ArticlePDF Available

Comparative study of extraction, purification and estimation of bromelain from stem and fruit of pineapple plant

Authors:

Abstract and Figures

Bromelain is a major proteinase, isolated from pineapple (Ananas comosus). In the plant, bromelain is accumulated in the entire plant to different extent and properties depending on its source. The objective of present study was to compare the amount and activity of bromelain present in stem and fruit of the plant. Bromelain was isolated from stems and fruit of adult pineapple plants by buffered aqueous extraction. Purification of enzyme was done by centrifugation, salt precipitation technique, dialysis, ion-exchange chromatography and estimation by Lowryûs method. Bromelain was assayed for its activity by hydrolysis of gelatin, represented by using gelatin digestion unit. The homogeneity of bromelain was confirmed by SDS-PAGE (sodium dodecylsulphate-polyacrylamide gel electrophoresis) analysis. It was found that stem bromelain had a better activity than fruit bromelain in gelatin digestion unit analysis. Moreover, ion exchange chromatography using diethylaminoethyl cellulose (DEAE) anion exchangers maintained the structural integrity of purified bromelain and thereby the product exhibited better proteolytic activity then crude extract.
Content may be subject to copyright.
Thai J. Pharm. Sci. 34 (2010) 67-76 67
Original article
Comparative study of extraction, purification and estimation of
bromelain from stem and fruit of pineapple plant
S. S. Gautam1, S. K. Mishra1, V. Dash1, Amit K. Goyal2 and G. Rath2*
1Kanak Manjari Institute of Pharmaceutical Sciences, Rourkela, Orissa, India
2Department of Pharmaceutics, ISF College of Pharmacy, Moga (Punjab), India
*Corresponding author: E-mail address: goutamrath@rediffmail.com
Abstract:
Bromelain is a major proteinase, isolated from pineapple (Ananas comosus). In the plant, bromelain is
accumulated in the entire plant to different extent and properties depending on its source. The objective of
present study was to compare the amount and activity of bromelain present in stem and fruit of the plant.
Bromelain was isolated from stems and fruit of adult pineapple plants by buffered aqueous extraction. Purification
of enzyme was done by centrifugation, salt precipitation technique, dialysis, ion-exchange chromatography
and estimation by Lowryûs method. Bromelain was assayed for its activity by hydrolysis of gelatin, represented
by using gelatin digestion unit. The homogeneity of bromelain was confirmed by SDS-PAGE (sodium
dodecylsulphate-polyacrylamide gel electrophoresis) analysis. It was found that stem bromelain had a better
activity than fruit bromelain in gelatin digestion unit analysis. Moreover, ion exchange chromatography using
diethylaminoethyl cellulose (DEAE) anion exchangers maintained the structural integrity of purified bromelain
and thereby the product exhibited better proteolytic activity then crude extract.
Keywords: Fruit bromelain; Gelatin digestion unit; Ion exchange chromatography; Lowryûs method; Stem
bromelain
68 S. S. Gautam et al.
Introduction
Bromelain is a mixture of protein-digesting
(proteolytic) enzymes found in pineapples (Ananas
comosus). Pineapple has been used for centuries to
treat indigestion and reduce inflammation [1]. In pineapple
plant, bromelain is accumulated in the entire part with
different extent and properties depending on its source.
Bromelain, which is derived from the stem and juice of
the pineapple, was first isolated from the plant in the
late 1800s. It is usually distinguished as either fruit
bromelain or stem bromelain depending on its source,
with all commercially available bromelain being derived
from the stem. The stem bromelain has the EC Number
EC 3.4.22.32 and that of the fruit bromelain is EC
3.4.22.33. It is approved to treat swelling and inflammation
following surgery, particularly sinus surgery. Bromelain
can be useful in treating a wide range of conditions, but
it is particularly effective in reducing inflammation
associated with infection, sinusitis, osteoarthritis and
cancer [2-7].
The primary component of bromelain is a sulfhydryl
proteolytic fraction. It also contains a peroxidase,
acid phosphatase, several protease inhibitors, and
organically-bound calcium. It is made up of 212 amino
acids and the molecular weight is 33 kDa [8, 9]. Bromelain
is stable at pH 3.0-6.5 and once it has combined with
its substrate, the activity is no longer susceptible to the
effect of the pH. The effective temperature range is
40 ÌC-65 ÌC with the optimum being 50 ÌC-60 ÌC. Bromelain
can be activated by calcium chloride, cysteine, bisulphate
salt, NaCN, H2S, Na2S and benzoate. However, bromelain
is usually sufficiently active without the addition of
activators. Bromelain is inhibited by Hg++, Ag+, Cu++,
antitrypsin, estatin A and B, iodoacetate [10].
Isolation, separation and purification of enzymes/
proteins can be performed using variety of chromatography,
electrophoretic, ultrafiltration, precipitation and other
procedures. Ion exchange chromatography is often very
useful in protein purification [11, 12]. This chromatographic
procedure uses the net charges of the molecules to
achieve their separation [13, 14]. Two commonly employed
ion exchange resins are the cationic and anionic resins,
carboxymethyl (CM) and diethylaminoethyl cellulose
(DEAE). Several works on ion exchange chromatography
have contributed to better understanding of the effects
of ionic resins, effect of velocity and elution solution for
the recovery of biomolecules [15].
In the present work, purification of bromelain-
containing juice extracted from pineapple (Ananas
comosus) plant stems and fruits were studied by
centrifugation technique at different rotational speed and
DEAE cellulose bed based ion-exchange chromatography
techniques. The effect of separation and purification
methods of bromelain activity and purity was studied by
gelatin digestion unit assay and SDS-PAGE (sodium
dodecylsulphate-polyacrylamide gel electrophoresis)
analysis.
Experimental
Materials
Pineapple stem was collected from Kerala
Agricultural University, Kerala, India. Herbarium of the
same was prepared and maintained at KMIPS, Rourkela,
Orissa bearing the voucher no. KMP-AN-COM-98.
Temperatures of 7 to 12 ÌC (45 to 55 ÌF) were maintained
for storage of pineapples for 14 to 20 days at 85 to
95% of relative humidity. DEAE cellulose (GeNei India Ltd.,
Bangalore) and all other reagents (Hi-Media Labs Ltd.,
Mumbai) used were of highest commercially available
purity.
