UNIVERSIDADE ESTADUAL PAULISTA

“JÚLIO DE MESQUITA FILHO”

Instituto de Ciência e Tecnologia

Campus de São José dos Campos

ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2024.e4039

Braz Dent Sci 2024 Jan/Mar; 27 (1): e4039

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in

any medium, provided the original work is properly cited.

Eﬀect of

Hamamelis virginiana

Persea americana

Cynara scolymus

and

Stryphnodendron barbatiman

M plant extracts on the phenotypic

expression of virulence factors in bioﬁlms of the

Candida albicans

Efeito de extratos vegetais de

Hamamelis virginiana, Persea americana, Cynara scolymus L e Stryphnodendron barbatiman M

sobre a expressão fenotípica dos fatores de virulência em bioﬁlmes de

Candida albicans

Juliana Guimarães dos SANTOS1 , Isabela AMÊNDOLA1 , Ellen Roberta Lima BESSA1 , Graziella Nuernberg BACK-BRITO1 ,

Luciane Dias de OLIVEIRA1 

1 - Universidade Estadual Paulista, Instituto de Ciência e Tecnologia, Escola de Odontologia, Departamento de Biociências e Diagnóstico

Bucal, São José dos Campos, SP, Brazil.

How to cite: Santos JG, Amêndola I, Bessa ERL, Back-Brito GN, Oliveira LD. Effect of

Hamamelis virginiana

Persea americana

Cynara scolymus

L and

Stryphnodendron barbatiman

M plant extracts on the phenotypic expression of virulence factors in biolms of the

Candida albicans

. Braz Dent Sci. 2024;27(1):e4039. https://doi.org/10.4322/bds.2024.e4039

ABSTRACT

Objective: Analyze the phenotypic expression of virulence factors in Candida albicans biolms against plant glycolic

extracts. Material and Methods: The biolms of Candida albicans (ATCC 18804) obtained from incubation for

48 hours were exposed for 5 minutes and 24 hours to different concentrations of glycolic extracts of Hamamelis

virginiana and Persea americana, Cynara scolymus L and Stryphnodendron barbatiman M, in order to verify the

antifungal activity of the proteinase, phospholipase and hemolysin. Results: All extracts were effective in reducing

biolm. In contact for 5 minutes. the extracts reduced 50% of the biolm. After 24 hours, the Persea americana

extract showed the biolm at 90%, followed by Cynara scolymus, which interrupted it at 85%, There was a change

in proteinase intensity after 5 minutes and 24 hours. with an average enzymatic activity of 0.69 compared to

the control of 0.49. Cynara scolymus was the extract with the highest mean concentration of 100 mg/ml; the

phospholipase intensity was changed with Stryphnodendron barbatiman being more effective in 24 hours compared

to the control (p< 0.0001). The hemolysin secretion was modied by Hamamelis virginiana (12.5 mg/ml) after

5 minutes of exposure, and in 24 hours. all extracts were capable to cause changes in secretion. Conclusion: The

tested extracts have antifungal potential in Candida albicans biolms, implying a signicant reduction in virulence

factors. Thus, these can be indicated as an alternative therapeutic tool to reduce the morbidity of these infections,

as in both investigated exposure times. they were able to reduce the

enzymatic secretion of the fungus.

KEYWORDS

Antifungal agents;

Candida albicans

; Infection; Plant extracts; Virulence factors.

RESUMO

Objetivo: Analisar a expressão fenotípica de fatores de virulência em biolmes de Candida albicans frente a

extratos glicólicos de plantas. Material e Métodos: Os biolmes de Candida albicans (ATCC 18804) obtidos a

partir de incubação de 48 horas foram expostos por 5 minutos e 24 horas a diferentes concentrações de extratos

glicólicos de Hamamelis virginiana e Persea americana, Cynara scolymus L e Stryphnodendron barbatiman M,

a m de vericar a ação antifúngica da proteinase, fosfolipase e hemolisina. Resultados: Todos os extratos

foram ecazes na redução do biolme. Em contato por 5 minutos. os extratos reduziram 50% do biolme.

Após 24 horas. o extrato de Persea americana apresentou o biolme em 90%, seguido de Cynara scolymus, que

o interrompeu em 85%. Houve mudança na intensidade da proteinase após 5 minutos e 24 horas, com uma

atividade enzimática média de 0,69 em comparação com o controle de 0,49. Cynara scolymus foi o extrato

Braz Dent Sci 2024 Jan/Mar; 27 (1): e4039

Santos JG et al.

