Antifungal Efficacy of Gallic Acid Extracted From Pomegranate Peel Against Trichophyton rubrum : In Vitro Case Study

Abstract

Objectives: Trichophyton rubrum is one of the main pathogens causing superficial dermatophytosis, producing symptoms such as skin itching and pain, which seriously affects the quality of life of patients. Pomegranate peel extract is rich in gallic acid (GA), which has been reported to have biological effects including antifungal activity. However, the morphological and molecular mechanisms underlying the effects of GA on T rubrum are not well understood. The objectives of this study were to determine the antifungal efficacy of GA extracted from pomegranate peel against T rubrum in vitro, and to explore the underlying molecular mechanisms. Methods: The effects of 0-, 0.5-, and 1 mg/mL GA in pomegranate peel extract on T rubrum was investigated by detecting cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Transmission electron microscopy (TEM) was used to analyze the ultrastructure of T. rubrum, and transcriptome sequencing was used to analyze the enrichment pathway of differentially expressed genes. The identification of biosynthesis-related and key genes in the pathways involved using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) technology. Terbinafine hydrochloride (TERB) as a positive control group. Results: Pomegranate peel extract has a GA content of 1.0 mg/mL. Compared with untreated group, following treatment with 1.0 mg/mL GA content is rich in pomegranate peel extract, and the microstructure of T rubrum is destroyed. TEM results show that the number of lipid droplets in T rubrum was significantly increased, mitochondrial vacuoles degenerated, the serosa were damaged, and the boundary of thallus was unclear. In addition, 1 mg/mL GA can significantly inhibit T rubrum proliferation, and its inhibition ability is better than TERB. Transcriptomics results show that GA can change the gene expression profile of T rubrum, specifically: The biosynthesis was blocked, drug resistance was weakened, the transport of ATP-binding cassette (ABC) drugs transporter was increased, and the mitogen-activated protein kinase (MAPK) pathway was significantly inhibited. Conclusions: Pomegranate peel extract is rich in GA, which strongly inhibited the growth of T rubrum and reduced its drug resistance. This extract is a promising natural antifungal agent for clinical use.

Introduction

Trichophyton rubrum is one from the most common pathogenic fungus, causing skin diseases such as tinea capitis, tinea corporis, tinea cruris, and tinea pedis.^1–3 These conditions cause patients to experience discomfort such as itching and pain, which can seriously affect their quality of life. ⁴ Fluconazole, itraconazole, voriconazole, amphotericin B, and terbinafine hydrochloride (TERB) are the most widely used clinical antifungal drugs.^5,6 However, increasing resistance of the pathogens to antifungal drugs, coupled with the potential toxicity and carcinogenicity of the drugs, ⁷ leads to the need for safe, hypoallergenic alternative drugs. Medicines and food from traditional Chinese medicine may be a good source of such alternative treatments.

Pomegranate is part of traditional Chinese medicine. ⁸ Gallic acid (GA; Figure 1A) is a polyphenolic organic compound; is present at relatively high levels in pomegranate peel extract; and has been used in food, biology, medicine, the chemical industry, and other fields.^9,10 GA can inhibit the growth of Staphylococcus aureus and Escherichia coli by affecting biofilms in vitro.^11,12 Some studies have found that GA can inhibit the growth of fungi such as Candida albicans and T rubrum, and T rubrum was the most sensitive fungus tested.^13,14 Notably, a recent study found that GA is a promising adjuvant to conventional amphotericin B in the treatment of cutaneous leishmaniasis. ¹⁵ These results indicate that GA has a certain ability to inhibit the growth of fungi. However, the mechanism of the inhibition of the growth of fungi by GA is very limited. Li et al. found a mechanism of action of GA to inhibit the growth of T rubrum by reducing ergosterol biosynthesis. ¹³ Liberato et al. found that GA can induce changes in membrane integrity and mitochondrial transmembrane potential, production of reactive oxygen species and externalization of phosphatidylserine to inhibit fluconazole resistant C albicans. ¹⁴ Therefore, it is necessary to analyze the characteristics of GA on the growth of T rubrum and elucidate its mechanism.

