Bioactive triterpenoids from Solanum torvum fruits with antifungal, resistance modulatory and anti-biofilm formation activities against fluconazole-resistant candida albicans strains.

Vulvovaginal candidiasis (VVC) is the second most common vaginal infection that affects women of reproductive age. Its increased occurrence and associated treatment cost coupled to the rise in resistance of the causative pathogen to current antifungal therapies has necessitated the need for the discovery and development of novel effective antifungal agents for the treatment of the disease. We report in this study the anti-Candida albicans activity of Solanum torvum 70% ethanol fruit extract (STF), fractions and some isolated compounds against four (4) fluconazole-resistant strains of C. albicans. We further report on the effect of the isolated compounds on the antifungal activity of fluconazole and voriconazole in the resistant isolates as well as their inhibitory effect on C. albicans biofilm formation. STF was fractionated using n-hexane, chloroform (CHCl3) and ethyl acetate (EtOAc) to obtain four respective major fractions, which were then evaluated for anti-C. albicans activity using the microbroth dilution method. The whole extract and fractions recorded MICs that ranged from 0.25 to 16.00 mg/mL. From the most active fraction, STF- CHCl3 (MIC = 0.25-1.00 mg/mL), four (4) known compounds were isolated as Betulinic acid, 3-oxo-friedelan-20α-oic acid, Sitosterol-3-β-D-glucopyranoside and Oleanolic acid. The compounds demonstrated considerably higher antifungal activity (0.016 to 0.512 mg/mL) than the extract and fractions and caused a concentration-dependent anti-biofilm formation activity. They also increased the sensitivity of the C. albicans isolates to fluconazole. This is the first report of 3-oxo-friedelan-20α-oic acid in the plant as well as the first report of betulinic acid, sitosterol-3-β-D-glucopyranoside and oleanolic acid from the fruits of S. torvum. The present study has demonstrated the anti-C. albicans activity of the constituents of S. torvum ethanol fruit extract and also shown that the constituents possess anti-biofilm formation and resistance modulatory activities against fluconazole-resistant clinical C. albicans isolates.

1 Introduction

Vulvovaginal candidiasis (VVC), the second most common vaginal infection affects about 70–75% reproductive-aged women worldwide [1]. This infection occurs due to the abnormal growth of Candida species in the mucous membrane of the female genital area with Candida albicans accounting for 85–90% of the fungal infection [2]. The condition can be recurrent and is associated with severe itching, dyspareunia and dysuria, vaginal discharge and vulvar erythema and oedema [3]. Although VVC is not life-threatening, it can lead to disruption of routine and daily social and occupational activities, neurological problems such as anxiety and depression, and even sexual complications [4]. The treatment options for vulvovaginal candidiasis are standard antifungal agents such as azoles, echinocandins and polyenes which are available in oral and/or vaginal formulations [5]. The azoles, particularly fluconazole and clotrimazole have emerged as the drugs commonly used in treating VVC due to their ease of administration and favourable pharmacokinetic profile. However, like other antifungal drugs, their extensive use has resulted in the emergence of resistant C. albicans rendering them ineffective in the treatment of VVC and leading to recurrent yeast infections in some cases [6, 7]. Additionally, vaginal clotrimazole has been shown to cause hypersensitivity reactions like burning, itching, erythema and some drug interactions [8]. The increased occurrence and associated treatment cost of VVC, in addition to the rise in the rate of resistance to current antifungal therapies has necessitated the need for the discovery and development of novel effective antifungal agents for the treatment of the disease.

Solanum torvum Swartz is a small Solanaceous tree with woody taproots that grows up to 5 m high. It is widely distributed in the tropical regions of the world and cultivated in Africa, Asia and the West Indies [9]. S. torvum has alternate leaves which are broadly ovate with entire or lobed margins [10]. The globular shaped edible fruits grow in clusters and have a bitter taste when cooked whereas the stems have scattered hooked prickles [11]. In Ghana, the leaves and fruits of S. torvum are boiled and used in the treatment of diabetes, hypertension, malaria and tuberculosis [12-14]. Leaf poultices are applied directly on skin lesions [15]. The fruit is also used a tonic and a haematinic for pregnant women and in the treatment of bacterial and fungal infections [16]. Phytochemical investigation of S. torvum has led to the isolation of several steroidal glycosides including steroidal alkaloids such as chlorogenone and neochlorogenone [17]. Torvosides A-M and sitosterol β-D-glucopyranoside were isolated from the leaves whereas astorvosides A-G were obtained from the roots [18].

The fruits of S. torvum have also yielded steroidal glycosides like 5α-pregn-16-en-3,20-dione-6α-ol-6-O-[α-L-rhamnopyranosyl-(1→3)-β-D-quinovopyranoside], 25(S)-26-O-β-D-glucopyranosyl-5α-furost-22(20)-en-3β,6α,26-triol-6-O-β-D-quinovopyranoside, 25(S)-26-O-β-D-glucopyranosyl-5α-furost-22(20)-en-3β,6α,26-triol-6-O-[α-L-rhamnopyranosyl-(1→3)-O-β-D-quinovopyranoside] and 5α-pregn-16-en-20-one-3β,6α-diol-6-O-[α-Lrhamnopyranosyl-(1→3)-β-D-quinovopyranoside] [19]; and phenolic compounds including methyl caffeate and isoflavonoid sulfate [20]. The fruits are reported to possess anti-ulcerogenic, anti-hypertensive, anti-inflammatory [10], antidiabetic [21], anticancer [22] and antibacterial activities [23].

Studies have also shown that the fruits of S. torvum possess antifungal activities against fluconazole-susceptible strains of C. albicans [24-26]. However, in those reports, the constituents responsible for the antifungal activity were neither isolated nor tested. The present study therefore sought to isolate and investigate the antifungal activity of the constituents of the fruits of S. torvum against fluconazole resistant clinical isolates of C. albicans.

