- Email: [email protected]
The use of Brazilian propolis for discovery and development of novel anti-inflammatory drugs
Accepted Manuscript The use of Brazilian propolis for discovery and development of novel antiinflammatory drugs Marcelo Franchin, Irlan Almeida Freires, Josy Goldoni Lazarini, Bruno Dias Nani, Marcos Guilherme da Cunha, David Fernando Colon, Severino Matias de Alencar, Pedro Luiz Rosalen PII:
S0223-5234(17)30496-8
DOI:
10.1016/j.ejmech.2017.06.050
Reference:
EJMECH 9545
To appear in:
European Journal of Medicinal Chemistry
Received Date: 1 May 2017 Revised Date:
7 June 2017
Accepted Date: 23 June 2017
Please cite this article as: M. Franchin, I.A. Freires, J.G. Lazarini, B.D. Nani, M.G. da Cunha, D.F. Colon, S.M. de Alencar, P.L. Rosalen, The use of Brazilian propolis for discovery and development of novel anti-inflammatory drugs, European Journal of Medicinal Chemistry (2017), doi: 10.1016/ j.ejmech.2017.06.050. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The use of Brazilian propolis for discovery and development of novel
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anti-inflammatory drugs
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Marcelo Franchina, Irlan Almeida Freiresb,*, Josy Goldoni Lazarinia, Bruno Dias Nania,
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Marcos Guilherme da Cunhaa, David Fernando Colonc, Severino Matias de Alencard,
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Pedro Luiz Rosalena.
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a
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Campinas, 901 Limeira Ave., 13414-903, Piracicaba, SP, Brazil.
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Piracicaba Dental School, Department of Physiological Sciences, University of
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b
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Drive, 32610, Gainesville, Florida, United States.
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c
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3900 Bandeirantes Ave., 14049-900, Ribeirão Preto, SP, Brazil.
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d
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Agriculture, University of São Paulo, Pádua Dias Ave., 13418-900, Piracicaba, SP,
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Brazil.
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Ribeirão Preto Medical School, Department of Immunology, University of São Paulo,
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Department of Agri-food Industry, Food, and Nutrition, ‘Luiz de Queiroz’ College of
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University of Florida College of Dentistry, Department of Oral Biology, 1395 Center
* Corresponding author:
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University of Florida College of Dentistry, Department of Oral Biology, 1395 Center
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Drive, 32610, Gainesville, Florida, United States.
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E-mail address: [email protected]
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Tel.: +1 352 328-9101
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Abstract
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Anti-Inflammatory drugs have been routinely used in the management of acute and
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chronic inflammatory conditions. Nevertheless, their undesirable side and adverse
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effects have encouraged the development of more selective, tolerable and efficacious
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drugs able to modulate the inflammatory process through distinct mechanisms than
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those of drugs currently available in the market, for instance, inhibition of leukocyte
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recruitment (chemotaxis, rolling, adhesion and transmigration). Natural products,
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including Brazilian propolis, have been considered a rich source of anti-inflammatory
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molecules due to a very complex phytochemical diversity. Brazil has at least thirteen
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distinct types of propolis and many bioactive compounds have been isolated therefrom,
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such as apigenin, artepillin C, vestitol, neovestitol, among others. These molecules were
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proven to play a significant immunomodulatory role through (i) inhibition of
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inflammatory cytokines (e.g. TNF-α) and chemokines (CXCL1/KC and CXCL2/MIP2);
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(ii) inhibition of IκBα, ERK1/2, JNK and p38MAPK phosphorylation; (iii) inhibition of
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NF-κB activation; and (iv) inhibition of neutrophil adhesion and transmigration (ICAM-
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1, VCAM-1 and E-selectin expression). In this review, we shed light on the new
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advances in the research of compounds isolated from Brazilian propolis from Apis
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mellifera bees as potentially novel anti-inflammatory drugs. The compilation of data
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and insights presented herein may open further avenues for the pharmacological
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management of oral and systemic inflammatory conditions. Further research should
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focus on clinical and acute/chronic toxicological validation of the most promising
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compounds described in this review.
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Keyword: Propolis; Apis mellifera bee; anti-Inflammatory; neutrophils; leukocyte.
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Abbreviations
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AP-1, activator protein-1; CXCL1/KC, chemokine ligand 1/KC, keratinocyte
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chemoattractant; CXCL2/MIP-2, chemokine ligand 2/MIP-2, macrophage inflammatory
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protein 2; CXCR2, chemokine receptor type 2; ERK, extracellular signal-regulated
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kinases; ICAM-1, intercellular adhesion molecules type 1; IKK, I kappa B kinase;
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IRAK, interleukin-1 receptor-associated kinase; IκBα, nuclear factor of kappa light
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polypeptide gene enhancer in B-cells inhibitor, alpha; JNK, c-Jun amino-terminal
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kinases; LPS, Lipopolysaccharides; MAPK, mitogen-activated protein kinases; Myd88,
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myeloid differentiation primary response 88; NETs, Neutrophil extracellular traps; NF-
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κB, factor nuclear kappa B; PECAM-1, platelet and endothelial cell adhesion molecule
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1; p38, p38 mitogen-activated protein kinases; TAK-1, transforming growth factor beta-
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activated kinase 1; TNF-α, tumor necrosis factor-alpha; TRAF6, tumor necrosis factor
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receptor-associated factor 6; TLR, toll-like receptors; VCAM-1 - vascular cell adhesion
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molecule 1
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Contents
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1. Introduction………………………………………………..…………………... 5
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2. Leukocyte recruitment in inflammation and targets for drug development
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.…………………………………………………………………………………. 6
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3. Brazilian propolis from Apis mellifera bees …………………………………..
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3.1.Botanical origin and classification of Brazilian propolis …………………… 9
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4. Compounds isolated from Brazilian propolis and their leukocyte
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recruitment
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……………………………………………………………. 13
87 88 89 90 91 92 93 94 95 96 97
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activity
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5. Conclusion
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inhibitory
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1. Introduction
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In recent years, a great amount of effort and research has been put forth for the
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development of anti-inflammatory therapies, particularly for the management of chronic
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conditions. One example of that are the relatively new drugs designed to block
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recruitment of leukocytes, especially neutrophils [1-2]. This novel strategy aims to
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prevent the deleterious and undesirable effects caused by proteolytic enzymes and free
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radicals released by neutrophils during an exacerbated inflammatory response [3-4].
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Hence, pharmacological studies have been carried out to discover and develop drugs
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capable of inhibiting not only the release and/or production of pro-inflammatory
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cytokines and chemokines, but also of proteins involved in rolling and transmigration of
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leukocytes into the inflammatory focus [2,5,6,7], as further discussed in this review.
