Investigation of the inhibitory activity of extracts, fractions and secondary metabolites of Silene spp. (Caryophylaceae) and Serratula cupuliformis (Asteraceae) on the «entry» of herpes simplex type 2 into sensitive cells of the Vero line

In vitro analysis of the inhibitory activity of extracts, fractions and secondary metabolites of plants of the genus Silene (Caryophylaceae) and Serratula cupuliformis (Asteraceae) to the «entry» of herpes simplex type 2 (HSV‐2) into sensitive cells of the Vero line.

Ethanol extracts and butanol fractions of Silene spp. and Serratula cupuliformis were prepared. The flavonoid shaftoside and the ecdysteroid 20‐hydroxyecdysone from Lychnis chalcedonica were isolated. For analysis of biologically active substances (BAS) the HPLC method was used. The samples were dissolved in distilled water or DMSO. The laboratory strain MS HSV‐2 was grown on Vero cell culture. In vitro analysis of the inhibitory activity of the herbal preparations on «entry» of HSV‐2 was performed in Vero cell culture by direct inactivation (neutralisation) of virions with 10³ PFU /ml.

Effective concentrations (EC50) have been identified in the range of 50 % from 2.12±0.47 to 180.99±49.24 μg/ml when preparations were dissolved in water and from 1.99±0.44 to 57.42±14.74 μg/ml when they were dissolved in DMSO. Such results for some samples is comparable to the EC50 of comparison preparations (dry ethanol extracts of spice of cloves, chaga and licorice root). The results obtained suggest the presence of BAS in the herbal preparations studied that act destructively on HSV‐2 virions and affect one of the main stages of its «life» cycle – the «entry» of the virus into sensitive cells.

1. Sait Mezhdunarodnogo komiteta po taksonomii virusov (ICTV, 2021) [Website of the International Committee on Virus Taxonomy]. Available at: https: ictv.global/taxonomy (accessed 01.12.2023)

2. Gatherer D., Depledge D.P., Hartley C.A., Szpara M.L., Vaz P.K., Benkő M., Brandt C.R., Bryant N.A., Dastjerdi A., Doszpoly A., Gompels U.A., Inoue N., Jarosinski K.W., Kaul R., Lacoste V., Norberg P., Origgi F.C., Orton R.J., Pellett P.E., Schmid D.S., Spatz S.J., Stewart J.P., Trimpert J., Waltzek T.B., Davison A.J. ICTV Virus Taxonomy Profile: Herpesviridae 2021. Journal of General Virology, 2021, vol. 102, no. 10, article id: 001673. DOI: 10.1099/jgv.0.001673

3. Connolly S.A., Jardetzky T.S., Longnecker R. The structural basis of herpesvirus entry. Nature Reviews Microbiology, 2021, vol. 19, no. 2, pp. 110–121. DOI: 10.1038/s41579‐02000448‐w

4. Jambunathan N., Clark C.M., Musarrat F., Chouljenko V.N., Rudd J., Kousoulas K.G. Two Sides to Every Story: Herpes Simplex Type‐1 Viral Glycoproteins gB, gD, gH/gL, gK, and Cellular Receptors Function as Key Players in Membrane Fusion. Viruses, 2021, vol. 13, no. 9, article id: 1849. DOI: 10.3390/v13091849

5. de Souza Carneiro V.C., Pereira J.G., de Paula V.S. Family Herpesviridae and neuroinfections: current status and research in progress. Memórias do Instituto Oswaldo Cruz, 2022, vol. 117, article id: e220200. DOI: 10.1590/007402760220200

6. Stamos J.D., Lee L.H., Taylor C., Elias T., Adams S.D. In Vitro and In Silico Analysis of the Inhibitory Activity of EGCGStearate against Herpes Simplex Virus‐2. Microorganisms, 2022, vol. 10, no. 7, article id: 1462. DOI: 10.3390/microorganisms10071462

7. Koelle D.M., Norberg P., Fitzgibbon M.P., Russell R.M., Greninger A.L., Huang M.‐L., Stensland L., Jing L., Magaret A.S., Diem K., Selke S., Xie H., Celum C., Lingappa J.R., Jerome K.R., Wald A., Johnston C. Worldwide circulation of HSV‐2 × HSV‐1 recombinant strains. Scientific Reports, 2017, no. 7, article id: 44084. DOI: 10.1038/srep44084

8. Grünewald K., Desai P., Winkler D.C., Heymann J.B., Belnap D.M., Baumeister W., Steven A.C. Three‐dimensional structure of herpes simplex virus from cryo‐electron tomography. Science, 2003, vol. 302, iss. 5649, pp. 1396– 1398. DOI: 10.1126/science.1090284

