16010825, 2020, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/odi.13476 by UFPI - Universidade Federal do Piaui, Wiley Online Library on [06/02/2023].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
| |
Received: 24 November 2019    Revised: 12 May 2020    Accepted: 21 May 2020

DOI: 10.1111/odi.13476

ORIGINAL ARTICLE

Bromelain reduces the non-alcoholic fatty liver disease and

periodontal damages caused by ligature-induced periodontitis

Even Herlany Pereira Alves1  | André dos Santos Carvalho1 |

Felipe Rodolfo Pereira Silva2 | Luiz Felipe Carvalho França1 | David Di Lenardo1 |
Any Carolina Cardoso Guimarães Vasconcelos3  | Hélio Mateus Silva Nascimento1 |
Víctor Lucas Ribeiro Lopes1 | Jefferson Soares Oliveira4 |
Daniel Fernando Pereira Vasconcelos1

1
Laboratory of Histological Analysis and
Preparation (LAPHis), Federal University of
Parnaiba Delta (UFDPar), Parnaiba, PI, Brazil
2
Department of Morphology, Federal
University of Amazonas, Manaus, Brazil
3
Medicine School, Education Institute of
Parnaiba Valley (FAHESP/ IESVAP - Afya),
Parnaíba, PI, Brazil
4
Laboratory of Biochemistry and Biology of
Microorganism and Plants (BIOMIC), Federal
University of Parnaiba Delta (UFDPar),
Parnaíba, PI, Brazil
Correspondence
Daniel Fernando Pereira Vasconcelos,
Universidade Federal do Delta do Parnaíba,
Campus Ministro Reis Veloso, Colegiado de
Biomedicina, Av. São Sebastião, 2819, Reis
Veloso, Parnaiba - PI 64204-035, Brazil.
Email: prof.dr.daniel.vasconcelos@gmail.com
Funding information
CNPq, Grant/Award Number: 455104/2014-
0
Abstract
Objective: The objective of this research was to evaluate the effects of bromelain
(derived from Ananas comosus) upon periodontitis in rats.
Materials and Methods: Twenty-four rats were separated into groups: control, peri-
odontitis, and bromelain treatment. Bromelain was administered daily by intraperi-
toneal injection for 20 days. Periodontitis was induced by ligature around the first
molars. Oral parameters and blood biomarkers were measured. The histopathological
evaluation of the hepatic tissue was performed. Bromelain treatment significantly re-
duced several oral inflammatory parameters, alveolar bone loss, and blood biomark-
ers compared to the rats on periodontitis.
Results: Treatment with bromelain improved the steatosis score. Bromelain used in
ligature-induced periodontitis in rats was able to reduce the oral inflammatory pa-
rameters Gingival Bleeding Index (GBI), tooth mobility (TM), probing pocket depth
(PPD), malondialdehyde (MDA), alveolar bone height (ABH) and gingival myeloper-
oxidase (MPO) and blood parameters (cholesterol, triglycerides, alanine aminotrans-
ferase, and aspartate aminotransferase). Bromelain treatment reduced the impact
of periodontitis, such as the reduction of hepatic steatosis and improvement in the
dosages of MDA and GSH.
Conclusion: Bromelain acts as a potential adjunct in the non-surgical treatment of
periodontitis and, consequently, reduces the impact of periodontitis, acting as anti-
inflammatory and antioxidant.

KEYWORDS

alveolar bone loss, anti-inflammatory, antioxidant, periodontal diseases, proteinase

© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved

Oral Diseases. 2020;26:1793–1802.  |

wileyonlinelibrary.com/journal/odi     1793

|
1794       ALVES et al.
1 |  I NTRO D U C TI O N
Periodontitis has been characterized as a multifactorial and chronic
inflammatory disease that affects millions of people worldwide
(Manji, Dahlen, & Fejerskov,  2018). The inflammatory condition is
caused by the accumulation of bacteria around the periodontal tis-
sues, resulting in an over immune–inflammatory response of the
organism in which several cytokines, growth factors, enzymes, and
inflammatory cells act resulting in periodontal destruction (Junior
et al., 2013). Although the bacteria are the initiating agent of peri-
odontitis due to their causing the release of inflammatory mediators
(Lee, Lee, Lee, Park, & Kim, 2018), genetic factors of the host pre-
dispose to a more intense inflammatory response with greater tissue
destruction (da Silva et al., 2016, 2018).
Specific bacteria, lipopolysaccharides (LPS), and their prod-
ucts induce inflammatory infiltrate with polymorphonuclear, neu-
trophil (PMN), and macrophage recruitment. Neutrophils release
the enzyme myeloperoxidase (MPO), a component considered as
antibacterial and that is actively involved in periodontitis (Nicu
& Loos,  2016). Some substances generated by the inflammation,
such as the reactive oxygen species (ROS) and cytokines produced
by activated phagocytes, are deleterious to tissues. ROS include
free radicals of oxygen and non-radical oxygen derivatives such
as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). The
imbalance between ROS and antioxidants in the body leads to
oxidative stress, making a significant contribution to a variety of
systemic changes that affect and cause kidney alterations (França
et al., 2017) and/or non-alcoholic fatty liver disease NAFLD (Dos
Santos Carvalho et al., 2017; Pessoa et  al.,  2018; Vasconcelos
et al., 2017; Vasconcelos et al., 2019).
Conventional periodontal therapy consists of scaling and
root planning to reduce the bacterial load and other harmful
components, aiming to maintain the health of the periodontal
tissues. Nonetheless, the recolonization of the subgingival area
by periodontopathogens and resistance acquired by some mi-
croorganisms can occur as the result of the failure of preventive
maintenance therapy, which can lead to recurrent disease. In this
way, studies with natural products that can aid in the control of
the growth of dental biofilms with both anti-inflammatory and an-
tioxidant properties and that prevent bone resorption are gain-
ing focus (Filho et  al.,  2018; da Silva et  al.,  2016). Bromelain is a
mixture of thiol endopeptidase and other components including
phosphatases, glycosidases, peroxidases, cellulases, glycopro-
teins, ribonuclease, and carbohydrates that are obtained from the
stem or fruit of pineapple (Ananas comosus). Bromelain has a broad
spectrum of enzyme activity over an ideal pH of 5.5–8.0 (Novaes
et al., 2016).
These enzymes are considered as the most active fraction,
which include 2% of the total proteins (Rathnavelu, Alitheen, Sohila,
Kanagesan, & Ramesh, 2016). It was demonstrated that the part of
the biological activity of bromelain cannot be due to a single pro-
teolytic fraction, and the useful effects of bromelain are likely due
to multiple factors (Maurer, 2001). These molecules from pineapple
16010825, 2020, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/odi.13476 by UFPI - Universidade Federal do Piaui, Wiley Online Library on [06/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Clinical relevance
Scientific rationale for the study: Periodontitis is an inflam-
matory disease that affects periodontal tissues and causes
systemic changes in the body such as the development of
non-alcoholic fatty liver disease (NAFLD).
Main findings: This study demonstrated that the adminis-
tration of bromelain in rats with induced periodontitis was
able to reduce inflammation and alveolar bone loss, as well
as minimize the NAFLD caused by it. These findings dem-
onstrate that bromelain can be potently used in the peri-
odontal treatment.
are well-known due to their clinical and therapeutic applications,
particularly in the inhibition of colorectal cancer cell proliferation
(Chang et  al.,  2019), burn debridement (Rosenberg et  al.,  2012),
improvement of antibiotic action (Ali, Mohammed, & Isa, 2015), an-
ti-inflammatory (Lee et al., 2018), antioxidant (Sahbaz et al., 2015),
antithrombotic, and fibrinolytic activities (Rathnavelu et al., 2016).
A previous study found that bromelain at a dose of 15  mg/kg
administered intraperitoneally was effective in reducing concentra-
tions of malonaldehyde (MDA) and elevated levels of glutathione
(GSH), which are markers of oxidative stress, thus demonstrating
antioxidant potential. In addition, bromelain has been shown to in-
hibit the growth of microorganisms involved in the progression of
periodontitis and to reduce neutrophil chemotaxis at the periodontal
site by 40% (da Silva et al., 2016).
Taking into account the anti-inflammatory activity of bromelain
and the lack of studies addressing its effect upon periodontitis, it
was hypothesized that bromelain can act as a possible therapeutic
approach for periodontitis (da Silva et al., 2016). In order to confirm
this hypothesis, the objective of this study was to investigate the
local and systemic effects of the treatment with bromelain (derived
from Ananas comosus) during periodontitis induced by ligation in
rats, considering that it is promising as a target for the investigation
of its osteogenic, anti-inflammatory, and oxidative stress in the peri-
odontal tissues.
2 | M ATE R I A L A N D M E TH O DS
2.1 | Animals
This study was approved by the Ethics Committee on Animal
Experimentation from the Federal University of Piauí under the pro-
tocol number of 233/16. Twenty-four female rats (150.6  ±  13.0  g,
Wistar, Rattus novergicus) were obtained. They were kept in cages
with water and feed ad libitum and placed in the laboratory for
30 days in alternating cycles of 12 hr of artificial lighting and 12 hr of
absence of light at 23°C ± 2°C, for setting the animals.

ALVES et al.
2.2 | Experimental design
Periodontitis was induced after intramuscular general anesthe-
sia with 15  mg/kg xylazine hydrochloride (Francotar-Virbac®,
Roseira, SP, Brazil) and 35 mg/kg ketamine (Francotar-Virbac®). A
nylon ligature 3-0 (Shalon®) was placed around the cervical region
of the right lower first molar of each rat and then tightly bound.
A similar model has been used previously in several other studies
(Dos Santos Carvalho et al., 2017; França et al., 2017; Vasconcelos
et  al.,  2019). The animals were divided into three groups: con-
trol group (n  =  8), which received no treatment or induction of
periodontal disease; periodontitis (n  =  8), and bromelain (n  =  8),
which received intraperitoneal treatment with bromelain (bro-
melain from pineapple stem, Sigma-Aldrich®, São Paulo, SP, Brazil)
15 mg/kg for 20 days + induction of periodontitis.
2.3 | Gingival bleeding index (GBI)
For the assessment of this index, the mandibular first molars were
probed in the area of the gingival sulcus or periodontal pocket for
10 s, using an adapted round-ended probe (Golgran) with a 0.2-mm
tip radius, before the euthanasia of the animals. After that, scores
between 0 and 5 were classified according to Liu et al. (2012).
2.4 | Probing pocket depth (PPD)
Three points were assessed, and the measurement was used (me-
siobuccal, midbuccal, and distobuccal; Liu et al., 2012).
2.5 | Evaluation of tooth mobility (TM)
The tooth mobility was evaluated by means of a score classification
as described previously (Xu & Wei, 2006).
2.6 | Myeloperoxidase (MPO) activity
MPO activity was assessed through the accumulation of neutrophil
in the gingival tissue (soft tissue) around the lower right first molar,
as described by Chaves et al. (2013).
2.7 | Measurement of alveolar bone height (ABH)
The ABH was evaluated to delimit the cementum–enamel junction
(CEJ), where three points were measured in the lingual part accord-
ing to Dos Santos Carvalho et al. (2017). These images as well as the
histopathological evaluation were measured using the image analy-
sis software (ImageJ v.1.48 Software) by 4 trained evaluators, and
the evaluation was performed blindly.
16010825, 2020, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/odi.13476 by UFPI - Universidade Federal do Piaui, Wiley Online Library on [06/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
|
      1795
2.8 | Histopathological evaluation of the mandible
The mandible samples were fixed in 10% formalin# and under-
went a process to remove the mineralized part in 10% nitric acid
(Dinâmica®). It was then embedded in paraffin to produce 6 microm-
eter cuts and stained with hematoxylin/eosin according to Toker,
Ozdemir, Eren, Ozer, & Sahin (2009).
2.9 | Neutrophil score
The neutrophil score of the right and left lower first molars was clas-
sified according to the following scores: 0 = physiological infiltrate
cell, 1 = slight infiltrate cell, 2 = moderate infiltrate cell, and 3 = se-
vere infiltrate cell (Vasconcelos et al., 2019).
2.10 | Histopathological evaluation of the liver with
hematoxylin/eosin and toluidine blue
The samples were removed, cut into fragments, and fixed in 10%
buffered formaldehyde. The histology was according to Dos Santos
Carvalho et al. (2017). Histologic evaluation of the hepatic tissue
(liver) and inflammation was graded (scored) according to Younossi
et al. (2005).
2.11 | GSH concentration
The GSH concentration for hepatic tissues was determined accord-
ing to the method described previously by Sedlak and Lindsay (1968).
2.12 | MDA level of the liver and gum
The MDA levels for liver and gum were determined using the method
described previously by Uchiyama & Mihara (1978).
2.13 | Serum alanine aminotransferase (ALT),
aspartate aminotransferase (AST), glucose,
triglycerides, and cholesterol
Serum contents of ALT, AST, glucose, triglycerides, and cholesterol
were measured with a commercial ELISA kit (Labtest®).
2.14 | Statistical analysis
The results were expressed as mean ± SD (standard deviation) and/or
median. Shapiro–Wilk test was used to verify the data distribution.
Differences between the three groups were analyzed using ANOVA
and the Bonferroni test for parametric data. Also, Kruskal–Wallis
 16010825, 2020, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/odi.13476 by UFPI - Universidade Federal do Piaui, Wiley Online Library on [06/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
|
1796       ALVES et al.