Figure 1.

Gallic acid (GA) content is rich in pomegranate peel extract. (A) Chemical structure of GA. (B) Determination of GA content in GA standard and pomegranate peel using high-performance liquid chromatography (HPLC).

Based on this, in the present work, we described the antifungal activity and mechanism of GA in pomegranate peel extract against T rubrum via detecting cell viability, ultrastructure with using transmission electron microscope (TEM), biological functions, and signaling pathways using transcriptomics, and the differential signaling pathway-related fungal growth key gene expressions in vitro.

Materials and Methods

Chemicals

Pomegranate peel was purchased from Bengbu Medicinal Materials Co., Ltd (Anhui, China) and GA (purity ≥98%) standard and TERB was obtained from Sigma-Aldrich (St. Louis, MO, USA). Milli-Q ultrapure water (Millipore), methanol, and acetonitrile were of chromatographic grade (Kermel); the rest of the required reagents were of analytical grade.

Extraction Method of GA From Pomegranate Peel Extract

The pomegranate peel was dried at 30°C, pulverized with a pulverizing machine, accurately weighed 1.0080 g, impregnated with 100 mL of 50% methanol aqueous solution for about 6 h, treated with ultrasonic (power 500 W, frequency 40 kHz) for 1 h, filtered, and the residue eluted with 30 mL of 50% methanol aqueous solution, combined with filtrate and washing solution. The dry matter was concentrated with a rotary evaporator, 100 mL of 50% acidified methanol solution (containing 1.2 mol /L hydrochloric acid and 0.2 g vitamin C) was added, reflux at 90°C, and then concentrated with a rotary evaporator to the dry matter, dissolved with methanol, in a constant-volume 100 mL volumetric flask. 10 mL was carefully absorbed into a 50 mL volumetric flask, the volume was fixed with methanol, and the filtrate was filtered with a 0.45 μm microporous filter membrane. The filtrate was used as the test solution.

The Content of GA in Pomegranate Peel was Determined by High-Performance Liquid Chromatography (HPLC)

After the GA standard was made into a solution with 50% methanol, a Waters e2695 HPLC system with a 2998 PDA detector (Waters Corporation) was used to load 10 µL each time, and GA was detected at 270 nm. The solution concentration is the abscissa (x-axis), and the peak area is the ordinate (y-axis). The standard curve of the GA response was determined using 10μL injections of 6 standards (6.637, 13.275, 26.550, 53.100, 106.200, and 212.400 μg/mL) using the equation. The pomegranate peel powder was dissolved in 50% methanol, sonicated for 30 min, and allowed to cool, then 50% methanol was added to the bottle and shaken, and finally the filtrate was obtained by suction filtration through a microporous membrane (0.45 μm) for several times, and the GA content is detected by the above HPLC conditions.

Fungal Strains and Growth Conditions

The strain of T rubrum (ATCC® 28190™) used in this study was purchased from the American Type Culture Collection (ATCC, USA; https://www.atcc.org/). T rubrum were grown on TTC-Shabao weak medium (Solarbio) at 25 °C to 28 °C. The growth of T rubrum was assessed by detecting the optical density (OD) at 612 nm with a microplate reader (Thermo Fischer Scientific).

Fungal Viability Assays

A solution of T rubrum was inoculated in 96-well plates. Either GA (0.5- or 1 mg/mL) and TERB (0.1μg/mL) was added to the experimental groups, and the control group was left untreated. Fungal growth was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kits (NJJCBIO, China). At 0, 24, 48, or 72 h, 10 µL of MTT was added to each well, the plates were incubated at 37 °C for 4 h, and the OD value at 570 nm was detected using a microplate reader (Thermo Fischer Scientific).

