Chemical Composition and Antifungal and Antibiofilm Effects of Vitex pseudo-negundo Essential Oil against Pathogenic Fungal Strains

Background Vitex pseudo-negundo is a plant of the Lamiaceae family that grows in different parts of the world and the vicinity of seasonal rivers in Iran. Methods The chemical composition of the Vitex pseudo-negundo essential oils was distilled and evaluated using gas chromatography/mass spectrometry. The antifungal activity of the essential oils against the fungal strains was analyzed by broth microdilution methods as suggested by the Clinical and Laboratory Standards Institute. Furthermore, the antibiofilm activity of the Vitex pseudo-negundo essential oils was assessed using the XTT reduction assay. Results Based on GC/MS analysis, the major components of the Vitex pseudo-negundo essential oils were α-pinene, α-terpinyl acetate, limonene, and (E)-caryophyllene. The growth of tested yeasts was inhibited at concentrations ranging from 2 to 64 μl/mL. Vitex pseudo-negundo fruit essential oil was the most effective in inhibiting yeast growth. Moreover, the essential oils exhibited antifungal activity against filamentous fungi strains. Additionally, the biofilm formation of Candida albicans was inhibited by the leaf, flower, and fruit of the essential oils. Conclusion Considering the significant antifungal activities of these essential oils, they can be considered a potential source for formulating novel agents to control fungal infections.


Introduction
Vitex pseudo-negundo belongs to the Lamiaceae family and is commonly known as the Chinese chaste tree or fve-leaved chaste tree.Vitex pseudo-negundo is typically characterized by a large aromatic square shrub and thickly whitish, containing a multitude of phytochemical compounds [1].
Tere are approximately 270 species of this genus which are distributed in regions around the world from the Mediterranean areas to Central Asia [2].Tis genus serves as an important natural source of traditional medicine in Western Asia, including Syria, Iran, Afghanistan, and other areas.In Iran, it can be found in Tehran, Karaj, Qom, Khorasan, Ahvaz, and Fars provinces [1,3,4].Recent studies have revealed that Vitex pseudo-negundo possesses various medicinal properties such as antimicrobial, anti-infammatory, and antioxidant activities.It has been used to treat several female disorders including endometriosis, abnormal menstrual cycles, menopausal dysfunction, and inadequate lactation.On the other hand, the incidence of fungal infections is currently on the rise, placing signifcant pressure on healthcare professionals [5,6].
One of the most important reasons for this increase is related to the rising number of patients with immune system defects resulting from changes in medical practice, including chemotherapy and immunosuppressive drugs.Additionally, invasive fungal infections (IFI) have signifcantly increased among immunosuppressant patients, especially among those who are HIV-infected [7].Te Candida genus comprises many species considered normal fora of mucosal surfaces.Tis species has been regarded as the most common pathogenic yeast and also the fourth cause of systemic infections in hospitalized patients.Furthermore, it is worth noting that recent clinical implications have highlighted the increasing involvement of non-Candida albicans species [8].
Among the flamentous fungi, Aspergillus species are known as common saprophytes found in various ecological niches.Among the 200 identifed species in this genus, A. fumigatus and A. favus are considered important human pathogenic species, causing various forms of the disease [9].Another group of signifcant flamentous fungi is dermatophytes, which are keratinophilic fungi encompassing three major types: Trichophyton, Epidermophyton, and Microsporum [10].Due to the widespread use of broad-spectrum antifungal drugs, there has been a noticeable increase in drug resistance [8,[11][12][13].Consequently, there is a high demand for the development of new and efective antifungal agents with low toxicity [14,15].Nowadays, there is a growing preference to alternative-based natural compounds with lower resistance rates [16].
Essential oils (EOs) as secondary metabolites obtained from the aerial part of plants exhibit various benefcial biological efects, including antimicrobial, anti-infammatory, antitumor, and analgesic activities [17][18][19].To our knowledge, there are a limited number of studies on the antibacterial activities of Vitex pseudo-negundo; however, there have been no reports on the antifungal and antibioflm activities of these mentioned EOs.Terefore, the present study was designed to characterize the chemical composition of the EO distilled from the leaf, fower, and fruit of Vitex pseudo-negundo, collected from the Fars province in Iran, and evaluate its antifungal and antibioflm activities.

