Effect of Cudrania tricuspidata on Cariogenic Properties and Caries-Related Gene Expression in Streptococcus mutans
Abstract
:1. Introduction
2. Results
2.1. UPLC-Q-TOF-MS Analysis Results of C. tricuspidata Extract
2.2. Inhibitory Effect on S. mutans Growth
2.3. Inhibitory Effect on S. mutans Biofilm Formation
2.4. Inhibitory Effect on S. mutans Adherence
2.5. Inhibitory Effect on Organic Acid and Free Calcium Production by S. mutans
3. Discussion
4. Materials and Methods
4.1. Material Extraction
4.2. Bacterial Culture
4.3. Biofilm Formation Assay
4.4. Scanning Electron Microscopic Analysis of S. mutans Biofilm Formation
4.5. Bacterial Adherence
4.6. Analysis of Organic Acid Production and Calcium Release by S. mutans
4.6.1. Analysis of Organic Acid Production
4.6.2. Analysis of Calcium Release
4.7. Real-Time Polymerase Chain Reaction (PCR) Analysis of S. mutans Virulence Genes
4.8. UPLC-Q-TOF-MS Analysis
4.9. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Seneviratne, C.J.; Zhang, C.F.; Samaranayake, L.P. Dental plaque biofilm in oral health and disease. Chin. J. Dent. Res. 2011, 14, 87–94. [Google Scholar] [PubMed]
- de Soet, J.J.; van Loveren, C.; van Lammens, A.J.; Pavicić, M.J.; Homburg, C.H.; ten Cate, J.M.; de Graaff, J. Differences in cariogenicity between fresh isolates of Streptococcus sobrinus and Streptococcus mutans. Caries Res. 1991, 25, 116–122. [Google Scholar] [CrossRef] [PubMed]
- Rabin, N.; Zheng, Y.; Opoku-Temeng, C.; Du, Y.; Bonsu, E.; Sintim, H.O. Biofilm formation mechanisms and targets for developing antibiofilm agents. Future Med. Chem. 2015, 7, 493–512. [Google Scholar] [CrossRef]
- van Houte, J. Role of micro-organisms in caries etiology. J. Dent. Res. 1994, 73, 672–681. [Google Scholar] [CrossRef]
- You, Y.O. Virulence genes of Streptococcus mutans and dental caries. Int. J. Oral. Biol. 2019, 44, 31–36. [Google Scholar] [CrossRef]
- Shemesh, M.; Tam, A.; Steinberg, D. Expression of biofilm-associated genes of Streptococcus mutans in response to glucose and sucrose. J. Med. Microbiol. 2007, 56, 1528–1535. [Google Scholar] [CrossRef]
- Yang, J.; Deng, D.; Brandt, B.W.; Nazmi, K.; Wu, Y.; Crielaard, W.; Ligtenberg, A.J.M. Diversity of SpaP in genetic and salivary agglutinin mediated adherence among Streptococcus mutans strains. Sci. Rep. 2019, 9, 9943. [Google Scholar] [CrossRef]
- Burne, R.A. Oral streptococci… products of their environment. J. Dent. Res. 1998, 77, 445–452. [Google Scholar] [CrossRef]
- Zhang, Q.; Ma, Q.; Wang, Y.; Wu, H.; Zou, J. Molecular mechanisms of inhibiting glucosyltransferases forbiofilm formation in Streptococcus mutans. Int. J. Oral Sci. 2021, 13, 30. [Google Scholar] [CrossRef]
- Lemos, J.A.C.; Brown, T.A., Jr.; Burne, R.A. Effects of RelA on key virulence properties of planktonic and biofilm populations of Streptococcus mutans. Infect. Immun. 2004, 72, 1431–1440. [Google Scholar] [CrossRef]
