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]


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ISSN- 22315640 (Print) www.asianpharmaonline.org
ISSN- 22315659 (Online) 0974-3618


RESEARCH ARTICLE
Clinical Study of Preventive Potentials of Consumption of Buah naga
[Cactaceae] Against ParacetamolInduced Hepatotoxicity as well as the
Other Associated Biological Effects

Dr. Ahmad Zubaidi Bin A. Latif
1
, Dr. Mainul Haque
2
, C. Shanmugasundaram
3
,
Dr. U.S. Mahadeva Rao
4
*

1
Dean, Faculty of Medicine and Health Sciences, Universiti Sultan Zainal Abidin, Malaysia.
2
Professor, Faculty of Medicine and Health Sciences, Universiti Sultan Zainal Abidin, Malaysia.
3
Assistant Professor, Department of Biochemistry, RKM Vivekananda College, Chennai, India.
4
Associate Professor, Faculty of Medicine and Health Science, Universiti Sultan Zainal Abidin, Malaysia
*Corresponding Author E-mail: raousm@gmail.com / raousm@unisza.edu.my

ABSTRACT:
Objective: Preliminary studies on the effects of dietary supplementation with Buah naga or Red pitaya fruit on some
biochemical, and haematological parameters and histological examinations of liver, were investigated in albino rats in
which liver damage was induced by paracetamol (PAM). Experimental Design: Thirty six rats were divided into six
groups (including hepatotoxic and non hepatotoxic), and were fed with 5 and 10% Buah naga fruit extract
supplemented diet. Methods: Weight changes and parameters including alanine transaminase(ALT), aspartate
transaminase(AST), total protein, glucose, total triglyceride(TG), total cholesterol, reduced glutathione(GSH), lipid
peroxidation(LPO), packed cell volume(PCV), total and differential leucocyte count were determined using standard
methods. Results of Biochemical Parameters: The consumption of Buah naga supplemented diet gain the weight of
the animals as well as alteration in the levels of glucose, protein, ALT and AST in the hepatotoxic groups towards
normalcy. The GSH level were significantly increased (p<0.05) while TG were reduced in hepatotoxic group fed
extract supplemented diet. Glucose was significantly reduced to near normal (p<0.05) in the two treated groups.
Similar results observed in cholesterol and LPO status. Results of Haematological Parameters: WBC, Hb, and PCV
were significantly reduced in hepatotoxic groups and refurbished in treated animals. The hepatotoxic control had
significant reduction in neutrophils count and recuperated to near normal in treated rats. Histological Studies: The
necrotic effects of paracetamol seen in the abnormal histological changes were gradually regenerated to its native
architecture in the hepatotoxic treated groups. Conclusion: Thus the present study conjectured that Buah naga
consumption prevent or treat the PAM induced hepatotoxicity and associated other deleterious effects.

KEY WORDS: Buah naga, Paracetamol, Lipid peroxidation, Necrosis, Hepatotoxicity.

INTRODUCTION:
Liver is a vivacious organ that functions as metabolic centre
for various nutrients such as carbohydrates, proteins and
lipids [1]. It also takes part in metabolism of drugs,
xenobiotics and excretion of their waste metabolites from
the body and protects the organs against various
toxicants [2].



Received on 05.01.2012 Accepted on 08.02.2012
Asian Pharma Press All Right Reserved
Asian J. Res. Pharm. Sci. 2(1): Jan.-Mar. 2012; Page 16-23

It is well established that liver injury is caused by various
toxicants [3] such as certain chemotherapeutic agents (anti-
tubercular drugs, anti-HIV drugs and some antibiotics),
carbon tetrachloride, thioacetamide, chronic alcohol
consumption (e.g., liver cirrhosis) and microbes.

As herbal based therapeutic drugs has been popularized
worldwide for the treatment of liver disorders by leading
pharmaceutical industries and is worthwhile to search safe
hepatoprotective agents [4]. Most of the liver protective
plants may contain various biologically active
phytochemicals in it. Recently, investigators have reported
Asian J. Res. Pharm. Sci. 2012; Vol. 2: Issue 1, Pg 16-23 [AJPSci.]


