Abstract

Polyphenols are potential neuroprotectors that increase lifespan and slow down aging. Red clover (Trifolium pratense L.) is a promising source of biologically active substances. Its extracts contain biochanin A and chlorogenic acid. This research used Caenorhabditis elegans to study the effect of polyphenols extracted from red clover callus cultures on SOD-3 and HSP-16.2 genes, as well as their anti-amyloid potential.
The chlorogenic acid and biochanin A (200, 100, 50, and 10 µM) with a purity of 95% were isolated from callus extracts of T. pratense L. The effect of polyphenols on SOD-3 and HSP-16.2 was assessed after 5 and 2 h of heat stress (35°C), respectively, using C. elegans N2 Bristol as model organism. The neuroprotective potential was measured by counting paralyzed nematodes after 18, 40, and 62 h of incubation.
The research established a dose-dependent effect between the concentration of biologically active substances and the percentage of paralyzed nematodes after 18 h of cultivation. The lowest paralysis phenotype count occurred at a concentration of 200 μM. The activity of 200 μM biochnin A was 1.18 times as high as that of a 200 μM chlorogenic acid solution. Biochanin A solutions increased SOD-3 expression by 3.7 times, compared to the control.
The biologically active substances exhibited relative neuroprotective activity and affected the expression of antioxidant defense gene in C. elegans.

Keywords

Aging, Alzheimer's disease, neuroprotectors, polyphenols, chlorogenic acid, biochanin A, Caenorhabditis elegans, β-amyloid peptide

FUNDING

The research was part state task FZSR-2023-0002 “Polyphenols from Siberian Federal District plants: assessment of molecular and spatial structure, characterization of biofunctional properties, and toxicological safety indicators using in vivo model systems”. It involved the Shared Use Center for Instrumental Analysis in Applied Biotechnology, Kemerovo State University (KemSU) .

