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Evaluation of effects on hepatocellular carcinoma cell line of Cocos nucifera: In vitro study

Evaluation of effects on hepatocellular carcinoma cell line of Cocos nucifera: In vitro study Acta Marisiensis - Seria Medica 2023;69(1):45-49 DOI: 10.2478/amma-2023-0004 RESEARCH ARTICLE Evaluation of effects on hepatocellular carcinoma cell line of Cocos nucifera: In vitro study 1 2* 3 Zeynep Tasdan , Gülçin Alp Avci , Emre Avci 1. Department of Molecular Biology and Genetics, University of Hitit, Corum, Türkiye 2. Department of Basic Medical Sciences, University of Health Sciences, Ankara, Türkiye 3. Department of Biochemistry, University of Health Sciences, Ankara, Türkiye Objective: Cancer is one of the most important diseases today. The use of chemical drugs, surgical operations, and transplants is very common in its treatment. In addition to these treatment methods, studies include the effects of natural and plant-derived substances. Vari- ous substances are used in these studies, which are called phytotherapy. The antioxidant activity and cytotoxic effects of Cocos nucifera on hepatocellular carcinoma HepG-2 and the mouse fibroblast L929 cell line were investigated in this study. Methods: In this study, in vitro cyto- toxic effects of C. nucifera at different concentrations (7.81-500 mg/ml) were investigated on the L929 Mouse Fibroblast cell line and HepG-2 Hepatocellular Carcinoma cell line. In addition to these studies, their antioxidant capacity was evaluated via spectrophotometric methods. In this work, different concentrations of C. nucifera were examined. Results: According to the results, C. nucifera had a cytotoxic effect in HepG-2 and ensured the proliferative effect of cells in the L929 cell line. Among C. nucifera extracts according to total antioxidant capacity results, C. nucifera extract was found to be the richest in antioxidants with 2.79 mmol/L, while the material with the lowest antioxidant capacity was determined to be C. nucifera milk. DPPH free radical scavenging activity results show the opposite. Conclusion: In line with the data we obtained, it is thought that C. nucifera can be used in liver cancer studies, and its antioxidant effect may play an important role in balancing against oxidative stress. Simultaneously, the data show that the exposure time and concentrations of the active substance are related to the cytotoxic effect. However, it may be considered that the use of C. nucifera water, extract, and milk in cancer patients may be supported by more comprehensive clinical studies. Keywords: Cocos nucifera, HepG-2, L929, cytotoxicity, antioxidant activity Received 9 September 2022 / Accepted 1 February 2023 Introduction Coconut (Cocos nucifera), which belongs to the  Areca- Today, plants maintain their place in the cosmetics, health, ceae family, is the fruit of the tropics. It has a supportive, pharmaceutical, textile, and various industrial fields. Its use strengthening effect on the defense and intestinal systems in the health and pharmaceutical industries started in an- and is very nutritious. C. nucifera is a product of the tro- cient times, and its history dates to the first ages [1,2,3]. pics. C. nucifera dates grow on sandy soils and are highly Recently, the production of plant-derived drugs has been tolerant of soils with high salt content. It prefers regions increasing in developing countries in the fields of medicine with plenty of sunlight and regular precipitation [7].  C. and biotechnology [3]. The use of plants in treatments over nucifera  is a large palm that reaches up to 30 meters in time is called “Phytotherapy” [2]. Phytotherapy is also used length. C. nucifera  is a hard-shelled fruit [8]. C. nucifera for treating cancer, which is one of the most important development progresses from leafy to fruition. It is an en- diseases today. The coconut ( Cocos nucifera) plant, which docarp in a third layer other than the exocarp and meso- we have chosen as a phytotherapy agent, has been used carp that make up the shell [8,9]. C. nucifera is very rich in many cancer studies [4,5].  Milk obtained from Cocos in protein, fat, carbohydrates, and fiber. It contains pan - nucifera fibers may be a source of new drugs with antitu - tothenic acid, pyridoxine, riboflavin, niacin, and thiamin, mor and anti-multidrug resistance activities. They tested as well as vitamins C, E, and K. C. nucifera, which is rich different forms of C. nucifera milk on the human erythro- in minerals, is first given sodium and potassium; it con - leukemia cell lines K562 and Lucena-1. They wrote that C. tains elements such as iron, calcium, copper, phosphorus, nucifera milk reduced multiple resistance and cell viability selenium, zinc, magnesium, and manganese. Additionally, by half in the Lucena-1 cell line [4]. The anticancer effect many other nutritional components beneficial for health of tripalmitin and trilaurine glycerides in C. nucifera oil are also included in its structure [10]. is known. Antioxidant-rich polyphenol compounds in C. The clear, sweet, and slightly acidic liquid in the inte - nucifera oil have anti-inflammatory and antioxidant