Extraction
Fresh pineapple stems parts were collected,
washed with 0.1% hydrogen peroxide solutions, peeled
off, cut into small pieces and weighed. The weighed
mass was found out to be 1,700 gm. Juice was collected
from the fresh pineapple stem part by homogenization,
in the presence of sodium acetate buffer solution and
was filtered. Five-hundred ml of filtrate were collected.
Benzoic acid/sodium benzoate was added as a
preservative at a concentration of 1 gm. per kg of stem.
The filtrate obtained was called as çcrude extracté, and
used as source of çstem bromelainé.
Purely ripe pineapple fruits were taken, cleaned
and made into small slices. The weighed mass was
600 gm. The juice was extracted using a homogenizer,
Thai J. Pharm. Sci. 34 (2010) 67-76 69
collected into a beaker and filtered. The filtered mass
was about 300 ml and 0.6 gm of sodium benzoate
was added. The filtrate was called as çcrude extracté,
and used as source of çcruit bromelainé.
Selection of substrate
Bromelain can be assayed by measuring digestion
action on gelatin and is expressed as GDU (Gelatin
digestion unit). Activity of one gram of bromelain is
approximately equivalent to 1,200 GDU. Gelatin was
chosen as the substrate for the analysis of activity of
bromelain [16].
Assay for crude stem bromelain by gelatin digestion
unit analytical method
Crude stem bromelain extract was used to
determine the rate at which gelatin (substrate) was
degraded. Various reagents were prepared such as gelatin
(5%) (reagent A) used as substrate, hydrogen peroxide
solution (3%) (reagent B), formaldehyde solution (37%)
(reagent C), 0.05N sodium hydroxide (reagent D), 100 mM
sodium acetate buffer with 2.6 M sodium chloride (reagent E).
One ml of crude stem extract was taken in a
beaker, designated as test solution. The pH was
maintained to 6.0 with 0.05 N NaOH. Twenty-five ml of
reagent A was added to the test solution and equilibrated
at 45 ÌC in a water bath. After 20 minutes of incubation
at 45 ÌC, 0.1 ml of reagent B was added and swirled.
The solution was incubated for an additional 5 minutes.
The beaker was removed from the water bath and pH
was adjusted to 6.0 with 0.05N NaOH. Ten ml of reagent
C was added with constant stirring. Titration was done
to pH 9.0 with 0.1 N NaOH. The titration volume of the
test solution was recorded. The blank solution was run
concurrently with the test solution. Firstly, 25 ml of reagent
A and 0.1 ml of reagent B was added and equilibrated
at 45 ÌC in water bath. After 20 minutes of incubation at
45 ÌC, 1.0 ml of bromelain solution was added, swirled
and incubated for an additional 5 minutes. The beaker was
removed from the water bath and pH 6.0 was adjusted
with 0.05 N NaOH. Ten ml of reagent C was further
added with constant stirring and the titration was done
to pH 9.0 with 0.1 N NaOH. The titration volume of the
blank solution was recorded (Table 1). The bromelain
content was calculated for the crude enzyme [16].
Units/gm enzyme
= (Volume of test-Volume of blank) (N) (14) x 1000 (1)
mg enzyme/RM
where N is normality of NaOH, 14 = mg nitrogen
per millimole nitrogen, mg enzyme = amount per
concentration of bromelain enzyme present in 1 ml of
crude extract, RM = reaction mix. The concentration of
enzyme was found out to be 0.7 mg/ml of crude extract.
Table 1 Assay for crude stem and fruit bromelain
Crude Crude Volume of
Sample Reagent A enzyme enzyme Reagent B Reagent C NaOH run
(ml) (ml) (ml) (ml) (ml) down (ml)
Stem bromelain
Blank 25 --- 1 0.1 10 7.00
Test 25 1 --- 0.1 10 8.05
Fruit bromelain
Blank 25 --- 1 0.1 10 9.40
Test 25 1 --- 0.1 10 10.55
Bromelain standard
Blank 25 --- 1 0.1 10 7.00
Test 25 1 --- 0.1 10 8.05
70 S. S. Gautam et al.
Purification
The crude extracts of stem and fruit bromelain
were centrifuged for 10 minutes at 2,000 rpm, 4,000
rpm and 6,000 rpm consecutively at 4 ÌC. The superna
tant was collected and enzyme assay was performed
as above (Table 2). Finally, samples were taken for
purification by using ammonium sulfate precipitation,
dialysis, and ion exchange chromatography.
Salt precipitation/salting out
For stem bromelain, ammonium sulfate precipitation
was carried out by adding 6.6 gm of ammonium sulfate
salt, pinch by pinch, to 15 ml supernatant taken after
centrifugation under ice cold conditions with continuous
stirring on a magnetic stirrer for 45 minutes. For fruit
bromelain, similar activity was carried out for same time
period. Stem and fruit bromelain sample solutions were
Table 2 Enzyme assay of centrifugated fraction and ion exchange eluate of stem and fruit bromelain
Volume of
Sample Reagent A Enzyme Enzyme Reagent B Reagent C NaOH run
(ml) (ml) (ml)
down (ml)
Stem bromelain (2000 rpm)
Blank 25 --- 1 0.1 10 7.50
Test 25 1 --- 0.1 10 8.50
Stem bromelain (4000 rpm)
Blank 25 --- 1 0.1 10 7.60
Test 25 1 --- 0.1 10 8.60
Stem bromelain (6000 rpm)
Blank 25 --- 1 0.1 10 7.40
Test 25 1 --- 0.1 10 8.42
Fruit bromelain (2000 rpm)
Blank 25 --- 1 0.1 10 7.50
Test 25 1 --- 0.1 10 8.47
Fruit bromelain (4000 rpm)
Blank 25 --- 1 0.1 10 7.80
Test 25 1 --- 0.1 10 8.62
Fruit bromelain (6000 rpm)
Blank 25 --- 1 0.1 10 7.7
Test 25 1 --- 0.1 10 8.43
Stem bromelain (2nd eluate)
Blank 25 --- 1 0.1 10 7.90
Test 25 1 --- 0.1 10 8.75
Stem bromelain (4th eluate)
Blank 25 --- 1 0.1 10 7.40
Test 25 1 --- 0.1 10 8.35
Fruit bromelain (2nd eluate)
Blank 25 --- 1 0.1 10 7.40
Test 25 1 --- 0.1 10 7.95
Fruit bromelain (4th eluate)
Blank 25 --- 1 0.1 10 7.90
Test 25 1 --- 0.1 10 8.50
Thai J. Pharm. Sci. 34 (2010) 67-76 71
incubated overnight at 4 ÌC. After incubation, the preci-
pitated enzymes were centrifuged at 10,000 rpm for 10
minutes at 4 ÌC. The pellet was collected and dissolved
in 10 ml of 10 mM Tris HCl buffer which was later
subjected to dialysis.