non-parametric test was used, followed by Dunn's test for multiple significant difference between the periodontitis and bromelain
comparisons, for non-parametric data. Differences were considered groups with p < .05 (Table 1).
significant when p < .05.

3.2 | Measurement of alveolar bone height (ABH)

3 |   R E S U LT S
3.1 | GBI, PPD, TM, MPO activity, and MDA in
gingival tissue
There was a 63.7% reduction in the gingival bleeding index of the
group treated with bromelain when compared with the periodontitis
group (p < .5; Figure 1a–c). There was a statistically significant dif-
ference in the PPD, considered as improvement, that is, a decrease
in the depth of the pocket of 38% in the groups treated when com-
pared to the periodontitis group. Tooth mobility showed a significant
decrease also when compared to the control group. The bromelain
group presented significant lower MPO dosage (59.7% reduction)
(p < .05) when compared to the periodontitis group (Figure 2g). The
result of the MDA dosage of the gingival tissue showed a statistically
The images obtained with a stereomicroscope (LUPETEC®, MRP 09)
in 30× magnification after the dissection and staining with meth-
ylene blue were analyzed and measured, demonstrating that there
was a statistically significant difference, with decrease of 31.3% in
loss of alveolar bone in the bromelain group (control, 2.1 ± 0.4; peri-
odontitis, 5.5 ± 0.8; bromelain, 3.8 ± 0.7; p < .05) compared to the
periodontitis group (Figure 1d–g).
3.3 | Histopathological evaluation of the mandible
Histopathological analysis of bone at the furcation region demon-
strated that bromelain, when used intraperitoneally, has the ability
to reduce bone loss caused by periodontitis. All photomicrographs

F I G U R E 1   (a) Clinical aspect of the normal gingiva of the control group, the arrow indicates the first molar and letter T shows the tongue.
(b) Clinical aspect of the periodontitis group, showing alteration in color, presence of intense edema and bleeding after probing. (c) Clinical
aspect of the group treated with bromelain 15 mg/kg, with improvement of gingival papilla staining, reduction of edema, and absence of
bleeding. ABH, (d) control group, without bone (β) loss, image (e) periodontitis group, intense bone (β) loss and image (f) representing the
periodontitis group treated with bromelain with moderate bone loss. (g) Means and standard deviation of the morphometric analyses of
alveolar bone height. (h) Illustration of the evaluation of the furcation region from the groups. The periodontitis group yielded higher values
of alveolar bone loss than did other groups. The treatment with bromelain prevented (p < .05) the alveolar bone loss in comparison with
the periodontitis group. The arrow indicates the first molar, the letter T indicates the tongue, and β illustrates the bone, *p < .05 indicates
periodontitis groups versus control group and †p < .05 indicates periodontitis groups versus bromelain group [Colour figure can be viewed at
wileyonlinelibrary.com]
 16010825, 2020, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/odi.13476 by UFPI - Universidade Federal do Piaui, Wiley Online Library on [06/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
ALVES et al. |
      1797

F I G U R E 2   (a) and (d) illustrate the control group with normal aspects of bone (β). (b) and (e) demonstrate severe bone loss at the
furcation region and intense infiltration of inflammatory cells (Arrow) in the periodontitis group. (c) and (f) illustrate the group treated with
bromelain, demonstrating prevention of bone loss at the furcation region and decrease in the infiltration of inflammatory cells compared
to the periodontitis group. (g) Shows the evaluation of MPO activity from the groups. The treatment with bromelain reduced (p < .05) the
neutrophil chemotaxis compared with the periodontitis group. (h) illustrates the neutrophil score from the groups. The treatment with
bromelain reduced (p < .05) the neutrophil score compared with the periodontitis group in the region of the lowers first molars. Hematoxylin
and eosin, 100× magnification. β, bone; δ, dentin, N, necrosis.*p < .05 indicates periodontitis groups versus the control group and †p < .05
indicates the periodontitis groups versus the bromelain group [Colour figure can be viewed at wileyonlinelibrary.com]

TA B L E 1   Oral parameters, means and standard deviation of the group treated with bromelain, there was a lower bone resorption
analyses: GBI, PPD, TM, and MDA when compared to the periodontitis group (control, 116.0 ± 5.1; per-

Groups iodontitis, 346.0 ± 15.0; bromelain, 242.0 ± 3.7; p < .05) (Figure 1h).

