In Vitro Osteogenic Differentiation and Antibacterial Potentials of Chalcone Derivatives

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00288

Chalcone derivatives have been investigated as therapeutic agents for the anticancer, antioxidant, and anti-inflammatory fields. In this study, we have synthesized four different types of chalcone derivatives and demonstrated in vitro bioactivities. We divided these derivatives into two groups of chalcones on the basis of similar substituents on the aromatic rings, and we tested cell viability and proliferation potentials, which indicated that the methoxy substituent on the A ring could enhance cytotoxicity and antiproliferation potential depending on the chalcone concentration. We also investigated osteogenic differentiation of C2C12 cells by ALP staining, the early marker for osteogenesis, which demonstrated that the chalcones could not only induce activity of BMP-2 but also inhibit the activity of noggin, a BMP antagonist. In addition, chalcone bearing hydroxyl groups at the 2-, 4-, and 6-position on the A ring inhibited treptococcus mutans growth, a major causative agent of dental caries. Therefore, we concluded that the chalcone derivatives synthesized in this research can be good candidates for therapeutic agents promoting bone differentiation, with an expectation of inhibiting S. mutans, in dentistry.

Full text of DOI:10.1021/acs.molpharmaceut.8b00288

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Article
In vitro osteogenic differentiation and
antibacterial potentials of chalcone derivatives
Daheui Choi, Jin Chan Park, Ha Na Lee, Ji-Hoi Moon, Hyo-won Ahn, Kwangyong Park, and Jinkee Hong
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00288 • Publication Date (Web): 16 Jul 2018
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In vitro osteogenic differentiation and antibacterial potentials of chalcone
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derivatives
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Daheui Choi1, Jin Chan Park2, Ha Na Lee2, JiꢀHoi Moon4,5, Hyoꢀwon Ahn3, Kwangyong
Park2,*, and Jinkee Hong1,*
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1 Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonseiꢀro,
Seodaemunꢀgu, Seoul, 03722, Republic of Korea
2 School of Chemical Engineering and Material Science, ChungꢀAng University, 84
Heukseokꢀro, Dongjakꢀgu, Seoul 06974, Republic of Korea
3 Department of Orthodontics, School of Dentistry, Kyung Hee University, 26 Kyungheedaeꢀ
ro, Dongdaemunꢀgu, Seoul 02447, Republic of Korea
4 Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee
University, 26 Kyungheedaeꢀro, Dongdaemunꢀgu, Seoul 02447, Republic of Korea
5 Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, 26
Kyungheedaeꢀro, Dongdaemunꢀgu, Seoul 02447, Republic of Korea.
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Abstract
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Chalcone derivatives have been investigated for therapeutic agent such as anticancer,
antioxidant and antiꢀinflammatory fields. In this study, we also have synthesized 4 different
type of chalcone derivatives and demonstrated in vitro bioactivities. We divided into 2 groups
of chalcones on the basis of similar substituents at aromatic rings and tested cell viability and
proliferation potentials, indicating that methoxy substituent on A ring could enhance
cytotoxicity and antiꢀproliferation potential depending on chalcone concentration. We also
investigated osteogenic differentiation of C2C12 cells by ALP staining, the early marker for
osteogenesis, which is demonstrated that the chalcones could not only induce activity of
BMPꢀ2, but also inhibit Noggin activity that is BMP antagonist. In addition, chalcone bearing
hydroxyl groups at the 2-, 4-, and 6-position on the A ring inhibited treptococcus mutans
growth, a major causative agent of dental caries. Therefore, we concluded that the chalcone
derivatives synthesized in this research can be good candidates for therapeutic agent
promoting bone differentiation, with an expectation of inhibiting S. mutans, in dentistry.
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Key words: Chalcone derivatives, cytotoxicity, osteogenic differentiation and antibacterial
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1. Introduction
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Chalcones (1,3ꢀdiarylꢀ2ꢀpropenꢀ1ꢀones) are one of the series of flavonoids, which are
produced by natural plants or chemical syntheses. The bioactivities of chalcone derivatives
have been discovered in diverse application fields, mainly for therapeutic agents, such as
antifungal1, antibacterial2, antiꢀinflammatory3, antioxidant4, anticancer5 or antiobesity
properties6. Chalcones also serve as intermediate for the biosynthesis of natural compounds,
especially openꢀchain precursors for flavonoids in nature7. Chalcones have a skeleton of α, βꢀ
unsaturated ketone with two phenyl rings. In previous studies, the bioactivies of chalcones
were found to be highly dependent on the modification of the functional groups of the phenyl
rings8, 9. Therefore, syntheses of chalcone derivatives are necessary to discover appropriate
biomedical applications and figure out the structure–activity relationship.
