nutrients

Review
Experimental Evidence for Therapeutic Potentials of Propolis
Priyanshu Bhargava 1,† , Debajit Mahanta 1,2,† , Ashish Kaul 1 , Yoshiyuki Ishida 3 , Keiji Terao 3 , Renu Wadhwa 1
and Sunil C. Kaul 1,4, *

1 AIST-INDIA DAILAB, National Institute of Advanced Industrial Science & Technology (AIST),
Tsukuba 305-8565, Japan; [email protected] (P.B.); [email protected] (D.M.);
[email protected] (A.K.); [email protected] (R.W.)
2 DBT-APSCS&T Centre of Excellence for Bioresources and Sustainable Development, Kimin 791121, India
3 CycloChem Co., Ltd., 7-4-5 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan;
[email protected] (Y.I.); [email protected] (K.T.)
4 Kaul-Tech Co., Ltd., Nagakunidai 3-24, Tsuchiura 300-0810, Japan
* Correspondence: [email protected]
† Equal first authors.

Abstract: Propolis is produced by honeybees from materials collected from plants they visit. It is
a resinous material having mixtures of wax and bee enzymes. Propolis is also known as bee glue
and used by bees as a building material in their hives, for blocking holes and cracks, repairing
the combs and strengthening their thin borders. It has been extensively used since ancient times
for different purposes in traditional human healthcare practices. The quality and composition of
propolis depend on its geographic location, climatic zone and local flora. The New Zealand and
Brazilian green propolis are the two main kinds that have been extensively studied in recent years.
Their bioactive components have been found to possess a variety of therapeutic potentials. It was
found that Brazilian green propolis improves the cognitive functions of mild cognitive impairments
in patients living at high altitude and protects them from neurodegenerative damage through its
antioxidant properties. It possesses artepillin C (ARC) as the key component, also known to possess
anticancer potential. The New Zealand propolis contains caffeic acid phenethyl ester (CAPE) as the
Citation: Bhargava, P.; Mahanta, D.; main bioactive with multiple therapeutic potentials. Our lab performed in vitro and in vivo assays
Kaul, A.; Ishida, Y.; Terao, K.; Wadhwa, on the extracts prepared from New Zealand and Brazilian propolis and their active ingredients.
R.; Kaul, S.C. Experimental Evidence We provided experimental evidence that these extracts possess anticancer, antistress and hypoxia-
for Therapeutic Potentials of Propolis. modulating activities. Furthermore, their conjugation with γCD proved to be more effective. In the
Nutrients 2021, 13, 2528. https:// present review, we portray the experimental evidence showing that propolis has the potential to be a
doi.org/10.3390/nu13082528 candidate drug for different ailments and improve the quality of life.

Received: 31 May 2021

Keywords: honeybee; propolis; caffeic acid phenethyl ester (CAPE); artepillin C (ARC); biomedical
Accepted: 19 July 2021
properties; natural drug; traditional healthcare
Published: 24 July 2021

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
1. Introduction
published maps and institutional affil-
iations. The term propolis is derived from the Greek words pro, meaning ‘in front of’ or ‘at the
entrance to’, and polis, meaning ‘community’ or ‘city’, and it describes a substance used
to defend the hive. Propolis, also known as ‘bee glue’, is the most important ‘chemical
weapon’ of honeybees. It is a brownish resinous material collected by bee workers from the
Copyright: © 2021 by the authors.
flora (leaf and bark of tree species including birch, poplar, pine, alder, willow and palm)
Licensee MDPI, Basel, Switzerland.
they live on or visit. Bees carry and accumulate the resinous exudates from these plants
This article is an open access article
in their hives where they are modified by their enzymes into propolis, which is used as a
distributed under the terms and construction material for the hives. It also prevents the spread of microbial (bacterial and
conditions of the Creative Commons fungus) infections and attacks from predators. Propolis was identified and used by humans
Attribution (CC BY) license (https:// since ancient times (since 300 BC) as a traditional medicine [1,2]. Egyptians benefited
creativecommons.org/licenses/by/ from the antiputrefactive properties of propolis in order to preserve their corpses from
4.0/). decomposition [3,4]. Propolis was historically recognized for its medicinal properties by

Nutrients 2021, 13, 2528. https://doi.org/10.3390/nu13082528 https://www.mdpi.com/journal/nutrients

Nutrients 2021, 13, 2528 2 of 23

the Greek and Roman physicians and was first employed as a mouth disinfectant and an
antiseptic and healing natural drug in the treatment of wounds [3]. It has been listed as
an official drug in London pharmacopeias of the 17th century and has since then been
used as a popular and regular remedy, either in a pure form or in combination with other
natural products in cosmetics/health industries [5]. Most recently, several research labs
have investigated the bioactive constituents of propolis, a wide range of their bioactivities
(including antioxidant, antibacterial, antifungal, anti-inflammatory, hepatoprotective and
anticancer) and their mechanisms of action as nutritional, cosmetic and pharmaceutical
benefits [6–8].

2. Propolis Constituents
Propolis has been classified into different types based on their geographic origin
and plant source. The main widely known types include Poplar (New Zealand, Europe,
North America, some regions of Asia), Green (Alecrim), Brazilian (Brazil), Birch (Rus-
sia), Red (Cuba, Brazil, Mexico), Mediterranean (Sicily, Greece, Crete, Malta) and Pacific
(Okinawa, Taiwan, Indonesia) propolis. New Zealand and Brazilian green propolis are
the two most prominent types that have attracted both scientific and commercial interest.
In the past, the quality of propolis was determined based on the local varieties of bee
species, botanical sources and climate conditions. In recent years, analytic technologies,
namely high-performance liquid chromatography (HPLC), thin-layer chromatography
(TLC), gas chromatography (GC), nuclear magnetic resonance (NMR), mass spectrometry
(MS) and gas chromatography–mass spectrometry (GC-MS), have been extensively used to
define chemotypes of propolis. With such highly refined analytic technologies, propolis
constituents such as sugars, hydrocarbons and minerals have also been identified and
regularly used for their profiling and branding.
More than 180 compounds, mainly polyphenols, have been identified as constituents
of varieties of propolis originating from different geographical regions. Polyphenol content
is often used as a criterion to evaluate the quality of propolis. The major polyphenols are
flavonoids, phenolic acids, esters, phenolic aldehydes and ketones. Volpi et al. [9] reported
12 different flavonoids, pinocembrin, acacetin, chrysin, rutin, catechin, naringenin, galangin,
luteolin, kaempferol, apigenin, myricetin and quercetin; two phenolic acids, cinnamic acid
and caffeic acid; and one stilbene derivative, resveratrol, in propolis extracts. Terpenes
(secondary metabolites) in propolis account for its resinous odor and are considered as a
criterion to distinguish between different types of propolis and sometimes even as a marker
of premium versus ordinary propolis. Other compounds in propolis include volatile
oils and aromatic acids (5–10%), waxes (30–40%), resins, balms and pollen grains that
offer the prime source of essential elements such as magnesium, nickel, calcium, iron and
zinc. It is also enriched in biometabolites (amino acids, sugars, nucleic acids and lipids),
minerals (Mg, Ca, I, K, Na, Cu, Zn, Mn and Fe), hydrocarbons (alkanes, alkenes, alkadienes,
monoesters, diesters, aromatic esters, etc.) and vitamins (B1, B2, B6, C and E) that are
essential for various structures and functions in living systems. Enzymes such as succinic
dehydrogenase, glucose-6-phosphate, adenosine triphosphate and acid phosphate have
been detected in a variety of propolis types. The chemical compositions of varieties of
propolis from different geographical and botanical niches show striking variability, posing
additional challenges in the application of propolis in health/cosmetic/pharmaceutical
industries [10–14].
The appearance and physical properties of propolis also vary according to its geo-
graphical location and vegetation. It is generally dark green or brownish in color, hard
at low temperature and soft at high temperature and melts at 60 to 70 ◦ C; some kinds
have melting temperature close to 100 ◦ C. For commercial use, propolis extracts are of-
ten prepared with suitable solvents, such as ethanol, methanol, chloroform, ether and
acetone, of which ethanol extract has been popular due to its high level of bioactivities.
Several kinds of propolis products including dentifrices, lozenges, mouth rinses, creams,
gels, cough syrups, wine, cake, powder, soap, chewing gums, tablets, candies, shampoos,
Nutrients 2021, 13, 2528 3 of 23

