Antibacterial and Cytotoxic Evaluation of Lespedeza cuneata Extract Against Periodontal Pathogens and Human Gingival Cells: A Novel Option for Periodontal Therapy

Abstract

Background: This study aims to evaluate the antibacterial effect of Lespedeza cuneata extract on Porphyromonas gingivalis (P. gingivalis), a principal bacterium in periodontal disease, and its impact on human gingival fibroblasts (HGFs). Methods: Dried Lespedeza cuneata was extracted using 70% ethanol, concentrated, and freeze-dried to obtain the Lespedeza cuneata extract in powder form. The antibacterial effect, indicated by the inhibition of P. gingivalis growth, was assessed by administering concentrations of 1, 3, 5, 10, 20, 30, and 40 mg/mL. After 24 h of anaerobic incubation, colony-forming units per milliliter (CFU/mL) were measured. Cytotoxicity on HGF cells was evaluated after treatment with WST-1 solution followed by incubation at 37 °C, 5% CO2 for 2 h. Cell morphology and proliferation were assessed using the Sulforhodamine B (SRB) assay. Results: The antibacterial effect of Lespedeza cuneata extract was concentration-dependent, with 99.98% inhibition observed at 5 mg/mL, 99.99% at 10 mg/mL, and no detectable CFUs were observed at 40 mg/mL under the tested conditions. Evaluating the change in growth rate of HGF cells showed a decrease in cell viability as the concentration increased, and the application of Lespedeza cuneata extract at 10 mg/mL was found to be a safe and effective concentration with a half-maximal inhibitory concentration (IC50). Conclusion: Based on the antibacterial effect, cytotoxicity, and safety profile of Lespedeza cuneata extract, it holds potential as a natural extract material for the prevention, improvement, or treatment of periodontal disease. Additionally, validation of the practical approach will be necessary via a clinical applicability evaluation.

Full text

Academic Editor: Serge Lavoie
Received: 26 November 2024
Revised: 22 December 2024
Accepted: 27 December 2024
Published: 29 December 2024
Citation: Yoon, H.-J.; Kim, G.-C.;
Nam, S.-H. Antibacterial and
Cytotoxic Evaluation of Lespedeza
cuneata Extract Against Periodontal
Pathogens and Human Gingival Cells:
A Novel Option for Periodontal
Therapy. Appl. Sci. 2025,15, 190.
https://doi.org/10.3390/app15010190
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/
licenses/by/4.0/).
Article
Antibacterial and Cytotoxic Evaluation of Lespedeza cuneata
Extract Against Periodontal Pathogens and Human Gingival
Cells: A Novel Option for Periodontal Therapy
Hyo-Ju Yoon 1, Gyoo-Cheon Kim 2,*,† and Seoul-Hee Nam 3, *,†
1Department of Dental Hygiene, School of Health, Masan University, Changwon-si 51217, Republic of Korea;
cloven79@naver.com
2
Department of Oral Anatomy, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea
3Department of Dental Hygiene, College of Health Sciences, Kangwon National University,
Samcheok-si 25949, Republic of Korea
*Correspondence: ki91000m@pusan.ac.kr (G.-C.K.); nshee@kangwon.ac.kr (S.-H.N.);
Tel.: +82-51-510-8243 (G.-C.K.); +82-33-540-3394 (S.-H.N.)
†These authors contributed equally to this work.
Abstract: Background: This study aims to evaluate the antibacterial effect of Lespedeza
cuneata extract on Porphyromonas gingivalis (P. gingivalis), a principal bacterium in peri-
odontal disease, and its impact on human gingival fibroblasts (HGFs). Methods: Dried
Lespedeza cuneata was extracted using 70% ethanol, concentrated, and freeze-dried to obtain
the Lespedeza cuneata extract in powder form. The antibacterial effect, indicated by the
inhibition of P. gingivalis growth, was assessed by administering concentrations of 1, 3,
5, 10, 20, 30, and 40 mg/mL. After 24 h of anaerobic incubation, colony-forming units
per milliliter (CFU/mL) were measured. Cytotoxicity on HGF cells was evaluated after
treatment with WST-1 solution followed by incubation at 37
◦
C, 5% CO
2
for 2 h. Cell mor-
phology and proliferation were assessed using the Sulforhodamine B (SRB) assay. Results:
The antibacterial effect of Lespedeza cuneata extract was concentration-dependent, with
99.98% inhibition observed at 5 mg/mL, 99.99% at 10 mg/mL, and no detectable CFUs
were observed at 40 mg/mL under the tested conditions. Evaluating the change in growth
rate of HGF cells showed a decrease in cell viability as the concentration increased, and the
application of Lespedeza cuneata extract at 10 mg/mL was found to be a safe and effective
concentration with a half-maximal inhibitory concentration (IC50). Conclusion: Based on
the antibacterial effect, cytotoxicity, and safety profile of Lespedeza cuneata extract, it holds
potential as a natural extract material for the prevention, improvement, or treatment of
periodontal disease. Additionally, validation of the practical approach will be necessary
via a clinical applicability evaluation.
Keywords: Lespedeza cuneate;Porphyromonas gingivalis; human gingival fibroblast; antibacterial
effect; cell viability
1. Introduction
Digestion and nutrient intake are fundamental requirements for health, with oral
health considered to be an integral part of overall health [
1
]. Common oral diseases
include dental caries and periodontal disease, characterized by their chronic nature that
continuously worsens once developed, making prevention crucial [
2
]. According to the 2023
Korean Dental Medical Yearbook report, among the top one-hundred common diseases
in 2022, nine were dental diseases, and, among them, gingivitis and periodontal disease
Appl. Sci. 2025,15, 190 https://doi.org/10.3390/app15010190