- Steinberg, D.; Moreinos, D.; Featherstone, J.; Shemesh, M.; Feuerstein, O. Genetic and physiological effects of noncoherent visible light combined with hydrogen peroxide on Streptococcus mutans in biofilm. Antimicrob. Agents Chemother. 2008, 52, 2626–2631. [Google Scholar] [CrossRef] [PubMed]
- Xu, X.; Zhou, X.D.; Wu, C.D. The tea catechin epigallocatechin gallate suppresses cariogenic virulence factors of Streptococcus mutans. Antimicrob. Agents Chemother. 2011, 55, 1229–1236. [Google Scholar] [CrossRef]
- Chi, B.R.; Jo, D.Y.; Cha, S.Y.; Chi, M.J.; Hye-won Jeong, H.W.; Kang, K.H. The effect on growth inhibition of S. mutans by lotus leaf and Dandelion extracts. J. Korea Acad.-Ind. Coop. Soc. 2011, 12, 5773–5778. [Google Scholar] [CrossRef]
- Jung, E.J.; Hong, S.J.; Choi, J.I.; Jeong, S.S.; Oh, H.N.; Lee, H.J.; Choi, C.H. In vitro growth inhibition of Streptococcus mutans by extract of prickly pear (Opuntia ficus-indica var. saboten Makino). J. Korea Acad. Oral Health 2010, 34, 28–35. [Google Scholar]
- Kang, S.Y.; An, S.Y.; Lee, M.W.; Kwon, S.K.; Lee, D.H.; Jeon, B.H.; You, Y.O.; Lee, M.W. Effects of Aconitum Koreanum extract on the growth, acid production, adhesion and insoluble glucan synthesis of Streptococcus mutans. J. Physiol. Pathol. Korean Med. 2015, 29, 27–32. [Google Scholar] [CrossRef]
- You, Y.O.; Yu, H.H.; Kim, Y.J.; You, M.S.; Seo, S.J.; Lee, L.; Lee, H.S. Effects of Caesalpinia sappan extracts on the growth, acid production, adhesion, and insoluble glucan synthesis of Streptococcus mutans. J. Korean Acad. Dent. Health 2003, 27, 277–288. [Google Scholar]
- Kim, H.S.; Lee, S.W.; Sydara, K.; Cho, S.J. Antibacterial and antibiofilm activities of Diospyros malabarica stem extract against Streptococcus mutans. J. Life Sci. 2019, 29, 90–96. [Google Scholar] [CrossRef]
- Choi, H.J.; Kim, C.T.; Do, M.Y.; Rang, M.J. Physiological activities of Cudrania tricuspidata extracts (Part I). J. Korea Acad.-Ind. Coop. Soc. 2013, 14, 3907–3915. [Google Scholar] [CrossRef]
- Kim, J.W.; Cho, N.M.; Kim, E.M.; Park, K.S.; Kang, Y.W.; Nam, J.H.; Nam, M.S.; Kim, K.K. Cudrania tricuspidata leaf extracts and its components, chlorogenic acid, kaempferol, and quercetin, increase claudin 1 expression in human keratinocytes, enhancing intercellular tight junction capacity. Appl. Biol. Chem. 2020, 32, 260–274. [Google Scholar] [CrossRef]
- Quang, T.H.; Ngan, N.T.; Yoon, C.S.; Cho, K.H.; Kang, D.G.; Lee, H.S.; Kim, Y.C.; Oh, H. Protein tyrosine phosphatase 1B inhibitors from the roots of Cudrania tricuspidata. Molecules 2015, 20, 11173–11183. [Google Scholar] [CrossRef]
- Jeon, S.M.; Lee, D.S.; Jeong, G.S. Cudraticusxanthone A isolated from the roots of Cudrania tricuspidata inhibits metastasis and induces apoptosis in breast cancer cells. J. Ethnopharmacol. 2016, 194, 57–62. [Google Scholar] [CrossRef] [PubMed]
