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Barleria prionitis L. extracts ameliorate doxorubicin-induced acute
kidney injury via modulation of oxidative stress, inflammation, and
apoptosis
Sachinthi S. Amarasiri
a
, Anoja P. Attanayake
b
,
*
, Liyanage D.A.M. Arawwawala
c
,
Lakmini K.B. Mudduwa
d
, Kamani A.P.W. Jayatilaka
b
a
Department of Medical Laboratory Science, Faculty of Allied Health Sciences, University of Ruhuna, Galle, Sri Lanka
b
Department of Biochemistry, Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka
c
Industrial Technology Institute, Colombo, Sri Lanka
d
Department of Pathology, Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka
article info
Article history:
Received 26 May 2022
Received in revised form
3 May 2023
Accepted 30 May 2023
Available online xxx
Keywords:
Barleria prionitis
Biomarkers
Doxorubicin-induced acute kidney injury
Nephroprotective mechanisms
In vitro antioxidant studies
abstract
Background and aim: Doxorubicin (DOX) is a chemotherapeutic drug with potential nephrotoxic effects
on patients who are on cancer chemotherapy. An interest has been observed in using natural products to
ameliorate the potential side effects of DOX. The present study is to investigate the cellular mechanisms
underlying the protective effects of Barleria prionitis L. (BP) (Acanthaceae) extracts, DOX-induced acute
kidney injury (AKI).
Experimental procedure: Hexane (25 mg/kg/day), ethyl acetate (80 mg/kg/day), n-butanol (70 mg/kg/
day), and water (120 mg/kg/day) extracts of BP, were administered to DOX-induced (5 mg/kg (2500
m
L/
kg), ip) Wistar rats for four consecutive weeks. At the end of the study, investigations were carried out for
the assessment of biomarkers of nephrotoxicity, oxidative stress, inflammation, and apoptosis.
Results: Treatments with BP extracts significantly reversed DOX-induced elevations in serum and urine
biochemical markers of nephrotoxicity (serum creatinine; 21e33%, blood urea nitrogen; 26e58%,
b
2
-
microglobulin; 19e22% and urine total protein; 47e67%). There was a reduction in the levels of tumor
necrosis factor-
a
, interleukin-1
b
, and malondialdehyde in kidney homogenates of rats treated with the n-
butanol extract (by 43, 62, and 24%) and water extract (by 57%, 85%, and 26%) (p <0.05). Immunohis-
tochemical expression of the pro-apoptotic B-cell associated X protein was reduced while the anti-
apoptotic B-cell lymphoma gene product 2 protein was increased in kidney tissues after the treat-
ments with BP extracts.
Conclusions: The selected BP extracts significantly ameliorated DOX-induced AKI. The findings would
open new vistas for the development of a drug using the BP extracts to minimize DOX-induced AKI in
cancer patients.
©2023 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier
Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
1. Introduction
Drug-induced acute kidney injury (AKI) has become a severe
health problem worldwide due to the wide use of pharmaceutical
agents with potential toxicities.
1
The high blood perfusion and the
functions of kidneys in drug metabolism make them highly sus-
ceptible to the direct toxic effects of xenobiotics.
2,3
However, the
clinical manifestations of drug-induced AKI are often unrecognized,
particularly in a short duration of drug exposure. This may cause
issues in the precise assessment of the incidence, severity, and
long-term consequences of kidney damage.
4
AKI extends to the late
diagnosis of kidney failure, particularly after the manifestation of
end-stage renal disease leading to significant mortality.
5
Further-
more, the survivors of AKI appeared to be predisposed to chronic
*Corresponding author. Department of Biochemistry, Faculty of Medicine, Uni-
versity of Ruhuna, Karapitiya, Galle, 80000, Sri Lanka.
E-mail address: anojaattanayake@med.ruh.ac.lk (A.P. Attanayake).
Peer review under responsibility of The Center for Food and Biomolecules,
National Taiwan University.
Contents lists available at ScienceDirect
Journal of Traditional and Complementary Medicine
journal homepage: http://www.elsevier.com/locate/jtcme
https://doi.org/10.1016/j.jtcme.2023.05.007
2225-4110/©2023 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Journal of Traditional and Complementary Medicine xxx (xxxx) xxx
Please cite this article as: S.S. Amarasiri, A.P. Attanayake, L.D.A.M. Arawwawala et al.,Barleria prionitis L. extracts ameliorate doxorubicin-
induced acute kidney injury via modulation of oxidative stress, inflammation, and apoptosis, Journal of Traditional and Complementary
Medicine, https://doi.org/10.1016/j.jtcme.2023.05.007
kidney disease due to maladaptive repair mechanisms following
AKI. A study revealed that about 50% of the patients who survived
AKI, had residual structural and functional defects which might
lead to subsequent episodes of kidney failure or a state of end-stage
renal disease.
6
Several potent therapeutic drugs have been found to exert neph-
rotoxic effects through different pathogenic mechanisms in different
segments of the nephron.
4,7
However, studies have shown that the
nephrotoxicity of chemotherapeutic drugs remains a significant
drawback to the successful treatment of cancers. Approximately
50e60% of patients who undergo cancer chemotherapy develop AKI
leading to increased morbidity and mortality.
8
Doxorubicin (DOX) is
one such chemotherapeutic drug with potential nephrotoxic effects
that can causetemporary or permanent kidney injury inpatients who
are on cancer chemotherapy.
