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Anticoagulant Activity of Pineapple (Ananas

comosus) Extract on Human Blood Samples

Article · September 2014

DOI: 10.7718/iamure.ijscl.v6i1.768

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Vol. 6 July 2014 Vol. 6 · June 2014
Print ISSN 2244-1557 • Online ISSN 2244-1565
International Peer Reviewed Journal
doi: http://dx.doi.org/10.7718/iamure.ijscl.v6i1.768
This Journal is produced by IAMURE Multidisciplinary Research,
an ISO 9001:2008 certified by the AJA Registrars Inc.

Anticoagulant Activity of Pineapple

(Ananas comosus) Extract on
Human Blood Samples
MARX P. CATALAN
ORCID No. 0000-0001-7286-7074
marxcatalan85@gmail.com
University of the Immaculate Conception Davao City, Philippines

FELICITAS C. AQUINO
ORCID No. 0000-0001-7799-075X
citas.aquino2014@gmail.com
Philippine Women’s University Malate Manila, Philippines

RENAN P. LIMJUCO
ORCID No. 0000-0002-1812-8673
ren02lim@gmail.com
University of the Immaculate Conception Davao City, Philippines

ABSTRACT

The abundance, availability and affordability of pineapple have triggered
the researcher to explore its potential for clinical laboratory applications. This
study was conducted to determine the viability of pineapple (Ananas comosus)
extract as laboratory anticoagulant and substitute for ethylenediamine tetraacetic
acid (EDTA). The data obtained from laboratory standard procedures were
subjected to one-way analysis of variance with Tukey’s test. Among the four
methods of extraction used, 95% ethanol and Expression gave a considerable
yield with respect to volume of the extract produced. With regard to the ability to
prevent blood coagulation, extract obtained through Expression with spiral twist
obtained positive results comparable for EDTA. In the observation for red blood

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IAMURE International Journal of Science and Clinical Laboratory

cell hemolysis, the different volumes of extracts produced results comparable

to EDTA. However, with regard to the efficiency in preventing red blood cell
crenation and clumping, 300ul extract did not have a significant difference with
EDTA; therefore, the results obtained were comparable. Preservation of the
staining reactions of the cell structures was also observed. Although significant
differences were recorded in each of the volumes used, 300ul produced results
that were somehow comparable to EDTA. The efficiency was recorded in the
evaluation of the extract’s efficiency in maintaining cell size and shape.

KEYWORDS

Medical Technology, anticoagulant property, pineapple extracts, calcium

chelator, EDTA, bromelain, hemolysis, staining reaction, morphology, true
experimental design,ANOVA, Tukey’s test, Philippines

INTRODUCTION

Through the years, the importance of herbal plants in the field of medicine
continues to gain popularity and has attracted researchers to conduct broader
studies regarding their efficacy as sources of antimicrobial drugs and medicines for
diseases like diabetes and cancer (Farnsworth et al., 1991). Since the development
of technology, one of the challenges being faced in the clinical laboratory is the
use of effective anticoagulants for performing diagnostic procedures. Majority
of the anticoagulants used in the clinical laboratory are expensive and relatively
toxic to human health. Thus, the need to study plants as potential sources of
anticoagulants is imperative since it gives hopes in the future use of organic
substances that are less expensive and non-toxic. Several studies on the efficacy
of plants as therapeutic anticoagulants have been conducted, but none was
performed to determine their efficacy as anticoagulants for laboratory diagnostic
procedures. Plants such as red onion (Alium cepa), and noni (Morinda citrifolia)
were successfully studied for their viability as therapeutic anticoagulants for
treatment of cardiovascular, coagulation and thrombotic disorders (Karim et al.,
2003; Yoon et al., 2003).
Since blood coagulation is a natural phenomenon and can occur in vitro,
blood samples collected for laboratory procedures particularly in the hematology
section are collected using anticoagulants to inhibit in-vitro coagulation and
thus making the blood samples usable for testing. Through the years, numerous