Dialysis
The above obtained solution was placed in a
dialysis bag and checked for the leakage of the sample
in it. The dialysis bag was then suspended in a beaker
containing 100 mM phosphate buffer-NaCl solution.
This setup was kept in refrigerator/cool conditions
overnight. This entire process was carried out for both
stem and fruit bromelain.
Ion exchange chromatography of purified bromelain
on DEAE cellulose
DEAE cellulose bed, of 1 cm thickness, was
prepared in a chromatography column and equilibrated
with 0.5 M sodium phosphate buffer solution (pH-8.0)
followed by eluting buffer ù1û i.e. 25 mM Tris HCl and 25
mM NaCl. The dialyzed sample of stem and fruit
bromelain was poured onto the column, from the sides,
without disturbing the DEAE cellulose bed and allowed
to settle. Enzyme was eluted using the first eluting
buffer i.e. 25 mM Tris HCl and 25 mM NaCl. Eluate was
collected in test tube. Elution was done at a flow rate of
1 ml/min. The same process of elution was carried out
using solutions 2, 3, 4, 5 and 6 containing 50 mM,
75 mM, 100 mM, 125 mM and 150 mM NaCl, respectively.
Besides the variable concentration of NaCl, all eluates
contained 25 mM Tris HCl. The dialyzed enzyme sample
was poured onto the column. The enzymes were then
eluted using eluting buffer ù2û (10 ml of 25 mM Tris HCl
and 50 mM NaCl). Eluates were collected in the same
test tubes. The process of elution was continued using
eluting buffers 3, 4, 5 and 6 contained 75 mM, 100 mM,
125 mM and 150 mM of NaCl, respectively. Finally,
ion-exchange eluates of stem and fruit bromelain
were assayed for their activity (GDU assay) as reported
above for crude isolates of bromelain for stem and
fruits.
Quantitative estimation of stem and fruit bromelain by
Lowryûs method
Concentration of proteins (bromelain) in stem and
fruit was determined by Lowryûs method as reported
previously with minor modifications [17]. Different dilutions
of BSA solution were prepared by taking BSA solution
(100 µg/ml) and distilled water as a standard in the
test tube. The final volume in each of the test tubes was
1 ml. The BSA range was 0.02 to 0.1 mg/ml. The 0.1 ml
of crude enzymes of stem and fruit bromelain, ion
exchange eluate 2nd and 4th of stem and fruit bromelain
enzymes were taken into test tubes 7, 8, 9, 10, 11 and
12 respectively and the final volume was made up to 1
ml with distilled water. These were unknown samples.
Five ml of alkaline copper sulphate reagent was added
to each tube and mixed well. These solutions were
incubated at room temperature for 10 mins. Then 0.5 ml
of FC (Folin Ciocalteau) reagent was added to
each tube and incubated in dark for 30 min. The
spectrophotometer analysis was performed and the
optical density i.e. absorbance was measured at 660
nm for all the samples. The absorbance was plotted
against protein concentration to get a standard calibration
curve. The absorbance of unknown sample was checked
and the concentration of the unknown sample was
determined.
Gel electrophoresis of isolated enzymes
Sodium dodecylsulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed with different
isolated/extracted enzymes (bromelain). The extracted
enzymes were concentrated and loaded onto a 3.5%
stacking gel and subjected to electrophoresis on a 12%
separating gel at 200 V (BioRad, Hercules, California,
USA) until the coomassie dye stained protein band
reached the gel bottom. SDS PAGE photograph of
isolated bromelain was obtained.
72 S. S. Gautam et al.
Results
Bromelain is a mixture of enzymes found naturally
in the juice and stems of pineapple. In the present
experiments, purification of bromelain-containing juice
extracted from pineapple stems and fruits by known
procedures comprising in general crushing the stems
and fruits in roll presses followed by pressing the
crushed mass to extract pineapple stem and fruit juice.
Juice was extracted from stem and fruit of pineapple
plant which were called as crude extract of the enzymes
and the activity of these crude extracts was checked
by the hydrolysis of gelatin which was estimated and
represented in the form of gelatin digestion units (GDUs).
Fruit-and stem-isolated bromelain activities were
determined and the comparative analysis is shown in
Table 4 and Fig. 1. The enzymatic activity of crude
extracts was found out to be 2,100 units/gm for stem
bromelain, whereas for fruit bromelain it was 1,450
units/gm (Fig. 1).
The crude extract was subjected to ammonium
sulfate precipitation to precipitate the enzyme. The
pellet was dissolved in 10 mM Tris HCl buffer and
subjected to dialysis to remove the salt and other ions
bound to the enzyme. Then the enzymes were purified
by anionic ion exchange chromatography, where the
resin used was DEAE cellulose. The stem and fruit
bromelain enzymes were eluted using different
concentrations of NaCl and Tris HCl buffer. From
the enzymatic assay of ion exchange eluates, it was
observed that maximum amount of NaOH was required
for eluate 2nd and 4th of stem and fruit bromelain.
The protein concentration (bromelain) of crude
extract, centrifuged fraction and ion-exchage eluates
of stem and fruit were determined by Lowryûs method
(Table 3). Protein concentrations of stem bromelain
were found to be 0.7 mg/ml, 0.08 mg/ml, and 0.14 mg/
ml for crude extract, eluate 2nd and 4th, respectively.