Plant Material and EOs
Isolation.Vitex pseudonegundo was collected at the fowering stage from the forest area in Fasa city, Fars province, Iran.Te leaves, fowers, and fruits of the plant were authenticated, and a voucher specimen (HSU 3005) was deposited in the Herbarium of Shiraz University, Shiraz, Iran.Fifty grams of the air-dried parts of Vitex pseudo-negundo was hydrodistilled for 3 hours using a Clevenger-type apparatus.Te obtained essential oils (EOs) were then dried over anhydrous sodium sulfate and stored in brown vials at 4 °C until further investigations [20].

Gas Chromatography/Mass Spectrometry (GC/MS).
Te GC/MS analysis was performed using an Agilent 7000 triple quad mass spectrometer (Agilent Technologies, USA) coupled with an Agilent 7890A gas chromatography (Agilent Technologies, USA) instrument.Te oven temperature was initially set at 70 °C and then ramped up to 280 °C, where it was held for 4 minutes.Te injector and auxiliary temperatures were maintained at 250 °C and 280 °C, respectively.Te division ratio was 1/50, and the carrier gas, helium, fowed at a rate of 1.2 ml/min.Te operating parameters of the mass spectrometer were as follows: ionization voltage at 70 eV and a mass range of 40-650 atomic mass units (amu) [16].Retention indices (RIs) were determined for all compounds using the retention times of n-alkanes (C6-C24) injected after the oil under the same conditions.Te compounds were identifed by comparing their RIs with those reported in previous studies (Adams, 2007) and by matching their mass spectra with the spectra in the Wiley library.and A. fumigatus (ATCC14110) were used for the evaluation of the EOs antifungal activities.Furthermore, the antifungal activities of the EOs were examined against clinical isolates of Microsporum gypseum, Epidermophyton foccosum, and Trichophyton rubrum also, 6 clinical isolates of C. albicans that were susceptible (3 isolates) and resistant (3 isolates) to fuconazole were studied.Also, fuconazole as a commercial antimicrobial was chosen as a control.

Determination of
Te mentioned yeasts and molds fungi were subcultured on Sabouraud dextrose agar (SDA, Merck, Germany) and potato dextrose agar (PDA, Merck, Germany), respectively.Te culture plates were incubated to inhibit the bacteria growth, and 50 mg/L chloramphenicol (Sigma-Aldrich, Germany) was added to the culture media of fungi [11].

Antifungal Susceptibility Tests.
Te determination of minimum inhibitory concentrations (MICs) of the EOs against the tested fungi was performed using the broth microdilution method, following the guidelines of the Clinical and Laboratory Standards Institute (CLSI).Serial dilutions of the EOs (ranging from 0.012 to 64 μl/mL) were prepared in 96-well microtiter plates using RPMI-1640 media (Sigma, St. Louis, USA).Te inoculum yeast suspensions were adjusted to a turbidity equivalent to a 0.5 McFarland standard (approximately 1-5 × 10 6 cells/mL) using a spectrophotometer at a wavelength (λ) of 600 nm.Evidence-Based Complementary and Alternative Medicine For molds, conidia were collected, and their suspensions were adjusted to a turbidity equivalent to a 0.5 McFarland standard (approximately 1-5 × 10 6 conidia/mL) using a spectrophotometer at a specifc wavelength (λ) of 530 nm.Working suspensions of the studied yeast and mold fungi were prepared in 1/1000 and 1/50 dilutions with RPMI, respectively.After adding 0.1 mL of the prepared inoculum to each well, the plates were incubated at 32 °C for 24-48 hours and at 25 °C for 48-72 hours for yeast and molds, respectively.Te wells of the frst column were considered as negative control, and there were also growth controls containing medium without EO.Te MIC was defned as the lowest concentration of EOs that showed visible growth reduction.Additionally, for the determination of the minimum fungicidal concentration (MFC), 10 µL of the media from the wells showing no visible growth was cultured on SDA for fungi.Te MFCs were defned as the lowest concentration of EOs showing no growth or fewer than 4 colonies.Each experiment was conducted in triplicate [20][21][22][23].

