Desai V T et al.

/ Journal of Pharmacy Research 2012,5(8),4084-4087

Research Article Available online through
ISSN: 0974-6943 www.jpronline.info
An Investigation Into the Heavy Metal Chelating Potential of
Ananas Comosus Fruit in Arsenic Intoxicated Rats
Desai V T*, Ganatra T H, Joshi U H, Desai T R, Tirgar P R
Department of Pharmacology, R. K. College of Pharmacy Kasturbadham, Rajkot-Bhavanagar High way, Rajkot, India – 360 020
Received on:09-05-2012; Revised on: 14-06-2012; Accepted on:22-07-2012

ABSTRACT
Background: Heavy metals are the major pollutants among others. They are the leading cause of occupational hazard for industrial workers as well as
residents in the proximity. Herbs with higher phenolic content have been noted to possess metal chelating activity. Objective: To determine heavy metal
chelating potential and organo-protective activity of Ananas comosus fruit in arsenic intoxicated rats. Experimental design: Arsenic chelating activity
and organo-protective effect of Ananas comosus fruit along with standard drug – dimercaprol, were assessed against low exposure of arsenic in albino
wistar rats. Result: At the end of 6 weeks in vivo trial, hematological parameters (WBC, RBC, Hb%, HCT%, As levels) and renal function parameters
(uric acid, creatinine) revealed significant benefits by the treatment as compared to disease-controlled group, which was confirmed by histopathological
assessments of liver and kidney. Conclusion: We concluded that Ananas comosus fruit has significant arsenic chelating and organo-protectve activities.

Key words: Arsenic, Phenol content, Ananas comosus, Chelating activity, Oragano-protection.

INTRODUCTION
Metals have played a critical role in industrial development and technological
advances. There is probably no material that has shaped the progression of
mankind more than metal. Hence metals are an inseparable part of this
advanced human race.[1, 2, 3]
Heavy metal can cause both acute and chronic toxicity depending upon the
duration and potency of exposure. Heavy metals can affect multiple organ
systems causing severe disorders such as encephalopathy,
hypopigmentation, hyperkeratosis, Proteinuria, lung cancer, osteomalacia,
hepatic cirrhosis, pulmonary fibrosis, Alopecia and many more.[4, 5]Among
many heavy metals, Arsenic (As) is found to be most dangerous as far as
human occupational hazards are concerned. Today, the rising number of
cases of occupational toxicity due to arsenic is a matter of utmost concern
for our state as well as our nation.[6, 7]
Current treatment available for management of Arsenic toxicity involves
mainly chelation therapy which uses various synthetic chelating agents
such as calcium disodium edetate, BAL, DMSA, desferoxamine and d-
penicillamine.[8] The selection of chelating agent depends on the nature of
metal intoxicated.[8] Despite the significant efficacy of these chelators, they
are less preferred by patients due to certain drawbacks. These synthetic
agents can produce adverse effects such as convulsions, bone marrow
depression, hypotension, cardiac arrhythmias, respiratory arrest, and
hypocalcemia, headache, nausea, stomach upset, vomiting. The route of
administration is mostly intravenous, so chronic use is painful. They are
costly in comparison with other drugs as well.[8, 9]
Phytochelatins have been detected in plants which interact with heavy
metals and neutralize their damaging consequences.[10] They also increase
*Corresponding author.
Desai Viral T.
“Anand”,
Guruprasad society,
Near Doshi hospital,
Gondal Road,
Rajkot – 360004
excretion of heavy metals. as the phenolic content of herb increases, its
antioxidant activity and metal chelating potential also increases.[11, 13] The
activity is believed to be proportional to the phenolic content in the extract
of leaves.[12] It is known that fruits are rich source of phenols. This food
component is part of our daily diet. If they are tested for chelating activity
against arsenic, positive outcome can be anticipated.
Ananas comosus fruit, belonging to the family Bromeliaceae, contains higher
phenolic content, and thus, we decided to evaluate arsenic chelating activity
of Ananas comosus (Pineapple) fruit and determine its chelation and organo-
protective activity.[13]
MATERIALS AND METHODS
Collection and authentification of fruits of Ananas comosus
The fruits of Ananas comosus were collected from the medicinal garden of
R. K. College of Pharmacy, Rajkot. The roots of Ananas comosus were cut
to small pieces. These fruits of Ananas comosus were authentified by
Department of Botany, Christ College, Rajkot.
Preparation of methanolic extract of fruits of Ananas comosus[14]
The collected fruits of Ananas comosus were subjected to dry to brittle
material at 60°C in hot air oven to remove moisture. The fruits were then
cut into small brittle pieces. Methanolic extract of Ananas comosus fruits
was prepared using Simple maceration and at the end of extraction, methanol
was evaporated to dryness which yielded a semi-solid product, which was
further subjected for evaluation of % W/W yield, colour, consistency and
pharmacological activities.
Folin-Ciocalteu’s test[15]
The Folin-Ciocalteu reagent is sensitive to reducing compounds including
polyphenols, producing a blue colour upon reaction. This blue colour was
measured spectrophotometrically.