Oral parameters Control Periodontitis Bromelain

GBI D
0.2 ± 0.4 3.7 ± 0.4* 1.3 ± 0.5† 3.4 | Neutrophil score
PPDB 0.1 ± 0.4 2.3 ± 0.5* 1.4 ± 0.5†
TMD 0 (0–1) 3 (2–3) * 1 (1–2) † Analysis of the neutrophil scores of the right and left lower first mo-
MDA B
0.01 ± 0.005 0.03 ± 0.002* 0.02 ± 0.006 † lars demonstrated that there was a reduction in the amount of these
† cells in the bromelain-treated group compared to the periodontitis
Note: Different symbols (* and ) indicate a statistically significant
difference (p < .05), *p < .05 indicates periodontitis groups versus group (control, 0.28  ±  0.18; periodontitis, 2.85  ±  0.14; bromelain,
control group, and †p < .05 indicates periodontitis groups versus 1.14 ± 0.14; p < .05; Figure 2h).
bromelain group. Malondialdehyde (MDA), Gingival Bleeding Index
(GBI), probing pocket depth (PPD), tooth mobility (TM). The Bonferroni
testB (parametric data) was used for multiple comparisons and Kruskal–
Wallis and Dunn testsD (non-parametric data) were used for multiple 3.5 | Histopathological evaluation of the liver with
comparisons. hematoxylin and eosin (HE)

are stained with hematoxylin and eosin. Figure 2a–c has 40× origi- The livers of the periodontitis group demonstrated histopathological
nal magnification, and Figure 2d–f has 100× original magnification. changes, such as microvesicular steatosis in the hepatocytes, loss of
Statistical analysis showed that in the region of bifurcation of the cord organization around the central vein, and blood congestion, in
 16010825, 2020, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/odi.13476 by UFPI - Universidade Federal do Piaui, Wiley Online Library on [06/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
|
1798       ALVES et al.

F I G U R E 3   Illustration of the liver histopathological evaluation from different groups. The images (a), (d), and (g) represent the hepatic
tissue of the control group without histological alterations. Around the central vein (CV) are hepatocytes in the form of normal cord. The
sinusoidal blood vessels are also presented in their normal conformation (arrowhead). The images (b), (e), and (h) show the liver of the
periodontitis group, demonstrating hepatocytes with steatosis (arrows) and alterations in the sinusoid blood vessels (arrowhead). The images
(c), (f), and (i) illustrate the bromelain group, where one can observe sinusoidal blood vessels without alterations, hepatocytes organized in cord
conformation without degeneration and absence of steatosis. Image (j) illustrates GSH levels in the hepatic tissue from different groups. The
treatment with bromelain increased (p < .05) the GSH levels compared with the periodontitis group. Image (k) shows the values of liver MDA
in the groups. The treatment with bromelain reduced (p < .05) the values of liver MDA in comparison with the periodontitis group. Images (a),
(b), (c), (g), (h), and (i) are at 600× original magnification; images (d), (e), and (f) are at 1,000× original magnification and they represent the area
outlined by the dotted rectangle in detail, demonstrating the histological characteristics mentioned. Hematoxylin–eosin (HE) stain for images
(a–f) and toluidine blue stain for images (g), (h) and (i); central vein, CV; the arrowhead indicates sinusoid blood vessel. *p < .05 indicates the
periodontitis groups versus the control group and †p < .05 indicates the periodontitis groups versus the bromelain group [Colour figure can be
viewed at wileyonlinelibrary.com]

contrast to livers of the control group, which presented normal his-
tological structure of liver tissues. Bromelain-treated mice had a re-
duction in the development of hepatic alterations compared to the
periodontitis group (Figure  3a–i; p  <  .05). Treatment with 15  mg/kg
bromelain significantly reduced the steatosis score compared to the
periodontitis group (Table 2).
bromelain groups, showing that bromelain was able to main-
tain endogenous antioxidant levels and reduce lipid peroxidation
(Figure 3j) (GSH: control, 398.5 ± 87.8; periodontitis, 113.5 ± 65.4;
bromelain, 575.8 ± 47.7; MDA: control, 0.009 ± 0.001; periodontitis,
0.017 ± 0.001; bromelain, 0.012 ± 0.0007; p < .05).

3.7 | Blood biomarkers: AST, ALT, glucose,

3.6 | GSH and MDA concentration of the liver triglycerides, and cholesterol

The GSH and MDA dosing results showed that there was a sta- However, there were significant differences between periodontitis
tistically significant difference between the periodontitis and and bromelain groups in the biochemical analysis of AST (p < .05), as
 16010825, 2020, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/odi.13476 by UFPI - Universidade Federal do Piaui, Wiley Online Library on [06/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
ALVES et al. |
      1799

