Investigation of phytochemical contents and anticancer, antioxidant, antimicrobial activities of Cucurbita pepo leaves

Abstract

Ethanol and acetone extracts of Cucurbita pepo leaves were in vitro analyzed to determine the vitamin contents, antioxidant, anticancer, and antimicrobial activity of the leaves. According to HPLC analyses, the leaves contained slight amounts of vitamin C, A, and E. The analysis for antioxidant activity revealed that both ethanol and acetone extracts exhibited high activity values against ABTS+•, OH•, and DPPH• radicals. The total phenols and flavonoid assay revealed high values for the extracts. The MTT test revealed that both ethanol and acetone extracts exhibited cytotoxic activity toward human breast cancer cells (MCF-7). But no significant activities observed against hepatocellular carcinoma cells (HepG-2). The antimicrobial activity assay revealed activity of the extracts towards selected Gram's positive and Gram's negative bacteria. These results have provided scientific motivation to use Cucurbita pepo leaves as a medicinal plant to treat oxidative and inflammatory-related diseases.

Keywords

• Cucurbita pepo
• antioxidant
• anticancer
• antimicrobial
• antiradical

References

1. Zeng, G., Wu, W., and Huang, W. (2019). Evaluation of nutritional and other functional qualitiesas well as dietary safety of pumpkin leaves, Journal of Food Safety and Food Quality, 70, 23-29.
2. Njeme, C. Goduka, N.I., George, G. (2014). Indigenous leafy vegetables (imifino, morogo, muhuro) in South Africa: A rich and unexplored source of nutrients and antioxidants, African Journal of Biotechnology, 13, 1933-1942.
3. Grubben, G.J.H. (2004). Plant Resources of Tropical Africa, Vegetables, PROTA Foundation. p.118-275.
4. Ko, J.Y., Ko, M.O., Kim, D.S., Lim, S. (2016). Enhanced production of phenolic compounds from pumpkin leaves by subcritical water hydrolysis, Preventive Nutrition and Food Science, 21, 132-137.
5. Dissanayake, H., Deraniyagala, S., Hettiarachchi, C., Thiripuranathar, G. (2018). The study of antioxidant and antibacterial properties of skin, seeds and leaves of the Sri Lankan variety of pumpkin, IOSR Journal of Pharmacy, 8, 43-48.
6. Kulczyński, B., Gramza-Michałowska, A., Królczyk, J.B. (2020). Optimization of extraction conditions for the antioxidant potential of different pumpkin varieties (Cucurbita maxima), Sustainability, 12, 1305.
7. Collin, F. (2019). Chemical basis of reactive oxygen species reactivity and involvement in neurodegenerative diseases, International Journal of Molecular Sciences, 20, 2407.
8. Rao, P.S., Kalva, S., Yerramilli, A., Mamidi, S. (2011). Free radicals and tissue damage: role of antioxidants, Free Radicals and Antioxidants, 1, 2-7.