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In vitro Chelating activity[15, 16]
Arsenic chelation was determined in vitro against MEAC (Methanolic extract
of Ananas comosus ) using UV spectrophotometric analysis at 556 nm.
Selection of animals
Either sex Wistar albino rats, weighing 220-280 g were used for the study.
The animals were procured from animal house, Department of
Pharmacology, R. K. College of Pharmacy, Rajkot, India. Animals were
housed at a temperature of 24±20?C and relative humidity of 30 – 70 %. A
light and dark cycle was followed. All animals were fed on standard balance
diet and provided with water ad libitum. All the experimental procedures
and protocols used in study were reviewed and approved by the
Institutional Animal Ethical Committee (IAEC) of R. K. College of
Pharmacy and care of laboratory animals was taken as per the guidelines of
Committee for the Purpose of Control and Supervision of Experiments on
Animals (CPCSEA), (Registration No. 1131/ac/ 07/CPCSEA).
Treatment protocol
For induction of arsenic metal toxicity, Arsenic trioxide (As2 O 3 was
administered 1 mg/kg, orally daily for 6 weeks. The treatment group received
MEAC Methanolic Extract of Ananas comosus) along with arsenic trioxide.
The experimental animals were divided into 4 groups, (n=6).
Group 1: Normal control received drinking water
Group 2: Disease control treated with Arsenic trioxide (1 mg/kg, i.p. for 15
times in 3 weeks)
Group 3: Standard control treated with Dimercaprol (BAL) (100 µg/kg,
p.o., per day) along with Arsenic trioxide
Group 4: Disease control treated with MEAC (500 mg/kg, p.o., per day)
along with Arsenic trioxide
All the studies were carried for a period of 6 weeks. Histopathology of
transverse sections of heart was performed to find any liver and kidney
damage occurred due to Arsenic toxicity.
RESULTS
The Methanolic extract of Ananas comosus showed total phenol content of
9.3 (expressed in mg gallic acid equivalent/g of extract).
The in vitro Arsenic chelating activity was performed using
UVspectrophotometer which shows remarkable chelating potential:
Extract Arsenic concentration (mg/ml)
Control 10
MEAC 7.5
Where, control = vehicle – distilled water,Stock solution = 10 mg/ml As2 O3
solution
Hematological as well as urine sample analysis was performed to evaluate
arsenic chelating potential of Ananas comosus fruit and histopathology
was also performed to assess organoprotective effect of same.
Hematological parameters
Blood samples were collected at the end of 6 weeks,and WBC, RBC, Hb%
and Hematocrit% were determined in whole blood, whil SGOT, SGPT and
As concentration were measured in separated serum in UV
spectrophotometer.
Effects of MEAC on WBC count
Rats intoxicated with arsenic showed significant reduction in serum WBC
level (p<0.001) as compared to normal control group. Both standard and
MEAC showed significant increase in serum WBC level when compared
with disease control group (p<0.001). (Table 1)
Journal of Pharmacy Research Vol.5 Issue 8.August 2012
Effects of MEAC on RBC count
Rats intoxicated with arsenic showed significant reduction in serum RBC
level (p<0.001) as compared to normal control group. Both standard and
MEAC showed significant increase in serum RBC level when compared
with disease control group (p<0.001). (Table 1)
Effects of MEAC on Hb%
Rats intoxicated with arsenic showed significant reduction in Hb% (p<0.001)
as compared to normal control group. Both standard and MEAC showed
significant increase in Hb% when compared with disease control group
(p<0.001). (Table 1)
Effects of MEAC on HCT%
Rats intoxicated with arsenic showed significant reduction in HCT%
(p<0.001) as compared to normal control group. Both standard and MEAC
showed significant increase in HCT% when compared with disease control
group (p<0.001). (Table 1)
Effects of MEAC on SGOT
Rats intoxicated with arsenic showed significant increase in SGOT (p<0.001)
as compared to normal control group. Both standard and MEAC showed
significant reduction in SGOT when compared with disease control group
(p<0.001). (Table 1)
Effects of MEAC on SGPT
Rats intoxicated with arsenic showed significant increase in SGPT (p<0.001)
as compared to normal control group. Both standard and MEAC showed
significant reduction in SGPT when compared with disease control group
(p<0.001). (Table 1)
Effects of MEAC on Serum Arsenic
Rats intoxicated with arsenic showed significant increase in serum arsenic
level (p<0.001) as compared to normal control group. Both standard and
MEAC showed significant reduction in serum arsenic level when compared
with disease control group (p<0.001). (Table 1)
Renal function tests
Blood samples were collected at the end of 6 weeks and kidney function
bio-markers- creatinine and uric acid were measured using ERBA kit in UV
spectrophotometer.
Effects of MEAC on Creatinine
Rats intoxicated with arsenic showed significant increase in creatinine
(p<0.001) as compared to normal control group. Both standard and MEAC
showed significant reduction in creatinine when compared with disease
control group (p<0.001). (Table 2)
Effects of MEAC on Uric acid
Rats intoxicated with arsenic showed significant increase in uric acid
(p<0.001) as compared to normal control group. Both standard and MEAC
showed significant reduction in uric acid when compared with disease control
group (p<0.001). (Table 2)
Histopathological changes in arsenic induced organ damage
Disease control showed more lesions in both kidney and liver structure.
This kind of lesions were comparatively less in normal control group,
standard group and in test group. (Figure 1)
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Table: 1: Protective effects of MEAC on serum bio-markers in arsenic intoxicated rats.
Group WBC RBC Hb% HCT% SGOT SGPT As
X 103 /µl X 106 /µl (U/L) (IU/L) mg/ml