Appl. Sci. 2025,15, 190 2 of 10
ranked second with 18.12 million patients, and the prevalence of periodontal disease, which
is the proportion of people who require periodontal disease treatment beyond periodontal
tissue disease treatment in adults, temporarily decreased from 35.5% in 2007 to 22.7% in
2012 but increased after 2013 to 30.5% in 2016–2018 [
3
]. Periodontal disease is a prevalent
disease that affects approximately 20–50% of the world’s population, with a high prevalence
in adolescents, adults, and the elderly, and it causes destruction of periodontal tissues such
as gingival bleeding, gingival recession, periodontal pocket formation, and alveolar bone
destruction, as well as tooth loss [
4
]. Periodontal disease is closely linked with various
systemic diseases, altering normal host responses and being reported to affect conditions
such as coronary artery disease, stroke, diabetes, preterm birth, low-birth-weight infants,
and respiratory diseases [5].
Periodontal disease is defined as an inflammation of the supporting tissues of the
teeth, caused by specific bacteria or groups of bacteria, and characterized by the progressive
destruction of periodontal ligaments and the formation and recession of periodontal pockets
in the alveolar bone [
6
]. Since periodontal disease progresses from gingivitis, preventing
gingivitis may prevent severe periodontitis [7].
In recent years, numerous studies have focused on the equilibrium between the
immune system and dental plaque as a foundational mechanism for periodontal disease.
Porphyromonas gingivalis (P. gingivalis), a Gram-negative anaerobic bacterium in dental
plaque, is involved in the onset of periodontitis, an inflammatory disease that damages
the supporting tissues of teeth, eventually leading to tooth loss [
8
]. Among the over
500 bacterial species in the oral cavity, P. gingivalis, which belongs to the “red complex” that
classifies bacteria that cause severe forms of periodontal disease, is known to be the most
important causative agent of periodontal disease. P. gingivalis inherently carries resistance
to certain antibiotics while tending to acquire resistance to many others, making it one
of the strongest and most virulent bacteria in the oral microbiome. When periodontal
disease occurs, the number of P. gingivalis in periodontal lesions increases, and, as the
disease progresses, P. gingivalis is frequently found infiltrating the sulcus epithelium [
9
–
12
].
Given the complex interplay between microbial pathogens like P. gingivalis and periodontal
disease and the limitations of the current antimicrobial treatments, exploring natural
substances such as Lespedeza cuneata, which may offer targeted antimicrobial activity with
fewer side effects, becomes particularly relevant.
In the treatment of periodontal disease, root surface scaling and root planing are com-
monly performed. However, there is a risk of recurrence if bacteria that cause periodontal
disease, such as P. gingivalis, are present or regrow in the treated area. Therefore, topical
or systemic administration of antimicrobial agents is sometimes added to enhance the
effectiveness of mechanical periodontal therapy [
13
]. However, the misuse and overuse
of antibiotics have led to changes in bacterial resistance genes, resulting in the emergence
of super bacteria that cannot be treated with the existing antibiotics, posing a major chal-
lenge in public health [
14
]. For these reasons, instead of prescribing antibiotics for the
onset of inflammatory diseases like periodontitis, it is recommended to use mouthwashes
with antimicrobial properties, such as chlorhexidine or benzidamine hydrochloride, which
can directly eliminate P. gingivalis, the Gram-negative oral anaerobic bacteria involved
in periodontitis [
15
]. However, chlorhexidine, while being effective in improving oral
health and reducing hospital-acquired infections, can cause discomfort, staining of teeth,
and a burning sensation. benzidamine hydrochloride offers analgesic, anti-inflammatory,
and microbial reduction effects but can irritate the oral mucosa, leading to mucosal tissue
damage, muted taste sensation, and tongue discoloration [15].
Due to these issues, interest is growing in mouth rinses containing natural substances
with fewer harmful components to the human body. An ideal antimicrobial agent within