chocolate bars, skin lotions and toothpaste are commercially available [15,16]. Furthermore,
polyphenol-rich varieties of propolis from different geographical regions are commonly
used as antioxidant and antiseptic remedies in traditional home-medicine systems.
A variety of extraction methods including hexane; alcoholic; and nonalcoholic mix-
tures of solvents such as polyethylene glycol (PEG)-400, water and olive oil have been
used to generate propolis extracts. Interestingly, there were no large differences observed
in the total content of phenolic compounds observed in different extracts. PEG–water
extracts showed significant amounts of naringenin; galangin; kaempferol; ferulic, caffeic
and p-coumaric acids; quercetin; and artepillin C. Many of these were tested to possess con-
siderably similar levels of antimicrobial activity against S. aureus, B. cereus, P. aeruginosa and
K. pneumoniae compared to the standard ethanol extract [17]. Ethanolic and hexane extracts
of Brazilian propolis were tested on methicillin-resistant Staphylococcus aureus (MRSA) for
antibacterial activity [18]. Initial comparison found more artepillin C (active component of
Brazilian propolis) content in ethanol extracts as compared to hexane extracts. In addition,
artepillin C showed bacteriostatic activity with membrane blebbing, resulting in antibacte-
rial activity against S. aureus and anaerobic bacterium Porphyromonas gingivalis [19]. Other
bioactive components, i.e., 3-prenyl-cinnamic acid allyl ester, 2-dimethyl-8-prenylchromene,
kaempferide, drupanin and p-coumaric acid also showed antibacterial activity against
S. saprophyticus, L. monocytogenes and E. faecalis [20]. Pinocembrin and apigenin, flavonoids
found in Chilean propolis, showed significant antibacterial activity against S. mutants as
compared to a mixture of polyphenols and chlorhexidine (MIC = 1.6 µg/mL) with MIC 1.4
µg/mL and 1.3 µg/mL, respectively [21]. Apigenin showed antibacterial activity against
several Gram-negative bacteria, namely P. aeruginosa, K. pneumoniae, S. enterica serotype Ty-
phimurium, P. mirabilis and E. aerogenes [22]. Quorum sensing is a bacterial communication
mechanism used to express various survival or virulence traits to enhance resistance to
various external stimuli. Propolis is rich in cinnamic acid and its derivatives, as they exist
in the green parts of plants and flowers. They inhibit cell growth by damaging bacterial cell
membranes and inhibiting ATPase, cell division and biofilm formation with anti-quorum
sensing activity [23].

3. Propolis Bioactivities
As discussed in the above section, different types of propolis differ in their chemical
constituents and bioactivities, which accordingly determines their therapeutic applications,
including antibacterial, antioxidant, anti-inflammatory, antifungal, antiviral, cardioprotec-
tive, hepatoprotective, neuroprotective and anticancer applications.

3.1. Infections
Microorganisms (bacteria, viruses, fungus, protozoans and other parasites), the root
cause of infectious human diseases, possess high genome instability and high adaptability
to environmental conditions. Based on these features, infectious agents are rather difficult
to treat with single-module synthetic drugs. The natural compounds and extracts derived
from propolis (economical and easily available) have been proved to be effective and useful
in not only treating, but also preventing infections.

3.1.1. Antifungal Activity

Fungal infections are often caused by microscopic fungi through air, soil, water
and plants. In the last few years, fungal diseases caused over 1.6 million deaths, and
over a billion people suffer from fungal diseases annually. Fungal diseases can also
cause major losses in agriculture and food production. Ota et al. [24] conducted efficacy
testing of propolis for antifungal activity using 80 strains of Candida yeasts including
Candida albicans, Candida tropicalis, Candida krusei and Candida guilliermondii. The antifungal
activity of propolis was shown in the following order of sensitivity: C. albicans > C. tropicalis
> C. krusei > C. guilliermondii. Antifungal activity of propolis has been demonstrated by
in vitro assay for human dentinal tubules that are commonly infected with Candida albicans.
Nutrients 2021, 13, 2528 4 of 23

Infected dental samples treated with propolis showed a reduction in infection as com-
pared to the control group [25]. In another study, 105 human teeth were infected with
Candida albicans for 2 days followed by treatment with propolis for 5 days. The treatment
inhibited ~99% of fungal growth and was as effective as 2% chlorhexidine and alcoholic
extract of Azadirachta indica [26].
Caffeic acid (an active component of propolis) showed remarkable antimycotic activity
towards Helminthosporium carbonum, a pathogenic fungus that causes corn leaf blight
disease. This fungus is a common inhabitant in dead or decayed leaves. Anti-H. carbonum
activity is important to induce resistance or production of inhibitory compounds in maize
as a defense mechanism. Major constituents of propolis such as 3-acetylpinobanksin,
pinobanksin-3-acetate, pinocembrin, p-coumaric acid and caffeic acid have been shown to
possess antifungal activity [27–29]. Pinocembrin was reported to possess activity against
Penicillium italicum, a widely reported postharvest fungal pathogen affecting citrus fruits.
P. italicum causes major menace through their spores that are airborne and appears as a
fine powder. Infected fruits are completely covered by white mycelium followed by green
and bluish spores, responsible for the decaying of citrus fruits. Pinocembrin has been
shown to stop mycelial growth by inhibiting respiration and resulting in an imbalance
in energy homeostasis. Based on these findings, pinocembrin has been proposed as a
promising bioactive compound of propolis for the treatment of P. italicum infections on
postharvest citrus fruit [30]. The flavonoids present in propolis have also been shown
to possess considerable fungicidal activity against a variety of fungi strains including
C. pelliculosa, C. albicans, C. famata and C. glabrata [31,32].

3.1.2. Antibacterial Activity

In recent years, bacterial infections of crops and food products have attracted the
attention of several health organizations. These microorganisms not only target crops but
also spread several foodborne diseases, including food poisoning, fever and gastroenteritis.
Propolis and its active components have also been shown to possess antimicrobial activity
and boost the activity of conventional synthetic antimicrobial compounds [33,34]. Based on
these reports, the antibacterial activity of propolis is often evaluated at two levels: (i) direct
action on the microorganisms and (ii) stimulation of the immune system. Several studies
showed that propolis and some of its flavonoid components inhibited bacterial (B. subtilis)
motility by dissipating the membrane potential and uncoupling the energy-transducing
cytoplasmic membrane [35]. The toxic effect of propolis was therefore concluded as a direct
effect on membrane permeability and membrane potential. Propolis was also found to
be synergistically effective with most of the antibiotics except polymyxin B and nystatin.
It was reported to affect a wide range of Gram-positive rods and have limited activity
against Gram-negative bacilli. Bulgarian propolis was shown to be active against most
anaerobic bacterial strains and other pathogens, including Clostridium, Bacteroides and
Propionibacterium species [36]. Furthermore, xanthochymol and oblongifolin found in
red propolis were reported to possess antimicrobial activity against multidrug-resistant
bacteria [37]. The differential activity towards Gram-positive and Gram-negative bacteria
was attributed to the structural differences in the outer bacterial membrane and the pro-
duction of hydrolytic enzymes in Gram-negative bacteria that cause degradation of the
active component of propolis [38]. Helicobacter pylori, a major factor for gastrointestinal
illness, contains the enzyme peptide deformylase that catalyzes the removal of the formyl
group from the N-terminus of nascent polypeptide chains. Investigations on the absorption
spectra and crystal structure have shown that CAPE, an active component of propolis,
is a competitive inhibitor of peptide deformylase. It can block the substrate–active site
interactions. As the action of this enzyme is essential for H. pylori survival, propolis CAPE
has been proposed as a promising therapeutic drug candidate [39]. Feeding of ethanolic
extract of propolis (300 mg/kg body weight) for 30 days was shown to be most effective
against Salmonella in infected BALB/c mice [40]. Furthermore, propolis enhanced the
activity of antimicrobial drug cefixime (acts by penetrating the monocytes and causes
Nutrients 2021, 13, 2528 5 of 23

inhibition of Salmonella typhimurium growth), showing significant inhibitory effect on the

growth of Salmonella in just 5 days [40]. These data have suggested the synergistic activity
of propolis and conventional synthetic antimicrobial agents.