Effect of Hamamelis virginiana, Persea americana, Cynara scolymus L and Stryphnodendron barbatiman M plant extracts on the phenotypic expression of virulence factors in biofilms of the Candida albicans

Santos JG et al. Eﬀect of Hamamelis virginiana, Persea americana, Cynara scolymus L

and Stryphnodendron barbatiman M plant extracts on the phenotypic

expression of virulence factors in bioﬁlms of the Candida albicans

INTRODUCTION

Candida albicans

is a dimorphic fungus that

can change its commensal form to an invasive.

opportunistic pathogenic form. This transition is an

important example of their phenotypic expression.

in which a switch between a unicellular and a

multicellular gene expression program occurs [1].

Its prevalence in hemocompatible isolates is

high. and it is mainly responsible for invasive

fungal infections (IFI) that affect hospitalized

and immunocompromised patients. especially

those who make prolonged use of broad-spectrum

antibiotics [2-4].

This species can cause supercial and invasive

infections with the serious potential to enter the

bloodstream and affect several organs. causing

harm to the patient and even exposing him to

the risk of death in the most severe forms of

dissemination [5-7]. Mortality rates caused by

C. albicans

vary worldwide from 19.6% to 67%.

In the United States, the pathogen is ranked

as the 4th most prevalent microorganism in

nosocomial infections that cause death. The ability

to induce disease is directly related to host defense

mechanisms and virulence factors associated with

the pathogen [8-12].

The pathogenicity of

C. albicans

is due to

its morphogenesis. its ability to form a biolm.

and tissue invasion by different mechanisms,

including endocytosis, active penetration induced

by hyphae, and the production and secretion of

hydrolytic enzymes such as phospholipases and

proteinases, in addition to hemolysin. which

causes erythrocyte hemolysis [13-15].

Aspartyl-proteinases (SAP) are encoded by

a family of 10 genes (SAP1–SSAP10) that play a

vital role in the virulence of

C. albicans

as well

as assist in the formation of hyphae, phenotypic

exchange, adherence, and degradation of host

tissue proteins. The enzyme acts on junction

intracellular proteins, as well as components such

as collagen and keratin, in addition to degrading

proteins associated with host defense such as

immunoglobulins and cytokines [16,17].

Phospholipases are a group of enzymes

located on the surface of the yeast and at the end

of the germ tube. They hydrolyze phospholipids in

fatty acids and other lipophilic substances during

the period of tissue invasion, causing damage to

the host’s epithelial cells [18-20].

In addition to acting on virulence. these

enzymes secreted by

C. albicans

also help in

the development of antifungal resistance to

conventional antibiotics. Due to the continuous

and early use of antifungals in the hospital

environment. resistance to this pathogen associated

with high mortality has been prevalent [21,22].

For this reason. alternative medicine has been

sought for drugs capable of combating this species,

acting mainly in its enzymatic production, in the

control of infection, and in the inhibition of biolm

formation, using herbal medicines obtained from

plant extracts, dry fruits, and essential oils.

A small plant, with golden yellow leaves. native

to North America called

Hamamelis virginiana

has

already been studied showing promising results in

the treatment of gastrointestinal and inammatory

skin diseases, also helping in the healing of wounds

and burns, as it is rich in tannins, flavonoids,

and proanthocyanidins. Another study identied

that it has resistance to degradation by bacterial

collagenase [23-26]. Another fruit with several

proven biological activities is

Persea americana

popularly known as avocado. which acts as

an antioxidant, antidiabetic, antihypertensive,

antimicrobial, antinociceptive [27-30].

com maior concentração média de 100 mg/ml; a intensidade da fosfolipase foi alterada com Stryphnodendron

barbatiman sendo mais efetivo em 24 horas em relação ao controle (p< 0,0001). A secreção de hemolisina

foi modicada por Hamamelis virginiana (12,5 mg/ml) após 5 minutos de exposição e em 24 horas. todos

os extratos foram capazes de causar alterações na secreção. Conclusão: Os extratos testados apresentam

potencial antifúngico em biolmes de Candida albicans, implicando em redução signicativa dos fatores de

virulência. Assim, estes podem ser indicados como uma ferramenta terapêutica alternativa para reduzir a

morbidade dessas infecções, já que em ambos os tempos de exposição investigados, eles foram capazes de

reduzir a secreção enzimática do fungo

PALAVRAS-CHAVE

Agentes anfúngicos;

Candida albicans

; Infecção; Extratos vegetais; Fatores de virulência.

Braz Dent Sci 2024 Jan/Mar; 27 (1): e4039

Santos JG et al.

Santos JG et al. Eﬀect of Hamamelis virginiana, Persea americana, Cynara scolymus L

and Stryphnodendron barbatiman M plant extracts on the phenotypic

expression of virulence factors in bioﬁlms of the Candida albicans

Cynara scolymus L

, known worldwide

as artichoke, is an herbaceous plant, native

to the Mediterranean region, and it is used in

medicine for the treatment of dyspeptic disorders

and having high antioxidant and anti-aging

properties [31,32].