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Newman and Cragg [8] reviewed the drugs approved by the FDA from 1981 to
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2014 and showed that natural products continue to play a highly significant role in drug
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discovery and development (DDD). They pointed out that molecules obtained from
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natural products, or derived therefrom, represent 62% of all small molecules approved
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by FDA over the last 34 years, and that 2% of these natural molecules are anti-
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inflammatory drugs.
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Brazil has great importance in this scenario for harboring approximately 22% of
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the plant species [9-10]. Among the most promising naturally-occurring agents,
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Brazilian propolis has been considered a rich source of well-known and mainly
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unexplored bioactive compounds with pharmacological properties [11-13]. Previously,
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we published a review in this journal describing the biological activities of Brazilian red
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propolis - one of the 13 types of propolis cataloged to date - and its isolated compounds
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against human diseases, including those of anti-inflammatory origin [11]. In addition to
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anti-inflammatory activity [14-18], crude extracts and bioactive molecules isolated from
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propolis samples have also proven to be promising antibacterial [19], antifungal [11],
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antibiofilm [20] and anticancer [21] agents. In this review, we present the new advances in the research of compounds
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isolated from Brazilian propolis from Apis mellifera bees as novel anti-inflammatory
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drugs whose mechanisms are associated with leukocyte recruitment inhibition.
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2. Leukocyte recruitment in inflammation and targets for drug development
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Anti-inflammatory drugs are one of the most commonly used drugs worldwide
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[22,23]. Despite their undeniable efficacy, this class of medicines has been recurrently
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reported to cause side effects, including gastric and renal issues [22]. A current
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challenge in the pharmaceutical industry consists in the development of more selective,
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albeit tolerable, drugs able to control the inflammatory process, for instance, those able
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to modulate leukocyte recruitment [2, 24].
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Leukocyte recruitment is a key defense strategy of the organism, as described in
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Fig. 1. Activation of this event may be triggered by microbial infection or due to a
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tissue injury without infection [25, 26]. Neutrophils are the main defense cells recruited
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in the inflammatory process, thereby being playing a crucial role in host defense [4, 27].
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Inflammatory mediators such as cytokines and chemokines drive the leukocyte
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recruitment process [27]. For instance, TNF-α controls the release of other
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inflammatory cytokines, regulates the expression of leukocyte adhesion molecules on
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endothelial cells as well as of the chemokines CXCL1/KC and CXCL2/MIP- 2, which
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induces neutrophil chemotaxis by binding to CXCR2-like receptors [4, 28].
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In addition to neutrophils, resident macrophages are also one of the major
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sources of inflammatory cytokines and chemokines [29]. Intracellular signaling
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pathways involved in regulating cytokine and chemokine production require a
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participation of proteins such as MAPK ERK1/2, JNK and p38 MAPK [30]. Among the
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pathways regulated by these MAPKs is the activation of the transcription factors AP-1
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and NF-κB [31-32]. Activation of NF-κB involves the signaling cascade Myd88/IRAK
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/TRAF6/ TAK-1/IKK followed by degradation of IκBα, activation of NF-κB and
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release of inflammatory mediators [33].
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Proteins such as integrins, selectins and immunoglobulins are critical for
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leukocytes to migrate into the injured tissue [34]. This process occurs in different
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stages. Upon release of inflammatory cytokines and chemokines, leukocytes perform a
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process called endothelial cell rolling, which involves the participation of the adhesion
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molecules named selectins (L-, P- and E-selectin) [35]. Subsequently, there is a strong
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adhesion of leukocytes mediated by integrins (β2-integrins), which interact with
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immunoglobulins, such as intercellular adhesion molecules type 1 (ICAM-1) [34,35].
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Leukocytes transmigrate into the inflammatory site through the intercellular junctions
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(diapedesis) between endothelial cells and also due to interaction with cell adhesion
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molecules, such as PECAM-1 [34,35]. In the end, when present in the tissue, leukocytes
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exert phagocytosis of the aggressive/foreign agent and release inflammatory cytokines
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[25, 36].
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The process of leukocyte recruitment during the inflammatory process has been
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often related to the onset and persistence of inflammatory diseases [3,37-40]. The
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occurrence of an exacerbated inflammatory response leads to accumulation of
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leukocytes (especially neutrophils) in the injured tissue, which causes deleterious effects
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to the inflamed site [3]. Based on that, there is a close relationship between the onset of
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inflammatory diseases and leukocyte recruitment, which encourages the development of
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new anti-inflammatory drugs targeting leukocyte recruitment-related events, namely:
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release of inflammatory cytokines and chemokines, expression of adhesion molecules,
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and chemotaxis [2,6,7].
Blood
PSGL1
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Integrin (high affinity)
Integrin (low affinity)
Tissue
PECAM-1 (CD31)
P-Selectin E-Selectin
Phagocytosis
NETs
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Degranulation
Integrin ligand VCAM-1 (ICAM-1)
Proteoglycan
CXCL1/KC, CXCL2/MIP-2 and TNF
p IκBα
IκBα
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p50 p65
p MAPKs
(p38)
p
ERK
TLRs
p
JNK
p
c-fos p50 p65 c-jun NF-κB p AP-1
Microorganism
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Tissue macrophages or other cels
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Figure 1. Neutrophil recruitment process. During an inflammatory process,
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neutrophils are recruited from the blood to the site of infection in response to
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CXCL2/MIP-2 or CXCL1/KC chemokines released by resident macrophages or other
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resident cells in response to TLRs ligands (e.g. microorganisms). This recruitment
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process begins with the neutrophils rolling over the vascular endothelium through
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transient interactions between PSGL1 and selectins expressed on endothelial cells.
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Next, neutrophils adhere firmly to the endothelial vascular cells due to the affinity
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maturation of neutrophil integrins induced by chemokines and their interactions with the
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ICAM-1 and VCAM-1 adhesion proteins. Finally, the transendothelial migration of
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neutrophils is mediated via PECAM-1 interaction. Once at the site of infection,
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transmigrated neutrophils can phagocytose or release NETs to kill the pathogens and
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avoid their spreading.
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3. Brazilian propolis from Apis mellifera bees
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The propolis (also known as bee glue) is a resinous substance that is not directly
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produced by the bees, but is collected by them from plants surrounding the beehive
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[41]. Most of the scientific research points to propolis derived from exotic Africanized
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Apis meliffera honeybees, which were introduced in Brazil during the European
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colonization and then Africanized during an accidental release of a population of
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Africanized bees in Sao Paulo state [42]. Although there are Brazilian propolis collected
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by other species of bees, such as those produced by stingless bees (Meliponini tribe.), A.
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meliffera propolis remains the most studied ones in Brazil and worldwide due to the
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quality-added value of this product in the market.