9. Wald A., Ericsson M., Krantz E., Selke S., Corey L. Oral shedding of herpes simplex virus type 2. Sexually Transmitted Infections, 2004, vol. 80, no. 4, pp. 272–276. DOI: 10.1136/sti.2003.007823

10. Krummenacher C., Baribaud F., de Leon M.P., Baribaud I., Whitbeck J.C., Xu R., Cohen G.H., Eisenberg R.J. Comparative usage of herpesvirus entry mediator A and nectin‐1 by laboratory strains and clinical isolates of herpes simplex virus. Virology, 2004, vol. 322, no. 2, pp. 286–299. DOI: 10.1016/j.virol.2004.02.005

11. Sait Vsemirnoi organizatsii zdravookhranéniya (VOZ) [Website of the World Health Organization, WHO)]. Available at: https: https://www.who.int/ru/news‐room/factsheets/detail/herpes‐simplex‐virus] (accessed 01.12.2023)

12. Awasthi S., Friedman H.M. An mRNA vaccine to prevent genital herpes. Translational Research, 2022, no. 242, pp. 56–65. DOI: 10.1016/j.trsl.2021.12.006

13. James C., Harfouche M., Welton N.J., Turner K.M., AbuRaddad L.J., Gottlieb S.L, Looker K.J. Herpes simplex virus: global infection prevalence and incidence estimates, 2016. Bulletin of the World Health Organization, 2020, vol. 98, no. 5, pp. 315–329. DOI: 10.2471/BLT.19.237149

14. Okonko I.O., Cookey T.I., Okerentugba P.O., FrankPeterside N. Serum HSV‐1 and ‐2 IgM in pregnant women in Port Harcourt, Nigeria. Journal of Immunoassay and Immunochemistry, 2015, vol. 36, no. 4, pp. 343–358. DOI: 10.1080/15321819.2014.952442

15. Wertheim J.O., Smith M.D., Smith D.M., Scheffler K., Kosakovsky Pond S.L. Evolutionary origins of human herpes simplex viruses 1 and 2. Molecular biology and evolution, 2014, vol. 31, no. 9, pp. 2356–2364. DOI: 10.1093/molbev/msu185

16. Kropp K.A., Srivaratharajan S., Ritter B., Yu P., Krooss S., Polten F., Pich A., Alcami A., Viejo‐Borbolla A. Identification of the Cleavage Domain within Glycoprotein G of Herpes Simplex Virus Type 2. Viruses, 2020, vol. 12, no. 12, article id: 1428. DOI: 10.3390/v12121428

17. Nath P., Kabir M.A., Doust S.K., Ray A. Diagnosis of Herpes Simplex Virus: Laboratory and Point‐of‐Care Techniques. Infectious Disease Reports, 2021, vol. 13, no. 2, pp. 518–539. DOI: 10.3390/idr13020049

18. Tronstein E., Johnston C., Huang M.‐L., Selke S., Magaret A., Warren T., Corey L., Wald A. Genital shedding of herpes simplex virus among symptomatic and asymptomatic persons with HSV‐2 infection. JAMA, 2011, vol. 305, no. 14, pp. 1441–1449. DOI: 10.1001/jama.2011.420

19. Gornalusse G.G., Valdez R., Fenkart G., Vojtech L., Fleming L.M., Pandey U., Hughes S.M., Levy C.N., Cruz E.J.D., Calienes F.L., Kirby A.C., Fialkow M.F., Lentz G.M., Wagoner J., Jing L., Koelle D.M., Polyak S.J., Fredricks D.N., McElrath M.J., Wald A., Hladik F. Mechanisms of Endogenous HIV‐1 Reactivation by Endocervical Epithelial Cells. Journal of Virology, 2020, vol. 94, no. 9, article id: e01904‐19. DOI: 10.1128/JVI.01904‐19

20. Huang Y., Song Y., Li J., Lv C., Chen Z.‐S., Liu Z. Receptors and ligands for herpes simplex viruses: Novel insights for drug targeting. Drug Discovery Today, 2022, vol. 27, no. 1, pp. 185–195. DOI: 10.1016/j.drudis.2021.10.004

21. Desai D., Londhe R., Chandane M., Kulkarni S. Altered HIV‐1 Viral Copy Number and Gene Expression Profiles of Peripheral (CEM CCR5+) and Mucosal (A3R5.7) T Cell Lines Co‐Infected with HSV‐2 In Vitro. Viruses, 2022, vol. 14, no. 8, article id: 1715. DOI: 10.3390/v14081715

