evaluation of cytotoxicity of sodium benzoate and fresh ... · green chili pepper (capsicum spp.)...

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( Received 25 August 2018; Accepted 11 September 2018; Date of Publication 12 September 2018 )

WSN 110 (2018) 1-12 EISSN 2392-2192

Evaluation of cytotoxicity of sodium benzoate and fresh and boiled green chili pepper (Capsicum

annuum L.) on human red blood cells

Klaudia Kopczyńska1, Katarzyna Król1, Alicja Ponder1,*, Ewelina Hallmann1,

Erni Erniati2, Franziska Zakaria Rungkat2

1SGGW, Faculty of Human Nutrition and Consumer Sciences, Department of Functional, Organic Food and Commodities,

159c Nowoursynowska Str., 02-776 Warsaw, Poland

2Fateta IPB, Faculty of Agricultural Technology, Jl. Raya Dramaga, Kampus IPB Dramaga Bogor, 16680 West Java, Indonesia

*E-mail address: alicja_ponder@sggw.pl

ABSTRACT

Peppers (Capsicum spp.) are a rich source of different bioactive compounds with potential

health-promoting properties. Pepper fruits have numerous uses in culinary preparations that make this

one of the most important vegetables and spices. This study investigated the evaluate the antioxidant

activity of green chili pepper extracts on human red blood cells by comparison of different extraction

method and treatments. Two solvents (water and methanol) was used to extract from green pepper

compounds (fresh and after boiling process). The cytotoxic effect of sodium benzoate and fresh and

boiled green chili pepper on human red blood cells was investigated. The obtained results show the

protective effect of the extract from hot chili peppers on the negative effect of sodium benzoate. This

effect was significantly dependent (p

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1. INTRODUCTION

Pepper (Capsicum annuum L.) belongs to the Solanaceae family and it is widely

cultivated in Asia, Africa, and Mediterranean countries. C. annuum fruits have numerous uses

in culinary preparations that make this one of the most important vegetables and spice [18].

There are many varieties of pepper which are characterized by different sizes and shapes.

Their maturation process is distinct and for human consumption their fruits are collected in

different stages of maturation. The green fruits which are immature and the red ones (full

mature) are the most used in culinary preparations. Depending on the flavor intensity and

texture, the culinary application changes from as a spice (hot chili pepper) as well as

vegetable (sweet pepper or bell pepper). More than 30 different pigments have been identified

in pepper fruits [14]. The color of sweet bell peppers is the major factor associated with

consumer purchasing decisions, green and red fruits being the most consumed in the world-

wide [21].

Figure 1. Pepper (Capsicum annuum L.).

Green chili pepper (Capsicum spp.) is a rich source of diverse bioactive compounds

such as phenols, flavonoids, vitamin C and capsaicinoids with potential health-promoting

properties [2,27]. Phenolic compounds, especially phenolic acids and flavonoids, are

considerably present in vegetables and fruits; thus, they are an integral part of the human diet.

These bioactive compounds have a really strong antioxidant effect and play an

important role in prevention of numerous diseases, such a cancer, ischaemic heart disease.

Furthermore, antioxidant properties were deeply investigated namely in inhibition of lipid

peroxidation [15]. Capsaicinoids and flavonoids have been shown to act as anti-cancer,

antioxidant, anti-inflammatory, anti-viral and anti-bacterial compounds [8,9,11,12].

Furthermore, processing of food (boiling, freezing) has a negative effect on content of

bioactive compounds in pepper [10].

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Figure 2A. Green chili pepper (Capsicum spp.).

Figure 2B. Green chili pepper (Capsicum spp.).

Sodium benzoate is food additives and preservatives commonly used in a different

product, including beverages, fruit juices, jams, sauces, and other preserves. The addition of

food preservatives such as sodium benzoate prevent the growth of bacteria and fungi [13,16].

However, there have been several studies related to synthetic antimicrobial agent like sodium

benzoate which can cause liver damage, mutagenicity and neurotoxicity [7]. Sodium benzoate

in combination with ascorbic acid may form benzene well known as strong carcinogen [17].

Therefore, the aim of the present study was to evaluate the protection property of green

chili pepper extracts on human red blood cells by comparison of different extraction method

and treatments.

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2. MATERIALS AND METHODS

2. 1. Pepper samples and extraction procedures

Fresh green chili pepper (Capsicum annuum L var. annuum) was bought from a local fruit market (Bogor, Indonesia).

