Hindawi
Journal of Toxicology
Volume 2020, Article ID 8859716, 17 pages
https://doi.org/10.1155/2020/8859716
Research Article
Clerodendrum volubile Ethanol Leaf Extract: A Potential
Antidote to Doxorubicin-Induced Cardiotoxicity in Rats
Olufunke Esan Olorundare,1 Adejuwon Adewale Adeneye ,2 Akinyele Olubiyi Akinsola,1
Daniel Ayodele Sanni,3 Mamoru Koketsu ,4 and Hasan Mukhtar5
1
Department of Pharmacology and Therapeutics, Faculty of Basic Medical Sciences, College of Health Sciences,
University of Ilorin, Ilorin, Kwara, Nigeria
2
Department of Pharmacology, Therapeutics and Toxicology, Faculty of Basic Clinical Sciences,
Lagos State University College of Medicine, 1-5 Oba Akinjobi Way, G.R.A., Ikeja, Lagos, Nigeria
3
Department of Pathology and Forensic Medicine, Faculty of Basic Clinical Sciences, Lagos State University College of Medicine,
1-5 Oba Akinjobi Way, G.R.A., Ikeja, Lagos, Nigeria
4
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido,
Gifu 501-1193, Japan
5
Department of Dermatology, University of Wisconsin-Madison, Medical Science Center, 1300 University Avenue, Madison,
WI 53706, USA
Correspondence should be addressed to Adejuwon Adewale Adeneye; adeneye2001@yahoo.com
Received 5 March 2020; Accepted 11 June 2020; Published 4 July 2020
Academic Editor: You-Cheng Hseu
Copyright © 2020 Olufunke Esan Olorundare et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Doxorubicin is widely applied in hematological and solid tumor treatment but limited by its off-target cardiotoxicity. Thus,
cardioprotective potential and mechanism(s) of CVE in DOX-induced cardiotoxicity were investigated using cardiac and oxidative stress markers and histopathological endpoints. 50–400 mg/kg/day CVE in 5% DMSO in distilled water were investigated
in Wistar rats intraperitoneally injected with 2.5 mg/kg DOX on alternate days for 14 days, using serum troponin I and LDH,
complete lipid profile, cardiac tissue oxidative stress marker assays, and histopathological examination of DOX-treated cardiac
tissue. Preliminary qualitative and quantitative assays of CVE’s secondary metabolites were also conducted. Phytochemical
analyses revealed the presence of flavonoids (34.79 ± 0.37 mg/100 mg dry extract), alkaloids (36.73 ± 0.27 mg/100 mg dry extract),
reducing sugars (07.78 ± 0.09 mg/100 mg dry extract), and cardiac glycosides (24.55 ± 0.12 mg/100 mg dry extract). 50–400 mg/kg/
day CVE significantly attenuated increases in the serum LDH and troponin I levels. Similarly, the CVE dose unrelatedly decreased
serum TG and VLDL-c levels without significant alterations in the serum TC, HDL-c, and LDL-c levels. Also, CVE profoundly
attenuated alterations in the cardiac tissue oxidative stress markers’ activities while improving DOX-associated cardiac histological lesions that were possibly mediated via free radical scavenging and/or antioxidant mechanisms. Overall, CVE may play a
significant therapeutic role in the management of DOX-induced cardiotoxicity in humans.
1. Introduction
Drug-induced cardiotoxicity remains the most widely
studied of drug-induced organ toxicities [1]. Several approved drugs and agrochemicals have been implicated in
these toxicities, including acetaminophen, gentamicin, rifampicin, carbon tetrachloride, and antineoplastic drugs [2].
Thus, accidental, acute overdose, or chronic use of some of
these drugs could result in multiorgan toxicities including
hepatotoxicity, nephrotoxicity, cardiotoxicity, and testicular/ovarian toxicity. These specific organs are known to be
mostly susceptible and are organs of metabolism and excretion of these drugs [3]. Despite extensive studies of these
organ toxicities, definitive therapeutic/prophylactic options
2
at ameliorating the deleterious effects of these drugs are still
limited. In view of this, research into the etiopathology and
development of alternative or complementary options from
traditional medicine is currently been encouraged [4]. Also,
the World Health Organization reported that over 80% of
the world population depends partly or wholly on medicinal
plant-derived pharmaceuticals [5].
Doxorubicin (DOX) is a broad-spectrum antibiotic
anthracycline with wide application in the clinical chemotherapeutic management of solid tumors such as breast,
lung, ovarian, and uterine cancers and leukemias [6].
Despite its incontrovertible efficacy, DOX is notorious for
its life-threatening toxicity profile such as neurotoxicity,
hepatotoxicity, hematotoxicity, and cardiotoxicity, thus
limiting its clinical use in cancer treatment. Life-threatening cardiomyopathy represents the cumulative doselimiting toxicity of the drug. DOX toxicities are majorly
attributed to the interplay of oxidative stress and free
radical formation from its highly reactive and toxic secondary metabolites [7].
In African traditional medicine, several medicinal plants
are reputed for effectively ameliorating the deleterious effects of drug poisoning on organs such as the liver and the
kidneys. These medicinal plants (in whole or parts) include
Phyllanthus amarus, Harungana madagascariensis, Carica
papaya leaves, Zingiber officinale rhizome, Vernonia
amygdalina leaves, and Garcinia kola seeds [8, 9]. However,
the folkloric therapeutic claims of some of these plants have
been scientifically verified and reported. For example, the
protective activities of various plant extracts of Phyllanthus
amarus [10–12], Harungana madagascariensis [13], Carica
papaya leaves [14, 15], Zingiber officinale rhizome [16–19],
Vernonia amygdalina leaves [20, 21], and Garcinia kola seeds
[22, 23] against drug-induced hepato- and nephrotoxicities
are well documented. Similarly, several studies have equally
reported ameliorative effects of medicinal plants against
drug-induced cardiotoxicity [24–28].
Clerodendrum volubile P. Beauv, popularly known as
white butterfly, belongs to the Lamiaceae family. In Nigeria,
the plant is locally known as “Marugbo” or “Eweta” among
the Ikale, Ilaje, and Apoi people of Ondo State (Southwest
Nigeria) and “Obnettete” among the Itsekiri and Urhobo
tribes (Niger Delta area of Nigeria) where it is often combined with other vegetables as a condiment and spice to
improve taste and aroma [29]. Clerodendrum volubile is
indigenous and ubiquitous to the riverine belts of the West
African tropical rainforest of Nigeria, Benin Republic,
Ghana, Cameroon, Burkina Faso, and Sierra Leone where it
is also grown as an ornamental plant [30, 31]. Due to its high
nutritive and ethnopharmacological value, Clerodendrum
volubile whole plant and its parts are used in the African
ethnomedicine in the local management of joint pains and
swellings, diabetes mellitus, gastric ulcer, obesity and hyperlipidemia, hypertension, and other heart diseases and
dropsy [31, 32]. However, some of these folkloric claims have
been scientifically validated and have been attributed to its
high polyphenol content such as ajugoside, pectolinarigenin,
protocatechuic acid, biochanin, and 5, 7, 4′-trimethoxykaempferol [31]. Clerodendrum volubile has also been
Journal of Toxicology
reported to exhibit potential role in the management of
human breast cancer by inhibiting cell cycle phases, especially the G0/G1 phase, cell proliferation, and decreasing the
expression of matrix metalloproteinase 9 enzymes [33], as
well as the ability of its extracts to scavenge free radicals,
especially reactive oxygen species, generated by human
breast cancer cell lines in vitro [33] and its antiproliferative
activity against prostate cancer cells [34]. Considering the
wide application and historical use of different plant parts of
Clerodendrum volubile in the traditional management of
heart diseases, the reported role of reactive oxidative stress in
the etiopathogenesis of doxorubicin-related heart disease,
and paucity of scientific validation of the plant use in the
management of drug-related heart diseases including druginduced cardiotoxicity, the present exploratory study is,
therefore, designed at evaluating the possible antidotal
potential of 100–400 mg/kg of the Clerodendrum volubile
ethanol leaf extract (CVE) in doxorubicin- (DOX-) induced
cardiotoxic rats for 14 days. In doing this, reliable cardiac
injury biomarkers (serum troponin I and LDH), indices of
cardiovascular disease, as well as histopathological studies of
CVE-DOX-treated cardiac muscles, were evaluated. In addition, the effect of CVE on SOD, CAT, GSH, GPx, GST, and
MAD activities in the DOX-treated cardiac tissues was
evaluated.