9. Ruiz-Núñez, B., Pruimboom, L., Dijck-Brouwer, D.A., Muskiet, F.A. (2013). Lifestyle and nutritional imbalances associated with Western diseases: causes and consequences of chronic systemic low-grade inflammation in an evolutionary context, Journal of Nutritional Biochemistry, 24, 1183-1201.
10. Zhao, Y., Wang, Y., Li, Y., Santschi, P., Quigg, A. (2017). Response of photosynthesis and the antioxidant defense system of two microalgal species (Alexandrium minutum and Dunaliella salina) to the toxicity of BDE-47, Marine Pollution Bulletin, 124, 459-469.
11. Rosenow, P., Jakob, P., Tonner, R. (2016). Electron–vibron coupling at metal-organic interfaces in theory and experiment, Journal of Physical Chemistry Letters, 7, 1422-1427.
12. Hariri, M., Maghsoudi, Z., Darvishi, L., Askari, G., Hajishafiee, M., Ghasemi, S., Khorvash, F., Iraj, B., Ghiasvand, R. (2013). Antioxidants intake is negatively correlated with risk of stroke in Iran, International of Preventive Medicine, 4, S248-S289.
13. Halliwell, B., Gutteridge, J.M.C., (1989). Free Radicals in Biology and Medicine, Clarendon Press., pp. 121-197.
14. EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies). Turck, D., Bresson, J.L., Burlingame, B., Dean, T., Fairweather-Tait, S., Heinonen, M., Hirsch-Ernst, K.I., Mangelsdorf, I., McArdle, H.J., Naska, A., Neuhauser-Berthold, M., Nowicka, G., Pentieva, K., Sanz, Y., Sjodin, A., Stern, M., Tome, D., Van Loveren, H., Vinceti, M., Willatts, P., Martin, A., Strain, J.J., Heng, L., Valtuena-Martinez, S., Siani. A. (2018). Guidance for the scientific requirements for health claims related to antioxidants, oxidative damage and cardiovascular health (Revision 1). EFSA Journal, 16, 5136, 21.
15. Brubaker, R.F., Bourne, W.M., Bachman, L.A., McLaren, J.W. (2000). Ascorbic acid content of human corneal epithelium, Investigative Ophthalmology & Visual Science, 41, 1681-1683.
16. Ojima, F., Sakamoto, H., Ishiguro, Y., Terao, J. (1993). Consumption of carotenoids in photosensitized oxidation of human plasma and plasma low-density lipoprotein, Free Radical Biology and Medicine, 15, 377-384.
17. Gaziano, J.M., Manson, J.E., Ridker, P.M., Buring, J.E., Hennekens, C.H. (1990). Beta carotene therapy for chronic stable angina, Circulation, 82.
18. Traber, M.G., Atkinson, J. (2007). Vitamin E, antioxidant and nothing more, Free Radical Biology and Medicine, 43, 4-15.
19. Li, J., Wang, H., Rosenberg, P.A. (2009). Vitamin K prevents oxidative cell death by inhibiting the activation of 12‐lipoxygenase in developing oligodendrocytes, Journal of Neuroscience Research, 87, 1997-2005.
20. Ohyashiki, T., Yabunaka, Y., Matsui, K. (1991). Antioxidant effect of vitamin K homologues on ascorbic acid/Fe(2+)-induced lipid peroxidation of lecithin liposomes, Chemical and Pharmaceutical Bulletin, 39, 976-979.
21. Ohsaki, Y., Shirakawa, H., Miura, A., Giriwono, P.E., Sato, S., Ohashi, A., Iribe, M., Goto, T., Komai, M. (2010). Vitamin K suppresses the lipopolysaccharide-induced expression of inflammatory cytokines in cultured macrophage-like cells via the inhibition of the activation of nuclear factor κB through the repression of IKKα/β phosphorylation, Journal of Nutritional Biochemistry, 21, 1120-1126.
22. Varsha, M.K.N.S., Thiagarajan, R., Manikandan, R., Dhanasekaran, G. (2015). Vitamin K1 alleviates streptozotocin-induced type 1 diabetes by mitigating free radical stress, as well as inhibiting NF-κB activation and iNOS expression in rat pancreas, Nutrition, 31, 214-222.
23. Cruz, J.B.F., Soares, H.F. (2011). Uma revisão sobre o zinco, Ensaios e Ciência: Ciências Biológicas, Agrárias e da Saúde, 15, 207-222.
24. Laghari, A.H., Memon, S., Nelofar, A., Khan, K.M., Yasmin, A. (2011). Determination of free phenolic acids and antioxidant activity of methanolic extracts obtained from fruits and leaves of Chenopodium album, Food Chemistry, 126, 1850-1855.
25. Espíndola, K.M.M., Ferreira, R.G., Narvaez, L.E.M., Silva Rosario, A.C.R., da Silva, A.H.M., Silva, A.G.B., Vieira, A.P.O., Monteiro, M.C. (2019). Chemical and pharmacological aspects of caffeic acid and its activity in hepatocarcinoma, Frontiers in Oncology, 9, 541.
