Antihemolytic activity of hydroalcoholic leaves and bark extracts from Rhamnus alaternus against AAPH induced hemolysis on human erythrocytes

Abstract

Rhamnus alaternus is a Mediterranean shrub that has been used in traditional medicine to treat various diseases. This study aimed to determine the phenolic composition, as well as antioxidant and antihemolytic activities of R. alaternus leaves (LRA) and bark (BRA), extracts against AAPH-induced hemolysis. The extraction yields were 19.8% and 18.2% for leaves and bark. Total polyphenols (88.1 ± 1.83 mg GAE/g) and condensed tannins (36.24 ± 5.44 mg CE/g) were higher in BRA extract than in LRA extract (80.22 ± 1.4 mg GAE/g and 23.48 ± 0.25 mg CE/g, respectively). However, LRA extract was found to be richer in total flavonoids (64.6 ± 2.6 mg QE/g) and flavones/flavonol (18.34 ± 1.65 mg QE/g) than BRA extract (39.87 ±0.58 mg QE/g and 10.08 ± 0.35 mg QE/g), respectively. The IC50 of DPPH and ABTS radical scavenging activity were 86.59 ± 2 µg/ml and 12.49 ± 0.29 µg/ml for LRA extract and 69.23 ± 2.14 µg/ml and 12.83 ± 0.13 µg/ml for BRA extract, respectively. Also, both extracts showed good reducing power with 157.09 ± 5.53 mg Asc E/g for LRA extract and 194.97 ± 1.46 mg Asc E/g for BRA extract. The hemolytic effect was tested on human erythrocytes, and both extracts did not have cytotoxic effects at low doses. To induce hemolysis, AAPH was used at a concentration of 200 mM with an incubation time of 4h. The antihemolytic activity of the two extracts showed that pretreatment of human erythrocytes with various doses significantly reduced AAPH-induced hemolysis in a dose-dependent manner. Indeed, at 200 µg/ml, the percentages of hemolysis inhibition were 99.41 ± 1.17% and 76.26 ± 12.03% for BRA and LRA extracts, respectively. BRA extract was more effective (IC50 = 106.70 ± 1.48 µg/ml) compared to LRA extract (IC50 = 148.64 ± 7.04 µg/ml). Our results demonstrate for the first time that R. alaternus attenuates AAPH-induced hemolysis and can be used to prevent and treat hemolytic anemias.

Keywords

• Rhamnus alaternus
• Polyphenols
• AAPH
• Hemolysis
• Antioxidant
• Erythrocytes

Kaynakça

1. Abate, L., 2019. Bioactivity profiling of four traditional medicinal plants leave extracts native to Alemsaga Forest, Ethiopia. Chemistry International, 5(4), 281-290.
2. Ahmed, J., Salim, K.A., Lim, L.B., Jama, A.M., 2019. Evaluation of Antioxidant Activity and Phytochemical Screening of Leaves, Barks, Stems and Fruits of Alphitonia philippinensis (Rhamnaceae) From Brunei Darussalam. Pharmacognosy Journal, 11(5), 951-961.
3. Athamena, S., Chalghem, I., Kassah-Laouar, A., Laroui, S., Khebri, S., 2010. Activité antioxydante et antimicrobienne d’extraits de Cuminum cyminum L. Lebanese Science Journal, 11(1), 69-81.
4. Ayala, A., Muñoz, M.F., Argüelles, S., 2014. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity, 2014, 360438.
5. Badior, K.E., Casey, J.R., 2018. Molecular mechanism for the red blood cell senescence clock. International Union of Biochemistry and Molecular Biology Life, 70(1), 32-40.
6. Balderrama-Carmona, A.P., Silva-Beltrán, N.P., Gálvez-Ruiz, J.C., Ruíz-Cruz, S., Chaidez-Quiroz, C., Morán-Palacio, E.F., 2020. Antiviral, antioxidant, and antihemolytic effect of Annona muricata L. leaves extracts. Plants, 9(12), 1650.
7. Ben Ammar, R., Bhouri, W., Sghaier, M.B., Boubaker, J., Skandrani, I., Neffati, A., Ghedira, K., 2009. Antioxidant and free radical-scavenging properties of three flavonoids isolated from the leaves of Rhamnus alaternus L.(Rhamnaceae): A structure-activity relationship study. Food Chemistry, 116(1), 258-264.
8. Ben Ammar, R., Bouhlel, I., Valenti, K., Sghaier, M.B., Kilani, S., Mariotte, A.M., Chekir-Ghedira, L., 2007. Transcriptional response of genes involved in cell defense system in human cells stressed by H2O2 and pre-treated with (Tunisian) Rhamnus alaternus extracts: Combination with polyphenolic compounds and classic in vitro assays. Chemico-Biological Interactions, 168(3), 171-183.