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anticoagulants have been developed to inhibit clotting. These anticoagulants

prevent in-vitro coagulation by inhibiting certain steps or components in the
coagulation cascade. For instance, the anticoagulant Heparin used in clinical
chemistry inhibits thrombin formation; sodium citrate for blood coagulation
and ethylenediamine tetraacetic acid (EDTA) inhibits or chelates calcium
(Mcpherson & Pincus, 2011).
To date, the most common anticoagulant used in hematology procedures is
Ethylenediaminetetraacetic acid (EDTA). Lavender – top tubes with EDTA are
used in collecting blood samples. It prevents clotting by chelating calcium that is
an important element in the clotting process. The use of EDTA as anticoagulant
is recommended by the International Council for Standardization in Hematology
(ICSH) as the anticoagulant of choice for blood cell counting and sizing because
they produce less shrinkage of red blood cells and less of an increase in cell volume
on standing. It is used in concentrations of 1.5 milligrams per milliliter (1ml) of
whole blood sample for solid EDTA (K2) (Turgeon, 2010), and 2% EDTA, 1-2
drops per milliliter (1ml) of whole blood (Sigma-aldrich). Using EDTA, medical
technologists can prepare good quality blood smear as long they are prepared in a
period of 2-3 hrs after the specimen was obtained. In addition, EDTA maintains
the cell structures and provides marked staining qualities. However, excessive
amounts can cause shrinkage of erythrocytes; thus, affecting tests results such as
the manual red blood cell count. Also EDTA prevents platelet clumping on the
slide making it easier to more accurately estimate platelet counts (Rodak et al.,
2012).
However, smears made from EDTA tubes that sit at room temperature for
more than 5 hours often have an unacceptable artifact on blood cells (echinocytic
red blood cells, spherocytes and necrobiotic leukocytes). If a tube is not filled to
its full volume of draw, it may lead to a very high EDTA concentration and can
cause red cell shrinkage, and also can cause membrane damage of blood cells.
However white blood cell (WBC) count can remain stable for at least three days
when EDTA anticoagulated tubes are stored at room temperature (Patel, 2009).
Pineapple (Ananas comosus) is a common fruit that is found all throughout the
world. In fact, it is the third most important tropical fruit in world production,
next to banana and citrus. Although pineapples nowadays are processed
commercially for juice and food flavors like pineapple tidbits sold in the market
in cans, 70% of the pineapple produced in the world is consumed as fresh fruit
(Bartholomew et. al, 2003). One of the essential chemical components present in
pineapple is the enzyme bromelain. It is a crude extract from pineapple fruit and

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IAMURE International Journal of Science and Clinical Laboratory

stem. Bromelain is known to have exceptional health benefits. It is most notable

for its effectiveness in the reduction of inflammation and decreasing swelling,
but scopes of its benefits are increasing (Bhattacharyya, 2008). An admirable
capability of bromelain is preventing blood coagulation. Bromelain is a natural
anticoagulant that works by breaking down the blood clotting protein fibrinogen
and by inhibiting platelet aggregation that is why clinical studies were conducted
to test the potency of pineapple as a therapeutic anticoagulant for the treatment
of thrombosis and other coagulation disorders (Taussig et al., 1988). Bromelain
is a cysteine protease, which means that it cleaves proteins made up of cysteine
residues. Since majority of animal and human proteins including the coagulation
proteins are made up of cysteine residues, bromelain can quickly destroy and
inhibit them (Gautam et al., 2010).
In 1972, Heinicke et al. reported in their observation that oral administration
of bromelain to healthy persons, particularly those with high platelet counts,
significantly lowers the thrombin-induced aggregation of platelets.
The effect of bromelain in the kinin system of the body was also determined
by Kelly, in 1996. Kallikerein system, bradykinin levels, and plasma exudation at
the inflammatory site were examined in rats. Bromelain caused a dose dependent
decrease of bradykinin levels at the inflammatory site and a parallel decrease of
the prekalikrein levels in the rat sera. From this study, the author concluded that
bromelain degrades high molecular weight kininogen (HMWK) and thereby
preventing its activation to become bradykinin. Furthermore, this study provided
a proof that bromelain is a potent down regulator of inflammation (Kelly, 1996).

OBJECTIVES OF THE STUDY

The study investigated the anticoagulant activity on human blood samples

of pineapple (Ananas comosus) extract as a safe and cost-effective alternative
to ethylenediamine tetra acid (EDTA) in the laboratory. Specifically, this
experimental research determined the approximate volume of the extract that
can prevent blood clotting, red blood cell hemolysis, crenation, clumping, and
also maintain cell size and shape and preserve normal staining reactions of the
different blood cells. Lastly, this investigation also tested whether there is a
significant difference in the results obtained with respect to the various volumes
of pineapple extract and the positive control.