Appl. Sci. 2025,15, 190 3 of 10
the oral environment should selectively target bacteria causing dental caries, periodontitis,
or halitosis over other normal oral microbiota while maintaining low toxicity to humans
and the environment [
16
]. Research is actively being conducted to develop antimicrobials
derived from natural substances that meet these criteria [
17
]. Recently, a study demon-
strated the efficacy of propolis as a natural antimicrobial product against Pseudomonas
aeruginosa (P. aeruginosa), a Gram-negative opportunistic pathogen that is highly resistant
to disinfectants, antibiotics, and host defense mechanisms due to various toxic factors [
18
],
and another study demonstrated the efficacy of MicroRepair (MicroR) and Punica granatum
L. (PomeGr) as an inhibitor of biofilm formation against P. aeruginosa,Staphylococcus aureus
(S. aureus), and Candida albicans (C. albicans) [
19
]. The continuous use of chemical antibiotics
for oral diseases can cause side effects such as hypersensitivity, skin and allergic diseases,
and can change the bacterial microbiome, leading to resistance, so alternative treatments
using natural substances are needed [
20
]. Given the complex interplay between microbial
pathogens like P. gingivalis and periodontal disease, and the limitations of the current an-
timicrobial treatments, exploring natural substances such as Lespedeza cuneata, which may
offer targeted antimicrobial activity with fewer side effects, becomes particularly relevant.
Lespedeza cuneata G. Don, known by various names such as bi-suri and samyeobcho,
is a perennial herb in the legume family, distributed in countries including Korea, Japan,
China, and Taiwan. It is utilized as a medicinal resource to protect liver and kidney
functions, enhance lung function, and improve blood circulation [
21
]. The main components
of Lespedeza cuneata include pinitol, tannin,
β
-sistosterol, avicularin, juglanin, trifolin,
quercetin, kaempferol, vitexin, and orientin [
22
,
23
], and it is reported to have antioxidant,
hypoglycemic, cell-protective, insulin-secretion-promoting, and antimicrobial effects [24].
Natural substances can exhibit a broad-spectrum, non-selective antimicrobial effect,
potentially disrupting the balance of normal oral microbiota and leading to the emergence
of resistant strains, thereby creating an oral ecosystem imbalance [
25
]. Therefore, an ideal
antimicrobial agent for use in the oral environment requires a substance with selective
antimicrobial efficacy against periodontal pathogens like P. gingivalis rather than that
against other normal oral microbiota, and low human and environmental toxicity. The
stability of Lespedeza cuneata must be verified to determine if it meets these requirements.
Periodontal connective tissue primarily consists of human gingival fibroblasts (HGFs),
which produce the fibers and connective tissue matrix, as well as DNA and proteins.
Through interactions with various infiltrating inflammatory cells, HGFs play a crucial role
in the development of periodontal disease and in maintaining and repairing periodontal
tissue during wound healing due to their regenerative capacity [26,27].
This study aims to evaluate the potential use of Lespedeza cuneata extract as a natural
oral antimicrobial agent for periodontal disease by assessing its antibacterial effect on
P. gingivalis, a representative bacterium of periodontal disease, and its impact on the normal
periodontal bacteria and HGFs within the mouth.
2. Materials and Methods
2.1. Extract Preparation
Lespedeza cuneata was purchased from Cheongmyeong Co., Ltd. (Goesan, Chungcheongbuk-
do, Republic of Korea). Ground Lespedeza cuneata was extracted with 70% ethanol using
a rotary vacuum evaporator (N-1300E.V.S. EYELA, Rikakikai Co., Ltd., Tokyo, Japan) at
60
◦
C for 12 h, after which the solution was filtered through a filter paper (Advantec No. 2,
Tokyo, Japan) to remove impurities to obtain a concentrated extract. The extract was then
freeze-dried using a freeze dryer (Ilshin Lab Co., Yangju-si, Republic of Korea) at
−
80
◦
C
and stored in powder form at −20 ◦C until use.

