Phytochemicals as Inhibitors of Candida Biofilm

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Current Pharmaceutical Design, 2016, 22, 1-24 1
REVIEW ARTICLE
Phytochemicals as Inhibitors of Candida Biofilm
Jayant Shankar Raut1 and Sankunny Mohan Karuppayil2*
1University Institute of Pharmaceutical Sciences (UIPS), UGC-Centre of Advanced Study, Panjab University, Chandigarh-160014,
India; 2School of Life Sciences (DST-FIST & UGC-SAP sponsored), SRTM University, Nanded, 431606, (MH), India
Abstract: Background: Candida biofilm and associated infections is a serious threat to the

large population of immunocompromised patients. Biofilm growth on prosthetic devices or
host tissue shows reduced sensitivity to antifungal agents and persists as a reservoir of in-
fective cells. Options for successful treatment of biofilm associated Candida infections are

restricted because most of the available antifungal drugs fail to eradicate biofilms.
Objective: Various plant actives are known to possess interesting antifungal properties. To
explore and review the potential of phytochemicals as a novel strategy against Candida
biofilms is the intent of present article. Method: Thorough literature search is performed to
A R T I C L E H I S T O R Y
identify Candida biofilm inhibitors of plant origin. An account of efficacy of selected phy-
tochemicals is presented taking into consideration their biofilm inhibitory concentrations.
Received: May 12, 2016 Results: This review discusses biofilm formation by Candida species, their involvement in
Accepted: May 31, 2016 Sankunny M. Karuppayil
human infections, and associated drug resistance. It gives insight into the biofilm inhibitory
potential of various phytochemicals. Based on the available reports including the work done in our laboratory,
DOI: 10.2174/1381612822666160601 several plant extracts, essential oils and phytomolecules have been identified as excellent inhibitors of biofilms
104721
of C. albicans and non-albicans Candida species (NACS). Conclusion: Selected phytochemicals which exhibit
activities at low concentrations without displaying toxicity to host are potential therapeutic agents against
biofilm associated Candida infections. In vivo testing in animal models and clinical trials in humans are required
to be taken up seriously to propose few of the phytochemicals as candidate drug molecules.
Keywords: Antimicrobial, antifungal, biofilm, Candida, drug resistance, medicine, phytochemical, plant molecule.
1. INTRODUCTION mucosal tissues and prostheses put inside the patient’s body, lead-
ing to biofilm formation [9]. Several species of Candida are known
Candida albicans and various non-albicans Candida species
(NACS) are important fungal pathogens of the humans. Over the to form biofilms on various prosthetic devices which often leads to
severe infections [10].
last three decades there has been a considerable rise in opportunistic
Candida infections, which coincides with the advances in medical Most of the available antifungals are either ineffective against
technology, increase in the immunocompromised population, and Candida biofilms or exhibit activity at very high concentrations
increased use of broad spectrum antibiotics, chemotherapy, immu- [11, 12]. Marked side effects due to toxicity restrict the use of high
nosuppression therapy and indwelling prostheses [1, 2]. Particu- dosages of the available antifungal drugs [13]. These limitations
larly, there is a notable increase in the biofilm associated Candida have necessitated a search for novel molecules with anti-biofilm
infections [3]. Biofilm is a community of microorganisms charac- potential. Plants are rich sources of bioactive molecules exhibiting
terized by cells which are irreversibly attached to a surface and various biological and pharmaceutical properties. Various phyto-
embedded in a matrix of extracellular polymeric substances. chemicals are known to possess strong antimicrobial/ antifungal
Biofilm cells (sessile cells) represent an altered phenotype and ex- activities [14]. Use of these phytochemicals against biofilms could
hibit differential gene expression compared to the free living be an excellent strategy [15, 16]. First half of this review discusses
(planktonic) cells [4]. Living as a community provides several eco- formation of Candida biofilms, consequences to the human health,
logical benefits to microorganisms; for example, protection from associated drug resistance and underlying mechanisms. The second
environmental stress, better acquisition of nutrients, metabolic co- part gives an account of various phytochemicals and their potential
operation, and persistence in unfavorable niches [5]. to mitigate Candida biofilms.
In their natural habitats, majority of the microorganisms live as
2. CANDIDIASIS
aggregated communities attached to a surface [4]. Human patho-
gens adapt the strategy of biofilm formation to survive the attack of 2.1. Candida as a Human Pathogen
defensive immune system, resist antimicrobial agents/antibiotics,
Candida species are an integral part of the human microbiome.
and hence readily colonize host tissues or indwelling prostheses [6].
Primary colonization of the human host by these yeasts is through
It has been predicted that in humans 80% of all microbial infections
acquisition of maternal flora in the perinatal period or through later
are biofilm related. The involvement of biofilms in infectious dis-
human contact [17]. Candida cells usually colonize the skin surface
eases, prostheses related infections, associated drug resistance and
or survive as a commensal on the mucosal membranes of oral cav-
options available to mitigate them, is being investigated all over the
ity, oropharyngeal region, urogenital tract and gastrointestinal tract
World [7, 8]. Candida readily adheres on moist surfaces and
of healthy individuals. The commensal association does not cause

any damage to the host unless the immune status or the microbiota
*Address correspondence to this author at the School of Life Sciences of the host is disturbed [18]. Under some predisposing conditions
(DST-FIST & UGC-SAP sponsored), SRTM University, Nanded, 431606, such as debilitated immune system or changes in normal microbiota
(MH), India; Tel: [phone]; E mail: [email protected] upon antibiotic therapy, Candida may turn pathogenic to invade

1381-6128/16 $58.00+.00 © 2016 Bentham Science Publishers
2 Current Pharmaceutical Design, 2016, Vol. 22, No. 00 Raut and Karuppayil
tissues and proliferate to cause infections/disease. Candidiasis, the of filaments. This results in formation of a mesh like network con-
infection caused by Candida, is prevalent among people with com- sisting of yeast, hyphae and pseudo-hyphae. In the last phase (18 to
promised immune status. For example, AIDS paients, cancer pa- 48 h) sessile cells deposit extracellular polymeric matrix (EPM)
tients undergoing chemotherapy and invasive clinical procedures, which is a characteristic feature of mature biofilms. A dense net-
patients under prolonged antibiotic therapy, and people in major work of hyphae, pseudohyphae and yeast cells, embedded in the
trauma cases or under prolonged stay in intensive care units [19]. EPM give a three dimensional community structure. In late matura-
tion phase, controlled dispersion of planktonic cells from the com-
Candida is a major fungal pathogen responsible for consider-
able morbidity and mortality. Worldwide, Candida species is esti- munity takes place. The cells released from biofilms move to dif-
mated to infect > 4, 00,000 people every year, with a high mortality ferent places to initiate a new biofilm cycle [31].
rate of 46 to 75% [20]. It is the fourth most common cause of noso-
3.2. Adhesion and Filamention as Important Events
comial (hospital-acquired) bloodstream infections and ranks third
among catheter related infections [19]. More than 20 species of Adhesion of blastospores to a solid surface is the first crucial
Candida have been found involved in human disease. Candida step in biofilm formation. Initial attachment is reversible and in-
albicans which is most commonly isolated from infections is the volves nonspecific interactions (like van der waals and electrostatic
predominant species. Medically important species other than albi- forces) between cells and a surface [32]. Cell surface hydrophobic-
cans i.e. non-albicans Candida species (NACS) mainly include ity and hydrophobic interactions play important role in this interac-
Candida glabrata, Candida parapsilosis, Candida tropicalis, Can- tion [33, 34]. It is followed by anchoring of cells on the substrate by
dida dubliniensis, Candida krusei, Candida guilliermondii, Can- means of specific cell surface molecules called adhesins [35]. Bind-
dida orthopsilosis, Candida rugosa, Candida lusitaniae and Can- ing through adhesins is irreversible i.e. cells will remain adhered
dida kefyr [21]. unless strong physical/chemical forces are acting.
