Food taboo of taking pineapple and milk at a time

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 (CCl 4 was suspended in corn oil, 20% v/v; treated 1.25 mL/kg), c) PMS1 and d) PMS2 groups. CCl 4 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.


Introduction
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. 1There 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. 2However, 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. 3Pineapple (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, 4he 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.

Preparation of pineapple and milk mixture solution
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).

Experimental animals
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 lightdark cycle.Food and water were available ad libitum before started the experiment.

Abstract
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.

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 72 th hours before sacrifice.Visual observations included checking mortality, rectal temperature, behavioral changes (weakness, aggressiveness, food or water refusal), diarrhea or loose feces, salivation, discharge from eyes and ears, noisy breathing and clonic convulsion were recorded at 0, 24, 48 and 72 hours.

Sample collection
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.

Hematological parameters
Whole blood was collected into the test tube containing the anticoagulant, ethylenediamine tetraacetic 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 manufacturer's protocol.

Histological analysis
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 light microscope.

Statistical analysis
Differences between groups were evaluated by analysis of variance (ANOVA) with the Student's ttest 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

Hematological parameters
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 controlgroup (Table III).

Serum biochemicals
Cytosolic enzymes (ALT, AST, lactate dehydrogenase 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 groups.

Histological analysis
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, apoptotic nuclei, occasional bi-nucleation, cellular infiltration, hemorrhage and congestion.Histological analysis of PMS treated rats was like vehicle-treated control rats.

Discussion
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 administration of CCl4, acute toxicity were occurred which were represented by the alteration of normal behavioral, significant increased of rectal temperature and at the end of experiment significant elevation of hematological (leucocyte, lymphocytes, monocytes, basophils and neutrophils) parameters, serum cytosolic enzymes (ALT, AST, lactate dehydrogenase 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 biochemical 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. 8However, many favorite foods and recipe are made mixing milk and pineapple in many countries. 9-11Moreover, pineapple and its constituents exert anti-oxidative, anti-inflammatory, anti-cancer, hepatoprotective and neuroprotective effects. As consequences of injury, cytosolic enzymes are leak out to the blood stream and increased their normal level. 19Like 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, 20Additionally, 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. 22oreover, TNF-α and IL-6 are considered as indications of hepatotoxicity experimental model of liver injuries. 6, 7Inflammatory cytokines and inflammation related blood cells were also not found in the PMS-treated groups which were extremely elevated in the toxic standard group when compared 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.

Conclusion
Evaluating the toxicity related clinical signs, hematological 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.

Figure 2 : 6 Figure 1 :
Figure 2: Effect of pineapple and milk mixed solution on serum inflammatory cytokines 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 tumor necrotic factor-alpha, IL-6 interleukin-6

Table I Observation of clinical sings and behavioral changes of normal and experimental rats
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,Bonferronipost hoc test following one -way ANOVA versus the normal control group; a p<0.05; and b p<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 (TableII).

Table II Effect of pineapple and milk mixed solution on body weight and rel- ative organ weight of rat
) 12 mL/kg body weight.b p<0.001,Bonferroni post hoc test following one-way ANOVA versus the normal control group; a p<0.05; b p<0.01; and b p<0.001;Bonferroni post hoc test following one-way ANOVA versus the CCl4 treated group; Data are expressed as means ± SEM