17
about hepatoprotective activity of alkaloids [5], polyphenols
[6], glycosides [7,8]), carotenoids [9,10], coumarins [11]
and flavonoids [12].

Paracetamol (Acetaminophen) is one of the most widely
used pharmaceutical analgesic and antipyretic which causes
liver toxicity and damage due to excessive use or overdose
[13]. Paracetamol induced-hepatotoxicty in experimental
animals as well as human subjects is widely recognized and
reported [14-16].

Studies have shown that several plants have anti-
hepatotoxic properties that can protect animals from
paracetamol toxicity [17-18]. Due to its high tolerance and
its availability, over-the-counter misuse and overdose of
paracetamol is common and well recognized all over the
world [19]. However, the widespread consumption of plant
based diets such as Hylocereus polyrhizus may mask the
apparent hepatotoxicity from the misuse of paracetamol.

Selected species of the genus Hylocereus, which consists of
climbing three-ribbed stems and mostly white, fragrant,
nightblooming flowers, have been recently developed as
fruit crops [20, 21]. According to the color of the skin and
pulp of the fruit(Fig.1), these species were named as white
pitaya (Hylocereus undatus Britt & Rose, red skin, white
pulp), red pitaya (Hylocereus polyrhizus, red skin and red
pulp), yellow pitaya (Hylocereus megalanthus, yellow peel
and white pulp) [22, 23].
Fig.1





The red pitaya otherwise called as Red Pitahaya,
Dragonfruit, Night blooming Cereus, Strawberry Pear,
Belle of the Night, Conderella Plant, was reported to offer
many health benefits including cancer chemoprevention,
anti-inflammatory, anti-diabetic and cardiovascular
mortality risk reducing properties [24-26]

The fruits of red pitaya [Hylocereus polyrhizus (Weber)
Britton & Rose] have recently drawn much attention both
for their economic value and potential health benefits.
Previous studies reported on the content of betacyanins in
both flesh and peels [27]. These deep redpurple pigments,
with their stable appearance in a broad pH range, have a
great potential as natural coloring agents for a wide array of
food [28]. In addition, recent studies have focused attention
on their antioxidant activity, suggesting that these pigments
may provide protection against certain oxidative stress-
related disorders [29].

Hence, the aim of this present study was to investigate the
preventive potentials of consumption of fruit of red pitaya
against PAM induced liver poison through biochemical,
and haematological effects, as well as histological changes.

MATERIALS AND METHODS:
Sample preparation and processing
Red pitaya, Hylocereus polyrhizus, used in this study was
obtained locally from four lots of fruits, which were washed
and stored at -20
0
C before analysis. The fruit was peeled
prior to analysis.

Formulation of experimental diets: Three isocaloric and
isonitrogenous experimental diets namely control diet, 5
and 10% Buah naga supplemented diets were formulated.
The diets were formulated from commercially available
feed grade feedstuffs including maize, corn flour, fishmeal,
groundnut meal, bone meal and vitamin premix. The
control diet was formulated without the inclusion of H.
polyrhizus while the Buah naga supplemented diets were
incorporated with 5 and 10% of H. polyrhizus pulp.

Experimental design and paracetamol-induced
hepatotoxicity: The experimental design involved random
distribution of six rats each into six experimental groups
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18
namely non-hepatotoxic control (Group I), PAM induced
hepatotoxic control (Group II), non-hepatotoxic fed 5%
Buah naga supplemented diet (Group III), non-hepatotoxic
fed 10% Buah naga supplemented diet (Group IV),
hepatotoxic fed 5% Buah naga supplemented diet (Group
V), and hepatotoxic fed 10% Buah naga supplemented diet
(Group VI). The animals in non-hepatotoxic control and
hepatotoxic control groups were fed control diet while those
in non-hepatotoxic fed 5% Buah naga supplemented diet
and hepatotoxic fed 5% Buah naga supplemented diet.
The animals in non-hepatotoxic fed 10% Buah naga
supplemented diet and hepatotoxic fed 10% Buah naga
supplemented diet. All the animals were given the various
feed and water ad libitum for 56 days. Prior to the end of
feeding, animals in hepatotoxic control, hepatotoxic fed 5%
Buah naga supplemented diet and hepatotoxic fed 10%
Buah naga supplemented diet groups were orally
administered daily with 3 g kg-1 b.wt. of PAM for seven
days while those in non-hepatotoxic control, non-
hepatotoxic fed 5% Buah naga supplemented diet and non-
hepatotoxic fed 10% Buah naga supplemented diet groups
were administered with the vehicle (distilled water) only.
Weight changes of animals in all the groups were recorded
throughout the experiment. All the experiments were
performed under standard animal husbandry conditions and
after the protocols had been approved by the animal ethics
committee of the department of Bio-sciences.