Control 5.833 ± 0.14 8.953 ± 0.085 15.78 ± 0.11 52.02 ± 0.18 119.27 ± 0.30 40.45 ± 0.54 1.28 ± 0.01
group
# # # # # #
DC 3.45 ± 0.111 7.541 ± 0.13 14.36 ± 0.14 44.57 ± 0.23 163.57 ± 0.51 60.64 ± 0.63 1.85 ± 0.02#
Std 6.45 ± 0.08*** 9.42 ± 0.11*** 15.81 ± 0.1*** 50.87 ± 0.52*** 120.61 ± 0.70*** 40.132 ± 0.44*** 1.09 ± 0.02***
MEAC 5.3 ± 0.22*** 9.41 ± 0.032*** 15.09 ± 0.07*** 50.77 ± 0.4*** 50.77 ± 0.4*** 40.442 ± 0.33*** 1.18 ± 0.01***
F 187.56 81.099 37.907 87.58 1411.90 409.90 348.764
df 23 (3, 20) 23 (3, 20) 23 (3, 20) 23 (3, 20) 23 (3, 20) 23 (3, 20) 23 (3, 20)
p <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Table 2: Protective effects of MEAC on urine bio-markers in arsenic and blood dyscrasia etc.[17] In view of these drawbacks of currently used
intoxicated rats. synthetic molecules, in the present study, we attempted to find herbal
Group Uric acid Creatinine (mg/dL) remedy for effective and safe chelation of arsenic.
Control group 5.89 ± 0.0012 2.35 ± 0.028
DC 6.14 ± 0.0029 # 5.726 ± 0.107# While reviewing literature, we observed that as the phenolic content of herb
Std 5.87 ± 0.017*** 1.649 ± 0.03***
MEAC 5.892 ± 0.0055*** 2.710 ± 0.04*** increases, its antioxidant activity and metal chelating potential also
F 493.89 344.301 increases.[18] Hence, we selected Ananas comosus fruit, rich in phenolic
Df 23 (3, 20) 23 (3, 20)
P <0.001 <0.001 content, as potential candidate for heavy metal chelating activity.
n=6 and results were shown as mean ± SEM
# In the in vivo study, Albino Wistar rats were divided in to normal control,
indicate significant difference in the data compared to normal control group
and the level of significance was p<0.001? significant. disease control, standard drug control and treatment groups. The disease
*** indicate significant difference in the data compared to disease control control rats were administered with arsenic trioxide metal suspension. The
group and the level of significance was p<0.001 equivalant to highly significant. standard control rats received As2 O3 along with dimercaprol and the
MEAC - Methanolic extract of Ananas cosmosus. treatment group rats were given the As2 O3 metal with extract of pineapple.
DC - Disease control group. Dimercaprol is currently use as a drug of choice for the cases of low exposure
Std.- Standard control group.
of heavy metal poisoning. [19] Thus, it was selected as standard drug for the
experiment. Due to suspected slow onset of toxic activity of arsenic upon
various systems of the body, the duration of treatment was as long as 6
weeks.