Protein concentration for centrifuged fractioned of stem
Table 3 Quantitative estimation of stem and fruit bromelain by Lowryûs method
Sample sample (ml) Distilled water (ml) Alkaline CuSO4 (ml) F.C. reagent (ml) O.D. at 660 nm
Blank --- 1.0 5 0.5 ---
BSA (20 µg/ml) 0.2 0.8 5 0.5 0.135
BSA (40 µg/ml) 0.4 0.6 5 0.5 0.292
BSA (60 µg/ml) 0.6 0.4 5 0.5 0.400
BSA (80 µg/ml) 0.8 0.2 5 0.5 0.535
BSA (100 µg/ml) 1.0 --- 5 0.5 0.635
Stem crude 0.1 0.9 5 0.5 0.475
Fruit crude 0.1 0.9 5 0.5 0.756
Stem bromelain (2000 rpm) 0.1 0.9 5 0.5 0.268
Stem bromelain (4000 rpm) 0.1 0.9 5 0.5 0.308
Stem bromelain (6000 rpm) 0.1 0.9 5 0.5 0.421
Fruit bromelain (2000 rpm) 0.1 0.9 5 0.5 0.536
Fruit bromelain (4000 rpm) 0.1 0.9 5 0.5 0.482
Fruit bromelain (6000 rpm) 0.1 0.9 5 0.5 0.362
Stem eluate 2 0.1 0.9 5 0.5 0.052
Stem eluate 4 0.1 0.9 5 0.5 0.096
Fruit eluate 2 0.1 0.9 5 0.5 0.258
Fruit eluate 4 0.1 0.9 5 0.5 0.088
Thai J. Pharm. Sci. 34 (2010) 67-76 73
bromelain at 2,000, 4,000 and 6,000 rpm were found to
be 0.87 mg/ml, 0.72 mg/ml and 0.52 mg/ml, respectively
whereas protein concentration of centrifuged fractioned
for fruit bromelain at 2,000, 4,000 and 6,000 rpm were
found to be 0.40 mg/ml, 0.51 mg/ml and 0.63 mg/ml
respectively. Protein concentration of fruit bromelain was
determined as 1.11 mg/ml, 0.39 mg/ml and 0.14 mg/ml
for crude extract, 2nd and 4th eluate of fruit bromelain,
respectively.
Enzymatic activity of crude extract for stem and
fruit bromelain obtained from centrifugation at different
rpm demonstrated that the maximum enzymatic activity
was observed at 2,000 rpm (GDU 3,500 units/gm) for
stem bromelain (P > 0.001), while maximum enzymatic
activity of fruit bromelain was found to be at 6,000 rpm
Figure 1 Proteolytic activity of isolated bromelain from stem and fruits of Ananas comosus. Significance was tested using one way
ANOVA and Tukey-kremer post test by comparing all the isolated extracts with crude stem bromelain. **indicates the most
significant methods (p < 0.01) for bromelain isolation.
(GDU 1,965 units/gm). Similarly, enzymatic activity of
2nd eluate for stem bromelain was 14,875 units/gm
and that of eluate 4th was 9,500 units/gm (P > 0.001).
For fruit bromelain, eluate 2nd was 1,974 units/gm and
that of eluate 4th was 6,000 units/gm. Comparative
study of proteolytic activity of different fractions from
stem and fruit bromelain showed stem bromelain was
found to be superior to fruit bromelain in hydrolyzing
the gelatin (Fig. 1).
The SDS-PAGE analysis showed that extracted
protein (bromelain) was electrophoretically pure
and stable during in-process isolation steps (Fig. 2).
The estimated molecular weight of bromelain was
around approximately 30 kDa (Fig. 2), as in previous
reports [8, 9].
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
Enzyme activity (GDU/gram)
Crude stem bromelain
Crude fruit bromelain
Stem bromelain (2000 rpm)
Stem bromelain (4000 rpm)
Stem bromelain (6000 rpm)
Stem bromelain (2
nd
elute)
Stem bromelain (4
th
elute)
Fruit bromelain (2
nd
elute)
Fruit bromelain (4
th
elute)
Fruit bromelain (2000 rpm)
Fruit bromelain (4000 rpm)
Fruit bromelain (6000 rpm)
**
**
**
**
74 S. S. Gautam et al.
Figure 2 SDS-PAGE electrophoresis of isolated bromelain. Lane 1: standard of molecular weight, from top to bottom, phosphorylase b
(94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and
alpha lactoalbumin (14.4 kDa); Lane 2: crude stem bromelain; Lane 3: crude fruit bromelain; Lane 4: 2nd eluate stem bromelain;
Lane 5: 4th eluate stem bromelain; Lane 6: 2nd eluate fruit bromelain; Lane 7: 4th eluate fruit bromelain.
Table 4 Proteolytic activity of isolated bromelain from stem and fruits of Ananas comosus
Sample Actual amount of Normality Conc. of
Enzyme activity
NaOH rundown (ml) of NaOH (N)
enzymes
(GDU/gram) (n=3)
(mg/ml)
Crude stem bromelain 1.05 0.1 0.70 2,100 ± 340
Crude fruit bromelain 1.15 0.1 1.11 1,450 ± 450
Stem bromelain (2000 rpm) 1.00 0.1 0.40 3,500 ± 480
Stem bromelain (4000 rpm) 1.01 0.1 0.51 2,772 ± 350
Stem bromelain (6000 rpm) 1.02 0.1 0.63 2,266 ± 330
Fruit bromelain (2000 rpm) 0.97 0.1 0.87 1,560 ± 500
Fruit bromelain (4000 rpm) 0.82 0.1 0.72 1,594 ± 500
Fruit bromelain (6000 rpm) 0.73 0.1 0.52 1,965 ± 400
Stem bromelain (2nd eluate) 0.85 0.1 0.08 14,875 ± 500
Stem bromelain (4th eluate) 0.95 0.1 0.14 9,500 ± 600
Fruit bromelain (2nd eluate) 0.55 0.1 0.39 1,974 ± 280
Fruit bromelain (4th eluate) 0.60 0.1 0.14 6,000 ± 290
Thai J. Pharm. Sci. 34 (2010) 67-76 75
Discussion
Bromelain is a general name for a family of
sulfhydryl-containing, proteolytic enzymes obtained from
Ananas comosus. The primary component of bromelain
is a sulfhydryl proteolytic fraction. It also contains a
peroxides, acid phosphatase, several protease inhibitors
and organically-bound calcium [18]. It is a cysteine
endopeptidase which specifically cleaves peptide bonds
at the carbonyl group as found in arginine or in aromatic
amino acids like phenylalanine or tyrosine. In the present
work comparative study of stem bromelain and fruit
bromelain has been performed. Juices were extracted
from stem & fruit of pineapple plant, which contained
the enzymes cysteine endopeptidase in stem bromelain
and aspartic endopeptidase in fruit bromelain [19].
The juice extracted was called as crude extract of the
enzymes and the activity of these crude extracts was
estimated by the hydrolysis of gelatin and represented
in the form of gelatin digestion units (GDUs).