2 Materials and methods

2.1 Drugs and chemicals

Fluconazole, voriconazole, chloramphenicol, Mueller-Hinton (MH) agar and Sabouraud Dextrose Agar (SDA) were obtained from Thermo Fisher (Oxoid Limited, Hampshire, UK). All the organic solvents were purchased from BDH laboratory supplies (Merck Ltd, Lutterworth, UK).

2.2 Plant material collection and processing

The fruits of S. torvum were bought from the local markets in Ho, Volta Region in August, 2020 and authenticated by Mr. Alfred Ofori at the Institute of Traditional and Alternative Medicine (ITAM), University of Health and Allied Sciences (UHAS) where voucher specimen has been deposited (Voucher specimen number: UHAS/ITAM/2021/FRCO2). The fruits were washed under running water, chopped into pieces, air-dried for a week and ground into coarse powder.

2.3 General procedures

Column Chromatography (CC) was performed using silica gel 60 (70–230 mesh; AppliChem, GmbH, Darmstadt, Germany) or sephadex LH-20 (25–100 μm; Amersham Biosciences) as stationary phases. Thin Layer Chromatography (TLC) was carried out using pre-coated silica gel 60 plates (0.25 mm thickness) incorporated with fluorescent indicator GF254. 1D and 2D NMR spectra were recorded at 25°C on a Bruker Avance-500 (500 MHz). Chemical shifts (δ) were expressed in parts per million (ppm) using tetramethylsilane (TMS) as internal standard and coupling constants (J) were measured in Hertz (Hz).

2.4 Extraction, fractionation and isolation of compounds

The air-dried powdered fruits of S. torvum (2.7 kg) were extracted by cold maceration with 70% ethanol (3 x 3 days) at room temperature with occasional agitation. The combined extracts were then concentrated under reduced pressure using the rotary evaporator to obtain a solid extract (STF, 174 g). Thereafter, 150 g of STF was suspended in distilled water and partitioned successively with hexane, chloroform (CHCl3) and ethyl acetate (EtOAc) and concentrated to obtain four respective major fractions: STF-hexane (8.1 g), STF-CHCl3 (45.7 g), STF-EtOAc (11.4 g) and STF-AQ (68.9 g) fractions [27].

STF-CHCl3 (32 g) was subjected to column chromatography on silica gel eluting with gradients of CHCl3: MeOH (v/v, from 90:1 to 1:1) to obtain 5 fractions: F1 (0.8 g), F2 (7.0 g), F3 (0.6 g), F4 (2.8 g), (3.5 g) and F5 (6.6 g) based on the TLC profiling [28]. Purification of fraction F5 (6.6 g) [Pet ether-EtOAc—6:4 to 0:1] using silica gel column chromatography afforded 110 fractions (20 mL each) bulked into 3 sub-fractions, F5A, F5B and F5C according to their TLC profiles. Sub-fraction F5A was further column chromatographed on sephadex LH-20 with CHCl3: MeOH (1:1) to yield compounds 1 (1.7 g, 0.063%) and 2 (1.3 g, 0.048%). Sub-fraction F5B was also column chromatographed on sephadex LH-20 using CHCl3: MeOH (1:1) as the eluent to give compound 3 (0.94 g, 0.035%). Compound 4 (0.75 g, 0.028%) was obtained after subjecting sub-fraction F5C to column chromatography on sephadex LH-20 using CHCl3: MeOH (1:1) as the eluent [29].

2.5 Antifungal testing

2.5.1 Fungal strains isolation and growth conditions

Clinical isolates of C. albicans were obtained from the Microbiology laboratory of the Ho Teaching Hospital, Ho, Ghana. They were primarily isolated from pregnant women who reported with VVC and were resistant to antifungal agents [30]. To ensure the purity of the Candida isolates, separate yeast colonies were sub-cultured on Sabouraud Dextrose Agar (SDA) with chloramphenicol before incubation at 37°C for 48 h. Identification of C. albicans was carried out by by culturing the isolates on HiCrome Candida Differential Agar (HiMedia Laboratories, India) at 35°C for 48 h for production of species-specific colours. C. albicans isolates appeared as light green coloured smooth colonies and were selected for the study. Confirmation using API ID 32C strips (Biomerieux, France) were carried out according to standard microbiological methods [30].

2.5.2 Fluconazole susceptibility test

Antifungal susceptibility testing of fluconazole (25 μg) was carried out by disc diffusion on Mueller-Hinton (MH) agar method as described by [31] with slight modifications. Briefly, with a sterile inoculating loop, an inoculum was prepared using distinct colonies of C. albicans isolates from the SDA plates which were transferred into a 5 mL test tube containing 0.85% sterile saline solution, and emulsified to form a suspension of turbidity equivalent to 0.5 McFarland standard as compared to a 0.5 McF PhoenixSpec Calibrator (Becton, Dickinson and Company, USA). Thereafter, the media lawn was seeded in three dimensions using sterile swabs dipped in prepared inoculum. Then fluconazole-loaded disks were aseptically placed on the lawn and incubated at 37°C for 24–48 h. The zone diameters of antifungal disks were determined using a measuring ruler. Zone diameter of ≥ 19 mm was considered sensitive, 15 to 18 mm dose-dependently susceptible, and ≤ 14 mm considered resistant. Subsequently, four strains of fluconazole resistant C. albicans isolates were used for the study and were designated as CA-1, CA-2, CA-3 and CA-4 respectively.

2.5.3 Evaluation of antifungal activity

The antifungal activity of STF and its major fractions; column fractions of the active STF-CHCl3 and isolated compounds was tested using the microbroth dilution method based on the CLSI documents M27—A3 as previously described [32]. Fluconazole and voriconazole were included as positive controls and 0.1% DMSO employed as a negative control. The experiment was carried out in triplicate.

