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Vol 14, Issue 12, 2021
Online - 2455-3891
Print - 0974-2441
OLEANOLIC ACID, A PROSPECTIVE PROTECTIVE AGENT AGAINST BRAIN ENERGY
METABOLISM AND OXIDATIVE DYSFUNCTIONS FOLLOWING HEXAVALENT CHROMIUM
EXPOSURE IN MICE
SUDIPTA PAL*
Nutritional Biochemistry and Toxicology Laboratory, Department of Human Physiology, Tripura University, Suryamaninagar, West
Tripura, Tripura, India. E-mail: sudiptapal@tripurauniv.ac.in
Received: 05 October 2021, Revised and Accepted: 10 November 2021
ABSTRACT
Objective: Effect of oleanolic acid against hexavalent chromium-induced altered brain energy metabolism associated with oxidative stress was
evaluated in the present study.
Methods: Swiss albino mice were divided into three groups, Control (n=6), chromium-treated (n=6), and oleanolic acid (OA) supplemented (n=6).
The chromium treated group was orally administered with K2Cr2O7 for 30 days at a dose of 10 mg/kg b.w/day. OA supplementation was given at a
dose of 5 mg/kg bw/day for the past 14 days of chromium treatment. Control group received the vehicle only. After the treatment, whole brain was
removed for examining the parameters such as pyruvic acid, free amino nitrogen, tissue protein, TCA cycle enzyme activities, NADH dehydrogenase
function, and oxidative stress markers.
Results: Significant decrease in cerebral pyruvic acid content associated with suppressed malate dehydrogenase and succinate dehydrogenase
activities were observed. The NADH dehydrogenase activity was inhibited owing to enhanced accumulation of chromium in cerebral tissue. Depletion
of proteins and increased free amino acid nitrogen were accompanied with inhibited cathepsin, pronase and trypsin activities, and increased
transaminase function. In addition, GSH content was decreased along with increased lipid peroxidation, oxidized GSSG content, TG/GSSG ratio,
carbonylated protein content, and tissue free hydroxyl radical formation. Superoxide dismutase, catalase, glutathione reductase, and glutathione
peroxidase were also inhibited by hexavalent chromium. Oleanolic acid supplementation was found to have significant protective effect against brain
metabolic and oxidative dysfunctions.
Conclusion: The present study elucidated therapeutic efficacy of oleanolic acid against hexavalent chromium toxicity in brain tissue of mice.
Keywords: Hexavalent chromium, Glycolysis, TCA cycle, Protease activity, Oxidative stress, Oleanolic acid, Natural antioxidant.
INTRODUCTION
Metal toxicity is one of the serious problems to living organisms as a
consequence of industrialization, globalization, pollution, and other
anthropogenic activities. Its extensive applications including plating,
painting, leather tanning, cementing and as anticorrosion agent
make it more available for human exposure [1]. In environment, it
exists in different valency states, among which hexavalent chromium
is hemotoxic, carcinogenic and mutagenic in nature [2,3]. Due to
solubility in water, hexavalent chromium is abundant in polluted
water. The maximum permissible limit for hexavalent chromium
in drinking water is 0.05 mg/l as per WHO recommendation. Other
common route of exposure is inhalation through paint dusts, aerosol
and color pigments [4]. Seven countries in the world namely South
Africa, India, Zimbabwe, Kazakhstan, Brazil, Finland, and Turkey
experience chromium exposure from human activities at stationary
point sources [5].
The toxic effect of hexavalent chromium exhibits when it rapidly crosses
the biological membrane, enters the cell and is converted to trivalent
chromium; otherwise, reduction of hexavalent chromium outside the
cell is found to be less toxic [6]. Respiratory distress is the most common
symptom for chromate sensitive workers acutely exposed to hexavalent
chromium. Other organ systems such as cardiovascular, hematological,
nervous, gastrointestinal, reproductive, immunological, and renal
systems are also adversely affected by chronic and acute chromium
exposure [6]. The molecular mechanism of hexavalent chromium
toxicity involves generation of free radicals and highly reactive
chromium intermediates [7]. Brain tissue is highly susceptible to
oxidative stress due its high lipid content and high oxygen consumption
for its metabolic purpose. Enhanced lipid peroxidation in brain tissue
of mammals was noted in an earlier study [8]. Chrome plating plant
workers when exposed to chromium trioxide fumes, often experience
headache, dizziness and weakness [6], indicating harmful neurological
effects of chromium in human. Further study confirmed that Cr (VI) is a
potent neurotoxic agent for the matured neuronal cell and altered brain
physiochemical functioning [9].
In a number of studies, it is established that over-exposure of
hexavalent chromium is associated with its significant deposition in
the rat hypothalamus, anterior pituitary, hepatic as well as muscular
tissue [10]. Excessive accumulation of this metalloid may lead to
metabolic complications in exposed organisms. Inadequate information
regarding detailed metabolic toxicity by hexavalent chromium raised
the curiosity about this compound and thus aimed at determining its
mechanism of action on metabolic profile. Limited evidences are there
in support of metabolic toxicity by Cr (VI) [11,12]. In a stressed tissue,
it is very much expected that metabolites involved in energy yielding
biochemical processes may be influenced by the stressor. Chromium,
being an oxidative stress producing metalloid may have certain adverse
effects on metabolic profile of the soft tissue like brain. The present
study thus tried to explore plausible mechanism related to TCA cycle,
oxidative phosphorylation and the protein metabolic efficacy along
with certain oxidative stress markers in the highly energetic cerebral
tissue in presence of excess hexavalent chromium.
© 2021 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/
licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ajpcr.2021v14i12.43311. Journal homepage: https://innovareacademics.in/journals/index.php/ajpcr
Research Article
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Natural antioxidants have important roles in ameliorating
environmental pollutant-induced cellular damage. Among certain
polyphenolic antioxidants, oleanolic acid (OA) has gained attention due
to its beneficial effect against metal/metalloid encouraged metabolic
toxicity in vivo [13]. Oleanolic acid, or 3β-hydroxyolean-12-en-28-oic
acid, is a pentacyclic triterpenoid found naturally in certain leaves,
fruits, and plants. Being a non-toxic and hepatoprotective agent against
chemical-induced fatty liver diseases, it attained its importance in
pharmacotherapy [14]. Protective functions of oleanolic acid such
as, antitumor, antidiabetic, hepatoprotective, anti-inflammatory,
antioxidant, and antibacterial activities were addressed in different
animal models [13,15-17].
