Mode of action of bioactive phytochemicals, plant secondary

The
Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs (A. Méndez-Vilas, Ed.)
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Mode of action of bioactive phytochemicals, plant secondary metabolites,
possessing antimicrobial properties
Heejeong Lee and Dong Gun Lee*
School of Life Sciences, College of Natural Sciences, Kyungpook National University, Daehak-ro 80, Buk-gu, Daegu 702701, Republic of Korea
* Corresponding author: email: dglee222@knu.ac.kr; Tel: 82-53-950-5373; Fax: 82-53-955-5522
Emergence of microorganism having drug resistance is a major global problem and it has heightened the need for new
therapeutics or alternation of drugs. Natural products from various plants have a potential control microbial growth in
diverse situations and in the specific case of disease treatment and numerous studies have aimed to describe the chemical
composition of these plant antimicrobials and the mechanisms involved in microbial growth inhibition. Phytochemicals,
non-proteinaceous secondary metabolites, are non-nutritive bioactive compounds as protective agents against external and
pathogenic stress.
Phytochemicals induce membrane permeabilization by forming pores or disrupting membrane integrity and induced lysis
of cell wall. Currently, reactive oxygen species was generated when disrupted balance with antioxidant by phytochemicals.
With this phenotype, the mitochondrial dysfunction was examined such as mitochondrial membrane depolarization,
cytochrome c release. These phenomenona are one of the programmed cell death. Antimicrobial activity and their
mechanism of action are described as the current knowledge on phytochemical sources.
Keywords: Phytochemicals; Mechanism of action; Membrane disruption; Apoptosis
1. Introduction
Plants deploy a wide array of secondary metabolites as specialized metabolites or natural products that facilitate
interactions with the environment [1]. Each plant species may contain hundreds of different secondary metabolites [2].
Secondary metabolites apparently the essential role as defense against herbivores, microbes, viruses or competing plants
and signal compounds to attract pollinating or seed dispersing animals. Therefore its adaptive characters that have been
subjected to natural selection during evolution are represented [2]. This largely untapped resource is considerable as the
strategy of the discovery of new therapeutic agents [3]. Plant extracts found in folk medicine, essential oils or isolated
compounds was determined by alkaloids, flavonoids, tannins and others [4]. Alkaloids are the largest group of plant
secondary metabolites comprising basically of nitrogen bases synthesized from amino acid building blocks with various
radicals replacing one or more of the hydrogen atoms in the peptidic ring, most contain oxygen [4]. Flavonoids are
phenolic substances containing one carbonyl group and have been reported to possess antiviral activity against a wide
range of viruses [5, 6]. Tannin is a group of polymeric phenolic substances and may be formed by condensations of
flavan derivatives which have been transported to woody tissues of plants [4].
Some phytochemicals have been reported to exert antimicrobial activity against yeast via different mechanism (Table
1). Generally, phytochemicals have diverse antimicrobial mechanisms including damaging cell wall, cytoplasmic
membrane and so on (Fig. 1) [4]. Lately, the cells undergoing phytochemical-induced cell death appear particular
phenomena including reactive oxygen species (ROS) accumulation, phosphatidylserine (PS) externalization, DNA
fragmentation, nuclear condensation mitochondrial membrane depolarization, metacaspase activation and cytochrome c
release (Fig. 1) [7, 8]. Exploiting the potential of phytochemical should facilitate the development of better
antimicrobial strategies which could efficiently control the human infectious diseases.
2. Disturbance of integrity of cell wall
The fungal cell wall is a dynamic structure that protects fungal protoplasts from external osmotic shocks and defines
fungal morphogenesis. Thus, changes in the organization or functional disruption of the cell wall induced by antifungal
agents are involved in fungal death [9, 10]. The sorbitol protection assay was explored the mode of on the integrity of
the fungal cell wall [9]. Drugs that act on the cell wall cause lysis of fungal cells in the absence of an osmotic stabilizer
(sorbitol), but their growth can continue in the presence of sorbitol. This assay is of use in the search of compounds that
directly inhibit the synthesis of cell wall constituents such as glycans, mannans, chitin or the regulatory mechanisms as
found in these studies of the effects of eugenol on cell walls. This would suggest that inhibiting fungal cell wall
synthesis or assembly is not altered when the chemical structure of eugenol is maintained [9]. Eugenol, the main
volatile compound of the buds and leaves of clove, is a phenyl prepanoid component of aromatic plants, the biological
properties of which are related to the presence of phenolic group. Its pharmacological activities have broad range of
spectrum such as anti-inflammatory, antioxidant, antimicrobial properties [9]. Ultrastructural changes of Candida
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albicans cells exposed to polyphenols showed features of cellular degeneration. Many blastospores exposed to both
catechins and flavins demonstrated extravasations of cellular contents and shriveled and deflated cells. Some yeast cells
showed ‘mulberry-like’ surface features that are likely to be a transitional phase between normality and total collapse of
the cell wall due to the effect of the polyphenols [10]. Inhibition of integrity of cell wall induced cell lysis because of
osmosis.