- Lim, J.W.; Jo, Y.H.; Choi, J.S.; Lee, M.K.; Lee, K.Y.; Kang, S.Y. Antibacterial activities of prenylated isoflavones from Maclura tricuspidata against fish pathogenic Streptococcus: Their structure-activity relationships and extraction optimization. Molecules 2021, 26, 7451. [Google Scholar] [CrossRef] [PubMed]
- Katsura, H.; Tsukiyama, R.I.; Suzuki, A.; Kobayashi, M. In vitro antimicrobial activities of bakuchiol against oral microorganisms. Antimicrob. Agents Chemother. 2001, 45, 3009–3013. [Google Scholar] [CrossRef]
- Choi, S.R.; You, D.H.; Kim, J.Y.; Park, C.B.; Kim, D.H.; Ryu, J.; Choi, D.G.; Park, H.M. Antimicrobial Activity of Methanol Extracts from Cudrania tricuspidata Bureau according to the Parts Harvested and Time. Korean J. Med. Crop. Sci. 2009, 17, 335–340. [Google Scholar]
- Geske, L.; Baier, J.; Joelle, C.; Boulos, J.C.; Efferth, T.; Opatz, T. Xylochemical synthesis and biological evaluation of the orchidaceous natural products isoarundinin I, bleochrin F, blestanol K, and pleionol. J. Nat. Prod. 2023, 86, 131–137. [Google Scholar] [CrossRef]
- Li, W.; Fu, J.R.; Zheng, L.J.; Ni, L.; Liu, J.Q.; Zhai, J.W.; Zhou, Z.; Wu, S.S. Two new bibenzyls from Pleione grandiflora (Rolfe) Rolfe and their antioxidant activity. Nat. Prod. Res. 2023, 37, 2486–2492. [Google Scholar] [CrossRef] [PubMed]
- Kim, D.C.; Quang, T.H.; Oh, H.; Kim, Y.C. Cudratricusxanthone L suppresses lipopolysaccharideinduced activation of BV2 and primary rat microglial cells by inhibiting JNK, p38 MAPK, and NF-κB signaling. Preprints 2018, 2018080197. [Google Scholar] [CrossRef]
- Lee, H.J.; Ryu, J.; Park, S.H.; Woo, E.R.; Kim, A.R.; Lee, S.K.; Kim, Y.S.; Kim, J.O.; Hong, J.H.; Lee, C.J. Effects of Morus alba L. and natural products including morusin on in vivo secretion and in vitro production of airway MUC5AC mucin. Tuberc. Respir. Dis. 2014, 77, 65–72. [Google Scholar] [CrossRef]
- Park, J.H.; Park, K.L.; Ho, H.S.; Kim, H.D.; Pyo, M.Y. Synthesis and antitumor activity of novel gericudranin E derivatives. J. Pharm. Soc. Korea 1999, 43, 559–565. [Google Scholar]
- Lee, B.W.; Kang, N.S.; Park, K.H. Isolation of antibacterial prenylated flavonoids from Cudrania tricuspidata. Appl. Biol. Chem. 2004, 47, 270–273. [Google Scholar]
- Dong, H.B.; Liao, L.; Yu, P.; Long, B.; Che, Y.; Lu, L.; Xu, B. Total syntheses and antibacterial evaluations of cudraflavones A-C and related Flavones. Bioorg Chem. 2023, 140, 106764. [Google Scholar] [CrossRef] [PubMed]
- Fujiwara, T.; Terao, Y.; Hoshino, T.; Kawabata, S.; Ooshima, T.; Sobue, S.; Kimura, S.; Hamada, S. Molecular analyses of glucosyltransferase genes among strains of Streptococcus mutans. FEMS Microbiol. Lett. 1998, 161, 331–336. [Google Scholar] [CrossRef]
- Dua, Y.; Li, G.; Li, X.; Jian, X.; Wang, X.; Xie, Y.; Li, Z.; Zhang, Z. Dietary Immunoglobulin Y by Targeting Both GbpB and GtfB Enhances the Anticaries Effect in Rats. Int. Dent. J. 2024, 74, 1298–1305. [Google Scholar] [CrossRef]
