ENZYME ACTIVITIES AND HISTOPATHOLOGICAL STUDIES OF SELECTED TISSUES IN ALBINO MICE TREATED WITH METHANOLIC LEAVES EXTRACT OF LUFFA CYLINDRICA (AN ANTIMALARIAL HERB) 1 2 3

Abstract

Introduction: Luffa cylindrica is a plant used in folk medicine of Nigeria to treat malaria. Safety evaluation of methanolic leaves extract of Luffa cylindrica was carried out in selected organs of mice at the doses of 50, 100, 200, 400 and 800 mg/kg body weight (b.w).
Methodology: Thirty albino mice were completely randomized into six groups consisting of five mice each. Animals in group A served as the
control and received 0.2 ml of distilled water while groups B, C, D, E and F received the same volume (0.2 ml) of 50, 100, 200, 400 and 800 mg/kg b.w. methanolic leaves extract of L. cylindrica respectively for 7 days orally. The activity of aspartate, alanine and gamma glutamyl
aminotransferases (AST, ALT, GGT), alkaline phosphatase (ALT) as well as histological study in selected organs (spleen, liver and kidney) of mice was assessed. Serum albumin, total and conjugated bilirubin, total protein, urea and creatinine concentrations were also evaluated.
Results: The extract significantly (p < 0.05) reduced the activities of ALT, AST and ALP in the selected organs and significantly (p < 0.05) increased their activities in the serum at all doses investigated. GGT activity increased significantly in the serum and decreased significantly in the liver and spleen at 200, 400 and 800 mg/kg b.w. A
significant increase (p < 0.05) was observed in urea, total and conjugated bilirubin concentrations while there was no significant alteration observed in the serum albumin, creatinine and total protein
concentrations of animals at all the doses compared to the control. Histological changes with evidence of multinucleated giant cells and periportal lymphocytic inflammation occurred in the spleen
and liver respectively at 100, 200, 400 and 800 mg/kg b.w.
Conclusion: The results suggest that consumption of L. cylindrica leaf extract may have deleterious effect on the kidney, spleen and liver.

Full text

ENZYME ACTIVITIES AND HISTOPATHOLOGICAL STUDIES OF
SELECTED TISSUES IN ALBINO MICE TREATED WITH METHANOLIC
LEAVES EXTRACT OF LUFFA CYLINDRICA (AN ANTIMALARIAL HERB)
1 2 3
*Saliu A.O ; Akanji M.A and Idowu O.A
1Department of Environmental Health, Faculty of Health Sciences,
National Open University of Nigeria, Abuja
2Department of Biochemistry, Faculty of Life Sciences, University of Ilorin, P.M.B 1515,
Ilorin, Kwara State, Nigeria
3Department of Biochemistry, College of Natural and Applied Sciences,
Oduduwa University, P.M.B 5533, Ile-Ife Osun State, Nigeria
Corresponding Author:
Email: id4phemy@gmail.com
ABSTRACT
Introduction: Luffa cylindrica is a plant used in
folk medicine of Nigeria to treat malaria. Safety
evaluation of methanolic leaves extract of Luffa
cylindrica was carried out in selected organs of
mice at the doses of 50, 100, 200, 400 and 800
mg/kg body weight (b.w).
Metho dolog y: Thirty albino mi ce were
completely randomized into six groups consisting
of five mice each. Animals in group A served as the
control and received 0.2 ml of distilled water while
groups B, C, D, E and F received the same volume
(0.2 ml) of 50, 100, 200, 400 and 800 mg/kg b.w.
methanolic leaves extract of L. cylindrica
respectively for 7 days orally. The activity of
aspar tate, alanine and gamma g lut amyl
aminotransferases (AST, ALT, GGT), alkaline
phosphatase (ALT) as well as histological study in
selected organs (spleen, liver and kidney) of mice
was assessed. Serum albumin, total and
conjugated bilirubin, total protein, urea and
creatinine concentrations were also evaluated.
Results: The extract significantly (p < 0.05)
reduced the activities of ALT, AST and ALP in the
selected organs and significantly (p < 0.05)
increased their activities in the serum at all doses
investigated. GGT activity increased significantly
in the serum and decreased significantly in the liver
and spleen at 200, 400 and 800 mg/kg b.w. A
significant increase (p < 0.05) was observed in
urea, total and conjugated bilirubin concentrations
while there was no significant alteration observed
in the serum albumin, creatinine and total protein
concentrations of animals at all the doses compared
to the control. Histological changes with evidence
of multinucleated giant cells and periportal
lymphocytic inflammation occurred in the spleen
and liver respectively at 100, 200, 400 and 800
mg/kg b.w.
Conclusion: The results suggest that consumption
of L. cylindrica leaf extract may have deleterious
effect on the kidney, spleen and liver.
K e y w or d s : L u ff a c y l i n d r i ca , a l a n i n e
aminotransferase, alkaline phosphatase, aspartate
aminot r a n s ferase a n d g a m m a g l u t amyl
aminotransferase
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Journal of Malaria Research and Phytomedicine