Appl. Sci. 2025,15, 190 4 of 10
2.2. Bacterial Strain and Growth Conditions
P. gingivalis (KCTC 5352), obtained from the Korean Collection for Type Cultures
(KCTC, Daejeon, Republic of Korea), was cultured in liquid media supplemented with
yeast extract (5 mg/mL), L-cysteine hydrochloride (5
µ
g/mL), hemin (1
µ
g/mL), vitamin
K1 (0.2
µ
g/mL), and tryptic soy broth (Difco, Detroit, MI, USA). It was also grown on tryptic
soy blood agar plates supplemented with 5% sheep blood under anaerobic conditions at
37
◦
C for 24 h. Subsequently, the bacteria were inoculated to achieve a concentration of
1×105colony-forming units per milliliter (CFU/mL).
2.3. Antibacterial Activity
To measure the inhibitory effect of Lespedeza cuneata extract on P. gingivalis growth,
extracts were prepared at concentrations of 1, 3, 5, 10, 20, 30, and 40 mg/mL and inoculated
into 100
µ
L aliquots of media containing P. gingivalis. As a control group, all conditions
were the same and only a medium without extract was used. After incubating the mixtures
anaerobically at 37
◦
C for 24 h, 1 mL of each concentration was streaked onto tryptic soy
blood agar plates to observe the bacterial morphology and distribution. Additionally, to
quantify CFU, dilutions were created, plated, and incubated anaerobically for 24 h, followed
by CFU counting to evaluate the antibacterial effect of Lespedeza cuneata.
2.4. Cell Culture Conditions
HGFs were purchased from the American Type Culture Collection. The cells were
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Grand Island, NY, USA),
supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and
1% penicillin/streptomycin (Gibco, Grand Island, NY, USA) at 37
◦
C and 5% CO
2
in a
humidified chamber. The cells were used after 4–6 passages.
2.5. Cell Proliferation Assay Using Water-Soluble Tetrazolium Salt (WST-1) Assay
To quantify the effects of Lespedeza cuneata extract concentration on cell growth and
proliferation, a Water-Soluble Tetrazolium Salt (WST-1) assay was conducted [
28
]. HGF
cells were seeded in 96-well plates at an initial density of 1
×
10
5
cells/cm
2
and incubated
at 37
◦
C for 24 h. They were then treated with extract concentrations of 1, 3, 5, 10, 20,
30, and 40 mg/mL for 3 h. After treatment, WST-1 solution was added to each well and
incubated at 37
◦
C with 5% CO
2
for 2 h. The absorbance was measured at 450 nm using an
ELISA reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA) to evaluate the
growth rate. To ensure statistical significance, all experiments were independently repeated
three times.
2.6. Cell Growth Assay Using Sulforhodamine B (SRB) Assay
The Sulforhodamine B (SRB) assay was used to assess cell proliferation and cytotoxicity
through protein staining [
29
]. HGF cells were seeded at a density of 1
×
10
5
cells/cm
2
in
35 mm culture dishes and incubated at 37
◦
C in a 5% CO
2
incubator for 24 h. Afterward, the
cells were treated with Lespedeza cuneata extract diluted in DMEM media at concentrations
of 1, 3, 5, 10, 20, 30, and 40 mg/mL for 24 h. The liquid medium was removed, and the cells
were fixed with 500
µ
L of 4% paraformaldehyde at room temperature for 30 min, washed,
and stained with 0.4% SRB solution. Non-specific staining was removed by washing
with 1% acetic acid to fully remove the SRB dye, and the plates were completely dried.
Morphological changes in the cells were imaged at 200 times magnification using an optical
microscope (OLYMPUS Optical Co., Melville, NY, USA) equipped with a digital camera.