- Krzysciak, W.; Jurczak, A.; Koscielniak, D.; Bystrowska, B.; Skalniak, A. The virulence of Streptococcus mutans and the ability to form biofilms. Eur. J. Clin. Microbiol. Infect. Dis. 2014, 33, 499–515. [Google Scholar] [CrossRef] [PubMed]
- Frostell, G. Dental plaque pH in relation to intake of carbohydrate products. Acta Odontol. Scand. 1969, 27, 3–29. [Google Scholar] [CrossRef] [PubMed]
- Köhler, B.; Birkhed, D.; Olsson, S. Acid production by human strains of Streptococcus mutans and Streptococcus sobrinus. Caries Res. 1995, 29, 402–406. [Google Scholar] [CrossRef]
- Park, B.I.; Jung, Y.W.; Kim, Y.H.; Lee, S.M.; Kwon, L.S.; Kim, K.J.; You, Y.O. Effect of the ethanol extract of propolis on formation of Streptococcus mutans biofilm. Int. J. Oral Biol. 2016, 41, 253–262. [Google Scholar] [CrossRef]
- O’Neill, E.; Hilary, H.; James, P.O. Carriage of both the fnbA and fnbB genes and growth at 37 °C promote FnBP mediated biofilm development in meticillin-resistant Staphylococcus aureus clinical isolates. J. Med. Microbiol. 2009, 58, 399–402. [Google Scholar] [CrossRef]
- Kwang, H.L.; Kim, B.S.; Keum, K.S.; Yu, H.H.; Kim, Y.H.; Chang, B.S.; Ra, J.Y.; Moon, H.D.; Seo, B.R.; Choi, N.Y.; et al. Essential oil of Curcuma longa inhibits Streptococcus mutans biofilm formation. J. Food Sci. 2011, 76, H226–H230. [Google Scholar] [CrossRef]
- Liljemark, W.F.; Bloomquist, C.G.; Germaine, G.R. Effect of bacterial aggregation on the adherence of oral streptococci to hydroxyapatite. Infect. Immun. 1981, 31, 935–941. [Google Scholar] [CrossRef]
- Jeong, S.I.; Kim, B.S.; Keum, K.S.; Lee, K.H.; Kang, S.Y.; Park, B.I.; Lee, Y.R.; You, Y.O. Kaurenoic acid from Aralia continentalis inhibits biofilm formation of Streptococcus mutans. Evid. Based Complement. Altern. Med. 2013, 2013, 160592. [Google Scholar] [CrossRef] [PubMed]
Peak No. | RT (min) | Tentative Identification | Mass [M − H]− or [M − HCOO]− | Neutral Mass (m/z) | Molecular Formula | Mass Error (ppm) |
---|---|---|---|---|---|---|
Observed (m/z) | ||||||
1 | 1.85 | Koaburaside | 331.1038 | 332.1107 | C14H20O9 | 1.1 |
2 | 2.46 | Asperuloside (isomer I) | 459.1157 | 414.1162 | C18H22O11 | 2.8 |
3 | 2.59 | Cichoriin | 339.0731 | 340.0794 | C15H16O9 | 2.9 |
4 | 2.59 | 2-Hydroxy-1,4-Naphthoquinone | 219.0304 | 174.0317 | C10H6O3 | 2.4 |
5 | 2.81 | Asperuloside (isomer II) | 459.1167 | 414.1162 | C18H22O11 | 4.9 |
6 | 3.02 | Cistanoside B | 859.2918 | 814.2895 | C37H50O20 | 4.7 |
7 | 3.07 | Viscumneoside V | 727.2123 | 728.2164 | C32H40O19 | 4.4 |
8 | 3.18 | Jionoside B1 | 859.2917 | 814.2895 | C37H50O20 | 4.6 |
9 | 3.26 | Tenuifoliside A | 727.2124 | 682.2109 | C31H38O17 | 4.5 |
10 | 4.36 | Khellin | 259.0625 | 260.0685 | C14H12O5 | 5 |
11 | 5.03 | Gericudranin E (isomer I) | 393.0989 | 394.1053 | C22H18O7 | 2.2 |
12 | 5.37 | Gericudranin E (isomer II) | 393.0996 | 394.1053 | C22H18O7 | 4.1 |
13 | 5.61 | Tianshic acid | 329.2342 | 330.2406 | C18H34O5 | 2.4 |