Ability of cells to bind to abiotic surfaces is crucial in device
2.2. Infections Caused By Candida related infections. Proteins and mannoproteins of Candida cell wall
Infections caused by Candida can be categorized into two play a role in binding to host tissues and abiotic surfaces. The pep-
types, the superficial candidiasis and deep seated systemic mycoses. tide portion, particularly the hydrophobic domain, of cell surface
The superficial infections (also called as thrush), are infections of mannoproteins may be involved in binding to plastic materials [36].
the mucosal surface at vaginal, oropharyngeal, esophageal and gas- The agglutinin like sequence (ALS) gene family is known to code
trointestinal sites. Mucosal candidiasis is common among immuno- for C. albicans adhesins. Out of the eight different proteins encoded
compromised patients and also occurs in healthy individuals. It is by ALS genes, Als3p protein is mainly involved in adhesion to plas-
responsible for considerable morbidity among patients [21]. For tic [37]. Adhesion of C. albicans cells to a surface was also found
example, 50 to 75% of women suffer from at least one episode of to induce activation of drug efflux pumps and play crucial role in
vulvovaginal candidiasis (VVC) and 5 to 8% of them (approxi- biofilm associated drug resistance [38].
mately 75 million) experience at least four episodes annually [22]. Yeast to hyphae morphogenesis and filamentation is another
There are at least 10 million cases of oropharyngeal candidiasis
important event in development and maturation of biofilm. Pres-
(OPC) in HIV/AIDS patients, cancer patients and other immuno- ence of multiple layers of filamentous forms is crucial to provide
compromised patients [19]. Although cured upon appropriate anti- strength and structural integrity to C. albicans biofilm network.
fungal therapy, superficial infections have considerable effects on
Mutants of C. albicans that are unable to form hyphae were ob-
the quality of life. The deep seated invasive infections are difficult served to form only basal layer of biofilm [39]. However, few of
to treat and pose a serious threat to patients [23]. the NACS which do not undergo dimorphic switching form yeast
Candidemia and disseminated candidiasis are the systemic in- only biofilms. Quorum sensing molecules like tyrosol and farnesol,
fections. Cells adhered to a surface invade the mucosal barriers and are supposed to regulate yeast to hyphae morphogenesis and the
gain entry to blood which may result into blood stream infection or biofilm formation [40].
candidemia. In patients with debilitated immune status candidemia
may lead to invasion and subsequent colonization of internal tis- 3.3. Quorum Sensing in Biofilms
sues. Colonization of important organs like lungs, kidney, heart, Candida biofilm is not just an aggregation of cells, but a dy-
liver, spleen and brain, leads to severe septicemia [24]. Dissemi- namic structure developed by cells interacting as a community [35,
nated candidiasis is an extremely serious condition involving 30 to 41]. Various diffusible molecules regulate behavior of individual
50% of mortality rate [19]. cells in a biofilm. A signal transduction process which involves the
production, release and response to signaling molecules secreted by
3. CANDIDA BIOFILMS
the microbial cells themselves, is called quorum sensing. The dif-
fusible molecules involved in quorum sensing are called autoinduc-
3.1. Biofilm Formation
ers or quorum sensing molecules (QSMs) [42]. QSMs accumulate
Candida colonizes mucosal surfaces of oral and nasal cavities, in the medium and may convey the message of physiological
gastrointestinal tract and urogenital tract to develop into a commu- changes to individual cells, in a density dependent manner. Al-
nity [25, 26]. It readily adheres to prostheses implanted in a patient though it is known that C. albicans biofilm is regulated through
and forms biofilm which may lead to device associated infections quorum sensing, the molecular mechanisms behind it are not fully
[10]. Biofilm formation by different Candida species and its conse- understood.
quences to human health are being studied by various researchers
Farnesol, a sesquiterpene molecule continuously produced dur-
[27, 28]. Candida albicans has emerged as a model for fungal
ing growth of C. albicans, acts as a QSM. Its accumulation in the
biofilms studies and contributed to our understanding of various
culture exerts various physiological effects on individual cells and
manifestations associated with biofilm growth [29].
also the community. For example, yeast to hyphae morphogenesis
Formation of C. albicans biofilm advances through three dis- and biofilm growth are prevented in the presence of exogenously
tinct stages such as, early, intermediate and maturation phases [30]. added farnesol [43, 44]. Tyrosol, another QSM in C. albicans, ex-
Early phase extends over 0 to 6 hours and shows adhesion of blas- erts an effect opoosite to that of farnesol. At low cell densities, it
tospores (yeast cells) to a surface (0 to 2 h), followed by formation reduces lag phase in the growth and promotes formation of hyphae
of micro-colonies and dimorphic transition to produce hyphal forms [40]. Few more molecules like nerolidol, isoamyl alcohol, dode-
(3 to 6 h). Intermediate stage (6 to 18 h) is characterized by increase canol, ethanol and acetaldehyde have been detected in Candida
in cell density, presence of multiple layers of cells, and elongation
Phytochemical Inhibitors of Candida biofilm Current Pharmaceutical Design, 2016, Vol. 22, No. 00 3
cultures. These are known to act as morphogenetic signaling bloodstream infections. Few studies have reported that involvement
molecules to inhibit filamentation [45, 46]. of C. albicans biofilm in human infections is significantly less fre-
quent than that of NACS biofilms [56].
3.4. Differential Gene Expression
Candida biofilms on human skin have been linked to the devel-
Northern blot analysis of C. albicans biofilm cells has showed opment of dermatologic conditions or diseases [60, 61], for exam-
that at least 325 genes are differentially expressed by the biofilm in ple, atopic dermatitis (AD) associated with Candida [62]. Usually,
comparison to the planktonic cells. About 214 of the genes, includ- C. albicans is the species found to colonize skin of AD patients
ing those involved in cell cycle, metabolism, DNA processing, [63]. Pathogenic fungal species are known to play a role in wound
protein synthesis, cell signaling and transport, are found to be over infections, particularly Candida biofilms have been associated with
expressed [47]. Genes involved in synthesis of aromatic amino the delayed healing of chronic wounds [64]. Molecular analysis of
acids, sulfur amino acids, ergosterol, sphingolipid and phospholipid microbial communities at the non-healing surgical wounds and
are significantly upregulated, indicating their importance in biofilm venous leg ulcers revealed involvement of Candida spp. [56, 65,
mode of growth. Increase in the expression of genes that control 66].
cell wall synthesis and organization, adhesion and filamentation is
Candida is one of the most frequent fungal colonizers in oral
also observed [47]. ALS gene family and genes for drug efflux
cavity. The species majorly detected in biofilm related oral infec-
pump proteins, CDR1 & CDR2, are found to be over-expressed in
tions are C. albicans, C. parapsilosis, and C. tropicalis [67]. Can-
the biofilm [48].
dida species are low pH tolerant yeasts and easily colonize gastro-
3.5. Biofilm Formation in NACS intestinal tract. Besides bacteria such as Helicobacter and Lactoba-
cillus, C. albicans have also been isolated from the sites of gastric
Various non-albicans Candida species (NACS) are capable of
ulceration. It is now being realized that the Candida biofilms may
biofilm formation, in vitro and in vivo. Biofilms formed by C.
play a decisive role in overall health, especially in patients where
glabrata, C. dubliniensis, C. parapsilosis, C. tropicalis, and C.
normal bacterial biota of GI tract is disturbed [68].
