reversed
the above changes to near normal values (p < 0.001), comparable to
silymarin treated group. None of the studied parameters under lipid
profile showed a dose-dependent response, except for HDL. Though the
lower dose of CA increased the HDL level compared to that of EtOH
treated group, a significant difference is observed when compared to
the Cont group (p < 0.05).
3.6. Changes in cytokine levels
Administration of EtOH resulted in significant increase in serum
TNF-α and IL6 levels compared to Cont group (p < 0.001). Fig. 4
below depicts that serum TNF-α and IL6 levels increased by 47% and
45% respectively in EtOH treated group. Co-treatment with CA caused a
marked decrease in serum TNF-α and IL6 levels. However, a significant
difference is observed among the higher and lower dose of CA, when
compared to Cont group. Only extract treated groups showed no
changes and the results were comparable to silymarin treated group.
3.7. Studies on histo-morphological and histo-chemical changes
Routine H-E and PAS stained sections of liver tissue in Fig. 5A and B
also confirm the protective effect of aqueous extract of CA. Liver sec-
tions of Cont group showed normal lobular architecture with central
vein and distinctly radiating hepatocytes. However, the EtOH treated
group showed clear signs of hepatic degeneration and necrosis,

74
M. Das et al. Journal of Ethnopharmacology 227 (2018) 69–81

Table 3(a)
Effect of aqueous extract of CA against ethanol induced changes in tissue oxidative stress markers.
Groups LPO (µmoles MDA/100 g tissue) Cu-Zn SOD (U/min/mg protein) Mn-SOD (U/min/mg protein) CAT (µmoles H2O2/min/mg protein)

Cont 51.83 ± 2.19 3.78 ± 0.75 6.94 ± 0.24 279.94 ± 18.55

EtOH 100.59 ± 4.12* 10.35 ± 1.1* 0.95 ± 0.26* 110.78 ± 12.87*
SILY + EtOH 55.13 ± 2.03**,***** 3.69 ± 0.38**,***** 6.53 ± 0.44**,***** 280.17 ± 13.64**,*****
CA125 + EtOH 59.39 ± 2.69**,***** 6.94 ± 0.40**,***** 4.52 ± 0.47**,***** 177.73 ± 24.57***,****
CA250 + EtOH 57.92 ± 4.95**,***** 4.38 ± 0.74**,***** 6.24 ± 0.61**,***** 244.08 ± 22.78**,*****
Only CA125 52.04 ± 2.91**,***** 4.25 ± 0.62**,***** 5.38 ± 0.42**,***** 264.42 ± 18.39**,*****
Only CA250 57.36 ± 1.69**,***** 3.96 ± 0.48**,***** 6.09 ± 0.33**,***** 272.58 ± 22.05**,*****

The values are expressed as Mean ± S.E.

* p ≤ 0.001 vs control.
** p ≤ 0.001 vs EtOH.
*** p > 0.05 vs EtOH.
**** p ≤ 0.01 vs control.
***** p > 0.05 vs control.

Table 3(b)
Effect of aqueous extract of CA against ethanol induced changes in xenobiotic detoxifying enzymes.
Groups GSH (mg/100 g tissue) GPx (µMoles GSH consumed/ GST (µMoles CDNB-GSH conjugate GR (µMoles NADPH utilized/ G6PD (U/mg protein)
min/mg protein) formed/min/mg protein) min/mg protein)

Cont 77.29 ± 5.26 7.14 ± 0.41 30.39 ± 1.37 97.19 ± 2.37 1.85 ± 0.11
EtOH 24.98 ± 2.50* 26.13 ± 0.84* 57.44 ± 1.42* 47.54 ± 1.38* 0.54 ± 0.05*
SILY + EtOH 77.54 ± 3.51**,**** 7.21 ± 0.61**,**** 29.50 ± 1.04**,**** 98.29 ± 1.82**,**** 1.86 ± 0.14**,****
CA125 + EtOH 48.84 ± 2.20**,*** 12.74 ± 1.57**,*** 27.86 ± 1.26**,**** 70.53 ± 3.36**,*** 1.15 ± 0.15**,***
CA250 + EtOH 73.09 ± 3.39**,**** 7.35 ± 0.61**,**** 28.91 ± 1.63**,**** 91.00 ± 2.46**,**** 1.55 ± 0.1**,****
Only CA125 75.71 ± 2.43**,**** 7.31 ± 1.24**,**** 26.47 ± 1.59**,**** 96.25 ± 2.52**,**** 1.91 ± 0.13**,****
Only CA250 72.37 ± 3.17**,**** 7.68 ± 0.74**,**** 28.30 ± 1.32**,**** 95.91 ± 1.63**,**** 1.81 ± 0.07**,****

The values are expressed as Mean ± S.E.

