Clinical effect of equol supplementation in the treatment of desquamative gingivitis with 1-year follow-up

Abstract

Purpose: Desquamative gingivitis (DG) is characterized by desquamative erosion, edematous erythema, and vesicle formation on the gingiva. Because of its prevalence in women during the pre- and postmenopausal period, its potential association with female hormones has been suggested. Equol is a soy isoflavone metabolite with a chemical structure similar to estrogen. Scientific evidence suggests that equol helps in alleviating menopausal symptoms. This study evaluated the clinical effect of a 12-month equol supplementation as a substitute for estrogen to alleviate DG symptoms.

Methods: The study enrolled 16 women with DG who regularly visited Nihon University School of Dentistry Dental Hospital. Urinary equol levels, periodontal tissue examination, O’Leary’s plaque control record, stimulated saliva flow rate, and gingival pain-related questionnaires were evaluated before and after the 12-month daily intake of 10 mg equol supplement.

Results: Equol supplementation led to a statistically significant improvement in bleeding on probing, visual findings, and reductions in the frequency and severity of gingival pain.

Conclusion: Urinary equol testing and equol supplementation may be novel treatment options for female patients with DG.

Introduction

Desquamative gingivitis (DG) is a distinctive and complex periodontal condition characterized by desquamative erosion, edematous erythema, and vesicle formation on the gingiva. Typically, DG develops in the labial and buccal gingiva, exhibiting irritation pain and contact pain [1,2,3]. The epithelium and underlying connective tissues undergo histopathological changes associated with DG [1]. The clinical symptoms of DG frequently resemble various dermatoses, including oral lichen planus (OLP), mucous membrane pemphigoid (MMP), and pemphigus vulgaris [1,2,3].

The current classification system for periodontal disease and condition includes, among nonplaque-induced gingival diseases, “autoimmune disease of the skin and mucous membranes” categorized as “inflammatory and immune conditions and lesions” [4]. Although potential causes include hormonal factors [5] , bacteria, and allergies, the exact etiology of DG remains unclear. Notably, DG can arise from multiple factors rather than being a standalone disease [1,2,3]. Consequently, DG has no definitive cure [3]. The main treatment options focus on plaque control and topical corticosteroid application to manage lesions, alleviate symptoms, and prevent disease progression [3,6,7]. However, because of gingival pain, patients with DG often struggle to maintain good oral hygiene, increasing the long-term risk of developing periodontal disease [2,8]. As DG is more common in women during the pre- and postmenopausal period, a potential association with reduced female hormone levels has been suggested [9,10]. Therefore, examining female hormones may provide valuable insights into the etiology and development of treatment methods for DG. Epithelial barrier dysfunction is involved in low estrogen levels, and estradiol (estrogen) reinforces the physical barrier of oral epithelial cells and protects against tumor necrosis factor-α-induced impairment of the epithelial barrier [11]. However, using hormone replacement therapy (HRT) for treating oral conditions can be challenging and may carry the risk of potential side effects.

According to epidemiological research, women who regularly consume soy foods may experience fewer symptoms of menopause, osteoporosis, cardiovascular disease, and cancer [12]. Equol, a metabolite derived from daidzein found in soybeans, has a chemical structure and estrogenic activity similar to estradiol [12,13]. It is absorbed into the intestines, allowing it to perform its biological functions in targeted organs [14]. Finally, any surplus equol is excreted through the urine [14].

By observing patients with DG during the pre- and postmenopausal period and the potential influence of low estrogen levels, equol production, as a substitute for estrogen, was hypothesized to be the key to curing the disease. However, no evidence showed a relationship between DG and equol level. Therefore, this study focused on equol as a pharmacologic therapeutic intervention and examined the clinical effect of equol supplementation on DG treatment.

Materials and Methods

Study participant selection

The study included 16 postmenopausal female patients with DG (aged 58.2 ± 9.3 years; number of teeth, 26.3 ± 3.5) who regularly visited Nihon University Dental Hospital for supportive periodontal therapy between 2020 and 2022. In this study, patients with DG were defined as those who had desquamative erosion, edematous erythema, and vesicle formation on the gingiva, with clinical symptoms such as irritation. Based on the clinical, histopathological, and immunological findings, the following diagnosis was established by oral and maxillofacial surgery specialists [4]: OLP, 10/16 ; MMP, 3/16 and unknown, 3/16. Furthermore, two patients identified with metal allergies through patch testing had as many metal components as possible removed from their mouths.