Earlier reports on structural and kinetic analyses
revealed that stem bromelain differed markedly in their
enzymatic activity from fruit bromelain. From the results,
it was concluded that the stem bromelain showed more
enzymatic activity than fruit bromelain. Crude fraction of
stem bromelain obtained from centrifugation at 2,000
rpm possess better proteolytic than fruit bromelain
collected at 6,000 rpm. After that the crude extract was
subjected to ammonium sulfate precipitation to precipitate
out the enzyme. The pellet was dissolved in 10 mM
Tris HCl buffer and subjected to dialysis to remove the
salt and other ions bound to the enzyme. Then the
enzymes were purified by anionic ion exchange
chromatography using DEAE cellulose.
The stem and fruit bromelain enzymes were eluted
using different concentrations of NaCl and Tris HCl buffer.
From the enzymatic assays of ion exchange eluates, it
was observed that maximum amount of NaCl was
required for eluate 2nd and 4th of stem and fruit bromelain.
Thus, ion exchange eluate 2nd of stem bromelain and
eluate 4th of fruit bromelain showed more activity
(P > 0.001). Proteolytic activity of different fractions from
stem and fruit bromelain follows the sequence as follows,
2nd eluate stem bromelain > 4th eluate stem bromelain > 4th
eluate fruit bromelain > stem bromelain at 2,000
rpm > crude stem bromelain > crude fruit bromelain.
From the above results we concluded that stem bromelain
was found to be superior to fruit bromelain in hydrolyzing
the gelatin.
However, it was possible to obtain pure biological
products, such as bromelain, with lower operational cost
using DEAE-cellulose resins, thus, decreasing overall
process cost. Moreover, single SDS-PAGE band of ion
exchange isolated bromelain protein also inferred the
integrity and purity of bromelain protein. Results of
poly-acrylamide gel electrophoresis of purified bromelain
components supports the earlier findings revealed,
the molecular weight of bromelain was approximately
30 kDa [20].
It has now been found that stability and activity of
purified solution of the enzyme is important, not merely
from the standpoint of purity or concentration of the
product, but also because presence of impurities is
accompanied by a decrease in enzymatic activity.
The present study provides ion exchange process
producing a purified bromelain having not only the
advantages of reduced ash and increased specific
activity, but also stability in solution which in turn means
stability of specific activity.
Conclusion
This work exhibited that the purification of bromelain
by ion exchange chromatography using DEAE anion
exchanger, served as an economical means for the
purification of bromelain from stem and fruit of pineapple
plant. Recovered extract maintain the structural integrity
and showing better proteolytic activity then crude extract
and centrifugal fraction. Study result inferred that the
stem bromelain possess better GDU activity over fruit
bromelain. Centrifugal fraction exhibited better enzymatic
activity over crude extract but lesser in comparison to
ion exchange eluates, suggested that this technique
needs to be optimized to produce bromelain with better
proteolytic activity. Moreover a lot of attempts are
required to be made to develop a simple, economical
and effective technique to produce bromelain of ultrapure
grade.
76 S. S. Gautam et al.
Acknowledgement
The authors are indebted to the staff members of
the Kanak Manjari Institute of Pharmaceutical Sciences,
Rourkela, Orissa, India and Dr. (Mrs.) M.R. Vishnu Priya,
Nitza Biologicals, Hyderabad, India for providing us the
guidance and required facilities for the research work.
The authors gratefully acknowledge to chairman of ISF
College of Pharmacy, Moga to provide facilities for carry
out part of research work.
References
[1] S. Gregory, and N. D. Kelly. Bromelain: a literature review
and discussion of its therapeutic applications, Alt. Med.
Rev. 1(4): 243-257 (1996).
[2] M. T. Murray, and J. E. Pizzorno. Bromelain. In: J. E. Pizzorno,
and M. T. Murray (eds.), Textbook of Natural Medicine,
Vol 1. (2nd ed.), Churchill Livingstone, Edinburgh, 1999,
pp. 619-622.
[3] S. J. Taussig, and S. Batkin. Bromelain, the enzyme
complex of pineapple (Ananas comosus) and its clinical
application: an update, J. Ethnopharmacol. 22: 191-203
(1988).
[4] J. N. Moss, C.V. Frazier, and G.J. Martin. Bromelains, the
pharmacology of the enzymes, Arch. Int. Pharmacodyn.
145:168 (1963).
[5] R. Guo, P. H. Canter, and E. Ernst. Herbal medicines for
the treatment of rhinosinusitis: a systematic review,
Otolaryngol. Head Neck Surg. 135(4): 496-506 (2006).
[6] G. Uhlig, and J. Seifert. The effect of proteolytic enzymes
(traumanase) on posttraumatic edema, Fortschr. Med. 99:
554-556 (1981).
[7] A. Gutfreund, S. Taussig, and A. Morris. Effect of oral
bromelain on blood pressure and heart rate of hypertensive
patients, Hawaii Med. J. 37(5): 143-6 (1978).
[8] R. M. Heinicke, and W. A. Gortner. Stem bromelain-a new
protease preparation from pineapple plants, Econ. Bot. 11(3):
225-234 (1957).
[9] T. Murachi, and H. Neuratii. Fractionation and specificity
studies on stem bromelain, J. Bio. Chem. 235(1): 99-107
(1960).
[10] T. Harrach, K. Eckert, H. R. Maurer, I. Machleidt, W. Machleidt,
and R. Nuck. Isolation and characterization of two forms of
an acidic bromelain stem proteinase, J. Protein Chem. 17(4):
351-61 (1998).
[11] D. M. Bollag. Ion-exchange chromatography, Methods Mol.
Biol. 36: 11-22 (1994).
[12] B. Paull, and P. N. Nesterenko. Novel ion chromatographic
stationary phases for the analysis of complex matrices,
Analyst 130(2):134-46 (2005).
[13] P. R. Levison. Large-scale ion-exchange column chroma-
tography of proteins comparison of different formats, J.
Chromatogr. B Analyt. Technol. Biomed. Life Sci. 790(1-2):
17-33 (2003).
[14] J. J. Holthuis, and R. J. Driebergen. Chromatographic
techniques for the characterization of proteins, Pharm.
Biotechnol. 7: 243-99 (1995).
[15] G. L. Volkov, S. I. Andrianov, E. S. Gavriliuk, T. V.
Goroshnikova, and A. Slominskii. Purification of biomolecules
by the method of the expanded bed adsorption
chromatography. III. Method optimization Applications,
Ukr Biokhim Zh. 77(2): 26-57 (2005).
[16] P. Moodie, Gelatin digestion unit analytical method.
Enzyme Development Corporation, New York, 2001.