14 | 6.27 | Cudratricusxanthone L (isomer I) | 327.0882 | 328.0947 | C18H16O6 | 2.3 |
15 | 6.35 | Licoricone | 427.1401 | 382.1416 | C22H22O6 | 0.7 |
16 | 6.46 | Mortatarin B (isomer I) | 455.1715 | 456.1784 | C25H28O8 | 0.8 |
17 | 6.59 | Mortatarin B (isomer II) | 455.1711 | 456.1784 | C25H28O8 | 0 |
18 | 6.7 | Mortatarin B (isomer III) | 455.1711 | 456.1784 | C25H28O8 | −0.2 |
19 | 6.73 | Cudratricusxanthone L (isomer II) | 327.0872 | 328.0947 | C18H16O6 | −0.6 |
20 | 6.82 | Cudratricusxanthone L (isomer III) | 327.0877 | 328.0947 | C18H16O6 | 0.8 |
21 | 6.85 | Cudratricusxanthone M | 413.1603 | 414.1679 | C23H26O7 | −0.7 |
22 | 6.97 | Cudratricusxanthone L (isomer IV) | 327.0877 | 328.0947 | C18H16O6 | 1 |
23 | 7.08 | Mortatarin C (isomer I) | 439.1757 | 440.1835 | C25H28O7 | −1.1 |
24 | 7.16 | Mortatarin C (isomer II) | 439.1763 | 440.1835 | C25H28O7 | 0.1 |
25 | 7.21 | Cudratricusxanthone L (isomer V) | 327.0872 | 328.0947 | C18H16O6 | −0.7 |
26 | 7.24 | Mortatarin C (isomer III) | 439.1756 | 440.1835 | C25H28O7 | −1.3 |
27 | 7.34 | Cudratricusxanthone L (isomer VI) | 327.0872 | 328.0947 | C18H16O6 | −0.6 |
28 | 7.46 | Kuwanon E (isomer I) | 423.1815 | 424.1886 | C25H28O6 | 0.5 |
29 | 7.54 | 5,7,3′,4′-Tetramethoxyflavone | 341.1040 | 342.1103 | C19H18O6 | 2.8 |
30 | 7.7 | Isoarundinin II (isomer I) | 395.1495 | 350.1518 | C22H22O4 | −1.2 |
31 | 7.82 | Isoarundinin II (isomer II) | 395.1505 | 350.1518 | C22H22O4 | 1.2 |
32 | 7.88 | Cudraflavenone A (isomer I) | 421.1657 | 422.1729 | C25H26O6 | 0.2 |
33 | 7.97 | Isoarundinin II (isomer III) | 395.1500 | 350.1518 | C22H22O4 | 0 |
34 | 8.01 | Cudraflavenone A (isomer II) | 421.1655 | 422.1729 | C25H26O6 | −0.4 |
35 | 8.02 | Kuwanon E (isomer II) | 423.1823 | 424.1886 | C25H28O6 | 2.4 |
36 | 8.09 | Bavacoumestan A | 351.0873 | 352.0947 | C20H16O6 | −0.4 |
37 | 8.22 | Isoarundinin II (isomer IV) | 395.1507 | 350.1518 | C22H22O4 | 1.6 |
38 | 8.24 | Cudraflavenone A (isomer III) | 421.1662 | 422.1729 | C25H26O6 | 1.2 |
39 | 8.35 | Cudraxanthone B (isomer I) | 393.1343 | 394.1416 | C23H22O6 | −0.1 |
40 | 8.42 | Kuwanon A | 419.1504 | 420.1573 | C25H24O6 | 1 |
41 | 8.55 | Isoarundinin II (isomer V) | 395.1500 | 350.1518 | C22H22O4 | 0 |
42 | 8.68 | Isoarundinin II (isomer VI) | 395.1501 | 350.1518 | C22H22O4 | 0.2 |
43 | 8.74 | Isoarundinin II (isomer VII) | 395.1516 | 350.1518 | C22H22O4 | 4.1 |
44 | 8.81 | Isoarundinin II (isomer IX) | 395.1520 | 350.1518 | C22H22O4 | 5 |
45 | 8.82 | Cudraxanthone B (isomer II) | 393.1347 | 394.1416 | C23H22O6 | 0.9 |
46 | 8.94 | Isoarundinin II | 395.1510 | 350.1518 | C22H22O4 | 2.6 |
47 | 9.04 | Cudraxanthone B (isomer III) | 393.1348 | 394.1416 | C23H22O6 | 1 |
48 | 9.19 | Isoarundinin II | 395.1503 | 350.1518 | C22H22O4 | 0.8 |
Con. (μg/mL) | pH (Before Incubation) | pH (After Incubation) |
---|---|---|
Control | 7.33 ± 0.04 a | 5.39 ± 0.10 a |
15 | 7.30 ± 0.02 a | 5.32 ± 0.12 a |
30 | 7.32 ± 0.02 a | 6.26 ± 0.04 b |
