doi: 10.15389/agrobiology.2024.1.106eng

UDC: 615.322:582.621:581.192



T.A. Krol1 , D.N. Baleev1, V.I. Ossipov1, 2

1All-Russian Research Institute of Medicinal and Aromatic Plants, 7, ul. Grina, Moscow, 117216 Russia, e-mail (✉ corresponding author),,;
2Department of Chemistry, University of Turku, Turku, Vatselankatu 2, 20014 Finland

Krol T.A.Х
Ossipov V.I.
Baleev D.N.

Final revision received August 16, 2023
Accepted October 31, 2023

The species Casuarina equisetifolia L. is widely used in forestry in many countries with a tropical climate. Extracts from the shoots of C. equisetifolia are known to be rich in phenolic compounds which play an important role in plant growth and development, as well as in adaptation to abiotic and biotic environmental factors. Additionally, they exhibit antiviral, antibacterial, anti-inflammatory, anti-tumor, neuroprotective, and other activities. In this study, the composition of phenolic compounds primarily consisting of monomeric ellagitannins was comprehensively investigated for the first time in the shoots of C. equisetifolia. The aim of this study was to investigate the composition and content of phenolic compounds in C. equisetifolia shoots using ultra performance liquid chromatography coupled with photodiode and mass spectrometric detectors (UHPLC-PDA-MS). The study focused on the green one-year-old photosynthetic shoots of the C. equisetifolia tree grown in the greenhouse of the All-Russian Institute of Medicinal and Aromatic Plants (VILAR, Moscow). Samples were collected in the first decade of July 2019. The shoots were frozen, lyophilized, and ground. A 15 mg specimens were extracted with 1 ml of 80 % acetone for 60 min at room temperature with constant stirring. The extract was centrifuged for 20 min at 14000 rpm and evaporated to dryness at 45 °C. The extraction was repeated two more times. The resulting dry extract was dissolved in 1 ml of deionized water for 60 min, centrifuged for 20 min at 14000 rpm, diluted five times with deionized water, and filtered. An ultra-high performance liquid chromatographic system (UHPLC, Acquity UPLC® 2.9.0, Waters Corporation, USA) with a photodiode array detector (190-500 nm) and triple quadrupole mass spectrometer (Xevo TQ, Waters Corporation, USA) was used for the analysis of phenolic compounds. Separation was carried out in an Acquity UPLC® BEH Phenyl column (2.1×100 mm, 1.7 µm, Waters Corporation, Ireland). Data analysis was performed using the DataAnalysis 4.0 software. Phenolic compounds were identified based on mass spectrometry data by determining the m/z value of the [M-H] ion and its m/z fragments. The content of different classes of phenolic compounds such as gallolyl-glucoses, ellagitannins, condensed tannins, flavonoids (quercetin and kaempferol derivatives) was determined using multiple reaction monitoring. The extract was found to contain 16 phenolic compounds, with 14 belonging to the class of hydrolyzable tannins and 2 to the class of flavan-3-ols. It was discovered that C. equisetifolia shoots accumulate monomeric ellagitannins with molecular masses ranging from 784 to 1068 Da, containing glucose as a polyol in either cyclic or linear form. Among the ellagitannins of C. equisetifolia, casuarinin, two isomers of pedunculagin, stachyurin, chebulic acid, casuarininin, and casuarictin were identified for the first time. Two compounds with a molecular mass of 1068 Da were preliminarily identified as isomers of pterocarinin A. Ellagic acid and its derivatives, ellagic arabinoside and ellagic rhamnoside, were also identified in shoots. The total content of phenolic compounds was 55 mg/g dry weight, with ellagitannins being the main phenolic compounds. Their content reached 42 mg/g, or 76 % of the total amount of all phenolic compounds. Galloyl-glucose and condensed tannins each accounted for 10 % of the total amount of all phenolic compounds. These findings suggest the potential use of C. equisetifolia shoots as a raw material for obtaining individual ellagitannins and studying their antiviral, anti-inflammatory, and anti-tumor activities.

Keywords: Casuarina equisetifolia L., Casuarinaceae, liquid chromatography, mass spectrometry, phenolic compounds, hydrolysable tannins, ellagitannins.