Appl. Sci. 2025,15, 190 5 of 10
2.7. Statistical Analysis
Statistical significance analysis for the antibacterial activity of P. gingivalis and HGF
cell proliferation was performed using SPSS v.24.0 (SPSS Inc., Chicago, IL, USA), employing
one-way ANOVA, with post hoc analysis conducted using the Duncan test at a significance
level of 0.05.
3. Results
3.1. Effect of Bacterial Growth Inhibition
As the concentration of the extract increased, a marked inhibition of P. gingivalis
growth was observed, with no bacteria detected at 40 mg/mL (Figure 1). Additionally,
as shown in Figure 2, quantification through CFU counts revealed statistically significant
differences, indicating that the antibacterial effect of Lespedeza cuneata extract increased
with concentration (p< 0.05). At 5 mg/mL, the antibacterial effect was 99.98%, and, at
10 mg/mL, it was 99.99%. There was no statistically significant difference in antibacterial
effect between the concentrations of 30 mg/mL and 40 mg/mL.
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
theplateswerecompletelydried.Morphologicalchangesinthecellswereimagedat200
timesmagnificationusinganopticalmicroscope(OLYMPUSOpticalCo.,Melville,NY,
USA)equippedwithadigitalcamera.
2.7.StatisticalAnalysis
StatisticalsignificanceanalysisfortheantibacterialactivityofP.gingivalisandHGF
cellproliferationwasperformedusingSPSSv.24.0(SPSSInc.,Chicago,IL,USA),
employingone‐wayANOVA,withposthocanalysisconductedusingtheDuncantestat
asignificancelevelof0.05.
3.Results
3.1.EffectofBacterialGrowthInhibition
Astheconcentrationoftheextractincreased,amarkedinhibitionofP.gingivalis
growthwasobserved,withnobacteriadetectedat40mg/mL(Figure1).Additionally,as
showninFigure2,quantificationthroughCFUcountsrevealedstatisticallysignificant
differences,indicatingthattheantibacterialeffectofLespedezacuneataextractincreased
withconcentration(p<0.05).At5mg/mL,theantibacterialeffectwas99.98%,and,at10
mg/mL,itwas99.99%.Therewasnostatisticallysignificantdifferenceinantibacterial
effectbetweentheconcentrationsof30mg/mLand40mg/mL.

Figure1.DifferencesingrowthinhibitioneffectsofP.gingivalisaccordingtotheconcentrationof
Lespedezacuneataextract.

Figure2.ComparisonofCFUagainstP.gingivalisaccordingtotheantibacterialeffectofLespedeza
cuneataextract.*Thesignificantdifferenceamonggroupsinone‐wayANOVA.Differentletters(a,
b,c,d,e,f,andg)indicatethepresentedsignificantresultoftheposthocDuncantest(p<0.05).
Figure 1. Differences in growth inhibition effects of P. gingivalis according to the concentration of
Lespedeza cuneata extract.
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
theplateswerecompletelydried.Morphologicalchangesinthecellswereimagedat200
timesmagnificationusinganopticalmicroscope(OLYMPUSOpticalCo.,Melville,NY,
USA)equippedwithadigitalcamera.
2.7.StatisticalAnalysis
StatisticalsignificanceanalysisfortheantibacterialactivityofP.gingivalisandHGF
cellproliferationwasperformedusingSPSSv.24.0(SPSSInc.,Chicago,IL,USA),
employingone‐wayANOVA,withposthocanalysisconductedusingtheDuncantestat
asignificancelevelof0.05.
3.Results
3.1.EffectofBacterialGrowthInhibition
Astheconcentrationoftheextractincreased,amarkedinhibitionofP.gingivalis
growthwasobserved,withnobacteriadetectedat40mg/mL(Figure1).Additionally,as
showninFigure2,quantificationthroughCFUcountsrevealedstatisticallysignificant
differences,indicatingthattheantibacterialeffectofLespedezacuneataextractincreased
withconcentration(p<0.05).At5mg/mL,theantibacterialeffectwas99.98%,and,at10
mg/mL,itwas99.99%.Therewasnostatisticallysignificantdifferenceinantibacterial
effectbetweentheconcentrationsof30mg/mLand40mg/mL.

Figure1.DifferencesingrowthinhibitioneffectsofP.gingivalisaccordingtotheconcentrationof
Lespedezacuneataextract.

Figure2.ComparisonofCFUagainstP.gingivalisaccordingtotheantibacterialeffectofLespedeza
cuneataextract.*Thesignificantdifferenceamonggroupsinone‐wayANOVA.Differentletters(a,
b,c,d,e,f,andg)indicatethepresentedsignificantresultoftheposthocDuncantest(p<0.05).
Figure 2. Comparison of CFU against P. gingivalis according to the antibacterial effect of Lespedeza
cuneata extract. * The significant difference among groups in one-way ANOVA. Different letters (a, b,
c, d, e, f, and g) indicate the presented significant result of the post hoc Duncan test (p< 0.05).
3.2. Cytotoxicity Effect on HGF Cell Viability
The cell viability affected by the Lespedeza cuneata extract was quantitatively assessed
using the WST-1 assay. It was found that, as the concentration of the Lespedeza cuneata
extract increased, the survival rate of the HGF cells decreased (Figure 3). Statistically,