9,10
The nephrotoxic effect of DOX is
attributed to the decline in oxidative, inflammatory, and apoptotic
renal profiles in several scientific reports.
11,12
In recent decades, can-
cer biologists have shown great interest in the clinical use of natural
products to complement the therapeutic and potential side effects of
cancer chemotherapy.
13,14
Natural products, particularly medicinal
herbs, possess superior structural and chemicaldiversity compared to
synthetic libraries, making them the best-known sources of novel
drugs and drug leads.
15
Barleria prionitis L. (BP) (Acanthaceae),
generally known as “Porcupine flower”, is one of the most commonly
used medicinal herbs in conventional medicine with scientifically
proven anticancer effects. Its pharmacological significance includes
antidiabetic, antioxidant, anti-inflammatory, anticataract, antihy-
pertensive, antiarthritic, antinociceptive, antibacterial, antifungal,
hepatoprotective, analgesic, antihelminthic effects, etc.
16,17
Phyto-
constituents such as iridoid diglucosides, secoiridoids, terpenoids,
triterpenes, phenolic acid, flavones, and phytosterols were isolated
from the plant. The antioxidant, anti-inflammatory, and free radical
scavenging properties were reported for the isolated phytocon-
stituents.
16
More importantly lupeol, vanillic acid, syringic acid, 6-
hydroxyflavone,
b
-sitosterol, and p-hydroxybenzoic acid exerted
potential anticancer effects.
16
The pharmacological significance in
cancer treatment is further upgraded with the synthesis of platinum
and palladium nanoparticles from BP.
18
The plant is among the common remedies used in the man-
agement of kidney-related diseases in traditional systems of
medicine.
19
however, studies on investigation of potential neph-
roprotective principles are quite limited. We previously reported
the efficacy of the standardized aqueous extract of the whole plant
of BP in a nephrotoxicity animal model.
20
Yet, the exact mecha-
nisms of nephroprotection by the plant have not been investigated
in that particular study. Investigation of possible protective mech-
anisms by which the plant reduces nephrotoxicity is an important
consideration in the process of developing potential therapeutics/
adjuvants to assist conventional cancer chemotherapy.
14
Therefore,
herein we investigated the possible cellular mechanisms underly-
ing the protective effects of selected extracts of BP on DOX-induced
AKI.
2. Materials and methods
2.1. Chemicals and reagents
All general reagents and chemicals, used in the study were
analytical grade and obtained from Sigma-Aldrich (USA).
2.2. Plant material and authentication
The whole plant materials of BP were collected in the flowering
stage, in March 2019, from Galle, Sri Lanka. The plant materials
were taxonomically identified and authenticated by Dr. N.P.T.
Gunawardena, Taxonomist at the National Herbarium, Sri Lanka. An
herbarium specimen (PG/2016/55/03) was deposited at the Mini
Herbarium, Department of Biochemistry, Faculty of Medicine,
University of Ruhuna.
2.3. Plant extraction
The plant material was oven-dried (40
C) up to a constant
weight and coarsely powdered. The dried BP powder was accu-
rately weighed, and sequential Soxhlet extraction was carried out
with hexane, ethyl acetate, n-butanol, and distilled water. The
extraction was continued with each solvent until the leachate
become colorless and the subsequent solvents were added
following the complete evaporation of the previous. The solvent
extracts were collected separately. The organic extracts were
concentrated by rotary evaporation (Buchi, B-480, UK) and dried in
a vacuum (40
C) to obtain the extracts of BP-hexane (BPH) (yield:
1.96% w/w), BP-ethyl acetate (BPEA) (yield: 6.51% w/w), and BP-n-
butanol (BPNB) (yield: 5.47% w/w). The aqueous extracts were
freeze-dried (20
C) to obtain the extract of BP-water (BPW)
(yield: 9.32% w/w).
2.4. Preliminary phytochemical screening
The presence of bioactive phytoconstituents including phenolic
compounds, tannins, flavonoids, steroid glycosides, alkaloids, ter-
penoids, coumarins, and saponins was tested in BPH, BPEA, BPNB,
and BPW extracts according to previously published methods.
21
2.5. Development of thin-layer chromatography (TLC) fingerprints
Thin-layer chromatography(TLC) profiles were developed for the
selected extracts of BP using the solvent systems,which showed fine
separations with a maximum number of components.
22
The solvent
systems of dichloromethane:cyclohexane:methanol (1:1:0.1) and
dichloromethane:cyclohexane:methanol:diethylamine
(1:0.8:0.1:0.3) were used in the development of TLC fingerprints.