effects rior of a  C. nucifera  (in the endocarp section) is referred in colorectal cancers, and provide improvements, thus hav- to as “Cocos nucifera juice.” This is considered by many to ing an anticancer effect [6]. be the “perfect drink” for its refreshing nature and natural- ness. C. nucifera water is sterile and has not been hit or in- jured. It is a ready-made source of clean drinking water in * Correspondence to: Gulcin Alp Avci E-mail: alp.gulcin@yahoo.com any possible case. Its sodium and potassium content make 46 Acta Marisiensis - Seria Medica 2023;69(1) it an ideal drink for rehydration. Owing to the nutritional yltiyazole 2-il)-2.5 diphenyltetrazilium bromide (MTT) properties of the ingredients contained in C. nucifera  wa- colorimetric analysis. For this purpose, 96 wells plates were ter, it has antioxidant, hepatoprotective, antifungal, anti- used and the experiment was carried out in a total volume microbial, antiviral, and anticancer effects [11,14]. C. nu- of 200 µL. 5x10 cells were cultured in each well of the cifera  milk is an opaque, milk-white liquid derived from 96 plates. DMEM and cells were used as positive controls, the grated meat of mature C. nucifera. The efficiency and while only DMEM was used as a negative control. C. nu- quality of C. nucifera milk from C. nucifera meat depends cifera  inner water, extract and milk concentrations were on the temperature of the added water the water ratio added as 500-250-125-62.5-31.25-15.62-7.81 mg/ml, [15]. C. nucifera milk is an emulsion that mainly contains and incubated for 24 and 48 h. The liquid in the incuba - lipid, carbohydrates, and proteins. C. nucifera oil is a type tion wells was aspirated (50 µL /well), followed by the cells’ of oil derived from the seed, succulent tissues, and milk of processing for 3 h at 37°C with MTT (Sigma Aldrich) so- the plant [12]. Its content is quite rich, and its use is very lution (10 µL 5 mg/mL PBS-phosphate-buffered saline). common. This study determined the antioxidant activity Finally, the cells were broken down with 100 µL DMSO and in vitro cytotoxic effects on Hepatocellular Carcinoma (Dimethyl sulfoxide). Absorbance was measured at 570 (HepG-2) cells and the Mouse Fibroblast (L929) cell line nm using an ELISA (Enzyme-Linked ImmunoSorbent of C. nucifera. Assay) microplate reader. The percentage of living cells determined by the equation % viability = (absorbing pro- Methods cessed cells/absorbing control cells) ×100 was determined. IC cell growth, the sample amount that provides 50% Preparation of C. nucifera inhibition, was calculated from a dose-response curve. The Four C. nucifera were used the study. A hole was made in cytotoxic effect of  C. nucifera internal water, extracts, and each C. nucifera and the inner water was poured into a controls was evaluated by comparing the IC values of sterile container. C. nucifera, whose juice was filtered, was samples for all cell lines. divided into two parts, and the fleshy part was separated from the shell part. C. nucifera milk was obtained from Determination of Antioxidant Activity these fragments (Figure 1). Total antioxidant capacity measurement The ELISA method was used to determine the antioxidant Cell Culture Procedure capacity of the samples. The data were determined by a Hepatocellular Carcinoma (HepG-2) cells and Mouse Fi- spectrophotometer reading at 600 nm. broblast (L929) cells were used in my study. Cells were cul- tured in Dulbecco’s Modified Eagles Medium (DMEM) DPPH (1,1-diphenyl 2-picril hydrazil) free radical sweeper supplemented with 10% Fetal Bovine Serum (FBS), 20µg/ activity determination ml penicillin/streptomycin (100 UI), and 1% L-glutamine DPPH• free radical removal activity was carried out ac- at 37 C in 5% CO cording to a variant of the Blois method (1958). DPPH (1.1-Diphenyl-2-picrylhydrazyl radical) was calculated MTT Colorimetric Test and weighed as 0.1 mM. The weighed amount was dis - The cytotoxic activity was assessed using 3-(4,5 dimeth- solved in ethanol and stored at +4 C in the dark. It was Fig. 1. Preparation of Cocos nucifera Acta Marisiensis - Seria Medica 2023;69(1) 47 applied to samples at twice the rate, and the sample was varies according to the water and milk it contains. We ob- incubated in the dark for 30 min at 37 C and measured at served that the concentration of the active substance ap- 517 nm. After the first measurement, it was incubated in plied to the cells changed with time. The highest vitality o th the dark at 37 C for another 30 min and measured at 517 was determined at the 24 hour with 191.37%, while the nm (Figure 2). lowest was determined at the 48th hour with 77.12%. It was discovered that the essence of C. nucifera on Statistical analysis HepG-2 hepatocellular carcinoma cells produced the best In the study, the data was presented as a mean and standard results of 250 mg/mL in 24 and 48 h. The best viability of deviation. The cell analysis data were calculated with the the inland water effect on cells was determined at 7.81 mg/ Excel (2016) program and assigned statistical importance. ml in 24 h and 62.5 mg/ml in 48 h. The percentage of the The IC and EC (half the highest