Collection of blood and tissue samples: At the end of the
feeding and administration of PAM, the animals from each
group were anesthetized and blood samples were collected
in labeled sample bottles with drops of Ethylene diamine
tetra acetic acid (EDTA). Serum samples were collected in
sample bottles without EDTA and allowed to clot before
being centrifuged at 5000 rpm for 10 min. The livers of the
animals in all the groups were promptly excised soon after
sacrifice and stored in 10% formyl saline.

Determination of biochemical parameters: Glucose,
aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) were determined using test kits.
Total triglyceride and cholesterol were determined using
test kits produced by Linear chemicals SL, Spain. Total
protein was determined by the method described by [30]
Lowry. Reduced glutathione was determined using the
method of [31] Ellman. Lipid peroxidation was determined
by the thiobarbituric acid reactive substances (TBARS)
method [32].

Determination of haematological parameters: Full blood
count and white blood cells (WBC) differentials were
determined according to methods described by [33]Dacie
and Lewis. The haemoglobin and haematocrit estimations
were also carried out [34]

Histological study: Histological examinations were done
on sections of liver tissues from animals in the different
groups according to procedures described by [35]Disbrey
and Rack.
Expression of results and statistical analysis: Results
were expressed as MeanStandard Error of Mean (SEM)
for triplicate determinations. A One-way Analysis of
Variance (ANOVA) for a completely randomized design
was used to analyse experimental data. Values were
considered significant at p<0.05.

RESULTS:
Effect on body weight: The effects of feeding Red pitaya-
supplemented diet on weight prior to and after induction of
hepatotoxicity with paracetamol are shown in Table 1. Prior
to the induction of hepatotoxicity, Red pitaya-supplemented
diet caused varying percentage weight gains in all the
groups which were not statistically significant when
compared to the corresponding controls. In contrast,
significant weight loss were recorded in group II after
induction of hepatotoxicity in groups except the non-
hepatotoxic group fed 5% Buah naga supplemented diet.

The effect on biochemical parameters: The effect of Red
pitaya-supplemented diet on the biochemical parameters in
non-hepatotoxic and hepatotoxic rats are presented in Table
2, the levels of the liver enzymes were not significantly
altered except AST that was significantly increased
(p<0.05) in the non-hepatotoxic group fed 5% Red pitaya-
supplemented diet. Serum ALT levels were markedly
elevated in group II and slight increase in group III while
the group VI favored steep fall and group V with moderate
decline in their levels w.ref.t. group I. Blood glucose
concentration was significantly reduced (p<0.05) in
hepatotoxic groups fed 5 and 10% Red pitaya-supplemented
diet w. ref. t. group II. There were slight fall in the protein
and increase in cholesterol levels in all the hepatotoxic
groups. Triglyceride concentration was significantly
increased (p<0.05) in the hepatotoxic group and reduced in
the hepatotoxic extract treated groups. Reduced glutathione
was significantly increased (p<0.05) only in the hepatotoxic
group fed 10% Red pitaya supplemented diet. There was
moderate to marked fall in levels in 5 and 10 % extract fed
hepatotoxic group animals (Group V & VI).

Effect on haematological parameters: The result of
feeding Red pitaya-supplemented diet on haematological
parameters in control and paracetamol-induced hepatotoxic
rats are presented in Table 3.The PCV was significantly
reduced (p<0.05) only in the hepatotoxic group fed Red
pitaya supplemented diet while neutrophils were also
reduced only in the hepatotoxic control.

