Abstract
Background and purpose: Vulvovaginal candidiasis (VVC), caused by
Candida albicans (C. albicans), is exacerbated by oxidative stress and
uncontrolled inflammation. Pathogens like C. albicans generate reactive
oxygen species (ROS) to enhance virulence, while host immune responses
further amplify oxidative damage. This study investigates the
antioxidant and antifungal properties of Hyssopus cuspidatus Boriss
volatile extract (SXC), a traditional Uyghur medicinal herb, against
fluconazole-resistant VVC. We hypothesize that SXC’s bioactive
volatiles counteract pathogen-induced oxidative stress while inhibiting
fungal growth and inflammation. Methods: GC-MS identified SXC’s major
bioactive components, while broth microdilution assays determined
minimum inhibitory concentrations (MICs) against bacterial/fungal
pathogens, and synergistic interactions with amphotericin B (AmB) or
fluconazole (FLC) were assessed via time–kill kinetics. Anti-biofilm
activity was quantified using crystal violet/XTT assays, and in vitro
studies evaluated SXC’s effects on C. albicans-induced cytotoxicity
(LDH release in A431 cells) and inflammatory responses (cytokine
production in LPS-stimulated RAW264.7 macrophages). A murine VVC model,
employing estrogen-mediated pathogenesis and intravaginal C. albicans
challenge, confirmed SXC’s in vivo effects. Immune modulation was
assessed using ELISA and RT-qPCR targeting inflammatory and
antioxidative stress mediators, while UPLC-MS was employed to profile
metabolic perturbations in C. albicans. Results: Gas
chromatography-mass spectrometry identified 10 key volatile components
contributing to SXC’s activity. SXC exhibited broad-spectrum
antimicrobial activity with MIC values ranging from 0.125–16 μL/mL
against bacterial and fungal pathogens, including fluconazole-resistant
Candida strains. Time–kill assays revealed that combinations of AmB-SXC
and FLC-SXC achieved sustained synergistic bactericidal activity across
all tested strains. Mechanistic studies revealed SXC’s dual antifungal
actions: inhibition of C. albicans hyphal development and biofilm
formation through downregulation of the Ras1-cAMP-Efg1 signaling
pathway, and attenuation of riboflavin-mediated energy metabolism
crucial for fungal proliferation. In the VVC model, SXC reduced vaginal
fungal burden, alleviated clinical symptoms, and preserved vaginal
epithelial integrity. Mechanistically, SXC modulated host immune
responses by suppressing oxidative stress and pyroptosis through
TLR4/NF-κB/NLRP3 pathway inhibition, evidenced by reduced caspase-1
activation and decreased pro-inflammatory cytokines (IL-1β, IL-6,
TNF-α). Conclusions: SXC shows promise as a broad-spectrum natural
antimicrobial against fungal pathogens. It inhibited C. albicans hyphal
growth, adhesion, biofilm formation, and invasion in vitro, while
reducing oxidative and preserving vaginal mucosal integrity in vivo. By
disrupting fungal metabolic pathways and modulating host immune
responses, SXC offers a novel approach to treating recurrent,
drug-resistant VVC.
Keywords: volatile extracts of Hyssopus cuspidatus Boriss, vulvovaginal
candidiasis, Candida albicans, biofilm, riboflavin, oxidant stress
1. Introduction
Vulvovaginal candidiasis (VVC), a prevalent gynecological infectious
disease, has emerged as a critical global public health challenge.
Epidemiological studies indicate that approximately 500 million women
worldwide are affected annually, with about 138 million cases
progressing to recurrent VVC (RVVC), leading to significant declines in
quality of life and increased mental health burdens [[40]1,[41]2]. The
pathogenesis of VVC is closely associated with the hyphal transition of
Candida species, with clinical manifestations including intense vulvar
itching, burning pain, and in severe cases, dyspareunia, dysuria, and
urinary frequency, all of which impair physiological function and
social engagement [[42]3]. Although azole antifungals (e.g.,
fluconazole) remain first-line therapies, the widespread emergence of
drug-resistant strains (linked to drug overuse and biofilm formation)
and adverse effects (e.g., hepatotoxicity, gastrointestinal
disturbances) severely limit their long-term efficacy [[43]4]. Notably,
5–10% of refractory VVC cases are attributed to azole-resistant C.
albicans [[44]5], underscoring the urgent need for novel, efficient,
low-toxicity antifungal strategies targeting drug-resistant strains.
In recent years, bioactive compounds derived from medicinal plants have
attracted significant attention due to their multifaceted mechanisms of
action and reduced propensity to induce resistance compared to
synthetic antimicrobials [[45]6,[46]7]. Among these, Hyssopus
cuspidatus Boriss (H. cuspidatus), a characteristic Uyghur medicinal
herb of the Lamiaceae family native to Central Asia and northwestern
China, stands out for its traditional use in treating cold-damp
respiratory disorders, attributed to its warming, expectorant, and
antitussive properties [[47]8]. While the pharmacological activities of
its congener Hyssopus officinalis (European hyssop) have been
extensively studied, H. cuspidatus exhibits distinct morphological and
phytochemical profiles due to geographic divergence (European–West
Asian vs. Central Asian arid zones) [[48]9,[49]10]. Critically, H.
cuspidatus exhibits significantly higher pharmacological
potency—particularly for anti-asthmatic effects—despite its markedly
lower biomass yield (roughly 1/10 of H. officinalis) [[50]11].
Previous studies demonstrate that fragrant extracts from H. cuspidatus
exhibit superior inhibitory effects against Staphylococcus aureus,
Escherichia coli, and C. albicans compared to its aqueous extracts
[[51]12]. However, the antifungal mechanisms of such essential oils
remain poorly understood. Our research group has developed a
specialized technique to optimize the extraction of volatile active
components from H. cuspidatus (SXC), with preliminary studies revealing
broad-spectrum antifungal activity, especially against C. albicans,
including fluconazole-resistant strains. Our work specifically
elucidates novel pathways—including riboflavin deprivation,
Ras1-pathway suppression, and host cell protection—unexplored in
earlier research.
Building on this, the present study systematically investigates the
mechanisms underlying SXC oil’s efficacy against drug-resistant C.
albicans and its therapeutic potential for VVC mice. Key research
priorities include elucidating its impact on hyphal transition, biofilm
formation, and modulation of host immune responses via antioxidants and
inhibiting pyroptosis pathways. This study aims to provide novel
insights for the development of safe and effective antifungal agents,
while establishing a theoretical basis for a phytomedicine-based
targeted therapeutic system for VVC.
2. Materials and Methods
2.1. Reagents and Chemicals
SXC was provided by Dingyuan Biomedical Technology (Tianjin, China).
Phosphate buffered saline (PBS), cell culture media,
penicillin–streptomycin, and fetal bovine serum (FBS) were obtained
from Thermo Fisher Scientific (Waltham, MA, USA). Amphotericin B and
fluconazole were obtained from MedChemExpress (Beijing, China).