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In the beehive, propolis is used for many purposes, e.g. cover the hive, repel and
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embalm parasites, kill microorganisms, and act as a thermal isolator [43-45]. Noting the
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importance of propolis for the bees, humans have also taken advantage of the benefits of
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these resins since ancient times. Egyptians used propolis for embalming corpses;
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propolis was also used as a healing agent by the Greek and Roman physicians; and in
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the second world war it was used as an antimicrobial and anti-inflammatory agent [45].
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3.1. Botanical origin and classification of Brazilian propolis
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The phytochemical composition of Brazilian propolis reflects the great
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biodiversity of Brazil itself. Unlike other countries, which have little variability in the
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chemical composition and botanical origin of the propolis, Brazil has at least thirteen
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different types of Brazilian propolis identified thus far [21,41,46]. Park et al [46] were
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the first group to investigate the chemical profiles of Brazilian propolis. These authors
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collected more than 2 thousand Brazilian propolis samples from different regions and
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states and classified them into 12 types based on their physicochemical characteristics.
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By comparing the chemical profile of propolis samples and their respective plant
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source, the authors were able to identify the botanical origin of three propolis types:
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type 3 (Poplar propolis), which is chemically similar to Poplar trees (Populus L.,
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Salicaceae family); type 6 (brown propolis), which is chemically similar to Hyptis
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divaricate tree; and type 12 (green propolis), which is similar to Baccharis
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dracunculifolia tree [46].
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After the identification of the first 12 types of Brazilian propolis, a new type was
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described in 2007, the red propolis (type 13) [21]. The chemical composition of red
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propolis samples was found to be similar to that of Dalbergia ecastophyllum trees,
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which indicates this plant as the botanical origin of red propolis [41]. However, other
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authors detected prenylated benzophenone compounds in Brazilian red propolis,
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molecules that are commonly founded in Clusia species, which indicates that other
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plants may be the source of Brazilian red propolis [47].
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The pharmacological use of Brazilian red propolis and its isolated compounds
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against human diseases, such as dental caries, candidiasis, cancer, skin wound,
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oxidative stress-related conditions, among others, were subject of a previous review
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published in this journal by our research group [11].
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In Table 1 we show the 13 types of Brazilian propolis from A. millifera and their
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collection sites and botanical origins. These thirteen categories are scientifically
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consolidated as Brazilian types of A. meliffera propolis, but other Brazilian propolis are
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being studied and hereafter might be accepted as new types of Brazilian propolis. This
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is the case of Southern organic propolis, which is produced strictly under organic
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conditions in Paraná and Santa Catarina States and has proven antimicrobial,
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antioxidant and anti-inflammatory properties [19]. This type of propolis is produced in a
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dense rainforest with great biodiversity. Accordingly, our unpublished chemical
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findings point to the presence of molecules that are not usually present in propolis
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samples, some of them completely unknown, suggesting that Southern organic propolis
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may be a new source of bioactive molecules. The physicochemical composition and
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biological potential of this type of propolis remain to be fully elucidated in further
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studies.
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Table 1. Types of Brazilian Africanized Apis mellifera propolis and their respective
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State in Brazil, botanical origins and most abundant and/or important chemical
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compounds. Botanical Origin
1
Rio Grande do Sul
Unknown
2
Rio Grande do Sul
Unknown
3 (Poplar propolis)
Paraná
4
Most abundant and/or important chemical compounds Triterpenoids (melliferone, moronic acid, anwuweizonic acid and betulonic acid)
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Type of Propolis
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References
Park et al [48] Ito et al [49]
Park et al [48] Alencar and Park [50]
Poplar trees (Populus L., Salicaceae Family)
Mainly flavonoids (chrysin, pinocembrin, pinobanksin, apigenin and galangin)
Park et al [46] Park et al [48]
Paraná
Unknown
Cinnamic acid and derivatives
Park et al [48] Alencar and Park [50]
5
Paraná
Unknown
Flavonoids and cinnamic acid derivatives
Park et al [48] Alencar and Park [50]
6 (Brown propolis)
Bahia
Hyptis divaricate
Prenylated benzophenone
Park et al [46] Park et al [48] Castro et al [51]
7
Bahia
Unknown
Flavonoids and benzoic acid derivatives
Park et al [48] Alencar and Park [50]
Pernambuco
Unknown
Flavonoids and benzoic acid derivatives
Park et al [48] Alencar and Park [50]
Pernambuco
Unknown
Cinnamic acid derivatives
Park et al [48] Alencar and Park [50]
10
Ceará
Unknown
Sesquiterpenes
Park et al [48] Alencar and Park [50]
11
Piauí
Unknown
Diterpenes
Park et al [48] Alencar and Park [50]
12
São Paulo
Baccharis dracunculifolia
Prenylated cinnamic acids
Paulino et al [15]
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Flavonoids and cinnamic acid derivatives
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Alagoas
Dalbergia ecastophyllum
and flavonoids
Park et al [46] Park et al [48]
Mainly isoflavonoids (formononetin, vestitol, neovestitol and daidzein) and chalcones
Bueno-Silva et al [16] Alencar et al [21] Silva et al [41] Oldoni et al [52]
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4. Compounds isolated from Brazilian propolis and their leukocyte recruitment
276
inhibitory activity
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There has been an increasing effort of the scientific community to obtain
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bioactive compounds able to modulate leukocyte recruitment in the inflammatory
279
process [53-55]. Brazilian propolis from Apis mellifera bees and its different types has
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been reported to be a promising source of bioactive compounds with this distinct anti-
281
inflammatory mechanism, as shown in Table 2.
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A promising anti-inflammatory compound named apigenin was identified in
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poplar propolis [46]. A recent study showed that apigenin decreases the release of pro-
284
inflammatory cytokines by THP-1 macrophages while inhibiting ERK 1/2 and NF-κB
285
activation [56]. In vivo, apigenin promoted a significant decrease of NF-κB expression
286
in the lungs of LPS-stimulated mice, thereby modulating neutrophil influx and
287
decreasing the accumulation of chemotactic factors [57]. Taken together, these reports
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indicate apigenin as a promising candidate for development of a new drug or a
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nutraceutical compound.
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Galangin is another compound identified in poplar propolis [46] also presenting
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relevant anti-inflammatory activity. Galangin was shown to reduce in vitro mRNA
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levels of inflammatory cytokines by LPS-activated macrophages, in addition to
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inhibiting ERK and NF-κB p65 phosphorylation [58]. When tested in human subjects
294
with rheumatoid arthritis, this compound significantly reduced neutrophil activation
295
[59].
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Similarly, pinocembrin, which was also identified in poplar propolis [46],
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showed inhibitory activity on pro-inflammatory cytokine synthesis (including TNF-α)
298
in macrophages by inhibiting the phosphorylation of IκBα, ERK 1/2, JNK and
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p38MAPK. In addition, pinocembrin attenuated an LPS-induced lung injury by
300
decreasing neutrophil influx into the inflammatory focus [60].
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Artepillin C is a compound commonly found in green propolis. This compound
302
was found to inhibit different signaling pathways of the inflammatory process [15].