Stryphynodendron barbatiman

is a plant native to the Brazilian Amazon but

can also be found in other Brazilian regions. Its

properties perform anti-inammatory, healing,

astringent, hemostatic, antimicrobial, antifungal,

and antiseptic functions [26].

The glycolic extracts of

H. virginiana

and

P. americana

C. scolymus

and

S. barbatiman

obtained excellent results regarding the antifungal

action. When used in low concentrations,

presenting low cytotoxicity for the host tissues.

Thus, knowing the antifungal potential of these

extracts, it becomes interesting to investigate their

effect on the phenotypic expression of virulence

factors in

C. albicans

. In particular, it is valuable

to understand the mechanism of action of these

extracts to inhibit the enzymatic and hemolytic

activity of this fungus when organized in a biolm.

MATERIALS AND METHODS

Standard strains of

C. albicans

(ATCC

18804) were used. kept in a freezer at -70°C. in

the Microbiology and Immunology Laboratory of

the Institute of Science and Technology of São

José do Campos/UNESP. reactivated in Dextrose

Sabouraud medium. and incubated for 48 hours

at 37°C. The glycolic extracts of

H. virginiana

P. americana

C. scolymus

L and the bark

S. barbatiman

M, were prepared in 20%

propylene glycol. obtained from the company

Mapric (São Paulo. SP. Brazil). with the due

reports and specications.

Minimum Inhibitory Concentration (MIC) and

Minimum Fungicide Concentration (MFC)

To determine the MIC. the method of

microdilution in broth was used by the Clinical

and Laboratory Standards Institute (CLSI),

standards M7- A6 and M27- A2. The inoculum

was prepared from the culture for 24 hours. in

sterile physiological solution (NaCl 0.9%) and

standardized in a spectrophotometer (Micronal)

with 0.380 absorbance at a wavelength of

530 nm.

The test was performed on a microplate

(KASVI. Parana. Brazil), where 100 μl of culture

medium (broth Mueller Hinton-Himedia, Mumbai,

India) and 100 μl of the extracts were added only

in the first column of wells, from where they

departed a series of 12 serial dilutions. and the

inoculants were added.

After incubation from 24 hours to 37 oC.

the MIC was determined in the rst well of the

microplate. which did not present turbidity.

indicative of microbial growth. For the

determination of the MFC of the extract. they were

inoculated into plates containing Sabouraud-

dextrose culture medium (Himedia) and 20 μl

of the MIC, as well as 20 μl of all other higher

concentrations. After 48 h of incubation at

37 oC, the drop in which colony growth was not

observed was determined as the MFC of the plant

extract to

C. albicans

Biolm formation

The biolms were formed in the background

of 24 well plates (KASVI. Parana. Brazil). For

this. the inoculum of the microorganism in

broth was prepared with yeast nitrogen base

(YNB-Himedia) supplemented with 100 mM

glucose. diluted 10 times in sterile distilled water.

For this, the microorganism was incubated at

37°C for 16 h. After this period. the inoculum

was centrifuged and washed twice with sterile

physiological solution (NaCl 0.9%) and made

into standard suspensions in a spectrophotometer

(B582, Micronal, São Paulo. Brazil) containing

107 UFC/ml in YNB broth (10x). The microbial

suspension (1 ml) was placed in the microplate

wells, and it was incubated for 1 h and 30 min

(37 °C under agitation of 75 rpm) for initial

adhesion. After that time, the wells were washed

twice with sterile physiological solution and

placed in 1 ml of BHI broth. The plate was then

incubated under the same conditions as the initial

adhesion for 48 hours; however, after 24 hours

of incubation, the culture medium was changed.

After the formation period of the biolm. this was

put in contact with the extract.

Exposure to plant extracts

After the biofilm of 48 hours, this was

exposed to the action of the glycolic extracts of

Hamamelis virginiana

Persea americana

Cynara

scolymus L

., and

Stryphnodendron barbatiman

M. Concentrations were determined by the CIM

and CFM. as shown in Table I and tested for

5 minutes and 24 hours.

Braz Dent Sci 2024 Jan/Mar; 27 (1): e4039

Santos JG et al.

Santos JG et al. Eﬀect of Hamamelis virginiana, Persea americana, Cynara scolymus L

and Stryphnodendron barbatiman M plant extracts on the phenotypic

expression of virulence factors in bioﬁlms of the Candida albicans

The wells were washed three times with

PBS and received 1 mL of each extract in the

three different concentrations. Five independent

experiments were performed, with 3 repetitions

each, totaling n = 15 for each experimental group.

The control group was maintained in sterile saline.