3. Cytoplasmic membrane damage
Phytochemicals are generally known to cause membrane disturbance resulting in the loss of membrane integrity [2].
The fungal cell membrane is a dynamic structure composed of a lipid bilayer where enzymes and transport proteins are
embedded [10]. Ergosterol, lipid steroid of fungal cell membrane, serves as an important regulator of membrane fluidity
[11]. Sterols inserted between phospholipids moderate its fluidity, the reduction in the content of ergosterol interferes
with the integrity and functionality of the cell membrane [9]. Eugenol is related to its lipophilic character in that they
increase the fluidity and permeability of the cell membrane of microorganisms [9]. In fact, these compounds interfere
with ion transport, unbalancing osmotic conditions in the membrane and making its associated [9]. Eugenol interferes
with the integrity of the cell membrane producing damage to the structure. Based on this, it became imperative to
determine which factors present in the membrane are involved in this activity [9].
The phytochemicals could be interacted with cell membrane. Liposome become a useful model for imitating
biological membranes and morphological changes in the artificial membrane model allow the study of structural and
physical changes occurring during interactions with antimicrobial agents [12]. Isoquercitrin, a dietary flavonoid, has
been shown that isoquercitrin has a wide range of therapeutic properties, including anti-inflammatory, antioxidant antiallergic activities and antifungal activity. Extent of membrane damage induced by isoquercitrin was investigated by
measuring the release of FITC-labeled dextran (FD) of various sizes (FD 4, FD 10 and FD 20) from liposomes
composed of phosphatidylcholine (PC): phosphatidylethanolamine (PE): phosphatidylinositol (PI): ergosterol [5:4:1:2
(w/w/w/w)], mimicking the membranes of C. albicans [5, 12]. After interacting to isoquercitrin, liposomes disturb the
structure, eventually leading to cell death [5].
4. ROS accumulation
Yeast cells generate ROS from intracellular metabolism of oxygen under normal physiological conditions, but cellular
damages induced by ROS could be prevented by repair molecular damages or degrade oxidized molecules and
antioxidant defense that neutralize the ROS. However, when yeast cells are exposed to specific stress conditions, the
levels of ROS exceed the antioxidant capacity of the cells [13]. The excess production of ROS, such as superoxide
anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH·), is observed in almost every apoptotic scenario and
plays an important role as early signal mediators of apoptosis [14-16]. Some phytochemicals induced generation of
ROS [17-22] and the ROS accumulation by the phytochemicals could be measured using DCFDA or DCFH-DA, which
is an oxidant sensitive fluorescent probe [17, 18]. Berberine, which is widely used for dyeing wool, is shown to elicit
anticancerous, antidiabetic, antibacterial, anti-atherosclerotic anti-inflammatory and neuroprotective properties [18].
Using DCFDA, a significant increase in intracellular ROS was observed in Candia albicans cells treated with berberine
(Fig. 2A) [18]. This observation shows the activity of berberine for generated ROS.
An accumulation of ROS appears to be a central phenomenon in most instances of programmed cell death (PCD)
[23]. Among them, O2- mainly generated from the leakage of electrons from the mitochondrial respiratory chain which
is a normal consequence of aerobic respiration. After that O2- is converted to H2O2 via detoxification by superoxide
dismutase [24]. H2O2 then reacts with ferrous ion to generate very reactive OH· via the Fenton and Haber-Weiss
reactions [25]. While O2- and H2O2 are relatively unreactive, the OH· reacts indiscriminately with cellular components
such as unsaturated fatty acids, amino acid residues and DNA [16, 26]. Several apoptosis-inducing phytochemicals have
reported to produce a significant OH· during apoptosis [19, 27-29]. It is important to detect OH· production during
apoptosis. 3'-(p-hydroxylphenyl) fluorescein (HPF) is used for selectively detecting OH. ·Representatively, reactive OH·
production was detected in the C. albicans cells treated with amentoflavone (Fig. 2B) [22]. The OH· were a large part of
the total ROS produced by amentoflavone (Fig. 2B), suggesting that OH· was closely related to ROS [22].