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When administrated in mice, it decreased paw edema and neutrophil influx into the
304
inflammatory site. In addition, artepillin C exhibited in vitro anti-inflammatory activity
305
(RAW 264.7 macrophages) by blocking activation of NF-κB [15].
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A recent study with the most recently cataloged type of propolis, the thirteenth
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type of Brazilian propolis or red propolis, showed that the crude extract and molecules
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isolated therefrom have potent anti-inflammatory activity by decreasing the neutrophil
309
influx [16]. Chemical analysis of Brazilian red propolis samples indicated the presence
310
of formononetin as the major compound. Formononetin showed inhibitory activity on
311
neutrophil migration and exhibited an antidematogenic effect in the paw edema assay
312
[61], in addition to blocking NF-κB activation [62].
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Two isoflavonoids with anti-inflammatory activity, vestitol and neovestitol, have
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also been isolated from Brazilian red propolis [63]. Acute and chronic inflammation
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models were used by our group to identify the specific anti-inflammatory mechanism(s)
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of these compounds. We found that neovestitol reduces neutrophil influx by decreasing
317
ICAM-1 expression in the mesenteric microcirculation during acute inflammation in
318
vivo [18]. As for chronic inflammation, neovestitol reduced joint damage in mice with
319
arthritis [18]. The second isoflavonoid, vestitol, reduced neutrophil migration in mice
320
induced by different inflammatory stimuli [17]. We demonstrated that vestitol inhibits
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release of the chemokines CXCL1/KC and CXCL2/MIP-2 by resident macrophages
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and, as a result, decreases rolling and adhesion of leukocytes [17]. It was further
323
determined that vestitol decreases neutrophil chemotaxis via calcium influx blockage.
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Collectively, neovestitol and vestitol are promising candidates to treat acute and chronic
325
inflammation and therefore could be useful for therapeutic as well as nutraceutical
326
purposes [17-18].
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Other
bioactive
compound
isolated
from
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Brazilian
red
propolis
is
isoliquiritigenin (ISQ), whose anti-inflammatory activity is well documented [52,64].
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ISQ inhibits adhesion of neutrophil by downregulating expression of ICAM-1, VCAM-
330
1 and E-selectin on endothelial cells [64]. These effects are thought to be related to
331
interference with translocation of p65 from cytoplasm to the nucleus, thereby reducing
332
NF-κB activation [64].
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Daidzein is another bioactive molecule that has aroused attention due to its
334
significant anti-inflammatory potential [11,65]. Administration of this molecule
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significantly reduced the number of neutrophils, release of inflammatory cytokines, the
336
number of TLR4 receptors and the activation of NF-κB upon LPS challenge [65].
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In summary, the study of these compounds as potential candidates for a
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mechanistically distinct anti-inflammatory therapy is relatively new and remains a
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fascinating topic for application in biomedical areas and dentistry. Notably, the proposal
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of a naturally-derived prototype drug whose mechanism differs from that of the drugs
341
currently available in the market is innovative and merit further non-clinical and clinical
342
investigation.
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Table 2. Isolated molecule from different types of propolis and its main anti-
352
inflammatory pathway. Main
propolis
anti-inflammatory pathway
Propolis type 3 (Poplar Propolis)
Inhibition of inflammatory cytokines, ERK 1/2 and NF-κB activation and of neutrophil migration
Park et al [46] Zhang et al [56] Cardenas et al [57]
Inhibition of neutrophil activation, inflammatory cytokines and ERK and NFκB p65 phosphorylation
Park et al [46] Jung et al [58] Santos et al [59]
Propolis type 3 (Poplar Propolis)
Inhibition of inflammatory cytokines (TNF-α), phosphorylation of IκBα, ERK1/2, JNK and p38MAPK and neutrophil migration
Park et al [46] Soromou et al [60]
Propolis type 12 (Green Propolis)
Inhibition of neutrophil migration and of activation of NF-κB
Paulino et al [15]
Apigenin
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Galangin
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Propolis type 3 (Poplar Propolis)
Reference
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Type of
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Molecule
Pinocembrin
Artepillin C
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ACCEPTED MANUSCRIPT Propolis type 13 (Red Propolis)
Inhibition of leukocyte migration and activation of NF-κB
Cavendish et al [61] Wang et al [62]
Propolis type 13 (Red Propolis)
Acute inflammation: Franchin et al [18] Reduces neutrophil migration by decreasing ICAM-1 Chronic inflammation: Decreases the clinical score and joint damage in a collagen-induced arthritis model
Propolis type 13 (Red Propolis)
Inhibition of neutrophil migration, rolling and adhesion; inhibition of CXCL1/KC and CXCL2/MIP2 levels and neutrophil chemotaxis via blocking calcium influx
Vestitol
Isoliquiritigenin
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Oldoni et al [52] Inhibition adhesion of neutrophil, ICAM-1, VCAM- Kumar et al [64] 1 and E-selectin expression and translocation of the p65 subunit of NF-κB
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Propolis type 13 (Red Propolis)
Inhibition of neutrophil migration, inflammatory cytokine release, TLR4 receptor and NF-κB activation
Freires et al [11] Feng et al [65]
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Propolis type 13 (Red Propolis)
Franchin et al [17]
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Neovestitol
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Formononetin
Daidzein
353 354
5. Conclusion
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The present review addressed the main aspects involved in leukocyte
356
recruitment during the inflammatory process and the possible targets for development of
357
novel anti-inflammatory drugs. Different bioactive compounds obtained from Brazilian
358
propolis were presented along with preclinical evidence (in vitro and in vivo studies) of
20
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their inhibitory activity on leukocyte recruitment and mechanism of action. The
360
compilation of data and insights presented herein may open further avenues for the
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pharmacological management of oral and systemic inflammatory conditions. Further
362
research should focus on clinical and acute/chronic toxicological validation of the most
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promising compounds isolated from Brazilian propolis.
364
Conflict of interest
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The authors declare no conflict of interest.
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Acknowledgments
The authors would like to thank the São Paulo Research Foundation (FAPESP,
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grants no. 2016/15563-9, 2015/26864-7 and 2016/02926-6) and the National Council
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for Scientific and Technological Development (CNPq, 310522/2015-3) for financial
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support.
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ACCEPTED MANUSCRIPT Highlights •
There is a close relationship between inflammatory diseases and leukocyte recruitment. New anti-inflammatory drugs target a more selective action on neutrophil
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migration.
Brazilian propolis is a rich source of anti-inflammatory molecules.
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Compounds isolated from Brazilian propolis inhibit neutrophil migration.