Essential Oil Extraction Methodology: Hyssopus cuspidatus Boriss
whole-plant material was coarsely chopped and mixed with distilled
water at a ratio of 1:4–5 (w/w). The mixture was soaked at 25–30 °C for
8–10 h, after which the water was drained. The hydrated biomass
underwent steam distillation using saturated steam delivered at 15–40
m/s. The resulting vapor was condensed, and the essential oil was
separated from the hydrosol by decantation. The oil was dehydrated over
anhydrous sodium sulfate and stored at 4 °C until use.
2.2. SXC Composition Analysis
A 10 µL aliquot of SXC essential oil was diluted with 990 µL of
n-hexane (100-fold dilution, v/v), followed by a second 10-fold
dilution (v/v) using 100 µL of the initial diluted solution mixed with
900 µL of n-hexane, yielding a total dilution factor of 1:1000 (v/v).
Chromatographic separation was performed on a DB-5MS fused-silica
capillary column (30 m × 0.25 mm × 0.25 µm; Agilent Technologies, Santa
Clara, CA, USA) with helium (99.999% purity) as the carrier gas at a
constant flow rate of 1.0 mL/min. The injection volume was 1 µL in
splitless mode, and the needle wash solution was n-hexane (HEX). The
oven temperature program initiated at 60 °C (held for 5 min), then
increased at 4 °C/min to 100 °C (held for 2 min), followed by a 6
°C/min increase to 240 °C (held for 5 min), and finally increased at 10
°C/min to 300 °C (held for 2.67 min). Mass spectra were acquired in
full-scan mode (mass range: m/z 50–500) using an electron ionization
(EI) source, with the ion source temperature maintained at 250 °C,
quadrupole mass analyzer at 150 °C, and transfer line temperature at
280 °C.
2.3. Bacterial Strains, Fungal Strains, and Growth Conditions
C. albicans (ATCC 64550), used in cell culture and mice infections, was
purchased from the American Type Culture Collection (ATCC). C. albicans
(ATCC 90028, ATCC 10231), C. tropicalis (ATCC 750), C. krusei (ATCC
6258), E. coli (ATCC 25922, ATCC 35128), S. aureus (ATCC 25923, ATCC
33591), E. faecalis (ATCC 29212), S. epidermidis (ATCC 12228, ATCC
51625), were also purchased from ATCC. All the bacterial strains were
routinely grown in Tryptic Soy Broth (TSB) (Hope Bio-Technology,
Qingdao, China) at 37 °C for 12 h at 200 rpm in the shaker (Eppendorf,
Hamburg, Germany), and all the fungal strains were grown in Sabouraud
Dextrose Broth (SDB) (Hope Bio-Technology, Qingdao, China) at 37 °C for
24 h at 200 rpm in the shaker.
2.4. Minimal Inhibitory Concentration (MIC) Determination
The antibacterial and antifungal activities of compounds were
determined according to the 2021 Clinical and Laboratory Standards
Institute drug sensitivity standard protocol [[52]13]. Bacteria (1 ×
10^6 CFU/mL) were incubated with the compounds in a 96-well plate for
12 h at 37 °C. The compounds were in two-fold dilutions, with final
concentrations of AmB 0.0625 μg/mL and FLC ranging from 2 to 64 μg/mL.
MICs were defined as the lowest concentrations of the compounds to
cause no growth of bacteria.
2.5. Checkerboard Assay
The synergistic effect of amphotericin B (AmB), fluconazole (FLC) and
SXC was determined by performing standard checkerboard broth
microdilution assays. AmB, FLC and SXC were serially diluted in
eight-fold steps. Optical density 600 nm (OD[600]) was examined after
incubation at 37 °C for 24 h. The FICI was calculated as shown below to
analyze the synergistic effect using the concentration with the highest
combination effects:
[MATH:
FICI = (MIC o
f A in <
mi>the combination/MIC o
f A alon
e)+(MIC of B in the
combinat
ion/MIC<
/mi> of B alone)
:MATH]
A synergistic effect is defined as FICI ≤ 0.5, while an antagonistic
effect is defined as FICI ≥ 4.0. In addition, an indifferent effect is
defined as 0.5 < FICI < 4.0.
2.6. Time–Kill Curves
Time–kill curves were examined for AmB and SXC alone or in combination
for different strains at 37 °C with an initial bacterial concentration
of 5 × 10^3 CFU/mL. A 200 μL aliquot at different time points (0, 2, 4,
8, 12, 24, 48 h) was obtained and plated on Yeast Extract Peptone
Dextrose Medium (YPD) agar plates after serial dilution. Fungal
colonies were counted.
2.7. Effect of SXC on Biofilm Formation by C. albicans and Preformed Biofilms
The amount of biofilm formation was quantified with crystal violet (CV)
staining [[53]14]. Briefly, for the biofilm formation assay, C.
albicans ATCC64550 suspension was prepared in Roswell Park Memorial
Institute (RPMI) 1640 medium at a concentration of 1 × 10^6 cells/mL
with 0.25, 0.5 and 1 µL/mL concentrations of SXC, 1% Tween 80 as the
control, and 200 μL added to the wells of sterile 96-well culture
polystyrene plates (LABSELECT, Beijing, China). The plates were
incubated statically at 37 °C, and at 12 h, 24 h, and 48 h, the medium
was aspirated. The wells were washed three times with PBS (pH 6.25).
Methanol (100 μL per well) was added to fix the samples for 15 min,
followed by air-drying at room temperature. A 0.1% (w/v) crystal violet
solution (100 μL per well) was added and incubated at room temperature
for 30 min. Excess stain was removed by washing three times with PBS.
Absolute ethanol (100 μL per well) was added and incubated for 1 h at
25 °C. The absorbance at 595 nm was measured using a microplate reader,
with RPMI-1640 medium serving as the blank control. The biofilm
formation rate was calculated using the following formula:
[MATH:
Biofilm forma
tion ra<
mi>te 100%=<
mo>(A595 Dru
g−A595 Blank)/(A
595 Tween 80−A595 Blank)
:MATH]
For the preformed biofilms, 200 μL of activated yeast cells in
suspension (1 × 10^6 cells/mL) was added to the wells of 96-well
plates. After incubation at 37 °C for 24 h, the plates were washed
three times with PBS. Then, 200 μL of RPMI 1640 medium containing 0.25,
0.5 and 1 µL/mL concentrations of SXC was added to the 96-well plates.
After incubation at 37 °C for 24 h, the metabolic activity of the
biofilm was determined with the XTT (Macklin, Shanghai, China)
reduction assay [[54]15]. The optical absorbance was measured at 450 nm
on the microplate reader (BIO-RAD, iMark, Boston MA USA).
