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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.50 No.4 pp.127-137
DOI : https://doi.org/10.11620/IJOB.2025.50.4.127

Antibacterial activity of Maclura tricuspidata against Streptococcus mutans

Eun-Sook Kim1,2, Yun-Seong Lee1,2, Jooyi Kang2, Yong-Ouk You1,2*
1Wonkwang Dental Research Institute, Wonkwang University, Iksan 54538, Republic of Korea
2Department of Oral Biochemistry, School of Dentistry, Wonkwang University, Iksan 54538, Republic of Korea
*Correspondence to: Yong-Ouk You, E-mail: hope7788@wku.ac.krhttps://orcid.org/0000-0002-7754-3033
July 9, 2025 September 30, 2025 October 15, 2025

Abstract


The present study evaluated the antibacterial efficacy of Maclura tricuspidata root (MTroot) extracts against the cariogenic bacterium Streptococcus mutans. The chloroform (CHCl3) fraction obtained from organic solvent partitioning of the MTroot ethanol extract was subfractionated using open-column chromatography on silica gel. Among the subfractions obtained, subfraction 3 (Fra-3) obtained in 1:1 (v/v) n-hexan:ethyl acetate solvent system exhibited the most potent antibacterial activity. Accordingly, the chemical composition was analyzed using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry in positive electrospray ionization mode. In addition, the antibacterial activity of Fra-3 against S. mutans was assessed at each stage of extraction. While the minimum inhibitory concentrations (MICs) of the CHCl3, EtOAc, n-butanol, and aqueous fractions against S. mutans were above 15, 120, 120, and 120 μg/mL, respectively, the MICs of the subfractions obtained from silica gel open-column chromatography using different n-hexane:ethyl acetate ratios (3:1, 1:1, and 1:3) were 3.0, 1.5, and 25.0 μg/mL, respectively. Fra-3 was tentatively found to contain 38 major compounds, including kuwanon C, kuwanon E, cudratricusxanthone L, morusin C, cycloartocarpesin, kuwanon A, mulberrofuranol, and moracin U, which accounted for 66.7% of the total ion response. Collectively, these findings suggest that kuwanon C, kuwanon E, and cudratricusxanthone L present in MTroots may serve as novel therapeutic targets to prevent dental caries caused by S. mutans.


Maclura tricuspidata , Dental caries , Streptococcus mutans , UPLC-Q-TOF-MS

초록

    © The Korean Academy of Oral Biology. All rights reserved.

    This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Introduction

    Among the bacteria present in dental plaque, Streptococcus mutans is recognized as the primary agent responsible for the development of dental caries. Common antimicrobials that possess antimicrobial activity against S. mutans include chlorhexidine (CHX), penicillin, erythromycin, and tetracycline [1,2]. However, these antimicrobials are associated with various adverse events, including oral mucosal desquamation, calcium deposition, digestive disturbances, hypersensitivity reactions, and the emergence of drug-resistant bacteria [3-5]. These issues have prompted research into the development of antimicrobials derived from natural sources with improved safety profiles [6-9].

