Potential Role of Bromelain in Wound Healing Application: A Review

Abstract

Bromelain is a proteolytic enzyme derived from the pineapple plant (Ananas comosus). Bromelain can be extracted from pineapple stems and fruits. Additionally, it can be derived from pineapple wastes such as the core, crown, and peel. Various extraction and purification methods such as reverse micellar system, aqueous two-phase system, chromatographic techniques, and membrane filtration have been used in order to produce high-quality bromelain. Bromelain has been used clinically since 1876 and was first introduced as a therapeutic agent in 1957. Bromelain has gained increasing acceptance and compliance among patients as a phytotherapeutic drug due to its safety and lack of undesirable side effects. Bromelain is regarded as a nutrient that promotes wound healing due to the presence of several closely related proteinases that exhibit anti-inflammatory, fibrinolytic, and debridement properties.

Full text

INTERNATIONAL JOURNAL OF INTEGRATED ENGINEERING VOL. 15 NO. 4 (2023) 1-18
© Universiti Tun Hussein Onn Malaysia Publisher’s Office
IJIE
Journal homepage: http://penerbit.uthm.edu.my/ojs/index.php/ijie
The International
Journal of
Integrated
Engineering
ISSN : 2229-838X e-ISSN : 2600-7916
*Corresponding author: nadirul@uthm.edu.my
2023 UTHM Publisher. All rights reserved.
penerbit.uthm.edu.my/ojs/index.php/ijie
1
Potential Role of Bromelain in Wound Healing Application: A
Review
Celine Ng1, Mohd Syahir Anwar Hamzah1, Nadirul Hasraf Mat Nayan1,2*
1Faculty of Engineering Technology,
Universiti Tun Hussein Onn Malaysia, Pagoh Higher Education Hub, Jalan Panchor, Pagoh, 84600, MALAYSIA
2Oasis Integrated Group, Institute of Integrated Engineering,
Universiti Tun Hussein Onn Malaysia, Parit Raja, 86400, MALAYSIA
*Corresponding Author
DOI: https://doi.org/10.30880/ijie.2023.15.04.001
Received 3 January 2022; Accepted 10 May 2023; Available online 28 August 2023
1. Introduction
Pineapple or Ananas comosus is grown in several tropical and subtropical countries, including India, China,
Kenya, Hawaii, South Africa, Malaysia, Philippines and Thailand [1]. It has been used as a medicinal plant in several
native cultures. Crude pineapple extract belongs to a group of proteolytic enzymes (proteases) and is under the
classification of cysteine proteases [2]. There are at least 4 distinct cysteine proteases identified from the crude extract
of pineapple: stem bromelain, fruit bromelain, ananain and comosain. Ananain and comosain only be found in the
pineapple stem [3]. The major proteases that are present in pineapple stem and fruit are bromelain. Ananain is the
second most abundant cysteine enzyme found in the pineapple stem where 5% of total protein is present, and the exact
amount of comosain that can be found in the pineapple stem is still unidentified. Figure 1 below shows the parts of the
pineapple plant.
Bromelain was chemically known since 1876 and was introduced as a therapeutic compound in 1957 when
Heinicke found it in high concentrations in the pineapple stem [4, 5]. Bromelain from pineapple fruit is called fruit
bromelain EC 3.4.22.33, whereas bromelain extracted from pineapple stem is called stem bromelain (EC 3.4.22.32) [6].
Besides the fruit and stem, bromelain can also be isolated in small amounts from pineapple wastes such as core, leaves,
crown, and peel [7]. Bromelain concentration is high in stem compared to fruit and is considered an inexpensive source
of bromelain.
Abstract: Bromelain is a proteolytic enzyme derived from the pineapple plant (Ananas comosus). Bromelain can
be extracted from pineapple stems and fruits. Additionally, it can be derived from pineapple wastes such as the
core, crown, and peel. Various extraction and purification methods such as reverse micellar system, aqueous two-
phase system, chromatographic techniques, and membrane filtration have been used in order to produce high-
quality bromelain. Bromelain has been used clinically since 1876 and was first introduced as a therapeutic agent in
1957. Bromelain has gained increasing acceptance and compliance among patients as a phytotherapeutic drug due
to its safety and lack of undesirable side effects. Bromelain is regarded as a nutrient that promotes wound healing
due to the presence of several closely related proteinases that exhibit anti-inflammatory, fibrinolytic, and
debridement properties.
Keywords: Bromelain, wound, anti-inflammatory, fibrinolytic, debridement