List of abbreviations
AKI acute kidney injury
BCL-2 B-cell lymphoma gene product 2
Bax B-cell associated X protein
BP Barleria prionitis
BPEA Barleria prionitis ethyl acetate extract
BPH Barleria prionitis hexane extract
BPNB Barleria prionitis n-butanol extract
BPW Barleria prionitis water extract
COX-2 cyclooxygenase 2
DOX doxorubicin
DPPH 1, 1
0
-diphenyl-2-picrylhydrazyl
FS fosinopril sodium
GAE gallic acid equivalents
GC-MS gas chromatography-mass spectrometry
IL-1
b
interleukin-1
b
NC normal control
QE quercetin equivalents
TLC thin-layer chromatography
TNF-
a
tumor necrosis factor-
a
S.S. Amarasiri, A.P. Attanayake, L.D.A.M. Arawwawala et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx
2
2.6. Gas chromatography-mass spectrometry (GC-MS) analysis
For further identification of the bioactive compounds, gas
chromatography-mass spectrometry (GC-MS) analysis on hexane
and ethyl acetate extracts of BP was carried out using an Agilent
6890 series gas chromatograph interfaced to the Agilent 5973 N
series mass selective detector, which was equipped with a 5%
phenyl methyl siloxane capillary column (HP-5 MS,
30 m 0.25 mm ID, 0.25
m
mfilm). Helium was used as the carrier
gas and an injection volume of 1
m
L was employed (split ratio of
10:1). The injector temperature was 270
C. The oven temperature
was programmed from 100
C (5 min hold time) to 240
C at a rate
of 4
C/min (40 min hold time). The samples were run for 40 min.
The MS source and MS quadrupole temperatures were set at 230
C
and 150
C, respectively. Parameters were scanned at 15e550 amu.
The interpretation was performed using the Wiley W9N08.LIB
database and NIST's.LIB.
23
2.7. Determination of the total polyphenol content and the total
flavonoid content
Total polyphenol and flavonoid contents of BPH, BPEA, BPNB,
and BPW were determined following the standard protocols of the
Folin-Ciocalteu method and aluminum chloride method respec-
tively.
24,25
Quantification was carried out concerning the corre-
sponding standard curves of gallic acid (y ¼3.1561x þ0.0269) and
quercetin (y ¼13.619x þ0.0024). The results are expressed in
terms of gallic acid equivalents of the dry weight of extract (mg
GAE/g) and quercetin equivalents of the dry weight of extract (mg
QE/g) for the total polyphenol and total flavonoid contents
respectively.
2.8. Determination of 1,1ˊ-diphenyl-2-picrylhydrazyl scavenging
activity
The total antioxidant activity of the selected plant extracts was
determined by 1,1ˊ-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging assay.
26
Trolox was used as the reference compound
and the results are expressed in terms of Trolox equivalents of the
dry weight of the extract (mg trolox/g).
2.9. Experimental animals
The experimental procedures were carried out in strict accor-
dance with the recommendations in the “Guide for the Care and
Use of Laboratory Animals”.
27
All procedures were reviewed and
approved by the Ethical Review Committee of the Faculty of Med-
icine, University of Ruhuna (Permit Number: 14.12.2015:3.1). Blood
collection and organ removal were performed under CO
2
eutha-
nasia (EUTH 2A CO
2
euthanasia chamber, Orchid Scientific, India).
2.10. Experimental model of AKI
Freshly prepared DOX (Doxorubicin hydrochloride) at a single
dose of 5 mg/kg in 0.9% saline (2500
m
L/kg) was injected intra-
peritoneally into fasted (8 h) animals to establish the AKI model in
Wistar rats. The dose of DOX was selected based on the literature
and pilot experiments.
28,29
2.11. Experimental protocol
Male Wistar rats weighing 150e175 g were randomly allocated
into seven equally sized groups, with six animals in each group.
Except for the control group (NC), DOX (5 mg/kg, ip) was injected
into experimental rats in all other groups such as the model group,
BPH treatment group, BPEA treatment group, BPNB treatment
group, BPW treatment group, and FS treatment group to induce
AKI. Twenty-four hours after DOX intoxication, treatment regimens
were commenced. The human therapeutic dose (12 g/day for an
adult human with 60 kg body weight) applied in Ayurveda medi-
cine was extrapolated to experimental rats to calculate the relevant
doses of plant extracts for administration based on the extraction
yields of each extract and the dose conversion factor (6.17) from
human to rat.
30
The BPH and BPEA extracts were suspended in corn
oil for administration. BPNB was dissolved in 3% poly-
vinylpyrrolidone and BPW was dissolved in distilled water. All
suspensions were stored at 4
C. The experimental rats of treatment
groups underwent oral gavage feeding of selected extracts of BP for
four weeks at the equivalent human therapeutic dose of BPH
(25 mg/kg/day), BPEA (80 mg/kg/day), BPNB (70 mg/kg/day), BPW
(120 mg/kg/day) and fosinopril sodium (FS, 0.09 mg/kg/day).
Normal control and model rats received an equivalent volume of
distilled water. The plant extracts, fosinopril, and distilled water
were administered in a volume of 0.2 mL/150 g using a stainless-
steel oral gavage tube for 28 consecutive days. The duration of
plant extract administration was defined considering the previ-
ously published reports on the nephroprotective potential of plant
extracts against DOX-induced kidney injury.
31,32
2.12. Blood sample collection and tissue dissection
Experimental rats were fasted (free access to water) before
dissection. Twenty-four hours following the last dose of treatment,
blood samples were collected by cardiac puncture, and serum was
separated for the subsequent assessment of the kidney functions.
The kidneys were removed from the sacrificed animals and bisec-
ted. One-half of each kidney was fixed in 10% formalin for the
subsequent assessment of histopathology and immunohisto-
chemistry. The remaining half was weighed and homogenized in
ice-cold phosphate-buffered saline (0.01 M, pH ¼7.4). Aliquots of
kidney homogenates were stored at 80
C for the determination
of oxidative and inflammatory markers.