INTRODUCTION
The use of medicinal plant to treat various diseases
is widely gaining acceptance because they are
accessible, cheaper, natural and are believed to be
1,2
devoid of side effects . There is also a wide
available scientific literatures affirming the
therapeutic effects exhibited by medicinal plants
as purported by traditional health practitioners
such as anti-diabetic, anti-microbial, antioxidant,
anti-tumor, anti-cancer, analgesic, anti-pyretic,
anti-malarial, anti-hyperlipidemic, the list is
endless. Despite the upsurge use of medicinal
plants to treat several ailments, thorough scientific
investigations on the safety of medicinal plants as
herbal remedy is exigent because researches have
shown that not all medicinal plants are safe, as they
produce toxic effect resulting from overdosing,
chronic consumption, allergies, contaminations
3,4
and interactions . Luffa cylindrica commonly
called Sponge gourd is a plant belonging to the
5
cucumbers family . It is widely cultivated in Asia,
South America and Africa where it has been used
6,7
for medicinal, industrial and cosmetic purposes .
Locally, L. cylindrica is used to treat malaria,
jaundice, tumors, leprosy, wounds, bleeding from
8,9
bowels or bladders and pain . The analgesic and
10,11 12 13
antipyretic sedative , anti-inflammatory , anti-
14 ,1 5
hyperglycemic and anti-diabetic , anti-
16 1 0
microbial , anthelmintic and stomachic ,
5,17 18
antioxidant and activities of the plant parts
have been evaluated hence the need to assess the
toxicity of the plant arise. This study is therefore
aimed to assess the effect of the administration of
methanolic extract of L. cylindrica leaf on renal
and hepatic biochemical parameters as well as
their histological changes.
MATERIALS AND METHODS
Plant collection and identification
Fresh leaves of Luffa cylindrica (L.) Roem were
collected from Suleja, Niger State at early hours in
the month of July, 2015. The plant was
auth entic ated at Nati o nal I nsti t ute f or
Pharmaceutical Research and Development
(NIPRD), Abuja with voucher number (6650)
deposited in the herbarium of the institute.
Experimental animals
Thirty (30) healthy albino rats of both sexes (180-
200 g) were obtained from the Animal House of the
Department of Pharmacology, National Institute
for Pharmaceutical Research and Development
Idu-Abuja, Nigeria. The animals were housed in
clean metabolic cages placed in well-ventilated
house conditions (temperature 23 ± 1˚C;
photoperiod: 12 h natural light and 12 h dark:
humidity: 45-50%). They were also allowed free
access to Platinum Feed Mills Company, Nigeria)
and tap water freed of contaminants.
Ethical approval
Ethical approval for using the experimental
animals was issued by the University of Ilorin
Ethical Committee on the use of experimental
animals.
Assay kits
The assay kits for creatinine, urea, bilirubin,
albumin, alkaline phosphatase (ALP), gamma
glutamyl transferase (GGT), alanine and aspartate
aminotransferases (ALT and AST respectively)
were obtained from Randox Laboratories Ltd. (Co.
Antrim, UK). All other reagents used were of
analytical grade and were commercial obtained.
METHODS
Preparation of extract
The leaves of L. cylindrica were air-dried and
pulverized with mechanical blender (Mazeda Mill,
MT 4100, Japan). 500 g of the pulverized leaves
was macerated in 1 litre of absolute methanol
(99%) for 72 h with concomitant shaking using a
stirrer. The resulting extract was filtered with
cotton wool and with Whatman (No. 1) paper
res p ecti vely. The filt r ate obta i ned was
concentrated in a rotary evaporator (RE-300B
o
model, China) at 60-65 C and was dried to a
constant weight on a water bath to give a yield of
28% (w/w). The extract was reconstituted into
doses 50, 100, 200, 400 and 800 mg/kg b.w.
Animal grouping and administration of extract
Thirty albino rats of both sexes were completely
randomized into six groups each consisting of five
animals, and were orally administered as follows:
8Volume 4 No 1, 2020