2. Materials and Methods
2.1. Plant Materials. Stock of fresh mature whole plants of
Clerodendrum volubile was purchased from Herbal Vendors
in the Isikan Market in Akure, Ondo State, Nigeria, in the
month of March 2019. Samples of the plant obtained were
subjected to botanical identification and referencing at the
University of Ilorin (UNILORIN) Herbarium, with the
voucher specimen number UILH/01/019/1254 allotted.
2.2. Extraction Process. Fresh leaves of Clerodendrum volubile were destalked from the whole plant and then gently
but thoroughly rinsed under running tap water and completely air-dried at the room temperature (28–33°C) until the
weight of the dried leaves was constant. The dried leaves
were then pulverized using the milling machine and kept in a
water- and air-tight container.
1.50 kg of the pulverized leaves was completely macerated in 8 liters of absolute ethanol at room temperature
for 5 days but intermittently shaken to ensure complete
dissolution. Thereafter, the solution was first filtered with
cotton wool and then with the 110 mm Whatman filter
paper. The resultant filtrate was then concentrated in vacuo
using a rotary evaporator (BUCHI Rotavapor Model
R-215, Switzerland) with the Vacuum Module V-801
EasyVac , Switzerland) set at a revolution of 70 rpm and a
temperature at 36°C before it was completely dried over a
waterbath preset at 40°C. The jelly-like, dark-colored residue left behind was weighed and stored in an air- and
water-proof container which was kept in a refrigerator at
4°C. From this stock, fresh solutions were made whenever
required.
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Journal of Toxicology
3
% yield �
weight of the crude extract obtained(g)
× 100.
weight of the starting pulverized dry leaf extracted (g)
2.3. Preliminary Qualitative Phytochemical Analysis. The
presence of saponins, tannins, alkaloids, flavonoids, anthraquinones, glycosides, and reducing sugars was detected
by the simple and standard qualitative and quantitative
methods described by Trease and Evans [35] and Sofowora
[36].
2.4. Quantitative Determination of Secondary Metabolites
in CVE
2.4.1. Preparation of Fat-Free Sample. 2 g of CVE was exhaustively defatted with 100 ml of diethyl ether using the
method earlier described by Edeoga et al. [37].
2.4.2. Total Phenol Quantification. The total phenolic content in CVE was spectrophotometrically determined using
the procedure previously described by Edeoga et al. [37].
2.4.3. Alkaloid Quantification. The alkaloid content of CVE
was determined using the method earlier described by
Harborne [38]. 5 g of CVE was weighed into a 250 ml beaker,
and 200 ml of 10% acetic acid in ethanol was added. The
resulting solution was covered and allowed to stand for 4
hours, filtered, and then the filtrate was concentrated on a
waterbath to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop-wise to the
concentrate until the precipitation was complete. The whole
solution was allowed to settle, and the precipitate was collected and rinsed with dilute ammonium hydroxide and
then filtered. The residue is the alkaloid, which was dried and
weighed.
2.4.4. Tannin Quantification. Tannin content of CVE was
estimated using the method by Van-Burden and Robinson
[39]. 500 mg of defatted CVE was weighed into a 50 ml
plastic bottle. 50 ml of distilled water was added and shaken
continuously for 1 hour on a mechanical shaker. This was
filtered into a 50 ml volumetric flask and made up to the
mark. Following this, 5 ml of the filtrate was pipetted out
into a test tube and mixed with 2 ml of 0.1 M FeCl3 in 0.1 N
HCl and 0.008 M potassium ferrocyanide. The absorbance of
the resulting mixture was measured at 120 nm within
10 min.
2.4.5. Saponin Quantification. Saponin was estimated by the
method previously used by Obadoni and Ochuko [40]. 2 g of
CVE was placed into a conical flask, and 10 ml of 20%
aqueous ethanol was added. The resulting mixture was
heated over a hot waterbath for 4 hours under continuous
stirring at 55°C. The mixture was filtered, and the residue was
re-extracted with another 20 ml of 20% ethanol. The combined filtrate was reduced to 4 ml over a waterbath at 90°C.
The concentrate was transferred into a 50 ml separating
(1)
funnel, and 2 ml of diethyl ether was added and shaken
vigorously. The aqueous layer was recovered, while the ether
layer was discarded. The extraction process was repeated one
more time, and n-butanol was added to the combined
aqueous portion. The resulting mixture was shaken and
washed twice with 1 ml of 5% aqueous sodium chloride and
filtered, while the resulting solution was heated over a
waterbath. After evaporation, the samples were dried in the
oven to a constant weight, and the saponin content was
calculated as the percentage of the extract.
2.4.6. Reducing Sugar Quantification. Reducing sugar
content in CVE was determined using the spectrophotometric method as described by Shaffer and Somogyi [41].
2.4.7. Quantitative Determination of Total Flavonoid Content
in CVE. Total flavonoids in CVE were estimated using the
method by Ordonez et al. [42]. To 1 ml of crude CVE,
equivalent 1 ml of 2% aluminum chloride in the ethanol
solution was added. After 1 hour of incubation at room
temperature (28°C) for color development, the absorbance
was measured at 420 nm using the Unico 2100 spectrophotometer (United Products and Instruments Inc.,
Shanghai, China). A golden yellow color indicated the
presence of flavonoids. Total flavonoid contents were calculated as the rutin hydrate (minimum 98%; Sigma
Chemicals Co., St. Louis, MO, USA) equivalent using the
mathematical equation described by Ordonez et al. [42].
®
2.4.8. Determination of Total Proanthocyanidin Contents in
CVE. Total proanthocyanidin (tannin) content in CVE was
estimated by the method of Sun et al. [43]. 0.5 ml of 50 mg/l
of the extract was mixed in 3 ml of 4% vanillin-methanol
solution and 1.5 ml of concentrated hydrochloric acid, and
the mixture was allowed to stand for 15 minutes at room
temperature (28°C) for color development. The absorbance
was measured at 500 nm using the Unico 2100 spectrophotometer (United Products and Instruments Inc.,
Shanghai, China). Total proanthocyanidin contents were
calculated as the catechin hydrate (minimum 98%) (Sigma
Chemicals Co., St. Louis, MO, USA) equivalent (mg/g) using
the mathematical equation described by Sun et al. [43].
®
2.4.9. Determination of Total Phenols in CVE. Total phenol
content in CVE was determined by the modified
Folin–Ciocalteu method of Wolfe et al. [44]. An aliquot of
each of CVE was mixed with 2.5 ml of the Folin–Ciocalteu
reagent (previously diluted with distilled water, 1 : 10 v/v)
and 2 ml of (75 g/l) of sodium carbonate. The tubes were
vortexed for 15 seconds and allowed to stand for 30 minutes
at 40°C for color development. Absorbance was measured at
765 nm using the Unico 2100 spectrophotometer (United
Products and Instruments Inc., Shanghai, China). This
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4
Journal of Toxicology
procedure was replicated thrice. Total phenolic content was
calculated and expressed as the mg/g rutin equivalent as
earlier described by Wolfe et al. [44].
2.5. In Vitro Antioxidant Profiling of CVE
2.5.1. Determination of DPPH-Scavenging Activity of CVE.
The effect of CVE was estimated using the method by LiyanaPathirana and Shahidi [45]. A solution of 0.135 mM 1, 1diphenyl-2-picrylhydrazyl (DPPH) (Sigma Aldrich, St.
Louis, USA) in methanol was prepared, and 1.0 ml of this
solution was mixed with 1.0 ml of methanol containing
0.2–1.0 mg of each extract. The reaction mixture was
vortexed thoroughly and left in the dark at room temperature for 30 min. The spectrophotometric absorbance of
the mixture was measured at 517 nm. The reference drug,
vitamin C (Sigma Chemicals Co., St. Louis, USA), used was
equally prepared at the same concentration, and the experiment was conducted in triplicate. The ability to scavenge the DPPH radical was calculated by the following
equation:
DPPH radical − scavenging activity (%) �
where Abscontrol � absorbance of DPPH radical + methanol
and Abssample � absorbance of DPPH radical + sample extract/standard.
2.5.2. Determination of Superoxide Anion and Nitric OxideScavenging Activities of CVE. Superoxide anion and nitric
oxide-scavenging activities of CVE were evaluated using the
method by Sreejayan and Rao [46]. In both assaying
methods, quercetin was used as the standard drug.