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MATERIALS AND METHODS

Research Design
This study utilized true experimental research design using positive and
negative controls. The researchers studied the anticoagulant activity of pineapple
extract using blood samples collected from Medical Laboratory Science (MLS)
students of the University of the Immaculate Conception. The blood samples
after being added to the plant extract were then observed macroscopically and
microscopically to address the objectives of the study.

Research site and specimen

The plant sample was purchased from Bankerohan Public Market, Davao
City. It was submitted to the University’s taxonomist for correct classification
and speciation and extraction of the plant samples was then conducted. All
experiments were carried out at the University of the Immaculate Conception
laboratory. Blood samples were collected from the students of the University of
the Immaculate Conception after securing them informed consents. Evaluation
of samples and results were done by fellow Registered Medical Technologists of
the University.

Instrumentation and experimentation

Informed consent with signatures from the Dean of the Medical Technology
Department; Dean of College and Director of Laboratories were given to the
30 selected healthy and willing students of the third year Medical Laboratory
Science of the University of the Immaculate Conception. Selection was done
based on their physical appearance (healthy and not sickly) to ensure ease in
blood collection and processing. After having their parents or guardians signed
the informed consent, 2 ml of blood samples were collected from them to be used
in the anticoagulant assay.
Standard procedures for plant extraction were used in this study. Four
methods were employed: 95% Ethanol extraction, Soxhlet using Analytical Grade
Petroleum Ether, Expression with Spiral Twist and Decoction. Two hundred
grams of pineapple chunks were macerated and immersed in 95% Ethanol for 48
hrs. After 48 hrs, the mixture was filtered using two layers of muslin cloth. The
extract obtained was subjected for rotary evaporation to remove the alcohol and
to concentrate the extract. In Soxhlet extraction, 200 g of pineapple were placed
in a specialized type of glass apparatus and was gradually exposed to petroleum

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IAMURE International Journal of Science and Clinical Laboratory

ether for 8 hrs. Expression with Spiral Twist involves grinding of pineapple
chunks using an osterizer blender, and squeezing it using two layers of muslin
cloth to express the juice. In decoction, the pineapple chunks were subjected to
boiling with water for 15 mins.
Anticoagulant assay was performed using various volumes of the extract (300
ul, 400ul, and 500ul), each amount mixed with 2 ml of whole blood. Three trials
with three replicates each were conducted by the researcher to ensure validity of
the results. The mixture was kept and observed for 1-3 hrs for anticoagulation.
The tubes were centrifuged in an hourly basis for 5 minutes at 3000 rpm to
observe for red blood cell hemolysis indicated by a red supernatant. Wet smears
were prepared using a glass slide and examined it under a microscope for the
presence of red cell clumping and crenation. In the observation of good staining
reactions, preservation of cell size and shape, wedge smears were prepared,
stained with Wright’s stain and examined it under a microscope using 1000x
magnification.

Data Analysis
The data gathered during the experimentation was analyzed using Analysis of
Variance (ANOVA) with post analysis in Tukey’s test. ANOVA was implemented
to test whether the various amounts of pineapple extracts would show significant
differences with one another and positive control in terms of the anticoagulant
activity on human blood samples. Tukey’s test was used to determine which of
the pairings were significantly different and which of them were not to establish
comparability of the pineapple extracts to the EDTA solution.

RESULTS AND DISCUSSION

Extraction Methods
Table 1 showed the different extraction methods used for pineapple. From
the results, the extraction methods that have higher yields of the extracts were
95% Ethanol Extraction and Expression using spiral-twist technique. Soxhlet
extraction obtained a low yield of 0.03 % . However, the yield for the Decoction
method was not determined since the solvent that is distilled water was difficult
to evaporate.

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Table 1. Results of the extraction method

Expression using
Soxhlet Extraction Extraction using Decoction using
Spiral – twist
using Petroleum Ether 95% Ethanol distilled water
technique
Not applicable since
the solvent (water)
0.03%/200g 3.51 ml/200g 98 ml/200
was not successfully
evaporated

Both the extracts obtained from 95% Ethanol extraction and Expression
techniques were tested for anticoagulation as shown in Figures 2 and 3 and
only Expression with spiral twist technique showed anti-coagulation. Probable
reason for this is that, since the active component of the pineapple fruit that has
anticoagulant property is the proteolytic enzyme bromelain, have been denatured
by 95% ethanol since alcohol is a potent denaturizing agent.