The plant extract, partitioned and column fractions were considered active when MIC was < 0.1 mg/mL, moderately active when MIC ranged from 0.1 to 0.5 mg/mL, and weakly active when MIC was from 0.5 to 1 mg/mL. Above 1 mg/mL they were considered inactive [33].

The antifungal activity of the isolated compounds was interpreted as follows: very strong bioactivity <3.515 μg/mL; strong bioactivity 0.003515–0.025 mg/mL; moderate bioactivity 0.026–0.10 mg/mL; weak bioactivity 0.101–0.5 mg/mL; very weak bioactivity 0.5–2 mg/mL; and no activity above 2 mg/mL [34].

The modulatory effect of the isolated compounds on either fluconazole or voriconazole was also determined. The Minimum Inhibitory Concentration (MIC) of fluconazole or voriconazole were determined in the absence or presence of the compounds (¼ MIC) respectively against the C. albicans isolates using the broth dilution procedure as earlier described. The modulatory factor (MF) which is a measure of the modulation effect of the compounds on the MIC of the antifungals was calculated as the ratio of the MIC of the antifungals alone to the MIC of the antifungals in the presence of the compounds.

2.5.4 Checkerboard assay

Interactions of the isolated compounds with either fluconazole or voriconazole were investigated using the broth microdilution checkerboard procedure modified from the EUCAST-AFST guidelines reference technique as previously described [35]. Final concentrations for all test samples ranged from 0.063 to 64 μg/mL. The mode of the interactions was measured by calculating the Fraction Inhibitory Concentration Index (FICI).

For calculation of the FICI, the FIC of each drug was first determined as follows: FICA was obtained by dividing the MIC of drug A when used in combination (MICCA) by the MIC of the drug when used alone (MICA). Similarly, FIC of drug B was obtained by dividing the MIC of drug B when used in combination (MICCB) by the MIC of the drug when used alone (MICB).

FICI was then calculated as follows:

Interactions were interpreted as follows: Synergism for FICI ≤ 0.5, Indifference FICI was > 0.5 to ≤ 4.0, and Antagonism FICI > 4.0 [36].

2.5.5 Biofilm inhibition assay

The tendency of the isolated compounds to inhibit biofilm formation by the strains of fluconazole resistant C. albicans isolates was investigated using the microplate crystal violet stain retention assay according to a previously described method [6] with slight modifications. Briefly, each of the compounds was dissolved in 50 μL of the Mueller Hinton broth in 96-well plates to obtain concentrations of 100, 30, 10 and 3 μg/mL. Thereafter 10 μL each of the C. albicans isolates containing 105 per mL microorganisms were added. As control, wells without compounds were included. The plates were then incubated at 37°C for 48 h after which the planktonic cells were aspirated and wells dried at 25°C. The attached cells were then stained with 0.1% crystal violet and incubated for 15 mins. The adherent microbial biofilm on the walls of each well was then reconstituted with 150 μL ethanol and the absorbance determined at 595 nm.

Percentage biofilm inhibition was calculated as follows:

Each strain and concentration were assayed in three wells on each plate. The experiment was also replicated thrice.

3. Results

3.1 Isolation and identification of compounds from the fruits of S. torvum

Phytochemical investigation of the partitioned CHCl3 fraction of STF led to the isolation of four (4) known compounds. They were identified on the basis of their 1H and 13C NMR spectra and in comparison, to reported literature as Betulinic acid (1), 3-oxo-friedelan-20α-oic acid (2), Sitosterol-3-β-D-glucopyranoside (3) and Oleanolic acid (4) (Fig 1). This is the first report of 3-oxo-friedelan-20α-oic acid (2) in S. torvum. The spectral data of the compounds are provided in the supporting information.

Fig 1

Structures of compounds isolated from the fruits of S. torvum.

3.2 Antifungal activity of S. torvum extract and partitioned fractions

STF and partitioned fractions were active against the C. albicans isolates to varying extent in the microbroth dilution assay with MIC ranging from 0.25 to 16 mg/mL (Table 1). STF demonstrated moderate to little activity against the fungal growth with MIC between 0.25 and 2 mg/mL. The most active fraction was STF-CHCl3 with an MIC of 0.25–1.00 mg/mL. The least active fractions were STF-hexane and STF-AQ. All the isolates showed resistance to fluconazole with MIC >0.064 mg/mL. Voriconazole had variable inhibitory activities on the isolates (MIC = 0.004–0.016 mg/mL).

Table 1

Antifungal activity of S. torvum ethanol fruit extract and fractions against clinical isolates of C. albicans.

Strain	Minimum Inhibitory Concentration (mg/mL)
	STF	STF-hexane	STF-CHCl3	STF-EtOAc	STF-AQ	FLC	VRC
CA-1	1.00	16.00	0.50	0.50	16.00	>0.064	0.008
CA-2	0.50	16.00	0.50	2.00	16.00	>0.064	0.016
CA-3	0.25	16.00	0.25	8.00	16.00	>0.064	0.008
CA-4	2.00	16.00	1.00	4.00	4.00	>0.064	0.004

STF: Ethanol extract of Solanum torvum fruits; STF-hexane, STF-CHCl3, STF-EtOAc and STF-AQ: hexane, chloroform, ethyl acetate and aqueous fractions respectively of ethanol extract of S. torvum fruits. FLC: Fluconazole; VRC: Voriconazole. Experiment was carried out in triplicate.

3.3 Antifungal activity of the column fractions of STF-CHCl3

STF-CHCl3 exhibited the highest antifungal activity among the fractions evaluated and was subsequently fractionated using column chromatography over silica gel to obtain five fractions (F1 –F5). The antifungal activities of the column fractions are presented in Table 2. They demonstrated varying effects on the C. albicans isolates with column fraction 5 (F5) exhibiting the greatest activity (MIC = 0.125–0.250 mg/mL) across the panel of isolates. The activity of voriconazole and fluconazole were as earlier indicated.

Table 2

Antifungal activity of the column fractions of STF-CHCl3 against clinical isolates of C. albicans.