The plates with the different concentrations of

the extract were incubated for 5 minutes at room

temperature and 24 hours at 37°C under agitation.

After the exposure time, the supernatant

was discarded, and the joined cells were washed

three times with 2 ml of sterile physiological

solution. The biolm was added to 1 ml of sterile

physiological solution, and the microorganisms

were disengaged by friction with the aid of a

disposable tip for 30 seconds.

Analysis of virulence factors

To evaluate the secretion of proteinase. the

medium recommended by Rüchel et al. (1982)

was prepared as follows: The rst medium (a) is

composed of 15 g of noble agar and 900 ml of

distilled water. The medium (B), composed of

2.5 g of liquid vitamin (Protovit), 11.5 g of yeast

carbon base (Sigma), and 2 g of bovine serum

albumine (Sigma), was sterilized by ltration

with a pore membrane of 0.22 μm diameter

(Millipore, Sao Paulo, Brazil). The medium A was

sterilized in an autoclave at 121 oC for 15 minutes

and cooled to 50 oC. Then the medium B was

added to the A, homogenized, and distributed

in sterile Petri dishes.

The strains obtained from the biofilm,

previously exposed to the extracts and incubated

for 24 hours in

Agar Sabouraud Dextrose

, were

sown, Minced, and equidistantly placed on

the plates, which remained incubated at 37 ºC

for 5 days. The production of proteinase was

veried by the formation of hyaline halo around

the colony, resulting from the hydrolysis of the

substrate.

For the evaluation of the production of

phospholipase. the proposed medium was used

by Polak. In 1000 ml of sterile distilled water,

10 g of peptone, 30 g of glucose, 57.3 g of sodium

chloride, 0.55 g of calcium chloride, and 20 g of

agar were dissolved. After sterilizing the medium

in an autoclave (121 ºC for 15 minutes) and

cooling it to 50 ºC, an emulsion of sterile egg

yolk without potassium teluritum was added to it.

The strains obtained from the biofilm.

previously exposed to the extracts. and incubated

for 24 hours in

Sabouraud

dextrose medium were

sown at equidistant points in the middle, and

after 4 days of incubation at 37 ºC, the formation

of a zone of yellowish color was observed around

the colonies.

To evaluate the hemolytic activity of the

strains exposed to the extracts. 1000 ML of

the dextrose

Sabouraud agar

culture medium

supplemented with 30 g glucose, pH 5.6,

after sterilization in an autoclave (121 oC for

15 minutes) were prepared, cooled to 50 oC, and

added to 70 mL of ram blood.

The strains obtained from the biolm and

incubated for 24 hours in

Sabouraud

dextrose

culture medium with chloramphenicol were

sown in this medium and incubated at 37 ºC

for two days. After that time, the presence of a

translucent halo around the colonies indicated

positive hemolytic activity.

The enzyme activity of proteinase (Pz),

phospholipase (PHz), and hemolysin (Hz) was

evaluated by the ratio between the diameter of

the colony and the diameter of the colony plus

the precipitation zone. The lower the value of

Pz, the greater the enzyme activity. The enzyme

activity was classified as negative (PZ = 1),

positive (0.64 ≥ PZ < 1), and strongly positive

(Pz < 0.64).

Statistical analysis

The data that presented a normal distribution

(Shapiro Wilks) were statistically analyzed by the

ANOVA method complemented by the Tukey

test, with a signicance level of 5% (P ≤ 0.05).

Table I - Concentration of plant extracts used for control of growth in the bioﬁlm of 48 hours of

C. albicans

Glycolic Extract Concentrations used in bioﬁlm (5 minutes and 24 hours)

Cynara scoymus

25 mg/mL 50 mg/mL 100 mg/mL

Hamamelis virginiana

L. 3.13 mg/mL 6.25 mg/mL 12.5 mg/mL

Persea americana

6. 25 mg/mL 12.5 mg/mL 25 mg/mL

Stryphynodendron barbatiman

25 mg/mL 50 mg/mL 100 mg/mL

Braz Dent Sci 2024 Jan/Mar; 27 (1): e4039

Santos JG et al.

Santos JG et al. Eﬀect of Hamamelis virginiana, Persea americana, Cynara scolymus L

and Stryphnodendron barbatiman M plant extracts on the phenotypic

expression of virulence factors in bioﬁlms of the Candida albicans

The results that did not present a normal

distribution were analyzed by the Kruskal-Wallis

test and the Dunns test, with a signicance level

of 5% (P ≤ 0.05).