22. Taylor M., Gerriets V. Acyclovir. In: StatPearls. Treasure Island (FL), StatPearls Publ., 2023.

23. Schalkwijk H.H., Snoeck R., Andrei G. Acyclovir resistance in herpes simplex viruses: Prevalence and therapeutic alternatives. Biochemical Pharmacology, 2022, no. 206, article id: 115322. DOI: 10.1016/j.bcp.2022.115322

24. Treml J., Gazdova M., Smejkal K., Sudomova M., Kubatka P., Hassan S.T.S. Natural products‐derived chemicals: Breaking barriers to novel anti‐HSV drug development. Viruses, 2020, no. 12, article id: 154. DOI: 10.3390/v12020154

25. Mohan S., Taha M.M.E., Makeen H.A., Alhazmi H.A., Bratty M.A., Sultana S., Ahsan W., Najmi A., Khalid A. Bioactive Natural Antivirals: An Updated Review of the Available Plants and Isolated Molecules. Molecules, 2020, vol. 25, no. 21, article id: 4878. DOI: 10.3390/molecules25214878

26. Zibareva L.N., Filonenko E.S., Chernyak E.I., Morozov S.V., Kotelnikov O.A. Flavonoids of some plant species of the genus Silene. Chemistry of plant raw materials, 2022, no. 3, pp. 109–118. (In Russian) https://doi.org/10.14258/jcprm.20220310592

27. Shen N., Wang T., Gan Q., Liu S., Wang L., Jin B. Plant flavonoids: Classification, distribution, biosynthesis, and antioxidant activity. Food Chemistry, 2022, no. 383, article id: 132531. DOI: 10.1016/j.foodchem.2022.132531

28. Zibareva L.N., Amosova E.N., Krylova S.G., Zueva E.P., Rybalkina O.Y., Plotnikov M.B., Aliyev O.I., Vasiliev A.S., Anishchenko A.M., Suslov N.I., Nesterova Yu.V., Povetyeva T.N., Afanasyeva O.G., Erst A.A., Razina T.G., Safonova E.A., Kiseleva E.A. Rasteniya rodov Silene L. i Lychnis L. (Caryophyllaceae): sostav khimicheskikh komponentov i biologicheskaya aktivnost' [Plants of the genera Silene L. and Lychnis L. (Caryophyllaceae): composition of chemical components and biological activity]. Tomsk, TSU Publ., 2021, 496 p. (In Russian)

29. Arif Y., Singh P., Bajguz A., Hayat S. Phytoecdysteroids: Distribution, Structural Diversity, Biosynthesis, Activity, and Crosstalk with Phytohormones. International Journal of Molecular Sciences, 2022, vol. 23, no. 15, article id: 8664. DOI: 10.3390/ijms23158664

30. Nie C., Trimpert J., Moon S., Haag R., Gilmore K., Kaufer B.B., Seeberger P.H. In vitro efficacy of Artemisia extracts against SARS‐CoV‐2. Virology Journal, 2021, vol. 18, no. 1, article id: 182. DOI: 10.1186/s12985‐021‐01651‐8

31. Каzachinskaya Е.I., Romanova V.D., Ivanоva A.V., Chepurnov А.А., Коnonova Y.V., Shaulo D.N., Romanyuk V.V., Shestopalov А.М. Inhibitory activity of dry ethanol extracts of Artemisia spp. on SARS‐CoV‐2 replication in vitro. South of Russia: ecology, development, 2022, vol. 17, no. 4, pp. 111– 129. (In Russian) DOI: 10.18470/1992‐1098‐2022‐4‐111‐129

32. Alvarez A.L., Habtemariam S., Juan‐Badaturuge M., Jackson C., Parra F. In vitro anti HSV‐1 and HSV‐2 activity of Tanacetum vulgare extracts and isolated compounds: an approach to their mechanisms of action. Phytotherapy Research, 2011, vol. 25, no. 2, pp. 296–301. DOI: 10.1002/ptr.3382

33. Álvarez Á.L., Habtemariam S., Moneim A.E.A., Melón S., Dalton K.P., Parra F. A spiroketal‐enol ether derivative from Tanacetum vulgare selectively inhibits HSV‐1 and HSV‐2 glycoprotein accumulation in Vero cells. Antiviral Research, 2015, no. 119, pp. 8–18. DOI: 10.1016/j.antiviral.2015.04.004

34. Zibareva L., Athipornchai A., Wonganan O., Suksamrarn A. Application of ultrasound to extraction of biologically active substances of some Serratula species. International Journal of Food and Biosystems Engineering. 2017, vol. 5, no. 1, pp. 31–37.