inhibitory concen- vitality of C. nucifera milk in cells was 15.62 mg/ml and 50 50 tration-half the maximally effective concentration) values 250 mg/ml respectively, in 24 and 48 h (Figure 4). were determined with the GraphPad Prism (9.0) program. Three versions of  C. nucifera were used in the experi- ments, and according to the results of the 24-hour appli- Results and discussion cation on the HepG-2 cell line, known as the cancerous C. nucifera oil was found to reduce liver damage, regenera- cell line, the average order of their vitality was CNS   > te hepatocytes, increase apoptosis, and, most importantly, CNS  > CNs  > CNS > CNS  >CNÖ  the cytotoxic- 24 48 24  24 48, reduce cancerous cells in studies on mice with liver damage ity ranking in IC values is CNS   > CNS   > CNS   > 50  24 24 48 and hepato-steatosis [16,17]. In studies like these, many CNS  > CNS  > CN milk (Table 1). 48 24 48  types of cancer have been studied. The oral cancer cell line The C. nucifera extract used in this study has a high oil KB and liver cell line HepG-2 emphasized that the effect content, and although it varies depending on the concen- was not uniform and varied in the study treatment with tration difference in the HepG-2 cell line, like the other different concentrations of pure C. nucifera oil, processed studies, significant results were found in terms of effect. C. nucifera oil and fractionated C. nucifera oil for 72 h. It C. nucifera juice has been evaluated in various liver cancer is stated in the literature that all oil types have suppressive studies. In some studies, it cannot directly prevent liver effects against two types of cancer, albeit at different rates cancer but can reduce the damage [19]. It has been de- and percentages [18]. The cytotoxic effects and antioxidant termined that it delays the progression of breast cancer by activity of C. nucifera at various concentrations on HepG- inducing apoptosis, suppressing metastasis, and activating 2 were investigated in cell culture studies, but L929 was antitumor immunity in the 4T1 breast cancer cell line. not as healthy as the control. The antioxidant activity and This study was conducted considering that, some concen - cytotoxic effects on HepG-2 cells and the L929 cell line of trations showed an effect [20]. The effect of C. nucifera C. nucifera were determined in our study. milk on cancer has not been evaluated in studies. And vari- In L929 cells, the best viability percentage for  C. nu- ous concentrations of C. nucifera milk were applied to the cifera  milk was determined at 250 mg/ml within 24 and HepG-2 hepatocellular carcinoma cell line, which was pre- 48 h. The concentration of C. nucifera internal water was pared in a concentrated form with water, and to the mouse 31.25 mg/ml after a 24 h treatment and 500 mg/ml after fibroblast cell line, L929, which was determined to be the a 48 h treatment..  The best viability  percentage was de - control. As a result, after the application, it was observed termined at 125 mg/ml in 48 h and 500 mg/ml in 24 h that C. nucifera milk and extract could be used as a good (Figure 3). activator in L929 cells, while it was also observed that it When the proliferation effect of C. nucifera in the L929 supported growth in HepG-2 hepatocellular carcinoma cell line is examined according to the average viability, it cells at some doses and suppressed it at others. Fig. 2. DPPH standard graphic 48 Acta Marisiensis - Seria Medica 2023;69(1) Fig. 4. Results of % viability in HepG-2 hepatocellular carcinoma Fig. 3. % Viability results in L929 Mouse Fibroblast cells of cells of A. Cocos nucifera extract; B. Cocos nucifera juice; A. Extract of Cocos nucifera; B. inland water of Cocos nucifera; C. Cocos nucifera milk. C. milk of Cocos nucifera Table 1. C. nucifera effect on cytotoxic Hepatocellular Carcinoma HepG-2 cells   Extract Inner water Milk   24 h 48 h 24 48 24 48 IC ±SD (μg/ml) 63.49±8.21 93.13±9.53 93.13±19.53 96.37±33.55 16.35±17.71 71.96±19.70 Table 2. The effect of C. nucifera on mouse fibroblast L929 cells   Extract Inner water Milk   24 h 48 h 24 48 24 48 IC ±SD (μg/ml) 99.6±10.7 137.2±12.0 39.0±19.4 97.8±12.6 132.6±14.38 7.8±5.6 Table 3. Antioxidant results and evaluation of C. nucifer (extract, inner water, and milk) Examples mmol/L  assessment C. nucifera extract 2.79 Very good C. nucifera milk 0.94 The very low antioxidant level C. nucifera inner water 1.91 normal The results of the substances that were made and mea - The DPPH activity results of the samples were obtained sured according to the procedure of the total antioxidant and compared with the antioxidant BHT (Butyl Hydroxy capacity measurement kit (Rel Assay Diagnostic, Turkiye), Toluene) and evaluated using the standards. It was found were interpreted based on the procedural reference values. that they have antioxidant values of C. nucifera extract The antioxidant effects of  C. nucifera were found in water, 21.40 mg/ml, C. nucifera internal water 22.04 mg/ml, and extract, and milk. Their levels were evaluated (Table 3). C. nucifera milk 22.14 mg/ml. According to the compar- The results are consistent, considering the studies. ison between them, the best activity was detected in C. Acta Marisiensis - Seria Medica 2023;69(1) 49 5. Enos RT, Velázquez KT, McClellan JL, et al. High-fat diets rich in nucifera milk, while the lowest activity was found in C. nu- saturated fat protect against azoxymethane/dextran sulfate sodium- cifera extract. Supporting the antioxidant data in the study, induced colon cancer. American Journal of Physiology-Gastrointestinal and Liver Physiology. 