    Traditionally, the bark of Maclura tricuspidata has been used to strengthen the body and promote overall health. The stem of M. tricuspidata improves blood circulation [10], the fruit and leaves (15–30 g) have been used to alleviate rheumatoid arthritis [11], and the root has been used to treat hepatitis, especially viral hepatitis [12]. In Korea, the Donguibogam (1613 A.D., Joseon Dynasty) recorded that the M. tricuspidata tree has been used to treat eczema, mumps, tuberculosis, bruises, and acute arthritis [12]. Over the years, numerous chemical compounds have been isolated from M. tricuspidata, with xanthones and flavonoids identified as the primary active ingredients [13-15]. Notably, M. tricuspidata exhibits potent physiological activities, including anti-inflammatory [16], anti-tumor [13], hepatoprotective [17], and anti-diabetic [18] properties. These activities are mainly attributed to the isoprenyl-substituted xanthones, including cudratricusxanthone A, cudraxanthone L and M, and macluraxanthone B. High-performance liquid chromatograph of the root bark of M. tricuspidata revealed the presence of cudratricusxanthones B, D, and F, and macluraxanthone B at concentrations of 0.017, 0.026, 0.025, and 0.071%, respectively [12]. Flavonoids are the most abundant class of compounds in M. tricuspidata, with over 120 flavonoids isolated to date, many of which feature prenylated and benzylated structures. Among these, cudraflavone B, a representative prenylated flavonoid isolated from the root, exhibits anti-inflammatory [19], hepatoprotective [20], and anti-tumor [21] effects. Euchrestaflavanones B and C also exhibit antibacterial effects against Staphylococcus aureus and Bacillus bacteria [22]. Thus, the components of M. tricuspidata exhibit inhibitory effects against various bacteria and viruses. Both flavonoids and xanthones derived from M. tricuspidata exhibit potent antibacterial activity against Gram-positive bacteria. Furthermore, components isolated from the fruit inhibit the growth of oral bacteria, suggesting their potential use as oral hygiene products and functional food ingredients [12].

    Therefore, the present study aimed to explore the antibacterial potential of M. tricuspidata roots (MTroots) against S. mutans, a cariogenic bacterium.

    Materials and Methods

    1. Material extraction

    Dried MTroots were purchased from the Kujibong farm, a local farm located in Jinan, Jeonbuk, Korea. A total of 400 g of the dried MTroots was extracted with 1,000 mL of 95% ethanol at room temperature for 72 hours. The extract was filtered and vacuum evaporated to yielding 20 g of concentrated extract. The ethanolic extract was suspended in distilled water and subjected to solvent partitioning with chloroform (CHCl3), ethyl acetate (EtOAc), and n-butanol (BuOH), yielding fractions weighing 5.00, 2.94, and 1.44 g, respectively. Subsequently, 5.00 g of the CHCl3 fraction was further fractionated using silica gel column chromatography, eluted with a gradient mixture of hexane and EtOAc (from hexane:EtOAc = 100:0 to 0:100), resulting in 5 subfractions (Fra-1, Fra-2, Fra-3, Fra-4, and Fra-5).

    2. Analysis of the inhibitory effects of the subfractions on S. mutans growth

    The inhibitory effect of MTroots extract on S. mutans growth was assessed by culturing the S. mutans standard strain (American Type Culture Collection 25175) in liquid medium, Brain Heart Infusion (BHI; Difco, BD Biosciences). After adding each concentration of MTroots extract to the BHI liquid medium, S. mutans was inoculated at 1 × 108 CFU/mL and incubated for 24 g at 37℃. Then, its absorption at a wavelength of 550 nm was assessed using an enzyme-linked immunosorbent assay reader (Molecular Devices). The control group did not contain MTroots extract, and the positive control was 0.05% CHX. Following the incubation process, the lowest antimicrobial concentration that did not result in growth was determined to be the minimum inhibitory concentration (MIC).

    3. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry analysis

    Fra-3, which exhibited the most potent antibacterial activity on dental caries, was analyzed on a Waters ACQUITY UPLC I-Class PLUS system (Waters Corporation), equipped with a Waters UPLC BEH C18 column (Waters, 100 × 2.1 mm, 1.7 μm particle size), at a column temperature of 40°C. The mobile phase consisted of acetonitrile (A) and water (B), both containing 0.1% formic acid. The elution procedure was as follows: A:B = 99:1 (0–1 minute), 50:50 (1–15 minutes), and 40:60 (15–17 minutes), followed by 1% B at 17–18 minutes and maintained at 1% B at 18–21 minutes. The flow rate was 0.3 mL/minute, and the injection volume was 2 μL. Mass spectrometric data were collected using a time-of-flight (TOF) analyzer with TurboIonSpray (AB Sciex) operating in positive ion mode. The specific conditions were as follows: nebulizing gas (N2), 55 psi; drying gas (N2), 45 psi; curtain gas, 35 psi; source temperature, 600°C; ion spray voltage floating, +5,500 V/−4,500 V; TOF-mass spectrometry (MS) scan m/z range, 50–1,500 Da; TOF-MS/MS scan m/z range, 25–1,000 Da; TOF-MS scan accumulation time, 0.25 s/spectra; and product ion scan accumulation time, 0.035 s/spectra. Secondary MS was obtained through information-dependent acquisition in high sensitivity mode, with a declustering potential of ± 60 V and a collision energy of 35 ± 15 eV. The information dependent acquisition setup parameters were as follows: isotopes within 4 Da were excluded, and up to 12 candidate ions were monitored per cycle. The data were processed using SCIEX OS software (ver. 3.0), applying multiple confidence criteria, including quality accuracy, retention time, isotopes, and matching use of compound libraries. The traditional Chinese medicine MS/MS Library in the SCIEX OS software was employed to identify the major compounds in the Modified Sijunzi Decoction according to first-order accurate mass and isotope distribution ratio.