Earlier studies revealed that oleanolic acid served as a protective agent
against sodium fluoride induced alteration in brain protein and nucleic
acid metabolism and was suggested as a prospective neuroprotective
agent against fluoride toxicity [13]. Oxidative damage caused by
sub-acute fluoride toxicity was appreciably corrected by oleanolic
acid in vivo, signifying its antioxidant property to improve cellular
functions [13]. Antioxidant efficacy of oleanolic acid was mediated by
its superoxide radical trapping activity, inhibition of xanthine oxidase
activity and metal ion chelating property [18]. Accordingly, the present
study aims at determining the effect of oleanolic acid supplementation
in hexavalent chromium treated brain tissue of mice, to evaluate
whether this triterpenoid has any efficacy to counteract chromium-
induced metabolic alteration in brain tissue, or not.
MATERIALS AND METHODS
Materials
All chemicals and reagents, such as hemoglobin, trichloroacetic acid
(TCA), dimethyl sulfoxide (DMSO), 5,5’-DTNB, potassium dichromate
(K2Cr2O7), bovine serum albumin (BSA), EDTA, H2O2, NADH, NADPH.Na2,
methanol, ethanol, Fat blue BB salt, glutathione (GSH), thiobarbituric
acid (TBA), and sodium carbonate, were of analytical grade and
purchased from Merck (India), SRL (India), Sigma–Aldrich (India).
Biochemical kits such as GPT, GOT were purchased from Coral clinical
systems. TG/GSSG colorimetric assay kit (Elabscience), distilled
water prepared by Millipore water purifier was used throughout the
experiment to avoid metal contamination.
Isolation and extraction of oleanolic acid from Neanotis wightiana
plant
N. wightiana is a medicinal plant found in Tripura, collected from Kalsi,
Jolaibari, South Tripura and identified by Prof. B.K. Datta, Taxonomist,
Department of Botany, Tripura University. Voucher specimen
(#BD/02/08) was deposited in the National Herbarium, Botanical
Survey of India, botanical garden, Howrah, West Bengal, India. The
aerial part of this plant was claimed to have therapeutic effect on liver
and brain damage by the local people of Tripura.
The plant extract was prepared by the method of Das et al. [19]. In
brief, the aerial part of N. wightiana was air-dried and extracted with
methanol (10Lx3, 1 week each) at room temperature. The extract was
concentrated with reduced pressure in vacuo to obtain a semi-solid
mass (400 g). The residue was then suspended in 125 ml of triple
distilled water and further extracted with hexane, chloroform, ethyl
acetate, and n-butanol (three times each, 200 ml). The ethyl acetate
extract (20.2 g) was column chromatographed through silica gel and
eluted with stepwise gradient of CHCl3/EtOAc (100:0, 90:10, 80:20,
70:30, 60:40, 50:50, 40:60, 20:80, 10:90 each 500 ml). The fraction
eluted with CHCl3/EtOAc (70:30) formed a gummy residue which was
again subjected to column chromatography with silica gel and finally
oleanolic acid (3β-hydroxyolean-12-ene-28-oic acid) extract is obtained.
Selection of animals
To conduct the present experiment, Swiss albino male mice (n=18)
weighing 35–40 g were purchased from Chakraborty Enterprise
(Reg.No.1443/PO/b/11/CPCSEA), Kolkata, India, an authorized
animal supplier nominated by CPCSEA, Ministry of Environment and
Forests, Govt. of India. Drinking water was given to the animals ad-
libitum throughout the treatment schedule. The mice were kept in the
treatment room with sustaining 22–25°C temperature and humidity
(50%) with alternate light and dark coverage for 12 h.
Experimental design
Total eighteen numbers of healthy and equal average sized body weight
(30–35 g) of mice were taken for the present study and divided into three
groups namely control group, Cr (VI)-treated group, and OA-supplemented
group, each having six (n=6) numbers of animals in each group.
Control group
Mice received drinking water orally.
Cr (VI)-treated group
The animals of this group were treated with Cr (VI) (as potassium
dichromate, K2Cr2O7 at a dose of 10 mg/kg b.w/day orally by oro-gastric
feeding needle for a period of 30 days). The dose had been selected on
the basis of a dose-dependent study performed earlier which revealed
that at the mentioned dose significant alteration in certain parameters
related to carbohydrate metabolism in hepatic tissue was found without
causing any casualty to the animals [20]. Moreover, the selected dose of
Cr (VI) was also used in earlier occasions [21,22].
OA-supplemented group
Hexavalent chromium as potassium dichromate was administered at
10 mg/kg bw/day orally for 30 days along with oleanolic acid (OA)
supplementation at a dose of 5 mg/kg bw/day for the past 14 days of
chromium treatment. The dose of oleanolic acid was selected on the
basis of earlier report [13].
Animal sacrifice
At the end of the treatment, the mice were sacrificed by cervical
dislocation as per the rules and regulations of the Institutional Animal
Ethical Committee. Ethical approval for the present work was received
by the Institutional Animal Ethical Committee, Tripura University
[Approval no. TU/IAEC/2015/XI/2-3 dated 28th July, 2015]. Thereafter,
whole brain was dissected out from the animals, washed in ice-cold
saline (0.9% NaCl), blotted dry, weighed and kept at −20°C until
biochemical analyzes were performed.
Preparation of tissue homogenate
The 5% (mass/volume) brain tissue homogenate was prepared in 0.1 M
phosphate buffer (pH 7.4) using all glass homogenizer and kept frozen
at -20oC until biochemical analyzes were performed.
Preparation of mitochondrial isolate for estimation of the TCA
cycle enzymes
In brief, 500 mg of the brain tissue was kept in 10 ml of sucrose buffer
[containing 0.25 M sucrose, 0.001M EDTA, 0.05 M Tris HCl (pH 7.8)] at
25°C for 5 minutes. It was then homogenized in ice-cold chamber (at 4°C)
for ten minutes at 1500 rpm using a Potter Elvenjem glass homogenizer.
The supernatant was recentrifuged at 4000 rpm for 5 minutes at 4°C. The
supernatant was further centrifuged at 14000 rpm for 20 minutes at 4°C.
The final supernatant was discarded and the pellet was re-suspended
in sucrose buffer and kept at -20°C for biochemical analyses. Freshly
prepared mitochondrial isolate was used for the experiment [23].
Morpho-physiological analyzes
Body weight and cerebro-somatic index (CSI)
The body weight of the mice of each group was taken from the first
day of treatment and recorded periodically until sacrifice. The organ
weight (whole brain) of the respective group of mice was noted after
sacrifice of the animals. The cerebro-somatic index (CSI) was calculated
as follows [24].