TEM

T rubrum suspension was added to a culture tube at a concentration of 2.0  ×  10⁶ CFU/mL. After culture with shaking for 10 h, GA was added to the experimental group at concentrations of 0.5 or 1 mg/mL, and TERB concentration was 0.1μg/mL. T rubrum suspension served as a negative control. The cells were collected via centrifugation at 3000 rpm for 5 min in 2.5% glutaraldehyde, at 4 °C overnight, and then fixed with 1% osmic acid. After fixation, cells were dehydrated with 30%, 50%, 70%, 80%, 95%, and 100% ethanol and propylene oxide. Immediately following embedding in propylene oxide, epoxy resin, and pure epoxy resin, the embedded block was trimmed and cut into 70-mm ultra-thin sections, stained with lead and uranium electrons, and observed using a transmission electron microscope (Olympus, Japan).

RNA-seq Experiments and Analysis

Total RNA was extracted from GA-treated and control cells, and its integrity was checked using RNA Nano 6000 Assay Kits from the Agilent Bioanalyzer 2100 System (Agilent Technologies). Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (New England Biolabs) according to the manufacturer's protocol. Different sets of samples were clustered on the cBot Cluster Generation System using the HiSeq X-Ten/NovaseqS4 PE Cluster Kit (Illumina) according to the manufacturer's instructions. After cluster generation, library preparations were sequenced on the Illumina HiSeq X-Ten/NovaseqS4 platform and 150 bp paired-end reads were generated. ¹⁶

To perform the RNA-seq experiments, T rubrum samples were divided into a control group and a GA-treated group. In the GA group, the concentration of GA applied was 1.0 mg/mL, the incubation time was 2 h, and the samples were washed twice with phosphate buffered saline (PBS). An equal volume of medium was added to the control group and incubated at 37 °C for 2 h.

HaCaT Cell Line

The HaCaT cell line was acquired from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd (Shanghai, China) The medium required for growth of the HaCaT cell line is high glucose Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (BI, BTG) at 37 °C with 5% CO₂.

Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

RNA was extracted from the control group, the GA group, and TERB group using E.Z.N.A Total RNA Kits I was purchased (Omega Bio-tek, Inc.) according to the manufacturer's instructions. The RNA was analyzed using RT-qPCR technology following TransScript® Green One-Step qRT-PCR SuperMix Kit instructions (TransGen Biotech Co. Ltd). It should be pointed out that the expression of the MEP4, Rpb2, Chs1, and SUB1 was directly detected in T rubrum, while the expression of the ABCB1, JUN, FOS, and MAPK14 was detected by mixing the T rubrum suspension with HaCaT cells. These primer sequences are shown in Table 1.

Table 1.

Primer Sequence.

Gene	Primer	Sequence
ABCB1	Forward	5′-GCAGCTGGAAGACAAATACACAA-3′
	Reverse	5′-CCCAACATCGTGCACATCA-3′
Rbp2	Forward	5′-TGCAGGAGCTGGTGGAAGA-3′
	Reverse	5′-GCTGGGAGGTACTGTTTGATCAA -3′
Chs1	Forward	5′-GGCCACAACGAAGCCTATGA-3′
	Reverse	5′-CAATCGGCCTGGGAGATG-3′
SUB1	Forward	5′-CGGTAGGGTTCTCCTGAGCA-3′
	Reverse	5′-GTTGAACAACACGGCTGCAT-3′
MEP4	Forward	5′-CACCTTCCCTGGCTCAAAAC-3′
	Reverse	5′-CGGTAGGGTTCTCCTGAGCA-3′
JUN	Forward	5′-AAGAACTCGGACCTCCTCACCTC-3′
	Reverse	5′-GCCCGTTGCTGGACTGGATTATC-3′
FOS	Forward	5′-TTACTACCACTCACCCGCAGACTC-3′
	Reverse	5′-GGGAATGAAGTTGGCACTGGAGAC-3′
MAPK14	Forward	5′-CTGGCTCGGCACACTGATGATG-3′
	Reverse	5′-GCCCACGGACCAAATATCCACTG-3′
Beta-tubulin	Forward	5′-AACATGATGGCTGCCACTGA-3′
	Reverse	5′-AAGATGGCAGAGCAGGTAAGGT-3