For the preformed biofilms, 1 mL of activated yeast cells in suspension
(5 × 10^3 cells/mL) was added to the wells of 12-well plates. After
incubation at 37 °C for 24 h, the plates were washed three times with
PBS. Then, 200 μL of RPMI 1640 medium containing 0.031, 0.062 and 0.125
µL/mL concentrations of SXC was added to the 12-well plates. After
incubation at 37 °C for 24 h, a 0.1% (w/v) crystal violet solution (100
mL per well) was added and incubated at room temperature for 20 min.
After discarding the staining solution, the plates were washed three
times with PBS, air-dried, and then imaged under a 10× inverted
microscope.
2.8. Cell Cultivation
The A431 cell line, obtained from Pricella, Wuhan, China, was cultured
in RPMI 1640 containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL
streptomycin (Thermo Fisher Scientific). The Mouse Mononuclear
Macrophages Cells (RAW) 264.7 cell line was provided by the Cell
Resource Center of Peking Union Medical College (Beijing, China) and
was cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 100
μg/mL streptomycin. All of the cells were maintained in a humidified
incubator with 5% CO[2]. The cell lines were checked to be negative for
mycoplasma.
2.9. LPS Stimulates RAW264.7 Cells
The RAW 264.7 cell suspension was inoculated into a 6-well plate at 1 ×
10^5/well, 2 mL per well for 24 h. The supernatant was discarded, and
SXC was added to the culture medium containing 10% FBS DMEM to prepare
0, 1, 20 nL/mL SXC solutions. LPS was added at the same time to bring
the final concentration to 750 ng/mL, with a no treatment group as a
control group, for 18 h. Following the stimulation period, the cells
and supernatant were collected for additional analysis to examine the
effects of the different treatments.
2.10. Lactate Dehydrogenase (LDH) Assay
For cell infections, A431 cells (1 × 10^5 cells/well) were seeded in
48-well plates for 12 h. C. albicans, grown in SDB broth to the
logarithmic phase, was added to the seeded cells at a multiplicity of
infection (MOI) of 10 with SXC (0, 1, 20 nL/mL) for 10 h. No infection
cells were used as the control group with RPMI 1640 medium supplemented
with 10% FBS. The levels of LDH released into the culture supernatants
were measured using a LDH cytotoxicity assay kit (Beyotime, Shanghai,
China). The absorbance signal of 490 nm was measured by a Microplate
Reader (BioTek, Windsor, VT, USA).
2.11. Enzyme-Linked Immunosorbent Assay (ELISA)
Cytokine concentrations in the vagina were measured using mouse TNF-α,
IL-6, IL-10, IL-1β, IL-18 and MDA ELISA kits (Bioswamp, Wuhan, China),
SOD and GSH ELISA kits (NJJCBIO, Nangjing, China) according to the
manufacturer’s instructions ([55]http://www.njjcbio.com/ (accessed on
14 May 2024) and [56]https://www.bio-swamp.com/ (accessed on 16 May
2024)). The content of SOD, GSH and MDA in the culture supernatants
were measured according to the manufacturer’s instructions.
2.12. Gram Stanning
A431 cells were seeded at a density of 1 × 10^5 cells/mL onto 20 mm
glass coverslips placed in 12-well plates, with 1 mL per well. Cells
were incubated overnight for 12 h at 37 °C, 5% CO[2]. Non-adherent
cells were removed by washing the coverslips three times with DPBS. C.
albicans, grown in SDB for 24 h, was added to the seeded cells at a
multiplicity of infection (MOI) of 10 with SXC (0, 1, 20 nL/mL) for 3
h. No infection cells were used as the NC group with RPMI 1640 medium
supplemented with 10% FBS. Coverslips were gently washed three times
with DPBS to remove non-adherent C. albicans. A Gram Staining Kit
(Beyotime, Shanghai, China) was used to detect C. albicans morphology
and A431 cells according to the manufacturer’s instructions. Stained
coverslips were examined under a 40× phase-contrast inverted
microscope.
2.13. VVC Model
Female Institute of Cancer Research (ICR) 8 week old mice were obtained
from Vital River Biotechnology (Beijing, China). All animals were
maintained in specific pathogen free facilities under standard
conditions (12 h light/12 h dark cycle, 22 ± 1 °C, 55 ± 5% relative
humidity, and free access to food and water). The mice were
acclimatized for 3 d before treatment.
Each mouse received a subcutaneous injection of estradiol benzoate (0.2
mg/mL) at a volume of 100 μL per mouse. Injections were administered
once every 2 d for a total of three administrations. After the
estradiol benzoate regimen, 60 mice were randomly selected for
infection. These mice were intravaginally inoculated with 10 μL of a
30% glycerol suspension of C. albicans (ATCC 64550) at a concentration
of 5 × 10^8 CFU/mL. The control group received an intravaginal
injection of an equivalent volume of sterile physiological saline. This
inoculation procedure was performed daily for 7 consecutive days. Group
Assignment and Treatment: After the 7 d inoculation period, the 60
infected mice were randomly divided into five experimental groups (n =
12 per group): model group, ciclopirox olamine group (CTZ, 150 μg/mL),
fluconazole group (FLC, 150 μg/mL), SXC-L group (5 μL/mL), and SXC-H
group (50 μL/mL). Each group received daily intravaginal administration
of 10 μL of the respective treatment for 7 consecutive days. External
vaginal signs were monitored daily throughout the treatment period. On
the day 8 post-treatment initiation, vaginal lavage fluid was collected
from all mice, diluted 10-fold, and analyzed using an Abbott Cell-Dyn
3700 hematology analyzer to quantify leukocyte counts. Subsequently,
the mice were euthanized, and vaginal tissues were harvested for
further analysis.
2.14. Hematoxylin and Eosin (H&E) Staining
The fixed vagina tissues were embedded in paraffin using standard
techniques. Longitudinal sections of 5 μm thickness were stained with
H&E stains (Servicebio, Wuhan, China) and photographed by optical
microscope (Olympus CKX41, Tokyo, Japan).
2.15. Reverse Transcription and Quantitative Polymerase Chain Reaction
(RT-qPCR)
For the mice and cells, total RNA was extracted from vagina tissues
using an RNA Purification Kit (Beyotime, Shanghai, China) and then
reverse transcribed with a HiFiScript cDNA Synthesis Kit for qPCR
(CWBIO, Taizhou, China). Real-time PCR was performed with UltraSYBR
Mixture (Low ROX) (CWBIO) on an Applied Biosystems 7500 Fast Real-Time
PCR System (Applied Biosystems, Carlsbad, CA, USA). The primers used
for PCR amplification are listed in [57]Supporting Information Table
S1.
For the C. albicans, total RNA was extracted from colon tissues using
an RNA Purification Kit (Beyotime, Shanghai, China). Real-time PCR was
performed with a UniPeak U+ One Step RT-qPCR SYBR Green Kit (Vazyme,
Nanjing, China) on an Applied Biosystems 7500 Fast Real-Time PCR
System. The primers used for PCR amplification are listed in
[58]Supporting Information Table S2. The fold changes in mRNA
expressions were normalized to GraphPad Prism 9 using the ΔΔCt method.