26. Siquet, C., Paiva-Martins, F., Lima, J.L., Reis, S., Borges, F. (2006). Antioxidant profile of dihydroxy-and trihydroxyphenolic acids-A structure–activity relationship study, Free Radical Research, 40, 433-442.
27. Magalhães, L., Ramos, I., Reis, S., Segundo, M. (2014). Antioxidant profile of commercial oenological tannins determined by multiple chemical assays, Australian Journal of Grape and Wine Research, 20, 72-79.
28. Gülçin, İ., Huyut, Z., Elmastaş, M., Aboul-Enein, H.Y. (2010). Radical scavenging and antioxidant activity of tannic acid, Arabian Journal of Chemistry, 3, 43-53.
29. Anderson, R.J., Bendell, D.J., Groundwater, P.W. (2004). Organic Spectroscopic Analysis, Royal Society of Chemistry.
30. Kalinovski, H.O., Berger, S., Braun, S. (1988). Carbon-13 NMR Spectroscopy. John Wiley & Sons.
31. Pihlaja, K., Kleinpeter, E. (1994). Carbon-13 NMR Chemical Shifts in Structural and Stereochemical Analysis.
32. Tauc, J., Menth, A. (1972). States in the gap, Journal of Non-Crystalline Solids, 8–10, 569-585.
33. Singh, D., Chaudhuri, P.K. (2018). Structural characteristics, bioavailability and cardioprotective potential of saponins, Integrative Medicine Research, 7, 33-34.
34. Man, S., Gao, W., Zhang, Y., Huang, L., Liu, C. (2010). Chemical study and medical application of saponins as anti-cancer agents, Fitoterapia, 81, 703-714.
35. Sparg, S.G., Light, M.E., Van Staden, J. (2004). Biological activities and distribution of plant saponins, Journal of Ethnopharmacology, 94, 219-243.
36. Hawkins, P.T., Poyner, D.R., Jackson, T.R., Letcher, A.J., Lander, D.A., Irvine, R.F. (1993). Inhibition of iron-catalysed hydroxyl radical formation by inositol polyphosphates: a possible physiological function for myo-inositol hexakisphosphate, Biochemical Journal, 294, 929-934.
37. Lee, S., Kim, M.-G., Ahn, H., Kim, S. (2020). Inositol pyrophosphates: signaling molecules with pleiotropic actions in mammals, Molecules, 25, 2208.
38. Shamsuddin, A.M., Yang, G.Y., Vucenik, I. (1996). Novel anti-cancer functions of IP6: growth inhibition and differentiation of human mammary cancer cell lines in vitro, Anticancer Research, 16, 3287-3292.
39. Pekdemir, M.E., Pekdemir, S., İnci, Ş., Kırbağ, S., Çiftci, M. (2021). Thermal, magnetic properties and antimicrobial effects of magnetic iron oxide nanoparticles treated with Polygonum cognatum, Iranian Journal of Science and Technology, Transactions A: Science, 45, 1579-1586.
40. 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, 1231-1237.
41. Halliwell, B., Gutteridge, J.M., Aruoma, O.I. (1987). The deoxyribose method: a simple “test-tube” assay for determination of rate constants for reactions of hydroxyl radicals, Analytical Biochemistry, 165, 215-219.
42. Brand-Williams, W., Cuvelier, M.E., Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity, LWT-Food science and Technology, 28, 25-30.
43. Keser, S. (2014). Antiradical activities and phytochemical compounds of firethorn (Pyracantha coccinea) fruit extracts. Natural Product Research, 28, 1789–1794.
44. Keser, S., Keser, F., Kaygili, O., Tekin, S., Demir, E., Turkoglu, I., Turkoglu, S., Parlak, A.E., Yilmaz, O., Karatepe, M., Sandal, S., Kirbag, S. (2018). Phytochemical compounds and antiradical, antimicrobial, and cytotoxic activities of the extracts from Hypericum scabrum L. Flowers. Natural Product Research, 34, 714–719.
45. Keser, S., Keser, F., Karatepe, M., Kaygili, O., Tekin, S., Turkoglu, I., Demir, E., Yilmaz, O., Kirbag, S., Sandal, S. (2019). Bioactive contents, in vitro antiradical, antimicrobial and cytotoxic properties of rhubarb (Rheum ribes L.) extracts. Natural Product Research, 34, 3353–3357.
46. Keser, S., Kak, O. (2021). In vitro antimicrobial, antiradical, anticancer evaluation, and phytochemical contents of endemic Scorzonera semicana DC. Journal of Food Processing and Preservation, 45, e15971.
47. Slinkard, K. Singleton, V.L. (1977). Total phenol analysis: automation and comparison with manual methods, American Journal of Enology and Viticulture, 28, 49-55.
48. Kim, D.O., Chun, O.K., Kim, Y.J., Moon, H.Y., Lee, C.Y. (2003). Quantification of polyphenolics and their antioxidant capacity in fresh plums, Journal of Agricultural and Food Chemistry, 51, 6509-6515.