krusei were found to be involved in various human infections [49,
50]. Similar to C. albicans, C. dubiliensis form complex biofilms 4.2. Device Associated Infections
consisting of a network of blastospores, pseudohyphae, and hyphae
Candida biofilms on the abiotic surfaces of indwelling medical
enclosed in EPM [51]. Candida tropicalis form heterogeneous
devices may cause device associated infections. In USA, around
biofilms consisting of yeast and hyphal form cells [52]. The EPM in
21% of catheter related infections are due to Candida. About 10 to
C. tropicalis biofilm shows reduced levels of hexosamine, protein,
12% of vascular catheter related blood stream infections in the ICU
phosphorous, but increased uronic acid content compared to that of
patients, involve Candida spp. [10]. Fungal infections of cardiovas-
C. albicans biofilms [53].
cular devices such as pacemakers, prosthetic heart valves, ventricu-
Candida glabrata readily form biofilms on biotic and abiotic lar assist devices and synthetic vascular grafts, and central nervous
surfaces, but show reduced thickness. Another characteristic feature system related devices as well as joint prostheses prominently in-
of C. glabrata biofilm is the absence of filamentous growth because volve Candida spp. Dental implants, urinary tract prostheses, the
it does not undergo dimorphic switching. Cells adhering to a sur- peritoneal membrane and catheter, and hemodialysis catheters are
face form layered clusters which are then enclosed in an extracellu- known to be colonized by C. albicans. It also colonizes internal
lar matrix. The EPM of C. glabrata biofilm contains higher concen- fixation devices, percutaneous sutures, tracheal and ventilator tub-
tration of carbohydrates and proteins in comparison to the biofilms ing, penile implants, hip implants and joint prostheses [10, 27].
of other Candida species [54, 55]. Similarly, C. parapsilosis was Strikingly, C. albicans ranks as the third leading cause of catheter
observed to form biofilms devoid of hyphae; hence, the three di- related infections [69]. More than 90% of the fungal prosthetic
mensional community possesses only layers of clustered yeast cells. valve endocarditis (PVE) cases are reported to be due to C. albi-
EPM of C. parapsilosis biofilm is prominently rich in carbohy- cans. Colonization of contact lenses by C. albicans results in mi-
drates, while protein content is comparatively less [54]. Although crobial keratitis, while growth on intrauterine devices causes in-
C. glabrata and C. parapsilosis biofilms does not consist of true flammation [10].
hyphae, the elongated yeast cells which resemble pseudohyphae are
Presence of indwelling prostheses is considered as a risk factor
observed [56].
for the development of C. glabrata infections. It readily forms
biofilm on venous catheters, prosthetic joints and peritoneal dialysis
4. BIOFILM ASSOCIATED INFECTIONS
systems [56]. Formation of C. glabrata biofilms on voice prosthe-
4.1. Native Tissue Infections ses may hamper normal function of the device [59]. Candida
parapsilosis was found to colonize indwelling catheters in neonates,
Colonization of mucosal layers, tissues and prostheses and for-
prosthetic knee, hip joint, and breast implants [56]. Colonization of
mation of a biofilm community is a survival strategy of Candida
percutaneous endoscopy gastronomy tubes by C. albicans and C.
[57]. It may lead to either asymptomatic persistence of the pathogen
tropicalis may contribute to degradation of the polyurethane to
or extensive overgrowth culminating in an infection [18]. Biofilm
cause diarrhea and sepsis [70].
related Candida infections have been observed in several native
tissues including oral soft tissues, teeth, skin, wounds, the middle
4.3. Biofilm as a Virulence Factor
ear, gastrointestinal tract, the urogenital tract, airway/lung tissue,
heart valves, and the eyes. Oral thrush, denture stomatitis, vaginitis, Compared to their planktonic counterparts, Candida biofilms
exhibit reduced susceptibility to available antifungal drugs [71, 72].
burn and wound infections caused by C. albicans are common ex-
Involvement of biofilm in an infection hampers the normal antifun-
amples of biofilm associated infections. Biofilms may be involved
in severe systemic infections such as candidaemia caused due to gal treatment procedures and results in clinical failure. In general,
biofilm community can withstand the attack of host immune system
native valve endocarditis [49, 58].
[59]. Therefore, Candida biofilms may act as reservoirs and release
Several NACS including C. glabrata, C. dubliniensis, C.
the infective cells to cause repeated episodes of infections. Moreo-
parapsilosis, C. tropicalis, and C. krusei have been observed to
ver, colonization of important biomedical-assist devices may com-
cause biofilm-associated infections [49, 50]. Candida parapsilosis
promise the normal function and lead to its failure [73]. It is re-
infections in neonates, transplant patients, and patients receiving
vealed that Candida clinical isolates which are able to form
parenteral nutrition may be biofilm related [59]. Candida parapsi-
biofilms, have significantly more contribution to hospital mortality,
losis and C. glabrata are responsible for 13- 24 % of Candida
costs of antifungal therapy, and increased length of hospital stay of
4 Current Pharmaceutical Design, 2016, Vol. 22, No. 00 Raut and Karuppayil
the patients [56]. Overall, biofilm formation is an important viru- biofilm efficacy of amphotericin B, anidulafungin, and flucytosine
lence factor in Candida. [83]. The observation confirmed that glucan mediated binding
and/or sequestering of drugs as an important mechanism in biofilm
5. DRUG RESISTANCE IN CANDIDA BIOFILMS mediated drug resistance.
Antifungal drugs available for the treatment of Candida infec-
5.2. Extracellular DNA
tions are mainly categorized to four classes such as 5-fluoro-
cytosine (5-FC), polyenes, azoles and echinocandins [74-76]. Un- Along with carbohydrates and proteins, the EPM of Candida
fortunately, the available arsenal of antifungal drugs is not very biofilms also consists of extracellular DNA. Interestingly, treatment
effective against biofilm growth of Candida. A characteristic fea- of biofilms with DNAse results in enhanced sensitivity of C. albi-
ture of Candida biofilms is resistance to available antifungal drugs cans biofilm to caspofungin and amphotericin B. It indicated that
including some of the widely prescribed drugs like fluconazole and extracellular DNA provides structural integrity and strength to EPM
amphotericin B [71, 77]. Susceptibility studies have revealed that and also contributes to antifungal drug resistance [80, 84]. How-
C. albicans biofilm may be up to 2,000 times more resistant to anti- ever, the exact mechanism involved in this is yet to be understood.
fungal drugs than the corresponding planktonic cells (Table 1).
Biofilms formed by NACS also show reduced susceptibility to anti- 5.3. Activation of Drug Efflux
fungal agents [27, 28, 71]. Biofilm associated resistance to drugs is ATP binding cassette (ABC) transporter super family (e.g.
considered as a multi factorial phenomenon. Various reasons con- CDR1 and CDR2) and the major facilitator (MF) class (e.g. MDR1)
tribute to decreased antifungal drug susceptibility of Candida are two main types of efflux pump proteins in C. albicans [85, 86].
biofilms. The major mechanisms which have been studied include Upregulation of the efflux proteins upon exposure to drugs is a
sequestration of drugs by EPM, enhanced drug efflux, high cell prominent mechanism of antifungal drug resistance in planktonic
density, presence of persister cells and activation of stress respon- cells [76]. Adhesion of C. albicans to a solid surface is sufficient to
sive pathway [3, 79]. activate expression of the genes encoding the efflux pumps [87].