[17] O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R.J. Randall.
Protein measurement with the Folin phenol reagent, J. Biol.
Chem. 193: 265 (1951).
[18] A. D. Napper, S. P. Bennett, M. Borowski, M. B. Holdridge,
M. J. Leonard, E. E. Rogers, Y. Duan, T. Richard, R. A.
Laursen, B. Reinhold, and S. L. Shames. Purification and
characterization of multiple forms of the pineapple-
stem-derived cysteine proteinases ananain and comosain,
Biochem. J. 301: 727-735 (1994).
[19] A. D. Rowan, D. J. Buttle, and A. J. Barrett. The cysteine
proteinases of the pineapple plant, Biochem. J. 266(3):
869-75 (1990).
[20] J. Scocca, and Y. C. Lee. The composition and structure of
the carbohydrate of pineapple stem bromelain, J. Biol. Chem.
244(18): 4852-4863 (1969).
... As such, they can also be further classified as cysteine proteinases, belonging to the common group of sulfhydryl proteolytic enzymes. Bromelain is also considered a thiol endopeptidase (internal clearing enzyme) [20], with the activity of all sulfhydryl endopeptidases depending upon the thiol group (SH) of a cysteine residue [3,21]. Stem bromelain has a stable secondary structure and shows activity in the pH range 7-10, with its activity being irreversibly lost above pH 10 [22]. ...
... The protease activity of SBM is comparatively higher than FBM [21,25,26] even though it possesses inferior specificity for peptide bonds. The optimal pH for SBM has been reported to be about 7 in numerous studies, whilst its optimal conditions range for demonstrating development is 50-60 • C [16,21,25,26]. ...
... The protease activity of SBM is comparatively higher than FBM [21,25,26] even though it possesses inferior specificity for peptide bonds. The optimal pH for SBM has been reported to be about 7 in numerous studies, whilst its optimal conditions range for demonstrating development is 50-60 • C [16,21,25,26]. The optimal pH condition for FBM, on the other hand, is 3-8, whereas the average maximum temperature is very wide, 37-70 • C [27,28]. ...
Article
Bromelain is a complex combination of multiple endopeptidases of thiol and other compounds derived from the pineapple fruit, stem and/or root. Fruit bromelain and stem bromelain are produced completely distinctly and comprise unique compounds of enzymes, and the descriptor “Bromelain” originally referred in actuality to stem bromelain. Due to the efficacy of oral administration in the body, as a safe phytotherapeutic medication, bromelain was commonly suited for patients due to lack of compromise in its peptidase efficacy and the absence of undesired side effects. Various in vivo and in vitro studies have shown that they are anti-edematous, anti-inflammatory, anti-cancerous, anti-thrombotic, fibrinolytic, and facilitate the death of apoptotic cells. The pharmacological properties of bromelain are, in part, related to its arachidonate cascade modulation, inhibition of platelet aggregation, such as interference with malignant cell growth; anti-inflammatory action; fibrinolytic activity; skin debridement properties, and reduction of the severe effects of SARS-Cov-2. In this paper, we concentrated primarily on the potential of bromelain’s important characteristics and meditative and therapeutic effects, along with the possible mechanism of action.
... Methanol extraction of these pieces was obtained using a shaker at 150 rpm, 45 o C for 48 h. The extract was then filtered, kept at 57-58 o C in Hot Air Oven until completely dried and stored at -20 o C for further use (Gautam et al., 2010). Before the experiments, the extract was further dissolved in water and DMSO in the ratio of 1:2 (concentration = 1 g/ml). ...
Article
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disorder, which affects joints and causes synovial inflammation. Tumor necrosis factor (TNF-α) is the major cytokine involved in pathogenesis. In our study, we tried to investigate the anti-inflammatory response of crude extract of pineapple stem using SW982 as synovitis model induced by TNF-α. Cell survivability was measured using SW982 cells treated with 10 ng/ml TNF-α with and without different concentrations of the extract. The expression level of different pro-inflammatory cytokines was analyzed by qPCR and protein expression levels were measured by western blot. Crude extract effectively reduced the levels of IL6, IL-1β, and P65 (Rel A) in SW982 cells. It works as an anti-inflammatory agent to put a barrier in the induction of inflammation. Pineapple stem extract effectively decreases the inflammation in SW982 cells and pro-inflammatory cytokine level. It may be considered as a medicinal plant extract to reduce inflammation in RA disease.
... One of the proteins that has been widely researched and is known to have many health benefits is bromelain. It is a mixture of various cysteine proteinases with similar amino acid sequences and is found in pineapple fruits and stems [1,[23][24][25]. Bromelain has been shown to reduce the expression of ACE-2 and TMPRSS2 in VeroE6 cells, as well as to significantly reduce the expression of the S-Ectodomain of SARS-CoV-2 [26]. ...
Article
Full-text available
Before entering the cell, the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) binds to the human angiotensin-converting enzyme 2 (hACE2) receptor. Hence, this RBD is a critical target for the development of antiviral agents. Recent studies have discovered that SARS-CoV-2 variants with mutations in the RBD have spread globally. The purpose of this in silico study was to determine the potential of a fruit bromelain-derived peptide. DYGAVNEVK. to inhibit the entry of various SARS-CoV-2 variants into human cells by targeting the hACE binding site within the RBD. Molecular docking analysis revealed that DYGAVNEVK interacts with several critical RBD binding residues responsible for the adhesion of the RBD to hACE2. Moreover, 100 ns MD simulations revealed stable interactions between DYGAVNEVK and RBD variants derived from the trajectory of root-mean-square deviation (RMSD), radius of gyration (Rg), and root-mean-square fluctuation (RMSF) analysis, as well as free binding energy calculations. Overall, our computational results indicate that DYGAVNEVK warrants further investigation as a candidate for preventing SARS-CoV-2 due to its interaction with the RBD of SARS-CoV-2 variants.
... Physiochemical properties of cysteine endopeptidases derived from pineapple plants [1,13,15,[22][23][24]. The activation energy of bromelain is 41.7 kcal/mol [23], and same can be activated by many chemical agents, including calcium chloride, cysteine, sodium cyanide, bisulfate salt, hydrogen sulfide, sodium sulfide and benzoate [13,36,37]. Stem bromelain is reversibly inhibited during reaction with organic mercury, ions of mercury and tetrathionate. ...