2.16. Untargeted Metabolomic Analysis
For untargeted metabolomic analysis, a C. albicans ATCC64550 suspension
was prepared in SDB at a concentration of 1 × 10^6 cells/mL, exposed to
0, 0.125, or 0.25 µL/mL SXC essential oil, and incubated at 37 °C for
24 h. The fungal suspension was centrifuged at 6000 rpm for 5 min, and
the pellet was washed three times with DPBS followed by centrifugation.
To extract metabolites, 100 µL H[2]O was added to the pellet and
vortexed, followed by 400 µL methanol, vortexing for 1 min, and
sonication for 5 min. The mixture was centrifuged at 13,300 rpm for 10
min, and 80 µL of the supernatant was transferred to an injection vial
for liquid chromatography-mass spectrometry (LC-MS) analysis.
Chromatographic separation was performed on a BEH-C18-5 cm column at 30
°C with a flow rate of 0.3 mL/min and an injection volume of 3 µL. The
mobile phase consisted of A (0.1% formic acid in acetonitrile) and B
(0.1% formic acid in water), with the following gradient: 0–2 min (5%
A, 95% B), 2–10 min (5% A to 100% A, 95% B to 0% B), 10–12 min (100%
A), 12–12.1 min (100% A to 5% A), and 12.1–13.5 min (5% A). Mass
spectrometry was conducted in ESI positive and negative ion modes with
full-scan acquisition (50–1000 Da, 1 sec/scan, continuum data format).
The scan timeline was set as 0–2 min (divert to waste), 2–12 min
(direct to source), and 12–13.5 min (divert to waste).
2.17. Statistical Analysis
Data were processed by GraphPad Prism 10.0 software (La Jolla, CA, USA)
and were presented as mean ± standard deviation (SD). One-way ANOVA was
used to compare the multiple groups to evaluate the statistically
significant variance. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. model
group.
3. Results
3.1. Chemical Composition Analysis of SXC via GC-MS
The chemical composition of SXC was systematically characterized using
gas chromatography-mass spectrometry (GC-MS). Quantitative analysis
revealed ten major constituents, which collectively accounted for
95.39% of the total mass ([59]Table 1). The dominant components
included beta-pinene (39.64%), pinocarvone (23.04%), and
iso-pinocarvone (12.90%), followed by alpha-phellandrene (9.31%),
limonene (2.22%), carvone (0.62%), myrtenal (3.10%), methyl myrtenyl
ether (3.20%), carveol (0.86%), and methyl myrtenate (0.50%). To ensure
robustness, we performed repeated analyses across multiple independent
batches of SXC material. These analyses consistently revealed highly
conservative chemical profiles.
Table 1.
Identification of chemical composition of SXC.
Serial Number Chemical Compound Peak Area Aspect Ratio CAS
1 Beta-pinene 14,484,451.87 0.3964 127-91-3
2 Alpha-phellandrene 3,403,795.41 0.0931 4221-98-1
3 Methyl myrtenyl ether 1,153,267.56 0.032 202527-57-9
4 Pinocarvone 8,419,063.42 0.2304 30460-92-5
5 Iso-pinocamphone 4,718,913.25 0.129 18358-53-7
6 (+)-Limonene 812,852.16 0.0222 5989-27-5
7 (+−)-Myrtenal 1,133,324.76 0.031 18486-69-6
8 (−)-Carvone 229,361.68 0.0062 6485-40-1
9 Methyl myrtenate 183,471.5 0.005 30649-97-9
10 Carveol 315,911.66 0.0086 99-48-9
[60]Open in a new tab
Our GC-MS analysis quantified ten major constituents comprising 95.39%
of the total composition, with β-pinene (39.64%), pinocarvone (23.04%),
and iso-pinocamphone (12.90%) as dominant antimicrobial terpenoids.
This profile aligns with literature on Xinjiang-sourced H. cuspidatus,
confirming its characteristic high pinocarvone/β-pinene ratio—a
chemotypic signature differing markedly from Mediterranean H.
officinalis. The geographical and seasonal specificity explains SXC’s
enhanced bioactivity [[61]16]: the arid climate and flowering stage
harvest optimize terpenoid synthesis, with β-pinene and pinocarvone
demonstrating strong antimicrobial and anti-inflammatory effects
[[62]17].
[63]Figure 1a presents the GC-MS chromatogram highlighting the
separation and identification of these ten compounds, while [64]Figure
1b illustrates their respective chemical structures. This comprehensive
profiling provides a foundation for understanding the bioactive
composition of SXC.
Figure 1.
[65]Figure 1
[66]Open in a new tab
GC-MS was used to identify the main chemical components in SXC. (a)
GC-MS chromatograms of ten compounds and a mixed standard solution of
SXC. (b) beta-pinene, alpha-phellandrene, methyl myrtenyl ether,
pinocarvone, iso-pinocarvone, limonene, myrtenal, carvone, methyl
myrtenate, and carveol, pinocarvone, isopinocamphone, (+)-limonene,
(+/−)-myrtenal, (−)-carvone, methyl myrtenate, and carveol. Chemical
structure of the ten components.
3.2. Inhibition of Bacterial and Fungal Virulence by SXC In Vitro
We found that SXC has a broad-spectrum anti-microbial effect. MIC of
SXC against bacteria and fungi, as determined by the broth
microdilution method, ranged from 0.125 to 16 µL/mL, which is rarely
seen in plant purifications ([67]Table 2). Further time–kill assays
demonstrated that the bactericidal effect of SXC oil against C.
albicans was dose-dependent, particularly for the drug-resistant
strains ([68]Figure 2a). This finding has significant clinical
relevance, given the challenges in managing refractory fungal
infections like Candida.
Table 2.
MICs of SXC.
Strains ATCC MIC (μL/mL)
Staphylococcus epidermidis ATCC12228 0.25
Staphylococcus epidermidis ATCC51625 4
Enterococcus faecalis ATCC29212 8
Staphylococcus aureus ATCC25923 4
Staphylococcus aureus ATCC33591 8
Escherichia coli ATCC25922 8
Escherichia coli ATCC35128 16
Candida albicans ATCC90028 0.125
Candida albicans ATCC10231 0.125
Candida albicans ATCC64550 0.25
Candida tropicalis ATCC750 0.25
Candida krusei ATCC6258 0.25
[69]Open in a new tab
Figure 2.
[70]Figure 2
[71]Open in a new tab
In vitro antifungal activity of AmB-SXC and FLC-SXC combinations
against C. albicans strains. (a) Time–kill curves of SXC against C.
albicans strains (n = 3). (b) Checkerboard analysis of AmB-SXC and
FLC-SXC combinations. OD600 values were measured using a microplate
reader and visualized as a color gradient (dark purple: growth; white:
no growth). Red boxes indicate combinations with the highest
synergistic activity. (c) Time–kill curves of C. albicans strains
treated with AmB-SXC and FLC-SXC combinations at fractional inhibitory
concentration index (FICI) concentrations (n = 3).