Effect on liver tissues: The histological features of the
liver of the animals in all the groups are shown in Plates 1-
6. The liver sections of the rats in the control and non-
hepatotoxic groups fed with Red pitaya-supplemented diet
(Plates 1-3) showed less disarrangement and degeneration
of hepatocytes, indicating marked preservation of hepatic
architecture. The liver sections of the rats in the control
hepatotoxic group (Plate 4) showed disarrangement and
degeneration of normal hepatic cells with intense
centrilobular necrosis, sinusoidal hemorrhages and
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19
dilatation. There was also inflammatory cell infiltrate in the
portal tracts. However, Plates 5 and 6 shows that the
intensity of centrilobular necrosis was less in the liver
sections of the hepatotoxic groups fed extract supplemented
diet indicating marked regeneration. In histopathological
examination of the liver tissues also revealed the dose-
dependent protection effect of extract in treatment groups
from liver damage, when compared with PAM toxicated
group. Change in liver histology, such as fatty liver change,
degeneration of central hepatic vein, hepatocyte
proliferation, necrosis, inflammation, lymphatic infiltration
and in sinusoidal irregularities were reduced by the
treatment of extract. Maximum protection from liver
damage in animals was observed at higher dose. All
biochemical findings of liver functions were supported by
the positive results of histopathological study

DISCUSSION:
Paracetamol (Acetaminophen or n-acetyl-p-aminophenol), a
commonly used analgesic drug has the potential to cause
centrilobular hepatic necrosis in experimental animals and
in humans [36-38]. Damage to the liver or hepatotoxicity,
does not result from paracetamol itself, but from one of its
metabolites, N-acetyl-p-benzoquinoneimine (NAPQI) [39].
NAPQI is a highly reactive toxic and cytotoxic intermediate
metabolite, which is damaging to cell components if not
detoxified by conjugation with glutathione (GSH). NAPQI
can rapidly react with reduced glutathione (GSH) and lead
to a 90% total hepatic GSH depletion in the cells and
mitochondria, which can result in hepatocellular death and
mitochondrial dysfunction [40].

Phytochemical products including plant herbs and extracts
have been used for centuries to promote liver health.
Although the exact mechanisms behind this protection are
uncertain, many theories have been proposed. Paracetamol
is being used extensively to investigate hepatoprotective
activity of different treatments on various experimental
animals [41]. It is selected as hepatotoxicant in inducing
injury to the liver as it is known to cause hepatotoxicity in
man and experimental animals when taken overdose [42].

Red pitaya is highly valued as a nutritious, medicinal and
therapeutic fruit across Malaysia. The major purpose of this
investigation was to study the effect of Red pitaya on
paracetamol- induced hepatotoxicity and some associated
parameters in rats. Aspartate transaminase (AST) and
alanine transaminase (ALT) were used as parameters for
assessing of liver toxicity, while total protein, triglycerides,
cholesterol and glucose were used as supplementary tests
for hepatic synthetic and other allied functions. Liver
histopathology served as the most important tool for
identifying and characterizing liver injury. The feeding of
Red pitaya-supplemented diet of different concentration (5
and 10%) showed a general non-significant (p<0.05)
increase in weight of animals. The increase in weight
indicates that the fruit of Red pitaya was not toxic to the
animals and could be attributed to their content of nutrients
such as proteins, carbohydrates, lipids, minerals and
vitamins which are needed for growth, body repair and
maintenance [43,44]. Thus, the fruit could be a valuable and
viable source of bioactive nutrients and non-nutrient
substances with potential hepatoprotective properties.

Increases in activities of liver enzymes such as alanine
transaminase and aspartate transaminase are roughly
proportional to the extent of liver tissue damage [45].
Generally the consumption of Red pitaya-supplemented diet
did not significantly (p<0.05) change any of the liver
enzymes in the animals which shows that Red pitaya does
not have any noticeable or apparent toxic effect on the liver.
Total protein levels are rough measures of protein status but
reflect major functional changes in liver functions [46]. In
this research there was a non-significant change (p<0.05) in
the protein level of hepatotoxic animals, which could be
due to stabilization in protein synthesis secondary to a
decreased amount and availability of mRNA in the liver and
this could indicate liver dysfunction [47].