2.6. Spectral Studies of Secondary Metabolites in CVE Using
Gas Chromatography-Mass Spectrometry. GC-MS analysis was performed using a 7820A gas chromatograph
coupled to a 5975C inert mass spectrometer (with triple
axis detector) and an electron-impact source (Agilent
Technologies, Santa Clara, CA 95051, USA). 0.5 g of CVE
was suspended in ethanol to make a concentration of
100 mg/ml (w/v), followed by filtration through Varian
Bond Elute C18 solid-phase extraction to remove impurities. The stationary phase of separation of the compounds
was carried out on a HP-5 capillary column coated with 5%
of phenyl methyl siloxane (30 m length × 0.32 mm
diameter × 0.25 μm film thickness) (Agilent Technologies,
Santa Clara, CA 95051, USA). The carrier gas used was GCgrade helium (99.999% purity) at a constant flow rate of
1.573 ml/min, an initial nominal pressure of 1.9514 psi, and
at an average velocity of 46 cm/s. One microliter (1 μl) of
the samples was injected in the split-less mode at an injection temperature of 260°C. Purge flow was 21.5 ml/min
at 0.50 min with a total gas flow rate of 23.355 ml/min; the
gas saver mode was switched on. The oven was initially
programmed at 60°C (1 min) and then ramped at 4°C/min
to 110°C (3 min), followed by temperature program rates of
8°C/min to 260°C (5 min) and 10°C/min to 300°C (12 min).
Run time was 56.25 min with a 3 min solvent delay. The
mass spectrometer was operated in the electron-impact
ionization mode at 70 eV with an ion source temperature of
230°C, quadrupole temperature of 150°C, and transfer line
temperature of 280°C. The mass spectrophotometer
AbScontrol − AbSsample
× 100,
AbScontrol
(2)
conditions are solvent delay of 3.00 min, gain factor of 1.00,
and resulting EM voltage of 1859, and scanning of possible
compounds was from m/z 30 to 550 amu at a 2.62 s/scan
rate. Using computer searches on a National Institute
Standard and Technology (NIST) 14 Mass Spectral Database and the Mass Spectral Search Program (Version 2.2)
and comparing, the spectrum obtained through GC-MS
compounds present in the CVE was identified. The spectrum of the unknown components was compared with the
spectrum of known components stored in the NIST library
to ascertain its chemical identity [47, 48].
2.7. Experimental Animals. Young adult male Wistar albino
rats (aged 8–12 weeks and body weight: 130–190 g) used in
this study were obtained from the Animal House of the
Lagos State University College of Medicine, Ikeja, Lagos
State, Nigeria, after an ethical approval (Protocol Identification Code: UERC/BMS/134 and UERC Approval Number:
UERC/ASN/2019/1703) was obtained from the University of
Ilorin Ethical Review Committee for Postgraduate Research.
The rats were handled in accordance with international
principles guiding the Use and Handling of Experimental
Animals [49]. The rats were maintained on standard rat feed
(Ladokun Feeds, Ibadan, Oyo State, Nigeria) and potable
water which were made available ad libitum. The rats were
maintained at an ambient temperature between 28 and 30°C,
humidity of 55 ± 5%, and standard (natural) photoperiod of
approximately 12/12 hours of alternating light and dark
periodicity.
2.8. Measurement of Body Weight. The body weights of rats
were taken on days 1 and 14 of the experiment and determined on a digital rodent weighing scale ( Virgo Electronic Compact Scale, New Delhi, India). The obtained
values were expressed in grams (g).
®
2.9. Induction of DOX-Induced Cardiotoxicity and Treatment
of Rats. Prior to commencement of the experiment, rats
were randomly allotted into 8 groups of 7 rats per group such
Journal of Toxicology
that the weight difference between and within groups was
not more than ±20% of the average weight of the sample
population of rats used for the study. However, the choice of
the therapeutic dose range of 100, 200, and 400 mg/kg/day of
CVE was made based on the result of the preliminary studies
conducted.
In this experimental repeated dose model which lasted
for 14 days, Groups I rats which served as the untreated
control were orally pretreated with 10 ml/kg/day of distilled
water but equally treated with 2.5 mg/kg of doxorubicin
hydrochloride ( Celondoxily Injection 50, CELON Laboratories PVT. Limited, Gajularamaram, Ranga Reddy District-500 090, Telangana State, India) dissolved in 0.9%
normal saline administered on alternate days for 14 days.
Group II rats were orally treated with 200 mg/kg/day of CVE
dissolved in 5% DMSO distilled water (CVE being only
partly soluble in water, DMSO an organosulfur polar aprotic
and inert solvent that readily dissolves both polar and
nonpolar compounds was used) but treated with 1 ml/kg of
0.9% normal saline administered intraperitoneally on alternate days for 14 days. Group III–Group VI rats were
orally pretreated with 50 mg/kg/day, 100 mg/kg/day,
200 mg/kg/day, and 400 mg/kg/day of CVE dissolved in 5%
DMSO distilled water 3 hours before treatment with 2.5 mg/
kg of doxorubicin in 0.9% normal saline administered intraperitoneally on alternate days for 14 days, respectively.
Group VII rats which served as the positive control group
were equally pretreated with 20 mg/kg/day of Vitamin C 3
hours before treatment with 2.5 mg/kg of doxorubicin in
0.9% normal saline administered intraperitoneally on alternate days for 14 days. Group VIII rats were the untreated
normal rats and were orally pretreated with 10 ml/kg/day of
distilled water 3 hours before treatment with 2.5 mg/kg of
doxorubicin in 0.9% normal saline administered intraperitoneally on alternate days for 14 days (Table 1) [50, 51]. The
choice of vitamin C was made because it is a standard
antioxidant agent, and its effect as a positive control was
compared with other treatment groups.
®
2.10. Blood Sample Collection. On the 14th day which was the
last day of the experiment, the rats were weighed and later
fasted overnight but drinking water was made available ad
libitum. Rats were sacrificed, and whole blood samples were
collected directly from the heart under inhaled diethyl ether
anesthesia. Blood samples were carefully collected with the
fine 21G Needle and 5 ml Syringe (Hangzhou Longde
Medical Products Co. Ltd., Hangzhou, China) without
causing damage to the heart tissues. The rats’ heart, liver,
kidneys, and testes were identified, harvested, and weighed.
2.11. Biochemical Assays. Blood samples obtained directly
from the heart chamber were allowed to clot and then
centrifuged at 5000 rpm to separate clear sera from the
clotted blood samples. The clear samples were obtained for
assays of the following biochemical parameters: serum
cardiac troponin I, LDH, TG, TC, and cholesterol fractions
(HDL-c, LDL-c, and VLDL-c). Serum lipids were assayed
5
using methods by Tietz [52], while serum troponin I and
LDH were estimated by standard bioassay procedures.
2.12. Calculation of AI and CRI. AI was calculated as LDL-c
(mg/dl) ÷ HDL-c (mg/dl) [53], while CRI was calculated as
TC (mg/dl) ÷ HDL-c (mg/dl) [54].
2.13. Determination of Antioxidant Activities in the Rat
Cardiac Tissues. After the rats were sacrificed humanely
under inhaled diethyl ether, the heart was harvested en bloc.
The heart was gently and carefully divided into two halves
(each consisting of the atrium and ventricle) using a new
surgical blade. The left half of the heart was briskly rinsed in
ice cold 1.15% KCl solution in order to preserve the oxidative
enzyme activities of the heart before being placed in a clean
sample bottle which itself was in an ice-pack-filled cooler.
This is to prevent the breakdown of the oxidative stress
enzymes in these organs.
2.13.1. Determination of SOD Activities in the Heart Tissues.
Superoxide dismutase activity was determined by its ability
to inhibit the auto-oxidation of epinephrine by the increase
in absorbance at 480 nm as described by Paoletti et al. [55].
Enzyme activity was calculated by measuring the change in
absorbance at 480 nm for 5 minutes.
2.13.2. Determination of CAT Activities in the Heart Tissues.
Tissue CAT activities were determined by the method described by Hadwan [56]. The specific activity of CAT was
expressed as U/ml.