2.13. Biochemical analyses
Serum concentrations of creatinine, total protein, albumin,
b
2
-
microglobulin, cystatin C, and blood urea nitrogen, were estimated
according to the manufacturer's protocol, in the assessment of
kidney function.
The test procedure supplied with each commercial kit was fol-
lowed to estimate the total antioxidant status, glutathione reduc-
tase, and glutathione peroxidase for the assessment of oxidative
stress in kidney homogenates. Lipid peroxidation was evaluated in
terms of the concentration of malondialdehyde per gram of protein
in kidney homogenates according to the thiobarbituric acid method.
The inflammatory cytokines, tumor necrosis factor-
a
(TNF-
a
), and
interleukin-1
b
(IL-1
b
) were quantified in kidney homogenates using
ELISA kits for the assessment of anti-inflammatory markers.
2.14. Histopathological assessment
Hematoxylin and eosin-stained kidney sections were used in
the morphological evaluation of features of AKI. The procedure in
Bancroft and Gamble
33
was followed for the preparation of histol-
ogy samples for hematoxylin and eosin staining. The histological
changes were examined by two independent investigators
including a consultant histopathologist who was blinded to the
experimental profile. The morphological evaluation was carried out
according to a semi-quantitative score system developed by the
investigators, based on the features observed, as mentioned.
S.S. Amarasiri, A.P. Attanayake, L.D.A.M. Arawwawala et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx
3
For each animal, 20 high-power fields (magnification: 400)
were examined in the kidney cortex as follows; tubular cell vacuo-
lization, loss of tubular brush border, nuclear pyknosis, and
glomerular congestion were quantified on a 4-point scale of
0¼none,1 ¼<25%, 2 ¼25e50%, 3 ¼50e75%, and 4 ¼>75% based on
the area affected. The score for glomerular congestion was derived as
the arithmetic mean of 100 glomeruli. Intertubular hemorrhage and
inflammatory cell infiltrations were assessed by the absence
(score ¼0) or presence (score ¼1) of the characteristic feature at
high power (magnification: 400). The presence of tubular casts
was scored on a scale of 0e2as0¼none,1 ¼1 cast, and 2 ¼>2 casts.
The overall histological score represents the sum of the mean values
of observed changes for the comparison between the groups.
2.15. Immunohistochemical staining
Immunohistochemical staining was carried out on 4
m
m thick
sections of formalin-fixed, paraffin-embedded kidney tissue on
poly-
L
-lysine-coated microscopic slides. Deparaffinised, rehydrated
tissue sections were blocked for endogenous peroxidase activity
and were incubated with proteinase K (Dako, S3020, Denmark) for
antigen retrieval for anti-Bax (Abcam, ab216494, Cambridge, UK).
Fig. 1. Thin layer chromatography fingerprints of Barleria prionitis at UV 254 nm with the solvent systems; dichloromethane:cyclohexane:methanol (1:1:0.1) (A) and dichlor-
omethane:cyclohexane:methanol:diethylamine (1:0.8:0.1:0.3) (B). H, EA, NB, and W, as mentioned in the Fig. were the hexane, ethyl acetate, n-butanol, and water extracts of
Barleria prionitis, respectively.
Table 1
Thin layer chromatography of the selected extracts of Barleria prionitis.
Solvent system Extract No. of
Spots
R
f
values
Dichloromethane: Cyclohexane: Methanol (1:1:0.1) BPH 17 0.04, 0.12, 0.15, 0.19, 0.22, 0.25, 0.38, 0.46, 0.47, 0.51, 0.59, 0.63, 0.69, 0.74, 0.84,
0.93, 0.97
BPEA 11 0.06, 0.09, 0.15, 0.22, 0.26, 0.37, 0.44, 0.57, 0.68, 0.74, 0.96
BPNB 04 0.51, 0.60, 0.75, 0.82
BPW e
Dichloromethane: Cyclohexane: Methanol: Diethylamine
(1:0.8:0.1:0.3)
BPH 13 0.05, 0.11, 0.18, 0.30, 0.43, 0.56, 0.63, 0.71, 0.80, 0.84, 0.88, 0.91, 0.95
BPEA 12 0.09. 0.12, 0.17 0.26, 0.32, 0.39, 0.50, 0.54, 0.61, 0.86, 0.89, 0.96
BPNB 07 0.06, 0.11, 0.19, 0.27, 0.62, 0.73, 0.96
BPW 03 0.11, 0.16, 0.18
S.S. Amarasiri, A.P. Attanayake, L.D.A.M. Arawwawala et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx
4
Heat-induced antigen retrieval was carried out by microwave
method (100
C for 30 min) in citrate (pH 6) and Tris-EDTA (pH 9)
buffers for anti-BCL-2 (Dako, M0887, Denmark) and anti-COX-2
(Dako, M3617, Denmark) respectively. After cooling and blocking,
the tissue sections were incubated with the primary antibodies;
Bax (1:100), BCL-2 (1: 25), and COX-2 (1:100) overnight (4
C).
Subsequently, the sections were incubated with horseradish
peroxidase-conjugated (Dako REAL EnVision detection system,
K4061, Denmark) secondary antibody (27
C, 2 h) and counter-
stained with hematoxylin. Brownish staining in the cellular cyto-
plasm under the light microscope was considered the positive
staining in all three primary antibodies.