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Bone has the intrinsic potency to regenerate and remodel as part of fracture in the response of
injury site. The bone homeostasis is maintained continually remodeling and restoring to
replace to the new bone through the adult life10. However, in case of large bone defects
caused by tumor, trauma or infection could be retarded to restore to original state, which is
still major healthcare challenge11, 12. In addition, for enhancement of bone regeneration,
allogeneic bone grafting which is obtained from living donors are implanted into patients,
which concerns to induce immunological reaction13. Therefore, intrinsic retention or
stimulation of osteogenic differentiation potential on injury site is required to be effective to
regenerate bone mass. Bone Morphogenetic Protein 2 (BMPꢀ2) is the member of subfamily
of the Transforming Growth Factorꢀβ (TGFꢀβ) which is wellꢀknown growth factor to induce
osteogenesis of preostoblast or stem cells14, most effective material in stimulation of bone
regeneration. However, there are some difficulties involved in using BMPꢀ2 for osteogenesis,
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such as high costs and low stability in reconstitution state15. In order to promote BMPꢀ2
activity and functions, some small molecules which are contained aromatic rings have been
developed, inhibiting degradation of genes that is involved in BMPꢀ2 signaling pathway16, 17
.
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For the osteogenic differentiation of chalcones to cells, in our knowledge, few publications
have been reported for synthetic chalcone derivatives. For example, X. R. Ortolan et al., have
been demonstrated the osteogenic potential of chalcone for bone defect in rat calvaria model,
showing bone repair on the injured site18.
In this study, we synthesized four different trihydroxychalcone derivatives shown in Figure
1b. Size and hydrophilicity of the chalcones were varied by introducing hydroxyl (OH) or
methoxy (OMe) groups on A and B ring. Osteogenic differentiation and antibacterial
properties of the chalcone derivatives, which were classified into two groups, group A and
group B, depending on the substituents on A ring, were investigated. We used C2C12 (mouse
myoblast cell line), mesenchymal precursor cells, to determine osteogenic differentiation
property of the trihydroxychalcone derivatives. We also examined their effect on the growth
of Streptococcus mutans, which is a major pathogenic agent of dental caries.
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2. Experimental section
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2.1. Synthesis of chalcone derivatives
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2’,4’,6’ꢀTrihydroxyacetophenone and 4’ꢀhydroxybenzaldehyde were purchased from Alfaꢀ
Aesar. Benzaldehyde (1), dimethyl sulfate, and chloromethyl methyl ether were purchased
from SigmaꢀAldrich Co. Ltd. (St. Louis, MO, USA). 2’ꢀHydroxyꢀ4’,6’ꢀ
di(methoxymethoxy)acetophenone (A) and 2’ꢀhydroxyꢀ4’,6’ꢀdimethoxyacetophenone (B)
were prepared by the reaction of 2’,4’,6’ꢀtrihydroxyacetophenone with chloromethyl methyl
ether or dimethyl sulfate in the presence of K2CO3 in acetone at 50 °C, as previously
described with a modification (Figure 2)19. 4’ꢀ(Methoxymethoxy)benzaldehyde (2) was
prepared, as previously described20.
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Trihydroxychalcone derivatives A1, A2, B1 and B2 were synthesized by Claisen–Schmidt
condensation of the corresponding acetophenones with benzaldehydes as following21. Ethanol
(200 mL) was added to acetophenone A or B (3.9 mmol), benzaldehyde 1 or 2 (4.3 mmol),
and KOH (4.0 mmol) in a roundꢀbottomed flask under nitrogen atmosphere. After stirring for
72 h at room temperature, the reaction mixture was quenched with 1 % aqueous HCl. The
organic solution was extracted with Ethyl acetate (EtOAc), washed with water and saturated
aqueous NaCl, dried over MgSO4, and concentrated under vacuum. The crude product was
purified by recrystallization from ethanol to yield the corresponding chalcones A1’, A2’, B1
and B2’ as a cubicꢀshaped yellow crystalline solid in 68.7, 65.2, 78.3 and 60.3% isolated
yields respectively. HCl (3 %, 100 mL) and ethanol (100 mL) were added to the chalcones
A1’, A2’ or B2’ (1.0 g). The solution was heated for 15 min at 70 °C and then cooled to room
temperature. The crude product was extracted with EtOAc, washed with water and saturated
aqueous NaCl, dried over MgSO4, and concentrated under vacuum. The product was purified
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by column chromatography to yield the corresponding 2’,4’,6’ꢀtrihydroxychalcone
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derivatives A1, A2 and B2 as a yellow powder in 78.5, 60.2 and 62.8 % isolated yields
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respectively. Structure confirmation after synthesizing was conducted via HꢀMNR, 13Cꢀ
MNR, FTꢀIR and UVꢀvis absorption in our previous report21.