    4. Statistical analysis

    All experiments were repeated three times, and the obtained results are presented as the average and standard deviation using SPSS (ver. 10.0; SPSS Inc.). The average values of the experimental and control groups were compared using an independent samples t-test. Statistical significance was indicated at p ＜ 0.05.

    Results

    1. Preparation of the MTroots extract

    From 400 g of dried MTroots, 20 g of 95% ethanol extract was obtained. Subsequently, the extract was subjected to solvent partitioning was performed using CHCl3, EtOAc, and n-BuOH, yielding fractions of 5.00, 2.94, and 1.44 g, respectively. A total of 5.00 g of the CHCl3 fraction was subjected to silica gel column chromatography (hexane:EtOAc = 100:0 to 0:100), yielding a total of 5 subfractions (Fra-1, Fra-2, Fra-3, Fra-4, and Fra-5) weighing 5.5, 61.0, 28.5, 26.0, and 5.0 mg, respectively (Fig. 1).

    2. Antimicrobial activity analysis of the MTroots extract against S. mutans

    The antibacterial activity of each fraction obtained from solvent partitioning of the ethanol extract was tested against S. mutans. The CHCl3 fraction exhibited an MIC greater than 15 μg/mL, whereas both EtOAc and n-BuOH exhibited MICs exceeding 120 μg/mL (Table 1, Fig. 2).

    3. Antibacterial activity of the subfractions of the CHCl3 fraction using silica gel column chromatography

    The CHCl3 fraction, which displayed the most notable antimicrobial activity among the solvent partition fractions, was further fractionated through a silica gel column chromatography procedure. Compared to the control, growth inhibition of S. mutans exceeded 50% at n-hexane:EtOH ratios of 3:1, 1:1, and 1:3, with corresponding MIC values of 3.0, 1.5, and 25.0 μg/mL, respectively. No inhibitory effect on growth was observed with 100% n-hexane, whereas an MIC of 50 μg/ mL was observed with 100% EtOH fraction. Therefore, the inhibitory effect of the fractions on S. mutans growth was confirmed.

    For the 100% n-hexane fraction, no antimicrobial activity was observed at concentrations below 800 μg/mL. In fractions with n-hexane:EtOH ratios of 3:1, 1:1, and 1:3, significant inhibitory effects were observed at concentrations of 3.0, 1.5, and 25.0 μg/mL, respectively. For the 100% EtOH fraction, significant inhibition of S. mutans growth was observed at a concentration of 50 μg/mL. The MICs were observed at 3.0, 1.5, and 25.0 μg/mL for n-hexane:EtOH ratios of 3:1, 1:1, and 1:3, respectively. No MIC was observed in 100% n-hexane fractions, whereas an MIC of 50 μg/mL was observed in 100% EtOH fraction.

    Minimum inhibitor concentrations of MTroots subfractions of the CHCl3 fraction against S. mutans (Table 2, Fig. 3).