5. Mitochondrial dysfunction
Mitochondria are important for the energetic status of the cell as well as the fatal organelles deciding death or not.
Mitochondrial features in mammals and yeast are present when apoptosis initiates: Apoptosis-inducing factor (AIF) and
cytochrome c release to the cytosol and breakdown of mitochondrial membrane potential [30]. The mitochondrial
activity was addressed by employing MTR-FM probe. MTR-FM probe diffuses passively across yeast cell membrane
and accumulates in active mitochondria, which can be visualized by enhanced fluorescence (Fig. 3A) [18]. Also,
depolarization of the mitochondrial membrane potential which has been considered a characteristic feature in the early
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stage of apoptosis [16] is detected by a 3,30 -dihexyloxacarbocyanine iodide [DiOC6(3)] that fluoresces differently in
healthy and PCD cells [22]. With this dye, loss of mitochondrial transmembrane potential was also detected in the C.
albicans cells treated with amentoflavone (Fig. 3B).
Furthermore, cytochrome c release from mitochondria to cytosol can cause apoptotic signals-induced mitochondrial
dysfunction [16, 31]. Cytochrome c which is located in the mitochondrial membrane is an essential component of the
respiratory chain and a well-known lethal factor involving the activation of caspase-9 in the intrinsic stages of apoptosis
in mammals [31, 32]. During the early phase of apoptosis, mitochondrial ROS production is stimulated and cytochrome
c is then detached from the mitochondrial inner membrane and can be extruded into the soluble cytoplasm through
pores in the outer membrane [32]. As a result of the mitochondrial electron transport system defect, the cytochrome c
could form an apoptosome-like structure and activate caspase subsequently [31, 33].
6. Caspase Activation
Caspases (cysteine-dependent aspartate-specific proteases) are they play a major role in the apoptotic signaling pathway
and typically activated in the early stages of apoptosis [34]. Yeast has caspase the metacaspase Yca1p, one ortholog of
mammalian caspases [31]. Apoptotic death scenarios have been shown to depend on Yca1p. This applies to oxygen
stress, where disruption of YCA1, metacaspase1, results in reduced cell death and decreased formation of apoptotic
markers. [31]. Metacaspase1 activation is thought to be associated with cytochrome c release. The only reported caspase
is metacaspase1 and that is considering involved in phytochemicals-induced cell death. Caspase activity can be assessed
using a detection marker, broad-spectrum caspase inhibitor FITC-VAD-FMK, which enters the cell and binds
specifically to the activated caspases in apoptotic cells [21, 35]. The C. albicans cells treated with amentoflavone
exhibited increased fluorescence intensity at all tested concentrations (Fig. 4). This result demonstrated that
amentoflavone significantly induced intracellular caspase activation [31]. Additionally, to determine the involvement of
metacaspases in curcumin-induced cytotoxicity in C. albicans, Northern-blot analysis that can detect expression of
CaMCA1 was performed (Fig. 5) [17].
7. Phenotypes of early and late apoptosis
PS is predominantly located on the inner leaflet of the plasma membrane and is translocated to the outer leaflet when
apoptosis is induced [9]. The PS exposure serves as a sensitive marker for early stages of yeast apoptosis like
mammalian cells. It can be detected with fluorescein isothiocyanate (FITC) labeled annexin V, which binds to
phosphatidylserine with high affinity in the presence of Ca2+ and then fluoresces [8]. The FITC-annexin V and
propidium iodide double staining method is widely used to discriminate between apoptotic and necrotic cells [32]. In C.
albicans cells treated with berberine, binding of FITC labeled annexin V increased, but the fluorescent intensity of
propidium iodide did not change (Fig. 6). This indicated that berberine led to the translocation of the membrane
phosphatidylserine from the inner leaflet to the outer leaflet of the plasma membrane without damaging the plasma
membrane permeability [18].
The DNA fragmentation and nuclear condensation are considered the representative phenomena in late-stage
apoptotic cells [36]. DNA fragmentation is detected in situ by the TUNEL assay, as 3-OH DNA ends labeled with
FITC-conjugated dUTP via terminal deoxynucleotidyltransferase can be visualized by fluorescence microscopy [21].