* p ≤ 0.001 vs control.
** p ≤ 0.001 vs EtOH.
*** p ≤ 0.05 vs control.
**** p > 0.05 vs control.

indicated by dilatation and congestion of central and portal veins,
lymphocyte infiltration and cell pyknosis. The extent of hepatic damage
is represented in Fig. 5D as percentage area of hepatic lesion. PAS
stained liver sections of EtOH group also showed depletion of glycogen
content, compared to Cont group. Co-treatment with aqueous extract of
CA at both the doses, along with EtOH maintained the normal cellular
architecture, almost comparable to Cont and silymarin co-treated
group. Moreover, tissue sections of only extract treated groups at both
125 mg/kg body weight and 250 mg/kg body weight showed no dete-
rioration of cellular morphology, thus indicating their non-toxic effect.
DNA content was demonstrated by staining liver sections with
Feulgen's reaction (Fig. 5C). Cont and extract treated liver showed
normal DNA content as depicted by the magenta colour. However,
EtOH treated group showed a reduction in DNA content, marked by
moderate magenta colour and faintly stained nuclei. Liver of rats re-
ceiving both EtOH and extract also portrayed DNA content similar to
that of cont group, thus preventing ethanol induced DNA damage.
3.7.1. Studies on collagen content and quantification of fibrosis by confocal
microscopy
Picro-Sirius red stained liver sections in Fig. 6A reveals that treat-
ment with EtOH causes deposition of collagen, specially around the
central vein, thus indicating fibrosis. However, co-treatment with
aqueous extract of CA at both the doses reduced collagen deposition,
thus preventing fibrosis. The collagen content of only extract treated
groups was also comparable to that of Cont group. Similar images
captured by confocal laser scanning microscope for the quantification
of fibrosis are shown in Fig. 6B. Fig. 6C depicts graphically the per-
centage volume of total collagen content. It is observed that treatment
with EtOH significantly increased (p ≤ 0.001) collagen content when
compared to Cont group, whereas, co-treatment with CA decreased the
collagen content to near normal level.

Table 4
Effect of aqueous extract of CA against ethanol induced changes in liver function marker enzymes.
Groups ALT (U/L) AST (U/L) ALP (U/L) Serum protein (g/dL)

Cont 27.50 ± 2.08 108.67 ± 5.05 205.17 ± 9.13 5.7 ± 0.12

EtOH 89.67 ± 3.74* 282.83 ± 5.40* 375.67 ± 4.52* 3.73 ± 0.27*
SILY + EtOH 28.47 ± 2.64**,*** 109.61 ± 4.13**,*** 203 ± 6.33**,*** 5.66 ± 0.23**,***
CA125 + EtOH 34.67 ± 2.93**,*** 146.03 ± 7.57**,*** 211.17 ± 9.93**,*** 5.58 ± 0.29**,***
CA250 + EtOH 30.83 ± 3.30**,*** 134.83 ± 8.73**,*** 209.00 ± 11.75**,*** 5.25 ± 0.29**,***
Only CA125 29.03 ± 3.22**,*** 107.51 ± 5.72**,*** 208.83 ± 8.15**,*** 5.62 ± 0.25**,***
Only CA250 28.16 ± 3.84**,*** 110.22 ± 5.51**,*** 207.67 ± 9.27**,*** 5.55 ± 0.16**,***

The values are expressed as Mean ± S.E.

* p ≤ 0.001 vs control.
** p ≤ 0.001 vs EtOH.
*** p > 0.05 vs control.

