There are many taboos, rumors, myths, and
misconceptions which are ingrained in the deve
-loping countries, most often due to underlying
cultural, political, educational, economical and
environmental factors that determine the
complex human behaviors, including food
consumption practices.1 There are many food
taboos also in Bangladesh among them, related
to pineapple fruits, have two food taboos: a)
Eating pineapple during pregnancy results
miscarriage and b) Eating milk and pineapple
together cause toxicity, even death. To clarify
about the first one, only one experimental study
had been done.2 However, to the best of our
knowledge, no experimental study regarding
this second food taboo was found.
Milk is regarded as a complete food in a human
diet. It provides all the nutrients essential for
the nourishment of the human body.3 Pineapple
(Ananas comosus) fruit is a good source of
various vitamins like A, B, C; minerals like
calcium, phosphorus, iron, and enzymes.1 It
also contains tannins, cardenolides, dienolides,
cardiac glycoside and flavonoids (bromelain).2,4
The individual food is healthy and non-toxic.
Why would it be toxic together? If so, what
types of toxicity (hepatotoxicity, neurotoxicity,
hemotoxicity, etc) it causes? Still, it remains
mysterious. Therefore, the purpose of this
study was to assess whether potential toxic
interactions occur between the pineapple and
milk following oral administration in rats by
evaluating the clinical signs, hematological and
biochemical parameters, gross and microscopic
findings by comparing with the normal control
and toxic standard group.
Materials and Methods
Preparation of pineapple and milk mixture
Pineapple fruits (Jinwon Trading, Co. Korea)
and pasteurized milk (Seoul milk®, Korea)
were bought from the local market of Guri City,
South Korea. Fruits were cut into small pieces
and weighed. Then juice was made by electric
blender without mixing water and suck by
mesh. Two types of pineapple and milk
solutions were made by mixing juice and milk
by the ratio of 1:1 (PMS1) and 2:1 (PMS2).
A total of 40 male Sprague-Dawley rats (375–
448 g, Orient Bio, Korea) were used for this
study. The rats were housed in an environment
with a controlled temperature (23 ± 2°C) and
humidity (50 ± 5%) with a 12 to 12 hours light-
dark cycle. Food and water were available ad
libitum before started the experiment. After 7
days acclimatization, rats were equally divided
into four groups (n=10): a) Normal control
group (treated with only vehicle 12 mL/kg
body weight), b) Toxic standard group (carbon
tetrachloride, CCl4), c) PMS1 group (treated
with PMS 1:1, 12 mL/kg body weight) and d)
PMS2 group (treated with PMS 2:1, 12 mL/kg
body weight). The design of CCl4-induced
| Original | Article |
The purpose of this study was to assess whether potential toxic interactions occur between the
pineapple and milk following oral administration in rats by comparing clinical signs,
hematological and biochemical parameters with the normal control and toxic standard groups.
Pineapple and milk solutions were made by 1:1 (PMS1) and 2:1 (PMS2) ratio, administered 12 mL/
kg body weight. Forty rats were equally divided into 4 groups treated for 3 days: a) normal control
(only vehicle treated); b) toxic standard (CCl4 was suspended in corn oil, 20% v/v; treated 1.25
mL/kg), c) PMS1 and d) PMS2 groups. CCl4 administration altered the normal behavior, changes
gross and microscopic morphology. Toxicity related hematological and serum biochemistry
changed significantly (p<0.05) than the normal group. However, all these clinical and pathological
changes were completely absent in PMS treated groups. These results suggest that taking
pineapple with milk is not toxic and this food taboo is wrong.
Food taboo of taking pineapple and milk at a time
Md. Mahbubur Rahman
toxicity such as dosage and timing of CCl4 were
adapted and modified as described previously.5 The
CCl4 was suspended in corn oil (20% v/v) and
administered orally (1.25 mL/kg body weight). The
control animals received saline (12 mL/kg) orally at
12 hours intervals up to 72 hours.
Lethality and behavioral observation
Visual physical examination of the rat was
performed carefully before starting the experiment
to ensure a good state of health. The rats were
closely observed for any indication of toxicity effect
within the treatment period and finally at the 72th
hours before sacrifice. Visual observations included
checking mortality, rectal temperature, behavioral
changes (weakness, aggressiveness, food or water
refusal), diarrhea or loose feces, salivation, dischar-
ge from eyes and ears, noisy breathing and clonic
convulsion were recorded at 0, 24, 48 and 72 hours.
After 72 hours of treatment, the rats were sacrificed
by anesthesia and the blood (collected caudal from
vena cava), stomach, liver, heart and kidney were
collected for gross and histological analysis.