REFERENCES

1. Abate G, Marziano M, Rungratanawanich W, Memo M, Uberti D. Nutrition and AGE-ing: Focusing on Alzheimer's disease. Oxidative Medicine and Cellular Longevity. 2017;2017. https://doi.org/10.1155/2017/7039816
2. Gonzalez-Freire M, Diaz-Ruiz A, Hauser D, Martinez-Romero J, Ferrucci L, Bernier M, et al. The road ahead for health and lifespan interventions. Ageing Research Reviews. 2020;59. https://doi.org/10.1016/j.arr.2020.101037
3. Bitto A, Wang AM, Bennett CF, Kaeberlein M. Biochemical genetic pathways that modulate aging in multiple species. Cold Spring Harbor Perspectives in Medicine. 2015;5. https://doi.org/10.1101/cshperspect.a025114
4. Rusek M, Pluta R, Ułamek-Kozioł M, Czuczwar SJ. Ketogenic diet in Alzheimer's disease. International Journal of Molecular Sciences. 2019;20(16). https://doi.org/10.3390/ijms20163892
5. Collins AE, Saleh TM, Kalisch BE. Naturally occurring antioxidant therapy in Alzheimer's disease. Antioxidants. 2022;11(2). https://doi.org/10.3390/antiox11020213
6. Wang Y, Wang K, Yan J, Zhou Q, Wang X. Recent progress in research on mechanisms of action of natural products against Alzheimer's disease: Dietary plant polyphenols. International Journal of Molecular Sciences. 2022;23(22). https://doi.org/10.3390/ijms232213886
7. Prosekov AYu, Ostroumov LA. Innovation management biotechnology of starter cultures. Food Processing: Techniques and Technology. 2016;43(4):64–69. (In Russ.). https://www.elibrary.ru/XELELB
8. Kharitonov DV, Kharitonova IV, Prosekov AYu. The concept of synbiotics and synbiotic dairy products development. Food Processing: Techniques and Technology. 2013;31(4):91–94. (In Russ.). https://www.elibrary.ru/RNIEON
9. Vesnina A, Prosekov A, Atuchin V, Minina V, Ponasenko A. Tackling atherosclerosis via selected nutrition. International Journal of Molecular Sciences. 2022;23(15). https://doi.org/10.3390/ijms23158233
10. McGrattan AM, McGuinness B, McKinley MC, Kee F, Passmore P, Woodside JV, et al. Diet and inflammation in cognitive ageing and Alzheimer's disease. Current Nutrition Reports. 2019;8:53–65. https://doi.org/10.1007/s13668-019-0271-4
11. Hu N, Yu J-T, Tan L, Wang Y-L, Sun L, Tan L. Nutrition and the risk of Alzheimer's disease. BioMed Research International. 2013;2013. https://doi.org/10.1155/2013/524820
12. Shen N, Wang T, Gan Q, Liu S, Wang L, Jin B. Plant flavonoids: Classification, distribution, biosynthesis, and antioxidant activity. Food Chemistry. 2022;383. https://doi.org/10.1016/j.foodchem.2022.132531
13. Dhakal S, Kushairi N, Phan CW, Adhikari B, Sabaratnam V, Macreadie I. Dietary polyphenols: A multifactorial strategy to target Alzheimer's disease. International Journal of Molecular Sciences. 2019;20(20). https://doi.org/10.3390/ijms20205090
14. Xin L, Yamujala R, Wang Y, Huan W, Wu W-H, Lawton MA, et al. Acetylcholineestarase-inhibiting alkaloids from Lycoris radiata delay paralysis of amyloid beta-expressing transgenic C. elegans CL4176. PloS ONE. 2013;8(5). https://doi.org/10.1371/journal.pone.0063874
15. Wu Y, Wu Z, Butko P, Christen Y, Lambert MP, Klein WL, et al. Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans. Journal of Neuroscience. 2006;26(50):13102–13113. https://doi.org/10.1523/JNEUROSCI.3448-06.2006
16. Nguyen TS, Alekseeva GM, Generalova YuE, Kaukhova IE, Sorokin VV. Determination of isoflavone content by HPLC in dried extract of Trifolium pratense L. Journal of Pharmaceuticals Quality Assurance Issue. 2020;27(1):48–53. (In Russ.). https://doi.org/10.34907/JPQAI.2020.60.61.006
17. Dyshlyuk LS, Osintseva MA, Kozlova OV, Fotina NV, Prosekov AYu. Antiradical and oxidative stress release properties of Trifolium pratense L. extract. Journal of Experimental Biology and Agricultural Sciences. 2022;10(4):852–860. https://doi.org/10.18006/2022.10(4).852.860
18. Temerdashev ZA, Chubukina TK, Vinitskaya EA, Nagalevskii MV, Kiseleva NV. Assessment of the concentrations of isoflavonoids in red clover (Trifolium pratense L.) of the Fabaceae family using extraction by different methods. Journal of Analytical Chemistry. 2021;76(9):819–831. (In Russ.). https://doi.org/10.31857/S0044450221090115
19. Bijttebier S, van der Auwera A, Voorspoels S, Noten B, Hermans N, Pieters L, et al. A first step in the quest for the active constituents in Filipendula ulmaria (meadowsweet): Comprehensive phytochemical identification by liquid chromatography coupled to quadrupole-orbitrap mass spectrometry. Planta Medica. 2016;82(6):559–572. https://doi.org/10.1055/s-0042-101943
20. Kapil A, Koul IB, Suri OP. Antihepatotoxic effects of chlorogenic acid from Anthocephalus cadamba. Phytotherapy Research. 1995;9(3):189–193. https://doi.org/10.1002/ptr.2650090307