Ng et al., Int. J. of Integrated Engineering Vol. 15 No. 4 (2023) p. 1-18
2
Fig 1 - Parts of the pineapple plant
2. Biochemistry of Bromelain
Bromelain is a mixture of different thiol endopeptidases and other components peroxidases, phosphatases,
cellulases, carbohydrates, several protease inhibitors and organically bound calcium [8,9]. Fruit bromelain and stem
bromelain possess different biochemical properties and compositions when compared. As mentioned, along with stem
bromelain and fruit bromelain, two other cysteine proteases are present in the pineapple, which are ananain and
comosain. The extracellular matrix turnover, antigen presentation, processing events, digestion, immunological
invasion, haemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins are just a few of
the cysteine roles protease play. It is important to differentiate the enzymes according to their types to maximize the
role of pineapple cysteine proteases. Therefore, to differentiate and characterize these 4 proteases, a few tests, including
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), mass spectroscopy, N-terminal amino acid
sequence analysis and monosaccharide composition analysis, had been conducted. To date, 8 basic proteolytically
active components have been detected in the stem bromelain. The two main components have been labelled as F4 and
F5. The proteinase considered the most active fraction had been designated as F9, which comprises about 2% of the
total proteins. These components were fractionated and isolated from the crude extract of pineapple stem by using
cation exchange chromatography and further purified by affinity chromatography [10]. A small amount of comosain
was isolated; however, the exact percentage remains unidentified. SDS-PAGE and mass spectroscopy were used to
determine the molecular weight of each component. The molecular weight of F4, F5, F9 and comosain is 24.4 kDa,
24.5 kDa, 23.4 kDa and 24.5 kDa, respectively [2, 10, 11].
Figure 2 presents the first 20 amino acid sequences of F4, F5, F9 and comosain. All these components
demonstrated a similar sequence; however, their differences can be distinguished in positions 9, 10 and 20. Since F4
and F5 were the main active components found, their protein sequence was considered the reference for comparison. In
F4 and F5, about 70% of the major sequence was started with valine (V), and about 30% of the minor sequence started
with an additional alanine (A) [12]. Compared with F9, the sequence differs at positions 10 and 20. At position 10,
tyrosine (Y) was substituted by serine (S), asparagine (N) was substituted by glycine (G) at position 20. The amino acid
sequence of comosain differs at positions 9 and 20. Aspartic acid (D) at position 9 was substituted by asparagine, while
at position 20, glycine substituted asparagine amino acid [11]. The 20 amino acids are listed in Table 1
Fig 2 - N-terminal amino acid sequences of cysteine proteases of the pineapple plant [11]

Ng et al., Int. J. of Integrated Engineering Vol. 15 No. 4 (2023) p. 1-18
3
Table 1 - List of 20 amino acids [18]
Name
Abbreviation
Three letter code
Single letter code
Alanine
Ala
A
Arginine
Arg
R
Asparagine
Asn
N
Aspartic Acid
Asp
D
Cysteine
Cys
C
Glutamic Acid
Glu
E
Glutamine
Gln
Q
Glycine
Gly
G
Histidine
His
H
Hydroxyproline
Hyp
O
Isoleucine
Ile
I
Leucine
Leu
L
Lysine
Lys
K
Methionine
Met
M
Phenylalanine
Phe
F
Proline
Pro
P
Pyroglutamatic
Glp
U
Serine
Ser
S
Threonine
Thr
T
Tryptophan
Trp
W
Tyrosine
Tyr
Y
Valine
Val
V
The results from monosaccharide composition analysis showed the different nature of F4, F5, F9 and comosain.
Fraction of F4 and F5 contained fucose, N-acetylglucosamine, xylose and mannose at the ration of 1:2:1:2 and
1.1:2:1:2, respectively. It is estimated that 50% of the proteins in F4 and F5 contain a carbohydrate chain. In addition,
comosain was found to have similar carbohydrate composition. F9, on the other hand, showed no monosaccharide was
detected. From the results of monosaccharide composition analysis, it can conclude that F4, F5 and comosain were
glycosylated whereas F9 was not glycosylated, whereas F9 was found to be unglycosylated [2, 10, 11, and 12].
Through characterizations' results of each fraction, it can be concluded that F4 and F5 represented stem bromelain,
while F9 represented ananain. The optimal pH for the F4 and F5 fractions is between 4.0 and 4.5, and for F9 close to a
neutral pH. The entire extract of bromelain has exhibited activity over a pH range of 4.5 to 9.8 [10]. F4 and F5 has
isoelectric point (pI) of 9.55, F9 is more than 10 [2, 14, 15].
For fruit bromelain, similar testing as stem pineapple's protein fractions was conducted for characterization. Fruit
bromelain has a molecular weight range of 28-31 kDa and not a glycoprotein, meaning it is not glycosylated. N-
terminal amino sequence analysis showed that fruit bromelain amino sequence started with alanine (A) [16], as shown
in Figure 2. The pI of fruit bromelain is 4.6 [17]. Table 2 summarizes the biochemistry properties of cysteine proteases
from the pineapple plant.
Table 2 - Summary of biochemistry properties of cysteine proteinase of the pineapple plant
Name
Fraction/
Abbreviation
Molecular
weight (kDa)
Isoelectric
point (pI)
Glycosylation
Stem
bromelain
F4 & F5
24.4 & 24.5
9.55
Glycosylated
Ananain
F9
23.4
˃10
Unglycosylated
Comosain
-
24.5
-
Glycosylated
Fruit
bromelain
-
28-31
4.6
Unglycosylated