Statistical Analysis

The results of all experiments are expressed as mean  ±  standard error of the mean (SEM, n ≥ 3). GraphPad Prism software Version 8.0 (GraphPad Software; www.graphpad.com) was used for statistical analysis. Nonparametric t-tests were used to identify differences between the 2 groups; P value <.05 were considered to be statistically significant. *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Results

GA Content of Pomegranate Peel Extract

In order to determine whether pomegranate peel extract is rich in GA, we first used HPLC to determine the GA standard. The results showed that GA produced characteristic peaks under excitation at a wavelength of 270 nm, without sharp peaks or tails. We then detected GA in pomegranate peel extract under the same conditions. Pomegranate peel extract showed characteristic peaks similar to those of the GA standard (Figure 1B). The standard curve of GA response was made by taking the solution concentration as the abscissae (x-axis) and the peak area as the ordinate (y-axis). The regression equation of GA was Y  =  28352X  +  8233.8 (r  =  0.9999). The results showed that GA was linear from 6.6375 to 212.40 ug/mL. Subsequently, the GA extract in pomegranate peel was diluted 20 times, and the HPLC detection showed that the peak area was 1432210. After substituting into the standard curve, the GA concentration of pomegranate peel extract was 1.005 mg/mL.

GA in Pomegranate Peel Extract Significantly Inhibited the Growth of T rubrum in vitro

To explore the effect of GA in pomegranate peel extract on T rubrum, we added 0.5 or 1 mg/mL of GA in pomegranate peel extract to T rubrum culture tubes in vitro, and TERB group as positive control group. In the control group, T rubrum grew in suspension and the medium exhibited flocculent turbidity. In the TERB group, the cells of T rubrum were significantly smaller in the culture tube. When GA was added, the cells dissipated and the medium became increasingly clear with the increase in GA concentration in pomegranate peel extract (Figure 2A). MTT assays, a measure of the metabolic activity of the cells, ¹⁷ also showed that when GA in pomegranate peel extract was added, the proliferation of T rubrum decreased significantly (***P  =  .008), and the inhibition of proliferation produced by 1 mg/mL GA was significantly stronger than that produced by 0.5 mg/mL GA and TERB group. Meanwhile, GA at 1 mg/mL in pomegranate peel extract was comparable to TERB in inhibiting the growth of T rubrum (Figure 2B; *P  =  .036).

Figure 2.

Effects of different concentrations of gallic acid (GA) in pomegranate peel extract and terbinafine hydrochloride (TERB) on the growth and ultrastructure of Trichophyton rubrum in vitro. (A) Effects of GA (a) (0.5 mg/mL), GA (b) (1 mg/mL) in pomegranate peel extract, and TERB (0.1μg/mL) on the growth of T rubrum. (B) Effect of GA in pomegranate peel extract and TERB on the proliferation of T rubrum as detected using MTT. (C) Effect of GA in pomegranate peel extract and TERB on the ultrastructure of T rubrum as examined using transmission electron microscope (TEM).

We used TEM to analyze the morphology of the control group, the TERB group, the 0.5 mg/mL GA group and the 1 mg/mL GA group. The surface of T rubrum in the control group was smooth, the cell structure was complete, the cytoplasm was uniform, and the growth was good. In the TERB group, lipid droplets were significantly larger, mitochondrial vacuoles were degenerate, the serosa were damaged, and the boundary of thallus was unclear. In the 0.5 mg/mL GA treatment group, the serosal membrane was thin, the structure was unclear, the hyphae were necrotic, and there were only a few vacuolated mitochondria and lipid droplets, the serious phenomenon were found in the 1 mg/mL group (Figure 2C). These results indicated that compared with TERB, 1 mg/ml GA in pomegranate peel extract had a stronger inhibitory effect on the growth of T rubrum.