2.2. Cell culture
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C2C12 (Mouse myoblast cell line) was obtained from National Cancer Center (Republic of
Korea). The cells were used within 15ꢀ17 passages. For media preparation, 10% of Fetal
Bovine Serum (FBS, WELGENE, Gyeongsanꢀsi, South Korea) and 1% of Antibiotics (Gibco,
Grand Island, NY, USA.) were added into the high glucose Dulbecco Modified Eagle
Medium (DMEM, Gibco, Grand Island, NY, USA.) as followed our previous work22. Cells
were seeded on 100 mm dishes and maintained at 37°C and 5% CO2. The medium was
changed every 3 days. Upon reaching cell confluency up to 95%, C2C12 was detached from
the dishes with 0.05% Trypsin (Gibco, Grand Island, NY, USA.) dissolved in 1× Phosphate
Buffered Saline (PBS, Gibco, Grand Island, NY, USA.).
2.3. Proliferation and viability test
For measuring cell proliferation depending on concentration of chalcone, the C2C12 was
seeded on well plate at the density of 1.3×102 cells/cm2. After 1 day of incubation, cell
culture media was replaced with chalcone (group A and B) treated medium. Dimethyl
sulfoxide (DMSO) dissolved chalcone (3 mg/mL) was diluted with cell culture media for
adjusting to 5 ꢀMꢀ500 ꢀM. The chalcone treated medium was used 5% FBS contained cell
culture media and changed every 3 days. The increased cell number was measured by 3ꢀ(4,5ꢀ
Dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀDiphenyltetrazolium Bromide (MTT) assay with a standard cell
number curve.
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In order to measure viability of C2C12 at high chalcone concentration, the cells were seeded
on well plate at the density of 1.3×104 cells/cm2. After 1 day of culture, chalcone solutions
were treated and cells were cultured for 24 hours. For checking DMSO effect on cells, we
also treated cytotoxicity of DMSO. The average amount of DMSO corresponded with each
chalcone concentration was employed to C2C12 for viability. The amounts of DMSO we
added are 8.34 ꢀL for 200 ꢀM, 20.87 ꢀL for 500 ꢀM, 41.74 ꢀL for 1 mM and 83.48 ꢀL for 2
mL in 5 mL of cell culture media. The viability was measured by MTT assay.
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2.4. Osteogenic differentiation of C2C12 and ALP staining
For inducing osteogenic differentiation, C2C12 was seeded on well plates at cell number of
1.3×102 cells/cm2. After 1 day of incubation, osteogenic differentiation medium which is
comprised of 100 ng/mL or 300 ng/mL of Recombinant Human Bone Morphogenetic
Proteinꢀ2 (BMPꢀ2, Genoss, Suwonꢀsi, South Korea) mixed with cell culture media containing
10% of FBS was replaced and maintained for 8 days to differentiate myoblast to osteoblast
linage. For chalcone (group A and group B) treated groups, 500 ꢀM of A1 and A2 and 250
ꢀM of B1 and B2 were treated to as described above. 100 mg/mL of BMPꢀ2 was added to
chalcone treated groups. The medium was replaced every 4 days. After 8 days of incubation,
cells were measured by Alkaline Phosphatase (ALP) staining for checking osteogenic
differentiation. In case of Recombinant Human Noggin (NOG, Peprotech, Rocky Hill, NJ,
USA) treatment to C2C12 for osteogenic differentiation, 5% of FBS containing cell culture
media was used. 50 ꢀM of group A and 25 ꢀM of group B were added to each well
with/without BMPꢀ2 (100 ng/mL) or NOG (100 ng/mL). The medium was changed every 4
days. After 8 days of incubation, Alkaline Phosphatase (ALP) staining was carried out.