−= ×
()
100
30 ( )
Weight of thewh ole brain g
Organo somatic index Day thtotal bod y weight g
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Biochemical analyses
Pyruvic acid level
The 5% brain tissue homogenate was centrifuged with 5% TCA at
3000 rpm for 10 minutes. The supernatant was treated with 0.5 ml
2,4-DNPH and 1 ml of distilled water and mixed vigorously for
approximately 3 minutes. Toluene was added and mixed by hand
shaking for a few minutes. It was then added with 2 ml of Na2CO3
and NaOH solution each to measure the optical density at 420 nm in
a spectrophotometer. The observed result was expressed as µg/g of
tissue [25].
Succinate dehydrogenase (SDH) activity
The SDH activity was measured spectrophotometrically by the reduction
of potassium ferricyanide (K3Fe (CN)6) at 420 nm [23]. One millilitre
of the assay mixture contained 0.05 M phosphate buffer (pH 7.4), 2%
BSA (w/v), 4 mM succinate, 2.5 mM K3Fe (CN)6 and a definite volume
of the mitochondrial isolate (enzyme source). The enzyme activity was
expressed as unit/min/mg of protein.
Malate dehydrogenase (MDH) assay
The assay mixture contained potassium phosphate buffer, 76 mM
oxaloacetic acid and 5 mM NADH at pH 7.4. The reduction of NADH
was measured at 340 nm for 5 minutes by observing change in optical
density at each 10 second interval. The enzyme activity was expressed
as mmoles of NADH oxidized/min/mg of protein [26].
Assay of NADH: Ubiquinone C oxidoreductase activity
The assay mixture consisted of 1 ml phosphate buffer, 0.1 ml K3Fe (CN)6
and 0.2 ml of mitochondrial suspension in a total volume of 3 ml with
distilled water. Freshly prepared 0.1% NADH solution was added just
before the addition of the mitochondrial isolate except the blank set.
The change in optical density was measured at 420 nm for 3 minutes.
The enzyme activity was expressed as mmoles of NADH oxidized/min/
mg of protein [27].
Protein carbonylation
Briefly, the samples were mixed with the same volume of 10 mM 2,4-
DNPH reagent prepared in 2.5 M HCl and incubated for half an hour at
room temperature by vigorous shaking at each 15 minutes interval. The
samples were then treated with 20% TCA and kept in ice for a while. It
was then centrifuged at low r.p.m, and the pellet was taken. The pellet
was washed with ethanol/ethyl acetate mixture (1:1 v/v) twice and
finally dissolved in 6% SDS and centrifuged again. The optical density
of the supernatant was recorded at 370 nm. The results were expressed
as nmoles of DNPH-incorporated/mg protein. The molar extinction
coefficient of 22,000/M/cm was used for the final calculation of the
carbonylated protein content [28].
Free amino nitrogen content
The tissue homogenate was mixed with 2/3 N H2SO4 and 10% sodium
tungstate for precipitation of tissue proteins. The protein free aliquot
was treated with cyanide-acetate buffer and 3% ninhydrin solution and
mixed thoroughly. The mixture was heated in a boiling water bath for
5 minutes. Immediately after cooling, isopropyl alcohol was added to
the mixture. A violet color was developed, the absorbance was read
at 570 nm in a spectrophotometer. Free amino nitrogen level was
expressed in mg/g of tissue [29].
Pronase activity
The 5% tissue homogenate (in 0.1M PB, pH 7.4) was mixed with
casein substrate and incubated at 40°C for 30 minutes. The reaction
was terminated by protein precipitating reagent. The mixture was
then centrifuged to obtain clear supernatant, and the absorbance
of which was read at 280 nm in an UV-visible spectrophotometer.
Pronase activity was expressed as nmoles of tyrosine formed/min/mg
protein [30].
Trypsin activity
A definite volume of 5% tissue homogenate was taken in a sample tube
and added with 2.5 ml of hemoglobin substrate, followed by incubation
at 25°C for 30 minutes. A buffer blank was prepared by taking
trichloroacetic acid (TCA), then followed by addition of tissue sample
and the substrate. After incubation, 5% TCA was added to the sample
tube to stop the reaction. All the tubes were centrifuged to obtain
protein free filtrate. The absorbance of the final aliquot was taken in
an UV-visible spectrophotometer at 280 nm. The enzyme activity was
calculated as nmoles of tyrosine produced/min/mg protein [31].
Cathepsin activity
The 5% tissue homogenate (in 0.1M PB, pH 7.4) was added with 4%
hemoglobin substrate and incubated at 37°C for 60 minutes. After
incubation, reaction was stopped by addition of 8% TCA. A buffer blank
was prepared in a same manner in which TCA was added before addition
of the tissue homogenate and the Hb substrate. All the tubes were then
centrifuged to obtain protein free clear supernatant. Absorbance of
the supernatant was taken at 280 nm wavelength using an UV-visible
spectrophotometer. Tissue cathepsin activity was expressed in terms of
nmoles of tyrosine produced/min/mg protein [32].
Glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate
transaminase (GOT) activities
The assay was done with the help of a standard kit (Coral clinical system,
Goa, India). After completion of overall reaction, readings were taken in
a spectrophotometer at 505 nm wavelength. The enzyme activity was
expressed in terms of units/g of tissue [33].
Reduced glutathione (GSH) content
Reduced glutathione content in brain tissue of mice was measured
using Ellman’s reagent [34]. The 5% tissue homogenate in 0.1 M PB,
pH 7.4 was mixed with 20% TCA containing 1 mM EDTA to precipitate
proteins. The supernatant was mixed with Ellman’s reagent and kept for
20 minutes at room temperature. The absorbance was read at 412 nm.
The GSH content was expressed in terms of µmoles/mg protein.
Tissue total glutathione/oxidized glutathione ratio (TG/GSSG)
The TG/GSSG ratio was estimated in the cerebral tissue of mice by
colorimetric assay kit (Elabscience) [35]. The estimation was based on
oxidation-reduction reaction using DTNB. GSSG was reduced to GSH by
glutathione reductase, and GSH was oxidized by DTNB to produced GSSG
and a yellow TNB. The amount of TNB represented total glutathione
(GSSG+GSH), the absorbance of which was read at 412 nm. The content
of GSSG was determined by first removing GSH from the sample with
suitable reagent followed by the above-mentioned reaction principle.
Tissue lipid peroxidation (LPO) level
In brief, the 5% tissue homogenate in 0.1 M PB, pH 7.4 was mixed
with 20% TCA and thiobarbituric acid. The reaction mixture was
then mixed with 1 mM EDTA. The mixture was heated at 80°C for
5 minutes. Absorbance of the final reaction mixture was taken at
533 nm in a spectrophotometer. The molar extinction co-efficient,
1.56x105 cm2/mmol of malondialdehyde was used to calculate the LPO
level [36].