TA B L E 2   Histopathological evaluation of the hepatic tissue
Groups
Parameters evaluated Control Periodontitis Bromelain
Steatosis score 0 (0–1) 2 (1–3)* 1 (0–1)†
Inflammation score 0 (0–1) 1 (0–1) 0 (0–1)
Necrosis score 0 (0–1) 1 (0–1) 0 (0–1)
*p < .05 indicates periodontitis groups versus control group and †p < .05
indicates periodontitis groups versus bromelain group. Kruskal–
Wallis and Dunn tests (non-parametric data) were used for multiple
comparisons.
well as a significant statistical difference in the dosage of cholesterol
between control and periodontitis groups and between periodonti-
tis and bromelain groups (p < .05; Table 3).
4 | D I S CU S S I O N
To the best of our knowledge, this is the first study to show bro-
melain with a positive effect upon ligature-induced periodontitis
in rats. Taking into account the documented therapeutic effect of
bromelain in different processes associated with periodontitis, we
speculated that the treatment with bromelain could improve the
clinical aspects of the disease, thus confirming our hypothesis (da
Silva et al., 2016). In the present studies, our results showed that the
treatment was able to reduce the oral parameters (GBI, PPD, and
TM), improving biochemical measurements of gingival tissue (MPO
and MDA) and alveolar bone loss, according the ABH evaluation. In
addition, after the evaluation of the hepatic alterations caused by
periodontitis, the rats that received bromelain presented improved
results such as significant changes in the MDA and GSH steatosis
score, total and HDL cholesterol, uric acid and ALT in serum, when
compared to the rats that were only periodontitis-induced. The lig-
ature-induced periodontitis model in rats was sufficient to trigger
the inflammatory process, developing edema, gingival inflammation,
alveolar bone loss, and also systemic effects such as the develop-
ment of microvesicular steatosis in the liver.
Before euthanasia, the clinical parameters of the GBI, PPD, and
TM were evaluated. The results obtained through the statistical anal-
ysis showed significant differences between the groups. The brome-
lain group presented improvement in GBI (63.7%), PPD (38%), and
TM compared to the group with periodontitis. Such clinical param-
eters were reduced, probably, in view of the potential of bromelain
activity on pro-inflammatory cytokines such as interferon gamma
(IFN-γ), tumor necrosis factor (TNF-α), and granulocyte-macrophage
stimulating factors (GMCSF), which are cytokines involved in the
development and progression of periodontitis (da Silva et al., 2016).
Another parameter assessed was MPO, extensively used as a bi-
ological marker to measure the infiltration of neutrophils to the site
of inflammation (Fitzhugh, Shan, Dewhirst, & Hale, 2008). Exposure
to specific proteolytic activity of bromelain in vitro has been shown
to remove a number of cell surface molecules that are vital to leuko-
cyte trafficking (Hale, Greer, & Sempowski, 2002). These cell surface
changes were correlated with the rapid re-expression of brome-
lain-sensitive CD62L/L-selectin molecules that mediate rolling and
minimal re-expression of CD128 after in vivo treatment. These data
demonstrate that bromelain can effectively decrease neutrophil mi-
gration to sites of inflammation and support the specific removal of
the chemokine receptor CD128 as a potential mechanism of action
(Fitzhugh et al., 2008). The anti-inflammatory activity of bromelain
may also involve a reduction in the expression of COX-2 (an import-
ant mediator selectively induced in inflamed tissue) on periodontal
tissue. The reduction in myeloperoxidase levels observed in our ex-
periments confirms the decrease in leukocyte migration in treated
animals (Junior et al., 2013).
Besides alterations in MPO levels, the oxidative stress is also
present in the periodontal tissue damaged. As a result of an imbal-
ance between ROS production and antioxidant levels, the release
of proteolytic enzymes and ROS is considered as the main aspect
of host response against bacterial antigen in individuals susceptible
to periodontitis (Dahiya et al., 2013). The results of the evaluation
of lipid peroxidation of the present study presented that the treat-
ment with bromelain leads to a significant preservation of parame-

TA B L E 3   Blood biomarkers, means and standard deviation of ters similar to the control group, indicating that the bromelain group
the analyses: AST, ALT, glucose, triglycerides, and cholesterol presented a decrease in the levels of the inflammatory process, thus
corroborating with Bernela, Ahuja, & Thakur (2016), Rathnavelu
Groups
et al. (2016) and Lee et al. (2018).
Blood biomarkers Control Periodontitis Bromelain In addition, administration of bromelain reduced bone re-
AST 91.0 ± 5.6 98.4 ± 32.4 111.0 ± 33.4 sorption, confirming the hypothesis previously published (da Silva

ALT 34.5 ± 10.6* 40.6 ± 12.4* 24.3 ± 7.1† et  al.,  2016), which identified bromelain as an anti-inflammatory
agent with a possible action on the decrease in the release of me-
Glucose 291.0 ± 44.5 235.5 ± 53.4 273.2 ± 55.6
diating inflammatory agents such as interleukins, prostaglandins E2,
Triglycerides 50.1 ± 27.6 67.9 ± 19.5 23.2 ± 37.4†
prostacyclins, and TNF-alpha, whose effects on osteoclasts stimu-
Cholesterol 63.5 ± 0.005 86.3 ± 23.5* 66.2 ± 6.6*
late bone resorption dependent on prostaglandin synthesis (PGs).
Note: Different symbols (*periodontitis group versus control group and This signaling also induces RANK expression in osteoclasts, leading
†periodontitis group versus bromelain group) indicate a statistically
to increased RANKL activation. IL-1 beta stimulates the growth and
significant difference (p < .05). Alanine aminotransferase (ALT);
aspartate aminotransferase (AST). The Bonferroni test (parametric data) differentiation of osteoclast precursor cells and mature osteoclast
was used for multiple comparisons.