3. Statistical data analysis
Statistical analysis was performed using SPSS version 22. The
samples were analyzed in triplicates. The biochemical parameters
of the in vivo and in vitro studies were expressed as mean ±SD.
Comparison of the same parameters among different groups was
done using a one-way analysis of variance (ANOVA) followed by
post hoc Fisher's least significant difference (LSD) multiple com-
parison tests. Pearson's correlation between the examined pa-
rameters was assessed. Statistical significance was assigned at
p<0.05.
4. Results
4.1. Screening of phytochemicals, TLC fingerprints, and GC-MS
chromatogram of B. prionitis
TLC fingerprints were developed for the selected extracts of BP
using two solvent systems based on the polarity of the extracts. The
results of the analysis of TLC fingerprint profiles are presented in
Fig. 1 and Table 1.
The preliminary phytochemical screening of BPH, BPEA, BPNB,
and BPW revealed the presence of phenolic compounds. In
contrast, alkaloids and coumarins were absent in all the tested
extracts. Flavonoids and terpenoids werepresent in BPNB and BPW.
In addition, tannins, steroid glycosides, and saponins were present
in BPW.
The GC-MS chromatograms and the different bioactive com-
pounds identified from BPH and BPEA are presented in Fig. 2A and B
and Table 2, respectively.
4.2. Antioxidant activity of B. prionitis
Since reactive oxygen species and oxidative stress serve as the
key factors in DOX-induced AKI, the total antioxidant potential of the
four selected extracts of BP was assessed byDPPH radical scavenging
assay in vitro. The total antioxidant activity through the DPPH assay
was varied in order of BPNB >BPW >BPEA >BPH, as expressed by
the equivalent antioxidant capacity of trolox, 53.24 ±1.19,
29.23 ±0.39, 11.19 ±0.22, and 9.03 ±0.23 mg trolox/g respectively.
Extracts with organic solvents; BPH (18.71 ±0.20 mg GAE/g), BPEA
(22.83 ±0.85 mg GAE/g), and BPNB (31.67 ±0.18 mg GAE/g) showed
a higher polyphenol content than that in the BPW extract
(14.62 ±0.21 mg GAE/g). Similarly, the respective organic extracts
also showed a high flavonoid content (37.42 ±1.22, 15.97 ±0.17, and
9.06 ±0.17 mg QE/g) compared to the BPW (5.64 ±0.14 mg QE/g).
Interestingly, as determined by the Folin-Ciocalteu method, the
BPNB, which showed the highest antioxidant activity by DPPH assay,
showed the highest polyphenol content.
4.3. Effect of B. prionitis on biochemical parameters of AKI
The effect of BP on biomarkers of AKI is shown in Fig. 3AeF. The
BPW showed the highest nephroprotection among all the plant
extracts considering both concentrations of creatinine (33%) and
Fig. 2. The GC-MS chromatograms of the hexane (A) and ethyl acetate (B) extracts of
Barleria prionitis.
Table 2
The chemical constituents identified from the hexane and ethyl acetate extracts of Barleria prionitis.
Name of the compound Hexane extract Ethyl acetate extract
Retention time (min) Relative percentage (%) Retention time (min) Relative percentage (%)
Phenol, 2,4-bis(1,1-dimethylethyl) 16.066 0.14 16.054 3.50
Dodecanoic acid 16.279 0.19 ee
Methyl tetradecanoate 21.928 1.36 ee
3-Methylene-1-hexadecene 24.818 1.45 24.806 3.28
7-Octadecyne ee 25.434 3.84
2-Hexadecen-1-ol 25.884 2.11 25.872 4.47
Methyl palmitate 27.163 21.84 27.021 30.94
1-Dodecanol, 3,7,11-trimethyl- 30.764 1.09 ee
9,12-Octadecadienoic acid (Z,Z)- 31.036 10.84 30.930 4.69
Cyclohexane 31.427 3.00 31.297 1.56
Palmitic acid 32.919 0.17 ee
S.S. Amarasiri, A.P. Attanayake, L.D.A.M. Arawwawala et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx
5
blood urea nitrogen (58%). The blood urea nitrogen values of the
experimental rats treated with BPW were significantly different
from the BPH, BPEA, and BPNB as well as from fosinopril (p <0.05).
On the contrary, the experimental rats treated with FS (52%)
showed a marked reduction in the concentration of cystatin C
compared to the four extracts of BP (29%, 25%, 16%, and 23%).
4.4. Effect of B. prionitis on DOX-induced oxidative stress in kidney
Treatment with the selected extracts of BP for four consecutive
weeks ameliorated DOX-induced oxidative stress significantly, by
recovering the depleted total antioxidant status and the activity of
glutathione reductase and glutathione peroxidase enzymes as
shown in Fig. 4AeC(p<0.05). The BPH (111%) and BPW (55%)
demonstrated a significant improvement in glutathione peroxidase
activity, compared to the NC rats (p <0.05). Similarly, BPEA (40%)
showed a significant increase in the glutathione reductase activity
compared to the NC rats (p <0.05). Notably, the experimental rats
of the FS treatment group did not show significant improvement in
the activity of glutathione reductase and glutathione peroxidase,
compared to the DOX-induced model group in the present study
(p >0.05). Experimental rats in the BPEA (52%) and BPNB (29%)
treatment groups showed a statistically significant increase in
glutathione reductase activity, compared to the experimental rats
in the FS treatment group (p <0.05). Furthermore, there was a
significant improvement in glutathione peroxidase activity in
experimental rats from all four BP treatment groups, compared to
the FS treatment group (p <0.05).