RESULTS

Determination of MIC and MFC

In the test of the microdilution in broth.

it was possible to determine the most effective

concentrations for controlling the growth

C. albicans

in its planktonic form. The

most effective extract was

H. virginiana

which inhibited growth and was a microbicide

when in concentrations above 6.25 mg/ml,

followed by

P. americana

(avocado), which

displayed inhibitory and microbicide capacity

in concentrations above 12.5 mg/ml. When

exposed to the extracts of

S. barbatiman

and

C. scolymus

(artichoke), no concentration

was capable of inhibiting the growth of

microorganisms.

To verify the action of the extract in biolm,

it was chosen to use the concentration before

the MIC and a concentration after (Table I) so

that the cells remain alive, and it is possible to

check the effect of the extracts on the secretion

of virulence factors.

Growth control in biolm

The biofilms of 48 hours of

C. albicans

were kept in contact with the glycolic extracts

for 5 min and 24 h, in different concentrations

(Table I). When in contact for 5 minutes, all the

tested extracts were able to reduce the biolm

by about 50% in relation to the control, as

shown in Figure 1 (A, C, E, and G). There is no

statistical difference being veried between the

different concentrations tested for each extract.

In 24 hours of contact. the extract of

C. scolymus

was able to reduce on average 55.3% in relation

to the control; the concentration of 100 mg/ml

showed a reduction near 85%, with a statistical

difference between the groups (p = 0.0008)

(Figure 1B).

The biolms of

C. albicans

. when treated

by the different concentrations of

H. virginiana

presented average reduction percentages of

54.85% to 5 min and 58.53% to 24 h. When

compared to the tested concentrations, there was

no statistical difference observed between them

for 5 min (p = 0.8408) and for 24 h (p = 0.3527)

of contact, as shown in Figure 1 C and D.

The reduction percentage of the glycolic

extract of

P. americana

in contact with the

biolms of

C. albicans

for 5 min was less than

50% in relation to the control (Figure 1 E),

with no statistical difference between the tested

concentrations (P = 0.7644). In the period

of 24 hours, the average reduction in relation

to the control was 55.17%, 88%, and 90% in

the different concentrations of 6.25 mg/ml,

12.5 mg/ml, and 25 mg/ml, respectively. In the

statistical analysis (p = 0.0777), no difference

was veried between the concentrations tested

(Figure 1F).

In 5-minute contact with the glycolic extract

S. barbatiman

. the biofilms of

C. albicans

showed a 42.4% reduction in relation to the

control, with no statistical difference between

the different concentrations tested (p =0.8534).

Already after 24 hours of exposure to the extract,

it was able to reduce the biolm by 57.4% in

relation to the control. When the concentrations

were compared between them, there was no

statistical difference (p = 0.1392).

Analysis of virulence factors

Table II brings the found values of expression

of the virulence factors found after exposure to

the different concentrations of the different

plant extracts of 5 minutes of exposure. The

most effective extract in reducing the expression

of proteinase was that of

C. scolymus

in the

concentration of 100 mg/ml. the production of

phospholipase, in turn, was better controlled by

the extract of

S. sarbatiman

Achieving the same

efciency in all the tested concentrations. The

production of hemolysin. was only altered by

exposure to the extract of

H. virginiana

at the

concentration of 12.5 mg/ml.

Table III shows the results obtained after

exposure to the extract for 24 hours, in which a

decrease in the expression of the most effective

virulence factors can be observed in relation

to the control. All tested extracts were able to

reduce the reduction of these factors, and the

extracts of

S. barbatiman

at 100 mg/ml were

more effective in reducing the reduction of

proteinase and phospholipase. The production

of hemolysin suffered more interference after

exposure to a concentration of 12.5 mg/ml of

H. virginiana

extract.

Braz Dent Sci 2024 Jan/Mar; 27 (1): e4039

Santos JG et al.

Santos JG et al. Eﬀect of Hamamelis virginiana, Persea americana, Cynara scolymus L

and Stryphnodendron barbatiman M plant extracts on the phenotypic

expression of virulence factors in bioﬁlms of the Candida albicans

Figure 1 - A-F. Reduction percental of the mature bioﬁlm of

C. albicans

after exposure to the glycolic extracts. for 5 minutes and 24 hours.

Legend: A. C. E. and G are referring to exposure to the extract for 5 minutes; B. D. F. and H are referring to exposure for 24 hours.

Braz Dent Sci 2024 Jan/Mar; 27 (1): e4039

Santos JG et al.