The exclusion criteria were as follows: (1) systemic or topical antibiotics within 3 months, (2) HRT, (3) aromatase inhibitors, (4) hepatitis C, and (5) soybean allergy.

This study was approved by the Nihon University School of Dentistry Institutional Review Board (EP19D0014) and was conducted in accordance with the 1975 Declaration of Helsinki, revised in 2013. After a full explanation of all aspects of the study, a signed consent form was obtained from all patients.

Urinary equol measurement

Patients were instructed to consume soy foods containing approximately 50 mg of isoflavones on the day before urinary collection. For instance, they could eat one pack of natto (fermented soybeans), half a block of tofu (soybean curd), or drink 200 mL of soy milk, each containing approximately 50 mg of isoflavones. The following morning, the first urine sample of at least 1 mL in a tube was collected at home and sent by the postal service.

Soycheck (Healthcare Systems Co., Ltd., Nagoya, Japan) is an immunochromatographic strip test. In this method, 4 µL of urine was added to 96 µL of an antiequol monoclonal antibody solution, and then 75 µL of the mixture was added to the strip [15]. Equol was measured and calculated using Immunomeasure (Aisin Seiki, Kariya, Japan) [15]. Patients whose equol level was ≥1 µmol/g cre in the spot urine sample were classified as equol producers [15]. The produced equol level was calculated by evaluating the creatinine level (µmol/g cre).

Treatment

All patients received periodontal therapy by periodontists and a periodontal specialized hygienist, including oral hygiene instructions, full-mouth scaling with hand instruments (Hu-Friedy, Chicago, IL, USA) or an ultrasonic scaler (Varios V9, NSK-Nakanishi, Kanuma, Japan), and teeth polishing with a rubber cup and chip at every visit. Patients had been instructed on the scrubbing technique using a soft to extrasoft bristle brush and free lauryl sulfide toothpaste [7]. Floss or rubber interproximal brushes have also been used for self-care plaque control. Patients were asked not to use any mouth rinses except 4% sodium gualenate hydrate (Azunol Gargle liquid, Rotonitten Co., Ltd., Nagoya, Japan). The same professional care and self-care were performed before initiating this study. Patients were seen for professional cleaning once a month for the first 2 months and every 2 months for the following 10 months during equol supplementation.

Clinical measurements and questionnaire survey

The clinical parameters included the following: measured probing depth (PD), percentage of sites of bleeding on probing (BOP), and O’Leary’s plaque control record (PCR). These parameters were assessed at six sites around the teeth using a CP11 probe (Hu-Friedy). The salivary flow rate was measured by chewing paraffin gum for 5 min. One proficient dental hygienist (A.K.) performed all clinical measurements at baseline and at 1, 6, and 12-month intervals.

A questionnaire survey was conducted to assess the frequency and severity of pain in the mouth (gingiva, buccal mucosa, and tongue), soy intake frequency, and oral hygiene habits. Pain severity was examined using the visual analog scale method [16].

Equol supplementation

Patients consumed four tablets (10 mg) of equol supplement, a product classified under the food category, daily for 12 months. In correlation with equol supplementation, progress of the symptoms in the gingiva, buccal mucosa, and tongue was monitored at baseline and at 1, 6, and 12-month intervals. Supplement safety has been verified through a range of tests, including genotoxicity, acute and chronic toxicity, and reproductive and developmental toxicity, except for those with a known soy allergy [17,18,19,20].

Statistical analysis

All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, version 4.0.0, Vienna, Austria) [21]. For longitudinal comparisons of interventional studies, the Friedman test was performed for intragroup comparison of the clinical measurements and equol level and pain severity in the gingiva, buccal mucosa, and tongue at different time points. For pairwise comparisons, the Bonferroni-correlated Wilcoxon signed-rank test was used. Spearman’s rank correlation coefficient was examined for each clinical measurement and equol level. For all tests, P < 0.05 was considered statistically significant.