However, all groups of experimental animals treated with BP
showed a significant attenuation in malondialdehyde formation
compared to the experimental animals of the model group (Fig. 4D)
(p <0.05). The experimental rats of the BPH and BPEA treatment
groups showed a significant reduction in malondialdehyde for-
mation, compared to the FS treatment group as well (p <0.05).
BPEA (30%) and BPNB (24%) showed the highest and lowest
attenuation in lipid peroxidation, respectively. A significant corre-
lation was observed in the activity of glutathione reductase and
lipid peroxidation (r ¼663, p ¼0.001) in the present study.
4.5. Effect of B. prionitis on DOX-induced inflammation in kidney
Treatment with the plant extracts exhibited a significant sup-
pression in the levels of TNF-
a
and IL-1
b
compared to the model
group (p <0.05). The results are shown in Fig. 4EeF. The BPW
showed superior results over the other three extracts of BP con-
cerning both TNF-
a
and IL-1
b
. A significant amelioration of TNF-
a
was observed with experimental rats in the BPW treatment group
compared to the BPH, BPEA, and FS treatment groups (p <0.05).
Fig. 3. Effect of Barleria prionitis extracts on the concentrations of serum creatinine (A); blood urea nitrogen (BUN) (B); serum total protein (C); serum albumin (D);
b
2
-microglobulin
(E); serum cystatin C (F) in DOX-induced AKI. Data represent mean ±SD, n ¼6. Compared with normal control group *p<0.05, **p<0.01; compared with model group #p <0.05,
##p <0.01 and compared with the FS treatment group :p<0.05, :: p<0.01. NC: normal control, DOX: doxorubicin. The treatment groups (DOX þBPH, DOX þBPEA,
DOX þBPNB, DOX þBPW, DOX þFS) consisted of experimental rats administered with hexane, ethyl acetate, n-butanol and water extracts of B. prionitis and fosinopril sodium,
respectively.
S.S. Amarasiri, A.P. Attanayake, L.D.A.M. Arawwawala et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx
6
Interestingly, the anti-inflammatory activity of the four selected BP
extracts was varied in the same order as BPW >BPB >BPEA >BPH
considering both TNF-
a
, and IL-1
b
values.
4.6. Effect of B. prionitis on DOX-induced histopathological damage
in the kidney
DOX administration resulted in marked kidney damage with an
average score of 12.9, as mentioned in Fig. 5A, which was signifi-
cantly attenuated by the treatment with BPH (19% decrease), BPEA
(14% decrease), BPNB (19% decrease), and BPW (20% decrease)
(p <0.05). Histological examination showed cytoplasmic vacuoli-
zation, the disappearance of the brush borders, nuclear pyknosis,
and hyaline casts in the renal tubules of the model group (Fig. 5B).
However, no statistically significant improvement in the overall
histological score was observed after treatment with fosinopril
(12% decrease) (p >0.05). The interstitial tissue showed very few
lymphocytic aggregates at low power ( 100 magnification),
however, it was difficult to quantify them at high power ( 400
magnification). Glomerular congestion and intertubular hemor-
rhage were the additional features observed. However, no evidence
of full-blown acute tubular necrosis or glomerulosclerosis was
observed in the kidney sections stained with hematoxylin and
Fig. 4. Effect of Barleria prionitis extracts on total antioxidant status (A); the activity of glutathione reductase (B); glutathione peroxidase (C) and lipid peroxidation (D), concen-
tration of tumor necrosis factor-
a
(TNF-
a
) (E) and interleukin-1
b
(IL-1
b
) (F) in kidney homogenates of DOX-induced AKI. Data represent mean ±SD, n ¼6. Compared with normal
control group *p<0.05, **p<0.01; compared with model group #p <0.05, ##p <0.01 and compared with the FS treatment group :p<0.05, :: p<0.01. NC: normal control,
DOX: doxorubicin. The treatment groups (DOX þBPH, DOX þBPEA, DOX þBPNB, DOX þBPW, DOX þFS) consisted of experimental rats administered with hexane, ethyl acetate, n-
butanol and water extracts of B. prionitis and fosinopril sodium, respectively.
Fig. 5A. Effect of Barleria prionitis extracts on overall histological score of kidney
sections stained with hematoxylin and eosin. Data represent mean ±SD, n ¼6.
Compared with normal control group *p<0.05, **p<0.01; compared with model
group #p <0.05, ##p <0.01 and compared with the FS treatment group :p<0.05,
:: p<0.01. NC: normal control, DOX: doxorubicin. The treatment groups
(DOX þBPH, DOX þBPEA, DOX þBPNB, DOX þBPW, DOX þFS) consisted of
experimental rats administered with hexane, ethyl acetate, n-butanol and water ex-
tracts of B. prionitis and fosinopril sodium, respectively.
S.S. Amarasiri, A.P. Attanayake, L.D.A.M. Arawwawala et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx
7
eosin in any of the experimental groups in the present study.
Representative photomicrographs of kidney sections of the BP
treatment groups are displayed in Fig. 5B.