Oxidative stress caused by Reactive Oxygen Species (ROS)
plays a central role in hepatotoxicity [48]. GSH is an
important antioxidant and free radical scavenger that has the
ability to combat Reactive Oxygen Species (ROS) in the
liver [49]. However, during paracetamol poisoning, NAPQI
depletes markedly hepatocellular levels of reduced
glutathione making the hepatocytes susceptible to its the
toxic effects [50]. The result of this study showed that there
was a significant increase (p<0.05) in the level of reduced
glutathione in the hepatotoxic animals fed with the 10%
Red pitaya-supplemented diet. This suggests that increase
in quantity of Red pitaya consumed may improve
hepatoprotection during paracetamol-induced toxicity [51,
52, and 29].

Excess reduction of hepatic glutathione concentration
follows paracetamol challenge and is associated with
heightened lipid peroxidation via free radical damage and
directly damages cells in the liver [36, 39]. There was a
stabilization in the level of lipid peroxidation of hepatotoxic
control animals and those fed Red pitaya-supplemented diet
which could be attributed to the presence of antioxidant
phytochemicals including phenolic substances, flavonoids
and anthocyanidins in the Red pitaya whose phenolic
structure favor their reaction with free radicals and Reactive
Oxygen Species (ROS)[53-55].

The reduction (p<0.05) in the levels of glucose in
hepatotoxic rats fed Red pitaya-supplemented diet suggests
that consumption of Red pitaya could reduce the blood
concentration of glucose in the case of hyperglycemia seen
in disease conditions such as diabetes with associated liver
damage.

There were also non-significant changes (p<0.05) in the
levels cholesterol in the groups. This supports research
suggesting that Red pitaya may have cholesterol reducing
properties that is important in preventing atherosclerosis
[56]. The increase in triglycerides of hepatotoxic animals
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20
that consumed 10% Red pitaya-supplemented diet may be
due to liver dysfunction that causes their excessive
production while their reduction in the non-hepatotoxic
group is attributed to the hypolipidemic properties of fruit
diets.

Haematological parameters namely PCV, WBC and
differentials were monitored in this study because of their
diagnostic significance and role in providing information
concerning haematological changes caused by paracetamol-
induced toxicity [57]. Most phytochemical constituents of
plant foods affect the immune system and other
haematological parameters [58]. The increase in Hb and
PCV (p<0.05) in hepatotoxic rats fed 10% Red pitaya-
supplemented diet reflects the results obtained from the
consumption of noni [59] and avocado [60]. This may be
due to the presence of Hematinic factors in Red pitaya such
as iron which plays a role in iron metabolism that increases
the level of PCV and synthesis of hemoglobin [61-63]. The
non-significant negative change in the level of white blood
cells of the animals could be attributed to a rare case of
hematologic side effects called thrombocytopenia often
associated with paracetamol overdose. The cell membrane
damage associated with inflammation results in leucocyte
release of lysosomal enzymes that can be injurious to
nearby cells [64]. Stimulation of neutrophils can lead to the
production of oxygen derived free radicals that produce
further cellular damage. The increase in the levels of
lymphocytes and neutrophils level of the hepatotoxic rats
and those fed Red pitaya-supplemented diet supports the
study done by [65] Duthie et al., which stated that
antioxidant phytochemicals that can be found in Red pitaya
are known to protect them. The phytochemical constituents
of Red pitaya which include flavonoids and phytosterol are
possible candidates that increase white blood cells.

The liver of non- hepatotoxic animals fed with Red pitaya-
supplemented diet showed normal histological features.
This result shows that the consumption of Red pitaya-
supplemented diet does not have any apparent toxicity on
the liver of rats. The necrotic effects of paracetamol seen in
the abnormal histological changes in the liver of the animals
is similar to that gotten by Hewawasam et al.[66] where
Epaltes divaricata plant extract against carbon tetrachloride
induced hepatotoxicity. However the consumption Buah
naga-supplemented diet reduced this necrosis indicating
some level of hepatoprotective and regenerative properties.