2.13.3. Determination of GSH, GPx, and GST Activities in the
Heart Tissues. The reduced glutathione (GSH) content in
the heart tissue was estimated according to the method
described by Rahman et al. [57]. To the homogenate, 10%
TCA was added and centrifuged. One millilitre of the supernatant was treated with 0.5 ml of Elman’s reagent
(19.8 mg of 5, 5-dithiobisnitro benzoic acid (DTNB) in
100 ml of 0.1% sodium nitrate) and 3.0 ml of phosphate
buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm.
Similarly, GPx and GST activities were determined using the
method by Faraji et al. [58] and Vontas et al. [59].
2.13.4. Determination of MDA Activities in the Heart Tissues.
Method by Buege and Aust [60] was adopted in determining
MDA activities in the cardiac tissue. One millilitre of the
supernatant was added to 2 ml of (1 : 1 : 1 ratio) the TCATBA-HCl reagent (thiobarbituric acid 0.37%, 0.24 N HCl,
and 15% TCA) boiled at 100°C for 15 minutes and allowed to
cool. Flocculent material was removed by centrifuging at
3000 rpm for ten minutes. The supernatant was removed,
and the absorbance was read at 532 nm against a blank.
MDA was calculated using the molar extinction for the
MDATBA-complex of 1.56 × 105 m−1·cm−1.
6
Journal of Toxicology
Table 1: Group treatment of rats.
Groups
Group
Group
Group
Group
Group
Group
Group
Treatments
10 ml/kg of distilled water p.o. for 14 days + 2.5 mg/kg of doxorubicin hydrochloride in 0.9% normal saline given i.p. on
I
alternate days for 14 days
200 mg/kg/day of Clerodendrum volubile ethanol leaf extract in 5% DMSO-distilled water p.o. for 14 days + 1 ml/kg of 0.9%
II
normal saline given i.p. on alternate days for 14 days
50 mg/kg/day of Clerodendrum volubile ethanol leaf extract in 5% DMSO-distilled water p.o. for 14 days + 2.5 mg/kg of
III
doxorubicin hydrochloride in 0.9% normal saline given i.p. on alternate days for 14 days
100 mg/kg/day of Clerodendrum volubile ethanol leaf extract in 5% DMSO-distilled water p.o. for 14 days + 2.5 mg/kg of
IV
doxorubicin hydrochloride in 0.9% normal saline given i.p. on alternate days for 14 days
200 mg/kg/day of Clerodendrum volubile ethanol leaf extract in 5% DMSO-distilled water p.o. for 14 days + 2.5 mg/kg of
V
doxorubicin hydrochloride in 0.9% normal saline given i.p. on alternate days for 14 days
400 mg/kg/day of Clerodendrum volubile ethanol leaf extract in 5% DMSO-distilled water p.o. for 14 days + 2.5 mg/kg of
VI
doxorubicin hydrochloride in 0.9% normal saline given i.p. on alternate days for 14 days
20 mg/kg/day of vitamin C in distilled water p.o. for 14 days + 2.5 mg/kg of doxorubicin hydrochloride in 0.9% normal saline
VII
given i.p. on alternate days for 14 days
Group
VIII
10 ml/kg/day of distilled water p.o. for 14 days + 1 ml/kg of 0.9% normal saline given i.p
2.13.5. Histopathological Studies of the Heart. Using the
remaining equally divided harvested heart, the right halves
of the six randomly selected rats from each treatment and
control groups were subjected to histopathological examinations, with the right ventricle being the most susceptible to
doxorubicin toxicity of the heart chambers. After rinsing in
normal saline, the dissected right half was preserved in 10%
formo-saline before it was completely dehydrated in absolute (100%) ethanol. It was then embedded in routine
paraffin blocks. From the embedded paraffin blocks, 4-5 μm
thick sections of the tissue were prepared and stained with
the hematoxylin-eosin stain. These were examined under a
photomicroscope (Model N-400 ME, CEL-TECH Diagnostics, Hamburg, Germany) connected with a host computer. Sections were illuminated with white light from a 12 V
halogen lamp (100 W) after filtering with a 520 nm monochromatic filter. The slides were examined for associated
histopathological lesions [61].
2.14. Statistical Analysis. Data were presented as mean± SEM of four and seven observations for the in vitro and in
vivo studies, respectively. Statistical analysis was done using
two-way analysis of variance, followed by a post hoc test,
Student–Newman–Keuls test, on GraphPad Prism Version
5. Statistical significance was considered at p < 0.05, p < 0.01,
and p < 0.001.
3. Results
3.1. %yield. Complete extraction of the pulverized dry leaf
sample of Clerodendrum volubile in absolute ethanol was
calculated to be 8.39%. The resultant residue was a dark
color, sticky, jelly-like, and sweet-smelling (bland) residue
which was not completely soluble in water but completely
soluble in methanol and ethanol.
3.2. Qualitative Phytochemical Analysis of CVE.
Phytochemical analysis of CVE showed the presence of
flavonoids, alkaloids, reducing sugars, and cardiac
glycosides, while saponins, tannins, phenols, phlobatannins,
steroids, and terpenoids were absent.
3.3. Quantitative Analysis of CVE. Table 2 shows the estimates of each of the secondary metabolites present in 100 mg
of CVE. As indicated in Table 2, CVE contains flavonoids
(34.79 ± 0.37 mg/100 mg of the dry extract), alkaloids
(36.73 ± 0.27 mg/100 mg of the dry extract), reducing sugars
(07.78 ± 0.09 mg/100 mg of the dry extract), and cardiac
glycosides (24.55 ± 0.12 mg/100 mg of the dry extract).
3.4. Spectral Studies of Secondary Metabolites in CVE Using
Gas Chromatography-Mass Spectrometry. Figure 1 depicts
the presence and relative abundance of thirty (30) different
secondary metabolites in CVE obtained through gas chromatography-mass spectrometry, while the relatively abundant
secondary metabolites present in CVE obtained through the
phytoscan based on the CAS Library search include 9,12,15octadecatrienoic acid (otherwise known as) (Z,Z,Z)-9,12,15octadecatrienoic acid and (Z,Z,Z)-9,12,15-octadecatrienal
(9.02%); urea, triethyl-urea (5.74%); 7-octadecenoic acid
methyl ester and 9-octadecenoic acid methyl ester (5.61%);
n-hexadecanoic acid (5.39%); ethyl-α-d-glucopyranoside,
ethyl-β-d-glucopyranoside, and methyl-β-d-arabinopyranoside (5.13%); 9,12,15-octadecatrienoic acid and (Z,Z,Z)-ethyl9,12,15-octadecatrienoate
(4.08%);
phytol
(3.28%);
hexadecanoic acid, methyl ester (3.25%); methyl tetradecanoate (2.18%); glycine, N,N-dimethyl-, methyl ester and N,Ndimethyl-3-methoxypropylamine (2.16%); hexadecanoic acid,
ethyl ester (2.03%); benzoic acid, 4-methoxy-benzoic acid,
and 4-methoxy-benzoic acid 4-methoxy-(1.77%); 4-acetylanisole and 3-methoxyacetophenone (1.56%); 6-hydroxy4,4,7α-trimethyl-5,6,7,7α-tetrahydrobenzofuran-2(4H)-one
(1.36%); guaifenesin and 2-cyclohexen-1-one, 4-hydroxy3,5, 6-trimethyl-4-(3-oxo-1-butenyl)-(1.32%); phthalic acid,
di(2-propylpentyl) ester, phthalic acid, di(oct-3-yl) ester,
and diisooctyl phthalate (1.19%); n-propyl 9,12,15-octadecatrienoate and 7,10,13-hexadecatrienoic acid (1.02%)
(Table 3).
Journal of Toxicology
7
Table 2: Quantitative analysis of the secondary metabolites in CVE
(mg/100 mg of the dry extract sample).
Secondary metabolite
Flavonoids
Alkaloids
Reducing sugars
Cardiac glycosides
Quantity (mg/100 mg of dry extract)
34.79 ± 0.37
36.73 ± 0.27
07.78 ± 0.09
24.55 ± 0.12
3.5. In Vitro Antioxidant Profiling of CVE
3.5.1. Determination of DPPH-Scavenging Activity of CVE.
Table 4 shows the in vitro DPPH-scavenging activities of
25 μg/ml, 50 μg/ml, 70 μg/ml, and 100 μg/ml of CVE in
comparison with those of corresponding doses of the
standard antioxidant drug (vitamin C) used. As shown, the
DPPH-scavenging activities of the extract were significantly (p < 0.001) dose related and comparable to those of
vitamin C.