9. Ben Ammar, R., Sghaier, M.B., Boubaker, J., Bhouri, W., Naffeti, A., Skandrani, I., Chekir-Ghedira, L., 2008. Antioxidant activity and inhibition of aflatoxin B1-, nifuroxazide-, and sodium azide-induced mutagenicity by extracts from Rhamnus alaternus L. Chemico-Biological Interactions, 174(1), 1-10.
10. Benchaachoua, A., Bessam, H.M., Saidi, I., Bel-abbes, S., 2018. Effects of different extraction methods and solvents on the phenolic composition and antioxidant activity of Silybum marianum leaves extracts. International Journal of Medical Science and Clinical Invention, 5(3), 3641-3647.
11. Benchiha, W., Mahroug, S., Aoued, L., Bouterfas, K., 2017. Activité antihépatotoxique des extraits des feuilles de Rhamnus alaternus L.(Rhamnaceae). Phytothérapie, 15(1), 10-15.
12. Benmeziane, F., Djamai, R., Cadot, Y., Seridi, R., 2014. Optimization of extraction parameters of phenolic compounds from Algerian fresh table grapes,(Vitis vinifera). International Food Research Journal, 21(3), 1061-1065.
13. Bessada, S.M., Barreira, J.C., Oliveira, M.B.P., 2015. Asteraceae species with most prominent bioactivity and their potential applications: A review. Industrial Crops and Products, 76, 604-615.
14. Bouchenak, O., Yahiaoui, K., Benhabyles, N., Laoufi, R., Toubal, S., El Haddad, D., Arab, K., 2020. Criblage phytochimique et évaluation du pouvoir antioxydant des feuilles de myrtus communis L. et Rhamnus alaternus L. Revue Agrobiologia, 10, 1749-1761.
15. Boussahel, S., Speciale, A., Dahamna, S., Amar, Y., Bonaccorsi, I., Cacciola, F., Cristani, M., 2015. Flavonoid profile, antioxidant and cytotoxic activity of different extracts from Algerian Rhamnus alaternus L. bark. Pharmacognosy Magazine, 11(Suppl 1), S102-S109.
16. Chansiw, N., Paradee, N., Chotinantakul, K., Srichairattanakool, S., 2018. Anti-hemolytic, antibacterial and anti-cancer activities of methanolic extracts from leaves and stems of Polygonum odoratum. Asian Pacific Journal of Tropical Biomedicine, 8(12), 580.
17. Chaouche, T.M., Haddouchi, F., Boudjemai, O., Ghellai, I., 2020. Antioxidant and hemolytic activity of Ziziphus jujuba Mill and Rhamnus alaternus L (Rhamnaceae) extracts from Algeria. Activité antioxydante et hémolytique des extraits de Ziziphus jujuba Mill et Rhamnus alaternus (Rhamnaceae) d’Algérie. Bulletin de la Société Royale des Sciences de Liège, 89, 1-14.
18. Chaves, N., Santiago, A., Alías, J.C., 2020. Quantification of the antioxidant activity of plant extracts: Analysis of sensitivity and hierarchization based on the method used. Antioxidants, 9(1), 76.
19. Cheok, C.Y., Salman, H.A.K., Sulaiman, R., 2014. Extraction and quantification of saponins: A review. Food Research International, 59, 16-40.
20. Çimen, M.B., 2008. Free radical metabolism in human erythrocytes. Clinica Chimica Acta, 390(1-2), 1-11.
21. Derouich, M., Bouhlali, E.D.T., Bammou, M., Hmidani, A., Sellam, K., Alem, C., 2020. Bioactive compounds and antioxidant, antiperoxidative, and antihemolytic properties investigation of three apiaceae species grown in the southeast of Morocco. Scientifica, 2020, 3971041.