For ALP staining, C2C12 was washed with PBS 3 times and fixed with 10% Formalin neutral
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buffer solution (Sigma, St. Louis, MO, USA) for 20 min at room temperature. The ALP
staining was carried out following Burstone’s staining protocol. Briefly, for preparation of
substrate working solution, 5 mg of Naphthol ASꢀMS phosphate (Sigma, St. Louis, MO,
USA) was dissolved in 250 ꢀL of Dimethylformamide (DMF, Daejung, Siheungꢀsi, South
Korea). The Trisꢀhydrochloric acid buffer (Santacruz Biotech., CA, USA) was adjusted at pH
8.74. Sequentially, 250 ꢀL of Naphthol ASꢀMS phosphateꢀDMF solution, 25 mL of Trisꢀ
hydrochloric acid buffer and 25 mL of DI water were mixed together. Finally, 30 mg of fast
red violet (Sigma, St. Louis, MO, USA) was added into the mixture and vortexed mildly
(substrate working solution). The substrate working solution was treated each well plate and
wait for 30 min at incubator for enough staining of cells to red color. After staining, the cells
were washed with PBS 3 times. In order to quantitively analyze ALP staining, the ALP
stained area deducted from microscopic images are were calculated using Image J software
(n=3).
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2.5. Antibacterial test
S. mutans GSꢀ5 was grown at 37 °C anaerobically (85% N2, 10% H2, and 5% CO2) in brain
heart infusion broth (BHI, Difco Laboratories, Detroit, MI, USA). To examine the effect of
the chalcone derivatives on the bacterial growth, a 24 h culture of S. mutans was adjusted to
OD600 of approximately 0.1 in fresh medium. The bacterial suspension was then dispensed
(100 ꢀL per well) into wells of polystyrene 96ꢀwell plates containing various concentrations
of the test agents diluted in BHI (100 ꢀL). The final concentrations of chalcone derivatives
ranged from 0.001 mM to 1.0 mM. The blank control comprised BHI medium without the
bacterial inoculum. An inoculated BHI medium without test agents (0 mM) was also included.
The plates were incubated under anaerobic condition at 37°C. At various time points between
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0 and 24 h, the OD600 was recorded for every sample (n=1).
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2.6. Statistical analysis
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Numerical data obtained by ALP staining, MTT assay, and cell proliferation analysis was
represented in a graph with mean value and standard deviation. Differences between two
groups were compared by twoꢀtailed, paired tꢀtest. Differences between several groups were
analyzed by 2ꢀway ANOVA. P values smaller than 0.05 was marked (*p <0.05, **p <0.01
and ***p <0.001) and greater than 0.05 was marked “NS” indicating nonꢀsignificance. All of
the experiments were repeated at least three times.
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3. Result and discussion
Effects of chalcone derivatives on the proliferation and viability (metabolic activity) of
C2C12 cells
To evaluate the effect of the chalcones on the cell proliferation, C2C12 cells were treated
with the chalcones at low concentrations (5 ꢀMꢀ50 ꢀM) and cell number was measured at 1,
3, 5 and 7 days. As shown in figure 3, all chalcones up to 25 ꢀM did not show the obvious
effect on the cell proliferation over the entire incubation period. Cell proliferation was also
unaffected by A1 and A2 chalcones at 50 ꢀM. On the other hand, C2C12 cells exposed to B1
and B2 at 50 ꢀM exhibited considerably reduced cell numbers after 5 days and, at 7 days, had
approximately 38% decrease in cell numbers. We also investigated the effect of the chalcones
on the viability of C2C12 cells using high concentrations of chalcones (200 ꢀMꢀ2 mM,
Figure 4) by measuring the cellular metabolic activity. For control, cells were incubated with
DMSO alone or without any supplement. The viability of C2C12 cells treated with 200 ꢀM
chalcones was above 90%, which indicates that the chalcone derivatives have no cytotoxicity
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up to 200 ꢀM. However, B1 and B2 at ≥ 500 ꢀM remarkably decreased cell metabolism to
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less than 10% of control levels.