Whole blood was collected into the test tube
containing the anticoagulant, ethylenediamine tetra-
acetic acid (EDTA). Hematological and biochemical
changes were measured by a fully-automated
hematology system (ADVIR 2120i, Siemens).
Blood samples for other biochemical analysis were
collected into plain sample tubes. The serum was
separated by centrifugation at 3,000 rpm for 10 min
and stored at –20°C until analysis. Serum creatinine
kinase, aspartate aminotransferase (AST), alanine
aminotransferase (ALT), lactate dehydrogenase,
total bilirubin, direct bilirubin, creatinine, blood
urea nitrogen and uric acid levels were determined
with a Hitachi 7180 instrument (Hitachi, Japan).
Serum concentration levels of TNF-α protein levels
were measured with TNF-α (rat) enzyme-linked
immunosorbent assay kit (ALPCO Diagnostics,
USA) and IL-6 were measured with rat IL-6 ELISA
(ALPCO Diagnostics, USA) according to the
For histological analysis, the liver and kidney were
dissected from all of the study groups at the end of
experiment period. The tissues were washed in
normal saline, cut into pieces of the desired size,
and fixed in 10% neutral buffered formalin solution.
After fixation, the samples were cleaned and
embedded in paraffin. Tissue sections of 5 µm
thickness were mounted on slides, stained with
hematoxylin-eosin (H-E), and examined under a
Differences between groups were evaluated by
analysis of variance (ANOVA) with the Student’s t-
test versus control group using Prism 5.03
(GraphPad Software Inc., USA).
Lethality and behavioral observation
Mortality was not observed in all the experimental
groups during the study period. After the
administration of CCl4, the rectal temperature was
increased significantly (p<0.01) from 48 hours to
end of the experiment and decreased the amount of
feed and water intake (Table I). Restlessness,
weakness and diarrhea also observed in toxic
standard group. However, all of these clinical signs
were absent in the normal control, PMS1 and PMS2
groups. Other clinical sings such as salivation,
discharge from eyes and ears, noisy breathing,
convulsion and tremor all were absent in all groups.
Organ and body analysis
There were no significant differences in the mean
body weight of rats among groups. The relative
changes of the weight of stomach, liver and kidney
to the body weight increased significantly than the
normal control group but the PMS administered
groups had no change. No remarkable changes
6 BSMMU J 2017; 10: 5-10
Observation of clinical sings and behavioral changes of normal and
0 33.1 ± 0.3 32.9 ± 0.5 33.4 ± 0.3 32.6 ± 0.3
24 33.0 ± 0.3 33.0 ± 0.6 33.5 ± 0.3 32.7 ± 0.4
48 32.8 ± 0.2 34.8 ± 0.6a 32.8 ± 0.3 32.6 ± 0.3
72 33.0 ± 0.3 36.0 ± 0.8b 33.5 ± 0.3 32.7 ± 0.3
Restlessness 0 - - - -
24 - + - -
72 - - -
Weakness 0 - - - -
24 - + - -
72 - +- - -
0 - - - - Diarrhea or
loose feces 24 - - - -
72 - + - -
PMS1 group treated with mixed solution (1:1) 12 mL/kg body weight and PMS2 group treated
with mixed solution (2:1) 12 mL/kg body weight. ap<0.05, Bonferroni post hoc test following one
-way ANOVA versus the normal control group; ap<0.05; and bp<0.01; Bonferroni post hoc test
following one-way ANOVA versus the carbon tetrachloride group. ‘+’ is presence of clinical sign
and ‘-’ is absence of clinical signs; Data are mean ± SD
were found in the weight of heart among all groups
There were significantly increased in leucocyte,
lymphocyte, neutrophil, basophil and monocyte in
the toxic standard group compared to the normal
group. But PMS1 and PMS2 groups showed no
statistically significant (p>0.05) difference in the
hematological parameters than the normal control-
group (Table III).
Cytosolic enzymes (ALT, AST, lactate dehydro-
genase and creatinine kinase), direct bilirubin, total
bilirubin, blood urea nitrogen, creatinine and uric
acid were markedly increased (p<0.001) in the CCl4
group than the normal control group (Figure 1).
However, no alteration was observed in the
pineapple and milk treated groups. As shown in
Figure 2, serum inflammatory cytokines (TNF-α
and IL-6) also significantly increased (p<0.001) in
the CCl4 group than the normal control group. But
no changes were found in the PMS administered
In the normal control rats, histological analysis
revealed normal hepatic and renal cells (Figure 3).