  1. Diouf D., Sy M.O., Gherbi H., Bogusz D., Franche C. Casuarinaceae. In: Compendium of Transgenic Crop Plants. C. Kole, T.C. Hall (eds.). Blackwell Publishing Ltd, 2009: 279-292 CrossRef
  2. Dörken V.M., Parsons R.F. Morpho-anatomical studies on the leaf reduction in Casuarina: the ecology of xeromorphy. Trees, 2017, 31: 1165-1177 CrossRef
  3. Li H.-B., Li N., Yang S.-Z., Peng H.-Z., Wang L.-L., Wang Y., Zhang X.-M., Gao Z.-H. Transcriptomic analysis of Casuarina equisetifolia L. in responses to cold stress. Tree Genetics & Genomes, 2017, 13: 7 CrossRef
  4. Wang Y., Zhang Y., Fan C., Wei Y., Meng J., Li Z., Zhong C. Genome-wide analysis of MYB transcription factors and their responses to salt stress in Casuarina equisetifolia. BMC Plant Biology, 2021, 21: 328 CrossRef
  5. Zhong C., Zhang Y., Wei Y., Meng J., Chen Y., Bush D., Bogusz D., Franche C. The role of Frankia inoculation in Casuarina plantations in China. Antonie van Leeuwenhoek, 2019, 112: 47-56 CrossRef
  6. Saranya K., Gowrie U. Phytochemical analysis and in vitro studies on antibacterial, antioxidant and anti-inflammatory activities using Casuarina equisetifolia bark extracts. International Journal of Pharmacy and Pharmaceutical Sciences, 2018, 10(1): 118-125 CrossRef
  7. Pawar A.R., Rao P.S., Vikhe D.N. Pharmacognostic, phytochemical, physico-chemical standardization of Casuarina equisetifolia stem-inner bark. Research Journal of Science and Technology, 2021, 13(3): 193-199 CrossRef
  8. Zhang L., Zhang S., Ye G., Qin X. Seasonal variation and ecological importance of tannin and nutrient concentrations in Casuarina equisetifolia branchlets and fine roots. Journal of Forestry Research, 2020, 31(5): 1499-1508 CrossRef
  9. Muhammad H.L., Garba R., Abdullah A.S., Adefolalu F.S., Busari M.B., Hamzah R.U., Makun H.A. Hypoglycemic and hypolipidemic properties of Casuarina equisetifolia leaf extracts in alloxan induced diabetic rats. Pharmacological Research-Modern Chinese Medicine, 2022, 2: 100034 CrossRef
  10. Muthuraj S., Muthusamy P., Radha R., Ilango K. Pharmacognostical, phytochemical studies and in vitro antidiabetic evaluation of seed extracts of Casuarina equisetifolia Linn. The Journal of Phytopharmacology, 2020, 9(6): 410-418 CrossRef
  11. Zhang S.-J., Lin Y.-M., Zhou H.-C., Wei S.D., Lin G.-H., Ye G.-F. Antioxidant tannins from stem bark and fine root of Casuarina equisetifolia. Molecules, 2010, 15(8): 5658-5670 CrossRef
  12. Okuda T., Yoshida T., Ashida M., Yazaki K. Tannis of Casuarina and Stachyurus species. Part 1. Structures of pendunculagin, casuarictin, strictinin, casuarinin, casuariin, and stachyurin. Journal of the Chemical Society, Perkin Transactions 1, 1983: 1765-1772 CrossRef
  13. Pratyusha S. Phenolic compounds in the plant development and defense: an overview. In: Plant stress physiology — perspective in agriculture. M. Hasanuzzaman, K. Nahar (eds.). Intechopen, 2022: 125-140 CrossRef
  14. Kumar S., Abedin M.M., Singh A.K., Das S. Role of phenolic compounds in plant-defensive mechanisms. In: Plant phenolics in sustainable agriculture. R. Lone, R. Shuab, A. Kamili (eds.). Springer, Singapore, 2020: 517-532 CrossRef
  15. Zhang S., He C., Wei L. Jian S., Liu N. Transcriptome and metabolome analysis reveals key genes and secondary metabolites of Casuarina equisetifolia ssp. incana in response to drought stress. BMC Plant Biology, 2023, 23: 200 CrossRef