Checkerboard analysis was employed to assess the efficacy of antibiotic
combinations compared to their individual activities, and the
fractional inhibitory concentration index (FICI) was used to reflect
the combined antibacterial effect. The results indicated that SXC, at
concentrations of 0.031–0.062 µL/mL, could exert an
antifungal-sensitizing effect on FLC and AmB, reducing the MIC of FLC
and AmB to at least 1/8 of its original value ([72]Table 3 and
[73]Table 4, [74]Figure 2b). FICIs were calculated using combination
concentrations with the highest synergistic activity, specifically the
combinations of 1/8–1/4 × MIC of AmB, 1/8 × MIC of FLC, and 1/4 × MIC
of SXC. Overall, all these results demonstrated that the combinations
of AmB-SXC and FLC-SXC exhibited significant synergistic activity in
vitro against the tested C. albicans strains.
Table 3.
MICs and FICIs of AmB with SXC against C. albicans strains.
MIC Alone Combined FICI
AmB
(μg/mL) SXC
(μL/mL) AmB
(μg/mL) SXC
(μL/mL)
C. albicans ATCC10231 0.0625 0.125 0.015 0.031 0.5
C. albicans ATCC90028 0.0625 0.125 0.015 0.031 0.5
C. albicans ATCC64550 0.0625 0.25 0.008 0.062 0.375
[75]Open in a new tab
Table 4.
MICs and FICIs of FLC with SXC against C. albicans strains.
MIC Alone Combined FICI
FLC
(µg/mL) SXC
(µL/mL) FLC
(µg/mL) SXC
(µL/mL)
C. albicans ATCC10231 4 0.125 0.5 0.031 0.375
C. albicans ATCC90028 2 0.125 0.25 0.031 0.375
C. albicans ATCC64550 64 0.25 8 0.062 0.375
[76]Open in a new tab
Time–kill assays were conducted to investigate the synergistic effects
of AmB, FLC, and SXC against all tested strains over time. The
concentrations of AmB, FLC, and SXC derived from FICI analyses were
utilized to construct time–kill curves. As shown in [77]Figure 2c,
monotherapy with AmB (1/8 or 1/4 × MIC), FLC (1/8 × MIC), or SXC (1/4 ×
MIC) induced fungal death within 0–4 h, followed by resumption of
stable fungal growth after 4 h, reaching approximately 10^10–10^11
CFU/mL at 48 h. In contrast, the AmB–SXC and FLC–SXC combinations
demonstrated pronounced growth inhibition across all three strains
throughout the 0–48 h observation period. Consequently, these
combinations exhibited significant synergistic activity against all
tested strains, manifesting a typical bactericidal pattern with
sustained suppressive effects.
3.3. SXC Alleviates Vaginal Lesions and Promotes Mucosal Repair in a VVC
Mouse Model
We initially established a VVC mouse model and validated the protective
effects of SXC, with the experimental timeline illustrated in
[78]Figure 3a. Briefly, VVC was established by priming with estradiol,
followed by Candida inoculation 1 week before treatment, and continuous
observed these animals for 8 days post drug application. We used the
drug-resistant strain ATCC 64550 to mirror refractory VVC cases. This
setup helps prove SXC’s efficacy in treatment-resistant scenarios. The
ATCC 64550 strain is resistant to FLC, so FLC treatment had no effect
([79]Figure 3b–f), while CTZ is effective and therefore served as a
positive drug.
Figure 3.
[80]Figure 3
[81]Open in a new tab
SXC attenuates vaginal damage and promotes mucosal repair in VVC mice.
(a) Schematic diagram of the animal experimental procedure. (b)
Representative external vaginal photographs of treated mice on day 1,
day 3, day 5, and day 8 following treatment. (c) Vaginal tissue
photographs of treated mice on day 8 post-treatment with C. albicans (n
= 4). (d) Statistical analysis of external vaginal inflammation scores
in treated mice on day 8 post-treatment (n = 6). (e) Leukocyte counts
in vaginal lavage fluid of treated mice on day 8 post-treatment (n =
5). (f) C. albicans colony counts in vaginal lavage fluid of treated
mice on day 8 post-treatment. (g) Protein expression levels of IL-10,
IL-6 and TNF-αin vaginal tissues measured via ELISA (n = 8). (h) mRNA
transcript levels of IL-10, IL-6 and TNF-α in vaginal tissues measured
via RT-qPCR (n = 8). Statistical significance is indicated as follows:
* p < 0.05, ** p < 0.01, *** p < 0.001, compared to the model group;
‘ns’ denotes not significant. (i) Representative histology sections
stained with H&E (magnification 40×; scale bar = 20 μm). The black
boxes in the upper figure indicate the regions of interest. The lower
figure shows a magnified view of these boxed areas. The arrow marks the
infiltration of immune cells.
VVC patients typically present with substantial vaginal discharge
accompanied by erythema and edema [[82]18]. To determine whether C.
albicans induced vaginal damage in mice, we documented vaginal
morphology across all treatment groups. Photographic observations on
days 1, 3, and 5 post-modeling revealed that mice in the control group
maintained normal vaginal architecture without erythema, mucus
secretion, ulceration, or hemorrhage. In contrast, the model group, CTZ
group, FLC group, SXC-L group, and SXC-H group exhibited varying
degrees of erythema, mucus secretion, ulceration, and hemorrhage
([83]Figure 3b), which were corroborated by vaginal pathology scores
and are quantified in [84]Figure 3d. At day 8 post-treatment, vulvar
erythema was substantially resolved in both the SXC-H and CTZ groups
([85]Figure 3b). Post-euthanasia vaginal tissue dissection revealed
pronounced edema in model group mice, which was alleviated to varying
extents in the treatment groups ([86]Figure 3c).
Simultaneously, ELISA and RT-qPCR assays revealed that the levels of
pro-inflammatory cytokines IL-6 and TNF-α in vaginal tissues of VVC
model mice were significantly elevated compared to the normal control
(NC) group, while the anti-inflammatory cytokine IL-10 was markedly
reduced. SXC treatment significantly reversed these alterations and
alleviated vaginal inflammation in VVC mice ([87]Figure 3g,h). We
quantified leukocyte infiltration in vaginal lavage fluid. The model
(MO) group demonstrated significant leukocytosis compared to NC, while
the SXC-H and CTZ groups exhibited marked leukocyte reduction
comparable to the MO group ([88]Figure 3e). Concurrent analysis of
fungal burden showed that the SXC-H and CTZ groups achieved significant
fungal load reductions, whereas the FLC group displayed no therapeutic
effect ([89]Figure 3f).