Conversely, CCl4-induced toxicity rats exhibited
extensive necrosis and loss of architecture of
hepatocytes, slight hydropic degeneration, apop-
totic nuclei, occasional bi-nucleation, cellular infil-
tration, hemorrhage and congestion. Histological
analysis of PMS treated rats was like vehicle-treated
The food taboo is that eating pineapple and milk
together induce toxicity but what types of toxicity
(hepatotoxicity, neurotoxicity, hemotoxicity etc) it
causes, still not known. So, a specific standard
group was not able to make. However, CCl4 is well
known toxic chemical. So, CCl4 was used as a toxic
standard in this experiment. After 4 times adminis-
tration of CCl4, acute toxicity were occurred which
were represented by the alteration of normal
behavioral, significant increased of rectal tempera-
ture and at the end of experiment significant
elevation of hematological (leucocyte, lymphocytes,
monocytes, basophils and neutrophils) parameters,
serum cytosolic enzymes (ALT, AST, lactate dehy-
drogenase and creatinine kinase), direct bilirubin,
total bilirubin, blood urea nitrogen, creatinine and
uric acid and serum inflammatory cytokines (TNF-α
and IL-6) were found. These are consistent with
other reports of CCl4-induced acute toxicity.5-7
Interestingly, no clinical signs, pathological or bio-
chemical changes related to toxicity were observed
in pineapple and milk (different doses) at a time
administered group. These indicated that this
mixture is non-toxic.
This is common food taboo in Bangladesh often
reported in the Bangladeshi newspaper.8 However,
many favorite foods and recipe are made mixing
milk and pineapple in many countries.9-11 Moreover,
pineapple and its constituents exert anti-oxidative,
anti-inflammatory, anti-cancer, hepatoprotective
and neuroprotective effects.12-16
Effect of pineapple and milk mixed solution on body weight and rel-
ative organ weight of rat
BBW (g) 419 ± 7 423 ± 5 418 ± 8 418 ± 5
FBW (g) 419 ± 6 419 ± 6 418 ± 8 419 ± 5
Stomach weight (%) 0.4 ± 0.0 0.5 ± 0.0a 0.4 ± 0.0 0.4 ± 0.0
Liver weight (%) 2.8 ± 0.1 3.1 ± 0.1a 2.7 ± 0.1 2.8 ± 0.1
Kidney weight (%) 0.3 ± 0.0 0.3 ± 0.0a 0.3 ± 0.0 0.3 ± 0.0
Heart weight (%) 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0
Feed (g)/kg BW/day 111.9 ± 3.2 31.3 ± 3.2b 118.1 ± 3.7 112.2 ± 4.0
Water (mL)/kg BW/day 120.2 ± 3.1 87.9 ± 4.1b 123 ± 3.2 127.6 ± 4.1
PMS1 group, treated with mixed solution (1:1) 12 mL/kg body weight and PMS2 group, treated
with mixed solution (2:1) 12 mL/kg body weight. bp<0.001, Bonferroni post hoc test following
one-way ANOVA versus the normal control group; ap<0.05; bp<0.01; and bp<0.001; Bonferroni
post hoc test following one-way ANOVA versus the CCl4 treated group; Data are expressed as
means ± SEM
Effect of pineapple and milk mixed solution on hematological param-
eters of rat
Red blood cell (×106/mL) 8.2 ± 0.3 8.3 ± 0.3 8.3 ± 0.4 8.1 ± 0.3
Hematocrit (%) 44.3 ± 0.4 45.6 ± 0.5 44.3 ± 0.3 44.5 ± 0.3
Hemoglobin (g/100 mL) 14.5 ± 0.3 14.8 ± 0.4 15.0 ± 0.3 14.6 ± 0.3
MCV (fL) 55.9 ± 0.8 56.2 ± 0.5 55.6 ± 0.6 55.0 ± 0.7
MCH (pg) 18.8 ± 0.2 19.1 ± 0.3 18.5 ± 0.2 18.5 ± 0.3
MCHC (mmol/L) 34.7 ± 0.3 34.8 ± 0.4 34.0 ± 0.9 35.3 ± 0.4
White blood cell (×103/mL) 11.6 ± 0.3 13.5 ± 0.5b 11.7 ± 0.3 11.7 ± 0.2
Neutrophils (%) 9.6 ± 0.3 11.8 ± 0.8a 9.4 ± 0.3 9.8 ± 0.3
Basophil (%) 0.5 ± 0.0 0.7 ± 0.1a 0.5 ± 0.0 0.6 ± 0.0
Eosinophil (%) 1.3 ± 0.0 1.3 ± 0.0 1.2 ± 0.0 1.3 ± 0.0
Lymphocyte (%) 88.8 ± 0.3 91.0 ± 0.8a 88.7 ± 0.3a 89.1 ± 0.3
Monocyte (%) 2.5 ± 0.0 2.6 ± 0.0a 2.6 ± 0.0 2.5 ± 0.0
Platelets (×109/L) 794 ± 8 785 ± 8 787 ± 6 794 ± 7
PMS1 group, treated with mixed solution (1:1) 12 mL/kg body weight and PMS2 group, treated
with mixed solution (2:1) 12 mL/kg body weight. ap<0.05, bp<0.01, Bonferroni post hoc test
following one-way ANOVA versus the normal control group. MCV, mean corpuscular volume;
MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration;
Data are expressed as means ± SEM
BSMMU J 2017; 10: 5-10 7
Any types of toxicity cause injury to the cells.17-18 As
consequences of injury, cytosolic enzymes are leak
out to the blood stream and increased their normal
level.19 Like this study, other studies have reported
that CCl4 intoxication elevated serum ALT, AST,