45 | 7.34 ± 0.06 a | 7.13 ± 0.04 c |
60 | 7.31 ± 0.04 a | 7.17 ± 0.07 c |
CHX (0.05%) | 7.34 ± 0.07 a | 7.15 ± 0.05 c |
Genes * | Primer Sequences (5’-3’) | |
---|---|---|
16S rRNA | Forward | CCTACGGGAGGCAGCAGTAG |
Reverse | CAACAGAGCTTTACGATCCGAAA | |
gbpB | Forward | ATGGCGGTTATGGACACGTT |
Reverse | TTTGGCCACCTTGAACACCT | |
spaP | Forward | GACTTTGGTAATGGTTATGCATCAA |
Reverse | TTTGTATCAGCCGGATCAAGTG | |
gtfB | Forward | AGCAATGCAGCCAATCTACAAAT |
Reverse | ACGAACTTTGCCGTTATTGTCA | |
gtfC | Forward | GGTTTAACGTCAAAATTAGCTGTATTAGC |
Reverse | CTCAACCAACCGCCACTGTT | |
gtfD | Forward | ACAGCAGACAGCAGCCAAGA |
Reverse | ACTGGGTTTGCTGCGTTTG | |
vicR | Forward | TGACACGATTACAGCCTTTGATG |
Reverse | CGTCTAGTTCTGGTAACATTAAGTCCAATA | |
relA | Forward | ACAAAAAGGGTATCGTCCGTACAT |
Reverse | AATCACGCTTGGTATTGCTAATTG | |
brpA | Forward | GGAGGAGCTGCATCAGGATTC |
Reverse | AACTCCAGCACATCCAGCAAG |
Function | Gene | Biological Roles |
---|---|---|
Adhesion | gbpB | Encode glucan binding proteins (GBP) A GBP in the cell membrane of S. mutans plays an important role in adherence of S. mutans to glucan molecules. |
spaP | Encodes cell surface antigen (SpaP) Adheres to salivary agglutinin glycoprotein (SAG) and proline-rich protein of the acquired pellicle on the tooth surface as a kind of surface fibrillar adhesin. | |
Formation of extracellular polysaccharide in biofilm | gtfB, gtfC, gtfD | Encode glycosyltransferase (GTF) B, GTF C, and GTF D. Synthesize glucan by polymerizing glucose. |
Regulation | vicR | Encodes putative histidine kinase. Regulates expression of gtfB, gtfC, and gtfD. |
Sugar uptake and metabolism | relA | Contributes to the regulation of glucose phosphotransferase system (PTS), the glucose uptake system of S. mutans |
Acid tolerance | brpA | Contributes to acid tolerance |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 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/).
Share and Cite
Kim, E.-S.; Jeong, J.-E.; Kim, Y.-H.; You, Y.-O. Effect of Cudrania tricuspidata on Cariogenic Properties and Caries-Related Gene Expression in Streptococcus mutans. Molecules 2025, 30, 1755. https://doi.org/10.3390/molecules30081755
Kim E-S, Jeong J-E, Kim Y-H, You Y-O. Effect of Cudrania tricuspidata on Cariogenic Properties and Caries-Related Gene Expression in Streptococcus mutans. Molecules. 2025; 30(8):1755. https://doi.org/10.3390/molecules30081755
Chicago/Turabian StyleKim, Eun-Sook, Ji-Eon Jeong, Young-Hoi Kim, and Yong-Ouk You. 2025. "Effect of Cudrania tricuspidata on Cariogenic Properties and Caries-Related Gene Expression in Streptococcus mutans" Molecules 30, no. 8: 1755. https://doi.org/10.3390/molecules30081755
APA StyleKim, E.-S., Jeong, J.-E., Kim, Y.-H., & You, Y.-O. (2025). Effect of Cudrania tricuspidata on Cariogenic Properties and Caries-Related Gene Expression in Streptococcus mutans. Molecules, 30(8), 1755. https://doi.org/10.3390/molecules30081755