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S0223-5234(17)30496-8
DOI:
10.1016/j.ejmech.2017.06.050
Reference:
EJMECH 9545
To appear in:
European Journal of Medicinal Chemistry
Received Date: 1 May 2017 Revised Date:
7 June 2017
Accepted Date: 23 June 2017
Please cite this article as: M. Franchin, I.A. Freires, J.G. Lazarini, B.D. Nani, M.G. da Cunha, D.F. Colon, S.M. de Alencar, P.L. Rosalen, The use of Brazilian propolis for discovery and development of novel anti-inflammatory drugs, European Journal of Medicinal Chemistry (2017), doi: 10.1016/ j.ejmech.2017.06.050. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The use of Brazilian propolis for discovery and development of novel
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anti-inflammatory drugs
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Marcelo Franchina, Irlan Almeida Freiresb,*, Josy Goldoni Lazarinia, Bruno Dias Nania,
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Marcos Guilherme da Cunhaa, David Fernando Colonc, Severino Matias de Alencard,
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Pedro Luiz Rosalena.
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a
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Campinas, 901 Limeira Ave., 13414-903, Piracicaba, SP, Brazil.
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Piracicaba Dental School, Department of Physiological Sciences, University of
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b
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Drive, 32610, Gainesville, Florida, United States.
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c
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3900 Bandeirantes Ave., 14049-900, Ribeirão Preto, SP, Brazil.
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d
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Agriculture, University of São Paulo, Pádua Dias Ave., 13418-900, Piracicaba, SP,
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Brazil.
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Ribeirão Preto Medical School, Department of Immunology, University of São Paulo,
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Department of Agri-food Industry, Food, and Nutrition, ‘Luiz de Queiroz’ College of
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University of Florida College of Dentistry, Department of Oral Biology, 1395 Center
* Corresponding author:
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University of Florida College of Dentistry, Department of Oral Biology, 1395 Center
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Drive, 32610, Gainesville, Florida, United States.
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E-mail address: [email protected]
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Tel.: +1 352 328-9101
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Abstract
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Anti-Inflammatory drugs have been routinely used in the management of acute and
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chronic inflammatory conditions. Nevertheless, their undesirable side and adverse
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effects have encouraged the development of more selective, tolerable and efficacious
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drugs able to modulate the inflammatory process through distinct mechanisms than
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those of drugs currently available in the market, for instance, inhibition of leukocyte
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recruitment (chemotaxis, rolling, adhesion and transmigration). Natural products,
35
including Brazilian propolis, have been considered a rich source of anti-inflammatory
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molecules due to a very complex phytochemical diversity. Brazil has at least thirteen
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distinct types of propolis and many bioactive compounds have been isolated therefrom,
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such as apigenin, artepillin C, vestitol, neovestitol, among others. These molecules were
39
proven to play a significant immunomodulatory role through (i) inhibition of
40
inflammatory cytokines (e.g. TNF-α) and chemokines (CXCL1/KC and CXCL2/MIP2);
41
(ii) inhibition of IκBα, ERK1/2, JNK and p38MAPK phosphorylation; (iii) inhibition of
42
NF-κB activation; and (iv) inhibition of neutrophil adhesion and transmigration (ICAM-
43
1, VCAM-1 and E-selectin expression). In this review, we shed light on the new
44
advances in the research of compounds isolated from Brazilian propolis from Apis
45
mellifera bees as potentially novel anti-inflammatory drugs. The compilation of data
46
and insights presented herein may open further avenues for the pharmacological
47
management of oral and systemic inflammatory conditions. Further research should
48
focus on clinical and acute/chronic toxicological validation of the most promising
49
compounds described in this review.
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Keyword: Propolis; Apis mellifera bee; anti-Inflammatory; neutrophils; leukocyte.
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Abbreviations
55
AP-1, activator protein-1; CXCL1/KC, chemokine ligand 1/KC, keratinocyte
57
chemoattractant; CXCL2/MIP-2, chemokine ligand 2/MIP-2, macrophage inflammatory
58
protein 2; CXCR2, chemokine receptor type 2; ERK, extracellular signal-regulated
59
kinases; ICAM-1, intercellular adhesion molecules type 1; IKK, I kappa B kinase;
60
IRAK, interleukin-1 receptor-associated kinase; IκBα, nuclear factor of kappa light
61
polypeptide gene enhancer in B-cells inhibitor, alpha; JNK, c-Jun amino-terminal
62
kinases; LPS, Lipopolysaccharides; MAPK, mitogen-activated protein kinases; Myd88,
63
myeloid differentiation primary response 88; NETs, Neutrophil extracellular traps; NF-
64
κB, factor nuclear kappa B; PECAM-1, platelet and endothelial cell adhesion molecule
65
1; p38, p38 mitogen-activated protein kinases; TAK-1, transforming growth factor beta-
66
activated kinase 1; TNF-α, tumor necrosis factor-alpha; TRAF6, tumor necrosis factor
67
receptor-associated factor 6; TLR, toll-like receptors; VCAM-1 - vascular cell adhesion
68
molecule 1
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Contents
73
1. Introduction………………………………………………..…………………... 5
75
2. Leukocyte recruitment in inflammation and targets for drug development
76
.…………………………………………………………………………………. 6
77
3. Brazilian propolis from Apis mellifera bees …………………………………..
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9
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3.1.Botanical origin and classification of Brazilian propolis …………………… 9
80
4. Compounds isolated from Brazilian propolis and their leukocyte
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SC
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recruitment
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……………………………………………………………. 13
87 88 89 90 91 92 93 94 95 96 97
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activity
…………………………………………………………………….
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5. Conclusion
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inhibitory
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1. Introduction
104
In recent years, a great amount of effort and research has been put forth for the
106
development of anti-inflammatory therapies, particularly for the management of chronic
107
conditions. One example of that are the relatively new drugs designed to block
108
recruitment of leukocytes, especially neutrophils [1-2]. This novel strategy aims to
109
prevent the deleterious and undesirable effects caused by proteolytic enzymes and free
110
radicals released by neutrophils during an exacerbated inflammatory response [3-4].
111
Hence, pharmacological studies have been carried out to discover and develop drugs
112
capable of inhibiting not only the release and/or production of pro-inflammatory
113
cytokines and chemokines, but also of proteins involved in rolling and transmigration of
114
leukocytes into the inflammatory focus [2,5,6,7], as further discussed in this review.
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Newman and Cragg [8] reviewed the drugs approved by the FDA from 1981 to
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2014 and showed that natural products continue to play a highly significant role in drug
117
discovery and development (DDD). They pointed out that molecules obtained from
118
natural products, or derived therefrom, represent 62% of all small molecules approved
119
by FDA over the last 34 years, and that 2% of these natural molecules are anti-
120
inflammatory drugs.