4.7. Effect of B. prionitis on DOX-induced tubular cell apoptosis and
inflammatory immuno-markers in kidney
The expression of the apoptosis-related proteins, BCL-2 and Bax
were assessed by immunohistochemistry for the evaluation of the
effect of selected plant extracts on DOX-induced apoptosis. The
immunostaining of the pro-apoptotic protein, Bax, was compara-
tively high and mainly cytoplasmic in the DOX-induced model
group (Fig. 6A). The staining was visible mainly on the luminal
surface of the proximal and distal tubular epithelial cells in rats of
the NC group and most of the treatment groups. Conversely, the
expression of the anti-apoptotic protein; BCL-2 in tubular epithelial
cells was comparatively high in rats of the NC group compared to
the model group (Fig. 6B). An uniformly positive cytoplasmic
expression of BCL-2 was observed in the NC group, whereas the
focal positivity was observed in the model group rats. The experi-
mental rats treated with plant extracts showed improved immu-
nostaining for BCL-2 compared to the model group. Notably, the
kidney sections of rats treated with BPH and BPW showed higher
expression of BCL-2 and a lesser expression of Bax compared to the
other two extracts. The anti-inflammatory potential of BP was
assessed in terms of the immunohistochemical expression of COX-2
in the present study. Treatment with the selected BP extracts for
four consecutive weeks caused a reduction in COX-2 expression.
The BPW showed lesser expression of COX-2 compared to the other
three extracts of BP in the present study. The immunohistochemical
expression of COX-2 is shown in Fig. 6C.
5. Discussion
Numerous experimental studies conducted over the past few
decades highlighted the value of phytomedicines as anticancer
agents as well as adjuvants or alternative therapeutics in amelio-
rating the potential side effects of chemotherapeutics.
14
Protective
Fig. 5B. Effect of Barleria prionitis extracts on photomicrographs of kidney sections stained with hematoxylin and eosin ( 400 magnification, Scale bar: 20
m
m) in DOX-induced
AKI. Cytoplasmic vacuolization (yellow arrow), disappearance of the brush borders (red arrow), nuclear pyknosis (blue arrow), tubular casts (green arrow), glomerular congestion
(black arrow) and intertubular hemorrhage (white arrow). NC: normal control, DOX: doxorubicin. The treatment groups (DOX þBPH, DOX þBPEA, DOX þBPNB, DOX þBPW,
DOX þFS) consisted of experimental rats administered with hexane, ethyl acetate, n-butanol and water extracts of B. prionitis and fosinopril sodium, respectively.
Fig. 6A. Effect of Barleria prionitis on the immunohistochemical expression of B-cell associated X protein (Bax) on DOX-induced AKI ( 400 magnification, Scale bar: 20
m
m). The
immunostaining of the pro-apoptotic protein, Bax was comparatively high and mainly cytoplasmic in the DOX-induced model group. Conversely, the staining was less and mainly
visible in the luminal surface of the proximal and distal tubular epithelial cells, in the rats of NC group and most of the treatment groups. NC: normal control, DOX: doxorubicin. The
treatment groups (DOX þBPH, DOX þBPEA, DOX þBPNB, DOX þBPW, DOX þFS) consisted of experimental rats administered with hexane, ethyl acetate, n-butanol and water
extracts of B. prionitis and fosinopril sodium, respectively.
S.S. Amarasiri, A.P. Attanayake, L.D.A.M. Arawwawala et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx
8
mechanisms of phytomedicines in terms of antioxidative, anticar-
cinogenic, anti-inflammatory, immunoprotective, etc. were found
to protect normal cells from chemotherapy-induced toxicities.
14
Treatment with the selected extracts of BP significantly amelio-
rated DOX-induced kidney injury, as shown by the improvements
in kidney function biomarkers.
Histopathological findings corroborated the results of the
biochemical parameters. None of the experimental groups showed
a full-blown picture of acute tubular necrosis or glomerular scle-
rosis. Sub-lethal changes of AKI were observed in experimental rats
of the model group which were ameliorated following the treat-
ment regimens.
Oxidative stress is considered a major mechanism in the path-
ogenesis of DOX-induced AKI.
12
Excessive production of reactive
oxygen species by exposure to DOX causes the corresponding
generation of oxidative stress, including the depletion of reduced
glutathione, the reduced level of antioxidant enzymes, and the
increased levels of malondialdehyde.
11,12
The same phenomenon
was observed in the present study; in fact, post-treatment with the
selected extracts of BP effectively reversed DOX-induced oxidative
stress to a normal degree. All four extracts of BP showed higher
antioxidant potential than those of fosinopril, considering the ac-
tivity of antioxidant enzymes; glutathione peroxidase, glutathione
reductase, and the level of malondialdehyde. FS is an angiotensin-
converting enzyme inhibitor and the use of the drug is a common
therapeutic approach in chronic kidney disease due to its long-term
nephroprotective effects.
34
In fact, angiotensin-converting enzyme
inhibitors demonstrate hypoalbuminemia by inhibiting the renin-
angiotensin system, thereby being the standard of care for pa-
tients with albuminuria.
35
Hence, fosinopril was used as the
reference drug in the present study, considering its beneficial ef-
fects in ameliorating proteinuria associated with DOX-induced AKI.
The potential nephroprotective effects of FS were further confirmed
by the observed attenuation of proteinuria in the FS treatment
group.