Results

Demographic characteristics of the patients are shown in Table 1. Only 3/16 (18.8%) patients were equol producers. Regarding oral hygiene habits, the participants reported brushing their teeth at least twice a day and using interproximal brushes or flossing at least once daily.

All 16 patients completed a 12-month equol supplementation program. However, two patients temporarily stopped their supplement intake, with a 2-month interval after a 3-month supplementation and a 6-month interval after a 6-month supplementation. Throughout the study, no side effects were observed in any patient. Table 2 shows clinical measurements before and after equol supplementation, revealing significant differences in mean PD, BOP, PCR, salivary flow rate, and equol levels.

In pairwise comparison (Fig. 1), significant differences were found in the mean PD between baseline and 12 months (P = 0.012) and between 1 and 12 months (P = 0.018). Significant differences were observed in BOP between baseline and different time points (1, 6, and 12 months, P < 0.001). Statistically significant differences between baseline and 6 months (P = 0.031) and between baseline and 12 months (P = 0.010) were also observed in PCR. No significant pairwise difference was observed in the salivary flow rate; however, a significant difference was found in the equol levels between baseline and different time points (1 month, P = 0.003; 6 and 12 months, P < 0.001). The correlation coefficient between BOP and equol level was −0.366 (P = 0.003: Fig. 2), that between BOP and PCR was 0.554 (P < 0.001), and that between PCR and PD was 0.322 (P = 0.009). No other correlations were found between the variables. Fig. 3 shows the changes in the gingiva and buccal mucosa after equol supplementation. Significant improvement in the visual findings of gingiva and buccal mucosa was observed after months of equol supplementation: reduction of desquamative erosion (case 1) and Wickham’s striae (case 4), along with improved healing of the gingiva and buccal mucosa that shed their outer layers of the oral stratified squamous epithelium (cases 2 and 3).

Questionnaire surveys on oral pain frequency and severity and dietary habits were conducted. The pain frequency in the gingiva, buccal mucosa, and tongue tended to change less often after equol supplementation than before equol supplementation (Table 3). A significant decrease in pain severity of the gingiva, buccal mucosa, and tongue was observed after 12 months of supplementation (P < 0.001; Table 4). Moreover, for pairwise comparisons, significant differences between baseline and 12 months (P = 0.012) and between 1 month and 12 months (P = 0.029) were observed in the severity of gingival pain, whereas no significant difference was observed in the buccal mucosa and tongue, respectively (Table 4). Regarding dietary habits, 9/16 patients could tolerate salty food intake at the baseline of this study. After the 12-month supplementation, an additional three patients developed tolerance. Moreover, 4/16 patients could tolerate spicy foods (chili pepper) at the start of this study. Following the 12-month supplementation period, an additional seven patients reported tolerance to spicy foods. An improvement from the baseline was observed. However, no statistically significant difference was observed in the soy intake frequency before and after 12 months of supplementation. Moreover, 14/16 patients stated that they were satisfied with the 12-month equol therapy.

Table 1 Demographic characteristics of the patients

Variable	Mean ± standard deviation (n = 16)
Age (years)	58.2 ± 9.3
Menopause age (years)	50.6 ± 2.4
Body mass index (kg/m2)	22.9 ± 3.9
Number of teeth	26.3 ± 3.5
	Number of people
Equol producers	3
OLP	2
MMP	0
unknown	1
Oral hygiene
twice a day	8
thrice a day	5
four times a day	3

Table 2 Comparison of the clinical measurements and equol levels before and after equol supplementation

Variable	n = 16
	baseline	1 month	6 months	12 months	P-value
Mean PD (mm)	2.0 (1.9-2.2)	2.0 (1.9-2.1)	2.0 (1.8-2.0)	1.9 (1.6-2.0)	0.003**
BOP (%)	12.4 (7.3-16.1)	4.1 (1.2-6.2)	2.7 (1.1-4.8)	1.9 (1.0-3.8)	<0.001**
PCR (%)	38.0 (29.2-55.4)	30.1 (23.9-35.2)	21.4 (14.5-30.8)	26.3 (20.5-34.0)	0.007**
Salivary flow rate (mL)	6.8 (5.8-8.3)	8.0 (6.4-8.3)	8.5 (6.9-11.1)	8.8 (6.3-9.5)	0.025*
Equol level (µmol/g cre)	0.4 (0-0.6)	35.5 (28.3-72.8)	44.1 (28.7-55.8)	29.7 (25.0-47.2)	<0.001**

All values are expressed as median (interquartile range)

The Friedman test was used to determine statistically significant differences before and after equol supplementation. *P < 0.05, **P < 0.01.