Group A (control) was administered with 0.2 ml of
distilled water while groups B, C, D, were
administered 50, 100, 200, 400 and 800 mg/kg
body weight of the extract respectively. The
administration was done repeatedly on daily basis
for 7 days using oropharyngeal cannula. The
animals were sacrificed 24 h after their 7 daily
doses of distilled water and extract.
Preparation of serum and tissue homogenates
Anima ls in eac h tre atment group were
anaesthetized on the last day of the experiment
with diethyl ether. Blood was collected by cardiac
puncture into dry clean sample bottles and the
blood was allowed to clot for 10 min. at room
temperature and then centrifuged using Uniscope
Laborat ory Cen trifu ge (Mode l SM8) B,
Surgrifriend Medicals, Essex, England at 350 ×g
for 15 min. The supernatant was immediately
transferred into clean sample bottles using a
Pasteur pipette to obtain the serum and was used
within 12 h of preparation for the enzymes assay.
The liver, kidney, and spleen of each animal were
removed, and were homogenized in ice cold 0.25
w
M sucrose solution (1:5 / ). The homogenates
v
were stored frozen overnight to preserve the
activity of the enzymes. Appropriate dilutions of
the homogenates (liver, kidney, spleen) and serum
were used for biochemical parameters.
Biochemical parameters
Serum total protein concentration was determined
19
by the method of Gornall et al. . The methods of
Doumas et al; Sherlock; Veniamin and Varkertzi;
20-23
Tietz et al. were used to determine the
concentrations of albumin, bilirubin, urea and
creatinine respectively while alkaline phosphatase
(ALP), gamma glutamyl aminotransferase (GGT),
aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) in the serum and tissue
homogenates of the animals were determined
24-26
using Wright, Szasz, Reitman and Frankel
methods respectively as described in the assay kits.
STATISTICAL ANALYSIS
Data obtained were subjected to one way Analysis
of Variance (ANOVA) and means were separated
by Duncan Multiple Range Test. Percentage data
were arcsine transformed before analysis.
Significant levels were tested at p < 0.05.
RESULTS
The effect of administration of methanolic leaves
extract o f L . cylindrica o n a s p a r t a t e
aminotransferase is presented in Fig. 1. A
significant decrease (p < 0.05) in the activity of
aspartate aminotransferase was observed in the
liver, kidney and spleen of rats at all the doses
compared to the control. Whereas, in the serum of
rats, activity of AST significantly (p < 0.05)
increased following the administration of the
extract at all the doses when compared to the
control. The extract at the end of the 7 days of
treatment, significantly (p < 0.05) decreased the
activity of alanine aminotransferase at 50, 100,
200, 400 and 800 mg/kg b.w in all the organs and a
concomitant decrease of ALT activity in the serum
when compared to the control (Fig. 2). GGT
activity decreased significantly (p < 0.05) at all
doses in the spleen but only at 200, 400 and 800
mg/kg b.w in the liver of rats, when compared to the
control. Contrary to what was observed in the liver
and spleen, GGT activity significantly (p < 0.05)
increased in the serum of rats at all the doses when
compared to the control (Fig. 3). Administration of
50, 100, 200, 400, 800 mg/kg b.w of the extract
caused a significant decrease (p < 0.05) in the
activity of ALP in the kidney and spleen of rats
when compared to the control. Similarly, a
significant decrease (p < 0.05) in ALP activity was
observed in the liver of rats that received the extract
doses of 100, 200, 400 and 800 mg/kg. This
decrease in ALP activity further resulted to a
concomitant increase in the activity of ALP in the
serum of the animals (Fig. 4). The effect of
administration of methanolic leaves extract of L.
cylindrica on kidney and liver function indices are
presented in Tables 1 and 2 respectively.
Concentration of urea significantly (p < 0.05)
decreased while there was no significant alteration
(p < 0.05) observed in the concentration of
creatinine in rats after treatment with the extract at
all the doses when compared to the control (Table
1). T he tota l a nd conj ugat ed bili rubi n
concentrations increased significantly (p < 0.05)
while there was no significant alteration (p < 0.05)
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Journal of Malaria Research and Phytomedicine