Appl. Sci. 2025,15, 190 6 of 10
there was no significant difference compared to the control group up to a concentration
of 3 mg/mL. The cell viability at concentrations of 1, 3, 5, 10, 20, 30, and 40 mg/mL
of Lespedeza cuneata extract was 99.99%, 93.82%, 78.84%, 58.41%, 46.96%, 32.23%, and
24.28%, respectively. Concentrations above 20 mg/mL were found to cause HGF cell death,
indicating a cytotoxic effect.
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
3.2.CytotoxicityEffectonHGFCellViability
ThecellviabilityaffectedbytheLespedezacuneataextractwasquantitativelyassessed
usingtheWST‐1assay.Itwasfoundthat,astheconcentrationoftheLespedezacuneata
extractincreased,thesurvivalrateoftheHGFcellsdecreased(Figure3).Statistically,there
wasnosignificantdifferencecomparedtothecontrolgroupuptoaconcentrationof3
mg/mL.Thecellviabilityatconcentrationsof1,3,5,10,20,30,and40mg/mLofLespedeza
cuneataextractwas99.99%,93.82%,78.84%,58.41%,46.96%,32.23%,and24.28%,
respectively.Concentrationsabove20mg/mLwerefoundtocauseHGFcelldeath,
indicatingacytotoxiceffect.

Figure3.ComparisonofsurvivalratesofHGFcellstreatedwithLespedezacuneataextract.*The
significantdifferenceamonggroupsinone‐wayANOVA.Differentletters(a,b,c,d,ande)indicate
thepresentedsignificantresultoftheposthocDuncantest(p<0.05).
3.3.MorphologyChangesinHGFCell
ChangesinthemorphologyofHGFcellswereobservedinaconcentration‐
dependentmanneraftertreatmentwithLespedezacuneataextract.AsshowninFigure4,
althoughthedensityandnumberofcellsdecreasedwithtreatmentupto10mg/mL,no
detachedordeadcellsweredetected.Morphologicalchangesbegantoappearinthose
cellstreatedwith20mg/mL,and,from30mg/mLonward,signsofnucleardamageand
cellcondensationindicatedanegativeimpactoncellgrowth.

Figure4.EffectsofvariousconcentrationsofLespedezacuneataextractoncellmorphologychanges
andproliferationinhibitionofHGFcellsculturedbySRBassay.
4.Discussion
Amongdentaldiseases,theprevalenceofperiodontaldiseaseincreases
progressivelyinadultsovertheageof30,withreportsindicatingthat60–90%of
individualsintheir40sand50sareaffected.Therefore,periodontaldiseaseistheleading
Figure 3. Comparison of survival rates of HGF cells treated with Lespedeza cuneata extract. * The
significant difference among groups in one-way ANOVA. Different letters (a, b, c, d, and e) indicate
the presented significant result of the post hoc Duncan test (p< 0.05).
3.3. Morphology Changes in HGF Cell
Changes in the morphology of HGF cells were observed in a concentration-dependent
manner after treatment with Lespedeza cuneata extract. As shown in Figure 4, although the
density and number of cells decreased with treatment up to 10 mg/mL, no detached or
dead cells were detected. Morphological changes began to appear in those cells treated with
20 mg/mL, and, from 30 mg/mL onward, signs of nuclear damage and cell condensation
indicated a negative impact on cell growth.
Appl.Sci.2025,15,xFORPEERREVIEW6of10

3.2.CytotoxicityEffectonHGFCellViability
ThecellviabilityaffectedbytheLespedezacuneataextractwasquantitativelyassessed
usingtheWST‐1assay.Itwasfoundthat,astheconcentrationoftheLespedezacuneata
extractincreased,thesurvivalrateoftheHGFcellsdecreased(Figure3).Statistically,there
wasnosignificantdifferencecomparedtothecontrolgroupuptoaconcentrationof3
mg/mL.Thecellviabilityatconcentrationsof1,3,5,10,20,30,and40mg/mLofLespedeza
cuneataextractwas99.99%,93.82%,78.84%,58.41%,46.96%,32.23%,and24.28%,
respectively.Concentrationsabove20mg/mLwerefoundtocauseHGFcelldeath,
indicatingacytotoxiceffect.