Tissue free hydroxyl radical formation
At first, all of the mice were treated with 30% DMSO (0.4 ml/100 g bw)
two hours before sacrifice of the animals. After sacrifice, 5% brain tissue
homogenate was prepared in triple distilled water for determination
of free hydroxyl radical generation [37]. The tissue homogenate was
added with 10 N H2SO4 to precipitate proteins. The filtrate was taken for
extraction of methane sulfinic acid, which was produced from DMSO by
the reaction with free hydroxyl radicals. The mixture was then treated
with fast blue BB salt to obtain a yellow color adduct, the absorbance of
which was measured in a spectrophotometer at 425 nm.
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Catalase (CAT) activity
The 5% brain tissue homogenate (in 0.1 M phosphate buffer, pH 7.4
containing 1% triton X-100) was taken for estimation of the catalase
activity [38]. The rate of degradation of H2O2 was measured as the
enzyme catalyzed reaction. The enzyme activity was expressed in terms
of micromoles of H2O2 utilized/minute/mg protein.
Superoxide dismutase (SOD) activity
The 5% brain tissue homogenate (in 0.1 M PB, pH 7.4) was centrifuged at
10,000 g for 10 minutes. The collected aliquot was taken for estimation
of the SOD activity [39]. The increase in rate of auto-oxidation of
hematoxylin in aqueous alkaline solution was noted after addition of
enzyme solution. The chromophore produced due to the reaction was
read at 560 nm in a spectrophotometer. The SOD activity was expressed
as units/minute/mg of protein.
Glutathione reductase (GR) activity
The assay medium contained 0.2 M phosphate buffer (pH 7.0 containing
2 mM EDTA), 20 mM GSSG and 2 mM NADPH and the tissue sample. The
enzyme activity was measured at 25°C at 340 nm wavelength in an UV-
visible spectrophotometer. The disappearance of NADPH was recorded
at 30 sec intervals and the activity was expressed in terms of NADPH
oxidized per minute per mg of protein [40].
Glutathione peroxidase (GPx) activity
The 10% brain tissue homogenate (in 0.1 M PB, pH 7.4) was centrifuged
at 10,000 g for 30 min at 4°C. The clear supernatant was used as sample
for estimation. The reaction mixture consisted of definite volume of 0.1
M PB, pH 7.4, 10 mM GSH, 0.2 units glutathione reductase and 50 µL of
the sample. The mixture was incubated at 37°C for 10 min followed by
addition of 1.5 mM NADPH. The absorbance was recorded at 340 nm
wavelength. The enzyme activity was represented as nmoles of NADPH
oxidized per min per mg protein [41].
Tissue protein content
The tissue protein content was estimated according to the method of
Lowry et al. [42]. Protein content was expressed as g/100 g of tissue.
Tissue Cr (VI) analysis
Tissue Cr (VI) content of brain tissue was determined using atomic
absorption spectrometer (Perkin Elmer A Analyst 700) according to the
method suggested by Sun and Liang [43]. Data were expressed as µg/g
tissue.
Statistical analyses of data
The obtained data were expressed as means±SEM. One-way ANOVA
was used for analyses of data followed by ‘multiple comparison t-test’
for comparison between the two groups. p<0.05 was considered
statistically significant.
RESULTS
There was insignificant change (6% decrease; p>0.05) in the body
weight of the Cr (VI)-treated mice compared to the control group at
the present dose and duration (Table 1). The cerebro-somatic index
(CSI) was significantly increased (32%, p<0.05) following Cr (VI)
treatment. Cr was accumulated in the brain tissue at high amount (ten
folds increase, p<0.001) in comparison to the respective control value
(Table 1). Oleanolic acid supplementation in chromium-treated mice
appreciably restored the changes in CSI (21.3% restoration, p<0.05)
and tissue chromium concentration (30.7% restoration, p<0.001),
when co-treated with hexavalent chromium (Table 1), whereas body
weight of the mice remained almost unaltered in the OA-supplemented
group.
It was revealed from the Fig. 1 that the malate dehydrogenase activity
was decreased by 48.2% (p<0.001) following exposure to hexavalent
chromium (p<0.001). OA supplementation was found to be protective
to some extent to restore the MDH activity in mice brain. The
counteraction was noted to be 35% in respect to the chromium-treated
group of mice (p<0.001).
The activity of the succinate dehydrogenase, another TCA cycle enzyme
was also decreased significantly (p<0.001) following exposure to
hexavalent chromium (Fig. 2). OA supplementation in chromium
exposed mice appreciably counteracted the brain SDH activity by
53.2% of the control value (p<0.001).
It was observed from Fig. 3 that the activity of mitochondrial complex 1
(NADH dehydrogenase) was reduced by 35.8% (p<0.01) in mice brain
owing the toxic effect of Cr (VI). Oleanolic acid supplementation in
chromium exposed mice partially counteracted the mitochondrial
complex 1 activity. The restoration was found to be 84.2% of the control
value.
Change in total protein content revealed that Cr (VI) treatment increased
total protein content in the cerebral tissue by 24.2% (p<0.05) (Table 2).
Oleanolic acid supplementation checked brain protein enhancement
Table 1: Body weight, CSI and bio-distribution of Cr (VI) in brain tissue of mice exposed to Cr (VI) with or without OA supplementation
Groups of animals (n=18) Final body weight (g) after 30 days Cerebro-somatic index Bio-distribution of Cr (VI) in brain tissue(µg/g
of tissue)
Control (n=6)
Cr-Treated (n=6)
OA-supplemented (n=6)
36.33±1.75
34.13±1.31#
35.26±1.06#
0.41±0.03
0.54±0.024pa*
0.425±0.018pb*
1.73±0.08
17.5±1.56pa***
12.12±1.84pb***
Figures in the parentheses indicate number of animals in each group (n=6). N represents total number of animals. Values are expressed as Means±SD. #p>0.05 (Not
significant), pa compared to control group and pb compared with Cr-treated group. *p<0.05; **p<0.01 and ***p<0.001 are considered statistically significant. Control
group received drinking water only, Cr-treated group was treated with hexavalent chromium at a dose of 10 mg/kg b.w./day for a period of thirty days and oleanolic acid
supplemented group received chromium at the mentioned dose and OA at a dose of 5 mg/kg b.w./day orally for last two weeks of chromium exposure.