21. Rashidi R, Rezaee R, Shakeri A, Wallace Hayes A, Karimi G. A review of the protective effects of chlorogenic acid against different chemicals. Journal of Food Biochemistry. 2022;46(9). https://doi.org/10.1111/jfbc.14254
22. Dmitrieva A, Vesnina A, Dyshlyuk L. Antioxidant and antimicrobial properties of squalene from Symphytum officinale and chlorogenic acid from trifolium pretense. AIP Conference Proceedings. 2022;2636(1). https://doi.org/10.1063/5.0104513
23. Ishida K, Yamamoto M, Misawa K, Nishimura H, Misawa K, Ota N, et al. Coffee polyphenols prevent cognitive dysfunction and suppress amyloid β plaques in APP/PS2 transgenic mouse. Neuroscience Research. 2020;154:35–44. https://doi.org/10.1016/j.neures.2019.05.001
24. Nabavi SF, Tejada S, Setzer WN, Gortzi O, Sureda A, Braidy N, et al. Chlorogenic acid and mental diseases: From chemistry to medicine. Current Neuropharmacology. 2017;15(4):471–479. https://doi.org/10.2174/1570159X14666160325120625
25. Singh SS, Rai SN, Birla H, Zahra W, Kumar G, Gedda MR, et al. Effect of chlorogenic acid supplementation in MPTP-intoxicated mouse. Frontiers in Pharmacology. 2018;9. https://doi.org/10.3389/fphar.2018.00757
26. Amato A, Terzo S, Mulè F. Natural compounds as beneficial antioxidant agents in neurodegenerative disorders: A focus on Alzheimer's disease. Antioxidants. 2019;8(12). https://doi.org/10.3390/antiox8120608
27. Tan JW, Kim MK. Neuroprotective effects of biochanin A against β-amyloid-induced neurotoxicity in PC12 cells via a mitochondrial-dependent apoptosis pathway. Molecules. 2016;21(5). https://doi.org/10.3390/molecules21050548
28. Biradar SM, Joshi H, Chheda TK. Biochanin-A ameliorates behavioural and neurochemical derangements in cognitive-deficit mice for the betterment of Alzheimer's disease. Human and Experimental Toxicology. 2014;33(4):369–382. https://doi.org/10.1177/0960327113497772
29. Youn K, Park J-H, Lee J, Jeong W-S, Ho C-T, Jun M. The identification of biochanin A as a potent and selective β-site app-cleaving enzyme 1 (BACE1) inhibitor. Nutrients. 2016;8(10). https://doi.org/10.3390/nu8100637
30. Park H-EH, Jung Y, Lee S-JV. Survival assays using Caenorhabditis elegans. Molecules and Cells. 2017;40(2):90–99. https://doi.org/10.14348/molcells.2017.0017
31. Amrit FRG, Ratnappan R, Keith SA, Ghazi A. The C. elegans lifespan assay toolkit. Methods. 2014;68(3):465–475. https://doi.org/10.1016/j.ymeth.2014.04.002
32. Nigon VM, Félix M-A. History of research on C. elegans and other free-living nematodes as model organisms. WormBook. 2017. pp. 1–84. https://doi.org/10.1895/wormbook.1.181.1
33. Vesnina AD, Dolganyuk VF, Dmitrieva AI, Loseva AI, Milentyeva IS. Evaluation of the geroprotective effect of squalene on the Caenorhabditis elegans model. Siberian Journal of Life Sciences and Agriculture. 2022;14(6):51–69. (In Russ.). https://doi.org/10.12731/2658-6649-2022-14-6-51-69
34. Shen P, Yue Y, Zheng Jo, Park Y. Caenorhabditis elegans: A convenient in vivo model for assessing the impact of food bioactive compounds on obesity, aging, and Alzheimer's disease. Annual Review of Food Science and Technology. 2018;9:1–22. https://doi.org/10.1146/annurev-food-030117-012709
35. Krishnan N, Konidaris KF, Gasser G, Tonks NK. A potent, selective, and orally bioavailable inhibitor of the protein-tyrosine phosphatase PTP1B improves insulin and leptin signaling in animal models. Journal of Biological Chemistry. 2018;293(5):1517–1525. https://doi.org/10.1074/jbc.C117.819110
36. Zhu Z, Yang T, Zhang L, Liu L, Yin E, Zhang C, et al. Inhibiting Aβ toxicity in Alzheimer's disease by a pyridine amine derivative. European Journal of Medicinal Chemistry. 2019;168:330–339. https://doi.org/10.1016/j.ejmech.2019.02.052
37. Limbocker R, Chia S, Ruggeri FS, Perni M, Cascella R, Heller GT, et al. Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes. Nature Communications. 2019;10. https://doi.org/10.1038/s41467-018-07699-5
38. Kato M, Chen X, Inukai S, Zhao H, Slack FJ. Age-associated changes in expression of small, noncoding RNAs, including microRNAs, in C. elegans. RNA. 2011;17:1804–1820. https://doi.org/10.1261/rna.2714411
39. Rangaraju S, Solis GM, Thompson RC, Gomez-Amaro RL, Kurian L, Encalada SE, et al. Suppression of transcriptional drift extends C. elegans lifespan by postponing the onset of mortality. eLife. 2015;4. https://doi.org/10.7554/eLife.08833.001
40. Gu J, Li Q, Liu J, Ye Z, Feng T, Wang G, et al. Ultrasonic–assisted extraction of polysaccharides from Auricularia auricula and effects of its acid hydrolysate on the biological function of Caenorhabditis elegans. International Journal of Biological Macromolecules. 2021;167:423–433. https://doi.org/10.1016/j.ijbiomac.2020.11.160