CONCLUSION:
The results of this study suggest that
1. Buah naga may be used in the treatment or prevention
of paracetamol-induced hepatotoxicity probably due to
its ability to preserve the natural integrity of
hepatocytes when challenged with hepatotoxicants.
2. It is also revealed the dose-dependent protection effect
of Buah naga fruit extract in treatment groups from
liver damage. i.e. the 10% extract exhibits better
therapeutic efficacy than the other one.
3. Further study is suggested to be carried out to help
unravel the precise mechanism(s) for paracetamol-
induced toxicity and the specific constituents of Buah
naga involved in hepatoprotection against liver poison.
4. It could also be speculated that the observed
hepatoprotective effects of Buah naga fruit extract
might be related to the rich phytochemicals such as
flavonoids, polyphenols, alkaloids, steroids, amino
acids, and vitamins with strong antioxidant properties.
5. The process of extraction and identification of active
principles responsible for the observed
pharmacological actions of Buah naga fruit through
bioactivity guided fraction is under progress to
understand the possible mechanism of action of Buah
naga fruits.



Table 1: Weight Change profile in Hepatotoxic and Non-Hepatotoxic Rat Administrated with Buah naga supplemented diet
Weight change Group I Group II Group III Group IV Group V Group VI
Prior to hepatotoxicity (%) 40.1 51.2 41.5 41.5 43.4 43.7
After hepatotoxicity (%) 40.1 -10.3 6.4 8 -6.1 -2.5

Table 2 : Biochemical Profile in Control and Experimental Group Rats Fed Buah naga Fruit Extract Supplemented Diet
Blood/serum parameter Group I Group II Group III Group IV Group V Group VI
Glucose ( mg/dL) 120.18.22 196.546.47 141.067.31 121.9110.52 152.018.96 129.5613.6
Protein (mg/mL) 6.960.14 4.720.90 6.860.30 6.990.01 5.010.31 5.910.76
GSH (mmol/mL) 0.01080.001 0.00960.006 0.01140.0052 0.01380.0016 0.01010.004 0.01470.092
Cholesterol (mg/dL) 180.161.21 292.895.47 176.538.01 164.644.77 274.013.77 214.899.83
TG (mg/dL) 165.726.01 243.896.99 169.881.001 153.868.01 222.493.68 196.876.77
LPO (mmol/mL) 13.510.93 24.111.01 13.011.96 12.950.001 20.010.86 17.361.52
AST (IU/L) 13.771.01 26.062.71 13.710.05 13.011.68 24.371.68 60.220.98
ALT (IU/L) 15.882.71 36.113.78 16.011.07 15.320.01 31.844.06 20.143.33


Table 3: Hematological Profile Status in Control and Experimental Groups of Rat Fed Buah naga Supplemented Diet
Parameter Group I Group II Group III Group IV Group V Group VI
Hemoglobin (g/dL) 13.110.67 10.881.11 13.890.01 13.961.63 11.371.79 12.830.11
Hematocrit (%) 39.331.84 32.062.04 39.120.05 39.560.50 35.011.69 38.070.13
Leukocyte (10/mm) 650088.61 500010.87 6500128.65 675060.17 5400121.06 610082.64
Lymphocyte (%) 24.261.76 31.001.76 24.150.79 23.991.67 29.010.19 26.792.86
Polymorphs (%) 62.731.95 50.981.66 62.110.78 64.971.44 54.723.01 59.872.01
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Plate 1. Non-hepatotoxic rat fed with control diet showing normal
architecture of liver tissues (MAG. X 100)


Plate 2. Section of liver of non-hepatotoxic rat fed with 5%Buah naga
extract supplemented diet (MAG. X 100)

Plate 3. Section of liver of non-hepatotoxic rat fed with 10%Buah naga
extract supplemented diet (MAG. X 100)

Plate 4. Section of hepatotoxic rat liver fed with control diet showing
congested central vien and vaculation of hepatocytic nuclei. (MAG. X
100)

Plate 5. Section of liver tissue from 5% Buah naga extract treated
hepatotoxic rat showing more or less normal architecture with
concentric arrangement of the hepatocytes around the central vein
(MAG. X 100)


Plate 6. Section of liver tissue from 10% Buah naga extract treated
hepatotoxic rat showing marked recvery towards normal architecture
with concentric arrangement of the hepatocytes around the central vein
(MAG. X 100)

Histological observation of hepatocytes of control and
experimental group of rats

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