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In consideration of viability on DMSO treated groups, A1 and A2 have nonꢀcytotoxicity
property over 2 mM. Overall, group A has nonꢀtoxic derivatives for all treated concentration,
whereas, group B has toxicity and suppress cell proliferation depending on increase of treated
concentration. These results are agreed with structural differences, group A and group B are
substituted hydroxyl groups and methoxy group at 2' and 4' positions of A benzene ring,
individually (figure 1a). The structural differences between group A and B could affect cell
survival and proliferation9. In the point of toxicology view demonstrated by previous study
on chalcone, the hydroxyl groups at benzene ring reduce toxicity of the synthesized chalcone
and methoxy groups at A ring increase antiꢀproliferative activity9. However, the substituents
on B ring could not be assigned any effects on cells. In addition, lipophilicity and
hydrophilicity on chalcone groups could be responsible for cell behavior; lipophilic chalcone
(high log P) shows rapid cellular uptake and accumulation, because cellular membrane is
comprised of phospholipids, resulting in negative influence on cell proliferation behavior and
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toxic effects9, 23
.
Osteogenic differentiation activity of chalcone derivatives on C2C12
To determine whether chalone derivatives have osteogenic inducing potency on C2C12, the
cells were incubated with chalcone derivatives and osteogenesis inducing growth factor,
BMPꢀ2 for 8 days. The C2C12 is mouse myoblast cell line, which has myoblastic properties,
also could differentiate to osteoblast linage by BMPꢀ2 treatment24. The BMPꢀ2 is responsible
for converting myoblastic differentiation pathway of C2C12 into osteogenic differentiation
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completely, however, these osteogenic properties are not heritable24. In this study, we
incubated chalcone groups and C2C12 together and demonstrated expression level of
Alkaline Phosphatase (ALP) which is one of the osteogenic activity marker. In figure 5 and 6,
we have employed chalcone derivatives and BMPꢀ2 to C2C12 and measured osteogenesis by
red ALP staining method. Cell survival rate was over 80% for 500 ꢀM of group A and 200
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ꢀM of group B, respectively, we assigned chalcone concentration within nonꢀcytotoxic
concentration level for osteoblastic differentiation. For high concentration of group B, the
chalcones suppressed cell proliferation although nonꢀcytotoxicity, which is an evidence of
cell proliferation inhibition ability for methoxy group on A ring. In overall, no or little
osteoblastic differentiation was observed for chalcone derivative groups, however, group A
chalcones could extremely increase osteogenic differentiation potential of C2C12 in the
presence of 100 ng/mL of BMPꢀ2, resulting in higher ALP stained levels than that of only
300 ng/mL of BMPꢀ2 treatment group (figure 5). Moreover, in case of group B treated with
BMPꢀ2, still osteogenic differentiation was occurred. To demonstrate synergic effects of
chalcone derivative combinations for osteogenic differentiation, we treated 2 different type of
chalcone derivatives with BMPꢀ2. In figure 6, ALP staining results was indicated for all
groups. In case of group A (A1+A2), the C2C12 proliferated as much as A1 and A2 treated
groups (figure 4), indicating that group A do not have cell proliferation inhibition property.
Furthermore, group A combination dramatically stimulated expression level of ALP as
compared to 100 ng and 300 ng BMPꢀ2 treatment group (figure 6B). However, the group B
treatment (B1+B2) cells could not grow well, severe cell death was occurred (data not
shown). Particularly as shown in ALP staining images for B2 treated with group A
components (A1+B2 and A2+B2), the proliferation number increased than B1 treated groups
(A1+B1 and A2+B1), revealing that higher lipophilicity can induce severe cytotoxicity and
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reduce cell proliferation during culture9. In addition, B2 treated groups were discovered that
osteogenic differentiation of C2C12 was induced without BMPꢀ2 that is not shown in other
groups and was significantly promoted than other groups with BMPꢀ2 (figure 6B). In
conclusion, for high concentration of chalcone derivatives, the chalcones could highly induce
osteogenesis of C2C12 in the presence of BMPꢀ2 within nonꢀcytotoxicity ranges. Once the
BMPꢀ2 is bound on BMP receptor (BMPR) in the cellular membrane, ERK and Smad1/5
signals are phospholylated, inducing that osteogenic differentiation genes are expressed in
nucleus25. Previous study regarding on licochalcone A, which is derived from licorice, one of
the widelyꢀused herb for traditional treatment, can induce ERK activation in preꢀosteoblastic
cells and C2C12, resulting in increase of ALP level26. In addition, some small molecules that
contain hydrogen bonding moieties and hydrophobic benzene ring have an osteogenic
inducing property to interact with Smurf1 and WW1 domain and inhibit Smad1/5
degradation16, 17. The canonical pathway to reduce activity of Smad1/5 are negatively
regulated by interaction with WW1 domain of Smurf1. The WW1 domain has hydrophobicity,
indicating that small molecules can bind with WW1 in Smurf1 and prevent domain from
interacting with Smad1/527. As reported in previous paper, the chalcone derivatives have a
hydrophobic benzene ring and act as Smurf1 inhibitor, might inducing activation of BMP
signal by Smad1/5 stream in the presence of BMPꢀ2. In addition, the licochalcone A like
structure in chalcone derivatives could influence on MAPK/ERK pathway to osteogenic
differentiation26.