3.5.2. Determination of NO-Scavenging Activity of CVE.
Table 5 shows the in vitro NO scavenging activities of 25 μg/
ml, 50 μg/ml, 70 μg/ml, and 100 μg/ml of CVE in comparison
with those of corresponding doses of the standard antioxidant drug (vitamin C). As shown, NO-scavenging activities
of the extract were significantly (p < 0.001) dose related and
comparable to those of vitamin C.
3.5.3. Determination of FRAP of CVE. Table 6 shows the in
vitro ferric-reducing activity power of 25 μg/ml, 50 μg/ml,
70 μg/ml, and 100 μg/ml of CVE in comparison with those of
corresponding doses of the standard antioxidant drug. As
shown, the FRAP activities of the extract were significantly
(p < 0.001) dose dependent and comparable to those of the
standard drug, vitamin C.
3.6. Effect of CVE on the Average Body Weight of DOX-Treated
Rats. Table 7 shows the effect of repeated intraperitoneal
DOX treatment and oral treatments with 50, 100, 200, and
400 mg/kg/day of CVE on the average body weight on day
1 and day 15 as well as percentage weight change (%∆wt.)
of rats repeatedly treated with doxorubicin and CVE for 14
days. As shown in Table 7, repeated intraperitoneal DOX
treatment was associated with significant and profound
(p < 0.0001) reduction in the weight gain pattern in the
DOX only treated (Group I) rats when compared to
untreated control (normal) rats (Group VIII). With repeated oral treatment of 50, 100, and 200 mg/kg/day of
CVE, there was a significant (p < 0.05 and p < 0.0001)
dose-dependent attenuation in the weight loss pattern in
the extract-treated rats (Table 7). Vitamin C also had a
similar effect on the weight gain pattern in the DOXtreated rats (Table 7).
3.7. Effect of CVE on the Cardiac Tissue Oxidative Stress
Markers (GSH, GST, GPx, SOD, CAT, and MDA) of DOXTreated Rats. Repeated intraperitoneal injection of DOX to
rats was associated with significant decreases (p < 0.05 and
p < 0.0001) in the activities of SOD, CAT, GPx, GST, and
GSH levels while significantly increasing (p < 0.001) MDA
activities (Table 8). However, repeated oral treatment with
CVE significantly (p < 0.05 and p < 0.0001) attenuated the
alterations in the activities of these enzyme markers in the
cardiac tissue (Table 8).
3.8. Effect of CVE on Cardiac Marker Enzymes (LDH and
Troponin I) of DOX-Treated Rats. Repeated intraperitoneal
injection of DOX resulted in significant increases (p < 0.05
and p < 0.0001) in the serum LDH and troponin I levels
when compared to that of untreated negative (control)
values (Table 9). However, oral pretreatments with
50–400 mg/kg/day of CVE significantly attenuated (p < 0.05
and p < 0.0001) increases in the serum LDH and troponin I
levels (Table 9). Oral pretreatment with 20 mg/kg/day of
vitamin C caused similar effects as CVE on the serum LDH
and troponin I (Table 9).
3.9. Effect of CVE on the Serum Lipid (TG, TC, HDL-c, LDL-c,
and VLDL-c) Level of DOX-Treated Rats. Repeated intraperitoneal DOX injections resulted in significant
(p < 0.0001) increases in the serum TG and VLDL-c
concentrations, while there were no significant alterations
in the serum HDL-c and LDL-c concentrations in the
treated rats when compared to those of untreated rats
(Table 10). However, in rats repeatedly pretreated with
50–400 mg/kg/day of CVE orally, there were significant
(p < 0.05 and p < 0.0001) dose-unrelated decreases in the
serum concentration of TG and VLDL-c without significant (p > 0.05) alterations in the serum concentration of
HDL-c and LDL-c when compared to DOX only treated
rats (Table 10). It is also noteworthy that vitamin C had
similar pattern of effects on the measured serum lipid
parameters (Table 10).
3.10. Effect of CVE on Cardiovascular Risk Indices (AI and
CRI) of DOX-Treated Rats. Repeated intraperitoneal injections with DOX resulted in a significant (p < 0.05)
increase in the CRI value, while they had no significant
effect on AI (Table 11). However, with pretreatment with
50–400 mg/kg/day of CVE orally, there were further
significant (p < 0.05) dose-unrelated decreases in the CRI
value without significant alterations in the AI values
(Table 11). Oral pretreatments with 20 mg/kg/day of vitamin C had a similar pattern on the AI and CRI as
exhibited by CVE (Table 11).
3.11. Histopathological Studies of the Effect of CVE on DOXTreated Heart. Figure 2 is a microphotograph of a crosssectional representative of DOX only treated heart
showing myocyte congestion with scanty pyknotic and
predominant hyperchromatic and meganuclei with interstitial fibrosis, suggestive of myocardial hypertrophy
when compared to normal untreated heart tissue with
normal cardiac architecture (Figure 3). However,
8
Journal of Toxicology
TIC: sample 1.D\data.ms
1.4e + 07
1.3e + 07
1.2e + 07
1.1e + 07
1e + 07
Abundance
90,00,000
80,00,000
70,00,000
60,00,000
50,00,000
40,00,000
30,00,000
20,00,000
10,00,000
4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
Time
Figure 1: Mass spectrometry of the Clerodendrum volubile ethanol leaf extract (CVE) indicating molecular weights of each of the secondary
metabolites and their relative abundance.
pretreatment with varying doses of CVE resulted in doserelated improvements in the histological distortions induced by DOX, especially at 200 mg/kg/day (Figure 4) and
400 mg/kg/day of CVE (Figure 5). On the contrary, there
were histological features of mild congestion and scattered cardiac myocyte necrosis in rats pretreated with
20 mg/kg/day of vitamin C, suggesting persisting histological lesions induced by doxorubicin treatment even in
the face of treatment with the standard antioxidant, vitamin C (Figure 6).
4. Discussion
DOX is a 14-hydroxylated congener of daunorubicin (the immediate biosynthetic DOX precursor) which acts by inhibiting
the progression of topoisomerase II, an enzyme which relaxes
supercoils in DNA for transcription, therefore inducing DNA
double-strand breaks, especially in rapidly dividing cells, and
resulting in DNA synthesis disruption [62]. DOX is known to
accumulate majorly in the liver, the kidneys, and the heart [63].
However, the heart is highly susceptible to doxorubicin toxicity
because of the high mitochondria-to-cardiomyocyte ratio,
making it more susceptible to oxidative damage especially from
semiquinone-type free radicals generated from DOX hepatic
metabolism [63, 64]. In addition, the heart has a low regenerative
capacity, compared to other body organs [63, 65]. These limiting
factors make the heart susceptible to DOX-induced off-target
cardiotoxicity, which is notoriously mediated by its highly reactive primary alcohol metabolite, doxorubicinol [66], leading to
overwhelming oxidative stress and a compromised endogenous
antioxidant system [67, 68]. The ensuing oxidative stress affects
the lysosomes, microfibrils, mitochondria, and the sarcoplasmic
reticulum [69], resulting in increased apoptotic cardiac myocytes
and eventual cardiac cell damage [70]. The resulting cardiac
damage is indicated by profound increases in the circulating
specific and reliable cardiac biomarkers such as CK-MB, AST,
Mb, IMA, PBNP, GPBB, troponin I, and LDH [71, 72]. Of these
cardiac biomarkers, cardiotoxicity in this study was reliably
measured using troponin I and LDH assays. Thus, the profound
elevation in the serum activities of cardiac troponin I and LDH
levels following doxorubicin treatment is a strong indication that
DOX-induced cardiotoxicity was reliably established. The literature has it that doxorubicin induces its toxicity through
generation of oxidative stress resulting in lipoperoxidation of the
cardiac muscle with the ultimate leakage of troponin I and LDH
into the serum [71–73]. Thus, our result is in complete agreement with reports of earlier studies by Al-Harthi et al. [73] and
Ammar El-Sayed et al. [74]. The DOX-related biochemical
changes were further corroborated by the histological features of
congested myocytes with scanty pyknotic and predominant
Journal of Toxicology
9
Table 3: Quantitative analysis of the secondary metabolites (PhytoScan) of the Clerodendrum volubile ethanol leaf extract (CVE) using gas
chromatography-mass spectrometry.