Ng et al., Int. J. of Integrated Engineering Vol. 15 No. 4 (2023) p. 1-18
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3. Bioavailability
The degree to which the targeted biological destination fully absorbs a medicine or substance is known as
bioavailability. The rate and percentage of a drug's initial dose that effectively reaches either its biological target or the
body fluid realm, where its intended targets have unrestricted access, is more appropriately referred to as
bioavailability. In bromelain cases, it is normally absorbed through the gastrointestinal tract before spreading
throughout the body. The highest concentration, up to 40% of the high molecular weight substances of bromelain, is
detected in the blood after 1-hour oral administration. In a study by Castell et al., (1997), the human body can absorb a
significant amount of bromelain. The result of the study shows that about 12 g/day of bromelain can be consumed
without causing any major side effects on the body [19]. In 2010, a study demonstrated that 3.66 mg/mL and 2.44
mg/mL of bromelain were stable and remained in artificial stomach juice and artificial blood after 4 hours of reaction
[20].
4. Toxicity and Side Effects
Bromelain is low toxicity, with LD50 greater than 10 g/kg. LD50 refers to the amount of a substance that will kill
50% (one-half) of a set of test animals when administered all at once. No immediate toxic reaction was observed when
bromelain was administrated in mice and rats at 37 mg/kg and 85 mg/kg, respectively, through intraperitoneal injection.
The result is similar for intravenous administration of 30 mg/kg bromelain to mice and 20 mg/kg bromelain to rabbits
[21]. Toxicity tests conducted on rats with 500 mg/kg/day of bromelain oral administered daily showed no toxic effect
and alteration towards food intake, growth, histology of heart, kidney and spleen. No carcinogenic and teratogenic
effects were observed in rats after administered dosages of 1500 mg/kg/day of bromelain [22]. Normal doses of 3000
FIP units/day given to humans over 10 days did not significantly affect blood coagulation parameters [23]. Table 3
summarizes the toxicity test conducted for bromelain.
Table 3 - Summary of toxicity test for bromelain
Experimental
Target
Amount, Administration
method
Observation
Ref.
Mice
37 mg/kg, intraperitoneal
injection
No immediate toxic reaction
21.
Rats
85 mg/kg intraperitoneal
injection
No immediate toxic reaction
21
Rabbits
20 mg/kg, intravenous
administration
No immediate toxic reaction
21
Mice
30 mg/kg, intravenous
administration
No immediate toxic reaction
21.
Rats
500 mg/kg/day, oral
administration
No side effect and changes towards
food intake, growth, histology of heart,
kidney and spleen
22.
Rats
1500 mg/kg/day
No carcinogenic and teratogenic effects
22
Human
3000 FIP units/day
Does not affect blood coagulation
parameters after 10 days being
administrated
23
5. Bromelain Extraction and Purification Techniques
Fruit bromelain can be easily extracted from the juice of pineapple through ultrafiltration [24], whereas stem
bromelain can be extracted through centrifugation, ultrafiltration, lyophilization [25] and two-step Fast Protein Liquid
Chromatography [26]. Generally, parts of the pineapple plant intended for bromelain extraction are collected and sent
for preliminary treatment: cleaning, peeling and size reduction. Then, they were homogenized or crushed for cell
disruption before removing debris through centrifugation or filtration. Once the extraction process is done, the crude
mixture consists of bromelain enzyme and then undergoes a purification process to eradicate impurities that may
interfere with bromelain that can hinder its application and reduce the specific activity of the enzyme [27]. The method
used for bromelain purification includes reverse micellar system (RMS), aqueous two-phase system (ATPS),
chromatographic techniques and membrane filtration. The overview of the extraction and purification process of
bromelain is shown in Figure 3.

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Fig 3 - Overview of extraction and purification process of bromelain [24]
5.1 Reverse Micellar System
A reverse micellar system (RMS) is an interesting and promising liquid-liquid extraction technique for the
downstream processing of biomolecules [28, 29]. Micelle is an aggregate of molecules possessing both polar and non-
polar regions. Reverse micelles are thermodynamically stable surfactant water droplets dispersed in organic solvents
[30]. This system provides simple, easily scalable, energy efficient and mild separation conditions for enzyme recovery
in active form. Only protein of interest will be entrapped in the core of the reverse micelle, whereas the impurities will
remain in the organic phase. In most cases, electrostatic and hydrophobic interactions between protein and reverse
micelles are considered the driving forces for the diffusion of solutes into the core of the reverse micelle [31]. RMS
consists of 2 stages: forward extraction and back extraction. Forward extraction is the process that involves the
diffusion of protein of interest from the aqueous phase into the reverse micelles in the organic phase, and the protein of
interest is then diffused back into the new aqueous phase from the reverse micelles during back extraction. Figure 4
shows the overview of forwarding and back extraction of RMS. 2 types of surfactant can be used in forwarding
extraction: cetytrimethylammonium bromide (CTAB) and sodium bis(ethylhexyl) sulfosuccinate (AOT). CTAB is a
type of cationic surfactant, whereas AOT is an anionic surfactant [29]. During forward extraction, in the organic phase,
AOT and CTOB needed isooctane as their solvent and co-solvent (n-butanol and n-hexanol) for CTOB. Due to CTAB
forming small micelles, co-solvents are needed to help the recovery action resulting from the extraction process [32].
AOT prefers forward extraction at the pH lower than the protein's isoelectric point. It is reported that AOT formed a
complex with bromelain, and white precipitates were observed at the aqueous-organic interphase at a pH lower than 4.2
[33]. Hence, AOT is not suggested to act as a surfactant in forwarding extraction. CTAB, on the other hand, works
better in the forward extraction of fruit bromelain since it has relatively low pI (4.6) with a wide range of pH stability
above pI.
In a study of comparison between AOT and CTAB surfactants in forwarding extraction of bromelain by
Hemavathi et al., (2007) CTAB was found to be the most suitable for the extraction of fruit bromelain concerning
activity recovery of 97.56% and 4.54 fold of degree of purification when employed as a 150mmolL−1
CTAB/isooctane/5% (v/v) hexanol/15% (v/v) butanol system [28]. RMS was applied to extract bromelain from
pineapple wastes such as core, crown, peel and extended stem. Bromelain extracted from pineapple core with a fairly
good activity recovery, 106% and 5.2 fold of purification was obtained using CTAB. Pineapple peel, extended stem and
crown yielded purification folds of 2.1, 3.5 and 1.7, respectively, with RMS extraction by a cationic surfactant (CTAB)
[29]. Several modifications in RMS have been studied to improve protein yield and purification fold. The affinity-based
reverse micellar extraction and separation technique to extract bromelain from pineapple wastes yielded an activity
recovery of 185.6% with 12.32-fold purification [15]. Ultrafiltration coupled with RMS to upgrade the efficacy of RMS
resulted in a purification fold of 8.9 and activity recovery of 95.8% for bromelain [34, 35].