lactate dehydrogenase, creatinine kinase, direct
bilirubin, and total bilirubin levels following acute
liver injury.5-7,18,20 Additionally, increases in serum
creatinine, blood urea nitrogen and uric acid levels
are all indicative of kidney toxicity. Both acute and
chronic CCl4 administration can alter the kidney
function and has been shown to reduce renal
function by promoting interstitial edema and
nephritis,21 which were absent in PMS administered
groups. Inflammation is a complex biological
response to injury as a result of different stimuli
such as pathogens, damaged cells, or irritants.22
Moreover, TNF-α and IL-6 are considered as
Figure 2: Effect of pineapple and milk mixed solution on serum inflammatory cyto-
NC, normal control group treated with only vehicle; TS group, toxic standard group, CCl4
treated group, 12 mg/kg body weight; PMS1 group, treated with mixed solution (1:1) 12 mL/
kg body weight and PMS2 group, treated with mixed solution (2:1) 12 mL/kg body weight. a:
p<0.001, Bonferroni post hoc test following one-way ANOVA versus the NC group. TNF-α tu-
mor necrotic factor-alpha, IL-6 interleukin-6
Figure 1: Effect of pineapple and milk mixed solution on serum biochemical changes
NC, normal control group treated with only vehicle; TS group, toxic standard group, CCl 4 treated group, 12 mg/kg body weight; PMS1 group, treated with
mixed solution (1:1) 12 mL/kg body weight and PMS2 group, treated with mixed solution (2:1) 12 mL/kg body weight. a: p<0.01, b: p<0.001, Bonferroni post hoc
test following one-way ANOVA versus the NC group. ALT alanine aminotransferase, AST aspartate aminotransferase, CK creatinine kinase, LDH lactate dehy-
drogenase, CRE creatinine, BUN blood urea nitrogen and UA uric acid
8 BSMMU J 2017; 10: 5-10
indications of hepatotoxicity experimental model of
liver injuries.6,7 Inflammatory cytokines and infla-
mmation related blood cells were also not found in
the PMS-treated groups which were extremely
elevated in the toxic standard group when compa-
red with normal.
It might be due to the absence of tissue injury in
PMS-treated groups which further confirmed by
histopathological examination. Microscopically, it
was found that necrosis of tissue and infiltration of
inflammatory in hepatic and renal tissues in the
CCl4 group, which was absent in the normal control
and PMS groups and showed normal architectures
of cells and tissues. The relative change in liver and
kidney weight to the body weight were increased
markedly in the toxic standard group, might be
attributed to increased infiltration of neutrophils,
edematous cellular space and increased protein
content due to tissue injuries or necrosis evidenced
by histopathology. These postmortem findings are
absent in PMS-treated group which furthermore
confirmed this mixture is not toxic. Logically,
altered behavioral changes and the high rectal
temperature were found in the CCl4 group while in
PMS-treated groups it was like normal and
indicating that this foods combination is safe.
Evaluating the toxicity related clinical signs, hema-
tological and biochemical parameters, gross and
microscopic findings propose that taking pineapple
and milk at a time is non-toxic. So, this food taboo
in Bangladesh is wrong.
All experimental protocols employed herein were
approved by the committee on the care of laboratory
animal resources, Knotus Pvt, Co., Ltd, Korea,
(Certificate number: IACUC 16-KE-104) and were
conducted in accordance with the Guide for the Care
and Use of Laboratory Animals published by the US
National Institute of Health (NIH Publication No. 85-
23, revised 1996).
Conflict of interest
Author has no conflict of interest.
This research was supported in part by the research
fund of Knotus Co. Ltd.
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Figure 3: Effect of pineapple and milk mixed solution on hepatic (left) and renal (right) micrographs
NC, normal control group treated with only vehicle; TS group, toxic standard group, CCl4 treated group; PMS1 group, treated with mixed solution (1:1) 12 mL/
kg body weight and PMS2 group, treated with mixed solution (2:1) 12 mL/kg body weight
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