Santos JG et al. Eﬀect of Hamamelis virginiana, Persea americana, Cynara scolymus L

and Stryphnodendron barbatiman M plant extracts on the phenotypic

expression of virulence factors in bioﬁlms of the Candida albicans

Table II - Production of virulence factors of

C. albicans

after 5 minutes of exposure to the extracts

Cynara scolymus

Control 100 mg/ml 50 mg/ml 25mg/ml Value of

Proteinase (Pz) 0.51 0.74* 0.66** 0.53 <0.0001

Phospholipase (Phz) 0.53 0.57 0.62* 0.60* <0.0001

Hemolysin (Hz) 0.46 0.50 0.47 0.43 0.0454

Hamamelis virginiana

Control 12.5 mg/ml 6.25 mg/ml 3.13 mg/ml Value of

Proteinase (Pz) 0.50 0.70* 0.68* 0.72* <0.0001

Phospholipase (Phz) 0.44* 0.55** 0.46* 0.50*** <0.0001

Hemolysin (Hz) 0.49 0.66* 0.50 0.49 <0.0001

Persea americana

Control 25 mg/ml 12.5 mg/ml 6.25 mg/ml Value of

Proteinase (Pz) 0.46 0.70* 0.65* 0.64** <0.0001

Phospholipase (Phz) 0.44 0.43 0.46 0.53* <0.0001

Hemolysin (Hz) 0.48 0.55 0.52 0.52 0.1365

Sphyrodendron barbatiman

Control 100 mg/ml 50 mg/ml 25mg/ml Value of

Proteinase (Pz) 0.51 0.72* 0.71* 0.54 <0.0001

Phospholipase (Phz) 0.52 0.62* 0.66* 0.62* <0.0001

Hemolysin (Hz) 0.49 0.47** 0.47** 0.45* <0.0001

*=p<0.5; **=p>0.5

Table III - Production of virulence factors of

C. albicans

after 24 hours of exposure to the extracts

Cynara scolymus

Control 100 mg/ml 50 mg/ml 25mg/ml Valor of

Proteinase (Pz) 0.45 0.78* 0.68** 0.71** <0.0001

Phospholipase (Pz) 0.49 0.64* 0.67* 0.67* <0.0001

Hemolysin (Hz) 0.46 0.64* 0.58*/** 0.55** <0.0001

Hamamelis virginiana

Control 12.5 mg/ml 6.25 mg/ml 3.13 mg/ml Valor of

Proteinase (Pz) 0.52 0.71* 0.72* 0.72* <0.0001

Phospholipase (Pz) 0.56 0.70* 0.67* 0.73* <0.0001

Hemolysin (Hz) 0.50* 0.70** 0.61*** 0.52* <0.0001

Persea americana

Control 25 mg/ml 12.5 mg/ml 6.25 mg/ml Value of

Proteinase (Pz) 0.54 0.75* 0.70* 0.70* <0.0001

Phospholipase (Pz) 0.53 0.61* 0.63* 0.61* <0.0001

Hemolysin (Hz) 0.50 0.68* 0.56** 0.57** <0.0001

Sphyrodendron barbatiman

Control 100 mg/ml 50 mg/ml 25mg/ml Value of

Proteinase (Pz) 0.45 0.76* 0.71*/** 0.68** <0.0001

Phospholipase (Pz) 0.50 0.74* 0.67*/** 0.64** <0.0001

Hemolysin (Hz) 0.46 0.63* 0.69* 0.60* <0.0001

*=p<0.5; **=p>0.5

Braz Dent Sci 2024 Jan/Mar; 27 (1): e4039

Santos JG et al.

Santos JG et al. Eﬀect of Hamamelis virginiana, Persea americana, Cynara scolymus L

and Stryphnodendron barbatiman M plant extracts on the phenotypic

expression of virulence factors in bioﬁlms of the Candida albicans

DISCUSSION

All around the world, there has been

observed a signicant increase in the incidence

of fungal infections, thus affecting global health.

That is why many research groups have included

in their studies objectives that aim to understand

biochemical and molecular characteristics that

interfere in the pathogenicity and fungal virulence,

as well as verify susceptibility and resistance

to antifungals, to propose new antimicrobial

alternatives. With the species of

Candida

spp.,

it has not been different, mainly with

Candida

albicans

, due to its high prevalence. Many studies

prioritizing the mentioned items have already

been reported in the literature [11,33-35].

According to the World Health Organization,

plant extracts or their active principles are used

by 80% of the world’s population. Due to the

growing use of herbal medicine, several studies

are being done to assess the antimicrobial activity

of these compounds [14,36,37]. The extracts

were used in inhibitory concentrations; after

all. the objective of the study was to evaluate

the production of virulence factors associated

with the invasiveness and perpetuation of the

microorganisms after exposure to the extracts,

allowing analysis later concerning the production

of proteinase, phospholipase, and hemolysin.

The present study was not the first to

evaluate the antifungal action of the extracts of

C. scolymus

H. virginiana

P. americana

, and

barbatiman

, and as expected, the glycolic extracts

of these plants, were able to reduce the biolm of

C. albicans

, corroborating with several authors.