Bioflm Preparation and Growth.
To form bioflms, standard strains of C. albicans (CBS 5982) were cultured on SDA plates.After 48 hours, one loop of C. albicans colonies was transferred to Sabouraud dextrose broth (SDB, Merck, Germany) and incubated for 24 hours at 32 °C on a shaker set at 100 rpm.Yeast cells were then harvested twice with sterile phosphate-bufered saline (PBS) and resuspended in RPMI 1640.Te cell concentration was measured spectrophotometrically, resulting in a concentration of 1 × 10 6 cells/mL at a wavelength (λ) of 600 nm.Serial dilutions of EOs (ranging from 0.012 to 64 μL/mL) were prepared in 96-well microtiter plates using RPMI 1640 medium.After adding 0.1 mL of yeast, the plates were incubated at 30 °C for 48 hours.Additionally, 200 μL of culture medium alone was used as a negative control, and RPMI with yeast without EOs served as a positive control [24].

Antifungal Activities of the EOs.
Te antifungal activities of the Vitex pseudo-negundo EOs against the tested fungi are shown in Table 2.
Based on the results, the leaf EO inhibited the growth of all the studied standard yeasts in the concentration range of 2-64 μl/mL.Also, the fower and fruit EOs also inhibited the growth of the tested yeasts in the concentration range of 4-32 μl/mL.Te leaf EO had a fungicidal activity in the range of 8-64 μl/mL on the tested yeasts, except for C. albicans and C. parapsilosis.In addition, all three EOs of leaf, fower, and fruit inhibited the growth of Cryptococcus neoformans at the concentration of 2, 4, and 4 μl/mL, respectively.
Te minimum inhibitory concentrations of leaf, fower, and fruit of Vitex pseudo-negundo EOs on the three clinically sensitive strains of C. albicans were 32-64 μl/mL, 16-64 μl/ mL, and 8-32 μl/mL.Also, the MIC of the leaf, fower, and fruit EOs on the three clinically resistant C. albicans strains were 32-64 μl/mL, 16-64 μl/mL, and 32 μl/mL, respectively (Table 2).No signifcant diference in MIC was observed between resistant and sensitive strains of C. albicans.Candida species are common causes of nosocomial infections which can produce a wide range of infections.In this study, the EOs of the Vitex pseudo-negundo organs had inhibitory efects on Candida yeast growth in the range of 2-64 μl/mL.Te EOs of the Vitex pseudo-negundo organs inhibited the growth of clinical fuconazole-resistant strains of C. albicans.Cryptococcus neoformans is one of the most important pathogenic fungi, particularly in AIDS patients.In this study, the EOs at concentrations of 2-4 μl/mL inhibited growth and at 8 μl/mL concentration resulted in the elimination of this yeast.All of the Aspergillus standard strains were resistant to Vitex pseudo-negundo leaf and fower.Te only fruit of the Vitex pseudo-negundo EO had a minimum inhibitory concentration on Aspergillus standard strains at a concentration of 64 μl/mL.In the current study, the growth of the clinical dermatophytes isolates   In this study, the EOs of Vitex pseudo-negundo exhibited antimicrobial activities against those examined fungal pathogens.Te broad-spectrum antimicrobial property of the EOs might be attributable to their main compounds and their synergic efects.Te chemical structures of major compounds of the Vitex pseudo-negundo EOs are shown in Figure 1.
α-Pinene (C 10 H 16 ) is one of the most abundant terpene in nature known with a turpentine odor and colorless liquid.α-Pinene is used as a powerful antimicrobial agent due to subcellular size and good permeability into the cell (partition coefcient of octanol to water with log P � 2.8) which induces toxic efects on microorganism membranes and prevents antimicrobial efux [30,31].It was also reported that α-pinene was capable of inhibiting phospholipase and esterase in yeasts [32].Tese unique biochemical and biophysical properties of α-pinene and high content α-pinene EOs make them outstanding antimicrobial agents used in disinfectants [32,33].Te high antimicrobial potency of EOs containing α-pinene was also seen in other studies.It was demonstrated that the MIC value of α-pinene was 360, 117, and 3125 μl/mL against Rhizopus oryzae, Cryptococcus neoformans, and C. albicans, respectively.Interestingly, synergistic activities of α-pinene antimicrobial drugs result in around an 8-fold reduction in MIC value [32].