Pk#
RT
Area (%)
(1)
3.601
2.16
(2)
7.464
5.74
(3)
8.579
0.77
(4)
9.878
1.56
(5)
10.895
1.77
(6)
12.691
0.50
(7)
13.113
5.13
(8)
(9)
(10)
(11)
13.528
13.684
13.887
14.089
2.18
0.95
0.81
1.36
(12)
14.268
1.32
(13)
14.539
0.88
(14)
14.747
0.67
(15)
(16)
(17)
15.278
15.636
15.827
3.25
5.39
2.03
(18)
16.682
5.61
(19)
16.780
3.28
(20)
17.046
9.02
(21)
17.196
4.08
(23)
18.004
0.62
(24)
19.575
0.54
(25)
19.824
1.19
(26)
20.771
1.02
(27)
(28)
(29)
(30)
21.527
22.832
25.137
25.801
0.68
0.55
0.68
0.52
Library/ID ref#
Glycine, N,N-dimethyl-, methyl ester
N,N-Dimethyl-3-methoxypropylamine
2-Methyl-allyl ethyl ether
Dimethylaminomethyl-isopropyl-sulfide
Urea, triethyl-urea
5-Hydroxymethylfurfural
5-Hydroxymethylfurfural
5-Hydroxymethylfurfural
4-Acetylanisole
3-Methoxyacetophenone
Benzoic acid
4-Methoxy-benzoic acid
4-Methoxy-benzoic acid 4-methoxy
Megastigmatrienone
1H-1,5-Benzodiazepine, 2,3,4,5-tetrahydro-2,2,4-trimethyl-phenol
2-(1,1-Dimethyl-2-propenyl)-3,6-dimethylEthyl-α-d-glucopyranoside
Ethyl-β-d-glucopyranoside
Methyl-β-d-arabinopyranoside
Methyl tetradecanoate
Germacyclopentane
Tetradecanoic acid
6-Hydroxy-4,4,7α-trimethyl-5,6,7,7 α-tetrahydrobenzofuran-2(4H)-one
Guaifenesin
2-Cyclohexen-1-one, 4-hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl)
Neophytadiene
Bicyclo[3.1.1]heptane
2,6,6-Trimethyl-, (1.alpha.,2.beta.,5.alpha.)9-octadecyne
9-Octadecen-1-ol
(Z)-6-Octen-1-ol, 3,7-dimethyl
Formate-6-octen-1-ol,3,7-dimethyl-, formate
Hexadecanoic acid, methyl ester
n-Hexadecanoic acid
Hexadecanoic acid, ethyl ester
7-Octadecenoic acid, methyl ester
9-Octadecenoic acid, methyl ester
(E)-9-Octadecenoic acid, methyl ester
Phytol
9,12,15-Octadecatrienoic acid
(Z,Z,Z)-9,12,15-Octadecatrienoic acid
(Z,Z,Z)-9,12,15-Octadecatrienal
9,12,15-Octadecatrienoic acid
(Z,Z,Z)-Ethyl 9,12,15-octadecatrienoate
9,12,15-Octadecatrienoic acid
Ethyl-9-hexadecenoate
Cyclopentadecanone, 2-hydroxyHexadecanoic acid
2-Hydroxy-1-(hydroxymethyl)ethyl ester
Phthalic acid, di(2-propylpentyl)ester
Phthalic acid, di(oct-3-yl) ester
Diisooctyl phthalate
n-Propyl 9,12,15-octadecatrienoate
7,10,13-Hexadecatrienoic acid, methyl ester
Methyl (Z)-5,11,14,17-eicosatetraenoate
Squalene
Gamma-tocopherol
2-(4-Fluoro-phenyl)-4-(3-methyl-benzylidene)-4H-oxazol-5-one
4-Dehydroxy-N-(4,5-methylene dioxy-nitrobenzylidene) tyramine
CAS#
8679 007148-06-3
8722 020650-07-1
1759 000557-31-3
14996 077422-33-4
21278 019006-59-8
11338 000067-47-0
11339 000067-47-0
11337 000067-47-0
25100 000100-06-1
25121 000586-37-8
26636 000100-09-4
26633 000100-09-4
26632 000100-09-4
56052 038818-55-2
56033 040358-38-1
56097 092617-73-7
72939 019467-01-7
60076 000709-50-2
35240 005328-63-2
104286 000124-10-7
36709 004554-75-0
91415 000544-63-8
61438 073410-02-3
62883 000093-14-1
85356 077846-84-5
138502 000504-96-1
17424 006876-13-7
111836 035365-59-4
128820 000143-28-2
51056 000105-85-1
51061 000105-85-1
130821 000112-39-0
117419 000057-10-3
144309 000628-97-7
155720 057396-98-2
155758 001937-62-8
155754 001937-62-8
155849 000150-86-7
138418 000463-40-1
138420 000463-40-1
123143 026537-71-3
165643 001191-41-9
165627 1000336-77-4
165642 001191-41-9
142080 054546-22-4
102369 004727-18-8
188252 023470-00-0
188251 023470-00-0
233419 1000377-93-5
233383 1000377-72-3
233361 000131-20-4
179097 1000336-79-4
124916 056554-30-4
177257 059149-01-8
243222 000111-02-4
245804 007616-22-0
140918 1000296-71-2
157264 1000111-66-9
Quality
86
80
50
50
50
93
70
62
94
76
96
95
93
99
70
62
74
53
50
74
53
97
93
72
64
99
55
53
70
70
70
98
99
99
99
99
99
98
99
99
91
99
99
99
89
70
90
87
91
80
74
93
91
90
99
95
55
53
Pk#: peak number, RT: retention time, area%: percentage area covered, library/ID ref#: library/identification number, and CAS#: chemical abstract scheme
number.
10
Journal of Toxicology
Table 4: In vitro DPPH-scavenging activity of 25–100 μg/ml of
CVE and vitamin C.
Graded doses (μg/ml)
25
50
75
100
c
c
CVE
33.41 ± 0.42 47.10 ± 0.63 60.69 ± 0.52 76.25 ± 0.32c
Vit. C 45.05 ± 0.48 56.55 ± 0.96c 70.45 ± 0.48c 89.83 ± 0.36c
Drug
A significant increase at p < 0.001.
c
Table 5: In vitro nitric oxide- (NO-) scavenging activity of
25–100 μg/ml of CVE and vitamin C.
Graded doses (μg/ml)
25
50
75
100
CVE
20.52 ± 0.34 49.32 ± 0.57c 64.97 ± 0.34c 78.57 ± 0.57c
Vit. C 47.38 ± 0.26 62.61 ± 0.10c 71.57 ± 1.32c 84.91 ± 0.53c
Drug
A significant increase at p < 0.001.
c
Table 6: In vitro FRAP activities of 25–100 μg/ml of CVE and
vitamin C.
Drug
CVE
Vit. C
25
0.13 ± 0.00
0.18 ± 0.00
Graded doses (μg/ml)
50
75
0.24 ± 0.00c
0.33 ± 0.00c
0.40 ± 0.00c
0.51 ± 0.00c
100
0.41 ± 0.00
0.66 ± 0.00
A significant increase at p < 0.001.
c
Table 7: Effect of repeated oral treatment with 50–400 mg/kg/day
of CVE on the average body weight of DOX-treated rats.
Group Body wt. on day 1 (g) Body wt. on day 14
I
159.9 ± 15.1
143.1 ± 19.0
II
178.3 ± 18.0
191.1 ± 20.6
III
144.2 ± 15.8
141.5 ± 25.2
IV
163.9 ± 08.0
153.0 ± 11.0
V
154.0 ± 19.6
151.6 ± 23.1
VI
165.2 ± 12.4
147.9 ± 12.8
VII
151.3 ± 12.9
146.0 ± 15.4
VIII
163.8 ± 16.8
187.4 ± 20.2
% ∆wt.