 

Fig. 1 Comparison of the clinical measurements before and after equol supplementation

All values are expressed as median (interquartile range). The Friedman test was used to determine statistically significant differences among baseline, 1 month (1 M), 6 months (6 M), and 12 months (12 M). Pairwise comparisons were performed using the Wilcoxon signed-ranked test (P-value adjustment method: Bonferroni). *P < 0.05; **P < 0.01

Fig. 2 Correlation coefficient between bleeding on probing and equol levels

Spearman’s rank correlation coefficient was used.

Fig. 3 Changes in the gingiva and buccal mucosa following equol supplementation

Case 1: Erosive area (>2 cm²) at baseline almost improved after 12 months. Case 2: The lesions on the anterior of the mandibular at baseline improved after 4 months. Case 3: Desquamative area on the labial gingiva at baseline improved after 8 months. Case 4: Wickham’s striae on the buccal mucosa (>1 cm²) reduced after 12 months

Table 3 Comparison of pain frequency in the gingiva, buccal mucosa, and tongue

Variable number (%)	n = 16
	baseline	1 month	6 months	12 months
Gingiva
always	6 (37.5)	4 (25)	3 (18.8)	3 (18.8)
often	6 (37.5)	5 (31.3)	4 (25)	2 (12.5)
sometimes	2 (12.5)	5 (31.3)	1 (6.3)	0 (0)
rarely	2 (12.5)	2 (12.5)	8 (50)	8 (50)
never	0 (0)	0 (0)	0 (0)	3 (18.8)
Buccal mucosa
always	3 (18.8)	3 (18.8)	2 (12.5)	1 (6.3)
often	1 (6.3)	1 (6.3)	0 (0)	0 (0)
sometimes	2 (12.5)	0 (0)	1 (6.3)	2 (12.5)
rarely	3 (18.8)	3 (18.8)	3 (18.8)	5 (31.3)
never	7 (43.8)	9 (56.3)	10 (6.3)	8 (50)
Tongue
always	1 (6.3)	1 (6.3)	0 (0)	0 (0)
often	2 (12.5)	1 (6.3)	0 (0)	0 (0)
sometimes	3 (18.8)	2 (12.5)	0 (0)	1 (6.3)
rarely	2 (12.5)	4 (25)	5 (31.3)	4 (25)
never	8 (50)	8 (50)	11 (68.8)	11 (68.8)

Table 4 Comparison of pain severity in the gingiva, buccal mucosa, and tongue

Pain severity was examined using the visual analog scale method (cm). All values are expressed as median (interquartile range). The Friedman test was used to determine statistically significant differences to compare pain severity before and after equol supplementation. Pairwise comparisons were performed using the Wilcoxon signed-ranked test (P-value adjustment method: Bonferroni). *P < 0.05, **P < 0.01

Discussion

The prevalence of DG is higher in women [4]. The peak incidence of this condition is typically recorded between the ages of 40 and 60 years [1]. In this study, DG developed around menopause in a significant majority of patients, accounting for 14/16 patients. Therefore, lower female hormone levels may be associated with DG development.

To maintain homeostasis, reproduction, development, and differentiation, female hormones, particularly estrogen, play significant roles. Therefore, a decreased estrogen level can lead to various pathological conditions [22]. HRT has long been regarded as the main treatment option for menopause. Despite its effectiveness, HRT often has several side effects. Equol supplementation has become an appealing alternative or complementary treatment to traditional HRT. Scientific evidence suggests that equol helps in alleviating menopausal symptoms, mitigating bone loss, and improving the lipid profile and arteriosclerosis status [12,15,18,20,23,24,25,26].