    4. Ultra-performance liquid chromatography coupled with quadrupole TOF-MS analysis

    Ultra-performance liquid chromatography coupled with quadrupole TOF-MS (UPLC-Q-TOF-MS) analysis of nhexane: EtOH = 1:1 (HEA11) resulted in the tentative characterization of 38 major compounds, including kuwanon C, kuwanon E, cudratricusxanthone L, mortatarin C, cycloartocarpesin, kuwanon A, mulberranol, and moracin U (Table 3, Fig. 4) [23-43].

    Discussion

    Dental caries is one of the leading causes of tooth loss. Despite continuous efforts to prevent and treat dental caries, its prevalence and incidence remain high. In response to these challenges, various natural products have been developed to aid in the prevention and treatment of both dental caries and periodontal disease. Numerous studies have demonstrated that certain natural products can inhibit the growth of S. mutans , a primary bacterium responsible for the development of dental caries. However, the mechanisms through which these natural compounds exert their effects vary. For example, Coptidis rhizoma extract inhibits both the growth and acid production of S. mutans [44]. In contrast, studies on the antimicrobial activity of M. tricuspidata remain limited. Nevertheless, antibacterial activity against Gram-positive bacteria has been reported for its CHCl3 extract [45]. More recently, extracts from M. tricuspidata leaves, ripe fruits, and unripe fruits exhibited antimicrobial effects against Streptococcus iniae, with prenylation of the isoflavone skeleton identified as a key contributor to the observed antimicrobial activity [46]. In addition, essential oils derived from M. tricuspidata fruits demonstrate antibacterial effects against Bacillus cereus, S. aureus, Listeria monocytogenes, Salmonella typhimurium, and Escherichia coli by disrupting bacterial cell membrane permeability [47]. The ethanol extract of M. tricuspidata exerts antibiofilm activity against S. mutans by suppressing the expression of biofilm-related genes. Notably, the major active constituents were identified as phenylpropanoids (14.19%), flavonoids (9.83%), and xanthones (6.54%) [48]. Similarly, extracts of Morus alba, a closely related species, also possess antimicrobial activity. For example, ethanol extracts of M. alba exhibit antibacterial efficacy against Porphyromonas gingivalis [49]. Kuwanon G, isolated from the methanol extracts of M. alba, exhibits antimicrobial activity against S. mutans [50]. To date, natural compounds with antimicrobial properties identified from M. alba include prenylated flavonoids such as kuwanon C, which has been extensively studied for its antiviral [51], antioxidant [52], and antiaging [53] activities. Overall, isopentenyl-substituted natural compounds display structural diversity and broad pharmacological activities, representing valuable resources for innovative drug development [54,55]. In the present study, analysis of the ethanol extract of MTroot using UPLC-Q-TOF-MS in negative ion mode identified isoarundinin II, cudratricusxanthone L, gericudranin E, kuwanon E, cudraflavenone A, cudraxanthone B, mortatarin B, and mortatarin C as major components of the extract. Notably, these compounds are isopentenyl-substituted natural products, which have been reported to exert antibiofilm effects against cariogenic S. mutans [48].

    In the present study, based on the antibiofilm inhibitory activity of the ethanol extract of MTroot against S. mutans, fractions (Fra-1–Fra-6) were analyzed for antimicrobial activity. Following solvent partitioning of the ethanol extract, the CHCl3 layer, which exhibited the strongest antimicrobial activity, was further fractionated using silica gel column chromatography. Among these, HEA11 was identified as the fraction with the most potent antimicrobial activity and the most active fraction. HEA11 was then subjected to component analysis using UPLC-Q-TOF-MS in positive ion mode. The total ion mass spectrum of the MTroot ethanol extract in positive ion mode is depicted in Fig. 3. UPLC-Q-TOF-MS analysis of antimicrobial constituents in the MTroot extract revealed the presence of 38 components, including kuwanon C, kuwanon E, cudratricusxanthone L, mortatarin C, cycloartocarpesin, kuwanon A, mulberranol, and moracin U, accounting for 66.70% of the base peak intensity peak (total BSI peak: 13,931,368). Kuwanon C, a prenylated flavone initially characterized in M. alba, possesses antioxidant and antimicrobial properties [56]. Various prenylated flavonoids, such as kuwanon C, E, G, H, J, and S, exhibit unique biological activities. Among them, kuwanon G exhibits antimicrobial activity against S. mutans and methicillinresistant S. aureus with an MIC of 12 μg/mL [57]. Kuwanon C demonstrates an antifungal activity against Candida albicans and Saccharomyces cerevisiae [58]. Kuwanon E exerts antibacterial effects [22,37], while cudraflavenone A inhibits biofilm formation [59]. Moreover, cudratricusxanthone L exhibits anti-neuroinflammatory activity [60]. Kuwanon A possesses antimelanoma [61] and antibacterial properties [62].