−10.7 ± 04.7c−
07.1 ± 04.1c+
−02.4 ± 10.5a+
−06.6 ± 04.8a+
09.0 ± 05.5c+
−10.6 ± 05.8
−03.5 ± 04.5a+
14.5 ± 06.2
A significant decrease at p < 0.0001 when compared to the untreated (normal)
negative control (Group VIII). c+A significant increase at p < 0.0001 when
compared to the untreated (DOX-treated) negative control (Group I). a+A
significant increase at p < 0.05 when compared to the untreated (DOX-treated
only) negative control (Group I).
c−
hyperchromatic and meganuclei with interstitial fibrosis, suggestive of myocardial hypertrophy. Again, this is in complete
agreement with the report by Kwatra et al. [67]. However,
profound attenuation in serum levels of troponin I and LDH
following oral pretreatment with 50–400 mg/kg/day of CVE is a
clear demonstration of the protective potential of the extract
against DOX-associated cardiotoxicity. Similarly, attenuations in
the serum troponin I and LDH were also corroborated by the
significant improvement in the histological architecture of the
CVE-treated heart muscle especially at the oral dose of 400 mg/
kg/day of CVE which showed no remarkable histological lesions
in the CVE-treated heart.
Another notable finding in this current study is that the
increased activities of the oxidative markers induced following
doxorubicin treatment evident from marked increases in lipid
peroxidation products (MDA) depleted reduced GSH levels
and decreased SOD and CAT activities. GSH is considered to
be the most important intracellular antioxidant system that is
utilized for the inactivation of lipid peroxides through the
activity of GPx, which generates GSSG as a byproduct [75]. It
also plays a vital role in conjugating GST, which detoxifies the
reactive substances of lipid peroxidation and other xenobiotics
[76]. Therefore, GSH depletion leads to loss of cellular integrity
and damage to macromolecules such as membrane lipids and
also accumulation of its oxidized form (GSSG) which further
leads to electrical and contractile dysfunction. DOX and its
metabolites cause depletion of the cardiac GSH level, resulting
in the consistent formation of oxygen-free radicals [77].
Similarly, SOD and CATplay a critical role in combating DOXinduced oxidative stress. Superoxide radicals generated at the
site of damage can alter the SOD and CAT levels which may
predispose to accumulation of the superoxide anion and in
turn damage the myocardium. In the present study, it is
suggested that DOX-induced massive free radical production
which utilized the myocardium oxidative machinery that
further leads to reduction in the activities of antioxidant enzymes via inhibition of enzyme protein biosynthesis. These
results are consistent with those previously reported by Li et al.
[78] and Mantawy et al. [79]. The fact that CVE treatment could
significantly reduce the DOX-induced lipid peroxidation, raise
the reduced GSH level, and increase the activities of SOD and
CAT strongly indicates the profound antioxidant property of
the extract which was further corroborated by the results of the
in vitro antioxidant study. Already, the antioxidant and free
radical-scavenging activities of CVE are well documented
[32, 80–82] Again, the literature has shown polyphenols
(consisting of flavonoids, alkaloids, tannins, and anthocyanins)
to freely scavenge free radicals in the body [83–86]. Among the
secondary metabolites identified, the most prevailing compounds which have been reported for eliciting potent antioxidant activities for other plant extracts were tetradecanoic
acid [87], n-hexadecanoic acid and hexadecanoic acid methyl
and ethyl esters [88], phytol [89], and gamma-tocopherol [90].
These antioxidant compounds are known to possess redox
properties through their free radical adsorption and neutralization [91, 92]. However, other reported pharmacological
activities attributable to the identified secondary metabolites
include anti-inflammatory, antimicrobial, cytotoxic, antihyperglycemic, hypocholesterolemic, and antiapoptotic activities [93, 94]. Thus, the presence of these identified secondary
metabolites in high amount as indicated by the results of
quantitative analysis of CVE undertaken in this study could be
responsible for the prominent antioxidant/free radical scavenging and hypocholesterolemic activities, as well as profound
improvement in the cardiovascular disease risk indices
recorded in this study.
Other findings of significance are the profound increases
in the serum TG, TC, and VLDL-c induced by DOX
treatment. These findings are in tandem with those previously reported where prolonged treatment of experimental
rats with DOX resulted in significant increases in the serum
lipids, especially cholesterol and triglycerides [95–97]. The
molecular mechanism of these lipid derangements has been
reported to be mediated via downregulation of PPARc,
Journal of Toxicology
11
Table 8: Antioxidant activities of 50–400 mg/kg/day of CVE in DOX-treated rat cardiac tissue.
Groups
I
II
III
IV
V
VI
VII
VIII
GSH
88.1 ± 5.3c−
62.8 ± 4.9f−
51.2 ± 2.8c−
77.6 ± 5.7
45.6 ± 4.2c−
65.7 ± 4.3c−
112.6 ± 2.3c+
113.5 ± 2.2
GST
20.4 ± 0.1c−
21.4 ± 0.9f−
20.8 ± 0.3f−
30.8 ± 0.7c+
21.5 ± 0.4f−
22.3 ± 0.8f−
21.9 ± 0.3f−
23.6 ± 0.4
Antioxidant parameters
GPx
SOD
81.7 ± 4.9c−
2.0 ± 0.2a−
69.4 ± 5.4
2.2 ± 0.1
51.5 ± 3.1f−
3.3 ± 0.2
81.4 ± 5.4
5.5 ± 1.1c+
f−
50.3 ± 4.6
4.0 ± 0.2a+
64.6 ± 4.0d−
2.8 ± 0.3
111.1 ± 2.3c+
5.3 ± 0.2c+
111.9 ± 2.2
2.5 ± 0.2
CAT
13.3 ± 1.7a−
11.3 ± 0.5
17.9 ± 1.1
35.6 ± 4.1c+
21.8 ± 1.6a+
19.6 ± 0.7
40.2 ± 1.7c+
19.8 ± 1.5
MDA
2.8 ± 0.1
2.1 ± 0.3
3.5 ± 0.4
4.3 ± 0.3b+
3.9 ± 0.2a+
6.4 ± 0.7c+
4.5 ± 0.5b+
2.4 ± 0.2
Significant decreases at p < 0.05 and p < 0.0001, respectively, when compared to the untreated negative (normal) control value. c+A significant increase at
p < 0.0001 when compared to the untreated negative (normal) control values. a+,b+Significant increases at p < 0.05 and p < 0.001, respectively, when compared
to the untreated negative (normal) control values. d−,f−Significant decreases at p < 0.05 and p < 0.0001 when compared to the untreated positive (doxorubicin
treated only) control values, respectively.
a−,c−
Table 9: Effect of 50–400 mg/kg/day of CVE on serum LDH and cardiac troponin I in DOX-intoxicated rats.
Treatment groups
I
II
III
IV
V
VI
VII
VIII
LDH (U/L)
4204 ± 637.1a+
3734 ± 251.5
2781 ± 657.5d–
2939 ± 184.1d–
2530 ± 189.2d–
2214 ± 340.1d–
2907 ± 204.4d–
3331 ± 227.5
Troponin I (ng/ml)
21.18 ± 7.72c+
3.29 ± 2.41f–
7.93 ± 7.03d–
10.63 ± 6.7d–
2.96 ± 1.71f–
3.53 ± 1.84f–
3.46 ± 1.71f–
4.11 ± 2.59
Significant increases at p < 0.05 and p < 0.0001, respectively, when compared to the untreated negative (normal) control values. d−,f−Significant decreases
at p < 0.05 and p < 0.0001, respectively, when compared to the untreated positive (doxorubicin treated only) control values, respectively.
a+,c+
Table 10: Effect of 50–400 mg/kg/day of CVE on the serum lipid profile of DOX-treated rats.
Groups
I
II
III
IV
V
VI
VII
VIII
TG (mmol/l)
1.3 ± 0.1c+
0.8 ± 0.2d−
0.8 ± 0.1d−
1.1 ± 0.1
0.8 ± 0.1d−
1.0 ± 0.1d−
0.9 ± 0.1d−
0.6 ± 0.1
TC (mmol/l)
2.3 ± 0.1a+
1.8 ± 0.2d−
2.3 ± 0.1
2.3 ± 0.1
2.3 ± 0.1
2.3 ± 0.2
2.1 ± 0.1
2.0 ± 0.1
Serum lipids
HDL-c (mmol/l)
0.6 ± 0.0
0.6 ± 0.0
0.7 ± 0.0
0.6 ± 0.0
0.6 ± 0.0
0.6 ± 0.0
0.6 ± 0.0
0.6 ± 0.0
LDL-c (mmol/l)
1.2 ± 0.1
0.9 ± 0.1d−
1.3 ± 0.1
1.2 ± 0.1
1.3 ± 0.1
1.2 ± 0.1
1.1 ± 0.1
1.1 ± 0.0
VLDC-c (mmol/l)
1.3 ± 0.1c+
0.4 ± 0.1f−
0.5 ± 0.1f−
0.5 ± 0.1f−
0.4 ± 0.0f−
0.5 ± 0.1f−
0.4 ± 0.0f−
0.3 ± 0.0
Significant increases at p < 0.5 and p < 0.0001, respectively, when compared to the untreated negative (normal) control value. d−,f−Significant decreases at
p < 0.05 and p < 0.0001 when compared to the untreated positive (doxorubicin treated only) control values, respectively.
a+,c+
Table 11: Effect of 50–400 mg/kg/day of CVE on the atherogenic index (AI) and the coronary artery index (CRI) in DOX-intoxicated rats.