10
observed in the albumin and total protein
concentrations of rats treated with methanolic
leaves extract of L. cylindrica at all the doses (50,
100, 200, 400 and 800 mg/kg b.w) administered
when compared to the control (Table 2).
Photomicrograph of the liver and spleen of rats
treated with 50, 100, 200, 400 and 800 mg/kg b.w
of the extract for 7 days is depicted in Plates 1-3.
His tolog ical cha n ges with evi d ence of
multinucleated giant cells and periportal
lymphocytic inflammation were observed in the
spleen and liver respectively at 200 400 and 800
mg/kg b.w while peritubular lymphocytic
infiltration was observed in the kidney of rats
administered 800 mg/kg b.w of the extract for 7
days.
S p e c i f i c A c t i v i t y o f A S T ( U / m g p r o t e i n )
C o n t r o l
5 0 m g / k g b .w . o f E x t r a c t
1 0 0 m g / k g b .w . o f E x tr a c t
2 0 0 m g / k g b .w . o f E x tr a c t
4 0 0 m g / k g b .w o f E x tr a c t
8 0 0 m g / k g b .w . o f E x tr a c t
Figure 1: Effect of administration of methanolic extract of Luffa cylindrica leaf on aspartate
aminotransferase activity in the liver, kidney, spleen and serum of mice
Values are means of 5 replicates ± SEM. Bars with different alphabets a, b, c, d and e means values are
significantly different (p < 0.05) from each other.
Volume 4 No 1, 2020

A Publication of Centre for Malaria Research & Phytomedicine 11
Journal of Malaria Research and Phytomedicine
S
p
e
c
i
f
i
c A c
t
i
v
i
t
y
o
f
A
L
T (
U /
m g p r o t e i n )
a
C o n t r o l
5 0 m g /k g b .w . o f E x tr a c t
1 0 0 m g /k g b .w . o f E x tr a c t
2 0 0 m g /k g b .w . o f E x tr a c t
4 0 0 m g /k g b .w o f E x tr a c t
8 0 0 m g /k g b .w . o f E x tr a c t
Figure 2: Effect of administration of methanolic extract of Luffa cylindrica leaf on alanine aminotransferase
activity in the liver, kidney, spleen and serum of mice
Values are means of 5 replicates ± SEM. Bars with different alphabets a, b, c and d means values are
significantly different (p < 0.05) from each other.
S p e c i f i c A c t i v i t y o f G G T ( U / m g p r o t e i n )
a
C o n t r o l
5 0 m g / k g b . w . o f E x t r a c t
1 0 0 m g / k g b . w . o f E x t r a c t
2 0 0 m g / k g b . w . o f E x t r a c t
4 0 0 m g / k g b .w o f E x t r a c t
8 0 0 m g / k g b . w . o f E x t r a c t
Figure 3: Effect of administration of methanolic extract of Luffa cylindrica leaf on gamma glutamyl
aminotransferase activity in the liver, kidney, spleen and serum of mice
Values are means of 5 replicates ± SEM. Bars with different alphabets a, b, c and d means values are
significantly different (p < 0.05) from each other.