Both, in vitro and in vivo biofilms, exhibit over expression of these
5.1. Drug Sequesteration by EPM transporter proteins, even in the absence of any drug. Upregulation
Biofilm community remains embedded in the matrix mainly of efflux pumps associated with early phases remain active even in
composed of carbohydrates, proteins, and nucleic acids [53, 80]. the mature stages of Candida biofilms [88], hence considered as an
Compared to the planktonic cells, sessile cells and the matrix con- important reason for biofilm associated drug resistance [89].
tains higher levels of (cid:1)-1, 3-glucans in their cell wall. The glucan
was observed to bind four- to five-fold more amount of drug than 5.4. High Cell Density
the concentrations required to inhibit planktonic growth. It mainly In the in vitro biofilm model for susceptibility testing, the cell
contributes to sequestering of antifungal azoles and polyenes [81, density ranges between 106 -108 cells/ml. The high cell density is
82]. Disruption of (cid:1)-1, 3-glucans by glucanase treatment leads to speculated to be responsible for reduced sensitivity to drugs. If the
increase in antifungal drug sensitivity of biofilms. Further, low biofilm community is dispersed, the cells with lower density exhibit
expression of glucan synthase gene was found to enhance the anti- increased sensitivity [3, 90]. Density dependent secretion of quo-
Table 1. Various drugs currently available for antifungal therapy and their efficiency against Candida albicans biofilms [104, 110,
242].
MIC ((cid:1)g/ml)
Drug
Planktonic Preformed/ Mature Biofilm
Amphotericin B 0.25 4 to 30
Amphotericin B- Liposomal preparation 0.06 0.25
Amphotericin B- Lipid Complex 0.06 0.25
Nystatin 1 16 to 32
Nystatin- Lipid Complex 0.06 16
Flucytosine (5-FC) 0.8 >420
Caspofungin 0.125 to 0.2 0.5
Micafungin 0.001 0.5
Fluconazole 0.25 >256
Voriconazole 8 >256
Ketoconazole 0.125 64
Ravuconazole 0.06 128
Chlorhexidine 8 8 to 32
Terbinafine 32 128
Phytochemical Inhibitors of Candida biofilm Current Pharmaceutical Design, 2016, Vol. 22, No. 00 5
rum sensing molecules, particularly farnesol, followed by modula- hibit biofilms of C. albicans was found to be much higher than MIC
tion of gene expression may be a reason for lowered drug suscepti- for planktonic growth [110]. Micafungin and caspofungin were
bility of biofilms [47, 91]. found effective against biofilms of C. albicans and C. glabrata; but,
they exhibited comparatively low activity against clinical isolates of
5.5. Persister Cells C. tropicalis or C. parapsilosis [111]. Anti-biofilm success of these
Persisters are phenotypically dormant cells which are tolerant to drugs over long term use is unknown, and their high cost and toxic-
the antimicrobial drugs [92]. Persister cells which are highly resis- ity issues are also a concern [112].
tant to antifungal agents have been observed in C. albicans biofilms Combination of polyenes and azoles was found useful against
[93]. These phenotypic variants of the wild type cells act as a reser- wound related biofilms. Candida biofilm infections of wounds and
voir to initiate a new biofilm cycle when favorable conditions pre- joints have been efficiently treated with a combination of liposomal
vail. Drug tolerant persister cells are an important reason for failure AMB and voriconazole or posaconazole. Combinatorial therapy is
of the antifungal treatment in clinical settings [94]. Biofilms of C. also applied to treat oral fungal biofilms like denture related stoma-
krusei and C. parapsilosis have been observed to harbor persisters titis and oral candidiasis [113]. Options of the antifungal drugs
and contribute to insensitivity to amphotericin B [95]. Molecular available against biofilm associated candidiasis are very few and
mechanism underlying the drug refractory characteristics of fungal treatment remains a challenge. In majority of cases complete re-
persisters is not investigated in detail. moval of colonized growth is not achieved which may result in
recurrent infections [28, 56]. Hence, prevention of biofilm growth
5.6. Activation of Stress Responsive Pathway
would be the best strategy [100; 101].
Adhesion of Candida cells to a substrate results in cell wall
stress and activation of various signaling cascades including the 7. PHYTOCHEMICALS AS ANTI-BIOFILM AGENTS
protein kinase C (PKC) pathway. Activation of such a stress re-
7.1. Plant products as Antimicrobial Agents
sponsive pathway in fungal cells turns them less sensitive to anti-
fungal drugs. Particularly, mitogen-activated protein kinase 1 (i.e. Plants have played an invaluable role in the human medication
Mkc1) of the PKC cascade is involved in drug resistance [38, 96]. since ancient times. Products of plant origin have given an invalu-
Activation of a heat shock protein, Hsp90, through calcineurin able platform for the search and synthesis of novel drugs [114].
pathway also contributes to azole and echinocandin resistance [96, About 25% of the drugs prescribed worldwide are obtained from
97]. Interestingly, inhibition of the calcineurin pathway or interven- plants. World Health Organisation (WHO) has considered phy-
tion of Hsp90 results in sensitization of C. albicans biofilm to vari- totherapy in its health programs for developing countries and has
ous antifungal drugs [98, 99]. suggested basic procedures for the validation of plant based medi-
cines [115]. Medicinal plants could be used as therapeutic resources
6. ANTIBIOFILM TREATMENT STRATEGIES for prevention and treatment of diseases in several forms. For ex-
Therapeutic strategy applied against device related Candida ample, in traditional home remedies they are used as herbal teas,
infections includes removal of the colonized device or inhibition of while phytopharmaceutical preparations are crude plant extracts or
sessile cells using antifungal drugs. Removal of medical devices fractions. The plant based drugs are pharmacologically active com-
suspected to harbor biofilm may help to reduce mortality in device pounds isolated through successive extraction and purification pro-
related Candida infections. However, removal strategy may not be cedures, characterized scientifically for efficacy, and approved for
applicabe in all cases because it involves risk and increases the cost safety and quality [115]. It is interesting to note that out of about
of treatment. For example, removal of infected prosthetic heart 250 drugs considered essential by the WHO, 11% are exclusively of
valves, pacemakers, and knee implants is complicated [100, 101]. plant origin [116].
The only option available in these situations is treatment with anti- Drugs synthesized by organic chemists and antibiotics domi-
fungal drugs. nated antimicrobial therapy for few decades; however, side effects
Antifungal drugs could be applied either for inhibition of sessile due to toxicity and emergence of drug resistant/multidrug resistant
cells or for eradication of the biofilm mass [102]. Antimicrobial pathogens have renewed the interest in plant derived antimicrobials.
lock therapy (ALT) is a procedure utilized for prolonged instillation Phytochemicals is an important source of innovative therapeutic
of a solution containing high concentrations of antimicrobial agents agents against infectious diseases and cancer [117, 118]. Plant ex-
within an infected intravascular catheter in attempt to sterilize the tracts, essential oils and their constituent molecules exhibit novel
catheter. The basic approach of ALT is to utilize extremely high antimicrobial and antifungal properties [14, 119]. Interestingly,
local concentrations of drugs in the “lock” solution that are 100 to phytochemicals have potential inhibitory activities against bacterial
1,000 fold higher than those used systemically [103]. Antifungal and fungal biofilms which are otherwise less responsive to antimi-
lock therapy is one of the initial options for the treatment of cathe- crobial therapy [16, 120].
ter related Candida infections [103]. Various in vitro studies have
indicated the efficacy of polyenes and echinocandins against C. 7.2. Plant Extracts and Candida Biofilms
albicans biofilm; hence, could be useful to treat the in vivo biofilms Practitioners of traditional systems of medicine like Ayurveda
[104]. For example, amphotericin B (AMB) and its liposomal form in India and Traditional Chinese Medicines (TCM), use plant ex-
are two agents commonly used for ALT purpose [101]. Similarly, tracts for the treatment of infectious diseases [114, 121]. Numerous
caspofungin has been used to deal with catheter related Candida reports are available which reveal antibacterial and antifungal po-
biofilms [105]. Although ALT using echinocandins inhibits biofilm, tential of plant extracts obtained using different organic solvents
it however fails to completely eradicate it [15, 106]. [122, 123]. Many of the extracts are known to be effective against
Catheter biofilm studies in animal models suggested that azoles Candida species [124, 125] and also inhibit Candida biofilms
are ineffective, but liposomal AMB reduces the C. albicans (Table 2).
biofilms significantly. AMB deoxycholate and caspofungin have 7.2.1. Prevention of Candida Albicans Biofilm
been observed to achieve 80 to 100% removal of C. albicans from
Several plant extracts have been described for their biofilm
catheters in rabbit models [107]. Candida related infective endo-
suppression effects, primarily through inhibition of events in early
carditis is difficult to treat and involves around 50% of mortality stages of biofilm development. For example, interefering with ad-
rates. It could be treated preferably with liposomal AMB or caspo-
hesion of Candida cells to a solid surface or inhibition of yeast to
fungin [108, 109]. The caspofungin concentration required to in-
hyphae morphogenesis leads to reduced biofilm growth. Schinus
6 Current Pharmaceutical Design, 2016, Vol. 22, No. 00 Raut and Karuppayil
Table 2. Plant extracts and their efficacies against biofilms of Candida albicans and NACS.