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Fig 4 - Overview of the reverse micellar system [29]
5.2 Aqueous Two-Phase System
An aqueous two-phase system (ATPS) is a liquid-liquid extraction method. It is based on incompatible and
immiscible two aqueous solutions. ATPS provides advantages such as low operational cost, highly selective, scalable,
non-toxic, reusable polymers and can withstand high biomass load [36, 37, 38, 39]. The most common biphasic system
is formed by two polymers (polymer/polymer) or a polymer with a salt (polymer/salt). Other types include alcohol with
salt (alcohol/salt) and polymer with ionic liquid (polymer/ionic liquid) [40, 41, 42]. In the polymer/polymer system, the
common material used is polyethylene glycol (PEG) and dextran. In the polymer/salt system, PEG was normally paired
with either phosphate, sulfate or citrate salt. An illustration of ATPS is shown in Figure 5. Water was utilized as both
phases' main component or solvent because it can form a gentle environment for biomolecules to separate and stabilize
polymers' structure and biological activities [43]. The phase-forming compounds must be solubilized above a critical
concentration in an aqueous solution to form 2 separate phases. In polymer/salt of ATPS, salts contain ions of different
hydrophobicity, and the hydrophobic ions force the portioning of counter ions to phase with higher hydrophobicity and
vice versa. The salting out effect moves the biomolecules (protein of interest) from the salt-rich phase to the polymer-
rich phase [44]. High recovery of enzymes can be achieved with ATPS due to the presence of polymer, especially PEG,
which caused the alteration in the structure of active sites of the enzymes [45]. ATPS had been applied to extract and
purify bromelain of crude extract of pineapple fruit, stem and its wastes.
A study investigated the optimum PEG and magnesium sulfate salt (MgSO4) concentration for bromelain
extraction from pineapple peels. It had been determined that 18% PEG with a molecular weight of 6000 / 17% MgSO4
is the optimum concentration that resulted in a high purification fold (3.44) and activity recovery (206%) [45]. ATPS of
PEG 1500/ potassium phosphate was employed to extract and purify fruit bromelain. The research results show that
18% PEG 1500 with 14% potassium phosphate yielded 228% activity recovery and 4-fold purity for fruit bromelain
extraction [46]. Another research composed of 14% PEG 1500/ 17.66 % potassium phosphate ATPS resulted in
89.65% fruit bromelain activity recovery and 2.8-fold purification [47]. A study by Coelho et al., (2012) focused on
stem bromelain extraction and purification with ATPS of polymer/salt method. Using 10.86% PEG 4000 and 36.21%
saturated ammonium sulphate yielded stem bromelain with 11.8-fold purification and 66.38% activity recovery [48].
Fig 5 - Illustration of the aqueous two-phase system [43]

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5.3 Chromatography Techniques
Chromatography is a separation technique to extract and purify the desired biomolecules. The biomolecules
mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material
called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate
[49]. The biomolecules' separation speed is affected by their molecular characteristics related to adsorption, partition
and affinity among their molecular weights. The purpose of applying chromatography is to achieve a satisfactory
separation within a suitable time interval and used as a quantitative analysis method [50]. Chromatography is a
relatively simple method where precise separation and purification are possible, a low sample volume is required, and it
works on a wide range of samples, including drugs, tissue extracts and food particles. Chromatography extraction and
purification techniques were used to extract bromelain from pineapples. These techniques include ion exchange
chromatography, gel filtration chromatography, affinity chromatography and high-speed counter-current
chromatography [34].
Two liquid chromatography steps for purified stem bromelains are ion exchange chromatography and gel filtration
chromatography. In the first step of the liquid chromatography ion exchange method, a glass column was packed with
caboxymethyl-celullose as a stationary phase. The crude extract of pineapple was allowed to move along the
carboxymethyl-cellulose resin. Then the experiment proceeded with gel filtration chromatography. Aliquots from ion
exchange chromatography were collected and submitted to a glass column filled with Sephadex G-50®. High recovery
of enzymatic activity (89%) and a purification factor of 16.93 were obtained from the two steps of liquid
chromatography [51]. High-speed counter-current chromatography (HSCCC) is a liquid-liquid extraction technique
based on hydrodynamic equilibration of the two-phase solvent system in the separation column. It is also recognized as
a hybrid technique of liquid-liquid counter current distribution and liquid chromatography [52]. In a study by Yin et al.,
(2013), HSCCC was coupled with the RMS consisting of 0.10 g/mL CTAB with isooctane and hexanol as solvent and
co-solvent, respectively. This study recovered a total of 3.01 g of fruit bromelain from 5.00 g of crude pineapple extract
in 200-minute run [53]. The immobilized metal affinity membrane (IMAM) was used to separate and purify the
mixture of bromelain and polyphenol oxidase. A microfiltration nylon membrane obtained activated membranes for
covalent immobilization of hydroxyethyl cellulose (HEC) with formaldehyde and zinc ions loaded on the membranes.
IMAM method successfully yielded 94.6 % bromelain activity recovery with 15.4-fold purification [54].
5.4 Membrane Filtration
Membrane filtration uses semi-permeable membranes to separate or purify the desired molecules based on size
differences [34]. The desired molecules will be diffused through the semi-permeable membrane. There are a few types
of membrane filtration: microfiltration, ultrafiltration, nanofiltration and reverse osmosis. Figure 6 below illustrates the
membrane filtration process. This purification method had been utilized to extract and purify bromelain from crude
pineapple extract. Membrane-based technology offers an alternative to producing high-quality purified bromelain in a
more efficient and sustainable process [55]. Membrane filtration of bromelain was applied either with two-step
filtration or a combination of membrane-based filtration with other purification techniques.
Simultaneous use of microfiltration and ultrafiltration to separate bromelain from pineapple pulp yielded 85%
activity recovery through microfiltration, and 10-fold purification was obtained by ultrafiltration [56]. Two-step
ultrafiltration with ceramic membrane was used to purify bromelain from pineapple's crude waste mixture. The
ultrafiltration was performed with 75 kDa and 10 kDa tubular ceramic membranes. In the first stage of ultrafiltration,
96.8% of enzyme recovery was achieved, and the purity of bromelain increased up to 2.5 fold in the second stage of
ultrafiltration [57]. The effect of diafiltration on two-stage ultrafiltration of bromelain from pineapple crude waste
mixture (crown. peel, and core) was investigated. The purpose of diafiltration was to dilute the bromelain in a diluent
followed by concentrating. 75 kDa and 10 kDa of tubular zirconium oxide were used as the membrane in the
ultrafiltration set-up. The diafiltration was introduced during the second stage of ultrafiltration. This combination of
methods resulted in 46 % of bromelain recovery, and the purity increased to 4.4-fold [58]. An integrated approach by
coupling RMS with ultrafiltration was studied to improve fruit bromelain extraction and purification. The RMS of
cationic surfactant 150 mM CTAB/80 % isooctane/ 5% n-hexanol/ 15% n-butanol (v/v) used for bromelain extraction
resulted in an activity recovery of 95.8% and purification of 5.9-fold. The purification of fruit bromelain increased to
8.9-fold after ultrafiltration with cellulose acetate membrane [35].