3.1.3. Antiviral Activity

Viruses, the small infectious agents that replicate only inside the living host cells,
cause several kinds of short-term (e.g., common cold, influenza, chickenpox, smallpox,
measles and cold sores) and long-term (e.g., chronic hepatitis, rabies, Ebola virus disease,
AIDS and several types of cancers) ailments. Several flavonoids have been shown to
possess inhibitory activity towards viruses such as herpes simplex virus (HSV-1 and
HSV-2), Sindbis virus, parainfluenza-3 virus, human cytomegalovirus and dengue virus
type 2 [41–43]. It has been observed that flavonoids are more active than flavones, and the
synergism of both compounds possibly presents better biological activity against various
ailments as compared to individual substances [44]. Propolis contains a large amount of
3-methyl-but-2-enyl caffeate, isopentyl ferulate and moronic acid constituents. Influenza A
(H3N2) virus, a respiratory pathogen, evades human immunity, preliminarily by acquiring
antigenic changes in the hemagglutinin (HA). Isopentyl ferulate, a constituent of propolis,
was shown to inhibit the infectious titer and activity of influenza virus A1 Hong Kong
(H3N2) in vitro [45]. Herpes simplex virus (HSV-1) and HSV-2 both have been reported
to cause oral and genital lesions due to autoinoculation. A minor constituent of propolis,
3-methyl-but-2-enyl caffeate, isolated from poplar buds was shown to possess anti-herpes
simplex virus type 1 activity in in vitro assays. Addition of propolis at 6.25 µg/mL to a
culture medium of infected Vero cells caused an effective reduction in the virus titer [46].
In a randomized study on a group of 90 men and women with recurrent chronic genital
herpes simplex virus (HSV) type 2, ointment of Canadian propolis was identified as more
protective than acyclovir and placebo ointments [5]. The replication of HSV-1 and HSV-2
was significantly suppressed in the presence of 25, 50 and 100 µg/mL of Hatay propolis.
The synergistic effect of Hatay propolis with acyclovir was found prominent on both HSV-1
and HSV-2 viruses [47].
In developing countries, acquired immunodeficiency syndrome (AIDS) is a pro-
nounced cause of death. The virus destroys the T cells after invasion and replicates
inside the host cells. Most conventional drugs are nucleoside-based and carry limitations
including adverse side effects, resistance and toxicity. Several flavonoids containing natural
products and caffeic acid derivatives such as chicoric, rosmarinic and lithospermic acids
have been shown to be active against HIV [48]. Anti-HIV activity in H9 lymphocytes
treated with triterpenoids, melliferone, moronic acid, anwuweizonic acid, betulonic acid
and four known aromatic compounds isolated from Brazilian propolis were evaluated.
Moronic acid showed significant anti-HIV activity (EC50 < 0.1 µg/mL) [49]. Propolis has
been shown to inhibit HIV expression in CD4+ lymphocytes and microglial cell cultures in
a concentration-dependent manner.
Canine distemper virus (CDV) is closely related to the measles virus, bovine pestivirus,
small ruminant pestivirus and phocine distemper virus. It causes multisystem disease
in dogs and other carnivores [50]. Mexican propolis and its three active pure flavonoids
quercetin, naringenin, and pinocembrin were tested for antiviral (CDV) activity in CCL-81
cells. The expression of virus nucleoprotein gene and cell viability measured to evaluate
antiviral activity endorsed that propolis and quercetin caused a significant decrease in gene
expression and increase in host cell viability, whereas naringenin and pinocembrin failed
to stop viral infection [51].
The rhinovirus (HRV) is the most common virus and the predominant cause of the
common cold. Kaempferol and coumaric acid, active ingredients of Brazilian propolis, were
found to be effective for treating HRV-2, HRV-3 and HRV-4 viruses in a HeLa cell culture
model. It was hypothesized that kaempferol and coumaric acid may inhibit viral entry into
the cells, generating protection from viral destruction and abating virus replication [52].
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The recent global pandemic of coronavirus disease 2019 (COVID-19), caused by a

novel coronavirus (severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-
nCoV)) lacks treatment modalities and has triggered repurposing of the existing natural
drugs, including propolis, that have been trusted for antimicrobial and antiviral activities
in traditional systems of home medicine. Several computational and molecular docking
studies have shown that the flavonoids in propolis (e.g., rutin, naringin, caffeic acid phenyl
ester, luteolin and artepillin C) have the potential to inhibit viral spike fusion to the host
cells. Interestingly, ethanolic extract of propolis and propolis liposomes have been shown to
inhibit (i) structural proteins of SARS-CoV-2 in vitro and (ii) SARS-CoV-2 infection in Vero
E6 cells when used in combination with naringin. Of note, nanocarrier-mediated delivery
of propolis extracts enhanced their antiviral activity. In a preclinical study, COVID-19
patients receiving green Brazilian propolis showed earlier recovery of symptoms and viral
clearance as compared to the group receiving standard treatment [53]. Several bioactive
compounds in propolis have been predicted to possess inhibitory effects on the entry of
virus to host cells by blocking the host cell receptors (ACE2, TMPRSS2), PAK1 signaling
pathways and immunoregulation of proinflammatory cytokines (including a reduction in
IL-6, IL-1 beta and TNF-alpha) [54]. Furthermore, propolis has been shown to be effective
in the treatment of various comorbidities (respiratory diseases, hypertension, diabetes and
cancer) that are high mortality risk factors for COVID-19 patients [55]. In an in silico study,
binding constants of 10 flavonoids commonly found in propolis (caffeic acid, caffeic acid
phenethyl ester, chrysin, galangin, myricetin, rutin, hesperetin, pinocembrin, luteolin and
quercetin) to ACE2 receptor were investigated by molecular docking. Rutin showed the
highest high binding energy (–8.04 kcal/mol), followed by myricetin, quercetin, caffeic acid
phenethyl ester and hesperetin, suggesting their inhibitory potential for COVID-19 [56].
Khayrani et al. [57] identified five compounds (namely glyasperin A, broussoflavonol F,
sulabiroins A, (2S)-5,7-dihydroxy-40 -methoxy-8-prenylflavanone and isorhamnetin) from
Sulawesi propolis that have the potential to inhibit the binding of ACE-2 and SARS-CoV-2.
Interestingly, the docking score of each of the test compounds was favorable as compared
to a known potent inhibitor (MLN-4760).
On the other hand, in silico studies investigated the binding potential of bioactive
compounds of propolis to SARS-CoV-2 proteins (Mpro and S2) that are essential for its
replication. Shaldam et al. [58] reported the binding affinity of 14 selected phenolics
and terpenes found in honey and propolis against the Mpro and RNA-dependent RNA
polymerase (RdRp) enzymes of the SARS-CoV-2 virus. Of these compounds, p-coumaric
acid, ellagic acid, kaempferol and quercetin showed the strongest interactions. In another
study, four compounds (neoblavaisoflavone, methylophiopogonone A, 30 -methoxydaidzin
and genistin) showed binding affinity similar to that of the positive control nelfinavir,
thus placing them as antiviral drug candidates [59]. Two compounds from Sulawesi
propolis, glyasperin A and broussoflavonol F, were shown to be potential drug candidates
for COVID-19 based on their high binding affinity to the catalytic sites of Mpro [60].
Kumar et al. [61] also reported that the binding affinity and energy of caffeic acid phenethyl
ester (CAPE) to the substrate-binding domain of Mpro were comparable to those of a
positive control, N3 protease inhibitor. Refaat et al. [62] reported in silico analysis where
rutin and CAPE were shown to possess the highest affinity to viral targets, 3CL-protease
and S1 spike protein. They also developed a liposomal formula of propolis that exhibited
significantly enhanced inhibition of viral replication as comparable to the viral inhibitory
effect of the potent antiviral drug remdesivir. These recent reports have rejuvenated
the interest in the bioactivities of propolis and its use as a valuable nutraceutical and
pharmaceutical ingredient.