To investigate the effects of SXC on tissue damage in VVC mice, we
performed H&E staining on vaginal tissue sections. Histological
examination revealed distinct morphological alterations among groups
([90]Figure 3i). The control group exhibited intact mucosal
architecture with resolution of pseudoestrus-induced squamous
hyperplasia and keratinization in the vaginal epithelium (VE) [[91]19].
In contrast, the model group demonstrated complete disruption of the
mucosal layer, characterized by VE keratinization loss, epithelial
desquamation, marked inflammatory cell infiltration, and concurrent
mucification with submucosal (SM) edema ([92]Figure 3i). SXC treatment
demonstrated dose-dependent histopathological improvement. The SXC-H
group showed partial restoration of both mucosal layer integrity and
keratinized epithelial stratification, although mild residual
mucification and SM edema persisted. The CTZ-treated group exhibited
restored keratinization in the VE but lacked discernible mucosal layer
reconstruction, with only slight SM edema remaining ([93]Figure 3i).
3.4. SXC Protects Against Vaginal Mucosal Injury by Inhibiting Oxidative
Stress and Pyroptosis Through the TLR4/NF-κB/NLRP3 Signaling Pathway
In mucosal inflammatory diseases, excessive ROS disrupt the mucosal
redox balance and induce oxidative stress, thereby promoting
pathological progression [[94]20,[95]21]. In the VVC model, C. albicans
invasion leads to increased cellular oxidative stress. Both in vivo and
in vitro experiments demonstrated that SXC administration, compared to
the model group, significantly inhibited the oxidative stress-induced
elevation of MDA and reduction of GSH and SOD. This alleviated
oxidative stress in vaginal and immune cells, thereby protecting
cellular mitochondria and thus enhancing the antioxidant capacity of
the vaginal mucosa ([96]Figure 4a,c).
Figure 4.
[97]Figure 4
[98]Open in a new tab
SXC treats VVC mice by exerting antioxidant effects and suppressing the
NLRP3 pathway to produce anti-inflammatory actions. (a) Levels of GSH,
SOD, and MDA in vaginal tissues (n = 7). (b) mRNA transcript levels of
TLR4, NF-κB, NLRP3, Caspase-1, IL-18 and IL-1β in vaginal tissues
measured via RT-qPCR (n = 8). (c) Levels of GSH, SOD, and MDA in the
supernatant of LPS-stimulated RAW264.7 cells (n = 6). (d) mRNA
transcript levels of inflammatory cytokines HO-1, NF-κB, NLRP3,
Caspase-1, IL-18 and IL-1β in LPS-stimulated RAW264.7 cells (n = 6).
Statistical significance is indicated as follows: * p < 0.05, ** p <
0.01, *** p < 0.001, compared to the model group; ‘ns’ denotes not
significant. (e) SXC suppresses oxidative stress and pyroptosis by
inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.
Upon pathogen stimulation (e.g., fungi), cytosolic pattern recognition
receptors (PRRs) assemble into multiprotein complexes that induce
Caspase-1 activation [[99]22]. Activated Caspase-1 cleaves pro-IL-1β
and pro-IL-18, facilitating their maturation and release [[100]23]. Our
results demonstrate that C. albicans infection upregulated mRNA
expression of TLR4, NLRP3, Caspase-1, NF-κB, IL-1β, and IL-18 in murine
vaginal tissues, which was significantly attenuated following SXC
intervention ([101]Figure 4b). We found that protein expression of
IL-1β and IL-18 were downregulated in murine vaginal tissues after
treatment with SXC ([102]Supplementary Figure S1). Complementary in
vitro studies confirmed that SXC modulates macrophage NLRP3 signaling
by suppressing LPS-induced NF-κB and IL-1β elevation, inhibiting NLRP3
inflammasome activation, and reducing Caspase-1 and IL-18 activation
([103]Figure 4d). Our study also revealed that SXC can stimulate the
production of HO-1, but no activation of Nrf2 was observed in vitro
([104]Figure 5b).
Figure 5.
[105]Figure 5
[106]Open in a new tab
SXC inhibits C. albicans biofilm formation and reduces its adhesion to
A431 cells. (a) Inhibitory effect of SXC on C. albicans biofilm
formation at 12 h and 24 h, assessed by crystal violet assay (n = 6).
(b) Disruptive effect of SXC on mature C. albicans biofilms (24 h-old),
measured by crystal violet assay (n = 6). (c) Metabolic activity of
mature C. albicans biofilms after SXC treatment, evaluated by XTT assay
(n = 6). (d) LDH release rate in cell culture supernatants (n = 6).
Statistical significance is indicated as follows: *** p < 0.001,
compared to the model group; ‘ns’ denotes not significant. (e) Effect
of SXC on biofilm-related gene expression. Expression of
biofilm-associated genes was detected via quantitative reverse
transcription polymerase chain reaction (RT-qPCR) (n = 3). C. albicans
untreated with SXC served as the NC group. Gene expression was
normalized to 18S rRNA levels in each sample, with NC group expression
set to 1 for each gene. (f) Gram staining of C. albicans hyphae
adherent to A431 cells. Scale bar = 50 μm. The arrow indicates the
formation of fungal hyphae.
Collectively, these results indicate that C. albicans infection
initiates macrophage inflammatory responses through the
TLR4/NF-κB/NLRP3 pathway, amplifying tissue inflammation via cascading
effects and leading to severe infection [[107]24]. However, SXC
treatment effectively interrupts this inflammatory cascade, thereby
reducing tissue inflammation.
3.5. SXC Inhibits C. albicans Hyphae and Biofilm Formation and Attenuates its
Adhesion to and Invasion of A431 Cells via the Ras1/cAMP/Efg1 Pathway
The formation of biofilms is frequently associated with the development
of fungal drug resistance; consequently, inhibiting fungal biofilm
formation represents a crucial strategy in antifungal drug development
[[108]25]. SXC inhibited C. albicans biofilm formation in a
dose-dependent manner by disrupting the multi-stage process—initial
adhesion (12 h), intermediate colonization (24 h), and maturation (48
h). ([109]Figure 5a,b and [110]Figure S4). CV staining assays revealed
that SXC significantly reduced the biomass of C. albicans biofilms
compared to the control group ([111]Figure 5a,b). Furthermore, the XTT
reduction assay demonstrated that SXC also diminished the metabolic
activity of C. albicans biofilms ([112]Figure 5c).
Biofilm formation encompasses multiple aspects, including adhesion
capacity and hyphal development, and is regulated by numerous
associated genes. To further investigate the potential antifungal
mechanisms of SXC, this study evaluated its impact on the expression of
biofilm-associated genes using RT-qPCR. In C. albicans, cAMP is
synthesized by the adenylate cyclase Cyr1 [[113]26]. Research indicates
that PKA is the sole component directly downstream of cAMP and upstream
of Efg1 in the cAMP-PKA signaling pathway, where it activates Efg1
through phosphorylation [[114]27]. Among the hyphal-related genes
assessed following SXC treatment, the expression of Ras1, Cyr1 and Efg1
was significantly downregulated ([115]Figure 5e), suggesting that SXC
might suppress hyphal development and biofilm formation in C. albicans
by downregulating the Ras1-cAMP-Efg1 signaling pathway. Additionally,
compared to the control group, SXC-treated samples exhibited
significant downregulation of several genes involved in adhesion and
hyphal formation, including Ece1, Ume6, Pde2, Als3, and Hwp1, while the
expression of Tup1, a negative regulator of biofilm formation, was
significantly upregulated ([116]Figure 5e).