In vitro studies of pure α-terpinyl acetate have not been performed yet.Chamaecyparis obtusa EO containing high amounts of α-terpinyl acetate was used for the treatment of urinary tract infection.Tey concluded that this EO showed strong antifungal activity against Pityrosporum ovale (MIC � 5 μl/mL) and Saccharomyces cerevisiae (MIC � 5 μl/ mL) [34].Elettaria cardamomum EO showed great potency against Gram-positive bacteria and fungi as compared to Gram-negative bacteria.Tis may be due to the high contents of α-terpinyl acetate as oxygenate monoterpene and its synergic efects [35].It was suggested that α-terpinyl acetate could participate in key hydrogen bond interactions with microorganisms and induce conformational cell changes.Besides, favorable log P (partition coefcient) value facilitates its partition into the lipid bilayer of the cell and induces its mode of action resulting in the death of the microorganism.
Te microbial inactivation mechanism of limonene (4isopropenyl-1-methylcyclohexene), as a natural hydracarbonic monoterpene, was due to sublethal damages on the outer membrane and an increase in the lethal efect.It should be noted that (+)-limonene can reach maximum cell permeability (∼90%) after 24 h incubation at pH 4.0 and 7.0 and induce inhibitory microorganism efects at very low concentrations [36].A more recent study indicated the MIC values of 3 mg/ml for limonene against Cryptococcus neoformans [37].Limonene is a major constituent of Citrus maxima (31.85% limonene) and Citrus sinensis (90.66% limonene).Tese EOs induced a promising fungitoxic spectrum against all tested fungi and caused 100% mycelial growth inhibition of the A. fumigatus, A. terreus, A. alternata, Fusarium oxysporum, and Trichoderma viride [38].
Te in vitro antifungal evaluations confrmed that the fruit exhibited higher inhibitory activity, with geometric means-minimal inhibitory concentration (GM-MIC) values of 8 μg/ml against tested yeast species compared with two other EOs by GM-MIC � 16 μg/ml of fower EO and GM-MIC � 17 μg/ml of leaf EO.However, the amount order of four major compounds in EOs is leaf (77.8%) > fower (72.3%) > fruit (67.3%).Tese data may indicate that the potency was related to not only the amounts of major compounds in the EOs but also the type and chemical structure of compounds [39].Moreover, the importance of miner compounds and their synergic efects should not be neglected.Statistical analysis confrmed that the amount of oxygen-containing monoterpenes and sesquiterpenes was 29.21% which belonged to the fruit followed by 21.75% of the leaf and 21.67% of the fower.Considering these facts, the antifungal activities of EOs were more related to the efects of oxygenated natural compounds on the tested strains.Recent studies demonstrated that oxygenated monoterpenes possess strong antifungal activity via form H-bound interactions with the fungal membrane, deplete the ATP pool, and fnally degrade microorganisms.Besides, the high solubility power of oxygenated monoterpenes in the media was another critical factor that justifes better antifungal activity of mentioned monoterpene [39].To the best of our knowledge, there are a few studies to evaluate the antibacterial efect of Vitex pseudo-negundo and none of them evaluate the antifungal and antibioflm activity of the mentioned EOs.In 2017, Balasubramani reported the considerable antibacterial activity of Vitex pseudo-negundo with MIC of 3.28, 21.26, and 21.07 μl/mL against Escherichia coli, Enterobacter aerogenes, and Enterococcus faecalis, respectively [40].

