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
Zeynep Tasdan1, Gülçin Alp Avci2*, Emre Avci3
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-
* Correspondence to: Gulcin Alp Avci jured. It is a ready-made source of clean drinking water in
E-mail: [Link]@[Link]
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. 5x103 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.
IC50 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 IC50 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 37oC in 5% CO2. 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 oC 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 37oC 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
the dark at 37o C for another 30 min and measured at 517 was determined at the 24th 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 IC50 and EC50 (half the highest inhibitory concen- vitality of C. nucifera milk in cells was 15.62 mg/ml and
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 CNS48 >
te hepatocytes, increase apoptosis, and, most importantly, CNS24 > CNs48 > CNS24 > CNS24 >CNÖ48, the cytotoxic-
reduce cancerous cells in studies on mice with liver damage ity ranking in IC50 values is CNS24 > CNS24 > CNS48 >
and hepato-steatosis [16,17]. In studies like these, many CNS48 > CNS24 > CN milk48 (Table 1).
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. 3. % Viability results in L929 Mouse Fibroblast cells of Fig. 4. Results of % viability in HepG-2 hepatocellular carcinoma
A. Extract of Cocos nucifera; B. inland water of Cocos nucifera; cells of A. Cocos nucifera extract; B. Cocos nucifera juice;
C. milk of Cocos nucifera C. Cocos nucifera milk.

Table 1. C. nucifera effect on cytotoxic Hepatocellular Carcinoma HepG-2 cells

Extract Inner water Milk
24 h 48 h 24 48 24 48
IC50±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
IC50±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

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