Article
Full-text available
Infectious diseases along with various cancer types are among the most significant public health problems and the leading cause of death worldwide. The situation has become even more complex with the rapid development of multidrug-resistant microorganisms. New drugs are urgently needed to curb the increasing spread of diseases in humans and livestock. Promising candidates are natural antimicrobial peptides produced by bacteria, and therapeutic enzymes, extracted from medicinal plants. This review highlights the structure and properties of plant origin bromelain and antimicrobial peptide nisin, along with their mechanism of action, the immobilization strategies, and recent applications in the field of biomedicine. Future perspectives towards the commercialization of new biomedical products, including these important bioactive compounds, have been highlighted.
... Bromelain is an enzyme extract with protease activity, which is found mainly in the pineapple plant (Ananas comosus) of the genus Bromeliaceae [6]. This extract can be obtained from both the stem and the fruit of the pineapple plant and contains as the main component a mixture of glycosylated proteolytic sulfhydryl enzymes [7][8][9][10][11]. The bromelain strain possesses different biochemical properties and compositions compared to fruit bromelain [12], the latter containing several thiol endopeptidases and also compounds such as peroxidases, acid phosphatase, glycoproteins, carbohydrates and organic complexed Ca 2+ [6,13]. ...
Article
Full-text available
The growing interest in the appearance and color of teeth has led to the emergence of a wide range of teeth whitening methods, both in dental offices and in patients’ homes. Concerns about the possible side effects or toxic effects of peroxide-based whitening gels leads to the identification of alternative whitening methods, based on natural compounds with mild action on tooth enamel and remineralizing effect. In this context, this study describes the preparation and in vitro analysis of whitening gels based on natural active agents—bromelain, quince and whey—using organic (polyacrylate, polyethylene glycol) and/or inorganic (silicate) excipients. Five natural products gels were prepared, containing bromelain extract, quince extract and whey, in various proportions. Two supplementary gels, one containing Lubrizol and another containing SiO2, were prepared. All gels were submitted for multiple in vitro analysis such as: SDS-PAGE analysis, UV-vis and FTIR spectroscopy, SEM microscopy, antibacterial activity on Streptococcus mutans ATCC 25175, Porphyromonas gingivalis ATCC 33277, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. The quince extract sample was the only one which completely discolored the blue dye on SDS-PAGE analysis. On the UV-vis spectra, the 303 nm band is assigned to an in situ modified form of bromelain. SEM images of gels containing SiO2 particles show evident marks of these particles, while the rest of the gels containing Lubrizol or whey are more uniform. Regarding antibacterial tests, the SiO2 gel samples did not show inhibition in any strains, but the other tested samples varied in the size of the inhibition diameter depending on the amicrobial strain tested; the protease activity of bromelain modulates the composition of the added whey proteins. Bromelain added as a nanoencapsulated assembly better preserves its integrity. The prepared gels showed antibacterial properties.
... Bromelain is a cysteine-type peptidase, extracted from the pineapple plant (Ananas comosus). Depending on its source, it is usually identified as either fruit bromelain or stem bromelain, and the latter is most commonly commercially available in the market [1]. Bromelain is composed of a complex mixture of proteases and non-protease enzymes of which stem bromelain constitutes approximately 80%. ...
Article
In-flow small angle X-ray scattering (SAXS) was used to probe in real-time (typically every second) the hydrolysis of whey protein isolate (WPI) by bromelain. The WPI concentration was 2.5%, the enzyme to substrate ratio was 1:10, and the enzymatic reaction was followed for 90 min at 50°C and pH 7. SAXS showed that the average size of WPI molecules was about 20 Å and that even at the completion of the enzymatic reaction some intact molecules of similar size remained; these are likely bromelain molecules and trace amount of BSA. SAXS allowed us to monitor the hydrolysis course through the calculation of the power-law exponent (P) and the Guinier scale factor (G) using a theoretical unified model fitting. The fitting exercise also indicated that bromelain hydrolysis transforms the globular WPI molecules into Gaussian polypeptides. The hydrolysis of WPI, which was completed within 40 min of hydrolysis, was confirmed by the degree of hydrolysis and turbidity measurements and by SDS-PAGE, which showed that bromelain has a broad specificity. This study demonstrates that SAXS is a powerful method to monitor in situ and real-time protein hydrolysis and can offer insights into protein structural changes that occur.
Article
Full-text available
Antioxidant phenolic compounds were extracted from fermented samples of Golden pineapple peels via an ultrasound method. The fermentation conditions to maximize the production of phenolic content and antioxidant activity were previously determined (pH: 5.5, T: 37.3 °C and 85% moisture content). A central composite design with 20 treatments was applied to evaluate the effect of the ethanol concentration, time, and temperature on the production of phenolic compounds and antioxidant activity of the extracts. The statistical analysis showed that the optimal conditions to produce extracts with high phenolic content and antioxidant activity were: 62 °C, 30 min and 58% ethanol. We obtained 866.26 mg gallic acid equivalents (GAE)/g d.m in total phenolic content and for antioxidant activity expressed as percentage inhibition, 80.06 ± 1.02% for ABTS and 63.53 ± 2.02% for DPPH, respectively. The bioactive compound profile in the extracts was identified and quantified using ultra-high performance liquid chromatography (UHPLC), this method showed the presence of rosmarinic acid, caffeic acid, vanillic acid, p-coumaric acid, ferulic acid, quercetin-3 glucoside, rutine, quercetin, kaempherol-3 glucoside and gallic acid, demonstrating the great potential of these by-products to obtain components that can benefit the consumer's health.
Article
The pineapple waste increased by the rise of production in pineapple, one of increasing pineapple waste is pineapple pulp (bromelain litter). The bromelain litter will be disadvantage for the environment, this matter could be solved by converting bromelain litter into compost. The composting process in this study used ligninolytic fungus (Trichoderma sp.). Composting technology which currently has rapidly developed is Aerated Compost Tea (ACT) or a derivate product of compost. The use of this study was to ensure the best incubation period of ACT bromelain litter which induced by Trichoderma sp. fungus on the growth of tomato (L. esculentum Mill.). The study was conducted using a completely randomized design (CRD) with 7 treatments and 3 replications, namely: P0= control (without ACT), P1= ACT bromelain 24 hours, P2= ACT bromelain 48 hours, P3= ACT bromelain 72 hours, P4= ACT bromelain and leaf litter 24 hours, P5= ACT bromelain and leaf litter 48 hours, and P6= ACT bromelain and leaf litter 72 hours. The variables observed were number of leaves and plant chlorophyll content. The data obtained were analyzed with ANOVA at the level of 5 % and tested for the Least Significant Difference (LSD) at the level of 5 %. The results of this study showed that ACT was induced by Trichoderma sp. fungus which effective for the vegetative growth of tomato (L. esculentum Mill.) is ACT bromelain litter with incubation period up to 72 hours (P3).