Figure3.ComparisonofsurvivalratesofHGFcellstreatedwithLespedezacuneataextract.*The
significantdifferenceamonggroupsinone‐wayANOVA.Differentletters(a,b,c,d,ande)indicate
thepresentedsignificantresultoftheposthocDuncantest(p<0.05).
3.3.MorphologyChangesinHGFCell
ChangesinthemorphologyofHGFcellswereobservedinaconcentration‐
dependentmanneraftertreatmentwithLespedezacuneataextract.AsshowninFigure4,
althoughthedensityandnumberofcellsdecreasedwithtreatmentupto10mg/mL,no
detachedordeadcellsweredetected.Morphologicalchangesbegantoappearinthose
cellstreatedwith20mg/mL,and,from30mg/mLonward,signsofnucleardamageand
cellcondensationindicatedanegativeimpactoncellgrowth.

Figure4.EffectsofvariousconcentrationsofLespedezacuneataextractoncellmorphologychanges
andproliferationinhibitionofHGFcellsculturedbySRBassay.
4.Discussion
Amongdentaldiseases,theprevalenceofperiodontaldiseaseincreases
progressivelyinadultsovertheageof30,withreportsindicatingthat60–90%of
individualsintheir40sand50sareaffected.Therefore,periodontaldiseaseistheleading
Figure 4. Effects of various concentrations of Lespedeza cuneata extract on cell morphology changes
and proliferation inhibition of HGF cells cultured by SRB assay.
4. Discussion
Among dental diseases, the prevalence of periodontal disease increases progressively
in adults over the age of 30, with reports indicating that 60–90% of individuals in their 40s
and 50s are affected. Therefore, periodontal disease is the leading cause of tooth loss in
adults, exposing the majority of individuals over 50 to the possibility of losing their teeth.
Tooth loss leads to difficulties in food intake, resulting in a loss of health and ultimately
a decline in quality of life [
30
]. To treat or manage periodontal disease, it is essential to
control P. gingivalis, a primary causative bacterium of the condition. P. gingivalis is an
anaerobic bacterium primarily present during the active phases of chronic and aggressive

Appl. Sci. 2025,15, 190 7 of 10
periodontitis, and animal studies have shown that it causes ulceration and necrosis [
31
],
forms phlegmonous abscesses, and activates splenic cells, promoting the bone resorption
capabilities of osteoclasts [32].
As a method to inhibit this bacterium, antimicrobial agents, either topically or sys-
temically, are sometimes used concurrently with non-surgical periodontal treatments to
enhance their effectiveness. However, prolonged use of chemical antimicrobials can lead
to resistance and an increase in pathogenic bacteria, prompting research into the clinical
application of various natural substances with antimicrobial effects as alternatives [
33
].
Research based on natural materials has evaluated the antibacterial effect of green tea
extract against Prevotella intermedia (P. intermedia), a causative bacterium of periodontal dis-
ease [
34
]. Phytoncides have also been reported to cause structural morphological changes
in P. gingivalis, demonstrating strong antimicrobial activity [
30
]. However, while research
on the antibacterial potential of natural extracts is ongoing, appropriate assessments of safe
concentrations through cytotoxicity evaluations have not yet been adequately conducted.
In a study by Nam et al., it was demonstrated that Lespedeza cuneate extract had an antibac-
terial effect on C. albicans, a causative agent of oral mucosal disease [35], and Streptococcus
mutans (S. mutans), a causative agent of dental caries [36].
Therefore, this study aimed to evaluate the practicality of utilizing Lespedeza cuneata
extract as an effective and safe antimicrobial agent for periodontal disease by assessing its
antibacterial effect against P. gingivalis, a representative periodontal bacterium, along with
evaluating its toxicity on HGFs, which are normal cells present in periodontal tissue. The
antibacterial effect of Lespedeza cuneata was shown to be concentration-dependent, with
CFU evaluation results indicating that higher concentrations of Lespedeza cuneata extract led
to greater antibacterial effects, demonstrating a 99.99% effectiveness at 5 mg/mL, and no
detectable CFUs were observed at 40 mg/mL under the tested conditions. Although there
have been no studies confirming the antibacterial effect of Lespedeza cuneata extract on peri-
odontal pathogens, research on C. albicans, a causative agent affecting oral mucosal diseases,
reported complete bacterial eradication at a concentration of 30 mg/mL [
35
]. Additionally,
studies involving S. mutans, the causative bacterium of dental caries, indicated a signif-
icant antibacterial effect at a concentration of 5 mg/mL, where no bacterial growth was
observed [
36
]. In our study, when Lespedeza cuneata extract was applied at 5 mg/mL against
P. gingivalis, a 99.98% inhibition rate was observed, and, at 10 mg/mL, a 99.99% inhibition
rate indicated significant antibacterial effects; however, complete bacterial eradication
occurred at a concentration of 40 mg/mL. This suggests that the anaerobic nature of
P. gingivalis, along with its ability to emit strong secretions of toxins such as ammo-
nia, hydrogen sulfide, amines, and lipopolysaccharides that degrade collagen [
37
], re-
quire higher concentrations for complete bacterial extinction compared to other oral-
disease-causing bacteria.
HGF plays a crucial role in the onset of periodontal disease through its relationship
with various infiltrating inflammatory cells and exhibits regenerative capabilities during
wound healing, thus playing an important role in the maintenance and repair of periodontal
tissues [
27
]. Therefore, a toxicity assessment was conducted to ensure safe application of
Lespedeza cuneata extract while minimizing its impact on HGF. The WST-1 analysis method
used in this study was developed to quantitatively assess cell proliferation and survival
across a wide range of pharmacological and functional applications [
38
]. When quantifying
cell viability using the WST-1 assay, it was found that concentrations of Lespedeza cuneata
extract above 20 mg/mL caused cell death in HGF cells, indicating a cytotoxic effect, and
suggesting that concentrations below 20 mg/mL may be appropriate. This study confirmed
that applying 10 mg/mL of Lespedeza cuneata extract did not affect cell viability. The
SRB analysis method is based on measuring cell protein content and is advantageous for