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For low concentration of chalcone derivatives, we have chosen the concentration within nonꢀ
cell proliferation inhibition range, which is 50 ꢀM for group A and 25 ꢀM for group B (figure
3). In figure S1 to S4, the treated chalcones did not block the cell growth. In low
concentration, we also measured the osteogenic inducing property on chalcone derivatives
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with BMPꢀ2. Compare with high concentration, the chalcone activity to induce BMPꢀ2
signaling is highly low, however, some chalcone derivatives (group A) have strong
osteogenic differentiation potency in the absence of BMPꢀ2 (figure S2 and S4). In addition,
the BMP antagonist Noggin (NOG), one of the BMP pathway regulator28, is used for
investigating whether NOG constrains BMP signaling pathway induced by chalcone
derivatives. In case of NOG treatment, the ALP levels are increased for all chalcone groups,
which is indicated that the chalcone derivatives might knockout the exogeneous NOG
effectively. The mechanism of inhibition of BMPꢀ2 activation by NOG is that the NOG can
bind with BMPꢀ2 or BMPꢀ4 and competitively constrain their interaction with BMP receptor
type 1A (BMPR1A)29, 30. Therefore, the inhibition of BMP/NOG interaction can induce
osteogenic differentiation on cells. The computational design of small molecules that inhibit
of NOG activity to bind with active site has been explored30, 31. The hydrophobic and
hydrophilic aromatic rings in small molecules can bind with BMP/NOG interaction site on
NOG with hydrogen bonding30. Interestingly, regarding on the role of NOG, previous report
has been demonstrated that the NOG dose not perform inhibition of BMP signaling in human
mesenchymal stem cells29. Furthermore, NOG and dexamethasone which has benzene rings
coꢀtreatment were revealed that more osteogenic differentiation was stimulated, which is
suggested that noggin may dimerize with small amount of BMPꢀ2, acting as a chaperone to
stimulate BMPꢀ2 pathway29. Consequently, we believed that the phenyl ring and methoxyꢀ
group substituted aromatic rings on chalcone derivatives bind with NOG to reduce activity
and induce BMPꢀ2 signaling simultaneously in the presence of chalcones.
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In this study, we have only measured ALP staining to check the differentiation property of
chalcone derivatives, indicating that the chalcones have a potential for preꢀosteogenic
differentiation of C2C12. Since osteogenesis is a process that takes place over several weeks
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or months, it may not be accurate to identify bone regeneration efficiency by shortꢀterm
osgeogenic markers. To fully demonstrate the osteogenic differentiation property for chalcone
derivatives, on further study, we will focus on confirmation of late marker expression and in
vivo test. The calcification of cells is strong signal to indicate osteogenic differentiation on
cells, which could be confirmed by Alizarin red, Von Kossa staining and quantitative analysis
of expression protein level. In our result, we have found that the chalcone compound can act
as an agent to stimulate osteogenesis when it is treated with BMPꢀ2, by stimulating BMPꢀ2
signaling pathway and blocking BMP antagonist as well. Furthermore, we have confirmed
that 2 types of blended chalcones improved ALP expression level and viability. For the future
work, it should be tested to demonstrate bone regeneration of chalcone derivatives by
implantation of scaffold with BMPꢀ2 and chalcone cocktails cultured with stem cells to bone
fracture model. We speculate the chalcones might increase bone regeneration and it could be
helpful for bone regeneration inducing agent.