Estrogen receptor (ER) α and ERβ are particularly well-known as an estrogen receptor. ERα is primarily distributed in female reproductive organs such as the mammary glands and uterus, and is involved in the physiological functions and characteristics of the female genitalia [27]. On the other hand, ERβ is distributed throughout various organs of the body. Due to this widespread distribution, ERβ is believed to play various physiological roles [27]. Equol is expected to exert estrogenic effects on various tissues without affecting reproductive organs since equol has a higher affinity for ERβ [27]. Furthermore, it has been shown to have tissue-specific effects and less influence on the uterus than estrogen preparations [28,29,30]. Additionally, equol may be associated with a lower risk of cancer in the breast [31].

No significant relationship was found between sex and genetic factors and equol production; however, an association was found between equol production with dietary habits (dietary fiber, soybeans, and seaweed) and frequency [12,23]. Only 20-30% of Westerners can produce equol, whereas 50% of Asians can because of their dietary habits [12,26]. Interestingly, in this study of 16 patients, only three demonstrated urinary equol levels above the threshold (1 µmol/g cre), defining them as equol producers, although all patients frequently consumed soy. By contrast, 14/20 (70%) women with PD ≤3 mm and normal gingiva without DG symptoms produced equol in the authors’ previous study (data not shown). Although 17 species of gut bacteria produce equol [24], their relationship and the mechanisms underlying their production remain largely unknown. According to previous research, blood levels of equol peaked within approximately 1-3 h (with a half-life of approximately 8 h) and were subsequently excreted in the urine within 12 h after equol supplementation [19,25], and thus, it is safe and nonaccumulative.

Epithelial cells of the oral mucosa normally have a quick turnover within 7-14 days [32]. Estrogen influences oral tissues via ERβ, affecting the oral mucosa and salivary glands [33]. ERβ is widespread across various cell layers of the oral stratified squamous epithelium [33], leading to the postulation that its effect on the oral epithelium might enhance normal epithelial turnover and the formation of physical barrier functions in the gingiva, buccal mucosa, and tongue [34,35]. After the 12-month supplementation, patients with DG were observed to have a significant BOP improvement. During equol supplementation, effective urinary equol levels (≥10 µmol/g cre) [25,36,37] were detected in all 16 patients. The correlation between BOP and the equol level was significant, despite the absence of correlations between each variable and the equol level. Given the high binding affinity of equol to ERβ [27], equol was assumed to affect ERβ in the oral mucosa, promoting normal keratinization and the formation of physical barrier function [11]. This activity was believed to improve gingival inflammation and reduce oral mucosal pain. Furthermore, equol supplementation improved the epithelium, which may have decreased PCR by reducing pain and increasing the area where patients can clean their teeth. These results revealed that equol supplementation improved the epithelium and reduced BOP. Moreover, reduced PCR may have decreased BOP and improved pain and visual findings. In patients who temporarily discontinued equol intake, an increase in BOP was observed despite a decrease in PCR. These findings suggest that equol supplementation-induced epithelial improvement could be the key to reducing BOP.

In this study, the salivary flow rate was increased by ≥1 mL in 10/16 patients after 12 months of equol supplementation. In 4/16 patients with DG, the salivary flow rate remained largely unchanged. However, in the other 2/16 patients with DG, the salivary flow rate decreased, which was attributed to one patient’s use of sleeping medication (Triazolam 0.5 mg, Teva Takeda Yakuhin Ltd, Nagoya, Japan) and another’s use of an antihistamine drug (Rupafin10 mg, Mitsubishi Tanabe Pharma Co., Ltd., Osaka, Japan) after the initiation of equol supplementation.

ERβ was found in the salivary glands, primarily the submandibular gland, where it is involved in the formation, maintenance, and physiological functions of the gland [30]. The salivary glands show significant sensitivity to drugs and hormones [33]. A previous report suggested the possibility of increased salivary secretion through isoflavone intake [38]. Estrogen administration increases the presence of ER in the adipocyte nuclei of the submandibular gland [30]. Therefore, equol supplementation is believed to increase ERβ in the submandibular gland, potentially leading to increased salivary secretion.

All patients suffered from DG for a period ranging from 6 months to 12 years (mean 4.3 years). In this study, the oral condition of all patients remained unresponsive to symptomatic treatments, including oral hygiene and corticosteroids. Before equol supplementation, to reduce gingival and buccal mucosal pain, corticosteroids were administered to 11 of 16 patients with DG. Of these, 7/11 patients with DG discontinued corticosteroid use because equol intake alleviated the pain. Another patient with DG stopped taking the corticosteroid because it exacerbated gingival exfoliation. For the remaining 3/11 patients with DG (MMP, n = 2; unknown, n = 1) who continued corticosteroid use, their usage frequency significantly decreased with equol supplementation, from every day to once monthly. In this study, none of the patients with OLP used corticosteroids after a 12-month equol supplementation.