Plant Minimum Biofilm Inhibitory Concentration* Biofilm Forming Candida species References
Acorus calamus (2 mg/ml) C. tropicalis [152]
Allium sativum 0.5 mg/ml; (2 mg/ml) C. albicans [243]
Alpinia officinarum 0.250 mg/ml C. albicans [130]
Anacardium occidentale (0.2 mg/ml) C. albicans [140]
Anadenanthera colubrina 31 (cid:1)g/ml C. albicans [129]
Cassia spectabilis 6.25 mg/ml C. albicans [136, 137, 139]
Croton urucurana 7 (cid:1)g/ml C. albicans [126]
Dodonaea viscosa var. angustifolia 1- 3 mg/ml C. albicans [133]
Helicteres isora 1- 2 mg/ml C. albicans [135]
Hibiscus sabdariffa 0.25- 1 mg/ml C. albicans [134]
Medicago sativa 0.5 mg/ml C. albicans [132]
C. albicans
Mentha piperita 0.38- 2.5 mg/ml [151, 198]
C. dubliniensis
Moringa oleifera 0.42 mg/ml; (0.42 mg/ml) C. albicans [142]
Myrtus communis 1- 4 mg/ml C. tropicalis [153]
Paeonia suffruticosa 0.125 mg/ml C. albicans [130]
0.512- 1.024 mg/ml; C. albicans;
Pimenta pseudocaryophyllus [150]
(0.512- 1.024 mg/ml) C. parapsilosis
Piper betle 0.78 mg/ml; (0.78 mg/ml) C. albicans [214]
Polygonum cuspidatum 0.39 mg/ml C. albicans [131]
Punica granatum 0.1 mg/ml; (0.250mg/ml) C. albicans [143]
Pongamia pinnata - C. parapsilosis [154]
Rosmarinus officinalis 1 mg/ml C. albicans [198]
Saponaria officinalis 0.5 mg/ml C. albicans [132]
3.12- 12.5 mg/ml;
Sansevieria aethiopica C. albicans [244]
(6.25- 25 mg/ml)
Schinus terebinthifolius 7 (cid:1)g/ml C. albicans [126]
Sida tuberculata (0.5 mg/ml) C. krusei [149]
Solidago virgaurea 0.25 mg/ml; (0.75 mg/ml) C. albicans [144]
Stryphnodendron adstringens 0.5 mg/ml; (1 mg/ml) C. albicans [145]
Syzygium aromaticum 25 (cid:1)g/ml C. albicans [128]
Vaccinium macrocarpon - C. glabrata [155]
Zataria multiflora (0.84 to 1.5 mg/ml) C. albicans [141]
*Values in parenthesis represent MICs against preformed/mature biofilm.

terebinthifolius (commonly known as aroeira or pepper-tree) is a methanol extract fraction, dissolved in hydoalcoholic solvent, re-
tree prominently occurring in South and Central America and tropi- duced formation of C. albicans biofilms by 49% and 47%, respec-
cal/subtropical regions of Africa. The leaf extracts of S. terebinthi- tively. SEM studies revealed absence of normal biofilm ultrastruc-
folius were found to exhibit good anti-biofilm activity. Merely 7 ture in treatment and confirmed the biofilm preventive capacity of
(cid:1)g/ml concentrations of ethyl acetate-methanol extract fraction and the extract. It was proposed that antiadherent activity of the S. tere-
Phytochemical Inhibitors of Candida biofilm Current Pharmaceutical Design, 2016, Vol. 22, No. 00 7
binthifolius extract results in less adhesion of cells to solid surface Polygonum cuspidatum is a perennial plant traditionally used
and subsequently less biofilm formation compared to the untreated for the treatment of skin burn, gallstone, hepatitis, inflammation,
control [126]. TLC analysis indicated the presence of phenolic chronic bronchitis, jaundice, amenorrhea, and high blood pressure
compounds, anthraquinones, alkaloids and terpenoids in the ex- [131]. At a concentration of 0.39 mg/ml the ethanolic root extract of
tracts. Although a single active compound remains to be revealed, P. cuspidatum significantly prevented biofilms at an early stage of
compound like apigenin could be responsible for antiadherent activ- development, probably through interference with adhesion of C.
ity of S. terebinthifolius [126]. albicans cells to polystyrene surface. The extract was also found to
The Croton urucurana, popularly known as dragon blood is a damage membrane integrity leading to cell death in C. albicans.
tree commonly found in Brazil, Argentina, Paraguay and Uruguay. Importantly, P. cuspidatum extract was devoid of any significant
hemolytic activity and holds a promise for treatment of biofilm
The plant is known to have antiinflammatory, antidiarrhoeal, anti-
haemorrhagic and antifungal properties. 7 (cid:1)g/ml concentration of related infections. The phenolic constituents in the extract may be
crude extract of stem bark dissolved in a hydroalcoholic solution responsible for the anti-Candida effect.
reduced C. albicans biofilm by 35%. The methanolic extract frac- Plant-derived saponins could be novel therapeutic agents
tion and ethyl acetate-methanol extract fractions inhibited Candida against biofilm infections or may support available drugs when
biofilms at 0.5 mg/ml [126]. Previously, a casbane diterpene iso- used in combination. Medicago sativa (also called alfalfa), a plant
lated from the ethanolic extract of Croton nepetaefolius was re- from the pea family Fabaceae, is an important forage crop. Alfalfa
ported to inhibit biofilm formation by pathogenic yeasts, including produces characteristic secondary metabolites, including coumarins,
C. albicans [127]. Activity of S. terebinthifolius and Croton isoflavones, naphthoquinones, alkaloids and saponins, and is known
urucurana at very low concentration is highly significant. These to exhibit various biological activities including anticancer and
two potential extracts need to be systematically analysed for their antimicrobial. Saponin rich fractions of M. sativa aerial parts and
constituents and in vivo efficacy as well as toxicity. root extract in aqueous methanol (80%) showed moderate activity
Flower buds of Syzygium aromaticum (known as clove) are one against C. albicans adhesion and biofilm formation, at 0.5 mg/ml.
