Activation of Apoptosis and Autophagy by Gratiola Officinalis Extract in Human Tumor Cell Lines

Natal’ya V. Polukonova
Saratov State Medical University named after V. I. Razumovsky, Russian Federation

Maria A. Baryshnikova
National Medical Research Center of Oncology named after N. N. Blokhin, Moscow, Russian Federation

Dmitry A. Khochankov
National Medical Research Center of Oncology named after N. N. Blokhin, Moscow, Russian Federation

Evgeniya V. Stepanova
National Medical Research Center of Oncology named after N. N. Blokhin, Moscow, Russian Federation

Eliso S. Solomko
National Medical Research Center of Oncology named after N. N. Blokhin, Moscow, Russian Federation

Anna V. Polukonova
Saratov State Medical University named after V. I. Razumovsky, Russian Federation

Dmitrij A. Mudrak
Saratov State Medical University named after V. I. Razumovsky, Russian Federation

Artem M. Mylnikov
Saratov State Medical University named after V. I. Razumovsky, Russian Federation

Alla B. Bucharskaya
Saratov State Medical University named after V. I. Razumovsky, Russian Federation

Galina N. Maslyakova
Saratov State Medical University named after V. I. Razumovsky, Russian Federation

Nikita A. Navolokin orcid (Login required)
Saratov State Medical University named after V. I. Razumovsky, Russian Federation

Paper #3449 received 1 Aug 2021; revised manuscript received 7 Oct 2021; accepted for publication 11 Oct 2021; published online 10 Nov 2021.

DOI: 10.18287/JBPE21.07.040307


The problem of creating antitumor drugs with new mechanisms of action that predominantly induce apoptosis is still topical. The extract of Gratiola officinalis is a potential antitumor agent containing mainly flavonoids. The aim of this research is to study the effects of Gratiola officinalis extract on activation of apoptosis and autophagy in breast adenocarcinoma SK-BR-3 and kidney carcinoma A-498 lines. Apoptotic activity of the extract was studied by flow cytofluorometry using Hoechst stain and double staining with annexin V plus propidium iodide. There was 96.3% of cells in SK-BR-3 culture in late apoptosis phase detected by flow cytofluorometry method at the extract concentration of 0.88 mg/ml, 86.3% of cells were in apoptosis by Hoechst stain. The concentration of 0.82 mg/ml caused apoptosis in half of the cells. The extract has cytoprotective activity at low concentration (0.0352 mg/ml). The cytoprotection mechanism is realized through the activation of autophagy. The maximum number of autophagosomes in kidney carcinoma cells is observed at the extract concentration of 0.056 mg/ml. Thus, Gratiola officinalisextract is able to block cytoprotective autophagy with increasing the extract concentration and to activate apoptosis in 85% of tumor cells. Detailed research should be continued to understand the mechanisms of antitumor activity of Gratiola officinalisextract.


autophagy; apoptosis; antitumor agents; Gratiola officinalis; antitumor agents; tumor; flavonoids

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1. E. D. Goldberg, A. B. Syrkina, “Guidelines for the preclinical study of agents with the ability to inhibit the process of metastasis and increase the effectiveness of cytostatic therapy of malignant tumors,” in The Guidelines for experimental (preclinical) study of new pharmacological substances, R.U. Khabriev (Ed.), Meditsina, Moscow (2005). ISBN: 5-225-04219-8.

2. D. B. Korman, “Targets and mechanisms of anticancer drugs,” Practical Medicine, 333 (2014).

3. V. M. Perelmuter, M. V. Zavjalova, S. V. Vtorushin, E. M. Slonimskaya, and O. V. Savenkova, “Association between the morphologic heterogeneity of infiltrating ductal breast carcinoma and various forms of tumor progression,” Siberian Journal of Oncology 3, 58–64 (2007).

4. G. Polier, J. Ding, B. V. Konkimalla, D. Eick, N. Ribeiro, R. Köhler, M. Giaisi, T. Efferth, L. Desaubry, P. H. Krammer, and M. Li-Weber, “Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1,” Cell death & disease 2(7), e182 (2011).

5. D. Patel, S. Shukla, and S. Gupta, “Apigenin and cancer chemoprevention: progress, potential and promise (review),” International Journal of Oncology 30(1), 233–245 (2007).

6. G. Seelinger, I. Merfort, U. Wolfle, and C. M. Schempp, “Anti-carcinogenic effects of the flavonoid luteolin,” Molecules 13(10), 2628–2651 (2008).

7. C. F. Tsai, W. L. Yeh, S. M. Huang, T. W. Tan, and D. Y. Lu, “Wogonin induces reactive oxygen species production and cell apoptosis in human glioma cancer cells,” International Journal of Molecular Sciences 13(8), 9877–9892 (2012).

8. H. Chung, Y. M. Jung, D. H. Shin, J. Y. Lee, M. Y. Oh, H. J. Kim, K. S. Jang, S. J. Jeon, K. H. Son, and G. Kong, “Anticancer effects of wogonin in both estrogen receptor-positive and -negative human breast cancer cell lines in vitro and in nude mice xenografts,” International Journal of Cancer 122(4), 816–822 (2008).