Figure 2C. Green chili pepper (Capsicum spp.).

Figure 3. Extraction and filtration of pepper samples.

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Thirty grams of fully mature peppers were analyzed fresh and after boiling process.

Boiling procedure was made by 50 g putting whole fresh peppers in 400 ml of boiling water

for 10 min, blotted dried and used. Methanol and water extract of peppers were used for the

analysis. To extract the compounds soluble in methanol and water, 50 g fresh plant var

annuum and boiled samples were chopped and soaked and mixed in methanol or water at

room temperature. The samples were extracted three times, with fresh methanol (or water)

each time, over period of 3 days. All the extracts were filtered. The combined filtrates were

concentrated on a rotary evaporator (Heidolph, Germany) under vacuum at 40 °C.

2. 2. Preparation of red blood cells (RBC)

This study has received ethical approval from the Research Ethics Committee of Bogor

Agricultural University, Indonesia. RBC were isolated from blood of a healthy volunteer who

signed the informed consent approval forms. The blood was centrifuged at 6500g for 10 min

to separate the erythrocytes. The cell pellets were collected and diluted 20 times with

phosphate-buffered saline (PBS). The cell viability should be 95% living. Cell calculation and

viability was done using hemacytometer and tryphan blue.

Figure 4. Cultures of red blood cells

2. 3. Cytotoxicity assay

Suspension of RBC cells (1×10 6 cells/ml), 80 µL were added in microplate 96 wells

and 20 μl of each extract (in three dilution C1: 0.385 mg/ml, C2: 0.75 mg/ml, C3: 1.25

mg/ml) were added as S1. In separated wells, 60 µL of RBC suspension was added in wells

and 20 μl of each extract and 20 μl sodium benzoate acid (BA) with concentration 10mg/ml

were added (S2). For S3, 80 µL of RBC suspension were added in wells and 20 μl of RPMI

that function as controls. The cultures were incubated at 37 °C and RH 90% for 2h. After 2

hours the cultures were added with 10 μl MTT (3-[4,5-dimetilthiazol-2yl]-2,5-difenil

tetrazolium bromide) at concentration 5 mg / ml. Two hours before the last incubation period,

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to Absorbance value was read using microplate reader at λ = 595 nm. The activity of extract

protection was expressed as the protection index (PI):

PI = absorbnce of sample

absorbance of control× 100% (1)

Figure 5. Distribution of components used in the study.

3. RESULTS AND DISCUSSION

Exposure to chemicals often results in toxicity to living organisms. This is not equal for

all cells. Toxicity of chemical compounds can be different for individual cell components.

Many organs, including the kidney, are capable to decomposition toxic chemicals to

less toxic compounds. The toxicity effects of chemicals in blood can be diminish by

phytochemical (plants substances which plants produced for self-defense). Polyphenols,

flavonoids as well as phenolic acids belong to this group. Using more than one method for

analyses of bioactive compounds gives more statistical significant results. [7]. In this paper

cytotoxicity assay using human red blood cells was used. That method has not been

documented previously [13,24]. In this study two kind of solvents (water and methanol) and

two kinds of chili pepper treatment (fresh and boiled) were used for testing survivability of

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blood cells and calculate for protection index (PI). Results from absorbance measurement are

presented in Table 1.

Table 1. Absorbance value of control and tested samples after 2 and 4 hours incubation.

methanol extract

2 hours incubation

Tested: RBC+E Tested:

RBC+E+BA

Tested:

RBC+BA

Control:

RBC+RPMI

fresh boiled fresh boiled fresh boiled

C1 2.586a 2.154a 2.418a 2.077b 1.474a 2.074b 1.459aA 2.403bA

C2 2.614a 2.254a 2.605a 2.066b 1.665a 2.353b 1.516aA 2.119bA

C3 2.362a 2.440a 2.468a 2.045b 1.254a 2.685b 1.556aA 2.088bA

4 hours incubation

C1 2.757a ***a 2.710a 3.410a 1.547a ***a 0.601aA ***bA

C2 2.012a ***a 3.436a ***a 1.668a ***a 1.633aA ***bA

C3 1.494a ***a 3.058a ***a 1.362a ***a 1.962aA ***bA

water extract

2 hours incubation

C1 3.032a 1.903b 2.943a 1.941b 2.879a 2.074b 2.810aA 2.403bA

C2 2.995a 2.023b 2.894a 2.015b 2.827a 2.163b 2.848aA 2.119bA

C3 3.083a 2.025b 3.001a 2.058b 3.228a 2.091b 2.921aA 2.088bA

4 hours incubation

C1 ***a ***a ***a 3.197 a ***a 3.455a ***aA ***bA

C2 ***a ***a *** a *** a ***a ***a ***aA ***bA

C3 ***a ***a 3.359 a *** a ***a ***a ***aA ***bA

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Means with different superscript capital letter in the same column were significantly different

(p≤0.05). means in different superscript small letter in the same row were significantly

different (p≤0.05).