  16. Ai D., Wang Y., Wei Y., Zhang J., Meng J., Zhang Y. Comprehensive identification and expression analyses of the SnRK gene family in Casuarina equisetifolia in response to salt stress. BMC Plant Biology, 2022, 22: 572 CrossRef
  17. Zhang L.H., Shao H.B., Ye G.F., Lin Y. M. Effects of fertilization and drought stress on tannin biosynthesis of Casuarina equisetifolia seedlings branchlets. Acta Physiologiae Plantarum, 2012, 34: 1639-1649 CrossRef
  18. Kiss A.K., Piwowarski J.P. Ellagitannins, gallotannins and their metabolites-the contribution to the anti-inflammatory effect of food products and medicinal plant. Current Medicinal Chemistry, 2018, 25(37): 4946-4967 CrossRef
  19. Olchowik-Grabarek E., Sękowski S., Kwiatek A., Płaczkiewicz J., Abdulladjanova N., Shlyonsky V., Swiecicka I, Zamaraeva M. The structural changes in the membranes of Staphylococcus aureus caused by hydrolysable tannins witness their antibacterial activity. Membranes, 2022, 12(11): 1124 CrossRef
  20. Kaneshima T., Myoda T., Nakata M., Fujimori T., Toeda K., Nishizawa M. Antioxidant activity of C-Glycosidic ellagitannins from the seeds and peel of camu-camu (Myrciaria dubia). LWT-Food Science and Technology, 2016, 69: 76-81 CrossRef
  21. Senobari Z., Karimi G., Jamialahmadi K. Ellagitannins, promising pharmacological agents for the treatment of cancer stem cells. Phytotherapy Research, 2022, 36(1): 231-242 CrossRef
  22. Engstrom M.T., Palijarvi M., Salminen J.P. Rapid fingerprint analysis of plant extracts for ellagitannins, gallic acid, and quinic acid derivatives and quercetin-, kaempferol-and myricetin-based flavonol glycosides by UPLC-QqQ-MS/MS. Journal of Agricultural and Food Chemistry, 2015, 63(16): 4068-4079 CrossRef
  23. Xu M., Liu P., Jia X., Zhai M., Zhou S., Wu B., Guo Z. Metabolic profiling revealed the organ‐specific distribution differences of tannins and flavonols in pecan. Food Science & Nutrition, 2020, 8(9): 4987-5006 CrossRef
  24. Wishart D.S., Feunang Y.D., Marcu A., Guo A.C., Liang K., Vázquez-Fresno R., Sajed T., Johnson  D., Li C., Karu N., Sayeeda Z., Lo E., Assempour N., Berjanskii M., Singhal S., Arndt D., Liang Y., Badran H., Grant J., Serra-Cayuela A., Liu Y., Mandal R., Neveu V., Pon A., Knox C., Wilson M., Manach C., Scalbert A. HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Research, 2018, 46(D1): D608-D617 CrossRef
  25. Plaza M., Batista A.G., Cazarin C.B.B., Sandahl M., Turner C., Östman E., Júnior M.R.M. Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study. Food Chemistry, 2016, 211: 185-197 CrossRef
  26. Nonaka G., Ishimaru K., Azuma R., Ishimatsu M., Nishioka I. Tannins and related compounds. LXXXV: Structures of novel C-glycosidic ellagitannins, grandinin and pterocarinins A and B. Chemical and Pharmaceutical Bulletin, 1989, 37(8): 2071-2077 CrossRef
  27. Jorge T.F., Tohge T., Wendenburg R., Ramalho J.C., Lidon F.C., Ribeiro-Barros A.I., Fernie A.R., Antonio C. Salt-stress secondary metabolite signatures involved in the ability of Casuarina glauca to mitigate oxidative stress. Environmental and Experimental Botany, 2019, 166: 103808 CrossRef
  28. Aher A.K., Pal S., Yadav S., Patil U., Bhattacharya S. Evaluation of antimicrobial activity of Casuarina equisetifolia frost (Casuarinaceae). Research Journal of Pharmacognosy and Phytochemistry, 2009, 1(1): 64-68.
  29. Yoshida T., Amakura Y., Yoshimura M. Structural features and biological properties of ellagitannins in some plant families of the order Myrtales. International Journal of Molecular Sciences, 2010, 11(1): 79-106 CrossRef