Although informative, this study has several limitations. Its relatively small sample size might limit its findings and broader applicability. Furthermore, drawing a definitive comparative conclusion is limited because of the absence of a control placebo group. However, evidence showed that patients’ quality of life significantly improved, although DG symptoms were not completely eradicated after a 12-month equol supplementation. Because equol is a food substance, not a drug, its effects require a comprehensive evaluation over time; hence, further long-term observation is essential.

As far as it is known, this is the first study of patients with DG to confirm the effectiveness of equol and assess its effects after a 12-month equol supplementation. Therefore, these findings provide preliminary but promising insights into the potential benefits of equol supplementation for patients with DG. However, to confirm these findings and further investigate the role and efficacy of equol, more extensive and rigorously designed studies are necessary.

This study found that the urinary equol levels in patients with DG increased after 12 months of equol supplementation, leading to significant improvements in BOP and visual findings and reductions in gingival pain. Moreover, equol supplementation helped in alleviating DG symptoms. Based on these findings, urinary equol testing and equol supplementation may provide new treatment options to improve the clinical symptoms of female patients with DG. Additionally, the authors propose a new diagnosis, menopause-related gingival lesions, for the female DG patients targeted in this study.

Abbreviations

BOP: percentage of sites of bleeding on probing; DG:desquamative gingivitis; ER: estrogen receptor; HRT:hormone replacement therapy; MMP: mucous membrane pemphigoid; OLP: oral lichen planus; PCR: O’Leary’s plaque control record; PD: probing depth

Ethical Statements

This study was approved by the Nihon University School of Dentistry Institutional Review Board (EP19D0014) and was conducted in accordance with the 1975 Declaration of Helsinki, revised in 2013. After a full explanation of all aspects of the study, a signed consent form was obtained from all patients.

The authors declare that they have no conflicts of interest to report. This study was supported by the SoyCheck test kit from Healthcare Systems Co., Ltd. (Nagoya, Japan); however, the company was not involved in data management.

Conflicts of Interest

The authors declare that they have no conflicts of interest to report. This study was supported by the SoyCheck test kit from Healthcare Systems Co., Ltd. (Nagoya, Japan); however, the company was not involved in data management.

Funding

This study was institutionally funded by the Department of Periodontology, Nihon University School of Dentistry, Tokyo, Japan.

Author Contributions

All authors have made substantial contributions to the conception and design of the study. AK, MS, SO, HS, KS, SM, NY, and SSwere involved in recruitment and data collection. AK, NS, and KS were involved in the data analysis. AK and NS were involved in data interpretation and drafting of the manuscript. All authors revised the manuscript critically. All authors have given final approval for this version to be published.

ORCID iD

¹⁾AK*: [e-mail], https://orcid.org/0009-0006-2484-2407

^2,3)NS: [e-mail], NA

¹⁾MS: [e-mail], NA

⁴⁾SO: [e-mail], https://orcid.org/0009-0000-3133-4300

⁴⁾HS: [e-mail], https://orcid.org/0000-0002-5379-0603

⁵⁾KS: [e-mail], https://orcid.org/0000-0002-6130-1259

^2,3)SM: [e-mail], https://orcid.org/0009-0007-6291-5897

^2,3)NY: [e-mail], NA

^2,3)SS: [e-mail], NA

Acknowledgments

The authors express their deepest gratitude to Mr. Yoshikatsu Hosoya and Ms. Yukari Kobayashi of Healthcare Systems, Inc., for their help with this study. The authors also thank Prof. Noboru Noma at Nihon University School of Dentistry for his cooperation in conducting this study. The authors are grateful to Dr. Seidai Murai, the late professor emeritus at Nihon University, for his valuable suggestions.

Data Availability Statements

The data supporting the findings of this study are available from the corresponding author upon reasonable request. The data are not publicly available because of privacy or ethical restrictions.

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