12
)
n
i
e
t
o
r
p
g
m
/
U
(
T
G
G
f
o
y
t
i
v
i
t
c
A
c
i
f
i
c
e
p
S
SpecificActivityofALP(U/mgprotein)
a
C o n t r o l
5 0 m g / k g b .w . o f E x t r a c t
1 0 0 m g / k g b .w . o f E x tr a c t
2 0 0 m g / k g b .w . o f E x tr a c t
4 0 0 m g / k g b .w o f E x tr a c t
8 0 0 m g / k g b .w . o f E x tr a c t
Figure 4: Effect of methanolic extract of Luffa cylindrica leaf on alkaline phosphatase activity in
the liver, kidney, spleen and serum of mice
Values are means of 5 replicates ± SEM. Bars with different alphabets a, b, c, d and e means values are
significantly different (p < 0.05) from each other.
DISCUSSION
The liver and kidney are vital organs of the body
which when in healthy state keeps the physiology
of the body balanced. However, an assault on the
integrity of both organs could trigger physiological
alterations that may result in pathological
conditions thereby affecting the well-being of an
organism. Renal-function indices such as urea and
creatinine, hepatic function indices such as
albumin, globulin, total and conjugated bilirubin
as well as biomarker enzymes like ALP, ALT and
AST are among the biochemical indices that can be
used to assess the functionality of the kidney and
27,28
liver . Biochemical investigation of hepatorenal
functional indices is important because kidney and
liver toxicity has been reported following the use
29,30
of chemical compounds or plant extracts . ALP,
ALT, AST and GGT are marker enzymes that are
present predominantly in tissues than in the serum.
When tissues are damaged, these enzymes leak out
of the plasma membrane of the tissues into the
extracellular fluid or serum and causing an upsurge
31
in serum concentration of these enzymes .
Therefore, serum enzyme assessments are
important tool in clinical diagnosis, providing
information on the effect and nature of pathological
damage to any tissue. The significant decrease (p <
0.05) in the activities of AST, ALT in the three
organs (kidney, liver and spleen), GGT in the liver
and spleen as well as ALP in the liver and kidney at
much higher dose especially 200, 400 and 800
mg/k b.w which resulted into a significant increase
(p < 0.05) of these enzymes in the serum may
indicate a compromise in the integrity or altered in
membrane permeability of the organs especially
the liver and kidney resulting in the loss of the
32
enzymes from the organs to the serum . This is
suggestive of the toxic effect of the extract on vital
organs. The GGT activity which concomitantly
increased in the kidney and serum could imply
physiological response to the effect of the extract
on the enzyme causing a de novo synthesis of the
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Journal of Malaria Research and Phytomedicine
33
enzyme molecule in the serum and kidney which
thus may have consequential effect on the amino
34
acid metabolism of the animals . Urea is a major
product of protein metabolism that is excreted in
35
kidney while creatinine is a product of muscle
metabolism that is also secreted by glomerular
36
filtration in the kidney and are both used to assess
the renal function. Though the extract did not show
any effect on the creatinine concentration
however, the significant decrease observed in the
serum urea concentration of rats does not imply a
healthy state of the kidney since activity of major
biomarker enzymes (ALT and AST) were found to
decrease in the kidney and increase in the serum,
thus could still suggest kidney dysfunction.
Similarly, the extract did not have a significant
effect on the serum albumin and total protein
concentrations of animals, the significant increase
in the total and conjugated bilirubin following the
administration of the extract may be an indication
of liver dysfunction caused by the extract. This
further buttress the alteration of the organs
integrity especially the liver since leakages of the
studied marker enzymes were observed from the
liver to the serum. Histological changes in tissues
are late manifestation of a chemical, physical,
mechanical or inflammatory assault on the tissue.
The presence of multinucleated giant cells (MGC)
in the spleen of mice administered methanolic
leaves extract of L. cylindrica at 100, 200, 400 and
800 mg/kg b.w but absent at doses of 50 mg/kg b.w.
for 7 days suggest the fusion of monocytes or
macrophages in the spleen since MGC originate
37
from fusion of monocytes or macrophages thus
imply that the extract had adverse effect on the
function and structure of the spleen at much higher
doses. Multinucleated giant cells (MGC) are a
common feature of granulomas that develop during
certain infections, the most prominent example
being tuberculosis, or as a consequence of foreign
38
body reactions . The photomicrograph of the liver
wh i c h r e v e a l s p e r i port a l l y m p h o c y t i c
inflammation in hepatocytes at 200, 400 and 800
mg/kg b.w following the administration of the
extract for 7 days further establishes the adverse
effect of the extract. Report has shown that cells
died as a result of necrosis or apoptosis when they
are challenged with toxins, noxious agents, or
39
injuries . Therefore, the various morphological
changes such as periportal inflammation in the
liver, multinucleated giant cells in the spleen and
peritubular lymphocytic infiltration in kidney
suggest that the extract adversely affect the normal
structural functioning of these vital organs at all the
doses investigated.