Fig. 1: Protective effect of oleanolic acid on chromium induced
decreased MDH enzyme activity in mice brain. Values were
expressed as Means±SEM Each bar represents mean value of
data from six number of mice. Asterisk indicates significant
difference level. pa compared with control group, pb compared
with chromium treated group. ***considered statistically highly
significant (p<0.001)
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partially (9.6% counteraction, p<0.05). Pyruvic acid content was
significantly decreased in brain tissue by 63.41% (p<0.01) due to Cr
(VI) toxicity (Table 2). Oleanolic acid supplementation moderately
counteracted (50% of the control value) chromium induced decrease
in brain pyruvic acid content. It was further noted that Cr (VI) exposure
increased the level of free amino nitrogen by almost three folds
(p<0.001); GPT and GOT activities were increased by 52.2% (p<0.001)
and 45% (p<0.001), respectively, in chromium exposed brain tissue
(Table 2). Administration of oleanolic acid in chromium-treated mice
appreciably restored the increased free amino acid nitrogen content as
well as transaminase enzyme activities toward normalcy.
Protein carbonylation was an important marker of the oxidative
damage of proteins. In the present study, carbonylated protein content
was significantly elevated after chromium treatment (Fig. 4). The
increase was recorded as 49.5% (p<0.01) of the control value. OA
supplementation partially restored the level of carbonylated proteins in
chromium-exposed mice brain. The restoration was found to be 23.1%
(p<0.05).
Different proteolytic enzyme activities of the brain tissue of mice were
significantly varied in response to the Cr (VI) toxicity (Fig. 5). Pronase,
cathepsin, and trypsin activities were decreased by 32.05% (p<0.01),
18.8% (p<0.05), and 58% (p<0.001), respectively after chromium
exposure. Oleanolic acid supplementation counteracted chromium-
induced change in pronase activity by 83%, cathepsin activity by 100%
and trypsin activity by 81% of the respective control values.
Cellular antioxidant status revealed that hexavalent chromium exposure
abruptly affected both enzymatic and non-enzymatic antioxidants after
scheduled duration of exposure (Table 3). It was revealed that brain
glutathione content was decreased by 41.15% (p<0.001) by hexavalent
chromium exposure. Oleanolic acid supplementation restored the
decreased GSH content by 55% in respect of the Cr (VI)-treated group.
Chromium exposure significantly increased (almost 99%, p<0.001)
the GSSG content in brain tissue of mice, which was restored by
28% (p<0.05) by oleanolic acid supplementation. Additionally, total
glutathione/oxidized glutathione ratio (TG: GSSG) was remarkably
decreased (47.3%, p<0.001) in chromium exposed mice brain.
Oleanolic acid supplementation partially restored the TG: GSSG ratio
(76% restoration) toward the control value. Chromium exposure
caused significant elevation of lipid peroxidation in cerebral tissue of
mice. The elevation was recorded as 55.8% in comparison to the control
value. Oleanolic acid appreciably checked chromium-induced increased
Table 2: Protective effect of oleanolic acid (OA) on Cr (VI)-induced alteration in brain total protein content, pyruvic acid level, free amino
acid nitrogen (FAAN) content and transaminase enzyme (GOT and GPT) activities
Groups of animals (N=18) Total protein
(g/100g tissue)
Pyruvic acid (µg/g tissue) FAAN (mg/g tissue) GOT activity (U/g tissue) GPT activity
(U/g tissue)
Control (n=6) 9.22±0.15 198.26±6.35 0.98±0.07 80.5±2.63 36.14±3.87
Cr-treated (n=6) 11.45±0.18p* 72.54±4.32pa** 2.96±0.34pa*** 117±4.34pa*** 55±1.88pa***
OA-Supplemented (n=6) 10.35±0.382pb* 100.12±2.36pb*** 1.65±0.08pb*** 93.83±1.42pb** 42.93±1.76pb***
Figures in the parentheses indicate number of animals in each group (n=6). N represents total number of animals. Values are expressed as Means±SEM. pa compared to
control group and pb compared with Cr-treated group. *p<0.05; **p<0.01 and ***p<0.001 are considered statistically significant. Control group received drinking water
only, Cr-treated group was treated with hexavalent chromium at a dose of 10 mg/kg b.w./day for 30 days and oleanolic acid supplemented group received chromium at
the mentioned dose and OA at a dose of 5 mg/kg b.w./day orally for past 2 weeks of chromium exposure.
Fig. 2: Counteractive effect of oleanolic acid on chromium
induced decrease in SDH enzyme activity in mice brain. Values
are expressed as Means±SEM. Each bar represents mean value
of data from six numbers of mice. Asterisk indicates significant
difference level. pa compared with control group, pb compared
with chromium treated group. ***Considered statistically highly
significant (p<0.001).
Fig. 4: Counteractive effect of OA against Cr (VI) induced elevated
carbonylated protein content in brain tissue of mice. Values
were expressed as Means±SEM. Each bar represents mean value
of data from six numbers of mice. Asterisk indicates significant
difference. pa compared with control group, pb compared
with chromium treated group. *and **Considered statistically
significant (p<0.05, p<0.01, respectively)
Fig. 3: Effect of Cr (VI) on mitochondrial Complex 1 (NADH
dehydrogenase) activity in brain tissue of mice with or without
oleanolic acid supplementation. Values were expressed as
Means±SEM. Each bar represents mean value of data from six
numbers of mice. Asterisk indicates significant difference. pa
compared with control group, pb compared with chromium
treated group. ***Considered statistically highly significant
(p<0.001) and **indicated p<0.01
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Pal
lipid peroxides in mice brain. The counteraction was found to be 24%
(p<0.01). Free hydroxyl radical production was markedly increased in
chromium intoxicated mice brain (118%, p<0.001). Oleanolic acid also
restored the free hydroxyl radical toward normalcy (Table 3).
Moreover, both the catalase and SOD activities were inhibited in mice brain
after exposure to hexavalent chromium (Table 4). The inhibition was
noted as 26.78% (p<0.01) in case of the catalase, and 42% (p<0.001) in
case of the SOD activity. Oleanolic acid supplementation showed significant
protective effect in restoration of these two potential antioxidant enzyme
activities toward their respective control values. Other than these, GR and
GPx activities were also significantly inhibited by hexavalent chromium
exposure at the present dose and duration. The decrease was recorded
as 54% (p<0.001) and 37.7% (p<0.01). Oleanolic acid supplementation
partially counteracted both of the GR and GPx activities toward normalcy.
DISCUSSION
Significant disorientation in metabolic homeostasis in the brain tissue
of mice was observed after 30 days of hexavalent chromium exposure at
sub-acute dose. Cr (VI), being a non-biodegradable toxicant, penetrates
the organisms through inhalation, drinking water and incidental
ingestion and thus affects crucial bio-molecules and enzymatic
functions [44]. In the present study, Cr (VI) treatment appeared to have
no significant effect on the body weight of experimental mice, indicating
that gain in the body weight was independent of chromium exposure at
the present dose and duration, but moderate increase in cerebro-somatic
index (CSI) was observed during post-sacrifice examination. This might
be due to over accumulation of elemental chromium in the brain tissue of
mice as evidenced by the present study. Bio-magnification of Cr generally
occurs in the organisms by occupational, environmental or accidental
exposure [45] that accounts for the toxic effect on the specific tissue of
exposed animals. Metabolic perturbation is proposed to be one of the
toxic manifestations of hexavalent chromium which may contribute
to functional abnormalities of the cell. The present study elucidated
significant alteration of glycolytic and TCA cycle intermediates as well as
certain protein metabolites after chromium exposure and also signified
the role of oleanolic acid, a plant-based pentacyclic triterpenoid, in
protection of that metabolic imbalance caused by hexavalent chromium.