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Brazil has great importance in this scenario for harboring approximately 22% of
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the plant species [9-10]. Among the most promising naturally-occurring agents,
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Brazilian propolis has been considered a rich source of well-known and mainly
124
unexplored bioactive compounds with pharmacological properties [11-13]. Previously,
125
we published a review in this journal describing the biological activities of Brazilian red
126
propolis - one of the 13 types of propolis cataloged to date - and its isolated compounds
127
against human diseases, including those of anti-inflammatory origin [11]. In addition to
128
anti-inflammatory activity [14-18], crude extracts and bioactive molecules isolated from
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propolis samples have also proven to be promising antibacterial [19], antifungal [11],
130
antibiofilm [20] and anticancer [21] agents. In this review, we present the new advances in the research of compounds
132
isolated from Brazilian propolis from Apis mellifera bees as novel anti-inflammatory
133
drugs whose mechanisms are associated with leukocyte recruitment inhibition.
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134 135
2. Leukocyte recruitment in inflammation and targets for drug development
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Anti-inflammatory drugs are one of the most commonly used drugs worldwide
138
[22,23]. Despite their undeniable efficacy, this class of medicines has been recurrently
139
reported to cause side effects, including gastric and renal issues [22]. A current
140
challenge in the pharmaceutical industry consists in the development of more selective,
141
albeit tolerable, drugs able to control the inflammatory process, for instance, those able
142
to modulate leukocyte recruitment [2, 24].
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Leukocyte recruitment is a key defense strategy of the organism, as described in
144
Fig. 1. Activation of this event may be triggered by microbial infection or due to a
145
tissue injury without infection [25, 26]. Neutrophils are the main defense cells recruited
146
in the inflammatory process, thereby being playing a crucial role in host defense [4, 27].
147
Inflammatory mediators such as cytokines and chemokines drive the leukocyte
148
recruitment process [27]. For instance, TNF-α controls the release of other
149
inflammatory cytokines, regulates the expression of leukocyte adhesion molecules on
150
endothelial cells as well as of the chemokines CXCL1/KC and CXCL2/MIP- 2, which
151
induces neutrophil chemotaxis by binding to CXCR2-like receptors [4, 28].
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In addition to neutrophils, resident macrophages are also one of the major
153
sources of inflammatory cytokines and chemokines [29]. Intracellular signaling
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pathways involved in regulating cytokine and chemokine production require a
155
participation of proteins such as MAPK ERK1/2, JNK and p38 MAPK [30]. Among the
156
pathways regulated by these MAPKs is the activation of the transcription factors AP-1
157
and NF-κB [31-32]. Activation of NF-κB involves the signaling cascade Myd88/IRAK
158
/TRAF6/ TAK-1/IKK followed by degradation of IκBα, activation of NF-κB and
159
release of inflammatory mediators [33].
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Proteins such as integrins, selectins and immunoglobulins are critical for
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leukocytes to migrate into the injured tissue [34]. This process occurs in different
162
stages. Upon release of inflammatory cytokines and chemokines, leukocytes perform a
163
process called endothelial cell rolling, which involves the participation of the adhesion
164
molecules named selectins (L-, P- and E-selectin) [35]. Subsequently, there is a strong
165
adhesion of leukocytes mediated by integrins (β2-integrins), which interact with
166
immunoglobulins, such as intercellular adhesion molecules type 1 (ICAM-1) [34,35].
167
Leukocytes transmigrate into the inflammatory site through the intercellular junctions
168
(diapedesis) between endothelial cells and also due to interaction with cell adhesion
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molecules, such as PECAM-1 [34,35]. In the end, when present in the tissue, leukocytes
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exert phagocytosis of the aggressive/foreign agent and release inflammatory cytokines
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[25, 36].
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The process of leukocyte recruitment during the inflammatory process has been
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often related to the onset and persistence of inflammatory diseases [3,37-40]. The
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occurrence of an exacerbated inflammatory response leads to accumulation of
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leukocytes (especially neutrophils) in the injured tissue, which causes deleterious effects
176
to the inflamed site [3]. Based on that, there is a close relationship between the onset of
177
inflammatory diseases and leukocyte recruitment, which encourages the development of
178
new anti-inflammatory drugs targeting leukocyte recruitment-related events, namely:
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release of inflammatory cytokines and chemokines, expression of adhesion molecules,
180
and chemotaxis [2,6,7].
Blood
PSGL1
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Integrin (high affinity)
Integrin (low affinity)
Tissue
PECAM-1 (CD31)
P-Selectin E-Selectin
Phagocytosis
NETs
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Degranulation
Integrin ligand VCAM-1 (ICAM-1)
Proteoglycan
CXCL1/KC, CXCL2/MIP-2 and TNF
p IκBα
IκBα
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p50 p65
p MAPKs
(p38)
p
ERK
TLRs
p
JNK
p
c-fos p50 p65 c-jun NF-κB p AP-1
Microorganism
p
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Tissue macrophages or other cels
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Figure 1. Neutrophil recruitment process. During an inflammatory process,
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neutrophils are recruited from the blood to the site of infection in response to
184
CXCL2/MIP-2 or CXCL1/KC chemokines released by resident macrophages or other
185
resident cells in response to TLRs ligands (e.g. microorganisms). This recruitment
186
process begins with the neutrophils rolling over the vascular endothelium through
187
transient interactions between PSGL1 and selectins expressed on endothelial cells.
188
Next, neutrophils adhere firmly to the endothelial vascular cells due to the affinity
189
maturation of neutrophil integrins induced by chemokines and their interactions with the
190
ICAM-1 and VCAM-1 adhesion proteins. Finally, the transendothelial migration of
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neutrophils is mediated via PECAM-1 interaction. Once at the site of infection,
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transmigrated neutrophils can phagocytose or release NETs to kill the pathogens and
193
avoid their spreading.
194
3. Brazilian propolis from Apis mellifera bees
195
The propolis (also known as bee glue) is a resinous substance that is not directly
197
produced by the bees, but is collected by them from plants surrounding the beehive
198
[41]. Most of the scientific research points to propolis derived from exotic Africanized
199
Apis meliffera honeybees, which were introduced in Brazil during the European
200
colonization and then Africanized during an accidental release of a population of
201
Africanized bees in Sao Paulo state [42]. Although there are Brazilian propolis collected
202
by other species of bees, such as those produced by stingless bees (Meliponini tribe.), A.
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meliffera propolis remains the most studied ones in Brazil and worldwide due to the
204
quality-added value of this product in the market.
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In the beehive, propolis is used for many purposes, e.g. cover the hive, repel and
206
embalm parasites, kill microorganisms, and act as a thermal isolator [43-45]. Noting the
207
importance of propolis for the bees, humans have also taken advantage of the benefits of
208
these resins since ancient times. Egyptians used propolis for embalming corpses;
209
propolis was also used as a healing agent by the Greek and Roman physicians; and in
210
the second world war it was used as an antimicrobial and anti-inflammatory agent [45].