3.1.4. Inflammation
Inflammation is largely defined as a biological response to an altered environment
categorized as stress (exogenous or endogenous) that causes cell/tissue damage, triggered
by physical (burns, bruises, radial damage, etc.), chemical (acid, alkali, allergens, etc.) and
Nutrients 2021, 13, 2528 7 of 23

biochemical (parasites, microorganisms, endotoxins) factors. It is often divided into acute

and chronic. In acute inflammation, immune system cells are activated and migrate to
the site of damage and release growth factors, cytokines, reactive oxygen species (ROS)
and reactive nitrogen species (RNS). Chronic inflammation occurs whenever the acute
inflammation is not successfully resolved. The inflammatory condition has been shown to
play a critical role in the pathogenesis of diseases, namely asthma, atherosclerosis, cancer,
Alzheimer’s and Parkinsonism [63,64]. A broad range of signaling pathways has been
reported to be associated with inflammation, namely nuclear factor kappa B (NFk-B),
activator protein (AP)-1, peroxisome proliferator-activated receptor (PPAR) and nuclear
factor erythroid 2-related factor 2 (Nrf2) transcription factors; mitogen-activated protein
kinases (MAPKs); protein tyrosine kinases (PTKs); tyrosine phosphatidylinositol 3-kinase
(PI3K)/Akt; and the ubiquitin–proteasome system. The identification and characterization
of phenolic compounds that modulate signaling events involved in inflammation are
important not only to validate the use of dietary phenolics in human health but also to find
new potential molecules that serve as templates for the further structural development of
safe and effective anti-inflammatory drugs. The anti-inflammatory properties attributed to
the phenolic compounds are often related to their antioxidant activity in addition to the
modulation of specific inflammation-signaling pathways [65].
Several studies have shown that flavonoids, polyphenolic compounds present in
propolis, possess anti-inflammatory capability [65,66]. The main reported mechanisms of
anti-inflammatory activity of propolis include (i) inhibition of cyclooxygenase (COX) and
subsequent inhibition of prostaglandin biosynthesis, (ii) free radical scavenging activity,
(iii) inhibition of nitric oxide synthesis, (iv) reduction in inflammatory cytokine levels and
(v) immunosuppressive activity [67–69]. Immunomodulatory assays including tests with
positive controls, such as lipopolysaccharide (LPS), concanavalin A (CON A), phorbol
myristate acetate (PMA) or cytokines (IFN-γ), have been performed to assess the efficacy
of propolis as an immunomodulator. The administration of 200 mg/kg ethanolic extract of
Brazilian propolis to mice for 3 days enhanced the innate immunity, activated the immune
response by upregulating the expression of toll-like receptors TRL-2 and TRL-4 and the
production of proinflammatory cytokines interleukin IL-2 and IL-6 by macrophages and
spleen cells, contributing to the recognition of microorganisms and activation of lympho-
cytes by antigen-presenting cells [70]. The effects of propolis extracts were investigated in
rat models of inflammation [71]. The arthritis index in the chronic inflammatory animal
model was suppressed upon treatment with propolis extract (50–100 mg/kg/day). In addi-
tion, physical weakness due to chronic disease state was improved in the propolis-treated
group in a dose-dependent manner. It was suggested that the anti-inflammatory activity of
propolis in both chronic and acute inflammation might be mediated by its inhibitory effect
on prostaglandin production [71]. Several studies have demonstrated that propolis acts
as a potent anti-inflammatory agent for chronic and acute inflammation conditions [72].
Propolis has also been shown to act on the nonspecific immune response by releasing
hydrogen peroxide and inhibiting the production of nitric oxide in a dose-dependent man-
ner [73]. Several studies have suggested that propolis extracts possess antioxidant activity
due to their high content of polyphenols, flavonoids, caffeic acid, ferulic acid, CAPE and
kaempferol. They block lipid peroxidation and protect cells from the damage caused by
free radicals and overabundance of inflammation [73,74].
In a human study, early on, the administration of propolis capsules (500 mg) for
2 weeks showed spontaneous and LPS-induced increase in proinflammatory cytokine
secretion capacity of peripheral blood leukocytes [75]. Another human study has reported
the prolonged effect of propolis supplementation on redox state. Male population supple-
mented with propolis demonstrated a reduction in free-radical-induced lipid peroxidation
as well as an increase in activity of superoxide dismutase [76]. Most recently, Chinese
and Brazilian propolis types were shown to alter lipoglycan, endotoxin-based inflamma-
tory cascade and nuclear factor kappa B (NF-kB) function [77–79]. In most studies, the
most popular bioactive compound of New Zealand propolis, CAPE, was shown to pos-
Nutrients 2021, 13, 2528 8 of 23

sess remarkable anti-inflammatory properties. It showed immunosuppressive activity by

inhibiting the early and late events of T-cell activation and the consequent release of cy-
tokines. Treatment with propolis led to a reduction in pulmonary inflammation and mucus
production. It caused an increase in in vitro differentiation and in vivo frequency of lung
myeloid-derived suppressor cells (MDSC) and CD4+ /Foxp3+ regulatory T cells, suggesting
its potential in the treatment of allergies and asthma [68]. It was also reported to inhibit
myeloperoxidase activity, NADPH oxidase, ornithine decarboxylase, tyrosine-protein ki-
nase and hyaluronidase in guinea pig mast cells [1]. Several bioactive compounds other
than CAPE have been assigned anti-inflammatory activity. These include pinocembrin,
chrysin and artepillin C [80–83].

3.2. Liver Function

The liver is a large glandular organ comprising 1/50 of the total weight of the adult
body. It synthesizes and secretes bile, lipoproteins and plasma proteins, including clotting
factors. It maintains a stable blood glucose level by taking up and storing glucose as
glycogen (glycogenesis), breaking this down to glucose when needed (glycogenolysis) and
forming glucose from non-carbohydrate sources such as amino acids (gluconeogenesis).
In conjunction with the spleen, the liver regulates the destruction of dying red blood
cells and the reclamation of their constituents. Hepatotoxicity of drugs or drug-induced
liver injury (DILI) is a major health problem accounting for more than 50% of acute liver
failure, including that caused by overdosing of drugs. The mechanism of DILI involves two
pathways, namely direct hepatotoxicity and adverse immune reactions. DILI is initiated
by the bioactivation of drugs to chemically reactive metabolites having the ability to
interact with cellular macromolecules leading to a variety of anomalies including protein
dysfunction, lipid peroxidation, DNA damage and oxidative stress. Such impairments in
subcellular structures and functions, accumulation of macromolecular damage and loss of
energy homeostasis often culminate in cell death and possible liver failure. Early diagnosis
and withdrawal of the suspected medication is the mainstay of treatment of DILI. Therapies
developed along the principles of synthetic medicine are often limited in efficacy, carry
the risk of adverse effects and are often expensive, especially for the developing world.
Therefore, treating liver diseases with plant-derived compounds has attracted research and
clinical interest [84–86].
Several studies have addressed the mechanism of the hepatoprotective activity of
propolis and its active ingredients. Propolis extracts exhibit significant cellular recovery,
as evidenced by the reversal of various biochemical indices and histopathology, altered
due to CCl4 toxicity. Owing to the antioxidative potential, propolis has been assigned
clinical importance and is useful for the treatment of metabolic syndrome and its associated
chronic diseases [66]. Early on, using D-GalN-induced hepatic injury model, it was shown
that ethanol extract of Brazilian and Cuban propolis types possess strong hepatoprotective
activity against paracetamol- and CCl4-induced liver injury in mice and rat models [87].
Ethanolic extracts of propolis also showed protective effect against alcohol-mediated liver
injury by preventing the elevations of total cytochrome P-450 enzymes, NADPH-dependent
cytochrome C reductase, aniline hydroxylation, 7-ethoxyresorufin (7-ERH) hydroxylation,
7-penthoxyresorufin (7-PRH) hydroxylation and lipid peroxidation. A study on Brazilian
propolis reported its protective effect against hepatic oxidative damage in rats with water-
immersion restraint stress (WIRS) in comparison with vitamin E (VE) [88]. Exposure of
vehicle-treated rats to 6 h of WIRS caused liver damage as indicated by levels of serum
alanine aminotransferase and aspartate aminotransferase; increased hepatic liquid peroxide
and NO(x) content and myeloperoxidase activity; and decreased hepatic non-protein
SH, ascorbic acid content and superoxide dismutase activity. Rats on fast were orally
administered with ethanol extract of Brazilian green propolis (BPEE10, 50 or 100 mg/kg)
and VE (250 mg/kg) at 30 min before the onset of WIRS. Prior administration of BPEE (50 or
100 mg/kg) or VE to the stressed rats protected against the hepatic damage and attenuated
the increased hepatic lipid peroxide, NOx and myeloperoxidase activities. These results
Nutrients 2021, 13, 2528 9 of 23