22. Drobatz, K.J., Hopper, K., Rozanski, E.A., Silverstein, D.C., 2018. Hemolytic anemia. In Wiley Blackwell (Eds), Textbook of Small Animal Emergency Medicine (pp. 419-443). John Wiley and Sons.
23. Elgailani, I.E.H., Ishak, C.Y., 2016. Methods for extraction and characterization of tannins from some Acacia species of Sudan. Pakistan Journal of Analytical & Environmental Chemistry, 17(1), 43-49.
24. Enneb, H., Belkadhi, A., Cheour, F., Ferchichi, A., 2015. Comparaison des composés phénoliques et du pouvoir antioxydant de la plante de henné (Lawsonia inermis L.). Journal of New Sciences, 20, 788-793.
25. Fibach, E., Dana, M., 2019. Red blood cells as redox modulators in hemolytic anemia. In Tombak Anil (Eds). Erythrocyte (pp. 49-63). IntechOpen publishers.
26. Fibach, E., Rachmilewitz, E., 2008. The role of oxidative stress in hemolytic anemia. Current Molecular Medicine, 8(7), 609-619.
27. Hmidani, A., Ajebli, M., Khouya, T., Benlyas, M., Alem, C., 2021. In vitro investigation of antioxidant and antihemolytic activities of three Lamiaceae species from Morocco. Beni-Seuf University Journal of Basic Applied Sciences, 10, 1-8.
28. Kosalec, I., Bakmaz, M., Pepeljnjak, S., Vladimir-Knezevic, S., 2004. Quantitative analysis of the flavonoids in raw propolis from northern Croatia. Acta Pharmaceutica, 54(1), 65-72.
29. Kosalec, I., Kremer, D., Locatelli, M., Epifano, F., Genovese, S., Carlucci, G., Končić, M.Z., 2013. Anthraquinone profile, antioxidant and antimicrobial activity of bark extracts of Rhamnus alaternus, R. fallax, R. intermedia and R. pumila. Food Chemistry, 136(2), 335-341.
30. Kumar, G., Karthik, L., Rao, K.V.B., 2011. Hemolytic activity of Indian medicinal plants towards human erythrocytes: an in vitro study. Elixir Applied Botany, 40, 5534-5537.
31. Liao, W., Chen, L., Ma, X., Jiao, R., Li, X., Wang, Y., 2016. Protective effects of kaempferol against reactive oxygen species-induced hemolysis and its antiproliferative activity on human cancer cells. European Journal of Medicinal Chemistry, 114, 24-32.
32. Moussi, K., Nayak, B., Perkins, L.B., Dahmoune, F., Madani, K., Chibane, M., 2015. HPLC-DAD profile of phenolic compounds and antioxidant activity of leaves extract of Rhamnus alaternus L. Industrial Crops and Products, 74, 858-866.
33. Nekkaa, A., Benaissa, A., Mutelet, F., Canabady-Rochelle, L., 2021. Rhamnus alaternus Plant: Extraction of Bioactive Fractions and Evaluation of Their Pharmacological and Phytochemical Properties. Antioxidants, 10(2), 300.
34. Nga, E.N., Nchinda, G., Mahi, L.L., Figuei, P.B., Mvondo, J.M., Sagnia, B., Adiogo, D., 2019. Phytochemical screening and evaluation of antioxidant power of hydro-ethanolic and aqueous leaves extracts of Annona muricata Linn (soursop). Health Sciences and Disease, 20(1), 29-33.