Cells exposed to -tomatine were exhibited intensive TUNEL fluorescence that mainly located around the edge of the
cells meaning fragmented DNA (Fig. 7) [21]. -Tomatine which is the major saponin in tomato has been reported to
inhibit the growth of some fungi lacking sterols in their cell membranes, suggesting that -tomatine possesses an
unknown fungicidal action [21
The chromatin condensation can be visualized by fluorescence microscopy after DAPI staining. DAPI, a cellpermeable dye, is commonly utilized to examine nuclear morphologic shapes at late-state apoptosis because of its
strong binding to the minor groove of A-T rich sequences of DNA with very high affinity [37]. DAPI staining of the C.
albicans cells treated with -tomatine showed more concentrated and split fluorescence intensity, indicating nuclear
condensation compared to that of untreated cells (Fig. 7) [21].
8. Biofilm inhibition
Biofilms are highly organised communities of cells, possessing unique developmental characteristics that are in stark
contrast to the characteristics of free-floating planktonic cells [38]. Fungal biofilms cells have resistance to many
antifungal drugs [38. 39]. Biofilm involves three steps: adhesion; biofilm growth and maturation [38, 39]. Carvacrol
demonstrated the strongest antifungal activity against C. albicans biofilms, with a MIC of 0.03%. Furthermore,
carvacrol, one of the terpenoid phenols, was shown to be effective regardless of the maturity of the biofilm [8]. As
shown in figure 8, biofilm development was inhibited and growth was predominantly composed of yeast cells and
pseudohyphae by baicalein [40].
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In conclusion, these studies help us understand an accurate antimicrobial mechanism of phytochemicals and the
apoptosis-inducing phytochemicals could be are worthy of further investigation for clinical applications.
9. Table
Table 1 The classification of phytochemicals by origins.
Structure of
compounds
Categories
Phytochemicals
origins
Mode of actions
References
Flavonoid
Amentoflavone
Selaginella
Induction fungal apoptosis
22
Flavonoid
Isoquercitirin
Starwart
Disruption of membrane
5
Flavonoid
Cathechin
Green tea
Cell wall damage
10
Terpenoid
Carvacrol
Oregano
Calcium stress, Biofilm inhibition
27, 41
9, 41-43
Terpenoid
Eugenol
Clove
Perturbation of cytoplasmic permeases,
Inhibition of ergosterol biosynthesis,
Interference with the integrity of the cell
membrane, Biofilm inhibition
Terpenoid
Caffeic acids
Tarragon
Biofilm inhibition
44
Polyphenol
Curcumin
Culinary
ROS, Inhibition of morphogenetic switch
and biofilm formation , Membrane pore
formation
12, 17, 45
Polyphenol
Resveratrol
Grape
Fungal apoptosis
19
Alkaloid
Berberine
Barberry
DNA binding, inhibition CDR1, Induction
fungal apoptosis
18, 46, 47
Saponin
-Tomatine
Tomato
Disruption of membrane, Induction fungal
apoptosis
22
Carotenoid
Lycopene
Tomato
Membrane damage, Induction fungal
apoptosis
20, 28, 48
CDR1 : Candida drug resistance gene
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10. Figures
Fig. 1 Phytochemicals of mode of action in yeast cells.
Fig. 2 (A) Generation of intracellular ROS was detected after staining with DCFDA. (a) Control cells and cells exposed to berberine
(BER) with ascorbic acid (AA) or not [18]. (B) The formation of hydroxyl radicals in C. albicans upon exposure to amentoflavone,
amphotericin B (AMB), and H2O2 using the dye HPF [22].
Fig. 3 (A) Inactivation of mitochondrial activity. MTR labeling was done in WT, HSF1 conditional mutant and HSF1 heterozygous
strains in presence and absence of BER [18]. (B) Depolarization of the mitochondrial membrane potential induced by treatment with
amentoflavone (a), AMB (b), and H2O2 (c) using DiOC6(3) [22].
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Fig. 4 Caspase activity measurement using FITC-VAD-FMK in situ marker. Untreated control (A) cells were treated with
amentoflavone (B), amentoflavone and thiourea (C) AMB (D) and H2O2 (E) [22].
Fig. 5 Transcript level of CaMCA1 in the absence and presence of curcumin (CUR). ACT1 mRNA levels were used as a loading
control [17].
Fig. 6 Phosphatidylserine externalization was shown by annexin V-FITC staining [18].
Fig. 7 DNA fragmentation by TUNEL assay and nuclear condensation by DAPI staining [21].
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Fig. 8 Confocal laser scanning microscopy showing on C. albicans biofilm. (A) Normal biofilm. (B) Cells were treated with
baicalein [40].
Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea
government (MSIP) (No. 2015R1A5A6001906).
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