|
1800       ALVES et al.
activity. IL-1 beta is the most active cytokine involved in the process
(da Silva et al., 2016).
It is noteworthy that bromelain may also have been acting in in-
hibiting the growth of periodontopathogens: Streptococcus mutans,
Enterococcus faecalis, Porphyromonas gingivali,s and Aggregatibacter
actinomycetemcomitans, thus confirming the in vitro study by
Praveen et al. (2014), which concludes that bromelain can be used as
an antibiotic in the treatment of periodontitis.
Periodontitis is not only a local disease, but it is also associated
with systemic alterations, such as liver disease and steatosis related
to the oxidative stress (França et al., 2017; Lee et al., 2018; Tomofuji
et al., 2007) . Oxidative stress is a disturbance of balance between
oxidants and antioxidant species, generated by periodontitis (Dos
Santos Carvalho et al., 2017; França et al., 2017; Pessoa et al., 2018;
Vasconcelos et al., 2017; Vasconcelos et al., 2019) and the presence
of bacteria that can fall into the bloodstream, releasing toxins, fim-
briae, and enzymes that reach the blood vessels, the liver tissue and
cause lesions (Komazaki et  al.,  2017; Ziebolz et  al.,  2017). The ex-
perimental period of 20  days is sufficient for the development of
steatosis in animals, as it has been shown in previous studies (Pessoa
et al., 2018; Vasconcelos et al., 2019).
Our results demonstrated that bromelain was able to reduce oxi-
dative stress in the liver. The results were observed through the dos-
ages of GSH, an intracellular antioxidant that works as electrophilic
detoxification and elimination of oxidants (Lee et al., 2018), and dos-
age of MDA, released by lipid peroxidation caused in most cases by
ERO (França et al., 2017).
Both parameters were significantly different between the bro-
melain and periodontitis groups, leading to a reduction in hepatic
steatosis. Other studies also showed that bromelain was able to re-
duce the oxidative stress by reducing the production of nitric oxide
and ROS by cells (Chaudhary, Agarwal, & Bist, 2018).
The ingestion of bromelain has been reported in the literature
and can reduce serum cholesterol, triglycerides, and the accumula-
tion of cholesterol and triglycerides in the liver, which may lead to a
reduction of hepatic steatosis, as observed in the bromelain-treated
group compared to the periodontitis group (Al-Otaibi, Virk, &
Elobeid, 2015). They also reported a decrease in the plasma levels
of AST and ALT, which are the most commonly used biochemical
markers for liver damage. They are transaminase enzymes present in
large quantities in hepatocytes, and when suffering an injury, these
enzymes fall into the blood, increasing their concentration (Al-Otaibi
et  al.,  2015; Nyblom et  al.,  2006). Our results showed that there
were significant alterations in ALT dosage.
Other studies could be conducted with bromelain from fruit
(ripe or green) or leaves of the pineapple and obtained by differ-
ent processes. That may influence the physicochemical properties
of biomolecules such as their isoelectric point, surface hydropho-
bicity, molar mass, and presence of glycoproteins. For instance,
the bromelain dtem used in this study has a reactive sulfhydryl
group, which is essential for catalytic activity (de Lencastre
Novaes et al., 2016). This property of the bromelain stem can re-
move essential cell surface molecules in leukocytes or reduce the
16010825, 2020, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/odi.13476 by UFPI - Universidade Federal do Piaui, Wiley Online Library on [06/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
migration of neutrophils to periodontal sites (da Silva et al., 2016),
according to our results.
After several experiments, the data gathered opened up per-
spectives for further studies with appropriate clinical trials to assess
the effect of bromelain in different pharmaceutical formulations as
an adjuvant treatment for periodontal disease in humans.
5 | CO N C LU S I O N
The treatment of animals with bromelain during ligature-induced
periodontitis was able to reduce oral inflammatory parameters (GBI,
TM, PPD, MDA, and gingival MPO) and alveolar bone loss charac-
teristic of the disease. It also improved blood parameters such as
cholesterol, triglycerides, and ALT and demonstrated the poten-
tial to reduce damage caused by periodontitis, as in this study. It
also reduced hepatic steatosis and improved dosages of MDA and
GSH. Therefore, bromelain appears as a potential adjunct in the
non-surgical treatment of periodontitis and, consequently, reduces
the systemic damage caused by it, acting as anti-inflammatory and
antioxidant.
AC K N OW L E D G E M E N T S
Study supported by CNPq (455104/2014-0 for DFPV) and Even
Herlany Pereira Alves and André dos Santos Carvalho (CAPES
scholarships).
C O N FL I C T O F I N T E R E S T
The authors declare that there is no conflict of interest.
AU T H O R S C O N T R I B U T I O N
Even Alves: Data curation; Formal analysis; Investigation;
Methodology; Validation; Writing-original draft; Writing-
review & editing. André Carvalho: Data curation; Formal analy-
sis; Investigation; Methodology; Validation; Writing-original
draft. Felipe Pereira da Silva Silva: Data curation; Formal analy-
sis; Investigation; Methodology; Validation; Writing-original
draft. Luiz França: Formal analysis; Investigation; Methodology;
Validation; Visualization; Writing-original draft. David Lenardo:
Formal analysis; Investigation; Methodology; Writing-original
draft. Any Carolina Vasconcelos: Investigation; Methodology;
Project administration; Supervision; Validation; Writing-original
draft; Writing-review & editing. Hélio Nascimento: Investigation;
Methodology; Writing-original draft. Víctor Lucas Ribeiro Lopes:
Data curation; Investigation; Methodology; Writing-original draft.
Jefferson Oliveira: Conceptualization; Formal analysis; Investigation;
Supervision; Writing-review & editing. Daniel Fernando Pereira
Vasconcelos: Conceptualization; Data curation; Formal analysis;
Investigation; Methodology; Project administration; Supervision;
Validation; Writing-original draft; Writing-review & editing.
All the authors contributed with the conception, design, ex-
periments, data acquisition, analysis, and interpretation; they also
drafted and critically revised the manuscript. All the authors gave