These results are promising because the main

death rates related to this microorganism are in

the production of biolms [26,38-42]. However,

no studies were found in the scientic literature

that associated the action of the extracts tested

in this study with the phenotypic expression of

the virulence factors of

C. albicans

, mainly the

hydrolytic enzymes produced by the different

species of

Candida

, which have stood out as

possible therapeutic targets, due to their decisive

role in the pathological process [13].

The production of proteinase was affected

by exposure to all the tested extracts; although

they were not capable of negative production,

there was a signicant reduction compared to

the control [43]. Different from this study, the

exhibition of 50 strains of

C. albicans

(all of

them producing proteinase and phospholipase),

isolated from patients with oral candidiasis. after

being subjected to liver transplant, to the crude

extract of the leaves of

Eugenia uniora

(Cherry),

was able to zero the secretion of these enzymes,

in 94% of the strains tested [44].

The effectiveness of the extract of the

leaf of

Pluchea dioscoridis

was analyzed in

the different expressions of proteinases; this

presented high potential against this enzyme

because the expression decreased by 90% and

40% for SAP1 and SAP10, respectively [45].

These characteristics attenuating the production

C. albicans

proteinase have also been observed

in studies with the oil of

Ocimun Sanctum

(Basil-Sacred), and similar results were observed

in reducing the production of proteinase. It was

also able to inhibit the expression of the SAP1 gene,

responsible for encoding these enzymes [46].

The same was seen after contact for 18 hours

with the methanolic extract of

Juglans regia

. in

which a reduction in the production of proteinase

to the control (Pz = 0.34) was observed in the

concentrations of 175 μl/mL (PZ = 0.45) and

350 μl/mL (Pz = 0.62) [47].

Regarding phospholipases. it can be seen

that

S. babatiman

extracts were more effective

in reducing phospholipase production when

compared to the highest tested concentrations.

The same happened when strains of

C. albicans

were isolated from patients with prosthetic

stomatitis for 30 min from the essential oil of

Origanum vulgare

. To evaluate the anti-enzymatic

activity, it was veried that the oil was able to

inhibit the production of phospholipase compared

to the control [37]. Different from this work,

which veried a statistically relevant reduction in

the secretion of phospholipase in microorganisms

that were exposed to extracts for 24 hours, the

results were similar to those observed after

exposure to the essential oil of

Junglans regia

which presented a reduction in the production

of phospholipase [47].

The avones are part of the avonoids group,

compounds with widely known biological activity

in traditional medicine. A avone, 2-phenyl-4h-

chromen-4-one, has been tested in the control of

the growth and enzyme activity of

C. albicans

, as

well as its cytotoxicity. Although the compound

presents a signicant reduction in the growth of

C. albicans

and low cytotoxicity, it wasn’t able

to alter the patterns of secretion of enzymes like

proteinase and phospholipase [48-50].

Braz Dent Sci 2024 Jan/Mar; 27 (1): e4039

Santos JG et al.

Santos JG et al. Eﬀect of Hamamelis virginiana, Persea americana, Cynara scolymus L

and Stryphnodendron barbatiman M plant extracts on the phenotypic

expression of virulence factors in bioﬁlms of the Candida albicans

Different concentrations of Dracorodim’s

percolation. a avonoid extracted from a plant

widely used in Chinese medicine, the

Daemonorops

draco

(Dragon’s Blood), were put in contact with

strains of

C. albicans

for 24 hours and were able

to inhibit the growth of the fungus as well as the

formation of the biolm and the morphological

transition. The active principle was also able to

inhibit the secretion of phospholipase concerning

the control (PHz = 0.6), compared to the different

concentrations tested (16, 32, and 64 mM), which

exhibited PHz statistically inferior to the control

(0.64, 0.68, and 0.69, respectively) [51]. Different

from this, the cetonic fractions of the aqueous

extract of

Buchenavia tomentosa

presented an

antifungal effect, but exposure to the extract for

1 hour was not able to change the production of

proteinase and phospholipase, with Pz avenge

from 0.45 to all groups [14]. Like these plants,

H. virginiana

has avonoid-rich properties as well.

About hemolysin secretion, it was possible

to verify in this work that the glycolic extract of

H. virginiana

was the only one capable of altering

the secretion of hemolysin at a concentration

of 12.5 mg/mL during a 5-minute exposure.

However, when the strains were exposed for

24 hours, it was possible to perceive a signicant

reduction in the production of hemolysin. Similar

results were obtained when strains of

C. albicans

resistant to different antifungals, were tested in

sub-MIC oil concentrations of

Carum copticum

and

Thymus vulgaris

[52,53].