49. Amaeze, O., Ayoola, G., Sofidiya, M., Adepoju-Bello, A., Adegoke, A., Coker, H. (2011). Evaluation of antioxidant activity of Tetracarpidium conophorum (Müll. Arg) Hutch & Dalziel leaves, Oxidative Medicine and Cellular Longevity, 2011, 976701.
50. Offiong, O.E., Martelli, S. (1997). Stereochemistry and antitumour activity of platinum metal complexes of 2-acetylpyridine thiosemicarbazones, Transition Metal Chemistry, 22, 263-269.
51. Karatepe, M. (2004). Simultaneous determination of ascorbic acid and free malondialdehyde in human serum by HPLC-UV, LC-GC North America, 22, 362-365.
52. Catignani, G.L, Bieri, J.G. (1983). Simultaneous determination of retinol and alpha-tocopherol in serum or plasma by liquid chromatography, Clinical Chemistry, 29, 708-712.
53. Dalkilic, S., Dalkilic, L.K., İnci, S., Korkmaz, İ., Kirbag, S. (2021). Investigation of cytotoxic effect of black mulberry (Morus nigra L.) fruit, Indian Journal of Traditional Knowledge, 20, 54-58.
54. Van Meerloo, J., Kaspers, G.J.L, Cloos, J. (2011). Cell sensitivity assays: the MTT assay, Methods in Molecular Biology, 731, 237-245.
55. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, Journal of Immunological Methods, 65, 55-63.
56. Gerlier, D., Thomasset, N. (1986). Use of MTT colorimetric assay to measure cell activation, Journal of Immunological Methods, 94, 57-63.
57. Erecevit Sönmez, P., Kırbağ, S., İnci, Ş. (2019). Antifungal and antibacterial effect of dodder (Cuscuta campestris) used for hepatitis treatment of mothers and newborn infants in province Mardin in Turkey, Yuzuncu Yil University Journal of Agricultural Sciences, 29, 722-730.
58. 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.
59. Van Jaarsveld, P., Faber, M., Van Heerden, I., Wenhold, F., van Rensburg, W.J., Van Averbeke, W. (2014). Nutrient content of eight African leafy vegetables and their potential contribution to dietary reference intakes, Journal of Food Composition and Analysis, 33, 77-84.
60. Dafam, D.G., Agunu, A., Dénou, A., Christopher, D., Kagaru, T.L.O., Ajima, U., Damos, J.N., Okwori, V.A. (2020) Determination of the ascorbic acid content, mineral and heavy metal levels of some common leafy vegetables of Jos Plateau State (North Central Nigeria), International Journal of Biosciences, 16, 389-396.
61. Zuorro, A., Iannone, A., Lavecchia, R. (2019). Water–organic solvent extraction of phenolic antioxidants from brewers’ spent grains, Processes, 7, 126.
62. Felhi, S., Daoud, A., Hajlaoui, H., Mnafgui, K., Gharsallah, N., Kadri, A. (2017). Solvent extraction effects on phytochemical constituents profiles, antioxidant and antimicrobial activities and functional group analysis of Ecballium elaterium seeds and peels fruits, Food Science and Technology, 37, 483-492.
63. Dar, P., Farman, M., Dar, A., Khan, Z., Munir, R., Rasheed, A., Waqas, U. (2017). Evaluation of antioxidant potential and comparative analysis of antimicrobial activity of various extracts of Cucurbita pepo L. leaves, Journal of Agricultural Science and Food Technology, 3,103-109.
64. Fakoya, S., Aderoboye, O.Y., Olusola, S.E. (2019). Phytobiotics effects of pawpaw (Carica papaya) leaves and fluted pumpkin (Telferia ocidentalis) leaves extracts against certain aquatic pathogens, Medicinal and Aromatic Plants, 8, 1000328.
65. Oboh, G., Nwanna, E., Elusiyan, C. (2006). Antioxidant and antimicrobial properties of Telfairia occidentalis (fluted pumpkin) leaf extracts, Journal of Pharmacology and Toxicology, 1, 167-175.
66. Nwanna, E.E., Oboh, G. (2007). Antioxidant and hepatoprotective properties of polyphenol extracts from Telfairia occidentalis (fluted pumpkin) leaves on acetaminophen induced liver damage, Pakistan Journal of Biological Sciences, 10, 2682-2687.
67. Yang, E.C., Hsieh, Y.Y., Chuang, L.Y. (2021). Comparison of the phytochemical composition and antibacterial activities of the various extracts from leaves and twigs of Illicium verum, Molecules, 26, 3909.
68. Balouiri, M., Sadiki, M., Ibnsouda, S.K. (2016). Methods for in vitro evaluating antimicrobial activity: a review, Journal of Pharmaceutical Analysis, 6, 71-79.