Abstract

Hyssopus officinalis L. is a valuable medicinal plant. Its extracts have good commercial prospects as biologically active substances. Biotechnological production methods that yield valuable secondary metabolites often involve callus cultures, which differ in qualitative and quantitative content of target substances. This research compared phenolic secondary metabolites, antioxidants, and antimicrobial activities in extracts of three callus cultures in vitro, native plants, micro-plants and a commercial preparation of H. officinalis.
The extracts were obtained using 70% ethanol. The content of phenolic compounds, flavonoids, hydroxycinnamic acids, and tannins was determined by standard methods. The method of high-performance liquid chromatography revealed the content of individual phenolic compounds. The antioxidant activity tests relied on DPPH, FRAP, and ABTS. The antimicrobial and fungicidal activity was assessed by the disc-diffusion method.
The Murashige-Skoog medium with 0.2 mg/L 6-benzylaminopurine and 1 mg/L 2,4-dichlorophenoxyacetic acid demonstrated a high growth index of callus crude biomass (11.6 ± 1.4). The same culture was rich in phenolic compounds (35.5 ± 1.2 mg-eq. gallic acid/g solids), hydroxycinnamic acids (82.5 ± 2.6 mg-eq. rosmarinic acid/g solids), tannins (49.6 ± 0.8 mg-eq. gallic acid/g solids), rosmarinic acid (20.25 ± 1.84 mg/g solids), and caffeic acid (1.48 ± 0.08 mg/g solids). Its extracts showed significant antioxidant activity according to DPPH (127.8 ± 5.6 mg-eq. ascorbic acid/g solids) and FRAP (15.9 ± 1.3 mg-eq. ascorbic acid/g solids). However, they had a rather weak antibacterial and fungicidal activity against Bacillus subtilis (7.2 ± 0.2 mm) and Candida albicans (7.0 ± 0.2 mm) at a concentration of 2 mg/disk. The antibacterial and fungicidal activity against Escherichia coli was equally weak at 1 mg/disk (7.0 ± 0.3 mm) and 2 mg/disk (7.3 ± 0.1 mm), respectively.
The extracts of H. officinalis callus cultures and micro-plants in vitro produced the best yield of secondary metabolites compared to the native plant extract. Biotechnological methods proved effective in obtaining valuable compounds from cell cultures.

Keywords

Callus, phenolic compounds, flavonoids, hydroxycinnamic acids, tannins, antioxidant activity, antimicrobial activity