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3.1. Botanical origin and classification of Brazilian propolis
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The phytochemical composition of Brazilian propolis reflects the great
215
biodiversity of Brazil itself. Unlike other countries, which have little variability in the
216
chemical composition and botanical origin of the propolis, Brazil has at least thirteen
217
different types of Brazilian propolis identified thus far [21,41,46]. Park et al [46] were
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the first group to investigate the chemical profiles of Brazilian propolis. These authors
219
collected more than 2 thousand Brazilian propolis samples from different regions and
220
states and classified them into 12 types based on their physicochemical characteristics.
221
By comparing the chemical profile of propolis samples and their respective plant
222
source, the authors were able to identify the botanical origin of three propolis types:
223
type 3 (Poplar propolis), which is chemically similar to Poplar trees (Populus L.,
224
Salicaceae family); type 6 (brown propolis), which is chemically similar to Hyptis
225
divaricate tree; and type 12 (green propolis), which is similar to Baccharis
226
dracunculifolia tree [46].
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After the identification of the first 12 types of Brazilian propolis, a new type was
228
described in 2007, the red propolis (type 13) [21]. The chemical composition of red
229
propolis samples was found to be similar to that of Dalbergia ecastophyllum trees,
230
which indicates this plant as the botanical origin of red propolis [41]. However, other
231
authors detected prenylated benzophenone compounds in Brazilian red propolis,
232
molecules that are commonly founded in Clusia species, which indicates that other
233
plants may be the source of Brazilian red propolis [47].
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The pharmacological use of Brazilian red propolis and its isolated compounds
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against human diseases, such as dental caries, candidiasis, cancer, skin wound,
236
oxidative stress-related conditions, among others, were subject of a previous review
237
published in this journal by our research group [11].
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In Table 1 we show the 13 types of Brazilian propolis from A. millifera and their
239
collection sites and botanical origins. These thirteen categories are scientifically
240
consolidated as Brazilian types of A. meliffera propolis, but other Brazilian propolis are
241
being studied and hereafter might be accepted as new types of Brazilian propolis. This
242
is the case of Southern organic propolis, which is produced strictly under organic
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conditions in Paraná and Santa Catarina States and has proven antimicrobial,
244
antioxidant and anti-inflammatory properties [19]. This type of propolis is produced in a
245
dense rainforest with great biodiversity. Accordingly, our unpublished chemical
246
findings point to the presence of molecules that are not usually present in propolis
247
samples, some of them completely unknown, suggesting that Southern organic propolis
248
may be a new source of bioactive molecules. The physicochemical composition and
249
biological potential of this type of propolis remain to be fully elucidated in further
250
studies.
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Table 1. Types of Brazilian Africanized Apis mellifera propolis and their respective
272
State in Brazil, botanical origins and most abundant and/or important chemical
273
compounds. Botanical Origin
1
Rio Grande do Sul
Unknown
2
Rio Grande do Sul
Unknown
3 (Poplar propolis)
Paraná
4
Most abundant and/or important chemical compounds Triterpenoids (melliferone, moronic acid, anwuweizonic acid and betulonic acid)
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State in Brazil
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References
Park et al [48] Ito et al [49]
Park et al [48] Alencar and Park [50]
Poplar trees (Populus L., Salicaceae Family)
Mainly flavonoids (chrysin, pinocembrin, pinobanksin, apigenin and galangin)
Park et al [46] Park et al [48]
Paraná
Unknown
Cinnamic acid and derivatives
Park et al [48] Alencar and Park [50]
5
Paraná
Unknown
Flavonoids and cinnamic acid derivatives
Park et al [48] Alencar and Park [50]
6 (Brown propolis)
Bahia
Hyptis divaricate
Prenylated benzophenone
Park et al [46] Park et al [48] Castro et al [51]
7
Bahia
Unknown
Flavonoids and benzoic acid derivatives
Park et al [48] Alencar and Park [50]
Pernambuco
Unknown
Flavonoids and benzoic acid derivatives
Park et al [48] Alencar and Park [50]
Pernambuco
Unknown
Cinnamic acid derivatives
Park et al [48] Alencar and Park [50]
10
Ceará
Unknown
Sesquiterpenes
Park et al [48] Alencar and Park [50]
11
Piauí
Unknown
Diterpenes
Park et al [48] Alencar and Park [50]
12
São Paulo
Baccharis dracunculifolia
Prenylated cinnamic acids
Paulino et al [15]
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Flavonoids and cinnamic acid derivatives
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Alagoas
Dalbergia ecastophyllum
and flavonoids
Park et al [46] Park et al [48]
Mainly isoflavonoids (formononetin, vestitol, neovestitol and daidzein) and chalcones
Bueno-Silva et al [16] Alencar et al [21] Silva et al [41] Oldoni et al [52]
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4. Compounds isolated from Brazilian propolis and their leukocyte recruitment
276
inhibitory activity
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There has been an increasing effort of the scientific community to obtain
278
bioactive compounds able to modulate leukocyte recruitment in the inflammatory
279
process [53-55]. Brazilian propolis from Apis mellifera bees and its different types has
280
been reported to be a promising source of bioactive compounds with this distinct anti-
281
inflammatory mechanism, as shown in Table 2.
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A promising anti-inflammatory compound named apigenin was identified in
283
poplar propolis [46]. A recent study showed that apigenin decreases the release of pro-
284
inflammatory cytokines by THP-1 macrophages while inhibiting ERK 1/2 and NF-κB
285
activation [56]. In vivo, apigenin promoted a significant decrease of NF-κB expression
286
in the lungs of LPS-stimulated mice, thereby modulating neutrophil influx and
287
decreasing the accumulation of chemotactic factors [57]. Taken together, these reports
288
indicate apigenin as a promising candidate for development of a new drug or a
289
nutraceutical compound.
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Galangin is another compound identified in poplar propolis [46] also presenting
291
relevant anti-inflammatory activity. Galangin was shown to reduce in vitro mRNA
292
levels of inflammatory cytokines by LPS-activated macrophages, in addition to
293
inhibiting ERK and NF-κB p65 phosphorylation [58]. When tested in human subjects
294
with rheumatoid arthritis, this compound significantly reduced neutrophil activation
295
[59].
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Similarly, pinocembrin, which was also identified in poplar propolis [46],
297
showed inhibitory activity on pro-inflammatory cytokine synthesis (including TNF-α)
298
in macrophages by inhibiting the phosphorylation of IκBα, ERK 1/2, JNK and
299
p38MAPK. In addition, pinocembrin attenuated an LPS-induced lung injury by
300
decreasing neutrophil influx into the inflammatory focus [60].
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Artepillin C is a compound commonly found in green propolis. This compound
302
was found to inhibit different signaling pathways of the inflammatory process [15].
303
When administrated in mice, it decreased paw edema and neutrophil influx into the
304
inflammatory site. In addition, artepillin C exhibited in vitro anti-inflammatory activity
305
(RAW 264.7 macrophages) by blocking activation of NF-κB [15].