Figure 2. Anticoagulant assay of extract obtained through 95% Ethanol

Figure 3. Anticoagulant assay of extract obtained through expression

with spiral twist

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IAMURE International Journal of Science and Clinical Laboratory

As shown in Figure 4, the extract used in the assay was tested for the pH.
Initial testing showed that it had the pH of 3. It was adjusted to pH 7 to coincide
with the blood’s physiologic pH which is neutral. Adjustment was done using
0.1N Sodium Hydroxide. Furthermore, the pH was retested after 24 hrs of
storage at 2-10OC and the result did not show a significant change in the pH.

Figure 4. pH adjustment of the extract

Determination of the Effective Volume of the Extract

The pineapple extract obtained through Expression with Spiral Twist was
tested for its anticoagulant activity using two milliliters of whole blood samples.
The different volumes used are the following: 10ul, 100ul, 200ul, 300ul, 400ul,
500ul. However, the blood sample with extracts volume of 10ul, 100ul and
200ul showed no anticoagulation. Only the 300 – 500ul of extracts showed
anticoagulation using 2 ml of blood. Additional assay was performed using 250ul
of the extract mixed with 2ml of blood in order for the researcher to observe
the transition between 200ul to 300ul. The result obtained was still negative for
anticoagulation.
Although the study failed to elucidate the main reason for this 100%
difference in the anticoagulant activity of pineapple extract based on the amount
used, it indicates that this characteristic is dose dependent, which implies that
the higher the volume of the extract, the higher the concentration of bromelain
enzyme capable of preventing blood coagulation.

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Figure 5. Additional observation of blood anticoagulation

using 250ul of the extract

Observation of Longevity Effect on Blood Anticoagulation
As shown by Table 2, among the seven volumes of extract used in the assay
only 300ul -500ul are capable of anticoagulation for a period of 3 hrs since they
register a grand mean of 3, in contrast to 10ul – 250ul which register at 1. These
findings mean that 300ul – 500ul have efficiency in anticoagulation similar to
EDTA.

Table 2. Results of the anticoagulant assay

Efficiency in Positive Negative
10ul 100ul 200ul 250ul 300 ul 400 ul 500 ul
Preventing Control Control
Blood Hours Hours Hours Hours Hours Hours Hours Hours Hours
Clotting 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Replicate 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1

Replicate 2 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1

Replicate 3 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1
Category
1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1
Mean
Grand
1 1 1 1 3 3 3 3 1
Mean

Legend:
1 – Blood is fully coagulated
2 - Blood is slightly coagulated
3 – Blood is not coagulated

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IAMURE International Journal of Science and Clinical Laboratory

Figure 6. Observation of Anticoagulant Activity of Pineapple Extract

Figure 6 showed the observation for the anticoagulant activity of pineapple

extract obtained through Expression with spiral twist technique. As what is shown
in the picture and in table 2, no statistical treatment was applied since the results
showed a 100% comparability with that of the positive control which is EDTA.
Since the anticoagulant activity of pineapple extract is attributed to the
proteolytic enzyme bromelain, the findings above showed that higher volumes
of the extract contain as sufficient amount of bromelain enough the prevent
blood coagulation by inhibiting blood fibrinogen and thrombin induced platelet
aggregation (Maurer, 2001).
Another study conducted by Kelly (1996) also revealed that bromelain
also affects other coagulation proteins. He established that the bromelain also
decreases the activity of high molecular weight kininogen (HMWK) which serves
as the anchor protein of the coagulation proteins (XIIa, IXa, XIa) in the damaged
epithelium; and coagulation factor X which is important in the generation of
thrombin needed for the polymerization of fibrinogen (Kelly,1996).
In addition, it was also found that the bromelain is helpful in regulating
blood clotting in vivo by hydrolyzing fibrin strands, and inactivating plasminogen
making it a possible alternative for treatment coagulation and thrombotic
disorders (Maurer, 2001).

Efficiency of the Extract in Preventing Red Blood Cell Hemolysis

As revealed in Table 3, all the volumes of the extract used have the same
efficiency with EDTA in preventing red blood cell hemolysis for a period of
3 hrs. These findings showed the enzyme bromelain present in the pineapple
fruit which is the active component that exhibits anticoagulation does not cause

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significant damage to the red blood cells and, therefore, is efficient in maintaining
the functional and structural integrity of the cells.