These findings on the enhancement of cellular antioxidant sta-
tus by BP extracts are in line with previous reports which showed a
potent antioxidant effect of BP.
16,17
According to published reports,
numerous phytoconstituents isolated from BP, including balar-
enone, pipataline, lupeol, vanillic acid, melilotic acid, 6-
Fig. 6B. Effect of Barleria prionitis on the immunohistochemical expression of B-cell lymphoma gene product 2 (BCL-2) on DOX-induced AKI ( 400 magnification, Scale bar:
20
m
m). The expression of the anti-apoptotic protein BCL-2 in tubular epithelial cells was comparatively high in rats of NC group compared to the model group. Uniformly positive
cytoplasmic expression of BCL-2 was observed in the NC group whereas focal positivity was observed in the model group rats. The experimental rats treated with plant extracts
showed enhanced immunostaining for BCL-2 compared to the model group. NC: normal control, DOX: doxorubicin. The treatment groups (DOX þBPH, DOX þBPEA, DOX þBPNB,
DOX þBPW, DOX þFS) consisted of experimental rats administered with hexane, ethyl acetate, n-butanol and water extracts of B. prionitis and fosinopril sodium, respectively.
Fig. 6C. Effect of Barleria prionitis on the immunohistochemical expression of cyclooxygenase 2 (COX-2) on DOX-induced AKI ( 400 magnification, Scale bar: 20
m
m). The
immunohistochemical expression of COX-2 was comparatively higher in the experimental rats of the model group compared to the rats of the NC group. The treatment groups
showed a reduction in the expression of COX-2 compared to the rats of the model group. NC: normal control, DOX: doxorubicin. The treatment groups (DOX þBPH, DOX þBPEA,
DOX þBPNB, DOX þBPW, DOX þFS) consisted of experimental rats administered with hexane, ethyl acetate, n-butanol and water extracts of B. prionitis and fosinopril sodium,
respectively.
S.S. Amarasiri, A.P. Attanayake, L.D.A.M. Arawwawala et al. Journal of Traditional and Complementary Medicine xxx (xxxx) xxx
9
hydroxyflavone,
b
-sitosterol, verbascoside, etc. possessed antioxi-
dant properties in the investigations of their potential bio-
activities.
16,17
As inflammation is closely related to the generation of
reactive oxygen species, anticancer drug-induced severe oxidative
stress, generally induces inflammatory responses and vice versa.
However, it is reported that the phytoconstituents with antioxidant
potential could ameliorate inflammation-related side effects in
chemotherapy.
14,36
The increased levels of TNF-
a
and IL-1
b
observed in the present study indicate an activation and increased
production of pro-inflammatory cytokines in the kidney, leading to
a state of inflammation in DOX-intoxicated rats. However, BP
treatment significantly reversed DOX-induced inflammatory
changes, suggesting that the nephroprotective effect of BP might
occur partly due to its anti-inflammatory properties. The immu-
nohistochemical findings on the expression of the inflammatory
mediator, COX-2, were comparable to the findings on TNF-
a
and IL-
1
b
. Accordingly, the present findings corroborate the anti-
inflammatory effects of the aqueous extract of the plant.
37
More-
over, phytoconstituents isolated from BP including, lupeol, vanillic
acid, 6-hydroxyflavone,
b
-sitosterol, p-hydroxybenzoic acid, ver-
bascoside exerted anti-inflammatory potential.
16
The phytochemi-
cals present in the BP extracts could inhibit the activity of enzymes
that mediate inflammation, such as decreasing the damage caused
by acute inflammation.
38
The presence of sub-lethal changes
related to acute kidney injury and the absence of inflammatory
infiltration in the present study are related to the potential prop-
agation of apoptotic cell death rather than necrotic cell death. This
was further corroborated by the immunohistochemical expression
of the pro-apoptosis protein, Bax, and the anti-apoptosis protein,
BCL-2. DOX administration caused up-regulation of Bax and down-
regulation of BCL-2, promoting apoptosis, as demonstrated in the
present study. The plant extracts showed better immunostaining
for BCL-2 and relatively lower expression of Bax, suggesting the
potential anti-apoptotic effects.
The present findings on the potential nephroprotective mech-
anisms of BPH, BPEA, BPNB, and BPW provide the basis for
bioassay-guided isolation of nephroprotective phytoconstituents.
Therefore, the present findings would open new vistas for the
development of new nutraceuticals to minimize DOX-induced AKI
in cancer patients.
6. Conclusions
The present study offered the first evidence that BPH, BPEA,
BPNB, and BPW significantly ameliorate DOX-induced AKI via
antioxidative, anti-inflammatory, and anti-apoptotic effects in
experimental rats. The findings of in vitro antioxidant studies
further substantiated the potential antioxidant effects of the plant
extracts. The BP extracts might serve as a potential therapeutic/
adjuvant for assisting DOX chemotherapy through ameliorating AKI
in cancer patients who are on chemotherapy. However, further
studies are required to confirm the clinical efficacy of the selected
extracts in DOX chemotherapy.
Funding
This work was supported by grants from the National Science
Foundation, Sri Lanka (RG/2016/HS -03) and UGC block grant from,
the University of Ruhuna (RU/PG- R/16/14).
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
Technical officers of the departments of Biochemistry and Pa-
thology, Faculty of Medicine, University of Ruhuna are acknowl-
edged for the technical assistance provided.
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