VVC represents an immunoinflammatory mucosal disorder caused by C.
albicans, where adherence of the pathogen to vaginal epithelial cells
initiates infection [[117]28]. Consequently, primary therapeutic
strategies for VVC focus on interfering with C. albicans adhesion
[[118]29]. Gram staining revealed abundant C. albicans hyphal
formations in the model group (as compared to the NC group), while SXC
treatment significantly reduced hypha-mediated adhesion to A431
epithelial cells ([119]Figure 5f). Furthermore, LDH release assays
demonstrated that SXC intervention effectively attenuated C. albicans
invasiveness, as evidenced by significantly reduced cellular LDH
release in treated groups ([120]Figure 5d).
3.6. Metabolic Reprogramming in C. albicans Induced by SXC Treatment
Volcano plot analysis revealed profound metabolic perturbations in C.
albicans following SXC exposure ([121]Figure 6a). [122]Figure 6b
illustrates the differential metabolite profiling, where each data
point corresponds to an individual metabolite plotted by
log[2]-transformed fold change (FC, x-axis) and −log[10]-transformed
p-value (y-axis) derived from statistical comparison between
SXC-treated and untreated control groups.
Figure 6.
[123]Figure 6
[124]Open in a new tab
Metabolic effects of SXC on Candida albicans. (a) Metabolomics heatmap
of C. albicans after SXC treatment (n = 6). (b) Metabolomics volcano
plot of C. albicans after SXC treatment (n = 6). (c) Content plot of
significantly different metabolites (n = 6).
Stringent statistical thresholds were applied to identify biologically
relevant changes: metabolites exhibiting log[2] FC > 1 (equivalent to
FC > 2 or FC < 0.5) with p < 0.05 were classified as significantly
dysregulated. Upregulated compounds meeting these criteria are
highlighted in red, while downregulated metabolites are denoted in
blue. Non-significant changes (p ≥ 0.05) appear as gray points
clustered near the log[2] FC = 0 baseline.
This analysis revealed that SXC induces extensive metabolic
reprogramming, with multiple metabolites demonstrating statistically
significant dysregulation compared to controls ([125]Figure 6a,b). SXC
treatment selectively upregulated the concentrations of
glutathionate^−, beta-Citraurol, dGDP and chitobiose, while levels of
lyrsinone, rubraflavone C, LysoPE(0:0/14:1(9Z)), ganoderenic acid A and
related compounds were markedly reduced ([126]Figure 6c). Chitobiose is
a key intermediate metabolite in the chitin pathway, essential for
maintaining cellular morphology and environmental stress resistance in
C. albicans [[127]30]. Glutathionate^− (reduced glutathione anion,
GSH^−) functions as a core antioxidant and redox homeostasis regulator
in fungal cells, to suppress the yeast-to-hyphal transition mediated by
the Efg1/cAMP-PKA signaling pathway [[128]31]. After SXC treatment,
chitobiose and glutathionate^− levels significantly increased in C.
albicans, suggesting that SXC may reverse the impaired hyphal formation
and biofilm development ([129]Figure 6c). LysoPE(0:0/14:1(9Z)) serves
as a substrate for acyltransferase (LPLAT) and is rapidly re-acylated
into intact phosphatidylethanolamine (e.g., PE (16:0/14:1)) via the
Lands cycle, thereby maintaining membrane integrity [[130]32]. Notably,
SXC treatment markedly reduced levels of LysoPE(0:0/14:1(9Z)) in C.
albicans ([131]Figure 6c). These findings confirm that SXC exerts
profound metabolic regulation through targeted modulation of specific
biochemical pathways.
3.7. SXC Modulates C. albicans Growth Through Riboflavin Metabolic
Reprogramming
Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database,
pathway enrichment analysis of differential metabolites detected in
positive and negative ionization modes was performed. Using
hypergeometric testing, the top 25 significantly enriched metabolic
pathways among the differential metabolites were screened, revealing
that SXC exerted a significant impact on the riboflavin metabolism
pathway in C. albicans ([132]Figure 7a). Further investigation
demonstrated that SXC treatment significantly reduced the levels of
riboflavin and FAD, while significantly increasing the level of reduced
riboflavin in C. albicans ([133]Figure 7b). Riboflavin is essential for
FAD, and FAD is required by succinate dehydrogenase, which plays a
central role in energy production within the mitochondrial respiratory
chain [[134]33]. We propose that SXC inhibits C. albicans growth and
exerts its antifungal effect by impairing energy metabolism through the
reduction of riboflavin synthesis in C. albicans.
Figure 7.
[135]Figure 7
[136]Open in a new tab
SXC modulates C. albicans growth and hyphal formation by affecting
riboflavin metabolism. (a) KEGG pathway enrichment analysis of cationic
and anionic metabolites in C. albicans. (b) Levels of riboflavin,
reduced flavin, and flavin adenine dinucleotide (FAD) in C. albicans
metabolites (n = 6). (c) C. albicans biomass after 24-h treatment: SXC
(0.5 μL/mL), Riboflavin (100 μg/mL), SXC (0.5 μL/mL) + riboflavin (100
μg/mL) supplementation (n = 3). Statistical significance is indicated
as follows: ** p < 0.01, *** p < 0.001, compared to the model group;
‘ns’ denotes not significant. (d) Crystal violet staining of hyphal
formation: SXC (0.031, 0.062, 0.125 μL/mL) treatment groups. SXC (0.125
μL/mL) + riboflavin (100 μg/mL) rescue group. Scale bar = 100 μm.
Riboflavin supplementation experiments revealed that 100 µg/mL
riboflavin rescued the fungicidal effect of 0.5 µL/mL SXC against C.
albicans ([137]Figure 7c). CV staining further showed that 100 µg/mL
riboflavin rescued the growth inhibitory effect of 0.125 µL/mL SXC on
C. albicans, although riboflavin supplementation had no significant
effect on the SXC-mediated inhibition of hyphal growth ([138]Figure
7d). Therefore, we propose that SXC inhibits C. albicans growth
primarily by reducing riboflavin synthesis, with a lesser impact on
hyphal formation.