35. Oreopoulou, A., Tsimogiannis, D., Oreopoulou, V., 2019. Extraction of polyphenols from aromatic and medicinal plants: An overview of the methods and the effect of extraction parameters. In Ronald Ross Watson (Eds). Polyphenols in plants (pp. 243-259). Academic Press publishers.
36. Phillips, J., Henderson, A.C., 2018. Hemolytic anemia: evaluation and differential diagnosis. American Family Physician, 98(6), 354-361.
37. Phrueksanan, W., Yibchok-anun, S., Adisakwattana, S., 2014. Protection of Clitoria ternatea flower petal extract against free radical-induced hemolysis and oxidative damage in canine erythrocytes. Research in Veterinary Science, 97(2), 357-363.
38. Quettier-Deleu, C., Gressier, B., Vasseur, J., Dine, T., Brunet, C., Luyckx, M., Trotin, F., 2000. Phenolic compounds and antioxidant activities of buckwheat (Fagopyrum esculentum Moench) hulls and flour. Journal of Ethnopharmacology, 72(1-2), 35-42.
39. Rafat, A., Philip, K., Muni, S., 2010. Antioxidant potential and content of phenolic compounds in ethanolic extracts of selected parts of Andrographis paniculata. Journal of Medicinal Plants Research, 4(3), 197-202.
40. Rai, M., Ali, M.U., Geller, C., 2020. Hemolytic Anemia: Sneaky Cause, Leaky Valve. Cureus, 12(5), 1-9.
41. Ramchoun, M., Sellam, K., Harnafi, H., Alem, C., Benlyas, M., Khallouki, F., Amrani, S., 2015. Investigation of antioxidant and antihemolytic properties of Thymus satureioides collected from Tafilalet Region, south-east of Morocco. Asian Pacific Journal of Tropical Biomedicine, 5(2), 93-100.
42. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26(9-10), 1231-1237.
43. Shah, P., Modi, H.A., 2015. Comparative study of DPPH, ABTS and FRAP assays for determination of antioxidant activity. International Journal of Research in Applied Science and Engineering Technology, 3(6), 636-641.
44. Tacherfiout, M., Petrov, P.D., Mattonai, M., Ribechini, E., Ribot, J., Bonet, M.L., Khettal, B., 2018. Antihyperlipidemic effect of a Rhamnus alaternus leaf extract in Triton-induced hyperlipidemic rats and human HepG2 cells. Biomedicine & Pharmacotherapy, 101, 501-509.
45. Wolfe, K., Wu, X., Liu, R.H., 2003. Antioxidant activity of apple peels. Journal of Agricultural and Food Chemistry, 51(3), 609-614.
46. Yang, H.L., Korivi, M., Lin, M.K., Chang, H.C.W., Wu, C.R., Lee, M.S., Hseu, Y.C., 2017. Antihemolytic and antioxidant properties of pearl powder against 2, 2′-azobis (2-amidinopropane) dihydrochloride-induced hemolysis and oxidative damage to erythrocyte membrane lipids and proteins. Journal of Food and Drug Analysis, 25(4), 898-907.
47. Yuan, X., Wang, J., Yao, H., 2005. Antioxidant activity of feruloylated oligosaccharides from wheat bran. Food Chemistry, 90(4), 759-764.
48. Zeouk, I., Ouedrhiri, W., Jiménez, I.A., Lorenzo-Morales, J., Bazzocchi, I.L., Bekhti, K., 2020. Intra-combined antioxidant activity and chemical characterization of three fractions from Rhamnus alaternus extract: Mixture design. Industrial Crops and Products, 144, 112054.