Article
Several in vitro studies along with some in vivo studies have shown the anti-cancer activity of bromelain against several types of cancer except cervical cancer. This study is focused to address the therapeutic effect of bromelain on a human cervical cancer cell line in vitro using HeLa cells. Here, the IC50 dose of bromelain enhanced Hela cell apoptosis at a concentration of 100 μg/ml after 48 h treatment, which is statistically significant compared to the dose of 50 μg/ml (p < 0.01). The percentage of live cells was reduced to about 48% and the percentage of apoptotic cells has increased to about 47% at the IC50 dose of bromelain with a 48 h exposure. Significant proportions of HeLa cells were observed to undergo apoptosis via the p53-dependent pathway in a ROS-independent manner at 100 μg/ml after 48 h treatment. In addition to that, bromelain also inhibited the formation of colonies and the migratory ability of HeLa cells. The present study reports that bromelain can act in an apoptotic pathway on human cervical cancer cell line HeLa.
Article
Full-text available
Biodegradability is an important property for soft robots that makes them environmentally friendly. Many biodegradable materials have natural origins, and creating robots using these materials ensures sustainability. Hence, researchers have fabricated biodegradable soft actuators of various materials. During microbial degradation, the mechanical properties of biodegradable materials change; these cause changes in the behaviors of the actuators depending on the progression of degradation, where the outputs do not always remain the same against identical inputs. Therefore, to achieve appropriate operation with biodegradable soft actuators and robots, it is necessary to reflect the changes in the material properties in their design and control. However, there is a lack of insight on how biodegradable actuators change their actuation characteristics and how to identify them. In this study, we build and validate a framework that clarifies changes in the mechanical properties of biodegradable materials; further, it allows prediction of the actuation characteristics of degraded soft actuators through simulations incorporating the properties of the materials as functions of the degradation rates. As a biodegradable material, we use a mixture of gelatin and glycerol, which is fabricated in the form of a pneumatic soft actuator. The experimental results show that the actuation performance of the physical actuator reduces with the progression of biodegradation. The experimental data and simulations are in good agreement (R 2 value up to 0.997), thus illustrating the applicability of our framework for designing and controlling biodegradable soft actuators and robots.
Article
Full-text available
The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.
Article
Full-text available
Two major components of pineapple bromelain, purified to electrophoretical homogeneity, appeared to have the same oligosaccharide group consisting of D-glucosamine, D-mannose, D-xylose, and L-fucose in ratios of 2:2:1:1. By exhaustive proteolysis of the bromelains, glycopeptides containing only Asx, Glx, and Ser were obtained. Periodate oxidation, methylation, and glycosidase digestion showed that the oligosaccharide chain has a highly branched structure in which all the neutral sugars are in nonreducing terminal positions and both N-acetyl-D-glucosamine residues occur in internal positions.
Article
This review describes the performance of various column designs available to process-scale users of low-pressure chromatography for protein purification. By carrying out a range of ion-exchange separations using Whatman microgranular ion-exchange celluloses we are able to compare and contrast the practical performance issues associated with several designs of axial and radial flow columns.
Article
First introduced as a therapeutic compound in 1957, bromelain's actions include: (1) inhibition of platelet aggregation; (2) fibrinolytic activity; (3) anti-inflammatory action; (4) anti-tumor action; (5) modulation of cytokines and immunity; (6) skin debridement properties; (7) enhanced absorption of other drugs; (8) mucolytic properties; (9) digestive assistance; (10) enhanced wound healing; and (11) cardiovascular and circulatory improvement. Bromelain is well absorbed orally and available evidence indicates that it's therapeutic effects are enhanced with higher doses. Although all of its mechanisms of action are still not completely resolved, it has been demonstrated to be a safe and effective supplement. (Alt Med Rev 1996;1(4):243-257)
Article
The proteolytic enzymes in this plant product, not yet in commercial production, may find application, as do similar agents from other sources, in the bating of hides, tenderizing of meat, chill-proofing of beer and other directions suggested in this article.
Article
The effect of oral bromelain upon blood pressure and heart rate of 19 patients has been studied. Dosage of bromelain up to twice the maximum recommended had no effect upon blood pressure or heart rate. When the dosage was increased up to 8 times the maximum recommended, the heart rate increased proportionately with the amount administered. Blood pressure however, remained unchanged. These findings suggest that oral bromelain is safe and add further evidence to the hypothesis that the effect of bromelain is related to its action on endogenous prostaglandins.
Article
After a short description of the uses of pineapple as folk medicine by the natives of the tropics, the more important new pharmaceutical applications of bromelain, reported between 1975 and 1978, are presented. Although the exact chemical structure of all active components of bromelain is not fully determined, this substance has shown distinct pharmacological promise. Its properties include: (1) interference with growth of malignant cells; (2) inhibition of platelet aggregation; (3) fibrinolytic activity; (4) anti-inflammatory action; (5) skin debridement properties. These biological functions of bromelain, a non-toxic compound, have therapeutic values in modulating: (a) tumor growth; (b) blood coagulation; (c) inflammatory changes; (d) debridement of third degree burns; (e) enhancement of absorption of drugs. The mechanism of action of bromelain affecting these varied biological effects relates in part to its modulation of the arachidonate cascade.
Article
The edema producing property of a proteolytic enzyme (bromelain), which was parenterally or intraduodenally applied, was investigated in a traumatically induced hindleg edema in rats. Under standardized conditions the hindlegs were squeezed by a wringer and swelling was volumetrically measured. Whereas after enteral application of bromelain a significant reduction of the edema could be observed, the parenteral application only resulted in a minimal therapeutic effect. Although enterally applied enzymes are thought to be degraded in the gut, the better results were obtained after enteral administration of bromelain. This supports the observation that also enzymes can be absorbed by the gut without loosing their biological properties.
Article
Ion-exchange chromatography allows the separation of proteins and peptides by taking advantage of their net charge. These macromolecules can also be concentrated by ion exchange either on a column or as a batch procedure (see Note 5). Although procedures for separating peptides or proteins vary according to each individual molecule, many basic rules apply to all ion-exchange purifications, and these generalized procedures will be described in this chapter.