Arsenic is mostly distributed in three systems viz., urinary system,

hematopoietic system and nervous system.[20] Due to this tissue distribution
it was decided to take serum and urine samples during the experiments. The
samples were collected at the end of the treatment.

The experiment was planned to assess protective action of Ananas comosus,

hence, preventive modality was used. To check the organo-protective activity
of Ananas comosus, we carried out the histopathology of liver and kidneys.
The damaging effects of the arsenic in disease control group were compared
Arsenic - Disease control group - Kidney. Arsenic - Disease control group - Liver. with the effects of treatment group.

The hematological parameters included WBC and RBC counts, hemoglobin

content, hematocrit, SGOT, SGPT and As level while urine analysis
parameters included creatinine and uric acid.

Arsenic – Treated with MEAC - Kidney. Arsenic – Treated with MEAC - Liver.
DISCUSSION
Our study was aimed at studying and solving the growing menace of heavy
metal toxicity as a serious occupational and pollution hazard. Arsenic is the
leading heavy metal pollutant among them.[ 1 6 ] It can cause serious
consequences such as renal and hepatic damage, severe inflammatory
reactions. Current drug treatment available is chelation therapy, but itself
cause adverse effects such as bone marrow depression, hyper pigmentation
Our results showed that the decreased levels of WBC in disease control
were increased significantly in treatment disease control group. The
hemoglobin content was also increased in treatment group. But the level of
arsenic was significantly decreased in serum due to its excretion in urine
because of the chelating activity of the herbal extract. The heavy metal
damaged liver and kidney by producing lesions which is indicated by the
increased level of SGPT, SGOT and creatinine in serum.
The histopathological studies of liver and kidney revealed that damaging
property of arsenic was significantly decreased as evident from reduced
lesions and scars on the sections of the slide of treatment group as compared
to the disease control group.
Thus, at the end of 6 week trial, results indicated that Ananas comosus was

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effective heavy metal chelator and possessed significant organo-protective
activity against arsenic toxicity.
CONCLUSION
From the results of present investigation, we concluded that fruit of Ananas
comosus (pineapple) contain high phenol content. Ananas comosus
(pineapple) fruit shows significant chelating activity and delivers significant
improvement in hematological as well as kidney function parameters.
Pineapple also showed higher organo-protective action. It is concluded that
daily or frequent use of Ananas comosus (pineapple) fruit in routine diet
can effectively work as a prophylaxis for chronic low dose exposure of
arsenic for industrial workers and residents.
This research work shows that greater outcomes can be anticipated from
further research work in this direction. The isolation of active constituents
from Ananas comosus (pineapple) fruit could be a mile stone in the existing
health hazard of heavy metal pollution.
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Source of support: Nil, Conflict of interest: None Declared
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