  30. Karlińska E., Masny A., Cieślak M., Macierzyński J., Pecio Ł., Stochmal A., Kosmala M. Ellagitannins in roots, leaves, and fruits of strawberry (Fragaria ½ ananassa Duch.) vary with developmental stage and cultivar. Scientia Horticulturae, 2021, 275: 109665 CrossRef
  31. Anstett D.N., Cheval I., D’Souza C., Salminen J.-P., Johnson M.T. Ellagitannins from the Onagraceae decrease the performance of generalist and specialist herbivores. Journal of Chemical Ecology, 2019, 45: 86-94 CrossRef
  32. Grellet-Bournonville C.F., Di Peto P.A., Cervino Dowling A.M., Castagnaro A.P., Schmeda-Hirschmann G., Diaz Ricci J.C., Mamaní A.I., Filippone M.P. Seasonal variation of plant defense inductor ellagitannins in strawberry leaves under field conditions for phytosanitary technological applications. Journal of Agricultural and Food Chemistry, 2021, 69(42): 12424-12432 CrossRef
  33. Ossipov V., Salminen J.-P., Ossipova S., Haukioja E., Pihlaja K. Gallic acid and hydrolysable tannins are formed in birch leaves from an intermediate compound of the shikimate pathway. Biochemical Systematics and Ecology, 2003, 31(1): 3-16 CrossRef
  34. Salminen J.-P. The chemistry and chemical ecology of ellagitannins in plant–insect interactions: from underestimated molecules to bioactive plant constituents. In: Recent advances in polyphenol research. A. Romani, V. Lattanzio, S. Quideau (eds.). Wiley, Hoboken, 2014: 83-113 CrossRef
  35. Hatano T., Kira R., Yoshizaki M., Okuda T. Seasonal changes in the tannins of Liquidambar formosana reflecting their biogenesis. Phytochemistry, 1986, 25(12): 2787-2789 CrossRef
  36. Yin T.-P., Cai L., Chen Y., Li Y., Wang Y.-R., Liu C.-S., Ding Z.-T. Tannins and antioxidant activities of the walnut (Juglans regia) pellicle. Natural Product Communications, 2015, 10(12): 1934578X1501001232 CrossRef
  37. Khalifa I., Zhu W., Nafie M.S., Dutta K., Li C. Anti-COVID-19 effects of ten structurally different hydrolysable tannins through binding with the catalytic-closed sites of COVID-19 main protease: an in-silico approach. Preprints, 2020: 2020030277 CrossRef
  38. Du R., Cooper L., Chen Z., Lee H., Rong L., Cui Q. Discovery of chebulagic acid and punicalagin as novel allosteric inhibitors of SARS-CoV-2 3CLpro. Antiviral Research, 2021, 190: 105075 CrossRef
  39. Kuo P.-L., Hsu Y.-L., Lin T.-C., Lin L.-T., Chang J.-K., Lin C.-C. Casuarinin from the bark of Terminalia arjuna induces apoptosis and cell cycle arrest in human breast adenocarcinoma MCF-7 cells. Planta Medica, 2005, 71(3): 237-243 CrossRef
  40. Yang L.-L., Lee C.-Y., Yen K.-Y. Induction of apoptosis by hydrolyzable tannins from Eugenia jambos L. on human leukemia cells. Cancer Letters, 2000, 157(1): 65-75 CrossRef
  41. Kim M., Yin J., Hwang I.H., Park D., Lee, E., Kim M., Lee M. Anti-Acne vulgaris effects of pedunculagin from the leaves of Quercus mongolica by anti-inflammatory activity and 5a-reductase inhibition. Molecules, 2020, 25(9): 2154 CrossRef
  42. Kwon D.-J., Bae Y.-S., Ju S.M., Goh A.R., Choi S.Y., Park J. Casuarinin suppresses TNF-a-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells. Biochemical and Biophysical Research Communications, 2011, 409(4): 780-785 CrossRef
  43. Puljula E., Walton G., Woodward M.J., Karonen M. Antimicrobial activities of ellagitannins against Clostridiales perfringens, Escherichia coli, Lactobacillus plantarum and Staphylococcus aureus. Molecules, 2020, 25(16): 3714 CrossRef







Full article PDF (Rus)