Cytotoxicity studies with L929 fibroblast cells established the
of the oldest traditional medicines and have been used as a remedy
safety of these Medicago sativa saponin fractions [132]. Similarly,
for dental disorders, respiratory disorders, headache and sore throat
[128]. Significant inhibition of biofilm formation in several Can- Saponaria officinalis (commonly known as soapwort), is a well
known source of saponins and has shown potential biofilm eradicat-
dida isolates by ethyl acetate extract of clove buds was achieved at
ing effect in C. albicans. However, its haemolytic activity is a ma-
considerably low concentration i.e. 25- 50 (cid:1)g/ml. It was found to
modulate the cell surface hydrophobicity of Candida cells which jor hurdle in the direct therapeutic use in humans.
probably results into less adhesion during early phase of biofilm. It Dodonaea viscosa var. angustifolia (from Sapindaceae family)
is well known that eugenol is an active constituent of clove oil, is found in many parts of the World including South Africa. Leaves
however it was absent in the ethyl acetate extract of S. aromaticum and the twigs possess many medicinal properties and have been
[128]. Authors proposed that a novel compound other than eugenol traditionally used to treat colds, fever, flu, sore throats, and oral
must be exerting the antibiofilm activity. Interestingly, biofilm thrush [133]. Acetone extract of dried leaves of D. viscosa var.
inhibitory concentration of the extract was non-hemolytic hence, angustifolia was found to inhibit yeast to hyphae dimorphism,
can be a potential candidate to be investigated further for clinical biofilm formation and extracellular matrix formation by sessile
applications. cells of C. albicans. The TEM analysis revealed that the effective
concentrations (1 to 3 mg/ml) affected the cell ultrastructure
Lima and coworkers (2014) showed that hydroethanolic (80%)
extract of Anadenanthera colubrina bark has excellent antibiofilm through damage to cell membrane and cell wall [133]. It was also
activities. Anadenanthera colubrina commonly known as ‘angico’ speculated that presence of phytosterols and tannins in D. viscosa
may cause cellular damage, including the cell wall destructuring.
is a tree occurring in Brazil, Bolivia, Argentina, Paraguay, and
Peru. Bark of this tree is widely used in folk medicine against respi- Since the plant does not display any cytotoxic effects, it has thera-
ratory problems, inflammation, diarrhea, cough, bronchitis, influ- peutic potential against biofilm associated infections and need to be
studied further.
enza, and toothache. The extract and its ethyl acetate fraction pre-
vented C. albicans biofilm at 31 (cid:1)g/ml concentration [129]. The The infusion of Hibiscus sabdariffa calyces is commonly con-
activities were found comparable to the activity of a standard drug, sumed as an herbal drink and is used in traditional medicines
nystatin. SEM analysis revealed that reduction in biofilm growth at against hypertension and urinary tract infections. The methanolic
low concentrations of extract was probably through inhibition of extract (0.25 to 1 mg/ml) of the calyces showed good biofilm pre-
yeast to hyphae conversion, which is an important event during C. ventive activity against uropathogenic strains of C. albicans [134].
albicans biofilm formation. The cell wall plays important role in Presence of proanthocyanidin compounds in the extract may be
nearly all cellular mechanisms and pathogenicity of Candida. De- responsible for antibiofilm effects.
structuring of cell wall and membrane damage at the higher concen-
Helicteres isora L., also known as Indian screw tree is used by
trations may be responsible for fungicidal effects of the A. colu- practitioners of the Indian traditional system of medicine (i.e.
brina bark extract [129]. Phytochemical analysis showed the abun-
Ayurveda) for the treatment of skin problems, dermatitis, eczema,
dance of phenolic compounds in the extract which may be respon-
acne, gastrospasm, cough, asthma and diabetes. Recently, the
sible for the antibiofilm activities. The extract did not affect growth methanol extract of H. isora L. fruits was showed to exhibit signifi-
of the host cells (keratinocytes), indicating its nontoxic nature and
cant inhibition of C. albicans biofilms at 1 to 2 mg/ml concentra-
suitability for in vivo applications.
tion [135]. Although exact mechanism of action involved in anti-
Paeonia suffruticosa, commonly known as Cortex Moutan and Candida activities of H. isora L. is unknown, there is a possibility
Alpinia officinarum, also called as Lesser Galangal, are TCMs that plant molecule mediated repression of adhesion and filamenta-
which have been found effective against C. albicans biofilms. Wa- tion associated genes (like ALS3, HWP1, TUP1, CRK1) ultimately
ter extracts of Paeonia suffruticosa root bark and Alpinia officina- results in impairment of biofilm development.
rum rhizomes prevented formation of biofilms at 0.125 and 0.250 Sangetha et al. (2009) have studied the anti-biofilm activity of
mg/ml concentrations, respectively [130]. For an extract these are methanol extract of Cassia spectabilis leaf in C. albicans at a con-
significantly low MICs and hence need to be characterized further
centration of 6 mg/ml [136]. Visualization of biofilm ultrastructure
for the active constituents and toxicity profile. by SEM and CSLM revealed the decreased density of sessile cells
[137], and TEM showed cellular disruption, upon treatement with
8 Current Pharmaceutical Design, 2016, Vol. 22, No. 00 Raut and Karuppayil
the active concentration of the extract. Further to it, the extract was Stryphnodendron adstringens (Mart.) is a Brazilian plant from
confirmed to be nontoxic through in vivo studies on mice and rec- Leguminosae family. It has good antimicrobial activity and is tradi-
ommended as safe [138]. Efforts to reveal the underlying mecha- tionally used in the treatment of vaginal infections and wounds. The
nisms of action showed a gradual leakage of K+ ions in C. albicans extract from stem bark was found to inhibit formation of C. albi-
cells exposed to the extract, probably due to membrane alterations cans biofilms and mature biofilms at 0.5 and 1 mg/ml concentra-
and increased permeability. Analysis of cell wall proteins con- tions, respectively [145]. In addition, it inhibited cells dispersed
firmed that C. spectabilis leaf extract interrupts the cell wall organi- from mature biofilm indicating the ability to prevent dissemination
zation and functions [139]. The multicomponent extract must be and spread of biofilms. Evaluation through SEM showed decrease
targeting important cellular functions and thus exhibit antibiofilm in biofilm cell density. Treatment with the subfractions of the ex-
effects. tract induced alterations in blastospores to make them dumbbell-
7.2.2. Inhibition of Mature Biofilms of Candida Albicans shaped, suggesting cellular budding as a target in C. albicans. S.
adstringens extract was found rich in proanthocyanidin polymeric
The activity against preformed biofilms is considered signifi-
tannins which are oligomeric flavonoids composed by derivatives
cant as they are very much recalcitrant to antimicrobial agents.
of catechin, epicatechin, and their gallic acid esters. Proanthocya-
Most of the available antifungal drugs fail to mitigate the Candida
nidin polymeric tannins may inhibit the Candida growth through
biofilms in their mature stage. Newer approaches to eradicate/ in-
inactivation of important cellular proteins. Various fractions of the
hibit a preformed biofilm at the site of infection are urgently re-
extract were found nontoxic to blood cells, nongenotoxic and safe
quired. Plant extracts were found to exert good Candida biofilm
even after chronic use in mice model [146], indicating there poten-
inhibitory activity. For example, ethanolic extract of Anacardium
tial as therapeutics in biofilm related infections of C. albicans.
occidentale (cashew) leaves inhibited 24 h mature C. albicans
7.2.3. Application of High Concentrations of Plant Extracts for
biofilms upon exposure to 0.2 mg/ml concentration for only 4 h. In
Short Time
addition, it was found to be biocompatible through cytotoxicity
studies on human fibroblast cells and V79 cell line [140]. A. occi- A short exposure to high concentrations of selected extracts was
dentale leaf extract could be developed into an effective natural found to inhbit Candida biofilms. These extracts are usually desir-
mouth rinse against oral and periodontal candidiasis. able as mouth rinses where only a short time is available for drug to
interact with the infective pathogen. For example, crude extract of
Zataria multiflora Boiss, a plant belonging to the family Labi-
ateae is distributed all over the World. It is rich in terpenoids and Equisetum giganteum L. plant (aerial parts) was found to possess
exhibits proven antimicrobial/antifungal properties. The aqueous potent effects on C. albicans adherence to heat-polymerized acrylic
resin. Biofilm formation was suppressed when the substrate was
and ethanolic extract of Z. multiflora inhibited mature biofilms of
C. albicans at 1.5 and 0.84 mg/ml concentrations, respectively pretreated with 16 mg/ml concentration for only10 minutes [147].