9. M. Li-Weber, “New therapeutic aspects of flavones: the anticancer properties of Scutellaria and its main active constituents wogonin, baicalein and baicalin,” Cancer Treatment Reviews 35(1), 57–68 (2009).

10. J. Peng, Q. Qi, Q. You, R. Hu, W. Liu, F. Feng, G. Wang, and Q. Guo, “Subchronic toxicity and plasma pharmacokinetic studies on wogonin, a natural flavonoid, in Beagle dogs,” Journal of Ethnopharmacology 124(2), 257–262 (2009).

11. D. Bown, Encyclopaedia of Herbs and their Uses. The Royal Horticultural Society, Dorling Kindersley Limited, London, UK (1995).

12. E. Launert, Edible and Medicinal Plants, Hamlyn, London (1981).

13. M. Zia-Ul-Haq, A. Kausar, S. A. Shahid, M. Qayum, S. Ahmad, and I. Khan, “Phytopharmacological profile of Gratiola officinalis Linn.: A review,” Journal of Medicinal Plants Research 6(16), 3087–3092 (2012).

14. M. Ahmad, N. Muhammad, J. Mehjabeen, N. Jahan, M. Ahmad, and S. Habib, “Pharmacological and biological evaluation of extracts from Gratiola officinalis L. (Scrophulariaceae),” Pakistan Journal of Pharmaceutical Sciences 25(3), 657–663 (2012).

15. N. Muhammad, M. Ahmad, Mehjabeen, N. Jahan, M. Ahmad, and Z. Khan, “Spasmogenic and spasmolytic activity of Gratiola officinalis Linn,” International Journal of Biology, Pharmacy and Allied Sciences 1(5), 636–644 (2012).

16. J. Lust, The Herb Book, Bantam Books, USA (1974).

17. G. Graves, Medicinal Plants, Bracken Books, Randam House, London (1996).

18. L. Ali, T. S. Rizvi, M. Ahmad, and F. Shaheen, “New iridoid glycoside from Gratiola officinalis (Scrophulariaceae),” Journal of Asian Natural Product Research 14(12), 1191–1195 (2012).

19. S. Boryczka, E. Bebenek, M. Jastrzebska, J. Kusz, and M. Zubko, “Crystal structure of betulinic acid-DMSO solvate,” Zeitschrift für Kristallographie 227(6), 379–384 (2012).

20. J. Rothenburger, E. Haslinger, “Caffeic acid glycoside esters from Gratiola officinalis L,” Liebigs Annalen der Chemie 11, 1113–1115 (1994).

21. R. J. Grayer-Barkmeijer, F. A. Tomas-Barberan, “8-Hydroxylated flavone-O-glycosides and other flavonoids in chemotypes of Gratiola officinalis,” Phytochemistry 34(1), 205–210 (1993).

22. S. Kumar, A.K. Pandey, “Chemistry and Biological Activities of Flavonoids: An Overview,” The Scientific World Journal 2013, 162750 (2013).

23. T. Rudolf, H. Annemarie, and U. Triterpene, “Gratiosid, ein Triterpenglykosid aus Gratiola officinalis L,” Berichte der deutschen chemischen Gesellschaft Weinheim 85 (11), 1067–1077 (1952).

24. L. I. Borodin, V. I. Litvinenko, and N. V. Kurinnaya, “New flavonoid C-glycosides from Gratiola officinalis,” Chemistry of Natural Compounds 6(1), 19–24 (1970).

25. V. I. Litvinenko, L. I. Borodin, and N. V. Kurinnaya, “Apigenin and its glycosides from Gratiola officinalis,” Chemistry of Natural Compounds 5(4), 328–329 (1969).

26. N. V. Polukonova, N. A. Durnova, M. N. Kurchatova, N. A. Navolokin, and A. G. Golikov, “Chemical analysis of the new biological active composition from medicative herb hedge-hissop (Gratiola Officinalis L.),” Khimija Rastitel’nogo Syr’ja 4, 165–173 (2013) [in Russian].

27. N. A. Navolokin, A. V. Ivlichev, D. A. Mydrak, and G. A. Afanasjeva, “Influence of flavonoid-containing extract (Gratiola Officinalis L.) on the content of vitamin E and intensity of peroxidation processes in the blood of rats with transplanted liver cancer PC-1,” Eksperimental’naya i Klinicheskaya Farmakologiy 80(10), 40–43 (2017) [in Russian].

28. N. A. Navolokin, D. A. Mydrak, N. V. Polukonova, and A. B. Bucharskaya, “Effects of Gratiola Officinalis L. extract containing flavonoids on pathomorphism of inoculated renal cancer in rats,” Eksperimental’naya i Klinicheskaya Farmakologiy 80(6), 19–23 (2017) [in Russian].