Antibioflm Activity of the EOs.
Bioflm formation is a vital virulence factor in the Candida genus pathogenicity.Previous studies indicated that the presence of Candida in the form of bioflms might be related to the increased resistance to antifungal therapies.In addition, the formation of bioflm by Candida species has an important role in resistance to antifungal drugs, and the creation of this bioflm structure protects microbial cells against the host's immune defense.
In the present study, inhibitory impact of the tested EOs on C. albicans bioflm formation was determined by the XTT method.As shown in Table 3, the Vitex pseudo-negundo EOs inhibited the bioflm formation of C. albicans.
Based on the results (Figure 2), EOs of leaf, fower, and fruit of this plant at concentrations of 0.25 μl/mL, 1 μl/mL, and 4 μl/mL respectively, caused 50% inhibition of C. albicans bioflm formation.Moreover, the Vitex pseudo- Tese actions may be related to the presence of a variety of constituents.
EOs are characteristically lipophilic and can penetrate the microbial cell membrane.Several studies have demonstrated that terpenes alter cell permeability by penetrating between the lipid bilayer membrane, producing holes and gaps in the membrane, disrupting lipid structure, and changing cell membrane fexibility [24,41].Tis phenomenon will in return result in major surface and morphological misshaping followed by reducing the adherence potency to the surface.Since the adherence process represents a major step in bioflm formation, therefore, reduced adherence will prevent bioflm formation.Also, it was reported that EOs components have an antibioflm    Evidence-Based Complementary and Alternative Medicine mechanism through the inhibition of glucan production and reduction of glucosyltransferase activity as well as downregulation of the glucans synthase genes [42].In the present study, it was observed that the aerial part of Vitex pseudonegundo in 64 μl/mL concentrations efectively inhibited the bioflm formation of C. albicans in comparison with untreated control.

Conclusions
Te data obtained in the present study showed that Vitex pseudo-negundo essential oils (EOs) have signifcant antimicrobial activity against the studied fungal species.Te Vitex pseudo-negundo fruit EO was the most efective in inhibiting yeast growth.Additionally, the bioflm formation of Candida albicans was inhibited by the EOs extracted from the leaf, fower, and fruit.It is noteworthy that, considering the increasing issue of microbial resistance, these antimicrobial agents can also be used to control fungal infections and bioflm formation on both biological and nonbiological surfaces.To build upon the demonstrated inhibition of bioflm production achieved through our research, further comprehensive investigations involving a larger number of microorganisms are necessary to expand this study in future research activities.

2
ATCC: American Type Culture Collection, CBS: Centraal Bureau voor Schimmelcultures, R: resistance, S: sensitive, MIC: minimum inhibitory concentration, and MFC: minimum fungicidal concentration.Evidence-Based Complementary and Alternative Medicine including Microsporum gypseum, Epidermophyton foccosum, and Trichophyton rubrum were inhibited by the Vitex pseudo-negundo leave and fruit EOs at a concentration range of 2-64 μl/mL.

6
Evidence-Based Complementary and Alternative Medicine negundo EOs almost completely inhibited the formation of bioflm by the C. albicans at a concentration of 64 μl/mL.

Figure 1 :
Figure 1: Chemical structures of some major compounds identifed in the EOs of Vitex pseudo-negundo in Iran.
EOs chemical compositions of aerial parts of Vitex pseudo-negundo (leaf, fower, and fruit) are shown in Table1.In the present study, 50, 55, and 56 compounds were identifed in leaf, fower, and fruit, comprising 98.84%, 98.82%, and 97.74% of the total composition of the EOs, respectively.

Table 2 :
Antifungal efects of the Vitex pseudo-negundo EOs on the yeast and mold strains based on broth microdilution method.

Table 3 :
Inhibition of C. albicans bioflm in the presence of diferent concentrations of Vitex pseudo-negundo by the XTT reduction assay.