75
M. Das et al.
Fig. 3. Effect of aqueous extract of CA against ethanol induced changes in Lipid Profile. The values are expressed as Mean ± S.E. *p ≤ 0.001 vs control, **p ≤ 0.001
vs EtOH, #p ≤ 0.05 vs control.
3.7.2. Studies on apoptosis detection by TUNEL assay
TUNEL stained liver sections in Fig. 7A below reveal the anti-
apoptotic property of green CA. Under confocal microscopy, frag-
mented DNA labelled with FITC was viewed green and the DAPI stained
nuclei were viewed blue. Co-localisation of both the blue and green
stain in the merged image was marked as TUNEL-positive apoptotic
cells. The number of TUNEL-positive apoptotic cells in the EtOH treated
group was found to be significantly high compared to the cont group.
However, co-administration with the aqueous extract of CA at both the
doses and with silymarin reduced the number of TUNEL positive
apoptotic cells. Besides, the higher dose was found to be more potential
in preventing apoptosis. The only extract treated group showed no
significant difference with the cont group. Fig. 7B below represents
graphically the apoptotic index of each group.
4. Discussion
In the present study, the phyto-constituents present in aqueous ex-
tract of Capsicum annum have been identified and the hepato-protective
role of this extract against ethanol induced oxidative stress and hepa-
totoxicity has been assessed.
Fig. 4. Effect of aqueous extract of CA against ethanol induced changes in Serum cytokine levels. The values are expressed as Mean ± S.E. *p ≤ 0.001 vs control,
**
p ≤ 0.001 vs EtOH, #p ≤ 0.05 vs control, nsp > 0.05 vs control.
76
Journal of Ethnopharmacology 227 (2018) 69–81
Lipid peroxidation has been reported to be the most prominent
destructive process in ethanol induced liver injury (Muriel, 1997). This
is in accordance to our study, which shows a marked elevation of LPO
measured in terms of MDA in hepatic tissue of EtOH treated group. Co-
treatment with aqueous extract of CA however restores the MDA level
to near normal values, therefore preventing LPO.
The body's first line of defence against oxidative stress includes
GSH, SOD and CAT that directly scavenges the free radicals and re-
active metabolites. The results of our present study demonstrate an
increase in cytosolic Cu-Zn-SOD along with a concomitant decrease in
mitochondrial Mn-SOD and CAT activity in EtOH treated group.
Increased Cu-Zn-SOD activity indicates an excess production of super-
oxide anion, which is being converted to H2O2. Time dependent leakage
of SOD from mitochondria to cytosol might explain the depletion of Mn-
SOD, thus indicating loss of mitochondrial membrane integrity.
Decrease in the activity of CAT also indicates H2O2 accumulation
within hepatic tissue leading to DNA damage and cell injury. The de-
crease in CAT activity in EtOH group might be either due to excessive
utilisation of the enzyme in scavenging H2O2 or, due to a decrease in
iron availability to the enzyme. Co-treatment with aqueous extract of
CA at both the doses or with silymarin prevented decrease in CAT
M. Das et al. Journal of Ethnopharmacology 227 (2018) 69–81

Fig. 5. Effect of aqueous extract of CA against ethanol induced changes in Histo-morphology and Histo-chemistry. 5A depicts H-E stained liver tissue sections (200×
magnification), 5 B depicts PAS stained liver tissue sections (200× magnification) and 5C depicts Fuelgen reaction for nuclear DNA detection in liver tissue sections
(400× magnification). The arrow heads in EtOH group indicate dilatation and congestion of central vein in H-E stained liver sections along with depletion of
glycogen and nuclear DNA content in PAS and Fuelgen reaction stained liver sections respectively. 5D represents the histological scores as percentage of hepatic
lesion of H-E stained sections. The values are expressed as Mean ± S.E. *p ≤ 0.001 vs control, **p ≤ 0.001 vs EtOH, ***p ≤ 0.01 vs EtOH, #p ≤ 0.05 vs control.