[141]. The main chemical components such as linalool, thymol, and Interestingly, it did not compromise the viability of human mono-
cytes or human palatal epithelial cells indicating biocompatibility of
carvacrol may be responsible for the observed activity. However,
the mechanism of the action remains to be investigated. the high concentration of the extract. It was speculated that the
acidic pH of the hydroalcoholic extract could be responsible for
Few more extracts have promising activity against mature
antiadherent effect on C. albicans biofilms. It was predicted that the
biofilms of C. albicans. Recently, a study by Onsare and Arora
phenolic (derivatives of caffeic and ferulic acids) and flavonoid
(2015) revealed ability of Moringa oleifera methanolic extract to
(heterosides derived from quercitin and kaempferol) constituents of
eradicate preformed biofilms at 0.42 mg/ml concentration. Treat-
the extract are responsible for the anti-Candida and anti-biofilm
ment with the extract caused decrease in cell mass as well as meta-
activity. E. giganteum extract may prove as a promising alternative
bolic activity of biofilms. Pretreatment of the cells was also found
for prevention of oral candidiasis and denture associated Candida
to reduce formation of biofilms. The extract interfered with the C.
biofilm.
albicans attachment to a substrate leading to reduced biofilm
Similarly, a very short exposure to high concentrations of Equi-
growth. Evaluation of safety established nonmutagenic and non-
setum arvense L., Glycyrrhiza glabra L., and Stryphnodendron
toxic nature of this flavonoid rich extract [142]. Antibiofilm activity
of M. oleifera should be analyzed further in the in vivo models to barbatimam Mart., extracts in propylene glycol were observed to be
lethal to sessile cells. Merely 5 minute treatment with 50 mg/ml
confirm therapeutic utility.
concentration of the extract inhibited mature biofilms of C. albicans
Methanolic extract of Punica granatum L. (i.e. pomegranate)
on acrylic resin. Hence, these extracts may find use against oral
fruit peel was observed to exhibit excellent antibiofilm activity in
candidiasis or denture stomatitis [148]. The extracts need to be
C. albicans. Concentrations in the range 0.1-0.25 mg/ml showed
analyzed for their active constituents because a pure molecule may
50- 100% inhibition of biofilm development. Confocal microscopy
exhibit the activity at lower concentration which is desirable.
analysis of pomegranate extract treated biofilm revealed significant
(60%) reduction in the surface area coverage and the thickness 7.2.4. Activity Against NACS Biofilms
compared to the untreated control. Inhibition of filamentous forms Compared to C. albicans less is done on the effects of plant
in the biofilm matrix due to prevention of hyphal morphogenesis is extracts on biofilms of NACS. Sida tuberculata, a plant found in
an important mode of action of pomegranate extract [143]. Interest- South America, has traditionally been consumed as an infusion or
ingly, preformed biofilms which are very resistant structures were tea. The leaf and root infusions of this plant were particularly found
eradicated by the extract. About 90% of the sessile growth was effective against mature biofilms of C. krusei. Very significant
inhibited at 0.25 mg/ml concentration [143]. The activity was con- inhibition of biofilm grown on central venous catheter surface was
founded to the pure components like ellagic acid present in the evident with 90 minute exposure of 0.5 mg/ml concentration of the
extract. infusions. Ecdysteroids, which is a marker compound of Sida Sp.,
have been considered responsible for the antifungal activity [149].
A herbaceous plant, Solidago virgaurea (Goldenrod), displays
substantial antimicrobial potential. Water extracts of two Solidago Ethanol extract, and ethyl acetate and aqueous fractions of a
subspecies i.e. virgaurea and alpestris reduced preformed biofilms Brazilian tree, Pimenta pseudocaryophyllus exhibited inhibition of
at the concentration of 0.75 mg/ml. Biofilm formation was sup- preformed biofilms of C. parapsilosis and C. albicans. MIC for
50
pressed at lower concentration of the extract i.e. 0.25 mg/ml. The sessile cells were observed to be in the range 0.512 -1.024 mg/ml,
inhibition is probably through the ability of saponin constituents to while MIC was at concentrations >1.024 mg/ml [150]. Injury to
80
interfere with filamentous growth which is an important component cell membrane and alteration of yeast metabolism (as demonstrated
of heterogeneous biofilm structure [144].
Phytochemical Inhibitors of Candida biofilm Current Pharmaceutical Design, 2016, Vol. 22, No. 00 9
by flow cytometry) appeared to be the mechanisms involved for Candida activities at comparatively higher concentrations. The
this inhibitory activity. activity observed may be due to the synergistic effect of more than
Mentha piperita (family Lamiaceae), found in Iran and many one active constituent molecules. The extracts must be analyzed for
other active pure components and its toxicity profile should be eva-
parts of the World has an economical value. Leaves and flowers of
peppermint are widely used in foods, cosmetics and medicinal in- luated, so that its potential for clinical use would be established.
dustry due to its flavor, fragerence and medicinal properties. 0.2 % Oils of Carum copticum and Thymus vulgaris have been known
of M. piperita methanol extract was observed to prevent biofilm to exhibit strong antifungal activities against Candida spp. These
formation by C. dubliniensis [151]. are also among the most efficient suppressors of C. albicans
biofilms. Tests on their efficacy showed that C. capticum oil pre-
In another study, two fractions of methanol extracts of A. cala-
mus rhizome were found to perfuse through preformed biofilms of vented biofilms of various isolates at concentrations ranging from
22- 180 (cid:1)g/ml; while, T. vulgaris antibiofilm activity was evident at
C. tropicalis and C. albicans, and eliminated sessile cells at 2
22- 90 (cid:1)g/ml [163]. Biofilms treated with these oils were found to
mg/ml concentration [152]. Shoaie and team (2013) studied Myrtus
communis antibiofilm activity in C. tropicalis to reveal concentra- be retarded and disorganized compared to untreated control
biofilms. SEM and TEM analysis of sessile cells revealed that cell
tion dependent prevention of biofilms at 1 to 4 mg/ml [153]. Sub-
wall and cell membrane, and other membranous structures are the
inhibitory concentrations (75%) of ethanol extract of Pongamia
pinnata roots was found to reduce initial adhesion of two NACS, C. target sites of these two EOs. The hydrophobic oils may easily dif-
fuse through extracellular matrix and exert greater membrane dam-
guilliermondii and C. dubliniensis and decreased biofilm formation
age in Candida. GC-MS analysis confirmed that thymol, the main
in C. parapsilosis. It was predicted to be due to reduction of the cell
surface hydrophobicity (CSH) [154]. active constituent of C. copticum and T. vulgaris oils exert similar
antibiofilm effects [163].