Appl. Sci. 2025,15, 190 8 of 10
confirming cell proliferation inhibition and cytotoxicity as it provides excellent linearity,
sensitivity, and stable results [
39
,
40
]. In this study, the extract affected changes in HGF cell
morphology in a concentration-dependent manner. Regarding treatments up to 10 mg/mL,
cell density and number decreased, but no changes in cell morphology were detected, and
detached or dead cells were also not observed. However, morphological changes began
to appear in cells treated with 20 mg/mL of Lespedeza cuneata extract, confirming that
10 mg/mL is a safe concentration.
Based on the results above, Lespedeza cuneata extract has shown antibacterial efficacy
against P. gingivalis, the causative bacterium of periodontal disease. For stable application,
using a concentration of 10 mg/mL, which corresponds to the half-maximal inhibitory
concentration (IC50), will not affect normal oral periodontal cells and may be safely and ef-
fectively applied as a natural antibiotic for the prevention and improvement of periodontal
disease without chemical side effects.
A limitation of this study is that only P. gingivalis, a representative bacterium causing
periodontal disease, was examined, indicating a need for further research on the antibacte-
rial effects against various other bacteria that contribute to the occurrence of periodontal
disease. Future studies will be needed to fractionate Lespedeza cuneata extract and evaluate
the most active components to determine their specific contribution. Additionally, clinical
studies applying Lespedeza cuneata extract within a safe concentration range in products
related to periodontal disease improvement, such as mouthwashes and toothpastes, will be
necessary to evaluate its practicality and clinical applicability.
This study not only demonstrates the antibacterial effect of Lespedeza cuneata extract
against P. gingivalis, a major contributor to periodontal disease, but also verifies its safe
functioning at clinically relevant concentrations, thus providing foundational data for
the development of periodontal treatment agents containing Lespedeza cuneata extract and
establishing a basis for practical application.
5. Conclusions
Lespedeza cuneata extract at a concentration of 10 mg/mL does not cause toxicity to
normal periodontal cells and exhibits effective antibacterial activity against periodontal
disease. It is a potential candidate for a new natural substance to replace the conventional
treatments for periodontal disease. Based on this study, we will confirm the antibacterial
effect on various bacteria that cause periodontal disease and perform a clinical efficacy and
safety evaluation through application of Lespedeza cuneate extract to verify the possibility of
using it as a substance for improving clinical periodontal disease.
Author Contributions: Conceptualization, G.-C.K. and S.-H.N.; data curation, H.-J.Y. and S.-H.N.;
methodology, S.-H.N.; resources, H.-J.Y. and G.-C.K.; supervision, G.-C.K. and S.-H.N.; validation,
S.-H.N. and H.-J.Y.; writing the original draft, H.-J.Y. and S.-H.N.; writing—review and editing,
S.-H.N. and G.-C.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The raw data supporting the conclusions of this article will be made
available by the authors on request.
Conflicts of Interest: The authors declare no conflicts of interest.

Appl. Sci. 2025,15, 190 9 of 10
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