2016;310(11): 906-919. Nevin and Rajamohan in their 2004 study determined 6. Illam SP, Narayanankutty A, Raghavamenon AC. Polyphenols of virgin that pure C. nucifera oil decreased total cholesterol, tri- Coconut oil prevent pro-oxidant-mediated cell death.  Toxicology mechanisms and methods. 2017; 27(6): 442-450. glycerides, phospholipids, low-density lipoprotein (LDL), 7. Elevitch CR, Manner HI. Artocarpus heterophyllus (Jackfruit). In: Species very-low-density lipoprotein (VLDL), and increased high- profiles for pacific island agroforestry: ecological, economic, and cultural density lipoprotein (HDL) cholesterol levels [12]. Addi- renewal. Agroforestry Net, Inc. Hawaii, USA, 2006,1-17. 8. Carr Mkv. The Water Relations and Irrigation Requirements of Coconut tionally, many studies support these results [21, 22]. (Cocos Nucifera): A Review. Experimental Agriculture. Cambridge University Press. 2011;47(1):27–51. Conclusion 9. Cheesman EE. Tropical crops: Monocotyledons Vols. 1 and 2, By J. W. Purseglove London: Longman (1972), pp. 334 and 273, £6.00 In line with the data we obtained, it is thought that C. and £5.00. Experimental Agriculture. Cambridge University Press; nucifera can be used in liver cancer studies, and its antioxi- 1973;9(3):287–. dant effect may play an important role in balancing against 10. Yong JW, Ge L, Ng YF, Tan SN. The chemical composition and biological properties of coconut (Cocos nucifera L.) water. Molecules. oxidative stress. Simultaneously, the data show that the ex- 2009;14(12):5144–5164 posure time and concentrations of the active substance are 11. Loki AL, Rajamohan T. Hepatoprotective and antioxidant effects of tender coconut water on carbon tetrachloride-induced liver injury in rats. related to the cytotoxic effect. However, it may be consid - Indian J Biochem Biophys. 2003;40(5):354-357. ered that the use of C. nucifera water, extract, and milk in 12. DebMandal M, Mandal S. Coconut (Cocos nucifera L.: Arecaceae): cancer patients may be supported by more comprehensive in health promotion and disease prevention. Asian Pac J Trop Med. 201;4(3):241-247.  clinical studies. 13. Lima EB, Sousa CN, Meneses LN, et al. Cocos nucifera (L.) (Arecaceae): A phytochemical and pharmacological review. Braz J Med Biol Res. 2015;48(11):953-964. Author contributions 14. Asghar MT, Yusof YA, Mokhtar MN, et al. Coconut (Cocos nucifera L.) GAA - Conceptualization, Data curation, Formal Analysis, sap as a potential source of sugar: Antioxidant and nutritional properties. Funding acquisition, Investigation, Methodology, Project Food Sci Nutr. 201930;8(4):1777–1787. 15. Patil U, Benjakul S. Coconut Milk, and Coconut Oil: Their Manufacture administration, Resources, Software, Supervision, Valida- Associated with Protein Functionality. J Food Sci. 2018;83(8):2019– tion, Visualization, Writing – original draft. ZT - Conceptualization, Data curation, Methodology, 16. Enos RT, Velázquez KT, McClellan JL, et. al. High-fat diets rich in saturated fat protect against azoxymethane/dextran sulfate sodium- Visualization, Investigation. induced colon cancer. Am J Physiol Gastrointest Liver Physiol. EA - Conceptualization, Data curation, Formal Analysis, 2016;310(11):906-919. Investigation, Methodology, Resources, Validation, Writ- 17. Narayanankutty A, Palliyil DM, Kuruvilla K, Raghavamenon AC. Virgin coconut oil reverses hepatic steatosis by restoring redox ing – original draft. homeostasis and lipid metabolism in male Wistar rats. J Sci Food Agric. 2018;98(5):1757–1764. 18. Verma P , Naik S, Nanda P , Banerjee S, Naik S, Ghosh A. İn Vitro Anticancer Conflict of interest Activity of Virgin Coconut Oil and its Fractions in Liver and Oral Cancer None to declare. Cells. Anticancer Agents Med Chem. 2019;19(18):2223–2230. 19. Salil G, Nevin KG, Rajamohan T. Arginine-rich coconut kernel protein modulates diabetes in alloxan-treated rats. Chem Biol Interact. References 2011;189(1-2):107-111. 1. Heinrich M, Barnes J, Gıbbons S, Williamson EM. Fundamentals of 20. Mohamad NE, Yeap SK, Abu N, et. al.  İn vitro  and  in vivo  antitumor Pharmacognosy and Phytotherapy. 3rd Edt., Churchill Livingstone, effects of coconut water vinegar on 4T1 breast cancer cells. Food Nutr. Edinburgh, 2004, 10-23. Res. 2019; 10:63-65. 2. Ötnü H, Akan H. Şanlıurfa’daki Eczanelerde ve Aktarlarda Fitoterapi 21. Nadeeshani R, Wijayaratna UN, Prasadani WC, Ekanayake S, Amaçlı Satılan Bitkiler. Kahramanmaraş Sütçü İmam Üniversitesi Tarım Seneviratne KN, Jayathilaka, N. Comparison of the basic nutritional ve Doğa Dergisi, 2020; 23(4): 947–965. characteristics of the first extract and second extract of coconut milk. 3. Ergün G, Şahin Z, Kara İ. Complementary Herbal Treatments in Dentistry. International Journal of Innovative Research in Science, Engineering and Akdeniz Medical Journal 2020;6(2): 145-153. Technology. 2015; 4 (10): 9516-9521. 4. Kaman D. İstanbul Eczaneleri Ve Aktarlarından Temin Edilen Cocos 22. Nevin KG, Rajamohan T. Influence of virgin coconut oil on blood nucifera (L.) Yağı Üzerine Farmakope Araştırmaları. Yüksek Lisans Tezi, coagulation factors, lipid levels and LDL oxidation in cholesterol-fed İstanbul Üniversitesi Sağlık Bilimleri Enstitüsü Farmakognozi Anabilim Sprague–Dawley rats e-SPEN Eur. e-J. Cli. Nutr. Meta. 2008;3(1):1-8. Dalı Fitoterapi Yüksek Lisans Programı, İstanbul, 2019, 1-145. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Marisiensis - Seria Medica de Gruyter