Strain	Minimum Inhibitory Concentration (mg/mL)
	F1	F2	F3	F4	F5	FLC	VRC
CA-1	1.00	2.00	0.50	4.00	0.125	>0.064	0.008
CA-2	16.00	16.00	16.00	16.00	0.125	>0.064	0.016
CA-3	16.00	4.00	8.00	16.00	0.125	>0.064	0.008
CA-4	8.00	8.00	4.00	8.00	0.250	>0.064	0.004

F1, F2, F3, F4 and F5: Column fractions of the chloroform-partitioned fraction of S. torvum ethanol fruit extract (STF-CHCl3). FLC: Fluconazole; VRC: Voriconazole. Experiment was carried out in triplicate.

3.4 Antifungal activity of isolated compounds

The antifungal activity of the compounds against the growth of the C. albicans isolates are displayed in Table 3. The compounds demonstrated antifungal activity at MIC from 0.016 to 0.512 mg/mL. 3-oxo-friedelan-20α-oic acid (2) recorded the highest inhibitory effects with MIC of 0.016 and 0.032 mg/mL against CA-1 and CA-2; and CA-3 and CA-4 respectively, followed by betulinic acid (1) [MIC = (0.032 mg/mL against CA-1 and CA-4) and (0.064 mg/mL against CA-2 and CA-3)] and oleanolic acid (4) [MIC = (0.032 mg/mL against CA-1) and (0.064 mg/mL against CA-2, CA-3 and CA-4)]. The least potent compound was sitosterol-3-β-D-glucopyranoside (3) with MIC of 0.512 mg/mL against the isolates (Table 3).

Table 3

Antifungal activity of isolated compounds against clinical isolates of C. albicans.

Strain	Minimum Inhibitory Concentration (mg/mL)
	1	2	3	4	FLC	VRC
CA-1	0.032	0.016	0.512	0.032	>0.0 64	0.008
CA-2	0.064	0.016	0.512	0.064	> 0.064	0.016
CA-3	0.064	0.032	0.512	0.064	> 0.064	0.008
CA-4	0.032	0.032	0.512	0.064	> 0.064	0.004

FLC: Fluconazole; VRC: Voriconazole. Experiment was carried out in triplicate.

3.5 Effect of the isolated compounds on the antifungal activity of fluconazole and voriconazole

3.5.1 Modulation effect

The modulatory effect of the isolated compounds at sub-inhibitory concentrations (¼ MIC) on the antifungal activity of fluconazole or voriconazole were investigated against the clinical isolates of C. albicans. The compounds affected the susceptibility of the C. albicans isolates towards fluconazole, albeit to varying extents. 3-oxo-friedelan-20α-oic acid (2) was the most effective modulator causing a considerable increase in the susceptibility of the C. albicans isolates to fluconazole (MF = > 16). The isolated compounds, however, reduced drastically the activity of voriconazole (Table 4).

Table 4

Minimum Inhibitory Concentration (MIC) of fluconazole and voriconazole in the absence or presence of the isolated compounds at ¼ MIC concentration against clinical isolates of C. albicans.

Test Sample	CA-1		CA-2		CA-3		CA-4
	MIC (mg/mL)	MF	MIC (mg/mL)	MF	MIC (mg/mL)	MF	MIC (mg/mL)	MF
FLC	>0.064		>0.064		>0.064		>0.064
FLC + 1	0.008	>8	0.016	>4	0.008	>8	0.008	>8
FLC + 2	0.004	>16	0.004	>16	0.004	>16	0.004	>16
FLC + 3	0.008	>8	0.016	>4	0.016	>4	0.032	>2
FLC + 4	0.008	>8	0.004	>16	0.008	>8	0.008	>8
VRC	0.008		0.016		0.008		0.004
VRC + 1	0.032	0.25	0.016	1.00	0.016	0.50	0.008	0.50
VRC + 2	0.032	0.25	0.064	0.25	0.016	0.5	0.032	0.13
VRC + 3	0.064	0.13	0.016	1.00	0.064	0.13	0.016	0.25
VRC + 4	0.032	0.25	0.016	1.00	0.064	0.13	0.064	0.06

FLC: Fluconazole; VRC: Voriconazole. Modulation factor (MF) = MIC (FLC or VRC)/MIC (FLC or VRC + modulator); n = 3.

3.5.2 Checkerboard assay

The interactions of the antifungal combinations of the isolated compounds with fluconazole or voriconazole assessed using the checkerboard assay are summarised in Table 5. The compounds did not induce any synergistic action with fluconazole in the clinical strains of C. albicans with the exception of compound 2 with an FIC Index of 0.38 against CA-1. Their combinations with voriconazole, however, resulted in mostly antagonistic action.

Table 5

Effect of the combined antifungal activity of isolated compounds and fluconazole or voriconazole by the checkerboard microbroth dilution assay.

Combinations	CA-1		CA-2		CA-3		CA-4
	FICI	INT.	FICI	INT.	FICI	INT.	FICI	INT.
With FLC
1	0.63	I	0.63	I	1.13	I	4.06	A
2	0.38	S	1.13	I	0.63	I	2.13	I
3	1.13	I	1.13	I	2.06	I	4.06	A
4	0.63	I	1.13	I	1.13	I	2.13	I
With VRC
1	6.00	A	4.25	A	4.25	A	5.00	A
2	6.00	A	2.00	I	9.00	A	9.00	A
3	8.00	A	3.00	I	4.25	A	4.00	I
4	5.00	A	1.50	I	2.50	I	4.00	I

FLC: Fluconazole; VRC: Voriconazole; INT: Interpretation, FICI: Fraction Inhibitory Concentration Index. S: Synergism for FICI ≤0.5, I: Indifference FICI was >0.5 to ≤4.0, and A: Antagonism FICI >4.0.