Cranberry (Vaccinium macrocarpon Aiton) is known to be
helpful in prevention of dental caries, hence was assessed against Khan and Ahmad (2012) have showed that biofilms of standard
strain as well as clinical isolate of C. albicans are sensitive to Syzy-
adhesion and biofilm formation by Candida species. Besides C.
albicans, it displayed a significant anti-adhesion activity against C. gium aromaticum (clove) and Cymbopogon citratus (lemongrass)
glabrata. Moreover, pretreatment of solid surfaces with this extract essential oil. Concentration dependent inhibition of biofilm forma-
tion was evident with a significantly low i.e. 22.5 and 25 (cid:1)g/ml
was found to prevent adhesion and subsequent biofilm formation by
Candida sp. Further, phytochemical investigation revealed that MIC against biofilms for lemongrass oil and clove oil, respectively.
cranberry juice subfractions enriched in proanthocyanidins contrib- These oils were also very effective in eradicating/killing the pre-
formed Candida biofilms. 180 (cid:1)g/ml concentration of lemongrass
ute to the prevention of C. glabrata biofilm [155].
oil significantly inhibited mature biofilms, while clove oil displayed
7.3. Essential Oils Against Biofilms similar effect at 100 (cid:1)g/ml of concentration [164, 165]. Microscopy
analysis revealed that inhibition of yeast to hyphae conversion may
Essential oils (EOs) are complex mixtures of low molecular
be responsible for prevention of biofilm growth. Whereas, shrink-
weight (< 500 daltons) compounds extracted by steam distillation,
age and lysis of sessile cells in mature biofilms indicated damage to
hydro-distillation or solvent extraction. These compounds are usu-
cell membrane (probably due to interference in sterol metabolism)
ally stored in oil and resin ducts, and glands of the plants. EOs usu-
may be the mechanism of action of these EOs and their active ter-
ally constitutes 20-100 different secondary metabolites belonging to
penoid components. On the basis of SEM analysis of Candida cells
different chemical classes, terpenoids and phenolics being the major
treated with EOs, it was predicted that hydrophobic and volatile
components [156,157]. Bioactivity of a particular EO is decided by
nature of oils may result in their increased uptake through extracel-
one or two main components, overall activity must be attributed to
lular polymeric matrix and better activity on biofilms compared to
the specific combination of constituent molecules [158]. Compo-
that of antifungal drugs [164].
nents of EOs are secondary metabolites involved in plant defense
which are synthesized in response to microbial, insect pest and The Australian native plant Melaleuca alternifolia (tea tree) has
herbivores attack. EOs possesses a range of biological properties been known to exert a broad spectrum antimicrobial activity. Tea
including antimicrobial, antiviral, antimutagenic, anticancer, anti- tree oil exhibits strong fungicidal activity against C. albicans. Vari-
oxidant, antiinflammatory, immunomodulatory, and antiprotozoal ous reports have revealed antibiofilm properties of tea tree oil
activities [157]. Use of EOs in traditional systems of medicine is [166]. Sub-lethal i.e. 0.016% concentration of the oil prevented
being practiced since ancient times in human history. They exhibit biofilms, probably through inhibition of adhesion to the surface,
excellent anti-microbial properties including anti-Candida activities prevention of yeast to hyphae morphogenesis and modulation of
[159]. Activities of EOs against drug resistant Candida biofilms are cell surface hydrophobicity. The effective concentration was not
being studied extensively [160, 161] (Table 3). cytotoxic to BEC and HeLa cells [167]. In addition, tea tree oil
inhibited growth of mature biofilm but at comparatively high con-
7.3.1. Activity Against Candida Albicans Biofilms
centrations (0.375%). Monoterpene components of this oil i.e. car-
The essential oils of Boswellia Species are traditionally used as
vacrol, geraniol, and thymol have been considered responsible for
mouthwash, in the treatment of cough and asthma and known to anti-Candida biofilm efficacy. SEM and propidium iodide uptake
have antimicrobial potential. An interesting activity of prevention studies by Ramage and coworkers showed that TTO and its compo-
of adhesion and biofilm formation in C. albicans was observed at a
nent, terpinen-4-ol affect membrane integrity and damage it leading
very low concentration of (< 1 (cid:1)g/ml) oleogum resin oil (i.e. Bos- to leakage of cellular contents [168]. However, studies by this
wellia rivae). This activity is comparable to the standard antifungal group with fibroblast and epithelial cell culture indicated toxicity to
drugs like amphotericin B. The EO also inhibited mature biofilms at
mammalian cells at biofilm inhibitory concentrations.
44 (cid:1)g/ml of concentration [162], which is vey promising. Boswellia
In a study where activities of 20 medicinal plants were studied
rivae oil was found to act as inhibitor of germ tube formation at
for their efficacy against several oral pathogens, Bersan et al.
concentrations as low as 0.12 (cid:1)g/ml. Hyphal formation is an essen-
(2014) observed that leaf oils of Mikania glomerata (Guaco), Lip-
tial component for the structural integrity of C. albicans biofilms,
pia sidoides Cham. (Rosemary), Aloysia gratissima (Brazilian lav-
hence intereference in yeast to hyphae morphogenesis resulted in
ender), prevent formation of C. albicans biofilms at the concentra-
debilitated biofilm. However, molecular mechanism of inhibition is
tion of 0.5 mg/ml [169]. Oils extracted from Cyperus articulatus L.
still unknown. Limonene is the major chemical component of B.
(Piprioca) bulbs, and Coriandrum sativum L. (Coriander) leaves
rivae oleogum resin oil and known to be an antifungal compound.
were found to be active against Candida biofilms at 0.25 mg/ml
However, available reports [16] indicate that it exhibited the anti-
each. The EOs needs to be characterized for their chemical
10 Current Pharmaceutical Design, 2016, Vol. 22, No. 00 Raut and Karuppayil
Table 3. Plant essential oils and their efficacy against Candida biofilms.
Essential oil
Minimum Biofilm Inhibitory Concentration* Biofilm Forming Candida species References
(Source/Plant Species)
Brazilian lavender
0.5 mg/ml C. albicans [169]
(Aloysia gratissima )
Oleogum resin oil
0.8 (cid:1)g/ml; (44 (cid:1)g/ml) C. albicans [162]
(Boswellia rivae)
Carum oil
22- 180 (cid:1)g/ml C. albicans [163]
(Carum copticum)
C. albicans,
Cinnamomum oil
0.250- 0.5 mg/ml; (1- 2 mg/ml) C. parapsilosis, [180]
(Cinnamomum zeylanicum)
C. orthopsilosis
C. albicans,
C. tropicalis,
Coriander oil (Coriandrum sativum L.) 31- 62 (cid:1)g/ml; (0.312- 0.625 mg/ml) C. krusei, [169, 170, 175]
C. rugosa,
C. dubliniensis
Croton cajucara oil (13 (cid:1)g/ml) C. albicans [171]
C. albicans,
Lemongrass oil (Cymbopogon citratus) 22.5- 45 (cid:1)g/ml; (180 (cid:1)g/ml) [164, 178]
C. dubliniensis
Lemongrass oil vapour (Cymbopogon
32 (cid:1)g/ml C. albicans [177]
citratus)
Piprioca oil
0.25 mg/ml C. albicans [169]
(Cyperus articulatus L.)
C. albicans,
Eucalyptus oil (Eucalyptus globulus) 39- 78 (cid:1)g/ml [161, 176]
C. glabrata
Clove oil (Eugenia caryophyl-
25- 100 (cid:1)g/ml; (100- 200 (cid:1)g/ml) C. albicans [161, 164, 206]
lus/Syzygium aromaticum)
C. albicans,
Juniperus virginiana oil 4 mg/ml; ( 8 mg/ml) C. parapsilosis, [180]
C. orthopsilosis
Rosemary oil (Lippia sidoides Cham.) 0.5 mg/ml C. albicans [169]
C. albicans,
Peppermint oil (Mentha piperita) 1 (cid:1)l/ml; 0.5- 1.16 % [161, 151]
C. dubliniensis
Peppermint oil (Mentha suaveolens) 0.78 mg/ml C. albicans [179]
Tea tree oil C. albicans,
0.625 mg/ml; 0.016 %; (0.375- 0.75%) [166, 167, 176]
(Melaleuca alternifolia) C. glabrata
Mikania glomerata oil 0.5 mg/ml C. albicans [169]
Ocimum americanum oil 3 % C. albicans [173]
Piper claussenianum oil 0.12- 0.25 %; (0.25- 0.5 %) C. albicans [172]
C. albicans,
Conehead Thyme oil (Thymbra capi-
(0.64 (cid:1)l/ml) C. glabrata, C.tropicalis, [181]
tata)
C. parapsilosis, C. guilliermondii