Evaluation of effects on hepatocellular carcinoma cell line of Cocos nucifera: In vitro study

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Acta Marisiensis - Seria Medica 2023;69(1):45-49 DOI: 10.2478/amma-2023-0004 RESEARCH ARTICLE Evaluation of effects on hepatocellular carcinoma cell line of Cocos nucifera: In vitro study 1 2* 3 Zeynep Tasdan , Gülçin Alp Avci , Emre Avci 1. Department of Molecular Biology and Genetics, University of Hitit, Corum, Türkiye 2. Department of Basic Medical Sciences, University of Health Sciences, Ankara, Türkiye 3. Department of Biochemistry, University of Health Sciences, Ankara, Türkiye Objective: Cancer is one of the most important diseases today. The use of chemical drugs, surgical operations, and transplants is very common in its treatment. In addition to these treatment methods, studies include the effects of natural and plant-derived substances. Vari- ous substances are used in these studies, which are called phytotherapy. The antioxidant activity and cytotoxic effects of Cocos nucifera on hepatocellular carcinoma HepG-2 and the mouse fibroblast L929 cell line were investigated in this study. Methods: In this study, in vitro cyto- toxic effects of C. nucifera at different concentrations (7.81-500 mg/ml) were investigated on the L929 Mouse Fibroblast cell line and HepG-2 Hepatocellular Carcinoma cell line. In addition to these studies, their antioxidant capacity was evaluated via spectrophotometric methods. In this work, different concentrations of C. nucifera were examined. Results: According to the results, C. nucifera had a cytotoxic effect in HepG-2 and ensured the proliferative effect of cells in the L929 cell line. Among C. nucifera extracts according to total antioxidant capacity results, C. nucifera extract was found to be the richest in antioxidants with 2.79 mmol/L, while the material with the lowest antioxidant capacity was determined to be C. nucifera milk. DPPH free radical scavenging activity results show the opposite. Conclusion: In line with the data we obtained, it is thought that C. nucifera can be used in liver cancer studies, and its antioxidant effect may play an important role in balancing against oxidative stress. Simultaneously, the data show that the exposure time and concentrations of the active substance are related to the cytotoxic effect. However, it may be considered that the use of C. nucifera water, extract, and milk in cancer patients may be supported by more comprehensive clinical studies. Keywords: Cocos nucifera, HepG-2, L929, cytotoxicity, antioxidant activity Received 9 September 2022 / Accepted 1 February 2023 Introduction Coconut (Cocos nucifera), which belongs to the  Areca- Today, plants maintain their place in the cosmetics, health, ceae family, is the fruit of the tropics. It has a supportive, pharmaceutical, textile, and various industrial fields. Its use strengthening effect on the defense and intestinal systems in the health and pharmaceutical industries started in an- and is very nutritious. C. nucifera is a product of the tro- cient times, and its history dates to the first ages [1,2,3]. pics. C. nucifera dates grow on sandy soils and are highly Recently, the production of plant-derived drugs has been tolerant of soils with high salt content. It prefers regions increasing in developing countries in the fields of medicine with plenty of sunlight and regular precipitation [7].  C. and biotechnology [3]. The use of plants in treatments over nucifera  is a large palm that reaches up to 30 meters in time is called “Phytotherapy” [2]. Phytotherapy is also used length. C. nucifera  is a hard-shelled fruit [8]. C. nucifera for treating cancer, which is one of the most important development progresses from leafy to fruition. It is an en- diseases today. The coconut ( Cocos nucifera) plant, which docarp in a third layer other than the exocarp and meso- we have chosen as a phytotherapy agent, has been used carp that make up the shell [8,9]. C. nucifera is very rich in many cancer studies [4,5].  