activity, thereby increasing peroxisomal lipid oxidation, phospholipid
synthesis and improving membrane integrity. This is also reflected by
altered concentration of Mn-SOD level with extract administration. This
also corroborates with the finding that CA extract can effectively pre-
vent ethanol induced hyperlipidemia.
EtOH treated group in this study also revealed a depletion of GSH
content along with GR and G6PD activity. An enhanced activity of GPx
and GST in the EtOH group may account for the depletion of GSH. GST
also plays a vital role in detoxification of the lipid hydro-peroxides and
toxic electrophiles by catalyzing their conjugation with GSH, thus,
contributing to the protection of the cellular integrity (Townsend and
Tew, 2003). Eventually, an increased oxidation of GSH along with re-
duced activity of GR and G6PD leads to an increased GSSG: GSH ratio.
Prior treatment with aqueous extract of CA or silymarin restores the
above changes to near normal values.
Enhanced activity of ALT and AST are obvious marker of hepatic
damage. Our results also show an increased activity of ALT, AST and
ALP in EtOH treated rats, which is being suppressed by co-treatment
with aqueous extract of CA at both the doses. Besides, a decline in the
serum protein content is also considered to be a useful index in asses-
sing the severity of cellular dysfunction in chronic liver diseases.
Stimulation of protein synthesis in rats co-treated with aqueous extract
of CA might serve as a contributory hepatoprotective mechanism,
leading to liver cell regeneration.
During ethanol metabolism, large amounts of reduced nicotina-
mide-adenine dinucleotide (NADH) is produced, thus, inhibiting Krebs
cycle and oxidation of fatty acid, which favours liver steatosis and
serum hyperlipidaemia (Rukkumani et al., 2002). This has been well
reflected in the increased body weight to liver weight ratio in EtOH
treated group. In addition, EtOH group also shows increase in the levels
of CHLS, TG, LDL and VLDL, along with a simultaneous decrease in HDL
level. However, co-treatment with aqueous extract of CA at both the
doses attenuates the above changes. The hypo-cholesterolemic activity
of extract treated animals may be attributed to increased hepatic

77
M. Das et al.
Fig. 6. Effect of aqueous extract of CA on collagen content. 6A depicts Picro-Sirius stained liver tissue sections of different groups (200× magnification). 6B depicts
Picro-Sirius stained liver sections viewed under confocal microscope and 6C depicts the graphical representation of collagen content in liver sections of different
group. The values are expressed as Mean ± S.E. *p ≤ 0.001 vs control, **p ≤ 0.001 vs EtOH.
clearance of cholesterol through elevated serum HDL levels, or to down
regulation of cholesterol bio-synthetic enzyme, HMG-CoA reductase
(Patil et al., 2011).
Ethanol induced activation of kupffer cells promotes release of pro-
inflammatory and anti-inflammatory cytokines, such as TNF-α and IL-6,
which is also involved in acute phase protein synthesis (Tilg and Diehl,
2000) and inflammation. These literatures coincide with our result,
which depicts increase in the levels of TNF-α and IL-6 in EtOH treated
group. Decrease in the levels of TNF-α and IL-6 on co-treatment with
aqueous extract of CA again portraits the hepatoprotective activity of
CA.
Moreover, histological studies of H-E, PAS and feulgen stained
sections also acclaim that aqueous extract of CA at both the doses can
prevent ethanol induced liver injury. Ethanol induced chronic hepato-
toxicity is also found to be associated with apoptosis of hepatocytes, as
is observed by the increase in the number of apoptotic cells in the EtOH
group. This is in accordance with the study conducted by Das et al.
(2011), which shows an increase in the number of apoptotic cells with
chronic exposure to ethanol. In the present study, ethanol induced cell
apoptosis has been prevented in a dose-dependent manner by co-ad-
ministration of aqueous extract of CA.
Thus, it can be abridged from our study that aqueous extract of
Capsicum annum possesses hepatoprotective activity against ethanol
induced hepatic lesion. The active component, capsaicin present in CA
is reported to confer hepatic protection against CCl4, lipo-poly-
saccharide and iron induced hepatotoxicity (Thriveni et al., 2017;
Hassan et al., 2011; Manjunatha and Srinivasan, 2006). Presence of β-
carotene (vitamin A precursor), lycopene, vitamin C and vitamin E in
78
Journal of Ethnopharmacology 227 (2018) 69–81
CA, as quantified in our previous study (Das et al., 2017), are well
known anti-oxidative agents. β - carotene exhibits antioxidant activity
by suppressing singlet oxygen, scavenging peroxide radicals, and di-
rectly reacting with peroxy radicals, thus stabilizing membrane lipids
from free radical attack (Bast et al., 1998).
Antioxidant vitamins like vitamin C and vitamin E act synergisti-
cally against oxidative stress-related diseases. Vitamin E functions as a
chain-breaking antioxidant which prevents the propagation of free ra-
dical reactions whereas, vitamin C is a part of the normal immune
mechanism in humans. Besides, GC-MS analysis of CA extract in our
present study reveal the presence of several phytoconstituents like 2,3-
Dihydroxy 1,4-dioxane, 2,6-Dimethylnapthol [1,2-b] furan-4,5-dione,
4-[5-(Methylsulfinyl)-1,3,4 thiadiazole-2-yl] pyridine etc. with reported
anti-oxidative and hepato-protective activity.
5. Conclusion and future aspects
The hepatoprotective capability of CA preserved the liver's status
quo in terms of its antioxidant status, and structural and functional
integrity as well, against chronic ethanol exposure. Moreover, low
doses of CA is found to possess very low or no toxicity. Therefore, it will
be worth investigating whether CA can be used as one of the hepato-
protective agents for the treatment of ALD. Further study to explore the
underlying molecular mechanism is also required to recognise the pa-
thogenesis responsible for ethanol induced hepatic damage. Isolation of
the bio-active components of CA, providing hepatic protection is also a
prerequisite for recommending CA as a nutritional supplement to
combat ALD.
M. Das et al. Journal of Ethnopharmacology 227 (2018) 69–81