Essential oils of different plants,

Melissa

citratus indica

Cymbopogon citratus

Pelargonium

graveolens

, and

Eugenia caryophyllata

, were placed

in contact with standard strains of

C. albicans

and subsequently evaluated for the secretion

of hemolysin, among other factors of virulence,

concluding that these oils have a negative impact

on the pathogenicity of

C. albicans

acting in

an inhibitory way in the production of these

factors [54]. However, another study demonstrated

that the essential oil of

Mentha piperita

, showed

results in all virulence factors of

C. albicans

and

the cinnamon bark oil exhibited high antifungal

activity, active against a pre-formed

C. albicans

biolm [55,56].

As well as other microorganisms,

Escherichia

coli

and

Staphylococcus aureus

have already been

evaluated, together with

C. albicans

exposed

P. americana

extract and

Cynara scolymus

acting against

Porphyromonas gingivalis

[57].

Other studies have already shown that tea. including

H. virginiana

, can decrease oral bacterial bioadhesion

and act as a photosensitizer in antimicrobial

photodynamic therapy (aPDT) [40,41].

Work numbers have been used to determine

the production of these virulence factors by

C. albicans

isolated from different infectious

outbreaks. Different species of

Candida

were

isolated from injuries associated with oral cancer,

chronic candidiasis, and atypical infections, as well

as from isolated species of asymptomatic patients.

When some virulence factors were checked, you

concluded that the isolated species of patients with

symptoms, irrespective of the type of lesion, showed

greater production of proteinase and phospholipase.

That factors are directly linked to the capacity of

this microorganism to invade the tissues of the host,

which leads to an increase in the morbidity of the

infectious process. Many of the samples isolated

from infections, whether they come from blood or

supercial infections, cause a lot of damage to the

host [10,53,58-63]. Considering the above. these

data reinforce that the presence of the production

of these virulence factors is directly related to the

capacity of these strains to cause disease.

These studies show the importance of

knowing the standard of production of these

virulence factors in different types of lesions since

their production is associated with factors inherent

to the host and the infectious site. Also, knowledge

of the virulence factors involved in pathological

processes can allow new therapeutic strategies to

be created and can control the infections caused

by this pathogen. Different factors are involved in

the action of these extracts. such as the amount of

tannins, avonoids, and anthocyanidins, among

other principles inherent to each plant. The use

of total extracts allows synergism between these

compounds. often enhancing your action.

CONCLUSION

This study demonstrated that the tested

extracts have antifungal potential in Candida

albicans biolms, having the ability to inuence

the decrease in the phenotypic expression of

virulence factors, reducing enzymatic secretion,

and reaching the proposed objectives. which can

be indicated as alternative therapeutic tools with

the objective of reducing the morbidity of these

infections. In both times tested, the secretion

of phospholipases, proteases, and hemolyzines

produced by

C. albicans

decreased.

Braz Dent Sci 2024 Jan/Mar; 27 (1): e4039

Santos JG et al.

Santos JG et al. Eﬀect of Hamamelis virginiana, Persea americana, Cynara scolymus L

and Stryphnodendron barbatiman M plant extracts on the phenotypic

expression of virulence factors in bioﬁlms of the Candida albicans

Acknowledgements

The authors are grateful to Juliana Guimarães

dos Santos for content contributions and effort to

carry out this research, Graziella Nuernberg Back

Brito for their critical review of the manuscript

and the Luciane Dias de Oliveira for effective

support during the development and writing of

these manuscript.

Author’s Contributions

JGS, IA: Software, Validation, Formal Analysis,

Investigation, Resources, Data Curation, Writing –

Original Draft Preparation. ERLB: Writing – Review

& Editing. GNBB: Conceptualization, Methodology,

Software, Validation, Formal Analysis, Investigation,

Resources, Data Curation, Writing – Original

Draft Preparation, Visualization, Supervision,

Project Administration, Funding Acquisition.

LDO: Conceptualization, Methodology, Software,

Validation, Visualization, Supervision, Project

Administration, Funding Acquisition

Conict of Interest

The authors have no proprietary, nancial,

or other personal interest of any nature or kind

in any product, service, and/or company that is

presented in this article.

Funding

This research did not receive any specic

grant from funding agencies in the public,

commercial, or not-for-prot sectors.

Regulatory Statement

This study was conducted in accordance with

all the provisions of ICT/CSJC – UNESP Ethical

Committee Agency. It does not require approval

from the ethics committee because the research

does not involve humans or animals.

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Ellen Roberta Lima Bessa

(Corresponding address)

Universidade Estadual Paulista, Instituto de Ciência e Tecnologia, Escola de

Odontologia, Departamento de Biociências e Diagnóstico Bucal, São José dos Campos,

SP, Brazil.

Email: ellen.bessa@unesp.br Date submitted: 2023 Sept 13

Accept submission: 2024 Jan 26