Milk obtained from Cocos in protein, fat, carbohydrates, and fiber. It contains pan - nucifera fibers may be a source of new drugs with antitu - tothenic acid, pyridoxine, riboflavin, niacin, and thiamin, mor and anti-multidrug resistance activities. They tested as well as vitamins C, E, and K. C. nucifera, which is rich different forms of C. nucifera milk on the human erythro- in minerals, is first given sodium and potassium; it con - leukemia cell lines K562 and Lucena-1. They wrote that C. tains elements such as iron, calcium, copper, phosphorus, nucifera milk reduced multiple resistance and cell viability selenium, zinc, magnesium, and manganese. Additionally, by half in the Lucena-1 cell line [4]. The anticancer effect many other nutritional components beneficial for health of tripalmitin and trilaurine glycerides in C. nucifera oil are also included in its structure [10]. is known. Antioxidant-rich polyphenol compounds in C. The clear, sweet, and slightly acidic liquid in the inte - nucifera oil have anti-inflammatory and antioxidant effects rior of a  C. nucifera  (in the endocarp section) is referred in colorectal cancers, and provide improvements, thus hav- to as “Cocos nucifera juice.” This is considered by many to ing an anticancer effect [6]. be the “perfect drink” for its refreshing nature and natural- ness. C. nucifera water is sterile and has not been hit or in- jured. It is a ready-made source of clean drinking water in * Correspondence to: Gulcin Alp Avci E-mail: alp.gulcin@yahoo.com any possible case. Its sodium and potassium content make 46 Acta Marisiensis - Seria Medica 2023;69(1) it an ideal drink for rehydration. Owing to the nutritional yltiyazole 2-il)-2.5 diphenyltetrazilium bromide (MTT) properties of the ingredients contained in C. nucifera  wa- colorimetric analysis. For this purpose, 96 wells plates were ter, it has antioxidant, hepatoprotective, antifungal, anti- used and the experiment was carried out in a total volume microbial, antiviral, and anticancer effects [11,14]. C. nu- of 200 µL. 5x10 cells were cultured in each well of the cifera  milk is an opaque, milk-white liquid derived from 96 plates. DMEM and cells were used as positive controls, the grated meat of mature C. nucifera. The efficiency and while only DMEM was used as a negative control. C. nu- quality of C. nucifera milk from C. nucifera meat depends cifera  inner water, extract and milk concentrations were on the temperature of the added water the water ratio added as 500-250-125-62.5-31.25-15.62-7.81 mg/ml, [15]. C. nucifera milk is an emulsion that mainly contains and incubated for 24 and 48 h. The liquid in the incuba - lipid, carbohydrates, and proteins. C. nucifera oil is a type tion wells was aspirated (50 µL /well), followed by the cells’ of oil derived from the seed, succulent tissues, and milk of processing for 3 h at 37°C with MTT (Sigma Aldrich) so- the plant [12]. Its content is quite rich, and its use is very lution (10 µL 5 mg/mL PBS-phosphate-buffered saline). common. This study determined the antioxidant activity Finally, the cells were broken down with 100 µL DMSO and in vitro cytotoxic effects on Hepatocellular Carcinoma (Dimethyl sulfoxide). Absorbance was measured at 570 (HepG-2) cells and the Mouse Fibroblast (L929) cell line nm using an ELISA (Enzyme-Linked ImmunoSorbent of C. nucifera. Assay) microplate reader. The percentage of living cells determined by the equation % viability = (absorbing pro- Methods cessed cells/absorbing control cells) ×100 was determined. IC cell growth, the sample amount that provides 50% Preparation of C. nucifera inhibition, was calculated from a dose-response curve. The Four C. nucifera were used the study. A hole was made in cytotoxic effect of  C. nucifera internal water, extracts, and each C. nucifera and the inner water was poured into a controls was evaluated by comparing the IC values of sterile container. C. nucifera, whose juice was filtered, was samples for all cell lines. divided into two parts, and the fleshy part was separated from the shell part. C. nucifera milk was obtained from Determination of Antioxidant Activity these fragments (Figure 1). Total antioxidant capacity measurement The ELISA method was used to determine the antioxidant Cell Culture Procedure capacity of the samples. The data were determined by a Hepatocellular Carcinoma (HepG-2) cells and Mouse Fi- spectrophotometer reading at 600 nm. broblast (L929) cells were used in my study. Cells were cul- tured in Dulbecco’s Modified Eagles Medium (DMEM) DPPH (1,1-diphenyl 2-picril hydrazil) free radical sweeper supplemented with 10% Fetal Bovine Serum (FBS), 20µg/ activity determination ml penicillin/streptomycin (100 UI), and 1% L-glutamine DPPH• free radical removal activity was carried out ac- at 37 C in 5% CO cording to a variant of the Blois method (1958). DPPH (1.1-Diphenyl-2-picrylhydrazyl radical) was calculated MTT Colorimetric Test and weighed as 0.1 mM. The weighed amount was dis - The cytotoxic activity was assessed using 3-(4,5 dimeth- solved in ethanol and stored at +4 C in the dark. It was Fig. 1. Preparation of Cocos nucifera Acta Marisiensis - Seria Medica 2023;69(1) 47 applied to samples at twice the rate, and the sample was varies according to the water and milk it contains. We ob- incubated in the dark for 30 min at 37 C and measured at served that the concentration of the active substance ap- 517 nm. After the first measurement, it was incubated in plied to the cells changed with time. The highest vitality o th the dark at 37 C for another 30 min and measured at 517 was determined at the 24 hour with 191.37%, while