A remarkable decrease in pyruvic acid content in mice brain was noted
after hexavalent chromium treatment, which might be due to less
supply of glucose to the cerebral tissue via circulation during chromium
toxicity. This is supported by the fact that chromium exposure at that
dose and duration produced hypoglycemia [20] that might aid less
substrate like glucose to the brain tissue for its metabolism. Adjustment
of the bio-energetic fluxes in different tissues might result from
malfunctioning of those proteins directly involved in the metabolic
pathways or due to altered substrate availability in those tissues [46].
Within the cellular environment, hexavalent chromium may be reduced
to trivalent Cr-ATP complex, the later acts as a competitive inhibitor
for various ATP dependent enzymes and several kinases involved in
glycolysis as well as TCA cycle [46,47]. Consequently, this might slow
down the rate of glycolysis, leading to less production of pyruvic acid.
This is supported by the earlier observation where decreased glycolytic
activity in hepatic tissue was observed after chromium exposure [20]. It
was further suggested that glucagon hormone, secreted in response to
hypoglycemia, inhibited hepatic L-isozyme of the pyruvate kinase thus
induced retardation of glycolytic activity in that tissue [47]. Similar
explanation may also be suggested for chromium-induced retardation
of brain glycolytic activity.
Table 3: Counteractive effect of OA on Cr (VI) induced alteration in reduced glutathione (GSH) and oxidized glutathione (GSSG) contents,
total glutathione/oxidized glutathione (TG: GSSG) ratio, lipid peroxidation (LPO) level and free hydroxyl radical generation in mice
brain
Groups of animals (n=18) GSH (µmoles/
mg protein)
GSSG (µmoles/
mg protein)
TG: GSSG ratio LPO (nmoles MDA/g tissue) Free OH radical
(µmoles/g tissue)
Control (n=6) 59.86±1.51 29.62±0.97 3.02±0.05 20.13±0.62 12.4±0.68
Cr-treated (n=6) 35.23±1.53pa*** 58.93±0.84pa*** 1.59±0.03pa*** 31.37±0.98pa*** 27.08±0.73pa***
OA-Supplemented (n=6) 54.61±1.29pb*** 42.3±1.46pb* 2.29±0.06pb*** 23.73±0.85pb** 19.03±0.79pb***
Figures in the parentheses indicate number of animals in each group (n=6). N represents total number of animals. Values are expressed as Means±SEM. pa compared to
control group and pb compared with Cr-treated group. *p<0.05; **p<0.01 and ***p<0.001 are considered statistically significant. Control group received drinking water
only, Cr-treated group was treated with hexavalent chromium at a dose of 10mg/kg b.w./day for a period of thirty days and oleanolic acid supplemented group received
chromium at the mentioned dose and OA at a dose of 5 mg/kg b.w./day orally for last two weeks of chromium exposure.
Table 4: Protective effect of OA on Cr (VI) induced changes in catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR)
and glutathione peroxidase (GPx) activities in mice brain
Groups of animals
(n=18)
CAT activity (µmoles H2O2
hydrolyzed/min/mg protein)
SOD activity
(U/mg protein)
GR activity (nmoles NADPH
oxidized/min/mg protein)
GPx activity
Control (n=6) 118.98±1.65 2.96±0.08 99.55±1.75 117.07±2.12
Cr-treated (n=6) 87.11±1.61pa** 1.72±0.051pa*** 45.75±1.63pa*** 72.97±1.86pa**
OA-Supplemented (n=6) 107.14±2.74pb** 2.27±0.08pb** 73.05±1.5pb** 91.78±1.67pb**
Figures in the parentheses indicate number of animals in each group (n=6). N represents total number of animals. Values are expressed as Means±SEM. pa compared
to control group, pb compared with Cr-treated group. **p<0.01 and ***p<0.001 are considered statistically significant. Control group received drinking water only,
Cr-treated group was treated with hexavalent chromium at a dose of 10mg/kg b.w./day for a period of thirty days and OA supplemented group received chromium at the
mentioned dose and OA at a dose of 5 mg/kg b.w./day orally for last two weeks of chromium exposure.
Fig. 5: Restorative effect of OA against Cr (VI) induced decreased
brain proteolytic enzyme activities. Values were expressed as
Means±SEM. Each bar represents mean value of data from six
numbers of mice. Asterisk indicated significant difference. pa
compared with control group, pb compared with chromium
treated group. **Represented p<0.01 and *, p<0.05
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On the other hand, hexavalent chromium exposure at the present dose
and duration exhibited significant effect on brain TCA cycle enzyme
activities. The MDH activity in the brain tissue of mice was significantly
decreased after Cr (VI) exposure. This enzyme not only helps in
providing energy through TCA cycle but also aids in gluconeogenesis
to produce glucose from the non-carbohydrate source [48]. Suppressed
mitochondrial MDH activity in brain tissue by Cr (VI) indicated slow
metabolic conversion of malate to oxaloacetate in the TCA cycle, thus
paid to low energy state in the cerebral tissue in consequence of toxic
manifestation [49]. Inadequate reimbursement by fermentation and
inhibition of cellular respiration by hexavalent Cr (VI) perturbed the
nucleotide pool and ultimately obliterated the energy homeostasis in
Cr (VI) intoxicated organs [49]. In addition, reduced glucose supply
to brain tissue might also contribute to less production of glycolytic
intermediates like pyruvic acid resulting in impairment of energy
yielding mechanism and consequent brain dysfunctions after Cr
(VI) intoxication. Moreover, mitochondrial dehydrogenases such
as NADH dehydrogenase (mitochondrial complex I) and succinate
dehydrogenase (mitochondrial complex II) were significantly inhibited
by hexavalent chromium at the present dose and duration. These
might lead to exhaustion of NADH pool in the stressed tissue [50] and
caused subsequent deterioration of mitochondrial energy production.
Consequently, energy deficient brain tissue might proceed through the
anaerobic process following Cr (VI) treatment. That impairment of the
SDH activity by Cr (VI) was also evident from the earlier observation in
hepatic tissue of mice [20,51].