indicate that BPEE protected against hepatic oxidative damage in rats exposed to WIRS,
possibly through its antioxidant and anti-inflammatory properties.
Some studies have also reported the protective effect of CAPE in various hepatotoxicity
models. Intraperitoneal (i.p.) administration of CAPE to 12-week-old female Wistar albino
rats (10 µmol/kg body mass; 6 days), prevented cisplatin-induced oxidative damage in
their liver by reducing ROS production and increasing antioxidative enzyme (such as
SOD, GSH-Px and CAT) activities, signifying the hepatoprotective effect of CAPE [89].
CAPE (10 µmol/kg, i.p.) treatment for 2 weeks also showed protective effects against
cholestatic oxidative stress and liver damage in common bile duct ligated rats [90]. It was
found that the treated cells, in bile duct ligated rats, exhibit a reduction in the level of
malondialdehyde (MDA) and an increase in superoxide dismutase (SOD) and glutathione
peroxidase (GPx) enzymes in liver tissues. Based on these data, the hepatoprotective effect
of CAPE was attributed to its antioxidative potential. Treatment with CAPE (10 mg/kg
body mass; 10-days, i.p.) to Wistar albino rats revealed protective effects on the aflatoxin
B1-induced hepatoxicity by modulating free radical production. In addition, it also caused
a reduction in total oxidant capacity (TOC) and glutathione S-transferase activities [91].
CAPE-treated aged rats also showed better ultrastructure of liver tissues and an increase in
tissue catalase (CAT) activity, suggesting its potential in delaying age-related hepatocellular
alterations [92].

3.3. Brain Function

The brain is the most complex organ of the human body, both in structural and func-
tional aspects. The neuron, its functional unit, is a specialized cell designed to transmit
information between different parts of the brain and to other organs. It controls the func-
tioning of all organs and all aspects of our cognitive behavior (thoughts, memory, speech,
physical reaction and response). Aging, a natural and indispensable life phenomenon
associated with functional decline and adaptability represents cumulative deteriorative
changes in macromolecules that are the by-products of normal cell and tissue functions.
These are highly impacted by internal and external stress factors. Most stresses evoke
adaptive responses. It has been established that the adaptability to stress declines with
age. Furthermore, exposure to severe or chronic stressors dysregulates adaptive and repair
pathways and results in an accelerated aging phenotype. Degenerative changes in neurons
in response to aging and metabolic, ionic and oxidative stresses result in protein aggrega-
tion, perturbation of energy homeostasis and mutations in nucleic acids [63]. Stress has
been shown to cause apoptotic death of neurons and contributes significantly to neurode-
generative disorders. Often, these changes are connected to old-age-associated pathologies
including loss of memory, amnesia, Alzheimer’s disease (AD) and Parkinson’s disease
(PD) [63].
A large variety of studies using different experimental models have shown that natural
products play a vital role in the protection of neuronal cells from neuroinflammation and
oxidative stress, associated with normal and chronic age-related diseases. A diverse array
of bioactive nutrients, present in natural products, have been predicted to play a significant
role in the prevention and cure of various neurodegenerative disorders. Interestingly,
polyphenols and flavonoids present in plants and other natural sources are becoming
popular as health supplements. These compounds can interact with reactive oxygen and
nitrogen species and terminate deteriorative chain reactions involved in oxidative and in-
flammation signaling involved in chronic diseases. For example, epigallocatechin-3-gallate
(EGCG) polyphenol has been shown to provide a neuroprotective effect and the ability to
resist amyloidosis. It showed a protective effect on damaged nerves, inhibited neuronal
apoptosis and inhibited oxidative stress injury after middle cerebral artery occlusion by
modulating PI3K/AKT/eNOS pathway [93]. Several preclinical and clinical studies have
also revealed the importance of natural compounds such as resveratrol (polyphenol) and
curcumin in stress protection against oxidative stress-induced neuronal damage, lipid
peroxidation and behavior changes [94,95].
Nutrients 2021, 13, 2528 10 of 23

Propolis has also been reported to possess neuroprotective properties in vitro and in vivo
through its antioxidant, anti-inflammatory and immunomodulatory effects [65,66,73,74,96].
Brazilian green propolis has been shown to protect SH-SY5Y neuroblastoma cells from
neurodegenerative damage by upregulating brain-derived neurotrophic factor (BDNF) and
activity-regulated cytoskeleton-associated protein (ARC), proteins involved in memory [97].
In this study, the pretreatment of SH-SY5Y cells with propolis extract significantly attenu-
ated hydrogen peroxide induced toxicity and reduced ROS and 8-oxo-20 -deoxyguanosine
levels (markers of oxidative damage in mitochondria and DNA, respectively). Brazilian
yellow propolis, rich in triterpenoids, has been shown to elicit anxiolytic and antidepressant
effects at low doses (~1 mg/kg). Ethanol extract of yellow propolis at 30 mg/kg was shown
to improve cognitive function in rats without any sedative effects [98]. Artepillin C, a
major component of Brazilian propolis, was also found to induce neurite outgrowth in
rat PC12m3 cells in which nerve growth factor (NGF)-induced outgrowth is impaired. By
molecular analyses, it was concluded that artepillin C induced activation of p38MAPK
through the ERK signaling pathway, responsible for the neurite outgrowth [99]. These
findings suggested the neuroprotective role of propolis and its therapeutic potential for the
prevention of age-related cognitive impairment and brain pathologies. Propolis phenols
are reported to possess selective permeability across the blood–brain barrier (BBB), and
their systematic elimination limits the therapeutic efficacy with regard to optimal brain
function [100]. On the other hand, several studies have suggested that CAPE and some
other propolis derivatives are capable of crossing the BBB in rat models [101]. CAPE was
shown to provide neuroprotection by inhibiting NF-kB function and reducing the release
of proinflammatory miRNAs (miRNA-125b, miRNA-146a and miRNA-155) from primary
human neuronal glial cells stressed with Alzheimer’s disease (AD) derived extracellular
fluid [102]. This finding suggested that CAPE acts as an anti-NF-kB agent and could be
a potential natural therapeutic drug candidate for the treatment of AD. In another study,
CAPE was examined for its effects on oxidative tissue damage in experimental autoimmune
encephalomyelitis (EAE), (a condition when the body’s immune system mistakenly attacks
healthy brain cells) in rats [103]. Treatment with CAPE significantly inhibited reactive
oxygen species (ROS) production induced by EAE and later ameliorated clinical symptoms
in rats, possibly through its anti-inflammatory effect by suppression of NF-kB at the tran-
scriptional level and catalytic activity of inducible nitric oxide synthase. One of the propolis
flavonoids, pinocembrin, has also been shown to protect SH-SY5Y neuronal cells from
glutamate stress by inhibition of cytochrome C and apoptotic proteins such as BAX and
p53 [104]. Pinocembrin was also found to cross the BBB and alleviate BBB injury in a global
cerebral ischemia/reperfusion model in rats [105]. These studies have supported the use of
propolis for brain health, especially to protect against age- and stress-related anomalies.