35. Zibareva L.N., Yeriomina V.I. Sposob uvelicheniya stepeni izvlecheniya ekdisteroidov iz rastitel'nykh ob"ektov [A method for increasing the degree of extraction of ecdysteroids fromplant objects]. Patent RF, no. 2472519C1, 2013.

36. Каzachinskaya Е.I., Zibareva L.N., Filonenko Е.S., Ivanova A.V., Коnonova Y.V., Chepurnov А.А., Shestopalov А.М. Investigation of the inhibitory activity of extracts, fractions and secondary metabolites of Silene spp. (Caryophyllaceae) and Serratula cupuliformis (Asteraceae) on the replication of SARS‐CoV‐2 coronavirus. South of Russia: ecology, development, 2023, vol. 18, no. 1, pp. 62–81. (In Russian) DOI: 10.18470/1992‐1098‐2023‐1‐62‐81

37. Razumov I.A., Kosogova T.A., Kazachinskaya E.I., Puchkova L.I., Shcherbakova N.C., Gorbunova I.A., Mikhailovskaya I.N., Loktev V.B., Teplyakova T.V. Antiviral activity of aqueous extracts and polysaccharide fractions obtained from mycelium and fruiting bodies of higher fungi. Antibiotiki i khimioterapiya [Antibiotics and chemotherapy]. 2010, vol. 55, no. 9–10, pp. 14–18. (In Russian)

38. Каzachinskaya E.I., Chepurnov A.A., Shelemba A.A., Guseinova S.A., Magomedov M.G., Коnonova Yu.V., Romanyuk V.V., Shestopalov A.M. Inhibitory activity of aqueous extracts of tea compositions, individual ingredients for their preparation and some plants against replication of Herpes simplex virus type 2 in vitro. South of Russia: ecology, development, 2022, vol. 17, no. 3, pp. 135–152. (In Russian) DOI:10.18470/1992‐1098‐2022‐3‐135‐152

39. Fukuchi K., Okudaira N., Adachi K., Odai‐Ide R., Watanabe S., Ohno H., Yamamoto M., Kanamoto T., Terakubo S., Nakashima H., Uesawa Y., Kagaya H., Sakagami H. Antiviral and Antitumor Activity of Licorice Root Extracts. In Vivo, 2016, vol. 30, no. 6, pp. 777–785. DOI: 10.21873/invivo.10994

40. Susloparov M.A., Glotov A.G., Glotova T.I. To study the effectiveness of the therapeutic and prophylactic effect of ultra‐low doses of antibodies to gamma interferon on an experimental mouse model of herpes virus infection. Antibiotiki i khimioterapiya [Antibiotics and chemotherapy]. 2004, vol. 49, no. 10, pp. 3–6. (In Russian)

41. Shapolova E.G., Lomovskii O.I., Kazachinskaya E.I., Loktev V.B., Teplyakova T.V. Antiviral Activity of Silicon Dioxide Composites with Polyphenols Obtained by Mechanochemical Method from Plant Raw Materials. Khimikofarmatsevticheskii zhurnal [Chemico‐pharmaceutical Journal]. 2016, vol. 50, no. 9, pp. 25–29. (In Russian)

42. Hassan S.T.S., Berchova‐Bimova K., Šudomova M., Malanik M., Smejkal K., Rengasamy K.R.R. In Vitro Study of Multi‐Therapeutic Properties of Thymus bovei Benth. Essential Oil and Its Main Component for Promoting Their Use in Clinical Practice. Journal of Clinical Medicine, 2018, no. 7, p. 283. DOI: 10.3390/jcm7090283

43. Fisenko V.P. Rukovodstvo po eksperimental'nomu (doklinicheskomu) izucheniyu novykh farmakologicheskikh veshchestv [Guidelines for experimental (preclinical) study of new pharmacological substances. In accordance with the order]. Moscow, Remedium Publ., 2000, 398 p. (In Russian)

44. Lopez‐Muñoz A.D., Rastrojo A., Martín R., Alcamí A. Herpes simplex virus 2 (HSV‐2) evolves faster in cell culture than HSV‐1 by generating greater genetic diversity. PLoS Pathogens, 2021, vol. 17, no. 8, article id: e1009541. DOI: 10.1371/journal.ppat.1009541

45. Burrel S., Deback C., Agut H., Boutolleau D. Genotypic characterization of UL23 thymidine kinase and UL30 DNA polymerase of clinical isolates of herpes simplex virus: natural polymorphism and mutations associated with resistance to antivirals. Antimicrobial Agents and Chemotherapy, 2010, vol. 54, no. 11, pp. 4833–4842. DOI: 10.1128/AAC.00669‐10