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Fig 6 - Illustration of membrane filtration
6. Injury-Induced Wound and Wound Healing Properties
Injury to the skin provides a unique challenge as wound healing is a complex process. This process's main
objective or goal is to completely restore the skin structure and functions. The wound healing process consists of three
interrelated phases: hemostasis and inflammation, proliferation and tissue remodelling. The first stage, hemostasis and
inflammation, occurs soon after the skin is damaged or injured. Hemostasis involves coagulation which causes bleeding
to stop and clotting of blood. Fibrinogen, one of the major components of the skin's connective tissues, leads to
coagulation of exudates and, together with the formation of a fibrin network, produces a clot in the wound, which stops
the bleeding [59]. Inflammation takes place simultaneously with hemostasis. Inflammation is also called a cleansing
phase, where some inflammatory cells involved in wound cleansing and defending infections enter the wound medium
and penetrate inside the dead cells [60, 61].
Proliferation is the second stage of the wound healing process. Proliferation aims to provide fast vascular regrowth
and closure to the wound by the generation of new tissue. Granulation tissues are formed and comprise a type III
collagen network that acts as a quick fix to close the wound and prevent infection [62]. This granulation tissue is
subjected to regression and is gradually replaced by stronger, long-strand type I collagen in the form of scar tissue after
its function is fulfilled. The final stage would be the remodelling of tissue. At this stage, fibroblasts completely cover
the surface of the wound as a new layer of the skin, and there is no evidence of the wound.
Nutrient deficiencies can impede wound healing, and several nutritional factors required for wound healing may
improve healing time and wound outcome [63]. Nutrients such as Vitamin A [64], Vitamin C [65], Vitamin E [66], zinc
[67], protein [68, 69], bromelain [64] and many more are essential in wound healing process.
7. Wound Healing Application of Bromelain
Over the years, various research and studies have been done to investigate the potential of bromelain as an
alternative enzyme in medicinal use and as nutritional support in wound treatment. In terms of wound healing
application, bromelain has proved itself as a useful proteolytic enzyme as it exhibits anti-inflammatory, fibrinolytic and
debridement activities. These will be discussed in further detail in the following section.
7.1 Anti-Inflammatory Agent
Inflammation is defined as the body's attempt at self-protection or the immune system's response to harmful
stimuli, such as pathogens, damaged cells, toxic compounds, or irradiation [70], and acts by removing harmful stimuli
and initiating the healing process [71]. Generally, there are 2 types of inflammation: acute inflammation and chronic
inflammation. Acute inflammation is a short-term process which starts rapidly in response to tissue injury or damage
due to trauma, microbial invasion or noxious compounds [72]. Chronic inflammation refers to a prolonged
inflammation that involves a progressive change in the type of cells present at the site of inflammation [73]. The
prolonged inflammatory response may result in deregulated differentiation and activation of keratinocytes, hindering
progress through normal stages of wound healing [74]. Wound healing disorders present as hypertrophic scars or non-
healing chronic wounds, also known as ulcers. Ulcers are considered the most prevalent wound healing problem in
humans. Most non-healing wounds fail to progress through the normal phases of wound repair and remain in a chronic
inflammatory state (prolonged inflammatory response). Thus, the transition from acute to chronic inflammation must
be avoided for the injury site to be treated in normal stages of wound healing without causing the wound to become
more severe and lead to ulcers.
Bromelain is had been clinically used as an anti-inflammatory agent in soft tissue injuries, chronic pain and
surgical wound care. The effectiveness of bromelain is studied in a clinical trial with a group of 60 patients that
undergo surgery for fixation of long bone fractures. 30 of them were treated with 90 mg of bromelain/tablet whereas