29. N. A. Navolokin, D. A. Mydrak, N. V. Polukonova, and S. A. Tychina, “Evaluation of the antitumor and anticachexia activity of Gratiola Officinalis L. extract in rats with transplanted sarcoma,” Siberian Journal of Oncology 5(1), 37–43 (2016) [in Russian].

30. N. V. Polukonova, N. A. Durnova, and N. N. Khakhulina, “Phytocenoses Gratíola Officinális L., productivity and its inventories of raw materials in the islands chardymskogo r. Volga Saratov region,” Bulletin of the Botanical Garden of the Saratov State University 14(2), 56–61 (2016) [in Russian].

31. N. V. Polukonova, N. A. Navolokin, and S. V. Raykova, “Anti-inflammatory, antipyretic and antimicrobial activity flavonoid-containing extract of Gratiola officinalis L,” Eksperimental’naya i Klinicheskaya Farmakologiy 78(1), 34–38 (2015).

32. N. V. Polukonova, M. N. Kurchatova, N. A. Navolokin, A. B. Bucharskaya, N. A. Durnova, and G. N. Maslyakova, “A new extraction method of bioflavanoids from poisonous plant,” Russian Open Medical Journal 3(3), 304 (2014).

33. N. A. Navolokin, D. A. Mydrak, A. B. Bucharskaya, O. V. Matveeva, S. A. Tychina, N. V. Polukonova, and G. N. Maslyakova, “Effect of flavonoid-containing extracts on the growth of transplanted sarcoma 45, peripheral blood and bone marrow condition after oral and intramuscular administration in rats,” Russian Open Medical Journal 6(3), 304 (2017).

34. N. V. Tkachenko, E. V. Bykova, A. B. Pravdin, N. A. Navolokin, N. V. Polukonova, A. B. Bucharskaya, D. A. Mudrak, and A. Y. Prilepskii, “Comparison of membrane-protective activity of antioxidants quercetine and Gratiola officinalis L. extract under conditions of photodynamic haemolysis,” Proceedings of SPIE 9917, 99170L (2016).

35. N. A. Navolokin, N. V. Polukonova, G. N. Maslyakova, A. B. Bucharskaya, and N. A. Durnova, “Effect of extracts of Gratiola officinalis and Zea mays on the tumor and the morphology of the internal organs of rats with trasplanted liver cancer,” Russian Open Medical Journal 1(2), 0203 (2012).

36. A. Mylnikov, N. Navolokin, D. Mudrak, N. Polukonova, A. Bucharskaya, and G. Maslyakova, “Tumor cell death visualization of renal cell carcinoma under the combined effect of the Gratiola officinalis extract and cyclophosphamide using fluorescent staining methods,” Journal of Innovative Optical Health Sciences 14(05), 2142004 (2021).

37. Z. Yang, R. A. Ghoorun, X. Fan, P. Wu, Y. Bai, and J. Li, “High expression of Beclin-1 predicts favorable prognosis for patients with colorectal cancer,” Clinics and Research in Hepatology and Gastroenterology 39(1), 98–106 (2015).

38. J. Y. Tang, E. Hsi, Y. C. Huang, N. C. Hsu, P. Y. Chu, and C. Y. Chai, “High LC3 expression correlates with poor survival in patients with oral squamous cell carcinoma,” Human Pathology 44(11), 2558–2562 (2013).

39. J. L. Liu, F. F. Chen, J. Lung, C. H. Lo, F. H. Lee, and Y. C. Lu, “Prognostic significance of p62/SQSTM1 subcellular localization and LC3B in oral squamous cell carcinoma,” British Journal of Cancer 111(5), 944–954 (2014).

40. K. Lai, S. Matthews, J. S. Wilmott, M. C. Killingsworth, J. L. Yong, N. J. Caixeiro, J. Wykes, A. Samakeh, D. Forstner, M. Lee, J. McGuinness, N. Niles, A. Hong, A. Ebrahimi, and C. S. Lee, “Differences in LC3B expression and prognostic implications in oropharyngeal and oral cavity squamous cell carcinoma patients,” BMC Cancer 18, 624 (2018).

41. D. H. Wu, C. C. Jia, J. Chen, Z. X. Lin, D. Y. Ruan, and X. Li, “Autophagic LC3B overexpression correlates with malignant progression and predicts a poor prognosis in hepatocellular carcinoma,” Tumor Biology 35(12), 12225–12233 (2014).

42. W. Wang, Q. L. Guo, Q. D. You, K. Zhang, Y. Yang, and J. Yu, “The anticancer activities of wogonin in murine sarcoma S180 both in vitro and in vivo,” Biological and Pharmaceutical Bulletin 29(6), 1132–1137 (2006).

43. S. Baumann, S. C. Fas, M. Giaisi, W. W. Muller, A. Merling, and K. Gulow, “Wogonin preferentially kills malignant lymphocytes and suppresses T-cell tumor growth by inducing PLCgamma1- and Ca2+-dependent apoptosis,” Blood, The Journal of the American Society of Hematology 111(4), 2354–2363 (2008).

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