Table 3. Observation of red blood cell hemolysis

Positive Negative
Efficiency in 300 ul 400 ul 500 ul
Control Control
Preventing
Hours Hours Hours Hours Hours
Hemolysis
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Replicate 1 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1
Replicate 2 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1
3
Replicate 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1

Category Mean 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1

Grand Mean 3 3 3 3 1

Legend:
1 – Marked hemolysis
2 – Slight hemolysis
3 – No hemolysis

Figure 7. Observation for the efficiency of the pineapple extract

to prevent red blood cell hemolysis

As shown in figure 7, within 3 replicates, the pineapple extract successfully

prevented red blood hemolysis as indicated by the presence of pale – yellowish
plasma (normal color) after centrifugation for 5 minutes at 3000rpm. Also, as
revealed by table 3, no statistical treatment was applied in this observation since
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IAMURE International Journal of Science and Clinical Laboratory

the result clearly indicated a 100% comparability with that of EDTA.

One of the requirements for a chemical substance to become an efficient
alternative for ethylenediamintetraacetic is the ability to preserve cellular structure
especially for those cells that are easily damaged when exposed to chemical agents
like the red blood cells.
Pineapple extracts successfully prevented hemolysis that makes it a potential
substitute for ethylenediaminetetraacetic acid. Red cells are prone to structural
damage and hemolysis when exposed to unfavorable conditions like acidic pH,
hypotonic solutions and toxic compounds (Rodak, 2012). Pineapple extract did
not cause red blood cell hemolysis. The pH was adjusted to neutral to coincide
with the blood’s physiologic pH. Also, with pineapple being rich in tannin
compounds, it was found to help prevent cell membrane damage brought about
by toxic chemical substances (Harold, 2004).

Observation of the extracts’ efficiency in preserving good staining reactions
with Wright’s Stain and in maintaining cell size and shape.
Table 4 showed the significant differences of the various parameters used in
the observation as shown by the p values. Using Wright’s stained smears obtained
from the blood samples with varying volumes of pineapple extracts, the efficiency
of the extract in preserving good nuclear, Cytoplasmic and granule staining
reactions were seen using a microscope. 10 OIO fields were scanned and the
results were recorded. Statistical analysis of the results using One Way ANOVA
and post hoc analysis using Tukey’s test, significant differences were observed in
the different volumes of the extract in comparison to the positive control.
As shown in Table 4, statistical treatment of the data obtained using Pairwise
comparison (Tukey’s test) revealed significant differences for the different
parameters observed when compared to the positive control. For the extract’s
efficiency in preventing red blood cell clumping, only 300 and 400ul of the
extract provided efficient results comparable to the positive control. In the
prevention of red blood cell crenation, 300ul extract volume produced results
that are comparable to EDTA.

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 Vol. 6 · June 2014

Table 4. Pairwise comparison of the p-Values of different volumes of pineapple

extract
Pairwise
p-value Decision Conclusion
Comparison
EDTA vs 300ul 1.000 Not Significant Comparable
Prevention of Red EDTA vs 400ul .476 Not Significant Comparable
Blood Cell Clumping EDTA vs 500ul .009 Significant Not Comparable
EDTA vs 300ul .113 Not Significant Comparable
Prevention of Red Blood EDTA vs 400ul .000 Significant Not Comparable
Cell Crenation
EDTA vs 500ul .000 Significant Not Comparable
EDTA vs 300ul .447 Not Significant Comparable
Preservation of Staining EDTA vs 400ul .002 Significant Not Comparable
Reaction for Nucleus
EDTA vs 500ul .070 Not Significant Comparable
EDTA vs 300ul .005 Significant Not Comparable
Preservation of Staining EDTA vs 400ul .024 Significant Not Comparable
Reaction for Cytoplasm
EDTA vs 500ul .061 Not Significant Comparable
EDTA vs 300ul .168 Not Significant Comparable
Preservation of Staining EDTA vs 400ul .973 Not Significant Comparable
Reaction for Granules
EDTA vs 500ul .001 Significant Not Comparable
EDTA vs 300ul .806 Not Significant Comparable
EDTA vs 400ul .279 Not Significant Comparable
Preservation of Cell Size
EDTA vs 500ul .966 Not Significant Comparable
EDTA vs 300ul .112 Not Significant Comparable
Preservation of Cell EDTA vs 400ul .002 Significant Not Comparable
Shape
EDTA vs 500ul .000 Significant Not Comparable

As regards the extract’s efficiency in maintaining good staining reactions, 300

and 500ul produced results are comparable to EDTA in terms of the observation
for nuclear staining reactions; for the cell cytoplasm, 500ul volume of the extract
produced results comparable to EDTA; for the cell granules, 300 and 400ul
produced comparable results. With regard to preservation of cell size, all of the
volumes of the extract produced comparable results, while only 300ul has the
efficiency similar to EDTA in terms of the preservation of cell shape.