the nm (Figure 2). lowest was determined at the 48th hour with 77.12%. It was discovered that the essence of C. nucifera on Statistical analysis HepG-2 hepatocellular carcinoma cells produced the best In the study, the data was presented as a mean and standard results of 250 mg/mL in 24 and 48 h. The best viability of deviation. The cell analysis data were calculated with the the inland water effect on cells was determined at 7.81 mg/ Excel (2016) program and assigned statistical importance. ml in 24 h and 62.5 mg/ml in 48 h. The percentage of the The IC and EC (half the highest inhibitory concen- vitality of C. nucifera milk in cells was 15.62 mg/ml and 50 50 tration-half the maximally effective concentration) values 250 mg/ml respectively, in 24 and 48 h (Figure 4). were determined with the GraphPad Prism (9.0) program. Three versions of  C. nucifera were used in the experi- ments, and according to the results of the 24-hour appli- Results and discussion cation on the HepG-2 cell line, known as the cancerous C. nucifera oil was found to reduce liver damage, regenera- cell line, the average order of their vitality was CNS   > te hepatocytes, increase apoptosis, and, most importantly, CNS  > CNs  > CNS > CNS  >CNÖ  the cytotoxic- 24 48 24  24 48, reduce cancerous cells in studies on mice with liver damage ity ranking in IC values is CNS   > CNS   > CNS   > 50  24 24 48 and hepato-steatosis [16,17]. In studies like these, many CNS  > CNS  > CN milk (Table 1). 48 24 48  types of cancer have been studied. The oral cancer cell line The C. nucifera extract used in this study has a high oil KB and liver cell line HepG-2 emphasized that the effect content, and although it varies depending on the concen- was not uniform and varied in the study treatment with tration difference in the HepG-2 cell line, like the other different concentrations of pure C. nucifera oil, processed studies, significant results were found in terms of effect. C. nucifera oil and fractionated C. nucifera oil for 72 h. It C. nucifera juice has been evaluated in various liver cancer is stated in the literature that all oil types have suppressive studies. In some studies, it cannot directly prevent liver effects against two types of cancer, albeit at different rates cancer but can reduce the damage [19]. It has been de- and percentages [18]. The cytotoxic effects and antioxidant termined that it delays the progression of breast cancer by activity of C. nucifera at various concentrations on HepG- inducing apoptosis, suppressing metastasis, and activating 2 were investigated in cell culture studies, but L929 was antitumor immunity in the 4T1 breast cancer cell line. not as healthy as the control. The antioxidant activity and This study was conducted considering that, some concen - cytotoxic effects on HepG-2 cells and the L929 cell line of trations showed an effect [20]. The effect of C. nucifera C. nucifera were determined in our study. milk on cancer has not been evaluated in studies. And vari- In L929 cells, the best viability percentage for  C. nu- ous concentrations of C. nucifera milk were applied to the cifera  milk was determined at 250 mg/ml within 24 and HepG-2 hepatocellular carcinoma cell line, which was pre- 48 h. The concentration of C. nucifera internal water was pared in a concentrated form with water, and to the mouse 31.25 mg/ml after a 24 h treatment and 500 mg/ml after fibroblast cell line, L929, which was determined to be the a 48 h treatment..  The best viability  percentage was de - control. As a result, after the application, it was observed termined at 125 mg/ml in 48 h and 500 mg/ml in 24 h that C. nucifera milk and extract could be used as a good (Figure 3). activator in L929 cells, while it was also observed that it When the proliferation effect of C. nucifera in the L929 supported growth in HepG-2 hepatocellular carcinoma cell line is examined according to the average viability, it cells at some doses and suppressed it at others. Fig. 2. DPPH standard graphic 48 Acta Marisiensis - Seria Medica 2023;69(1) Fig. 4. Results of % viability in HepG-2 hepatocellular carcinoma Fig. 3. % Viability results in L929 Mouse Fibroblast cells of cells of A. Cocos nucifera extract; B. Cocos nucifera juice; A. Extract of Cocos nucifera; B. inland water of Cocos nucifera; C. Cocos nucifera milk. C. milk of Cocos nucifera Table 1. C. nucifera effect on cytotoxic Hepatocellular Carcinoma HepG-2 cells   Extract Inner water Milk   24 h 48 h 24 48 24 48 IC ±SD (μg/ml) 63.49±8.21 93.13±9.53 93.13±19.53 96.37±33.55 16.35±17.71 71.96±19.70 Table 2. The effect of C. nucifera on mouse fibroblast L929 cells   Extract Inner water Milk   24 h 48 h 24 48 24 48 IC ±SD (μg/ml) 99.6±10.7 137.2±12.0 39.0±19.4 97.8±12.6 132.6±14.38 7.8±5.6 Table 3. Antioxidant results and evaluation of C. nucifer (extract, inner water, and milk) Examples mmol/L  assessment C. nucifera extract 2.79 Very good C. nucifera milk 0.94 The very low antioxidant level C. nucifera inner water 1.91 normal The results of the substances that were made and mea - The DPPH activity results of the samples were obtained sured according to the procedure of the total antioxidant and