3.4. Cardiac Function

The heart, an essential organ for pumping blood to the rest of the body, is controlled
by a high degree of coordination of electrical signals that start in the right atrium. Aging
is associated with a critical reduction in the control of such electrical signals and is often
associated with progressive decline in numerous physiological processes, increased risk
of heart attack and other organ pathologies. It has been well established that the free
radicals produced during normal metabolic processes damage subcellular structures and
macromolecules, including DNA, proteins, lipids and carbohydrates, and contribute to age-
related tissue dysfunctions. Aging and oxidative stress have remarkable effects on the heart,
leading to an increase in various cardiovascular diseases (CVDs), namely cardiomyopathy,
hypertrophy, atherosclerosis, hypertension, myocardial infarction and stroke. Free radical
species such as singlet oxygen, hydrogen peroxide and hydroxyl radical produced by
the partial reduction of oxygen are highly unstable, reactive and toxic for all tissues and
can severely perturb heart functioning. Studies on isolated perfused hearts revealed that
even brief exposures to oxygen radicals lead to a decrease in high-energy phosphates and
a loss of contractile function and cause structural abnormalities [106]. Several studies
Nutrients 2021, 13, 2528 11 of 23

have documented oxidative stress as a root cause of insulin resistance, β-cell dysfunction,
impaired glucose tolerance and cardiotoxicity.
Natural products offer unique structural and chemical diversity and have been shown
to alleviate oxidative stress-induced diseases, including cardiovascular diseases (CVDs),
by regulating the NRF2/antioxidant-responsive element (ARE) pathway [107]. Typically,
natural products consist of several constituents that perform their actions independently
or through synergistic complementarity, making them multitarget drugs. Epidemiological
studies have demonstrated that nutritional habits such as the consumption of food rich
in natural bioactive substances have been associated with a longer life expectancy and
a significant decrease in the incidence and prevalence of CVD [108]. In a recent report,
phenolic compounds derived from natural products olive oil (hydroxytyrosol) and red
wine (resveratrol) have been reported to enhance nuclear translocation of Nrf2 (a protein
involved in oxidative stress management) and reduce the expression of miRNA-146a (a
noncoding regulator of inflammation response). Kubota et al. [109] examined the changes
in cardiovascular parameters of hypertensive rats following a 4-week diet of Brazilian
propolis extracts and found a significant reduction in systolic blood pressure, suggesting
their antihypertensive effect. Molecular characterization revealed that tri-caffeoylquinic
acids (CQAs: 3,4-diCQA, 3,5-diCQA and 3,4,5-triCQA), characteristic components of
ethanol extract, at a dose of 10 mg/kg showed good antihypertensive activity and hence
were recommended for the prevention and treatment of hypertension. In a study on mice
on a cholesterol-rich diet, it was revealed that all types of propolis (red, green or brown)
can successfully diminish cholesterol and elevated high-density-lipoprotein cholesterol
concentrations and sclerotic lesions [110]. When low-density lipoprotein (LDL) receptor
knockout mice (LDLr-/-) were treated with Brazilian red propolis (250 mg/kg/day), they
showed decreases in the levels of triacylglycerol (TAG), total cholesterol and non-high-
density-lipoprotein cholesterol [110]. An ethanolic extract of Brazilian red propolis has
been reported to enhance ATP-binding cassette transporter gene-1 (ABCA1), a regulator
of cellular cholesterol promoter activity in THP1 macrophages [111]. In addition, in vivo
data in C57BL/6 mice treated with 50 mg/kg extract once a day for 4 weeks supported the
in vitro findings and showed an increase in plasma HDL, but not LDL, cholesterol. Taken
together, in vitro and in vivo studies have suggested propolis as a potential candidate for
the prevention of atherosclerosis [112]. Administration of propolis in diabetic rats led to a
decrease in the levels of blood glucose (FBG), fructosamine (FRU), malonaldehyde (MDA),
nitric oxide (NO), nitric oxide synthetase (NOS), total cholesterol (TC), triglyceride (TG),
low-density-lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol
(VLDL-C) in serum of fasting rats. The data suggested that propolis could control and
modulate blood glucose, metabolism and blood lipids, leading to a decrease in output
of lipid peroxidation, and scavenge the free radicals in rats with diabetes mellitus [113].
Doxorubicin-induced myocardiopathy is a consequence of free-radical-induced oxidative
stress. The effect of intraperitoneal administration of propolis (50 and 100 mg/kg) was
studied on cardiomyopathy induced by doxorubicin (10 mg/kg, i.v.) in rats. Thiobarbituric
acid reactive substances (TBARS), serum creatine phosphokinase (CK), aspartate amino-
transferase (AST) and blood and tissue glutathione (GSH) in the heart are estimated to
assess the status of heart muscle. It was observed that the doxorubicin treatment elevated
the levels of CK, AST, GSH and TBARS as a result of cardiotoxicity; however, pretreatment
with propolis significantly reduced the levels of these parameters [114].
Flavonoids present in propolis are iron chelators and can therefore decrease the iron-
dependent production of free radicals. Thus, chelation raises the level of scavenging activity
of flavonoids. Pinocembrin, a flavonoid from propolis, is shown to affect cardiovascular
diseases based on its ability to regulate apolipoprotein E (ApoE; a protein involved in the
metabolism of fats) and reduce rho kinase (regulates shape and movement of cells) [115]. It
was demonstrated that the combination treatment of simvastatin (a lipid-lowering medica-
tion) with pinocembrin for 14 weeks significantly reduced serum lipid levels, improved
endothelial function and reduced atherosclerosis symptoms in ApoE-/- mice [116]. Platelet
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aggregation has been shown to contribute to atherosclerosis and atherothrombosis, com-

plex multifactorial chronic inflammatory diseases initiated by the accumulation of fat and
other macromolecules in the arterial walls. CAPE (15 and 25 µM) significantly inhibited
collagen-stimulated platelet aggregation [117]. In addition, CAPE was also shown to pro-
tect the cardiac tissues against oxidative stress induced by hyperthyroidism in rats [118].
It was reported that CAPE exhibited cardioprotective effects in short-term myocardial
ischemia in rats through the reduction in xanthine oxidase (XO) and adenosine deaminase
(ADA) activities and antioxidant effects.