Ng et al., Int. J. of Integrated Engineering Vol. 15 No. 4 (2023) p. 1-18
9
the other 30 patients were treated with standard anti-inflammatory drugs. The volume of the operated limb was
measured, and the starting volume value on the 1st post-operative day was 100%. On the 14th post-operative day, the
average volume of the operated limb for the patients treated with bromelain tablet reduced by 12% compared to the
other 30 patients with only a 9% reduction in average volume. A significant reduction in pain and swelling with
accelerated healing was observed in the bromelain-treated patients [75]. Rhinoplasty is a surgery that changes the shape
of the nose for appearance changes or breath, or both purposes. In a random and placebo-controlled study, it has been
proved that orally administered bromelain minimized edema, pain and swelling after the surgery [76]. In a clinical trial
on patients undergoing cataract surgery, it was demonstrated that bromelain was orally administered 2 days prior to
surgery and 5 days post-operatively, resulting in significant inflammation and pain reduction [77]. The anti-
inflammatory activity of bromelain is closely related to the Kinin system. Bromelain reduces High Molecular Weight
Kinin (plasma kininogen), thus inhibiting the production of bradykinin, an agent that induces inflammation, pain and
swelling [78]. Administration of bromelain before surgery can reduce the average days for the complete disappearance
of pain and post-surgery inflammation [79]. Nowadays, bromelain is used to treat post-surgical wounds and help lessen
the pain and swelling.
7.2 Role of Fibrinolysis
Fibrinolysis is a process to prevent fibrin clots from growing and allows the body to clear fragments of clots safely.
Fibrinolysis occurs by converting plasminogen to plasmin to degrade fibrin into soluble fibrin degradation products
(FDP). Plasminogen is activated by either two primary serine proteases, which are tissue plasminogen activator (tPA)
or urokinase-type plasminogen activator (uPA) [80, 81]. tPA is synthesized and released by endothelial cells, whereas
uPA is produced by monocytes, macrophages and urinary epithelium. Due to high concentrations of specific inhibitors
such as plasminogen activator inhibitor 1 (PAI-1), both tPA and uPA have short half-lives in circulation, about 4-8
minutes. Since tPA and plasminogen bind at the same binding site of fibrin (ogen), the zymogen and its activator are
brought into close proximity, resulting in efficient local generation of plasmin [82]. The uPA, on the other hand, has a
lower affinity for plasminogen in which it does not require fibrin as a cofactor, and under normal conditions, uPA
appears to act mainly in extravascular locations. Circulating serine protease inhibitors or serpins neutralize plasminogen
and plasmin activators in excess concentration [83]. Serpins that are important in fibrinolysis are plasminogen activator
inhibitor-1, plasminogen activator inhibitor-2 (PAI-2) and α2-antiplasmin (A2AP) [84, 85, 86]. PAI-1 released into the
circulation from endothelial cells, platelets, and other cells rapidly inhibit tPA and uPA. Plasmin and A2AP bind with a
stoichiometry of 1:1, at which point both become inactive. Plasmin is protected from A2AP inhibition when bound to
fibrin, allowing fibrinolysis to proceed [87]. Fibrinolytic activities are inhibited by thrombin activated inhibitor (TAFI),
a non-serpin inhibitor activated by thrombomodulin-associated thrombin. TAFI removes C-terminal lysine and arginine
residues on fibrin, thus decreasing the number of available plasminogen binding sites, slowing down the plasmin
generation and stabilizing the clots [88, 89]. Figure 7 shows the illustration of the fibrinolysis process.
Bromelain is an effective fibrinolytic agent and prevents blood from coagulation. It influences blood coagulation
by exaggerating the transformation of plasminogen to plasmin, inhibiting fibrin synthesis, a protein involved in blood
clotting [34, 90]. In vitro and in vivo studies have suggested that bromelain is an effective fibrinolytic agent as it
stimulates the conversion of plasminogen to plasmin, resulting in increased fibrinolysis by degrading fibrin [91].
Bromelain increased the fibrinolytic activity in a dose-dependent manner in a study on an inflammatory animal model
[92]. It is found that bromelain prolonged prothrombin and partial thromboplastin time and decreased the adenosine
phosphate (ADP) induced platelet aggregation in a dose-dependent manner [93]. The fibrinolytic activity of bromelain
has been attributed to enhancing the conversion of plasminogen to plasmin, which degrade the fibrin and limits the
spread of the clotting process.
Fig 7 - Illustration of fibrinolysis process [89]