Fig. 7. Effect of aqueous extract of CA on apoptosis. 7A depicts liver tissue sections of different treatment groups, stained with TUNEL (green) and counterstained
with DAPI (blue) respectively at 400×. Co-localisation of both the blue and green stain in the merged image represents the TUNEL-positive apoptotic cells. 7B shows
the graphical representation of the apoptotic index of different groups. The values are expressed as Mean ± S.E. *p ≤ 0.001 vs control, **p ≤ 0.001 vs EtOH,
***
p ≤ 0.01 vs EtOH, #p ≤ 0.05 vs control.

79
M. Das et al.
Fig. 7. (continued)
Acknowledgment
The first author highly acknowledges the grant received from UGC-
Major Research Project [F. No. 42-625/2013 (SR)], Govt. of India. The
second author acknowledges the grant received from INSPIRE pro-
gramme, Department of Science and Technology, Govt. of India. All
authors greatly acknowledge Dr. Pranabes Nath, faculty of Rammohan
College for providing constant co-operation and guidance throughout
the study. The authorities of Rammohan College and Bose Institute,
Kolkata are also acknowledged for providing necessary facility to carry
out the study. Special thanks are also due to Sri Sumanto Ghosal for
providing technical assistance.
Declaration of interest
The authors declare that there is no conflict of interest.
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Glossary
Alcoholic liver disease: It is a term that encompasses the liver manifestations of alcohol
overconsumption, including fatty liver, alcoholic hepatitis, and chronic hepatitis with
liver fibrosis or cirrhosis.
Antioxidant: It is a molecule that inhibits the oxidation of other molecules. Oxidation is a
chemical reaction that can produce free radicals, leading to chain reactions that may
damage cells.
Apoptosis: It is a process of programmed cell death that occurs in multicellular organisms.
Fibrosis: It is the formation of excess fibrous connective tissue in an organ or tissue in a
reparative or reactive process.
Oxidative stress: It reflects an imbalance between the systemic manifestation of reactive
oxygen species and a biological system's ability to readily detoxify the reactive in-
termediates or to repair the resulting damage. Disturbances in the normal redox state
of cells can cause toxic effects through the production of peroxides and free radicals
that damage all components of the cell, including proteins, lipids, and DNA.
Oxidative stress from oxidative metabolism causes base damage, as well as strand
breaks in DNA
Xenobiotic: It is a foreign chemical substance found within an organism that is not
naturally produced by or expected to be present within. It can also cover substances
that are present in much higher concentrations than are usual. Specifically, drugs
such as antibiotics are xenobiotics in humans because the human body does not
produce them itself, nor are they part of a normal food.