3.5. Antioxidant Activity

Living cells require oxygen to produce energy. It is a substrate for oxidative phospho-
rylation and ATP generation via the electron transport chain in mitochondria. Reactive
oxygen species (ROS) are the indispensable product of this life-sustaining process. These
are highly reactive molecular entities that cause structural and functional deteriorative
changes and are regulated by the enzymatic and nonenzymatic antioxidant cellular de-
fense mechanisms. The primary antioxidant enzymes are superoxide dismutase (SOD),
catalase (CAT) and glutathione peroxidase (GPx). Age-associated decline in antioxidative
enzymes has been shown to yield accumulation of oxidative damage as measured by
the increase in the level of reactive oxygen species (ROS) and reactive nitrogen species
(RNS). Mitochondria are the main organelles responsible for the production of ROS during
physiological and pathological states [119]. Polyphenols, lignans, stilbenes, flavonoids,
phenolic acids and derivatives of cinnamic (e.g., ferulic, coumaric and caffeic) and benzoic
acids (e.g., gallic acids and hydroxybenzoic acids) have been shown to possess physio-
logically relevant antioxidant properties. The antioxidant capacity of propolis has also
been related to some of its physiological effects, including chemoprevention. Cuban red
propolis showed a protective effect against allyl alcohol induced liver injury in a mouse
model [87]. The flavonoids in propolis are powerful antioxidants, capable of scavenging
free radicals, and protect cells from a variety of macromolecular damage. In 2005, de Lima
et al. showed that the aqueous extract (administered orally at 0.01%, 0.03%, 0.1% and
0.3% in the drinking water; approximately equivalent to 12, 34, 108 and 336 mg/kg body
weight/day dose) of Brazilian propolis was able to protect against 1,2-dimethylhydrazine-
induced DNA damage in the rat colon [120]. The antioxidant activity of propolis has also
been connected to its protective effect against heavy metal toxicity [121], cardiovascular
anomalies, and brain pathologies, as described in the above sections. An in vivo study
conducted on 47 healthy men and women to evaluate the effects of propolis supplemen-
tation on antioxidative status and red blood cells revealed that as compared to women,
men were more benefited by intake of propolis supplements and showed a significant
reduction in lipid peroxidation [76]. Turkish propolis has been shown to inhibit H2 O2 -
induced DNA damage in culture fibroblasts [122]. Antioxidant properties in propolis
have been assigned to several of its components, including CAPE, quercetin, kaempferol,
pinocembrin, flavones and flavonols [123,124]. Interestingly, an aqueous extract of propolis
was reported to be more effective than the ethanol extracts due to the higher polyphenol
content. The effect of pinocembrin on amyloid-beta (Aβ) accumulation, a key factor in
Alzheimer’s disease pathology, was examined. APP/PS1 transgenic mice treated with
pinocembrin for 3 months showed protection against the cognition decline without altering
Aβ burden and oxidative stress. Instead, a significant level of neurovascular protection
(maintenance of neuropil ultrastructure, preservation of microvascular function, reduction
in glial activation and levels of inflammatory mediators) was observed [125]. CAPE was
also shown to provide stress protection against oxidative stress induced by H2 O2 and
scopolamine in neuronal cell culture and animal models [126,127]. A study examined
the effect of CAPE on methotrexate-induced testicular toxicity in rats. Whereas the level
of lipid peroxidation was higher in the methotrexate group than in the control group,
the CAPE-treated group showed decrease [128]. CAPE was found to stabilize hypoxia-
inducible factor-1 (HIF-1α), a major transcriptional regulator of hypoxia (a condition of
oxygen deprivation), by inhibiting factor inhibiting hypoxia (FIH), which inhibits HIF-1α
Nutrients 2021, 13, 2528 13 of 23

by hydroxylation at ASN803 [129,130]. These studies have suggested the strong potential
of propolis in modulation of oxidative stresses and protection against a variety of oxidative
stress-related diseases.

3.6. Anticancer Activity

Cancer, a disease of uncontrolled cell proliferation, is a highly complex and hetero-
geneous disorder. In sharp contrast to normal cells, cancer cells escape the replicative
senescence process and undergo infinite division and malignant transformation resulting
in tumors (masses of cells) at any site of the body, and they often migrate from the primary
site to secondary distant sites in the process of metastasis. Recently, it has been shown
that aging and cancer are highly correlated, and that the incidence of cancer increases with
age. Often, the innate tumor suppressor mechanisms that regulate cellular senescence
are either lost or functionally inactivated in cancer cells. Hence, aging is considered as a
pro-tumorigenic state that contributes to cancer development by physiological alterations
in response to stress, DNA damage and inflammation [64]. Although significant and
rapid developments have been witnessed in cancer diagnostics and treatment in the past
decade, the highly invasive cancers still have high mortality and treatment failure and
hence warrant new treatment modalities. Furthermore, the current chemotherapeutic drugs
are highly expensive (costing approximately USD 100,000 per person); cause toxicity and
adverse side-effects, such as hormonal imbalance, hair loss, low immune response, fatigue
and nausea; and are often limited by drug resistance [131,132]. In this context, natural
compounds with anticancer activity have gained a focus of research and drug development.
Indeed, over 60% of the current anticancer drugs were derived in one way or other from
natural sources [133]. Taxol, vinblastine and camptothecin have been derived from plants
and comprise a large portion of current-day pharmaceutical agents, most notably in the
area of chemotherapy. In addition to compounds from terrestrial plants, compounds from
marine sources (e.g., ecteinascidin 743, halichondrin B and dolastatins), microbes (e.g.,
bleomycin, doxorubicin and staurosporin) and slime molds (e.g., epothilone B) have been
shown to possess remarkable cancer chemotherapeutic potential [134]. Natural products
have been shown to combat cancer by a variety of mechanisms. In several cases, they do
not kill cancer cells directly, but boost the immune system to eliminate them [135]. Based
on these aspects, mechanisms of anticancer activities in natural compounds have been
gaining popularity in research. Many plant-derived polyphenols such as resveratrol, green
tea polyphenols, piperine, flavopiridol, curcumin, genistein and caffeic acid phenethyl
ester have been investigated for their cancer-preventive and chemotherapeutic proper-
ties [136,137]. Several studies have summarized the anticancer potential of natural bee
products or their active components as an alternative treatment of human tumors [138].
Using a variety of human cancer-derived cell lines, several studies have reported anti-
cancer activity in varieties of propolis from different geographical regions that vary in
their bioactive constitution [139–145]. Furthermore, multimodal mechanisms of action
for different propolis types and their components have been reported. These include
immunomodulation and antitumor resistance [70,146,147], activation of apoptotic signal-
ing [80,83,143,148–150], inhibition of cancer cell migration and invasion [143,151–153] and
drug resistance [154,155].
The major components of propolis such as CAPE (New Zealand) and ARC (Brazil-
ian) have been shown to cause cell-cycle arrest, inhibition of matrix metalloproteinases,
cell migration, metastasis and angiogenesis. Galangin, cardanol, nemorosone, chrysin
and other compounds of propolis are reported to block the rapid division of tumor
cells. Several studies have reported the selective toxicity of CAPE and its derivative
compounds on cancer/transformed cells [138,143,152,156,157]. CAPE was shown to in-
hibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumors and therefore proposed
as an anticancer drug for the treatment of gastric carcinoma, hepatoma and colorectal
carcinoma [138,158]. The studies on a wide range of human cancer cell lines have re-
ported that CAPE causes cytotoxicity to most with IC50 of 10–20 µM; some cell lines
Nutrients 2021, 13, 2528 14 of 23

showed high sensitivity (IC50 1–5 µM), whereas some others showed low sensitivity
(IC50 70–100 µM) [143,150–152].
Mortalin/GRP75, a mitochondrial chaperone, is enriched in a large variety of can-
cer cells. It has been shown to contribute to carcinogenesis in multiple ways, including
inactivation of the function of tumor suppressor p53 protein, deregulation of apoptosis
and promotion of cell migration and epithelial-to-mesenchymal transition (EMT) progres-
sion [150,159–163]. In several studies, the interaction of mortalin and p53 in cancer was
found as a new therapeutic target for treatment. Several small molecules that downregulate
mortalin or target mortalin–p53 interaction resulting in reactivation of tumor-suppressor
p53 protein have been shown to possess anticancer potential [150,164–168]. On these lines,
CAPE-treated cells showed growth inhibition/apoptosis mediated by downregulation of
mortalin at transcriptional and translational levels and activation of p53 function, which
was supported by both computational and experimental approaches [143,150]. Since CAPE
was found to be unstable and degraded by secreted esterases, a CAPE–γ-cyclodextrin
(γCD; produced from starch by the process of enzymatic conversion and well known for its
use in pharmaceutical formulation of oral products to increase the bioavailability of poorly
water-soluble or unstable drugs) complex was prepared [143,152]. In vitro and in vivo
experiments demonstrated an enhanced anticancer activity of the CAPE–γ-cyclodextrin
complex [143,152]. Various stress conditions such as high temperature and metal stress
are known to cause protein misfolding and aggregation by shifting the conformation equi-
librium more towards the aggregation state due to protein–protein interaction through
their hydrophobic regions [169,170]. We investigated the antistress potential of nontoxic
doses of CAPE by recruiting arsenite and high-temperature-induced protein aggregation
in cells exogenously transfected with either green fluorescence protein (GFP) or luciferase
proteins, respectively [130]. The system allowed direct observations of protein aggregation.
Interestingly, we found that whereas control cells possessed expected diffused cytoplasmic
fluorescence of GFP protein, arsenite-treated cells showed its strong aggregation. Protein
deaggregation was seen in the cells recovered in CAPE-supplemented medium. In a similar
assay, heat-induced aggregation of luciferase was used as a model. Whereas heat-induced
aggregation of luciferase led to a decrease in quantitative reporter assays, cells treated
with CAPE showed recovery (Figure 1). Furthermore, low nontoxic doses of CAPE that
showed antistress and protein deaggregation activities also showed prohypoxia effect [130],
suggesting its use in the treatment of metal and hypoxia stress-driven pathologies.