4. Discussion
4.1. Broad-Spectrum Antimicrobial Activity of SXC Essential Oil
This study confirms that SXC essential oil exhibits potent
broad-spectrum antibacterial and antifungal activities. Notably, SCX
demonstrates significant inhibitory effects against diverse bacterial
strains, aligning with prior reports on its antimicrobial efficacy
[[139]12]. Its antifungal activity is particularly evidenced by
effective suppression of C. albicans hyphal transition and biofilm
formation, along with marked reduction of fungal burden in a VVC animal
model. These results highlight the dual capacity of SXC oil to
concurrently impede fungal proliferation and virulence-associated
morphological switching. As a mechanistic conclusion, SXC employs a
dual therapeutic strategy against VVC by simultaneously addressing
symptomatic relief and treating the root causes ([140]Figure 8).
Figure 8.
[141]Figure 8
[142]Open in a new tab
Mechanistic conclusion. SXC employs a dual therapeutic strategy against
VVC by simultaneously addressing symptomatic relief and treating the
root causes. For symptomatic management, SXC exhibits direct antifungal
activity through inhibiting Candida replication, hyphal development and
biofilm formation. At the same time, it mitigates host inflammation, to
alleviate mucosal irritation. Beyond symptom control group, SXC targets
root causes by disrupting fungal energy metabolism—reducing riboflavin
availability to starve pathogens, downregulating Ras1-mediated
hyphal/biofilm pathways, and counteracting oxidative stress and
pyroptosis in host cells. This dual-action approach offers a
comprehensive therapeutic profile that combines rapid symptom
alleviation with sustained anti-relapse potential, making SXC a
potential remedy for clinical refractory VVC.
4.2. Antifungal Mechanisms of Action
The antifungal mechanism of SXC oil combines direct fungicidal effects
with profound metabolic disruption. At the cellular level, it
effectively inhibits biofilm development, reduces fungal proliferation,
and blocks the critical hyphal transition phase in C. albicans—a
morphological shift essential for tissue invasion [[143]34].
Simultaneously, SXC executes targeted metabolic sabotage by interfering
with fungal energy production pathways ([144]Figure 8).
This metabolic intervention initiates with suppression of riboflavin
biosynthesis—a pathway vital for fungal survival—potentially linked to
SXC-mediated inhibition of two key enzymes: riboflavin kinase and GTP
cyclohydrolase [[145]35]. Enzymatic inhibition depletes intracellular
FAD and FMN, which are essential cofactors for mitochondrial
respiration and fatty acid oxidation, thereby impairing lipid
β-oxidation and oxidative phosphorylation, ultimately starving fungi of
energy [[146]36]. Exogenous riboflavin supplementation in C. albicans
systems rescued colony growth but failed to restore hyphal formation
defects, indicating distinct regulatory networks governing
proliferation versus morphogenesis.
SXC downregulates genes in the Ras1-cAMP-Efg1 pathway, likely reducing
cAMP and ATP levels, which subsequently represses hyphae-associated
genes (e.g., ECE1 and HWP1). Inactivation of this pathway disrupts C.
albicans growth, energy metabolism, yeast-to-hypha transition, and
biofilm formation [[147]37].
The metabolic perturbation induced by SXC represents a novel antifungal
paradigm. By targeting fungus-specific metabolic vulnerabilities rather
than conventional cellular structures, this strategy may circumvent
common resistance mechanisms. Reduced FAD/FMN levels not only
compromise energy metabolism but also disrupt redox homeostasis and
detoxification pathways, further jeopardizing fungal survival
[[148]38].
4.3. Antioxidant-Derived Host Protection
VVC is characterized by superficial fungal infection, vaginal mucosal
damage, and fungal bioburden [[149]39]. In mucosae, ROS overload alters
redox balance, triggering oxidative stress and signal transduction
cascades that perpetuate inflammation and cellular damage [[150]40].
This study demonstrates that SXC restores the balance between defensive
and invasive factors by: suppressing oxidative stress: reducing the
destructive mediator MDA while elevating protective factors SOD and
GSH; modulating inflammation: inhibiting NLRP3 inflammasome activation
via Toll-like receptor recognition of C. albicans β-glucans, thereby
blocking NF-κB-mediated release of IL-1β/IL-18; and attenuating
pyroptosis: preserving vaginal mucosal integrity by curtailing
inflammation-associated programmed cell death [[151]41].
In a murine vaginitis model, SXC conferred therapeutic benefits beyond
direct antifungal activity, including host-protective effects. It
preserved mucosal integrity while mitigating pathological immune
responses: histological assessment revealed attenuated neutrophil
infiltration, and vaginal lavage fluid showed diminished oxidative
stress markers. This immunomodulatory profile delivers dual therapeutic
advantages—resolving infection-associated tissue damage while
reinforcing host defenses against fungal resistance.
4.4. Treatment of Both Symptoms and Root Causes
SXC demonstrates compelling antifungal potential through its dual
mechanism of action: directly disrupting fungal replication while
dynamically modulating host-pathogen interactions. This approach
simultaneously inhibits symptoms (e.g., Candida replication, biofilm
formation, hyphal development, and tissue inflammation) and root causes
(e.g., by depriving energy production, downregulating Ras1-mediated
pathways, and counteracting oxidative stress and pyroptosis in host
cells). These properties align with emerging paradigms in antifungal
development that prioritize synchronous targeting of microbial
physiology and host-protective pathways [[152]42]. Like many natural
products with anticancer and analgesic properties [[153]43,[154]44],
SXC shows similar broad-spectrum efficacy that targets both symptoms
and underlying causes, indicating promising potential in these areas.
4.5. Limitations and Future Directions
Despite the significant advancements revealed in this study, several
critical considerations warrant emphasis to contextualize findings and
guide subsequent investigations. While SXC shows promising synergistic
effects with conventional antifungals in vitro, these interactions
require validation within dynamic host–pathogen interaction
models—particularly in chronic infection settings. The current fungal
panel, although inclusive of clinically relevant species, remains
relatively restricted; expanding evaluations to multidrug-resistant and
emerging fungal pathogens (e.g., Candida auris, azole-resistant
Aspergillus spp.) would better establish SXC’s clinical relevance
across diverse etiological contexts. Furthermore, the specific
bioactive compounds within SXC and their synergistic mechanisms remain
unclear and require systematic phytochemical and mechanistic profiling.
As seen in studies of other natural products [[155]45], elucidating the
precise composition and functional pathways of complex natural extracts
is essential to unlocking their therapeutic potential.
5. Conclusions
SXC emerges as a promising natural antimicrobial with broad-spectrum
activity against fungal pathogens. Its multimodal mechanism of action
simultaneously targets: (1) virulence attenuation through inhibition of
hyphal morphogenesis, (2) metabolic disruption via riboflavin
biosynthesis blockade, and (3) host immunomodulation by tempering
inflammatory responses. Demonstrated efficacy across preclinical models
supports its future clinical therapeutic development, particularly for
topical formulations against VVC. This plant-derived agent presents a
sustainable alternative to conventional antifungals, addressing the
critical need for novel therapies in an era of escalating antimicrobial
resistance.
Acknowledgments