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10
7.3 Debridement Agent
Debridement is the procedure involved in removing devitalized tissue such as necrotic tissue. Debridement is
considered a major component of wound management to prepare the wound bed for reepithelization [94]. The purpose
of debridement is to transform a chronic wound into an acute wound and initiate the healing process. Devitalized tissue,
such as necrotic tissue, serves as a source of nutrients for bacteria that can cause infection in the wound, making the
wound more severe [95]. Devitalized tissue also acts as a barrier for reepithelization, preventing applied topical
compounds from directly contacting the wound bed to provide their beneficial properties [96]. Several types of
debridement can be applied for the removal of devitalized tissue. These include autolytic debridement, biological
debridement, mechanical debridement, surgical debridement and enzymatic debridement.
Mechanical debridement is a non-selective type of debridement, which means that it will remove both devitalized
tissue and debris as well as viable tissue. This method involved mechanical force such as wet-to-dry dressing, wound
irrigation, and wound scrubbing [97]. The wet-to-dry dressing and wound scrubbing require using a sponge, brush or
gauze to remove devitalized tissues from the wound bed. Mechanical debridement will have a higher risk for bleeding
and peri-procedural pain. Mechanical debridement is applied on acute and chronic wounds with moderate to large
amounts of necrotic tissue, regardless of the presence of an active infection.
Surgical debridement uses a sharp instrument such as a scalpel and curettes to remove devitalized tissue on the
wound. It is the most rapid and effective method but is considered the most aggressive method [98]. Surgical
debridement can be performed at the bedside, wound care centre or operating room depending on the adequacy of
anaesthesia and the ability to control perioperative complications. This debridement should be done by skilled, trained,
qualified and licensed healthcare professionals. Surgical debridement is similar to mechanical debridement, where it
will have a higher risk of bleeding and possible general complication from the anaesthesia [99].
Autolytic debridement is the lysis or breakdown of damaged tissue at a wound site by the body's natural defence
system, in which endogenous phagocytic cells and proteolytic enzymes digest specific components of body tissues or
cells [100]. This type of debridement requires a moist environment and a functional immune system. Therefore,
moisture retentive dressings such as hydrocolloids, hydrogels, alginates and transparent films are encouraged to support
the maintenance of moisture and provide optimal conditions for the body's natural enzymes to activate wound
debridement [101]. Autolytic debridement induces softening of necrotic tissue and eventual separation from the wound
bed. It will take a few days for the tissue to be removed. The effectiveness is mandated by the amount of devitalized
tissue removed and the actual wound size.
Biological debridement uses sterile larvae of Lucilia sericata (species of green bottle fly) or maggot therapy to
remove the devitalized tissue. It is more suitable to be applied on the large wound where a painless removal of
devitalized tissue is needed. The larvae or maggots release proteolytic enzymes that contain secretions and excretions
that dissolve necrotic tissue from the wound bed [102]. A study has shown that complete debridement by free-range
maggots therapy took 14 days, whereas autolytic hydrogel debridement took 72 days to complete debridement.
Enzymatic debridement is described as a selective method of removing devitalized tissue using exogenous
proteolytic enzyme, fibrinolytic enzyme and collagenase [97]. Enzymatic debridement provides faster than autolytic
debridement, but slower when compared to mechanical and surgical methods [103]. Nowadays, many enzymes are
commercially available and being promoted as an alternative to surgical methods, and bromelain is one of them and is
the most employed [6, 104]. Table 4 summarizes the advantages and disadvantages of the debridement method
mentioned above.
Bromelain shows debridement properties as evidenced by the hydrolysis of devitalized wound tissue in studies in
vitro and in vivo without apparent effects on the surrounding normal tissue [78]. Burns are characterized by forming an
eschar, which is made up of burned and traumatized tissue. Eschar also serves as a medium for bacterial growth,
resulting in infection to the injury site and the neighbouring undamaged tissues [56]. Burns are common injuries
associated with significant morbidity and mortality, often leading to disfigurement and dysfunction due to scarring.
Tropical bromelain (35% in a lipid base) has complete debridement on experimental burns on rats in 2 days, compared
to collagenase, which required 10 days, with no side effects and damage to adjacent burned tissue [94]. Bromelain
contains escharase, which is responsible for this tremendous effect. Escharase is non-proteolytic and has no hydrolytic
enzyme activity against normal protein substrates or glycosaminoglycan substrates [91]. The efficacy of enzymatic
debridement of deeply burned hands with bromelain-containing gel, Debrase® is being studied. Debrase® is in the
form of lyophilized dry powder, can be activated by a hydrating vehicle gel or saline, applied and covered the wound
with an occlusive dressing for 4 hours. A total of 69 patients with deep burned wound hand was being assessed and
needed surgical procedure for skin grafting before enzymatic debridement. After application of Debrase®, only 36.2%
(25 out of 69 patients) require skin grafting for the treated wounded area. Moreover, the actual burn area that required
skin grafting was 1.0 ±0.7% total body surface area, a decrease of 40% from the area initially estimated (1.4 ± 0.8%
total body surface area) as deep and requiring surgery. This study showed that the enzymatic debridement reduced the
number of patients with burns who needed an operative procedure and skin grafting for the wounded area. [105]. In a
study by Bavata et al., (2019), the efficiency of bromelain-loaded chitosan nanofibers for burn wound repair was
investigated in an animal model. The burn healing effect of chitosan-2% w/v bromelain nanofiber was studied in the