3.6 Inhibition of biofilm formation

The isolated compounds demonstrated a concentration-dependent inhibition on biofilm formation in the fluconazole resistant clinical isolates of C. albicans(Fig 2). The percentage biofilm inhibition of the compounds ranged from 21 to 79%. Compound 2 gave the highest biofilm inhibitory effect against the isolates followed by compounds 4 and 1 respectively. CA-3 was the least susceptible to the test compounds.

Fig 2

Biofilm formation inhibitory effect of compounds 1–4 (A-D) in the Clinical isolates of C. albicans (CA-1, CA-2, CA-3 and CA-4).

4. Discussion

STF demonstrated moderate to weak antifungal activity against the fluconazole-resistant clinical isolates with MIC ranging from 0.25 to 2.00 mg/mL [33]. The antifungal activity of STF in this study corroborates the findings of Obiang et al [25] who showed that the ethanol fruit extract of S. torvum possess antifungal activity against fluconazole-sensitive reference strains (ATCC 10231 and ATCC 90028) and clinical isolates of C. albicans. Evaluation of the solvent-partitioned fractions of STF revealed that its antifungal activity resided in the chloroform fraction (STF-CHCl3). Whereas the other fractions gave very high MICs (2.00–16.00 mg/mL) indicating no antifungal activity, STF-CHCl3 recorded low MIC values of 0.25 mg/mL against CA-3, 0.50 mg/mL against CA-1 and CA-2; and 1.00 mg/mL against CA-4 respectively indicating moderate activity which also compared well with the whole fruit extract (Table 1).

Following a bioassay-guided fractionation of STF-CHCl3, four (4) known compounds were isolated from the fruits of S. torvum. They were obtained from the most active column fraction, F5 (MIC = 0.125–0.250 mg/mL) and identified based on their NMR analyses and by comparing their spectral data to reported literature as betulinic acid (1) [37], 3-oxo-friedelan-20α-oic acid (2) [38], sitosterol-3-β-D-glucopyranoside (3) [39] and oleanolic acid (4) [40]. To the best of our knowledge, this is the first-time report of Betulinic acid (1), sitosterol-3-β-D-glucopyranoside (3) and oleanolic acid (4) from the fruits of S. torvum, having been previously isolated from its aerial parts [41, 42]. Betulin was shown to be present in the fruits [43], however, its oxidative derivative betulinic acid was not detected. We are also reporting the presence of 3-oxo-friedelan-20α-oic acid (2) in the plant, S. torvum for the first time, although the compound has previously been reported in a number of plant species [44-46].

With the exception of sitosterol-3-β-D-glucopyranoside (3) (MIC of 512 μg/mL against all isolates), all the isolated compounds demonstrated greater antifungal activity compared to the fraction they were isolated from with MIC that ranged from 16 to 64 μg/mL. The strong to moderate antifungal activity of the compounds which were greater compared to their “parent” fraction suggests that the bioassay-guided fractionation and purification of the plant extract led to the isolation of some of the potent antifungal constituents of S. torvum.

Betulinic acid (1) has previously been reported to inhibit the growth of fluconazole-sensitive strains of C. albicans at MIC of 16 μg/mL [47]. Elsewhere, the compound effectively inhibited C. albicans secreted aspartic proteases (SAP), one of the most virulent factors in candida infection, at 6.5 μg/mL [48]. The low MIC of betulinic acid (1) against the fluconazole-resistant C. albicans isolates [(32 μg/mL against CA-1 and CA-4) and (64 μg/mL against CA-2 and CA-3)] reported in this study substantiates its potent antifungal activity. Favel et al [49] reported that triterpenoid saponins with oleanolic acid (4) as the aglycone demonstrated moderate antifungal activity whiles Eloff and co-workers [50] identified oleanolic acid (4) as the major antifungal constituent of Melianthus comosus against some plant pathogenic fungi through a bioactivity-guided isolation study. Oleanolic acid (4) and its oxime ester derivatives were also shown to inhibit C. albicans Glucosamine-6-phosphate synthase as a possible mechanism of antifungal activity [51]. The considerable antifungal activity of oleanolic acid (4) against the fluconazole-resistant C. albicans isolates [MIC = (32 μg/mL against CA-1) and (64 μg/mL against CA-2, CA-3 and CA-4)] may have also occurred through the same mechanism of action.

The antifungal activity of the five constituents including sitosterol-3-β-D-glucopyranoside (3), isolated from the stem of Jatropha maheshwarii were evaluated through the disc diffusion method. The result showed the constituents had weak antifungal activity in comparison to the whole extract and through a synergistic or additive effect, contributed to the activity of the J. maheshwarii stem [52]. Thus, the weak antifungal activity observed in the present study for sitosterol-3-β-D-glucopyranoside (3) lends support to that study.

This is the first report of the antifungal activity of 3-oxo-friedelan-20α-oic acid (2). It demonstrated the highest growth inhibitory effect [(16 μg/mL against CA-1 and CA-2) and (32 μg/mL against CA-3 and CA-4)] against the C. albicans isolates lending credence to the anti-C. albicans activity of friedelane-type triterpenoids [53].

Antifungal combination therapy (ACT) has been suggested as a promising strategy in overcoming resistance in recurrent candida infections. Again, the combination of antifungal agents with different mechanisms of action can increase efficacy and extend the spectrum of activity of the combined agents leading to synergism [54]. Furthermore, combination of different antifungal compounds can also reduce toxicity by reducing doses and improve pharmacokinetic profiles of one or both agents [3]. Hence, we investigated the antifungal combination activity of the isolated compounds with fluconazole or voriconazole by the checkerboard microbroth dilution assay. The combination of fluconazole with 3-oxo-friedelan-20α-oic acid (2) resulted in a synergistic action (FICI = 0.38) against CA-1, however, the predominant interaction observed in the C. albicans isolates for the combinations of the isolated compounds with the drug is indifferent. Even though the compounds failed to produce any synergistic effect against the clinical strains of C. albicans with fluconazole, at sub-inhibitory concentrations (¼ MIC), they significantly potentiated the antifungal effect of fluconazole (MF ranging from > 2 to > 16 folds) against them. The modulatory effect of natural products has been suggested to occur by their alteration of fungal cell wall to facilitate the access of antifungal agents to the fungal membrane or by inhibition of fungal efflux pumps [35]. The combinations of the isolated compounds with voriconazole mostly resulted in antagonism. This was further buttressed in the modulatory assay where MIC of voriconazole against the C. albicans isolates was increased by 2–16 folds indicating reduced antifungal activity.