41. Karpushina MV, Suprun II, Lobodina EV. Aplication of biotechnological methods in the nursery industry. Fruit Growing and Viticulture of South Russia. 2021;(71):116–130. (In Russ.). https://doi.org/10.30679/2219-5335-2021-5-71-116-130
42. Dyshlyuk LS, Fedorova AM, Loseva AI, Eremeeva NI. Callus cultures of Thymus vulgaris and Trifolium pratense as a source of geroprotectors. Food Processing: Techniques and Technology. 2021;51(2):423–432. https://doi.org/10.21603/2074-9414-2021-2-423-432
43. Gamborg OL, Miller RA, Ojima O. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research. 1968;50(1):151–158. https://doi.org/10.1016/0014-4827(68)90403-5
44. Faskhutdinova ER, Sukhikh AS, Le VM, Minina VI, Khelef MEA, Loseva AI. Effects of bioactive substances isolated from Siberian medicinal plants on the lifespan of Caenorhabditis elegans. Foods and Raw Materials. 2022;10(2):340–352. https://doi.org/10.21603/2308-4057-2022-2-544
45. Malca-Garcia GR, Liu Y, Nikolić D, Friesen JB, Lankin DC, McAlpine JB, et al. Investigation of red clover (Trifolium pratense) isoflavonoid residual complexity by off-line CCS-qHNMR. Fitoterapia. 2022;156. https://doi.org/10.1016/j.fitote.2021.105016
46. Drake J, Link CD, Butterfield DA. Oxidative stress precedes fibrillar deposition of Alzheimer's disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model. Neurobiology of Aging. 2003;24(3):415–420. https://doi.org/10.1016/S0197-4580(02)00225-7
47. Dostal V, Link CD. Assaying β-amyloid toxicity using a transgenic C. elegans model. Journal of Visualized Experiment. 2010;44. https://doi.org/10.3791/2252
48. Fedorova AM, Dyshlyuk LS, Milentyeva IS, Loseva AI, Neverova OA, Khelef MEA. Geroprotective activity of trans-cinnamic acid isolated from the Baikal skullcap (Scutellaria baicalensis). Food Processing: Techniques and Technology. 2022;52(3):582–591. (In Russ.). https://doi.org/10.21603/2074-9414-2022-3-2388
49. Sonani RR, Singh NK, Awasthi A, Prasad B, Kumar J, Madamwar D. Phycoerythrin extends life span and health span of Caenorhabditis elegans. AGE. 2014;36. https://doi.org/10.1007/s11357-014-9717-1
50. Leite NR, de Araújo LCA, da Rocha PS, Agarrayua DA, Ávila DS, Carollo CA, et al. Baru Pulp (Dipteryx alata Vogel): Fruit from the Brazilian savanna protects against oxidative stress and increases the life expectancy of Caenorhabditis elegans via SOD-3 and DAF-16. Biomolecules. 2020;10(8). https://doi.org/10.3390/biom10081106
51. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25(4):402–408. https://doi.org/10.1006/meth.2001.1262
52. Dimitriadi TA, Burtsev DV, Dzhenkova EA, Kutilin DS. Differential expression of microRNAS and their target genes in cervical intraepithelial neoplasias of varying severity. Advances in Molecular Oncology. 2020;7(2):47–61. (In Russ.). https://doi.org/10.17650/2313-805X-2020-7-2-47-61
53. Martorell P, Llopis S, Gonzalez N, Ramón D, Serrano G, Torrens A, et al. A nutritional supplement containing lactoferrin stimulates the immune system, extends lifespan, and reduces amyloid β peptide toxicity in Caenorhabditis elegans. Food Science and Nutrition. 2016;5(2):255–265. https://doi.org/10.1002/fsn3.388
54. Qin Y, Chen F, Tang Z, Ren H, Wang Q, Shen N, et al. Ligusticum chuanxiong Hort as a medicinal and edible plant foods: Antioxidant, anti-aging and neuroprotective properties in Caenorhabditis elegans. Frontiers in Pharmacology. 2022;13. https://doi.org/10.3389/fphar.2022.1049890
55. Gutierrez-Zetina SM, González-Manzano S, Ayuda-Durán B, Santos-Buelga C, González-Paramás AM. Caffeic and dihydrocaffeic acids promote longevity and increase stress resistance in Caenorhabditis elegans by modulating expression of stress-related genes. Molecules. 2021;26(6). https://doi.org/10.3390/molecules26061517
56. Haridevamuthu B, Guru A, Murugan R, Sudhakaran G, Pachaiappan R, Almutairi MH, et al. Neuroprotective effect of Biochanin A against Bisphenol A-induced prenatal neurotoxicity in zebrafish by modulating oxidative stress and locomotory defects. Neuroscience Letters. 2022;790. https://doi.org/10.1016/j.neulet.2022.136889
57. Zhou Y, Xu B, Yu H, Zhao W, Song X, Liu Y, et al. Biochanin A attenuates ovariectomy-induced cognition deficit via antioxidant effects in female rats. Frontiers in Pharmacology. 2021;12. https://doi.org/10.3389/fphar.2021.603316
58. Singh L, Kaur N, Bhatti R. Neuroprotective potential of biochanin-A and review of the molecular mechanisms involved. Molecular Biology Reports. 2023;50:5369–5378. https://doi.org/10.1007/s11033-023-08397-2
59. Socała K, Szopa A, Serefko A, Poleszak E, Wlaź P. Neuroprotective effects of coffee bioactive compounds: A review. International Journal of Molecular Sciences. 2021;22(1). https://doi.org/10.3390/ijms22010107