Gene Expression Profiles of T rubrum were Altered After GA Treatment in Pomegranate Peel Extract

We compared the expression of genes of T rubrum treated with 1 mg/mL GA in pomegranate peel extract with that of a control group. There were 852 genes differentially expressed between the GA group and the control group, 367 of which were upregulated and 485 downregulated (Figure 3A). Principal component analysis showed that there were large differences between the untreated and treated groups, but not within the groups (Figure 3B). Gene Ontology (GO) enrichment ¹⁸ analysis showed that genes involved in the component membrane, antioxidant, hydrolase activity, RNA silencing, pyknosis, oxidoreductase activity, and membrane of GA-treated thallus were upregulated, whereas genes involved in response to drug, single-organism metabolic process, cytochrome complex assembly, single-organism process, nucleotide binding, and tRNA aminoacylation were downregulated (Figure 3C). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway ¹⁹ analysis showed that after the treatment with GA, the genes involved in ATP-binding cassette (ABC) transportation, biosynthesis of secondary metabolites, and antioxidant metabolism in the T rubrum thallus were significantly upregulated, whereas those involved in metabolic pathways, glutathione metabolism, aminoacyl tRNA biosynthesis, mitogen-activated protein kinase (MAPK) signaling pathway, tryptophan metabolism, and fatty acid metabolism were downregulated (Figure 3D).

Figure 3.

Transcriptomic analysis of biological enrichment of genes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of differentially expressed genes in Trichophyton rubrum after gallic acid (GA) in pomegranate peel extract treatment. (A) Histogram of the number of differentially expressed genes after GA treatment. (B) Principal component analysis (PCA) plots of untreated and GA-treated groups. (C) Gene Ontology (GO) enrichment analysis of differentially expressed genes in GA-treated fungus compared with untreated fungus. (D) KEGG pathway analysis of differentially expressed genes in GA-treated fungus compared with untreated fungus.

GA Treatment Decreased the Expression of Genes Related to Biosynthesis in T rubrum

Rpb2 and Chs1 are the most stable and highly expressed genes of T rubrum. ²⁰ RT-qPCR results showed that compared with the untreated group, the mRNA expressions of Rpb2 and Chs1 of T. rubrum were significantly decreased in the GA treated group (**P < .01). In addition, we found that the GA in pomegranate peel can reduce SUB1 and MEP4 mRNA expression in T rubrum (Figure 4A; **P < .01). These results show that GA treatment decreased the expression of genes related to biosynthesis in T rubrum.

Figure 4.

Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) technology was used to analyze the changes of Trichophyton rubrum biosynthesis-related genes and the mitogen-activated protein kinase (MAPK) pathway in HaCaT cell line in in TERB, GA-treated, and untreated groups. (A) The expression of biosynthesis-related genes in T rubrum. (B) The expression of key genes of MAPK signaling pathway in HaCaT cell line after GA intervention in pomegranate peel extract.

GA Treatment Reduced ABC, and MAPK Pathway-Related Genes in HaCaT Cell Line

We used transcriptome sequencing to investigate the expression of ABC- and MAPK-related genes after the application of TERB, and GA in pomegranate peel extract. qRT-PCR results showed that after treatment with 1 mg/mL of GA and 0.1μg/mL of TERB, the expression of ABCB1 in HaCaT cell line was significantly increased (****P < .0001), whereas the expression of JUN, FOS, and MAPK14 was significantly reduced (Figure 4B, ****P < .0001).

Discussion

T rubrum is one of the most common fungi that skin infections.^1–3 At present, fluconazole, itraconazole, and other drugs are used for the treatment of fungal skin infections, but due to drug resistance, toxicity, and other unfavorable factors, there is an urgent need for alternative drugs with high safety. ²¹ Treatments used in traditional Chinese medicine may provide a good alternative. Pomegranate peel extract is rich in polyphenolic bioactive substances such as GA, which has anti-inflammatory and bacteriostasis effects. ²² In this study, we found that pomegranate peel is rich in GA, which could inhibit the growth and destroy the structure of T rubrum in vitro. In addition, GA treatment changes the gene expression profile of T rubrum and reduced the expression of genes related to biosynthesis.