ALVES et al.
final approval and agreed to be accounted for all aspects of the
work.
Daniel Fernando Pereira Vasconcelos, Jefferson Soares de
Oliveira contributed with the conception, design. Even Herlany
Pereira Alves, André dos Santos Carvalho, Felipe Rodolfo Pereira da
Silva, Luiz Felipe de Carvalho França, David Di Lenardo, Any Carolina
Cardoso Guimarães Vasconcelos, Hélio Mateus Silva Nascimento,
Víctor Lucas Ribeiro Lopes, and Daniel Fernando Pereira Vasconcelos
contributed with experiments, data acquisition, analysis, and in-
terpretation. Even Herlany Pereira Alves, Any Carolina Cardoso
Guimarães Vasconcelos, and Daniel Fernando Pereira Vasconcelos
also drafted and critically revised the manuscript. All the authors
gave final approval and agreed to be accounted for all aspects of
the work.
ORCID
Even Herlany Pereira Alves  https://orcid.
org/0000-0001-7566-1282
Any Carolina Cardoso Guimarães Vasconcelos  https://orcid.
org/0000-0001-7235-5159
Daniel Fernando Pereira Vasconcelos  https://orcid.
org/0000-0002-3331-452X
REFERENCES
Ali, A. A., Mohammed, A. M., & Isa, A. G. (2015). Antimicrobial effects
of crude bromelain extracted from pineapple fruit (Ananas como-
sus (Linn.) Merr.). Advances in Biochemistry, 3(1), 1–4. https://doi.
org/10.11648​/j.ab.20150​3 01.11
Al-Otaibi, W. R., Virk, P., & Elobeid, M. (2015). Ameliorative potential of
stem bromelain on lead-induced toxicity in Wistar rats. Acta Biologica
Hungarica, 66, 149–160. https://doi.org/10.1556/018.66.2015.2.2
Bernela, M., Ahuja, M., & Thakur, R. (2016). Enhancement of anti-in-
flammatory activity of bromelain by its encapsulation in katira
gum nanoparticles. Carbohydrate Polymers, 143, 18–24. https://doi.
org/10.1016/j.carbp​ol.2016.01.055
Chang, T. C., Wei, P. L., Makondi, P. T., Chen, W. T., Huang, C. Y., & Chang,
Y. J. (2019). Bromelain inhibits the ability of colorectal cancer cells
to proliferate via activation of ROS production and autophagy. PLoS
One, 14, e0210274. https://doi.org/10.1371/journ​al.pone.0210274
Chaudhary, B., Agarwal, S., & Bist, R. (2018). Invulnerability of bromelain
against oxidative degeneration and cholinergic deficits imposed by
dichlorvos in mice brains. Frontiers in Biology, 13, 56–62. https://doi.
org/10.1007/s1151​5-018-1479-1
Chaves, L. S., Nicolau, L. A., Silva, R. O., Barros, F. C., Freitas, A. L., Aragao,
K. S., … Medeiros, J. V. (2013). Antiinflammatory and antinocicep-
tive effects in mice of a sulfated polysaccharide fraction extracted
from the marine red algae Gracilaria caudata. Immunopharmacology
and Immunotoxicology, 35, 93–100. https://doi.org/10.3109/08923​
973.2012.707211
da Silva, F. R. P., Vasconcelos, A. C. C. G., Alves, E. H. P., Junior, P. V. O.,
de Oliveira, J. S., & Vasconcelos, D. F. P. (2016). Bromelain: A po-
tential strategy for the adjuvant treatment of periodontitis. Dental
Hypotheses, 7, 88. https://doi.org/10.4103/2155-8213.190483
da Silva, F. R. P., Vasconcelos, A. C. C. G., França, L. F. C., Di Lenardo,
D., Nascimento, H. M. S., & Vasconcelos, D. F. P. (2018). Association
between the rs1143634 polymorphism in interleukin-1B and chronic
periodontitis: Results from a meta-analysis composed by 54 case/
control studies. Gene, 668, 97–106. https://doi.org/10.1016/j.
gene.2018.05.067
16010825, 2020, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/odi.13476 by UFPI - Universidade Federal do Piaui, Wiley Online Library on [06/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
|
      1801
Dahiya, P., Kamal, R., Gupta, R., Bhardwaj, R., Chaudhary, K., &
Kaur, S. (2013). Reactive oxygen species in periodontitis. Journal
of Indian Society of Periodontology, 17, 411–416. https://doi.
org/10.4103/0972-124X.118306
de Lencastre Novaes, L. C., Jozala, A. F., Lopes, A. M., de Carvalho
Santos-Ebinuma, V., Mazzola, P. G., & Pessoa, J. A. (2016). Stability,
purification, and applications of bromelain: A review. Biotechnology
Progress, 32, 5–13. https://doi.org/10.1002/btpr.2190
Dos Santos Carvalho, J., Cardoso Guimarães Vasconcelos, A. C., Herlany
Pereira Alves, E., dos Santos Carvalho, A., da Silva, F. R. P., de
Carvalho França, L. F., … Medeiros, J. V. (2017). Steatosis caused
by experimental periodontitis is reversible after removal of ligature
in rats. Journal of Periodontal Research, 52(5), 883–892. https://doi.
org/10.1111/jre.12459
Filho, M. D. S., Medeiros, J. V. R., Vasconcelos, D. F. P., Silva, D. A.,
Leodido, A. C. M., Fernandes, H. F., … Pinto, G. R. (2018). Orabase for-
mulation with cashew gum polysaccharide decreases inflammatory
and bone loss hallmarks in experimental periodontitis. International
Journal of Biological Macromolecules, 107, 1093–1101. https://doi.
org/10.1016/j.ijbio​mac.2017.09.087
Fitzhugh, D. J., Shan, S., Dewhirst, M. W., & Hale, L. P. (2008). Bromelain
treatment decreases neutrophil migration to sites of inflamma-
tion. Clinical Immunology, 128, 66–74. https://doi.org/10.1016/j.
clim.2008.02.015
França, L. F. C., Vasconcelos, A., da Silva, F. R. P., Alves, E. H. P., Carvalho,
J. S., Lenardo, D. D., … Vasconcelos, D. F. P. (2017). Periodontitis
changes renal structures by oxidative stress and lipid peroxida-
tion. Journal of Clinical Periodontology, 44, 568–576. https://doi.
org/10.1111/jcpe.12729
Hale, L. P., Greer, P. K., & Sempowski, G. D. (2002). Bromelain treatment
alters leukocyte expression of cell surface molecules involved in
cellular adhesion and activation. Clinical Immunology, 104, 183–190.
https://doi.org/10.1006/clim.2002.5254
Junior, R. F. A., Souza, T. O., de Moura, L. M., Torres, K. P., de Souza, L.
B., Alves, M. S., … de Araujo, A. A. (2013). Atorvastatin decreases
bone loss, inflammation and oxidative stress in experimental peri-
odontitis. PLoS One, 8, e75322. https://doi.org/10.1371/journ​
al.pone.0075322
Komazaki, R., Katagiri, S., Takahashi, H., Maekawa, S., Shiba, T.,
Takeuchi, Y., … Izumi, Y. (2017). Periodontal pathogenic bacteria,
Aggregatibacter actinomycetemcomitans affect non-alcoholic fatty
liver disease by altering gut microbiota and glucose metabolism.
Scientific Reports, 7, 13950. https://doi.org/10.1038/s4159​8-017-
14260​-9
Lee, J. H., Lee, J. B., Lee, J. T., Park, H. R., & Kim, J. B. (2018). Medicinal
effects of bromelain (Ananas comosus) targeting oral environment
as an anti-oxidant and anti-inflammatory agent. Journal of Food and
Nutrition Research, 6, 773–784. https://doi.org/10.12691​/jfnr-6-12-8
Liu, R., Li, N., Liu, N., Zhou, X., Dong, Z. M., Wen, X. J., & Liu, L. C. (2012).
Effects of systemic ornidazole, systemic and local compound ornida-
zole and pefloxacin mesylate on experimental periodontitis in rats.
Medical science monitor: International Medical Journal of Experimental
and Clinical Research, 18, BR95–BR102. https://doi.org/10.12659​/
msm.882514
Manji, F., Dahlen, G., & Fejerskov, O. (2018). Caries and periodonti-
tis: Contesting the conventional wisdom on their aetiology. Caries
Research, 52, 548–564. https://doi.org/10.1159/00048​8948
Maurer, H. R. (2001). Bromelain: Biochemistry, pharmacology and med-
ical use. Cellular and Molecular Life Sciences, 58, 1234–1245. https://
doi.org/10.1007/PL000​0 0936
Nicu, E. A., & Loos, B. G. (2016). Polymorphonuclear neutrophils in
periodontitis and their possible modulation as a therapeutic ap-
proach. Periodontology, 2000(71), 140–163. https://doi.org/10.1111/
prd.12113