14
Table 1: Effect of administration of methanolic leaves extract of L. cylindrica on urea and creatinine of
mice
Treatment group Urea (mg/dl) Creatinine (mg/dl)
Control
30.90 ± 2.23a
14.63 ± 0.80a
50 mg/kg b.w
27.76 ± 1.85b
14.85 ± 0.41a
100 mg/kg b.w
28.51 ± 1.61b
14.46 ± 0.63a
200 mg/kg b.w
27.30 ± 1.35b
14.72 ± 0.80a
400 mg/kg b.w
26.72 ± 0.44c
14.65 ± 0.35a
800 mg/kg b.w
27.47 ± 1.08b
14.94 ± 0.73a
Values are means of 6 replicates ± SEM. Values with different alphabets a, b and c down the column
means values are significantly different (p < 0.05) from each other.
Table 2: Effect of administration of methanolic leaves extract of L. cylindrica on liver function indices
and total protein of mice
Treatment group Albumin
(g/dl)
To tal Bilirubin
(mg/dl)
Conjugated
Bilirubin (mg/dl)
To tal
Protein
Control 1.62 ± 1.02a 2.09 ± 1.07a 0.28 ± 1.01a 4.69 ± 1.13a
50 mg/kg b.w
1.49 ± 2.02a
1.23 ± 1.04b
1.70 ± 3.01b
4.64 ± 1.09a
100 mg/kg b.w
1.76 ± 1.05a
2.36 ± 3.06c
1.84 ± 3.03c
4.42 ± 1.14a
200 mg/kg b.w
1.47 ± 2.02a
2.69 ± 1.05c
1.86 ± 1.02c
4.58 ± 1.08a
400 mg/kg b.w
1.56 ± 3.05a
2.62 ± 2.03c
1.77 ± 2.22b
4.34 ± 2.06a
800 mg/kg b.w
1.49 ± 1.06a
3.94 ± 1.02d
1.70 ± 1.02b
4.48 ± 1.09a
Values are means of 6 replicates ± SEM. Values with different alphabets a, b, c and d down the column
means values are significantly different (p < 0.05) from each other.
Volume 4 No 1, 2020

CONCLUSION
Methanolic leaves extract of L. cylindrica caused
alteration in hepatorenal biochemical indices in
the studied rats, the study therefore suggest that the
consumption of this extract in managing disease
conditions like malaria in folk medicine may have
deleterious effect on vital organs of the body
particularly the spleen, liver and kidney at higher
dose.
A Publication of Centre for Malaria Research & Phytomedicine 15
Journal of Malaria Research and Phytomedicine
B
AC
D E F
MGC MGC
MGC
Plate 1: Photomicrograph of the spleen of mice administered various doses of methanolic extract of Luffa
cylindrica leaf for 7 days. A-F: Control, 50, 100, 200, 400 and 800 mg/kg b.w respectively (x 400). MGC
= Multinucleated giant cells, WP = White pulp; RP = Red pulp.

16
A B C
D E F
HP
BD
HA
PT
HV
SLS
PLI
PLI
PLI
Plate 2: Photomicrograph of the liver of mice administered various doses of methanolic extract of
Luffa cylindrica leaf for 7 days. A-F: Control, 50, 100, 200, 400 and 800 mg/kg b.w respectively (x
400). HV = Hepatic vein, PT = Portal tract, BD = Bile ductile, LI = Lymphocytic infiltration, PLI =
Periportal lyphotic inflammation, SLS = Spillage of lymphocytes.
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A Publication of Centre for Malaria Research & Phytomedicine 17
Journal of Malaria Research and Phytomedicine
PTLI
A B C
D E F
Plate 3: Photomicrograph of the kidney of mice administered various doses of methanolic extract of Luffa
cylindrica leaf for 7 days. A-F: Control, 50, 100, 200, 400 and 800 mg/kg b.w respectively (x 400). GL =
Glumeruli, TU = Tubules, PTLI = Peritubular lymphocytic infiltration.
Conflict of Interest
The authors declare there are no conflicts of interest
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