C. albicans is a robust biofilm-forming organism which presents serious therapeutic complications [55]. Its ability to form biofilms contributes greatly to its resistance and recurrent infections [56]. With the limited therapeutic options for Candida biofilms available, there is a pressing need for the discovery and development of new anti-C. albicans biofilm agents. The isolated compounds demonstrated considerable concentration-dependent inhibition of biofilm formation in the C. albicans isolates (Fig 2). A number of plant constituents particularly phenolics and triterpenoids have been shown to demonstrate anti-candida biofilm activity by impairing adhesion of planktonic cells on abiotic surfaces, reducing biofilm metabolic activity or promoting biomass degradation of mature biofilms [57]. We report for the first time the anti-biofilm formation of the isolated compounds in C. albicans. Some of these compounds however, have been known to exert this effect in some bacteria. Betulinic acid (1) significantly decreased biofilm formation in Pseudomonas aeruginosa at sub-MIC levels and have also been shown to inhibit biofilm formation in Cryptococcus neoformans [58, 59] whiles oleanolic acid (4) has demonstrated anti-biofilm activity across a wide range of Gram-positive and Gram-negative bacteria [60, 61]. The anti-biofilm activity of the isolated compounds may contribute in part to the efficacy of S. torvum fruits in the treatment of recurrent vaginal infections caused by C. albicans.

The present study has demonstrated the anti-C. albicans activity of the constituents of S. torvum ethanol fruit extract and shown that the constituents possess anti-biofilm formation and resistance modulatory activities against fluconazole-resistant clinical C. albicans isolates.

5. Conclusion

In this study, we report the antifungal activity of S. torvum fruit extract, fractions and some constituents on the growth of fluconazole-resistant strains of C. albicans. The compounds demonstrated considerably higher antifungal activity than the extract and fractions and caused a concentration-dependent anti-biofilm formation activity. In particular, 3-oxo-friedelan-20α-oic acid (2) reported in the plant for the first time significantly decreased the fluconazole-resistance levels of the C. albicans isolates.

1H NMR spectrum of compound 1.

(PDF)

Click here for additional data file.

DEPT Q NMR spectrum of compound 1.

(PDF)

Click here for additional data file.

COSY spectrum of compound 1.

(PDF)

Click here for additional data file.

HSQC spectrum of compound 1.

(JPG)

Click here for additional data file.

HMBC spectrum of compound 1.

(PDF)

Click here for additional data file.

1H NMR spectrum of compound 2.

(PDF)

Click here for additional data file.

13C NMR spectrum of compound 2.

(PDF)

Click here for additional data file.

DEPT 135 spectrum of compound 2.

(PDF)

Click here for additional data file.

HSQC spectrum of compound 2.

(PDF)

Click here for additional data file.

HMBC spectrum of compound 2.

(PDF)

Click here for additional data file.

1H NMR spectrum of compound 3.

(PDF)

Click here for additional data file.

13C NMR spectrum of compound 3.

(PDF)

Click here for additional data file.

DEPT 135 spectrum of compound 3.

(PDF)

Click here for additional data file.

HSQC spectrum of compound 3.

(PDF)

Click here for additional data file.

HMBC spectrum of compound 3.

(PDF)

Click here for additional data file.

1H NMR spectrum of compound 4.

(PDF)

Click here for additional data file.

DEPTQ spectrum of compound 4.

(PDF)

Click here for additional data file.

COSY spectrum of compound 4.

(PDF)

Click here for additional data file.

HSQC spectrum of compound 4.

(PDF)

Click here for additional data file.

HMBC spectrum of compound 4.

(PDF)

Click here for additional data file.

(PDF)

Click here for additional data file.

19 Aug 2021

PONE-D-21-19484

Bioactive triterpenoids from Solanum torvum fruits with Antifungal, Resistance Modulatory and Anti-Biofilm Formation Activities against Fluconazole-Resistant Candida albicans strains

PLOS ONE

Dear Dr. Waikhom,

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1. The name of the botanist that identified the plant should be stated.

2. In Section 2.4, please add the percentage on the compounds following the weight.

3. Tables 1 and 2 expressed the results in mg/mL, whereas Table 3 in ug/mL. Please standarize the dimensions.

4. The word triplicate is not plural.

5. Please add a statistical analysis in Fig. 2.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: It is an interesting study but requires major changes before being accepted for publication

Vulnerable points:

The whole (crude) is not a promising antifungal since the MIC were light, so the entire manuscript needs to be revised.

Why has the study focused just on C. albicans isolates? It needs justification, as the non-Candida albicans species have increased in the last decades.

To draw relevant conclusions from just four C. albicans isolates, in my opinion, is very bold.

The English should be improved, maybe at the discretion of the editor.

Specific points

Abstract

It is missing results details regarding the antifungal activity,

What do the numbers in parentheses written after the compounds mean? Please delete this

The obtained results did not support the conclusion “justifying its use as an antifungal agent in the treatment of vulvovaginal candidiasis….” Lines 21-22

Introduction

Line 4: rephrase this sentence

Line 27: delete the word “ opportunistic”

Line 29: replace "The condition" by VVC

Line 35: the reference [5] is unsuitable for this statement, please replace it.

Line 62: Add the words of S. torvum after “The fruits”

Objectives: Rewrite it focusing on the purified compounds (isolation and their antifungal activity) it is the most relevant in the present study.