First, we explored the optimal conditions for extracting GA from pomegranate peels, and found that the solubility of methanol in the semi-polar solvent was good, and the interference peaks of HPLC were less, so methanol was the best solvent for extracting GA from pomegranate peel. Nishant et al also found that methanol was the best solvent for pomegranate peel extraction. ²³ In addition, the antioxidant vitamin C was added to the extraction solvent to reduce oxidation during GA concentration and purification. ²⁴ Finally, we found the GA content in pomegranate peel was measured as 1.005 mg/mL, a finding that provides the basis for the chemical extraction and use of GA in biopharmaceuticals.

Next, we evaluated the effect of GA in pomegranate peel extract on T rubrum in vitro. TERB is the common antifungal drugs in clinic, which can inhibit the activity of squalene epoxidase and interfere with ergosterol synthesis in T rubrum. ²⁵ It was found that after TERB treatment, the “flocculent mass” of T rubrum. became smaller, while 1 mg/mL GA showed better antifungal effect, which showed that the “flocculent mass” basically disappeared, and the medium was clear and transparent. Li et al. also found that GA can inhibit fungi in vivo, ¹³ although they did not study the effect of GA on the morphology of T rubrum. TEM is a large-scale analytical technique, which is widely used to observe the morphology and composition of the surface ultrastructure of various solid substances. ²⁶ The TEM results showed that, compared with the untreated group, lipid droplets were increased, mitochondrial vacuoles were degenerated, the serosa was damaged, and the boundary was unclear in the TERB group, while treated with 1 mg/mL GA, the serous membrane of T rubrum was damaged more seriously, and the mitochondrial vacuoles were more obvious. The complete cell wall and mitochondrial morphology of T rubrum are the basis of its growth and infection. ²⁷ These results showed that GA in pomegranate peel extract can significantly inhibit the growth and destroy the structure of T rubrum and the antifungal ability was stronger than TERB.

However, the molecular mechanism by which GA in pomegranate peel extract inhibits T rubrum is still unclear. We used transcriptome sequencing to clarified potential mechanisms. Compared with the untreated group, antioxidant and hydrolase activity, RNA silencing, pyknosis, and oxidoreductase activity were enhanced in the GA-treated group, indicating that GA in pomegranate peel extract can promote cell membrane damage and increase fungal necrosis. We also found that after GA treatment, the response to drugs, single-organism metabolic process, cytochrome complex assembly, single-organism process, nucleotide binding, and tRNA aminoacylation of T rubrum were weakened, which suggested that GA in pomegranate peel extract can significantly reduce the metabolism, biosynthesis, and drug resistance of T rubrum. This finding provides evidence for GA in pomegranate peel extract as a treatment for T rubrum infection.

Finally, we explored the changes in KEGG pathways and related molecules following GA in pomegranate peel extract treatment. ABCB1 is a member of a drug transporter family. It has been reported that the upregulation of ABCB1 can reduce the drug resistance of bacteria. ²⁸ Our results also suggest that the expression of ABCB1 was significantly increased after the application of GA, suggesting that GA in pomegranate peel extract can reduce the resistance of T rubrum and increase its sensitivity. MEP4 and SUB1 are key enzymes involved in ergosterol biosynthesis in T rubrum.^29,30 We found that GA could significantly reduce the expression of MEP4 and SUB1, indicating that GA in pomegranate peel extract inhibited the biosynthesis of T rubrum. Finally, we also found that GA can downregulate the expression of MAPK signaling through the key genes JUN, MAPK14, and FOS and that MAPK activation is mainly involved in the inflammatory response, proliferation, and other processes,^31,32 which indicates that GA in pomegranate peel extract can significantly inhibit the induction of T rubrum infection, activation, invasion, and other biological behaviors.

Conclusions

In conclusion, the current research results support that GA is a promising natural antifungal compound. In vitro analysis showed that GA could significantly inhibit the growth of T rubrum, destroy the structure of T rubrum, and its inhibitory activity was higher than TERB. Therefore, optimizing the extraction process of GA from pomegranate peel, conducting animal experimental research, and gradually transiting to clinical research, GA may become a substitute for current antifungal drugs.