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IAMURE International Journal of Science and Clinical Laboratory

Figure 8. Wet preparation for the microscopic examination for the efficiency of
the pineapple extract to prevent red blood cell clumping

Figure 9. Wet preparation for the observation of the extract’s efficiency

in preventing red blood cell clumping

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 Vol. 6 · June 2014

Figure 10. Wright stain blood smear for the evaluation of the extract’s efficiency
in maintaining good staining reactions and preservation of cell size and shape

CONCLUSIONS

Pineapple extract is a viable anticoagulant and substitute for ethylenediamine

tetratacetic (EDTA) since it successfully inhibited blood coagulation for a period
of three hours and produced results with the same efficiency to that of EDTA
in terms of preventing the hemolysis, red blood cell clumping and crenation,
preservation of good staining reactions and maintaining cell size and shape.
Although significant differences were observed, 300ul produced a relatively
consistent result in the various parameters observed compared to 400 and 500ul.
The extract was also relatively stable for 4 hrs without chemical preservation, and
when stored at 2-8oC. The comparability of the 300ul extract with the EDTA in
terms of the different variables investigated was established by the statistical tests
specifically ANOVA and Tukey’s test.
The results obtained from this study strongly support its efficacy as a substitute
for EDTA in hematologic testing. In addition, since pineapple is readily available,
this alternative can be cost effective and easy to prepare since pineapple is grown
all throughout the country and can be bought at cheaper price. Furthermore,
pineapple extract is non-toxic, therefore, does not cause health hazards to
laboratory personnel and to the environment.

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IAMURE International Journal of Science and Clinical Laboratory

RECOMMENDATIONS

From the conclusions drawn, the study recommends the following:

The enzyme bromelain, which is responsible for the anticoagulant activity of
pineapple, should be isolated to come up with concentrations similar to EDTA.
Also, it is important to eliminate other chemical constituents in pineapple that
may have an effect to various blood components that may significantly affect the
results.
It is important to explore other parts of the pineapple plant, especially those
that go to waste such as the crown, the peel, core and leaves for their potential
source of bromelain to increase its practicality.
It is also suggested to examine the effect of bromelain anticoagulant when
tested using the various parameters in complete blood count using manual and
automated techniques and explore the possibility of bromelain as anticoagulant
for other laboratory tests including possibilities as substitutes for sodium citrate
for blood coagulation and for Heparin for clinical chemistry tests.
Further studies must also focus on the research on the appropriate preservative
for bromelain to improve its shelf life for further use, and to prevent rapid
deterioration.

LITERATURE CITED

Maurer, H.R.
2001 Bromelain: biochemistry, pharmacology and medical use. Cellular and
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FJ4SVz

Bartholomew, R.E., Paull, & Rohrbach, K.G.

2003 The Pineapple Botany, Production and Uses, p 2-3. Cabi Publishing.
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Bhattacharya, B.K.
2008 Bromelain: An overview. Natural Product Radiance 7(4): 359 – 363.
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Fansworth, Norman R. and Soejarto, Djaja D.

1991 Global Importance of Medicinal Plants. Cambridge University Press.

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Gautam S.S., Mishra, S.K., Dash, V., Goyal, Amit K., & Rath, G.
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Heinicke, RM, Van der Wal M., & Yokoma, M.M.

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Karim, M.A.A., Noor, S., Zainina, S., Rahayu, E., Tohit, M., Faridah, I.,
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Kelly, G.S.
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McPherson, R.A., & Pincus, M.

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2009 Why is EDTA the anticoagulant of choice for hematology use?. Tech Talk
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Rodak, B.F., Fritsma, G. A., & Keohane, E.

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Yoon, SJ., Yu, MA., Pyun, YR.,Hwang, JK., Chu, DC., Juneja, LR.,& Murao,
PA.,
2003 The Nontoxic mushroom Auricularia Auricula contains a polysaccharide

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with anticoagulant activity mediated by antithrombin. Thrombosis

Research 112.3:151-158. Retrieved on December 14, 2013 from http://
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Taussig, Steven J.
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