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Antibacterial property of chalcone derivatives
It has been known that antibacterial property of chalcone derivatives primarily depends on
substitution of functional groups and the lipophilicity2,32. In the present study, we assessed
whether each of the chalcone derivatives inhibits the growth of S. mutans, a Gram-positive
facultative anaerobic bacterium that is a major pathogen of dental caries33. A1 (0.5 and 1.0
mM) resulted in approximately 30% growth reduction compared to the control at 24 h (figure
7). Meanwhile A2, B1 and B2 showed no inhibitory effect at the concentrations between
0.001 and 1.0 mM (data not shown). These results indicate that hydroxyl groups on A ring
confer the antibacterial effect of A1 chalcone. Especially, hydroxyl group at 2-position on the
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A ring, a dominant functional group, may contribute to stabilizing of the molecular structure
of the chalcones, as reported previously8. Notably, A2 bearing an additional hydroxyl group
on the B ring showed no antibacterial effect. It is possible that the hydroxyl group on 4ꢀ
position of the B ring decreases lipophilicity of the chalcone, resulting loss of antibacterial
activity of A2 due to its poor interaction with bacterial membrane32. The methoxy
substituents in B1 and B2 seems to reduce activation of chalcone.
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4. Conclusion
In this study, we have synthesized 4 kinds of chalcone derivatives and found that the
chalcones have osteogenic differentiation and antibacterial properties. Substitutions
(hydroxyl and methoxy group) on A and B rings on chalcones make the different toxicity and
proliferation potentials. In case of group A chalcones, there were no cytotoxicity and antiꢀ
proliferation effect within 2 mM and 50 ꢁM, respectively. In the presence of BMPꢀ2, high
concentrations of the chalcones (at 200 ꢀM and 500 ꢀM) significantly induced osteogenesis
of C2C12. At low concentrations (at less than 50 ꢀM) the chalcones might inhibit NOG
activity which is BMP antagonist and express ALP levels at low chalcone concentrations.
Specially, the A1 chalcone exerted antibacterial effect against S. mutans. In conclusion, the
chalcone derivatives synthesized in this study can be used for bone regeneration, with an
additional effects of inhibiting S. mutans, in the field of dentistry.
Acknowledgement
This research was supported by a grant of the Korea Health Technology R&D Project
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through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of
Health & Welfare, Republic of Korea (HI14Cꢀ3266) and Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by the Ministry of Science
and ICT (NRFꢀ2017R1E1A1A01074343). This study was also supported by the ChungꢀAng
University Graduate Research Scholarship in 2016.
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Supporting Information
ALP staining results are displayed on Supporting information. This material is available free
of charge via the Internet at http://pubs.acs.org.
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Figure 1. A) Structure of basic chalcone. B) Structures of chalcone derivatives used in this
study. The chalcones are simplified as group A and group B chalcones.
O
OH
O
OH
O
OH
O
3% HCl
EtOH
R'
KOH/EtOH
RO
OR
RO
OR
R'
RO
OR
R'
A
B
1
2
A1'
A2'
B1
A1
A2
B1
B2
: R = MOM
: R = CH3
: R' = H
: R' = OMOM
: R = MOM, R' = H
: R = MOM, R' = OMOM
: R = CH3, R' = H
: R = H,
: R = H,
: R = CH3, R' = H
: R = CH3, R' = OH
R' = H
R' = OH
B2'
: R = CH3, R' = OMOM
Figure 2. Structural illustration of preparation of Chalcone derivatives
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Figure 3. Cell proliferation curve of C2C12 in the presence of 5 to 50 ꢀM of chalcone
derivatives (Low concentration). Statistical significance (*p < 0.05) is determined
comparison with control.
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Figure 4. Cell viability (metabolic activity) of C2C12 in the presence of high amount of
chalcone concentrations. Statistical significance (*p < 0.05, **p <0.01 and NS) is determined
in comparison with DMSO control.
Figure 5. (A) Alkaline Phosphatase (ALP) staining of C2C12 after 8 days of incubation with
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chalcone derivatives or BMPꢀ2. (B) Quantatively analyzed ALP stained area (%) of each
group calculated from ALP staining images. Statistical significance (*p < 0.05 and **p <0.01)
is determined in comparison with 300 ng BMPꢀ2.
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Figure 6. (A) Alkaline Phosphatase (ALP) staining of C2C12 after 8 days of incubation with
combination of chalcones or BMPꢀ2. (B) Quantatively analyzed ALP stained area (%) of each
group calculated from ALP staining images. Statistical significance (*p < 0.05, **p <0.01 and
***p <0.001) is determined by tꢀtest.
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Figure 7. Antibacterial property of chalcone. S. mutans was incubated with A1 for 1 day to
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Molecular Pharmaceutics
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