Figure 1. Schematic diagram showing the effect of low and high doses of propolis extract (CAPE and
artepillin C, in particular) on cell proliferation, stress-induced protein aggregation and protein misfolding.

Brazilian propolis has been shown to activate the immune system and possess direct
antitumor activity [171]. Artepillin C (ARC) was shown to exhibit a cytotoxic effect and
inhibit the growth of human and murine malignant tumor cells in vitro and in vivo. It was
also found to cause significant damage to solid tumors and leukemic cells [83,101]. Apop-
tosis, abortive mitosis and massive necrosis were identified by histological observation
Nutrients 2021, 13, 2528 15 of 23

after intratumor injection of 500 mg/kg BW of ARC three times a week. Cytotoxic effects
of ARC were most noticeable in carcinoma and malignant melanoma in human tumor
cells xenografts grafted and transplanted into nude mice [83,139,152]. Similar to CAPE,
ARC has been shown to activate p53 tumor-suppressor protein by abrogating its complex
with mortalin (p53-inactivating protein) possessing the anticancer activity [130] (Figure 1).
However, as compared to CAPE, ARC showed low efficacy (IC50 275 µM), and hence a
unique superficial extract of green propolis (GPSE) containing a multitude of bioactive
components was generated. As expected, GPSE exhibited higher cytotoxicity in in vitro
cytotoxicity assays. Furthermore, its conjugate with γCD showed better activity both in
in vitro and in vivo assays. The effects of low nontoxic doses of ARC and GPSE have not
been reported as yet and warrant studies with suitable cell culture and animal models.
Table 1 summarizes various bioactive components and bioactivities in different kinds of
propolis discussed in the above sections.

Table 1. Types of propolis and their active components, bioactivities and mechanisms.

Propolis (Types) Active Components Bioactivities Mechanisms Refs.

• Immunosuppression and
inhibitor of early and late
events in T-cell
receptor-mediated T-cell
Anti- activation
• NF-kB blocking [65–74]
inflammatory
• Inhibition of NADPH oxidase
and tyrosine-protein kinase
• Suppression of lipoxygenase
pathway of arachidonic acid
metabolism
• Free radical scavenging
activity
1. Caffeic acid
• HIF-1α stabilization and
2. Caffeic acid phenethyl ester
stress protection
3. Quercetin
Antioxidant • Reduction in [87,120–127]
New Zealand 4. Naringenin
hyperthermia-induced
5. Flavanones
survival inhibition, necrosis
6. Flavones
• Production and maintenance
7. Phenolic acids
of superoxide dismutase
activity

Antitumor • Inhibition of TPA, [138–145,156,157]

mortalin–p53 interaction
• Inhibition of vascular
Antimetastatic endothelial growth factor [151–153]
(VEGF) production

Hepatoprotective • Upregulation of SOD, GPx [88–92]

enzymes
Antibacterial • Inhibition of bacterial motility [33–40]
Antiviral • Inhibition of viral replication [52,55,60,61]

Anti- • Inhibition of NF-k B by

1. 3,5-diprenyl-4-hydroxycinnamic acid, proinflammatory cytokines [65–74]
inflammatory
3-prenyl-4-dihydrocinnamoloxycinnamic acid suppression
2. 2,2-dimethyl-6-carboxyethenyl-2H-1-
benzopyran Antioxidant • Free radical scavenging [120,125]
3. Triterpenoids activity
4. Moronic acid • Activation of immune system
5. Anwuweizonic acid Antitumor • Activation of p53 [139,143]
6. Betulonic acid • Inhibition of migration
7. Artepillin C
Brazilian 8. PM-3 (3-(2-dimethyl-8-(3-methyl-2- Antiviral • Anti-HIV activity in H9 [49,53]
butenyl)benzopyran)-6-propenoic lymphocytes
acid) Hepatoprotective • Upregulation of SOD, GPx [66,88]
9. Baccharin enzymes
10. Drupanin
11. Lignans • Antimicrobial activity against
12. 3,4-dicaffeoylquinic acid Bacillus cereus, Enterobacter
13. Prenylated p-coumaric acid Antimicrobial erogenous and Arthroderma [33–40]
14. Isoflavones benhamiae
15. Terpenes • Bacteriostatic, bactericidal
activity
Nutrients 2021, 13, 2528 16 of 23

Table 1. Cont.

Propolis (Types) Active Components Bioactivities Mechanisms Refs.

Antioxidant • Free radical scavenging [141]

activity
Taiwanese 1. Prenylated flavanones • Induction of apoptosis in
2. Propolin A, B and C human melanoma cells
Antitumor [141]
• Inhibition of xanthine oxidase
activity
• Antiproliferative activity
1. Flavones Antiproliferative towards colon 26-L5 [141]
Chinese 2. Flavanones effect carcinoma cells
3. Flavans
• Cytotoxicity in cervical cancer [78,141]
Radical
Canadian 1. Chalcones scavenging
• Radical scavenging activity
[5]
against DPPH
activity
• Antimicrobial activity against
1. Flavanones Gram-positive and
Greek Antimicrobial [3]
2. Terpenes Gram-negative bacteria,
pathogenic fungi
1. Isoflavonoids
Cuban 2. Prenylated benzophenones Antioxidant • Free radical scavenging [87]
3. Terpenes activity

4. Conclusions
Propolis has been used in folk medicines for centuries. Due to various therapeutic and
health-beneficial activities, it is among the top few natural products that have been used,
maintained, utilized and popularized over a long period of time. Flavonoids, polyphenolic
compounds, flavones, flavanones, phenolic acids and their esters are the pharmacologically
active molecules of propolis. These active molecules have shown broad arrays of biological
effects, such as antibacterial, antioxidant, anti-inflammatory, antifungal, anticancer and
antihypertensive activities. Propolis has been extensively investigated for its various biolog-
ical and health-beneficial properties, and it is attracting further interest among researchers
aiming to further understand the activities and mechanism(s) of action of propolis alone
and in complex with other compounds. There has been an increase in consumer interest
in propolis-containing products that has led to the development of newer products in the
health supplement and cosmetic industries. The traditional knowledge of the utilization of
propolis by the diverse indigenous populace offers substantial support to its health and
therapeutic merits. However, precise and targeted drug development is yet to take place.
The foremost requirement for drug development involves preclinical investigations using
defined resources of propolis from diverse phytogeographical locations and its constituents
in defined and established extraction protocols. With these biotechnological and pharmaco-
logical advances, propolis, its defined extracts and its active components have the potential
to serve as useful resources for natural, economical and easily available therapeutic drugs
for stress, age-related pathologies and cancer.

Author Contributions: Conceptualization, S.C.K. and R.W.; writing—original draft preparation, P.B.
and D.M.; writing—review and editing, P.B., D.M., A.K., Y.I., K.T., R.W. and S.C.K. All authors have
read and agreed to the published version of the manuscript.
Funding: This study was partly supported by the funds granted by AIST (Japan) and Department of
Biotechnology (DBT), Government of India: Grant No. BT/BI/14/042/2017.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare that they have no competing interests.
Nutrients 2021, 13, 2528 17 of 23

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