REFERENCES

1. Kumar V, Kaur N, Kaur A, Wadhwa P. Phytochemistry and Pharmacology of Indian Traditional Plant Hyssop (Hyssopus officinalis L.): A Review. The Natural Products Journal. 2023;13(4):e110822207418. https://doi.org/10.2174/ 2210315512666220811153919
2. Sayyahi J, Mobaiyen H, Jafari B, Jafari-Sales A. Antibacterial effects of methanolic extracts of Reum ribes L. and Hyssopus officinalis on some standard pathogenic bacteria. Jorjani Biomedicine Journal. 2019;7(3):34–44. https://doi.org/ 10.29252/jorjanibiomedj.7.3.34
3. Judžentienė A. Hyssop (Hyssopus officinalis L.) Oils. In: Preedy VR, editor. Essential Oils in Food Preservation, Flavor and Safety. Academic Press; 2016. pp. 471–479. https://doi.org/10.1016/B978-0-12-416641-7.00053-5
4. Tahir M, Khushtar M, Fahad M, Rahman MA. Phytochemistry and pharmacological profile of traditionally used medicinal plant Hyssop (Hyssopus officinalis L.). Journal of Applied Pharmaceutical Science. 2018;8(7):132–140. https:// doi.org/10.7324/JAPS.2018.8721
5. Zayova E, Geneva M, Stancheva I, Dimitrova L, Petrova M, Hristozkova M, et al. Evaluation of the antioxidant potential of in vitro propagated hyssop (Hyssopus officinalis L.) with different plant growth regulators. International Journal of Phytomedicines and Related Industries. 2018;10(4):295–304. https://doi.org/10.5958/0975 6892.2018.00044.8
6. Borrelli F, Pagano E, Formisano C, Piccolella S, Fiorentino A, Tenore GC, et al. Hyssopus officinalis subsp. aristatus: An unexploited wild-growing crop for new disclosed bioactives. Industrial Crops and Products. 2019;140:111594. https:// doi.org/10.1016/j.indcrop.2019.111594
7. Ozyigit II, Dogan I, Hocaoglu-Ozyigit A, Yalcin B, Erdogan A, Yalcin IE, et al. Production of secondary metabolites using tissue culture-based biotechnological applications. Frontiers in Plant Science, 2023;14:1132555. https://doi.org/10.3389/ fpls.2023.1132555
8. Efferth T. Biotechnology applications of plant callus cultures. Engineering. 2019;5(1):50–59. https://doi.org/10.1016/ j.eng.2018.11.006
9. Bulavin IV, Ivanova NN, Mitrofanova IV. In vitro regeneration of Hyssopus officinalis L. and plant genetic similarity. Doklady Biological Sciences. 2021;499:109–112. https://doi.org/10.1134/S0012496621040013
10. Soheili S, Miri SM, Ghazijahani N. Callus Induction from In Vitro Cultured Leaf, Hypocotyl and Root of Hyssopus officinalis. In The First National Conference on the Application of Advancedchemical and Agricultural Research for Development of Medicinal Plants; 2021.
11. Morovatti S, Zebarjadi A, Bahraminejad S, Nadhaphy A. Effect of plant growth regulators on callogenesis and regeneration of Hyssopus officinalis in in vitro conditions. Agricultural Biotechnology Journal. 2021;13(3):187–204. https:// doi.org/10.22103/jab.2021.16756.1277
12. Maslova E, Gulya N, Perelugina T, Semykina V, Kalashnikova E. Introduction of Hyssopus officinalis L. into in vitro culture to optimize the conditions for obtaining callus tissues and microclonal propagation as a promising metod of innovative agrobiotechnologies BIO Web of Conferences. 2021;30:05006. https://doi.org/10.1051/bioconf/20213005006
13. Skrzypek Z, Wysokińska H. Sterols and triterpenes in cell culture of Hyssopus officinalis L. Zeitschrift für Naturforschung C. 2003;58(5–6):308–312. https://doi.org/10.1515/znc-2003-5-602
14. Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum. 1962;15(3):473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
15. Sadat-Hosseini M, Soleimani A. Callus Induction, Shoot and Root Regeneration in Hyssopus officinalis using Sodium Nitroprusside and Plant Growth Regulators. Journal of Medicinal plants and By-product. 2023. https://doi.org/10.22034/ JMPB.2023.363600.1611
16. Babich O, Sukhikh S, Pungin A, Astahova L, Chupakhin E, Belova D, et al. Evaluation of the Conditions for the Cultivation of Callus Cultures of Hyssopus officinalis Regarding the Yield of Polyphenolic Compounds. Plants. 2021;10(5):915. https://doi.org/10.3390/plants10050915