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11
induced burn wounds in rats for 21 days. The results showed that chitosan-2% w/v bromelain nanofiber was more
efficient to heal burned skin than chitosan nanofiber alone in the animal model tested [106].
Table 4 - Summary of 5 debridement methods
Debridement
method
Mechanism of action
Advantages
Disadvantages
Mechanical
Uses mechanical force
such as wet-to-dry
dressing, wound irrigation
and wound scrubbing
Fast method
Painful
High risk of bleeding
Non-selective as it
removed both devitalized
and viable tissue
Surgical
Uses scalpel and curettes to
devitalized tissue on the
wound, can be done at the
bedside, wound care centre
or in the operating room
Fast
Effective
Selective
Aggressive method
Painful
High risk of bleeding
High cost
Autolytic
Uses the body's enzymes
and moisture to rehydrate,
soften and remove
devitalized tissue
Low risk of side effects
Slow process
Biological
Application of green bottle
fly larvae and maggots to
remove damaged tissue
Selective and fast methods
Suitable for large wound
Not suitable for all patients
Higher cost than autolytic
debridement
Enzymatic
Removing devitalized
tissue by using exogenous
proteolytic enzyme,
fibrinolytic enzyme and
collagenase
Selective
Commercially available
Painless and minimal blood
loss
High cost
Slower than mechanical
and surgical method
8. Kinin System and Clotting Cascade
The Kinin system is a plasma and tissue proteolytic system which consists of blood proteins that play a role in
inflammation, blood pressure control, coagulation and pain [107]. The Kinin system can be said to be involved in the
wound healing process. Blood coagulation is a process where a clot is formed to stop the bleeding at the injury area.
The clotting process is broken into 2 stages: primary and secondary haemostasis [108, 109]. Haemostasis is defined as
the arrest of bleeding, comes from Greek, haemo meaning blood and stasis meaning to stop [110]. The formation of a
weak platelet plug is achieved in primary haemostasis. A platelet plug is formed to temporarily protect from
haemorrhage until further stabilization of fibrinogen to fibrin via thrombin occurs in secondary hemostasis. Secondary
hemostasis involves the clotting factors acting in a cascade to stabilize the weak platelet plug. Platelets alone are not
enough to secure the damage in the vessel wall. A clot must be formed at the site of injury [111]. The formation of a
clot depends upon several substances called clotting factors. Roman numerals I designate these factors through XIII,
which activate each other, known as the clotting cascade. This cascade results in fibrinogen, a soluble plasma protein,
cleaving into fibrin, a non-soluble plasma protein. The fibrin proteins aggregate to form a clot [107].
The clotting cascade is triggered through 2 major pathways: intrinsic and extrinsic. The intrinsic pathway is
activated by trauma inside the vascular system and is activated by platelets, exposed endothelium, chemicals or
collagen. Clotting factors that are involved in this pathway are Factors XII, XI, IX, VIII [111]. On the other hand,
external trauma activates the extrinsic pathway that causes blood to escape from the vascular system. Factor VII is the
clotting factor involved in the extrinsic pathway [112, 113]. Both pathways meet and finish the pathway of clot
production in what is known as the common pathway. The common pathway involves factors I, II, V, and X. When a
surface (wound) is contacted by collagen or platelets, the kinin system and clotting cascade will be activated by
stimulating the conversion of Hageman factor to an active protease, Factor XIIa. The presence of Factor XIIa causes
the conversion of plasma prekallikrein into kallilrein and continues the intrinsic path of the clotting cascade by
converting Factor XI to its active form. In the autocatalytic loop, Kallikrein accelerates the activation of the Hageman
factor, which continues to potently activate both the kinin system and clotting cascade. In addition, Kallikrein cleaves
High Molecular Weight Kinin (HMWK) to produce bradykinin. Bradykinin is a mediator of inflammation, where it
stimulates both pain and vascular permeability, causing them to increase. The clotting cascade will convert fibrinogen
to fibrin, a protective matrix around the injury that inhibits tissue drainage, promotes edema, and blocks blood flow [8]
as illustrated in Figure 8.

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12
Fig 8 - Overview of Kinin system and clotting cascade [8]
8.1 Effect of Bromelain on Kinin System and Clotting Cascade
Many studies and research have been conducted to investigate the effect of bromelain on the kinin system,
especially on plasma kallikrein, bradykinin levels and plasma exudation at the inflammatory site. Most of the studies
were done on laboratory rats. 5 mg/kg and 7.5 mg/kg of bromelain caused the dose-dependent decrease of bradykinin
levels at inflammatory sites and prekallikrein levels in sera [114]. With a single injection of 10 mg/kg of bromelain, the
levels of HMWK and prekallikrein were markedly decreased in rat plasma [115]. Rats treated with bromelain show a
reduction in Factor X and prothrombin, which are needed for the activation of fibrinogen to fibrin through a common
pathway of the intrinsic and extrinsic cascade. These studies show that bromelain inhibits the generation of bradykinin
at the inflammatory site via depletion of the plasma kallikrein system and limiting the formation of fibrin by reducing
clotting cascade intermediates [21]. These activities result in a significant reduction in enema and pain and, at the same
time, enhance the circulation to the injured site.
Vessel repair begins after a clot is formed, starting with the conversion of plasminogen to plasmin. The function of
plasmin is to degrade the fibrin into smaller components which can be removed by monocytes and macrophages [116].
Bromelain had been shown to stimulate the conversion of plasminogen to plasmin, resulting in increased fibrinolysis in
rats. Thus, this minimizes venos stasis, facilitates drainage, increases permeability and restores the tissue's biological
continuity [21]. Table 5 below summarises bromelain's effect on the selected system and component.

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13
Table 5 - Summary of bromelain's effect on the Kinin System and the clotting cascade
Mediator/Enzyme
Function/Action
Effect of bromelain
Bradykinin
Pain mediator and vascular
leakage
Decrease
High Molecular Weight Kinin
(HMWK)
Precursor of bradykinin
Decrease
Prekallikrein
Produce Kallikrein
Decrease
Factor X
Clotting factor
Decrease
Prothrombin
Clotting factor, the precursor to
thrombin
Decrease
Plasminogen
Precursor to plasmin
Activate
9. Conclusion
Extensive research and studies have established bromelain's efficacy and efficiency in biomedical applications,
most notably wound healing. Due to its low toxicity and lack of adverse effects on the body when consumed, it has
become a common alternative phytotherapeutic enzyme among patients. Bromelain can also be extracted from
pineapple wastes such as the crown, core, peel, and leaf. Researchers are modifying and improving extraction and
purification methods to increase yield and purification fold and produce high-quality bromelain at a lower cost.
Bromelain extracted from pineapples has been suggested to have a promising role and play a significant role in wound
healing due to its anti-inflammatory, fibrinolytic, and debridement properties.
Acknowledgement
The authors would like to express their deepest appreciation to the Ministry of Higher Education Malaysia
(MOHE) through Fundamental Research Grant Scheme (FRGS/1/2022/TK09/UTHM/03/7) for funding this research.
This research is also supported by Universiti Tun Hussein Onn Malaysia (UTHM) through Geran Penyelidikan
Pascasiswazah (GPPS-Vote number H599) and Industrial Grant (Vote number M025).
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