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A recent study with the most recently cataloged type of propolis, the thirteenth
307
type of Brazilian propolis or red propolis, showed that the crude extract and molecules
308
isolated therefrom have potent anti-inflammatory activity by decreasing the neutrophil
309
influx [16]. Chemical analysis of Brazilian red propolis samples indicated the presence
310
of formononetin as the major compound. Formononetin showed inhibitory activity on
311
neutrophil migration and exhibited an antidematogenic effect in the paw edema assay
312
[61], in addition to blocking NF-κB activation [62].
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Two isoflavonoids with anti-inflammatory activity, vestitol and neovestitol, have
314
also been isolated from Brazilian red propolis [63]. Acute and chronic inflammation
315
models were used by our group to identify the specific anti-inflammatory mechanism(s)
316
of these compounds. We found that neovestitol reduces neutrophil influx by decreasing
317
ICAM-1 expression in the mesenteric microcirculation during acute inflammation in
318
vivo [18]. As for chronic inflammation, neovestitol reduced joint damage in mice with
319
arthritis [18]. The second isoflavonoid, vestitol, reduced neutrophil migration in mice
320
induced by different inflammatory stimuli [17]. We demonstrated that vestitol inhibits
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release of the chemokines CXCL1/KC and CXCL2/MIP-2 by resident macrophages
322
and, as a result, decreases rolling and adhesion of leukocytes [17]. It was further
323
determined that vestitol decreases neutrophil chemotaxis via calcium influx blockage.
324
Collectively, neovestitol and vestitol are promising candidates to treat acute and chronic
325
inflammation and therefore could be useful for therapeutic as well as nutraceutical
326
purposes [17-18].
327
Other
bioactive
compound
isolated
from
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Brazilian
red
propolis
is
isoliquiritigenin (ISQ), whose anti-inflammatory activity is well documented [52,64].
329
ISQ inhibits adhesion of neutrophil by downregulating expression of ICAM-1, VCAM-
330
1 and E-selectin on endothelial cells [64]. These effects are thought to be related to
331
interference with translocation of p65 from cytoplasm to the nucleus, thereby reducing
332
NF-κB activation [64].
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Daidzein is another bioactive molecule that has aroused attention due to its
334
significant anti-inflammatory potential [11,65]. Administration of this molecule
335
significantly reduced the number of neutrophils, release of inflammatory cytokines, the
336
number of TLR4 receptors and the activation of NF-κB upon LPS challenge [65].
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In summary, the study of these compounds as potential candidates for a
338
mechanistically distinct anti-inflammatory therapy is relatively new and remains a
339
fascinating topic for application in biomedical areas and dentistry. Notably, the proposal
340
of a naturally-derived prototype drug whose mechanism differs from that of the drugs
341
currently available in the market is innovative and merit further non-clinical and clinical
342
investigation.
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Table 2. Isolated molecule from different types of propolis and its main anti-
352
inflammatory pathway. Main
propolis
anti-inflammatory pathway
Propolis type 3 (Poplar Propolis)
Inhibition of inflammatory cytokines, ERK 1/2 and NF-κB activation and of neutrophil migration
Park et al [46] Zhang et al [56] Cardenas et al [57]
Inhibition of neutrophil activation, inflammatory cytokines and ERK and NFκB p65 phosphorylation
Park et al [46] Jung et al [58] Santos et al [59]
Propolis type 3 (Poplar Propolis)
Inhibition of inflammatory cytokines (TNF-α), phosphorylation of IκBα, ERK1/2, JNK and p38MAPK and neutrophil migration
Park et al [46] Soromou et al [60]
Propolis type 12 (Green Propolis)
Inhibition of neutrophil migration and of activation of NF-κB
Paulino et al [15]
Apigenin
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Galangin
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Propolis type 3 (Poplar Propolis)
Reference
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Type of
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Molecule
Pinocembrin
Artepillin C
19
ACCEPTED MANUSCRIPT Propolis type 13 (Red Propolis)
Inhibition of leukocyte migration and activation of NF-κB
Cavendish et al [61] Wang et al [62]
Propolis type 13 (Red Propolis)
Acute inflammation: Franchin et al [18] Reduces neutrophil migration by decreasing ICAM-1 Chronic inflammation: Decreases the clinical score and joint damage in a collagen-induced arthritis model
Propolis type 13 (Red Propolis)
Inhibition of neutrophil migration, rolling and adhesion; inhibition of CXCL1/KC and CXCL2/MIP2 levels and neutrophil chemotaxis via blocking calcium influx
Vestitol
Isoliquiritigenin
SC
Oldoni et al [52] Inhibition adhesion of neutrophil, ICAM-1, VCAM- Kumar et al [64] 1 and E-selectin expression and translocation of the p65 subunit of NF-κB
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Propolis type 13 (Red Propolis)
Inhibition of neutrophil migration, inflammatory cytokine release, TLR4 receptor and NF-κB activation
Freires et al [11] Feng et al [65]
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Propolis type 13 (Red Propolis)
Franchin et al [17]
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Neovestitol
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Formononetin
Daidzein
353 354
5. Conclusion
355
The present review addressed the main aspects involved in leukocyte
356
recruitment during the inflammatory process and the possible targets for development of
357
novel anti-inflammatory drugs. Different bioactive compounds obtained from Brazilian
358
propolis were presented along with preclinical evidence (in vitro and in vivo studies) of
20
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their inhibitory activity on leukocyte recruitment and mechanism of action. The
360
compilation of data and insights presented herein may open further avenues for the
361
pharmacological management of oral and systemic inflammatory conditions. Further
362
research should focus on clinical and acute/chronic toxicological validation of the most
363
promising compounds isolated from Brazilian propolis.
364
Conflict of interest
365
The authors declare no conflict of interest.
367
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Acknowledgments
The authors would like to thank the São Paulo Research Foundation (FAPESP,
369
grants no. 2016/15563-9, 2015/26864-7 and 2016/02926-6) and the National Council
370
for Scientific and Technological Development (CNPq, 310522/2015-3) for financial
371
support.
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E. Kolaczkowska, P. Kubes, Neutrophil recruitment and function in health and
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C.R. Mackay, Moving targets: cell migration inhibitors as new anti-
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[7]
I. Mitroulis, V.I. Alexaki, I. Kourtzelis, A. Ziogas, G. Hajishengallis, T.
399
Chavakis, Leukocyte integrins: role in leukocyte recruitment and as therapeutic
400
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ACCEPTED MANUSCRIPT Highlights •
There is a close relationship between inflammatory diseases and leukocyte recruitment. New anti-inflammatory drugs target a more selective action on neutrophil
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migration.
Brazilian propolis is a rich source of anti-inflammatory molecules.
•
Compounds isolated from Brazilian propolis inhibit neutrophil migration.
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•