compared with the antioxidant BHT (Butyl Hydroxy capacity measurement kit (Rel Assay Diagnostic, Turkiye), Toluene) and evaluated using the standards. It was found were interpreted based on the procedural reference values. that they have antioxidant values of C. nucifera extract The antioxidant effects of  C. nucifera were found in water, 21.40 mg/ml, C. nucifera internal water 22.04 mg/ml, and extract, and milk. Their levels were evaluated (Table 3). C. nucifera milk 22.14 mg/ml. According to the compar- The results are consistent, considering the studies. ison between them, the best activity was detected in C. Acta Marisiensis - Seria Medica 2023;69(1) 49 5. Enos RT, Velázquez KT, McClellan JL, et al. High-fat diets rich in nucifera milk, while the lowest activity was found in C. nu- saturated fat protect against azoxymethane/dextran sulfate sodium- cifera extract. Supporting the antioxidant data in the study, induced colon cancer. American Journal of Physiology-Gastrointestinal and Liver Physiology. 2016;310(11): 906-919. Nevin and Rajamohan in their 2004 study determined 6. Illam SP, Narayanankutty A, Raghavamenon AC. Polyphenols of virgin that pure C. nucifera oil decreased total cholesterol, tri- Coconut oil prevent pro-oxidant-mediated cell death.  Toxicology mechanisms and methods. 2017; 27(6): 442-450. glycerides, phospholipids, low-density lipoprotein (LDL), 7. Elevitch CR, Manner HI. Artocarpus heterophyllus (Jackfruit). In: Species very-low-density lipoprotein (VLDL), and increased high- profiles for pacific island agroforestry: ecological, economic, and cultural density lipoprotein (HDL) cholesterol levels [12]. Addi- renewal. Agroforestry Net, Inc. Hawaii, USA, 2006,1-17. 8. Carr Mkv. The Water Relations and Irrigation Requirements of Coconut tionally, many studies support these results [21, 22]. (Cocos Nucifera): A Review. Experimental Agriculture. Cambridge University Press. 2011;47(1):27–51. Conclusion 9. Cheesman EE. Tropical crops: Monocotyledons Vols. 1 and 2, By J. W. Purseglove London: Longman (1972), pp. 334 and 273, £6.00 In line with the data we obtained, it is thought that C. and £5.00. Experimental Agriculture. Cambridge University Press; nucifera can be used in liver cancer studies, and its antioxi- 1973;9(3):287–. dant effect may play an important role in balancing against 10. Yong JW, Ge L, Ng YF, Tan SN. The chemical composition and biological properties of coconut (Cocos nucifera L.) water. Molecules. oxidative stress. Simultaneously, the data show that the ex- 2009;14(12):5144–5164 posure time and concentrations of the active substance are 11. Loki AL, Rajamohan T. Hepatoprotective and antioxidant effects of tender coconut water on carbon tetrachloride-induced liver injury in rats. related to the cytotoxic effect. However, it may be consid - Indian J Biochem Biophys. 2003;40(5):354-357. ered that the use of C. nucifera water, extract, and milk in 12. DebMandal M, Mandal S. Coconut (Cocos nucifera L.: Arecaceae): cancer patients may be supported by more comprehensive in health promotion and disease prevention. Asian Pac J Trop Med. 201;4(3):241-247.  clinical studies. 13. Lima EB, Sousa CN, Meneses LN, et al. Cocos nucifera (L.) (Arecaceae): A phytochemical and pharmacological review. Braz J Med Biol Res. 2015;48(11):953-964. Author contributions 14. Asghar MT, Yusof YA, Mokhtar MN, et al. Coconut (Cocos nucifera L.) GAA - Conceptualization, Data curation, Formal Analysis, sap as a potential source of sugar: Antioxidant and nutritional properties. Funding acquisition, Investigation, Methodology, Project Food Sci Nutr. 201930;8(4):1777–1787. 15. Patil U, Benjakul S. Coconut Milk, and Coconut Oil: Their Manufacture administration, Resources, Software, Supervision, Valida- Associated with Protein Functionality. J Food Sci. 2018;83(8):2019– tion, Visualization, Writing – original draft. ZT - Conceptualization, Data curation, Methodology, 16. Enos RT, Velázquez KT, McClellan JL, et. al. High-fat diets rich in saturated fat protect against azoxymethane/dextran sulfate sodium- Visualization, Investigation. induced colon cancer. Am J Physiol Gastrointest Liver Physiol. EA - Conceptualization, Data curation, Formal Analysis, 2016;310(11):906-919. Investigation, Methodology, Resources, Validation, Writ- 17. Narayanankutty A, Palliyil DM, Kuruvilla K, Raghavamenon AC. Virgin coconut oil reverses hepatic steatosis by restoring redox ing – original draft. homeostasis and lipid metabolism in male Wistar rats. J Sci Food Agric. 2018;98(5):1757–1764. 18. Verma P , Naik S, Nanda P , Banerjee S, Naik S, Ghosh A. İn Vitro Anticancer Conflict of interest Activity of Virgin Coconut Oil and its Fractions in Liver and Oral Cancer None to declare. Cells. Anticancer Agents Med Chem. 2019;19(18):2223–2230. 19. Salil G, Nevin KG, Rajamohan T. Arginine-rich coconut kernel protein modulates diabetes in alloxan-treated rats. Chem Biol Interact. 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Journal

Acta Marisiensis - Seria Medica – de Gruyter

Published: Mar 1, 2023

Keywords: Cocos nucifera; HepG-2; L929; cytotoxicity; antioxidant activity

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