Methods

Lines 98-101: The authors should supply more details on the purification of Fraction F5, originating the compounds, also cite a reference, and so on

Lines 106-107: The growth in Sabouraud Dextrose Agar doesn't ensure the purity of the Candida isolates since this culture medium does not differentiate the colonial morphology between the species of this genus.

Line 116: it is written "ml", while elsewhere, as in line 142 it is "mL", standardize it in the entire manuscript, please

Line 126: the definition of the acronym STF .... ethanol extract (STF) is repeated other times, for instance, line 184 and others, please from the first definition (line 116) mention only STF

Line 155: This experiment is confusing, if the crystal violet test was performed in microplates, why were the compounds diluted in test tube sets? Next, line 159 again "The tubes were then incubated at …..", please rewrite this paragraph, clarifying the biofilm inhibition assay.

Results

Line 171: Is it missing some letters in the first word?

Fig 1: the caption is incomplete, it needs to be more informative, mentioning the compound names here instead of putting numbers every time compounds are cited in the text, as it makes reading confusing and tiring

Line 191: add the information “ethanol extract” into the title of table 1

Lines 185-187: The results are confusing, are the two sentences contradictory? Or is the second one just an interpretation? If yes, please move the sentence “STF was considerably active against the fungal growth with MIC between 0.25 and 2 mg/mL” to the methods session.

Line 229: Is the sentence “The isolated compounds, however, drastically reduced the activity of voriconazole” correct? I think it increased the activity of voriconazole Rephrase it, please

Discussion

The first paragraph should be deleted because it is confusing for instance (why was cited the reference 29?), and not relevant.

Line 271: What does “ considerable antifungal activity” mean? Please mention the reference used to define the breaking points in the interpretation of your results

Line 277: I disagree with the statement "low MIC values", as according to a recent systematic review by Alves et.al 2021 (https://doi.org/10.1155/2021/6653311), the values presented in Tables 1 and 2 (crude extract and fractions respectively) reveal only weak or no bioactivity. Please, clarify and discuss your data.

Line 342: This statement is true, but it needs a reference

Lines 360-361: The sentence “The reported activity justifies its use as an antifungal agent in the treatment of vulvovaginal candidiasis among the Ghanaian populace” must be deleted since it is not supported by data of the current study.

Conclusion

Lines 368-371: This subject is not a conclusion of the current study, maybe is possible to include it in the discussion session

Reviewer #2: Dear authors,

Solanum torvum is endemic in southern Mexico and South America, it is a widely studied plant. In their work they propose a possible therapeutic tool for antifungal treatment, making it a valuable proposal.

Best regards

**********

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Reviewer #1: Yes: Terezinha Inez Estivalet Svidzinski

Reviewer #2: No

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23 Oct 2021

The Editor-In-Chief

PLOS ONE

Cambridge, UK

Dear Madam,

RESPONSE TO REVIEWERS’ COMMENTS

We hereby in this letter provide responses to the queries raised by the academic editor and reviewers of our manuscript titled “Bioactive triterpenoids from Solanum torvum fruits with Antifungal, Resistance Modulatory and Anti-Biofilm Formation Activities against Fluconazole-Resistant Candida albicans strains” submitted to your revered journal PLOS One. All datasets generated for this study are included in the manuscript and/or the supplementary files.

Kindly find below our responses.

We hope it will receive a favourable consideration.

Thank you.

Yours faithfully,

Sayanika Devi Waikhom (Ph.D)

(Corresponding Author)

Academic Editor

1. The name of the botanist that identified the plant should be stated.

We have included the name of the Botanist who identified the plant.

Section 2.2, line 77

2. In Section 2.4, please add the percentage on the compounds following the weight.

The percentage weight of the compounds per weight of the dried powdered material have been included.

Section 2.4, lines 103-105

3. Tables 1 and 2 expressed the results in mg/mL, whereas Table 3 in ug/mL. Please standardize the dimensions.

The units of the all the tables have been standardized to mg/mL

Section 3.4 and Table 3.

4. The word triplicate is not plural.

The spelling of triplicate has been corrected.

Lines 136, 207, 220 and 232

5. Please add a statistical analysis in Fig. 2.

The analysis we conducted on the figure 2 was to determine the percentage biofilm inhibition. Since we are not comparing the inhibition to a control. we cannot carry out any further statistical analysis other than comparing the percentage inhibitions of the compounds to each other in terms of which one was higher and lower.

Reviewer 2

General comment

1. Why has the study focused just on C. albicans isolates? It needs justification, as the non-Candida albicans species have increased in the last decades.

We agree to the fact that non-albicans candida species have increased in the recent years, however we decided to focus on C. albicans in this study because despite the increase in the other species of candida, still Most Candida infections in

people are caused by Candida albicans [1]. The group however in other studies are investigating the effect of natural products on NAC such as C. glabrata and auris. We tried to justify the reason for our study on C. albicans in lines 28-29 and we provided an additional reference to support our justification.

2. To draw relevant conclusions from just four C. albicans isolates, in my opinion, is very bold.

We selected the four C. albicans strains as they covered a range of diameters considered as fluconazole-resistant which are ≤ 14 mm.

3. The English should be improved, maybe at the discretion of the editor.

We have improved the general English of the paper.

ABSTRACT

1. It is missing results details regarding the antifungal activity

We have included details regarding the antifungal activity in the abstract

Line 13-16

2. What do the numbers in parentheses written after the compounds mean? Please delete this

The number in parentheses represent the number assigned to the compounds in figure 1. This was done so readers could match the structure of the compound to the name. We have however deleted this as requested.

3. The obtained results did not support the conclusion “justifying its use as an antifungal agent in the treatment of vulvovaginal candidiasis….” Lines 21-22

The conclusion of the abstract has been re-written focusing on the isolated compounds. The statement “justifying its use as an antifungal agent in the treatment of vulvovaginal candidiasis….” has been deleted.