17. Bibi A, Khan MA, Adil M, Mashwani ZUR. Production of callus biomass and antioxidant secondary metabolites in black cumin. The Journal of Animal and Plant Sciences. 2018;28(5):1321–1328.
18. Padhi EMT, Liu R, Hernandez M, Tsao R, Dan Ramdath D. Total polyphenol content, carotenoid, tocopherol and fatty acid composition of commonly consumed Canadian pulses and their contribution to antioxidant activity. Journal of Functional Foods. 2017;38:602–611. https://doi.org/10.1016/j.jff.2016.11.006
19. Sevket ALP, Ercisli S, Jurikova T, Cakir O, Gozlekci S. Bioactive Content of Rose Hips of Different Wildly Grown Rosa dumalis Genotypes. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2016;44(2):472–476. https://doi.org/10.15835/ nbha44210432
20. Štefan MB, Rodríguez VJ, Blažeković B, Kindl M, Vladimir-Knežević S. Total Hydroxycinnamic Acids Assay: Prevalidation and Application on Lamiaceae Species. Food Analytical Methods. 2013;7:326–336. https://doi.org/10.1007/ s12161-013-9630-8
21. Feduraev P, Skrypnik L, Nebreeva S, Dzhobadze G, Vatagina A, Kalinina E, et al. Variability of Phenolic Compound Accumulation and Antioxidant Activity in Wild Plants of Some Rumex Species (Polygonaceae). Antioxidants. 2022;11(2):311. https://doi.org/10.3390/antiox11020311
22. Skrypnik L, Grigorev N, Michailov D, Antipina M, Danilova M, Pungin A. Comparative study on radical scavenging activity and phenolic compounds content in water bark extracts of alder (Alnus glutinosa (L.) Gaertn.), oak (Quercus robur L.) and pine (Pinus sylvestris L.). European Journal of Wood and Wood Products. 2019;77:879–890. https://doi.org/10.1007/ s00107-019-01446-3
23. Skrypnik L, Feduraev P, Golovin A, Maslennikov P, Belov N, Matveev M, et al. Biotechnological Potential of Different Organs of Mistletoe (Viscum album L.) Collected from Various Host Tree Species in an Urban Area. Plants. 2022; 11(20):2686. https://doi.org/10.3390/plants11202686
24. Milentyeva IS, Fedorova AM, Larichev TA, Altshuler OG. Biologically active compounds in Scutellaria baicalensis L. callus extract: Phytochemical analysis and isolation. Foods and Raw Materials. 2023;11(1):172–186. https://doi.org/ 10.21603/2308-4057-2023-1-564
25. Klančnik A, Piskernik S, Jeršek B, Možina SS. Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts. Journal of Microbiological Methods. 2010;81(2):121–126. https://doi.org/10.1016/ j.mimet.2010.02.004
26. Bertani G. Lysogeny at Mid-Twentieth Century: P1, P2, and Other Experimental Systems. Journal of Bacteriology. 2004;186(3):595–600. https://doi.org/10.1128/jb.186.3.595-600.2004
27. Espinosa-Leal CA, Puente-Garza CA, García-Lara S. In vitro plant tissue culture: means for production of biological active compounds. Planta. 2018;248:1–18. https://doi.org/10.1007/s00425-018-2910-1
28. Tungmunnithum D, Thongboonyou A, Pholboon A, Yangsabai A. Flavonoids and Other Phenolic Compounds from Medicinal Plants for Pharmaceutical and Medical Aspects: An Overview. Medicines. 2018;5(3):93. https://doi.org/10.3390/ medicines5030093
29. Milentyeva IS, Vesnina AD, Fedorova AM, Ostapova EV, Larichev TA. Chlorogenic Acid and Biohanin A from Trifolium pratense L. Callus Culture Extract: Functional Activity In Vivo. Food Processing: Techniques and Technology. 2023;53(4):754–765. (In Russ.). https://doi.org/10.21603/2074-9414-2023-4-2475; https://elibrary.ru/GGYCQG
30. Sun W, Shahrajabian MH. Therapeutic Potential of Phenolic Compounds in Medicinal Plants–Natural Health Products for Human Health. Molecules. 2023;28(4):1845. https://doi.org/10.3390/molecules28041845
31. Pizzi A. Tannins: Prospectives and Actual Industrial Applications. Biomolecules. 2019;9(8):344. https://doi.org/ 10.3390/biom9080344
32. Sharifi-Rad J, Quispe C, Kumar M, Akram M, Amin M, Iqbal M, et al. Hyssopus Essential Oil: An Update of Its Phytochemistry, Biological Activities, and Safety Profile. Oxidative Medicine and Cellular Longevity. 2022; 2022(1):8442734. https://doi.org/10.1155/2022/8442734