|
1802       ALVES et al.
Novaes, L. C. L., Jozala, A. F., Lopes, A. M., Santos-Ebinuma, V. C.,
Mazzola, P. G., & Junior, A. P. (2016). Stability, purification, and ap-
plications of bromelain: A review. Biotechnology Progress, 32, 5–13.
https://doi.org/10.1002/btpr.2190
Nyblom, H., Bjornsson, E., Simren, M., Aldenborg, F., Almer, S., & Olsson,
R. (2006). The AST/ALT ratio as an indicator of cirrhosis in patients
with PBC. Liver International: Official Journal of the International
Association for the Study of the Liver, 26, 840–845. https://doi.
org/10.1111/j.1478-3231.2006.01304.x
Pessoa, L. S., Silva, F. R. P., Alves, E. H., Franca, L. F., di Lenardo, D.,
Carvalho, J. S., … Vasconcelos, D. F. P. (2018). One or two ligatures
inducing periodontitis are sufficient to cause fatty liver. Medicina
Oral, Patologia Oral Y Cirugia Bucal, 23, e269–e276. https://doi.
org/10.4317/medor​al.22204
Praveen, N. C., Rajesh, A., Madan, M., Chaurasia, V. R., Hiremath, N. V., &
Sharma, A. M. (2014). In vitro evaluation of antibacterial efficacy of
pineapple extract (Bromelain) on periodontal pathogens. Journal of
International Oral Health: JIOH, 6, 96–98.
Rathnavelu, V., Alitheen, N. B., Sohila, S., Kanagesan, S., & Ramesh, R.
(2016). Potential role of bromelain in clinical and therapeutic appli-
cations. Biomedical Reports, 5, 283–288. https://doi.org/10.3892/
br.2016.720
Rosenberg, L., Krieger, Y., Silberstein, E., Arnon, O., Sinelnikov, I. A.,
Bogdanov-Berezovsky, A., & Singer, A. J. (2012). Selectivity of a bro-
melain based enzymatic debridement agent: A porcine study. Burns:
Journal of the International Society for Burn Injuries, 38, 1035–1040.
https://doi.org/10.1016/j.burns.2012.02.011
Sahbaz, A., Aynioglu, O., Isik, H., Ozmen, U., Cengil, O., Gun, B. D., &
Gungorduk, K. (2015). Bromelain: A natural proteolytic for intra-ab-
dominal adhesion prevention. International Journal of Surgery, 14,
7–11. https://doi.org/10.1016/j.ijsu.2014.12.024
Sedlak, J., & Lindsay, R. H. (1968). Estimation of total, protein-bound,
and nonprotein sulfhydryl groups in tissue with Ellman's reagent.
Analytical Biochemistry, 25, 192–205. https://doi.org/10.1016/0003-
2697(68)90092​- 4
Toker, H., Ozdemir, H., Eren, K., Ozer, H., & Sahin, G. (2009).
N-acetylcysteine, a thiol antioxidant, decreases alveolar bone loss
in experimental periodontitis in rats. Journal of Periodontology, 80,
672–678. https://doi.org/10.1902/jop.2009.080509
Tomofuji, T., Ekuni, D., Yamanaka, R., Kusano, H., Azuma, T., Sanbe,
T., … Takata, T. (2007). Chronic administration of lipopolysaccha-
ride and proteases induces periodontal inflammation and hepatic
16010825, 2020, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/odi.13476 by UFPI - Universidade Federal do Piaui, Wiley Online Library on [06/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
steatosis in rats. Journal of Periodontology, 78, 1999–2006. https://
doi.org/10.1902/jop.2007.070056
Uchiyama, M., & Mihara, M. (1978). Determination of malonaldehyde pre-
cursor in tissues by thiobarbituric acid test. Analytical Biochemistry,
86, 271–278. https://doi.org/10.1016/0003-2697(78)90342​-1
Vasconcelos, A., Vasconcelos, D. F. P., Silva, F. R. P., Franca, L. F. C., Alves,
E. H. P., Lenardo, D. D., … de Oliveira, A. P. (2019). Periodontitis
causes abnormalities in the liver of rats. Journal of Periodontology, 90,
295–305. https://doi.org/10.1002/JPER.18-0226
Vasconcelos, D. F., Silva, F. R. P., Pinto, M. E., Santana, L. A., Souza, I. G.,
Souza, L. K. M., … Oliveira, J. S. (2017). Decrease of pericytes is asso-
ciated with liver disease caused by ligature-induced periodontitis in
rats. Journal of Periodontology, 88, e49–e57. https://doi.org/10.1902/
jop.2016.160392
Xu, Y., & Wei, W. (2006). A comparative study of systemic subantimi-
crobial and topical treatment of minocycline in experimental peri-
odontitis of rats. Archives of Oral Biology, 51, 794–803. https://doi.
org/10.1016/j.archo​ralbio.2006.03.018
Younossi, Z. M., Baranova, A., Ziegler, K., Del Giacco, L., Schlauch, K.,
Born, T. L., … Chandhoke, V. (2005). A genomic and proteomic study
of the spectrum of nonalcoholic fatty liver disease. Hepatology, 42,
665–674. https://doi.org/10.1002/hep.20838
Ziebolz, D., Schmalz, G., Kauffels, A., Widmer, F., Widmer, K., Slotta, J. E.,
… Kollmar, O. (2017). Periodontal pathogenic bacteria and aMMP-8
findings depending on periodontal conditions of patients before and
after liver transplantation. Clinical Oral Investigations, 21, 745–752.
https://doi.org/10.1007/s0078​4-016-1821-4
How to cite this article: Alves EHP, Carvalho ADS, Silva FRP, et
al. Bromelain reduces the non-alcoholic fatty liver disease and
periodontal damages caused by ligature-induced periodontitis.
Oral Dis. 2020;26:1793–1802. https://doi.org/10.1111/
odi.13476