Line 20-23

INTRODUCTION

1. Line 4: rephrase this sentence

Unclear how the reviewer wants this sentenced rephrased.

2. Line 27: delete the word “ opportunistic”

“Opportunistic” has been deleted

Line 28

3. Line 35: the reference [5] is unsuitable for this statement, please replace it.

Appropriate reference has inserted

Reference [5]

4. Line 62: Add the words of S. torvum after “The fruits”

Line 62: Add the words of S. torvum after “The fruits”

Line 57

OBJECTIVES

Rewrite it focusing on the purified compounds (isolation and their antifungal activity) it is the most relevant in the present study.

The objectives have been re-written to focus on the isolation of the purified compounds and their subsequent testing.

Line 67-69

METHODS

1. Lines 98-101: The authors should supply more details on the purification of Fraction F5, originating the compounds, also cite a reference, and so on

Further details have been supplied on the purification of fraction F5.

Line 99-106

2. Lines 106-107: The growth in Sabouraud Dextrose Agar doesn't ensure the purity of the Candida isolates since this culture medium does not differentiate the colonial morphology between the species of this genus.

We have included how we identified and selected the C. albicans isolate

Line 113-116

3. Line 116: it is written "ml", while elsewhere, as in line 142 it is "mL", standardize it in the entire manuscript, please

The unit has been standardized and written as mL throughout the entire manuscript

4. Line 126: the definition of the acronym STF .... ethanol extract (STF) is repeated other times, for instance, line 184 and others, please from the first definition (line 116) mention only STF

The repeated definition of the acronym STF in the text has been deleted.

Line 132, 182, 195, 197, 277, 278, 281

5. Line 155: This experiment is confusing, if the crystal violet test was performed in microplates, why were the compounds diluted in test tube sets? Next, line 159 again "The tubes were then incubated at …..",please rewrite this paragraph, clarifying the biofilm inhibition assay.

The paragraph and the experiment have been re-written with clarity.

Section 2.5.5 lines 167-174

RESULTS

1. Line 171: Is it missing some letters in the first word?

The missing letter has been included

Line 182

2. Fig 1: the caption is incomplete, it needs to be more informative, mentioning the compound names here instead of putting numbers every time compounds are cited in the text, as it makes reading confusing and tiring

As we stated earlier, the numbers before the names of the compounds are there to connect their structures to their names in fig 1.

Using the numbers when the compounds are mentioned is a conventional practice of dealing with isolated compounds as seen in a number of journals including PLOS One. An example is this publication [2]. The caption of fig 1 has been modified.

Figure 1

3. Line 191: add the information “ethanol extract” into the title of table 1

We have added the ethanol to the information relating to the title of table 1

Line 202

4. Lines 185-187: The results are confusing, are the two sentences contradictory? Or is the second one just an interpretation? If yes, please move the sentence “STF was considerably active against the fungal growth with MIC between 0.25 and 2 mg/mL” to the methods session.

The first statement was a general summary on table 1 focusing on STF and its fractions. The second statement highlights the activity of STF alone before the 3rd statement finalizes on the most active fraction. Since it is a result, we do not think it will be appropriate to move it to the methods sections.

5. Line 229: Is the sentence “The isolated compounds, however, drastically reduced the activity of voriconazole” correct? I think it increased the activity of voriconazole Rephrase it, please

The statement is correct as MIC values correlates indirectly with antimicrobial activity. The lower the MIC value the higher the activity. From the results, the MIC of voriconazole was lower than when it was in combination with the isolated compounds therefore voriconazole alone had a higher activity than when it was combined with the compounds. It is for this reason we indicated that isolated compounds reduced the activity of the voriconazole

DISCUSSION

1. The first paragraph should be deleted because it is confusing for instance (why was cited the reference29?), and not relevant.

The first paragraph served as an introduction to the discussion. We have however deleted

2. Line 271: What does “considerable antifungal activity” mean? Please mention the reference used to define the breaking points in the interpretation of your results

We have re-written that aspect of the discussion and provided reference used to define the breaking points. We have also indicated that in the methods sections

Line 278-279

3. Line 277: I disagree with the statement "low MIC values", as according to a recent systematic review byAlves et.al 2021 (https://doi.org/10.1155/2021/6653311), the values presented in Tables 1 and 2 (crude extract and fractions respectively) reveal only weak or no bioactivity. Please, clarify and discuss your data.

We agree based on the break points and have deleted that statement. We have reviewed the publication you sent and have revised the discussion accordingly

Line 281-286

4. Line 342: This statement is true, but it needs a reference

A reference has been provided.

Line 347, reference [55]

5. Lines 360-361: The sentence “The reported activity justifies its use as an antifungal agent in the treatment of vulvovaginal candidiasis among the Ghanaian populace” must be deleted since it is not supported by data of the current study.

The statement has been deleted

CONCLUSION

1. Lines 368-371: This subject is not a conclusion of the current study, maybe is possible to include it in the discussion session

We have deleted that subject line

Submitted filename: Response to reviewers .docx

Click here for additional data file.

22 Nov 2021

Bioactive triterpenoids from Solanum torvum fruits with Antifungal, Resistance Modulatory and Anti-Biofilm Formation Activities against Fluconazole-Resistant Candida albicans strains

PONE-D-21-19484R1

Dear Dr. Waikhom,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Horacio Bach

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I carefully read the authors' responses and changes made to the new version of the manuscript. I realized that the vast majority of suggestions were accepted, improving the quality of the text. Therefore, I consider it suitable for publication in PLOS ONE. Morevoer, in my opinion the manuscript meets all requirements and recommendations mentioned above.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Terezinha Inez Estivalet Svidzinski

17 Dec 2021

PONE-D-21-19484R1

Bioactive triterpenoids from Solanum torvum fruits with Antifungal, Resistance Modulatory and Anti-Biofilm Formation Activities against Fluconazole-Resistant Candida albicans strains

Dear Dr. Waikhom:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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PLOS ONE