Treatment groups
I
II
III
IV
V
VI
VII
VIII
AI
2.1 ± 0.1
1.6 ± 0.2
2.0 ± 0.2
1.9 ± 0.3
2.1 ± 0.1
2.0 ± 0.1
1.8 ± 0.1
1.1 ± 0.0
CRI
4.0 ± 0.2a+
3.2 ± 0.2e−
3.5 ± 0.2d−
3.9 ± 0.3
3.8 ± 0.1d−
3.8 ± 0.2d−
3.8 ± 0.2d−
3.5 ± 0.1
a+
A significant increase at p < 0.05 when compared to the untreated negative (normal) control values. d−,e−Significant decreases at p < 0.05 and p < 0.01,
respectively, when compared to the untreated positive (doxorubicin treated only) control values, respectively.
12
Figure 2: A cross-sectional representative of a DOX-intoxicated
heart showing congested myocytes with scanty pyknotic and
predominant hyperchromatic and meganuclei with interstitial fibrosis, suggestive of myocardial hypertrophy (×400 magnification,
hematoxylin-eosin stain).
Journal of Toxicology
Figure 5: A photomicrograph of a cross-sectional representative of
a DOX-intoxicated rat heart treated with 400 mg/kg/day CVE
showing near normal myocytes with very scanty meganuclei indicating no remarkable histological changes (×400 magnification,
hematoxylin-eosin stain).
Figure 3: A cross-sectional representative of the normal rat heart
showing normal cardiac architecture (×400 magnification, hematoxylin-eosin stain).
Figure 6: A photomicrograph of a cross-sectional representative of
a doxorubicin-intoxicated rat heart treated with 20 mg/kg/day
vitamin C showing mild congestion and scattered myocyte necrosis
(×400 magnification, hematoxylin-eosin stain).
Figure 4: A cross-sectional representative of a DOX-intoxicated rat
heart treated with 200 mg/kg/day CVE showing mildly congested
myocytes with occasional meganuclei suggestive recovery from
doxorubicin toxicity (×400 magnification, hematoxylin-eosin stain).
mainly the white adipose tissue receptor, which regulates the
expression of glucose and fatty acid transporters and plays a
crucial role in lipid storage and glucose metabolism [98].
Thus, the downregulation of PPARc inhibits blood glucose
and lipid clearance, thereby causing hyperglycemia and
hyperlipidemia [99]. Research studies have equally shown
that derangements in the lipid profile predispose to cardiovascular diseases and increase the risk of major cardiovascular diseases such as ischemic heart disease and
thrombotic stroke [100–103] which was corroborated in the
present study by the profound increased CRI value, which
itself is a reliable indicator of cardiovascular diseases, especially ischemic heart diseases. Drugs have also been reported to cause dyslipidemia-related heart diseases
including doxorubicin [103, 104]. It is equally well documented in the literature that a direct relationship exists
between hypercholesterolemia and hyperlipidemia and
atherosclerosis which is considered as a major cause of
cardiovascular disease, especially coronary heart disease
[105–107]. Thus, drugs including medicinal plants with
Journal of Toxicology
hypolipidemic/antihyperlipidemic activities could equally be
considered antiatherosclerotic and cardioprotective
[108–110]. Thus, the hypolipidemic/antihyperlipidemic activity of CVE observed in this study is in tandem with those
previously reported for Clerodendrum volubile and related
species [32, 80, 111–114]. The fact that CVE profoundly
lowered the serum TG, VLDL-c, and CRI is a further
demonstration of the cardioprotective potential of CVE
against DOX-induced cardiotoxicity and DOX-induced
dyslipidemia which is similar to an earlier report by Kulkarni
and Viswanatha-Swamy [115]. Thus, it is plausible for CVE
to be lowering serum lipids and improving CRI value either
by promoting its clearance from the body or inhibiting its de
novo biosynthesis. Again, the serum lipid-lowering effect of
CVE is attributable to its high polyphenolic content which
has been widely reported to attenuate dyslipidemia in different experimental models [79, 116, 117].
5. Conclusions
Overall, it can be safely concluded that CVE offers protection
in DOX-induced cardiotoxicity that was mediated via free
radical-scavenging activity/antioxidant mechanism and
improvements in the cardiovascular disease risk indices.
Abbreviations
AI:
AST:
CAT:
CK-MB:
CRI:
CVE:
DMSO:
DOX:
DPPH:
DTNB:
GPBB:
GPx:
GSH:
GST:
FRAP:
HCl:
HDL-c:
IMA:
i.p.:
KCl:
LDH:
LDL-c:
MAD:
Mb:
NO:
PBNP:
p.o.:
PPARc:
SEM:
SOD:
TBA:
Atherogenic index
Aspartate transaminase
Catalase
Creatine kinase
Coronary artery index
Clerodendrum volubile ethanol leaf extract
Dimethyl sulfoxide
Doxorubicin
1,1-Diphenyl-2-picrylhydrazyl
5,5-Dithiobisnitro benzoic acid
Glycogen phosphorylase isoenzyme BB
Glutathione peroxidase
Reduced glutathione
Glutathione S-transferase
Ferric-reducing activity power
Hydrochloric acid
High-density lipoprotein cholesterol
Ischemic modified albumin
Intraperitoneal
Potassium chloride
Lactate dehydrogenase
Low-density lipoprotein cholesterol
Malondialdehyde
Myoglobin
Nitric oxide
Probrain natriuretic peptide
Per os
Peroxisome proliferator-activator receptor
gamma
Standard error of the mean
Superoxidase dismutase
Thiobarbituric acid
13
TC:
TCA:
TG:
UNILORIN:
UV:
Vit. C:
VLDL-c:
Total cholesterol
Tricarboxylic acid
Triglyceride
University of Ilorin
Ultraviolet
Vitamin C
Very low-density lipoprotein cholesterol.
Data Availability
The data used to support the study can be available upon
request to the corresponding author.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Authors’ Contributions
Olufunke Esan Olorundare designed the experimental
protocol for this study and was involved in the manuscript
writing; Adejuwon Adewale Adeneye supervised the research, analyzed data, and wrote the manuscript; Akinyele
O. Akinsola is an M.Sc. student in Olufunke Esan Olorundare’s laboratory who performed the laboratory research.
The initial and ongoing work of isolating phytochemical
principles from Clerodendrum volubile was performed in the
laboratory of Mamoru Koketsu; Hasan Mukhtar is the
collaborator in the USA who read through the manuscript.
Acknowledgments
The authors appreciate the technical assistance provided by
the Process Manager, Dr. Obehiaghe A. King, the Laboratory
Manager, Dr. Sarah John-Olabode, and other staff of the
Laboratory Services, AFRIGLOBAL MEDICARE, Mobolaji
Bank Anthony Branch Office, Ikeja, Lagos, Nigeria, in
assaying for the serum cardiac biomarkers and lipid profile.
Similarly, the technical support of staff of the LASUCOM
Animal House, for the care of the Experimental Animals
used for this study, and Mr. Sunday O. Adenekan of
BIOLIFE CONSULTS in the area of oxidative stress marker
analysis is much appreciated. In the same vein, the technical
assistance of Mr. Innocent Okoye, a Senior Technologist in
the Department of Oral Pathology and Medicine, Faculty of
Dentistry, Lagos State University College of Medicine, Ikeja,
Lagos, in the area of preparation of the slide for the histopathological studies is duly acknowledged. This research
was funded by the Tertiary Education Trust Fund (TETFUND) Nigeria, through its National Research Fund
(TETFUND/NRF/UIL/ILORIN/STI/VOL.1/B2.20.12), as a
collaborative research award to both Professors Olufunke
Esan Olorundare and Hasan Mukhtar.
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