Tissue protein content and proteolytic enzyme activities such as
pronase, cathepsin, and trypsin were declined in cerebral tissue due to
Cr (VI) toxicity. Reduction of protein in other biological samples such
as plasma by sub-acute dose of hexavalent chromium was reported
earlier [52]. Protein depletion in the tissue designated for physiological
adjustment in response to the changed metabolic situation that might
lead to stimulation of proteolysis and utilization of protein degradation
products for energy metabolism. Protein depletion might also be due to
oxidative degeneration of native proteins by heavy metals or metalloids
like chromium. This is supported by the earlier study of Das and Pal [53],
which revealed that lead (Pb) caused enhancement of carbonylated
protein content within hepatic and skeleto-muscular tissue, thereby
modulated protein metabolic efficacy of those tissues. Similar
observation was also noted in the present study where chromium
exposure significantly elevated carbonylated protein content in the
brain tissue of mice. In addition, heavy metals also induce abnormal
glycosylation of tissue proteins [54]. Uncharacterized glycosylated
or post-translated proteins lose their ability to achieve programmed
function of cellular integrity [55] and may thereby encourage tissue
metabolic disorders. Breakdown of tissue protein in consequence of
hexavalent chromium toxicity increased the free amino acid nitrogen
content in other tissues too [56]. The present study further established
that the sub-acute Cr (VI) exposure elevated free amino nitrogen content
in the cerebral tissue significantly. That increased level of free amino
acid nitrogen might be due to mobilization of free amino acids through
the circulation to meet the demand of energy or to provide substrates
for the synthesis of new proteins to cope up with the repairmen of
intoxicated nervous tissue due to Cr (VI) toxicity. The present study is in
conformity with the previous report of Shil and Pal [56], which revealed
a remarkable decrease in total protein content with increased activities
of transaminases such as GOT and GPT in hepatic and muscular tissue
of Cr (VI) treated animals. Increased availability of substrates like free
amino acids might contribute to enhanced transaminase activities to
promote gluconeogenesis to compensate hypoglycemia as suggested
by earlier reporters [56]. Moreover, increased transaminase activities
indicated degenerative changes in the brain tissue of mice which might
promote leakage of intracellular enzymes and other metabolites.
The present study further depicted that the activities of important
proteases such as trypsin, cathepsin, and pronase in mice brain were
decreased after sub-acute Cr (VI) exposure. Cathepsin and pronase
are the vital lysosomal proteolytic enzymes that accomplish protein
degradation and facilitate enrichment of amino acid pool in various
tissues for maintaining crucial metabolic attributes such as cellular
repair, energy generation, and utilization of degraded products. It was
already established that hepatic and muscular proteins and proteolytic
enzymes were sensitive to hexavalent chromium poisoning [56]. Cr
(VI), being a toxic metalloid, was widely suspected to impose organ
toxicity, genotoxicity, chromosomal aberration, mutational changes
and DNA-DNA cross strand, which might prevent enzyme synthesis
or might enhance depletion of metabolic intermediates from the
respective tissue [57]. Less availability of specific substrates in the
experimental tissue might be one of the causative factors of Cr (VI)
induced retardation of proteolytic enzyme activities. Alteration in
physicochemical properties of proteins might be involved in excess
production of reactive oxygen species, and Cr (VI), being a free radical
generator [10], encouraged oxidative stress mechanism. These in turn
might cause reduced level of desired tissue proteins for proteolytic
enzyme actions. Enhanced formation of carbonylated proteins in
the cerebral tissue of mice might explain oxidative stress mediated
damage of functional proteins, and its less availability for normal tissue
metabolic activities. Thus, short-term exposure of Cr (VI) exhibited
significant impact on brain metabolic activities by modulating certain
important enzymes and basic parameters of protein metabolism.
In addition, hexavalent chromium had been reported to modify
oxidative stress parameters in different organ systems [58,59]. It
was found to reduce brain glutathione (GSH) content in association
with enhanced oxidized glutathione (GSSG) level, lipid peroxide and
free hydroxyl radical formation at the present dose and duration.
Within the cellular environment, hexavalent chromium was reduced
to trivalent form by different reducing agents; GSH was one of
them [60]. So, Cr (VI) used to utilize GSH for its own metabolism and
thereby decreased intracellular GSH pool. During that process free
hydroxyl radicals were generated which might lead to DNA strand
break [60]. As glutathione normally helps in protection of tissue from
oxidative damage, depletion of this endogenous antioxidant promotes
oxidative stress and damages cellular macromolecules such as DNA,
protein, and lipids. Over production of free hydroxyl radicals was also
found in the current experimental setup after hexavalent chromium
intoxication, which might contribute to damage of cellular proteins
and lipids. Elevation of brain tissue oxidized glutathione (GSSG), an
indicative marker of oxidative stress, was pronounced in the chromium
exposed animals (Table 3). This might be due to decreased activities of
glutathione reductase and glutathione peroxidase in the experimental
tissue of mice after hexavalent chromium exposure. As a result, the
total glutathione and oxidized glutathione ratio (TG: GSSG) was also
significantly decreased in chromium-treated mice brain, indicating
imbalance in production and deposition of reactive oxygen species
(ROS). Malondialdehyde, a marker of lipid peroxidation was more in
concentration in the Cr (VI)-treated mice, as observed in the present
study. This is in conformity with the earlier reports [58,61]. Brain
tissue, due to its high content of polyunsaturated fatty acids and high
oxygen consumption, is susceptible to oxidative stress, and thus pay
to tissue degenerative changes. In addition, hexavalent chromium was
found to inhibit certain antioxidant enzyme activities such as catalase,
SOD, glutathione reductase and glutathione peroxidase at the present
dose and duration. Changes in antioxidant enzymes in different animal
cells by hexavalent chromium were observed in earlier studies [62,63].
Inhibition of those antioxidant enzymes by chromium increased the
chance for generation of superoxide radicals and hydrogen peroxide
which promoted oxidative brain tissue damage.
To minimize the adverse effects of hexavalent chromium, several
natural and synthetic antioxidants had been tried. Among them plant
polyphenols gained attention for the decades due to their potential
therapeutic effects without having major side effects. Oxidative
stress and apoptotic changes in lung epithelial cells by hexavalent
chromium were prevented by apple juice [64]. It was reported that
quercetin, a dietary flavonoid as a protective agent against Cr (VI)
mediated malignant cell transformation and ROS production [65].