    In this study, the identified compounds, including kuwanon C, cycloartocarpesin, and moracin U, exhibited only limited antibacterial activity against S. mutans . Therefore, additional comprehensive investigations are necessary to clarify the precise antibacterial mechanisms of these compounds against S. mutans. Nonetheless, these findings provide a strong rationale for continued research into prenylated natural compounds from M. tricuspidata as potential therapeutic candidates to prevent dental caries.

    Funding

    This research was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education [RS-2020-NR054824].

    Conflicts of Interest

    Yong-Ouk You is an editorial board member of the journal, but was not involved in the review process of this manuscript. Otherwise, there is no conflict of interest to declare.

    Figure

    IJOB-50-4-127_F1.jpg

    Schematic illustration of the extraction procedure from Maclura tricuspidata roots.

    EtOH, ethyl alcohol; CHCl3, chloroform; EtOAc, ethyl acetate.

    IJOB-50-4-127_F2.jpg

    Antibacterial activity of solvent fractions obtained from ethyl alcohol extracts of Maclura tricuspidata roots via the solvent partitioning method against Streptococcus mutans. (A) Bacterial growth in solvent fraction CHCl3. (B) Bacterial growth in solvent fraction EtOAc. (C) Bacterial growth in solvent fraction n-butanol. (D) Bacterial growth in solvent fraction aqueous.

    OD, optical density; CHX, chlorhexidine; CHCl3, chloroform; EtOAc, ethyl acetate.

    *p < 0.05 indicates statistical significance as determined by an independent samples t-test for the mean values different from the control group.

    IJOB-50-4-127_F3.jpg

    Antibacterial activity against Streptococcus mutans of subfractions of the CHCl3 fraction using silica gel column chromatography. (A) Bacterial growth on Hexan 100%, a subfraction of the CHCl3 fraction. (B) Bacterial growth on Hexan:EtOAc = 3:1, a subfraction of the CHCl3 fraction. (C) Bacterial growth on Hexan:EtOAc = 1:1, a subfraction of the CHCl3 fraction. (D) Bacterial growth on Hexan:EtOAc = 1:3, a subfraction of the CHCl3 fraction. (E) Bacterial growth on EtOAc 100%, a subfraction of the CHCl3 fraction.

    OD, optical density; CHX, chlorhexidine; EtOAc, ethyl acetate; CHCl3, chloroform; MIC, minimum inhibitory concentration.

    *p < 0.05 indicates statistical significance, as determined using an independent samples t-test for the mean values different from the control group. Red ring is MICs.

    IJOB-50-4-127_F4.jpg

    Base peak intensity chromatogram of the HEA11 fraction obtained from the CHCl3 extract, analyzed using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry in positive ion mode.

    ES, electrospray; TOF, time-of-flight; MS, mass spectrometry; BPI, base peak intensity; HEA11, n-hexane:EtOH = 1:1; CHCl3, chloroform.

    Table

    Minimum inhibitor concentrations of Maclura tricuspidata roots extracts against Streptococcus mutans

    CHCl3, chloroform; EtOAc, ethyl acetate; BuOH, butanol; ATCC, American Type Culture Collection.

    Minimum inhibitor concentrations of Maclura tricuspidata roots subfractions of the CHCl3 fraction against Streptococcus mutans

    EtOH, ethyl alcohol; ATCC, American Type Culture Collection.

    Compounds identified tentatively in Maclura tricuspidata roots using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry in positive ion mode

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