46. Ding L., Jiang P., Xu X., Lu W., Yang C., Li L., Zhou P., Liu S. T‐type calcium channels blockers inhibit HSV‐2 infection at the late stage of genome replication. European Journal of Pharmacology, 2021, vol. 892, article id: 173782. DOI: 10.1016/j.ejphar.2020.173782

47. Zverev V.V., Makarov O.V., Khashukoeva A.Z., Svitich O.A., Dobrokhotova Y.E, Markova E.A., Labginov P.A., Khlinova S.A., Shulenina E.A., Gankovskaya L.V. In vitro studies of the antiherpetic effect of photodynamic therapy. Lasers in Medical Science, 2016, vol. 31, no. 5, pp. 849–855. DOI: 10.1007/s10103‐016‐1912‐0

48. Cardozo F.T.G.S., Larsen I.V., Carballo E.V., Jose G., Stern R.A., Brummel R.C., Camelini C.M., Rossi M.J., Simões C.M.O., Brandt C.R. In vivo anti‐herpes simplex virus activity of a sulfated derivative of Agaricus brasiliensis mycelial polysaccharide. Antimicrobial Agents and Chemotherapy, 2013, vol. 57, no. 6, pp. 2541–2549. DOI: 10.1128/AAC.02250‐12

49. Luganini A., Sibille G., Mognetti B., Sainas S., Pippione A.C., Giorgis M., Boschi D., Lolli M.L., Gribaudo G. Effective deploying of a novel DHODH inhibitor against herpes simplex type 1 and type 2 replication. Antiviral Research, 2021, vol. 189, article id: 105057. DOI: 10.1016/j.antiviral.2021.105057

50. Hassan S.T.S, Švajdlenka E., Berchová‐Bímová K. Hibiscus sabdariffa L. and Its Bioactive Constituents Exhibit Antiviral Activity against HSV‐2 and Anti‐enzymatic Properties against Urease by an ESI‐MS Based Assay. Molecules, 2017, vol. 22, no. 5, p. 722. DOI: 10.3390/molecules22050722

51. Sangboonruang S., Semakul N., Sookkree S., Kantapan J., Ngo‐Giang‐Huong N., Khamduang W., Kongyai N., Tragoolpua K. Activity of Propolis Nanoparticles against HSV2: Promising Approach to Inhibiting Infection and Replication. Molecules, 2022, vol. 27, no. 8, article id: 2560. DOI: 10.3390/molecules27082560

52. Cheng H.‐Y., Lin T.‐C., Yang C.‐M., Wang K.‐C., Lin C.‐C. Mechanism of action of the suppression of herpes simplex virus type 2 replication by pterocarnin A. Microbes and Infection, 2004, vol. 6, iss. 8, pp. 738–744. DOI: 10.1016/j.micinf.2004.03.009

53. Reichling J., Neuner A., Sharaf M., Harkenthal M., Schnitzler P. Antiviral activity of Rhus aromatica (fragrant sumac) extract against two types of herpes simplex viruses in cell culture. Die Pharmazie–An International Journal of Pharmaceutical Sciences. 2009, no. 64, pp. 538–541.

54. Benzekri R., Bouslama L., Papetti A., Hammami M., Smaoui A., Limam F. Anti HSV‐2 activity of Peganum harmala (L.) and isolation of the active compound. Microbial Pathogenesis, 2018, vol. 114, pp. 291–298. DOI: 10.1016/j.micpath.2017.12.017

55. Churqui M.P., Lind L., Thörn K., Svensson A., Savolainen O., Aranda K.T., Eriksson K. Extracts of Equisetum giganteum L and Copaifera reticulate Ducke show strong antiviral activity against the sexually transmitted pathogen herpes simplex virus type 2. Journal of Ethnopharmacology, 2018, vol. 210, pp. 192–197. DOI: 10.1016/j.jep.2017.08.010

56. Donalisio M., Cagno V., Civra A., Gibellini D., Musumeci G., Rittà M., Ghosh M., Lembo D. The traditional use of Vachellia nilotica for sexually transmitted diseases is substantiated by the antiviral activity of its bark extract against sexually transmitted viruses. Journal Ethnopharmacology, 2018, vol. 213, pp. 403–408. DOI: 10.1016/j.jep.2017.11.039

57. Benzekri R., Limam F., Bouslama L. Combination effect of three anti‐HSV‐2 active plant extracts exhibiting different modes of action. Advances in Traditional Medicine, 2020, vol. 20, pp. 223–231. DOI: 10.1007/s13596‐020‐00430‐0