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Phytochemicals from Eugenia dysenterica leaf extract was found to
have antioxidant property against Cr (VI)-induced oxidative stress
and cytotoxicity [66]. Moreover, chemo-preventive effect of green
tea extract against Cr (VI)-induced genotoxicity was also evaluated
earlier [67]. In addition, curcumin, rutin, alpha-tocopherol, vitamin
C, N-acetylcysteine, ginger, and vitamin E were reported to have
chemo-preventive properties against Cr (VI)-induced genotoxicity
or carcinogenicity [68]. The present study further evaluated the
protective efficacy of oleanolic acid, isolated from N. wightiana, a
medicinal plant collected from the South district of Tripura, North East
India against hexavalent chromium toxicity. This chemical compound
is a triterpenoid having antioxidant property. OA supplementation in
Cr (VI)-treated mice appreciably counteracted chromium-induced
decreased pyruvic acid content, elevated free amino acid nitrogen and
transaminase enzyme activities such as GOT and GPT. It was found that
OA partially checked over deposition of elemental chromium within the
brain tissue of mice and thus moderately reduced the cerebro-somatic
index. A healthy tissue can fight better against any toxicant mediated
damage and give protection to diseases. Beneficial effects of oleanolic
acid against sodium fluoride-induced brain metabolic dysfunctions and
nucleic acid depletion were investigated earlier [13]. OA was found to
have almost similar efficacy as vitamin C in restoration of DNA and RNA
contents and free amino acid nitrogen level in different brain regions
of rat as evidenced by their studies. Moreover, antioxidant power of
OA was found to be comparable with other commercial antioxidants
by using ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-
picrylhydrazyl (DPPH) and lipid peroxidation inhibition assays [69].
It was further observed that OA appreciably counteracted the
decreased TCA cycle enzyme activities such as MDH, SDH as well as
mitochondrial NADH dehydrogenase enzyme function. These might
help in energizing the brain cells of mice to restore its normal metabolic
functioning that was threatened by hexavalent chromium. These might
be due to protective effect of OA on antioxidant defence system of the
brain tissue and certain drug metabolizing enzymes that neutralize
the harmful effects of chromium on the experimental animals. This is
supported by the earlier observation [70], which revealed appreciable
beneficial effect of OA against ethanol-induced hepatotoxicity by
stimulating certain antioxidant and drug metabolizing enzymes. In
addition, neuroprotective functions of this triterpenoid were evaluated
in the frontal cortex and hippocampus of mice brain in terms of release
of brain derived neurotropic factor and few neurotransmitters [71].
OA, being an antioxidant, exhibited significant amelioration against
hexavalent chromium-induced alteration in oxidative damage of
proteins to reduce the elevated level of carbonylated protein content
in chromium exposed cerebral tissue of mice. Earlier investigation
revealed remarkable protective effect of oleanolic acid pre-treatment
to check lipid peroxidation and to stimulate antioxidant enzyme
functions that were disturbed by ischemic injury [72]. The present
study further exhibits that depleted reduced glutathione content was
restored appreciably, whereas increased oxidized glutathione content,
free hydroxyl radical production and malonaldehyde level were
counteracted by OA supplementation, indicating potential antioxidant
efficacy of OA against hexavalent chromium induced oxidative stress.
In addition, decreased total glutathione/oxidized glutathione ratio
by chromium was also partially restored by OA supplementation.
Moreover, the inhibitory effects of hexavalent chromium on potential
antioxidant enzyme activities such as SOD, catalase, glutathione
reductase, and glutathione peroxidase were partially withdrawn by OA
supplementation at the present dose and duration. These observations
are in conformity with the several earlier studies where OA was used
as an ethno-medicine to minimize the complications of oxidative
injury mediated diseases [73]. An in vitro study established increased
the production of glutathione and the expression of key antioxidant
enzymes by OA [74]. Antioxidative properties of OA in different brain
regions of rats were evaluated earlier against sodium fluoride-induced
oxidative and metabolic dysfunctions [13]. Such protective effects of OA
might also be helpful in restoration of suppressed proteolytic enzyme
activities like pronase, trypsin and cathepsin following hexavalent
chromium exposure. Partial restoration of total protein content in mice
brain by OA was co-operative in maintaining the substrate availability
for normal functioning of the proteases. Thus, OA supplementation
exhibited significant counteractive effects against Cr (VI) induced
metabolic and oxidative dysfunctions in mice brain.
CONCLUSION
The current observation was made on ethno-medicinal efficacy of
oleanolic acid, a pentacyclic triterpenoid, isolated from N. wightiana,
against hexavalent chromium induced brain metabolic and oxidative
stress. Cr (VI) exposure at sub-acute dose for a period of thirty days
produced diminution of pyruvic acid content in brain tissue indicating
retardation of glycolytic activity in that tissue. In addition, Cr (VI)
exposure significantly altered the TCA cycle enzyme activities through
suppression of MDH, SDH, and NADH dehydrogenase activities showing
impairment of energy production through mitochondrial respiratory
chain. Moreover, protein depletion was also an important adverse effect
of Cr (VI) toxicity. Alteration in proteolytic enzyme activities such as
trypsin, cathepsin, and pronase was in accordance with the availability
of their specific substrates. Enhanced transamination was associated
with excess production of free amino acid nitrogen in chromium
stressed brain tissue. It is suggested that a compensatory adjustment in
between carbohydrate and protein metabolites was initiated after short-
term chromium exposure that may aid glucose from non-carbohydrate
source such as protein to meet the demand of energy in Cr (VI) stressed
brain tissue. In addition, perturbation of endogenous antioxidants and
antioxidant enzymes along with over production of lipid peroxides and
free hydroxyl radicals is the underlying mechanism of oxidative stress,
induced by hexavalent chromium toxicity. Oleanolic acid appreciably
checked chromium-induced metabolic and oxidative anomalies and
restored crucial metabolites and enzyme activities including MDH, SDH,
NADH dehydrogenase, transaminases, proteases, SOD, catalase, GR and
GPx toward normalcy. Thus, OA may serve as a prospective protective
agent against hexavalent chromium induced metabolic and oxidative
dysfunctions in mice brain.
ACKNOWLEDGMENT
The author acknowledges the Co-ordinator, State Biotech Hub, Tripura
University for providing instrumental facility to carry out the present
work. I am thankful to the H.O.D, department of Human Physiology to
use the laboratory and chemicals from the departmental facility. The
author is also thankful to the Chairman, Pollution Control Board, Tripura
for giving permission to use the atomic absorption spectrophotometer
for metal detection. I am also thankful to Dr. Kanu Shil for his support in
partial evaluation of data.
AUTHOR’S CONTRIBUTION
The author has made substantial contribution to carry out the present
work. The concept, design of the study, evaluation of raw data and whole
write up of the manuscript are made by the author in her own capacity.
CONFLICT OF INTEREST
There is no conflict of interest.
AUTHOR’S FUNDING
The present work is self-financed. No funding agency is involved in the
present work.
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