*** no cell detection.

RBC + E – red blood cells + extract

RBC + E + BA – red blood cells + pepper extract + sodium benzoate

RBC + BA – red blood cells + sodium benzoate

C1 – concentration of pepper extract: 0.375 mg/ml

C2 – concentration of pepper extract: 0.750 mg/ml

RBC + RPMI – red blood cells + RPMI medium

Positive absorbance result means there are still living cells. Basing on results from

Table 1 it can be seen that almost whole cells from every sample were dead after 4 hours

incubation, but they were still alive after 2 hours. Statistical significant differences between

samples from other type of processing after 2 hours and both solvent was obtained (p≤0.05). It

is interesting how absorbance value was changed in RBC + BA samples, what can clearly

show impact of sodium benzoate for blood cells. The obtain data show positive effect of

added extract in protection against sodium benzoate. Although in some case results were

changed rapid (RBC + BA after 4 hours in C1). Results after 2 hours were higher than after 4

hours, where most cells were dead, because normally RBC has very short life time in culture

[25]. Hu et al. [26] studied lymph node cells isolated from mice treated with different

concentrations of sodium benzoate and compared to control cells they founded that sodium

benzoate can caused change the structure of lymphocytes and damage the cell membrane.

Particularly important data in present study was absorption value after 4 hours of boiled

pepper in C3 (B + E + BA, water extract). After this incubation only, the highest

concentration was able to protect red blood cells against sodium benzoate.

PI was calculated and presented in Table 2. Statistical analysis was obtained statistically

significant differences between solvent in the highest concentration of pepper extract (C3 =

1.250 mg/ml). Results show distinct trends for fresh pepper depending on the solvent in every

concentration. The obtained PI results changed unregularly in case of methanol, but in case of

water and fresh sample PI was stable. Boiled peppers with water extract gave extreme results

of activity for C1 (1,019% - maximum value) and for C3 (0,018% - minimum value). Samples

tasted after 4 hours of incubation by spectrophotometric method were not alive in case of C3

extract.

The results obtained were dependent on whether aqueous or methanol extract was used

for the treatment. One of the most important issues is choosing the proper solvent for a

specific group of biologically active compounds. Wide range of solvents like acetone and

methanol or hexane were used in other research with chili peppers. The smallest capability for

pepper extract was obtained for acetone [2,3]. Presented PI results show that water has the

highest capability than methanol for C1 and C3. Another research also investigated an extract

from chili pepper extracted with methanol [22]. It has shown strong antioxidant and

antidiabetic properties and the inclusion of hot pepper in a full-day diet promotes maintaining

high quality of health. Hot peppers contain high quantity of bioactive compounds, which was

strongly correlated with antioxidant activity. The most important bioactivity effect has

contents of capsaicinoids, carotenoids, flavonoids and phenolics [2]. Most studies show that

the most widely investigated are flavanoid [5,6,20].

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Flavanoid content in peppers are around 0,66 mg/g which is compound in the highest

content after capsaicin, but in other research peppers are considered as a source of plant

phenolics [1]. Special property of phenolics is role in modulating oxidative\ stress, which

takes important place in protection against cancer [13,23]. Others reported the most active

compounds from chili pepper is only capsaicin. Already after 24 h, the use of capsaicin in

relation to tumor cells showed a significant reduction in cell division. [4]. The results obtained

indicate that the biologically active compounds contained in chili pepper have a protective

effect on cells treated with sodium benzoate. The mechanism of RBC death by sodium

benzoate still need further study. Furthermore, excess uptake of benzoic acid is eliminated

from body as hypuric acid, but stillcan cause the negative effects of many organs in people of

all ages [25].

Table 2. Antioxidant activity of the pepper extract in three concentrations on human blood

cells, expressed in PI (Protection index) after 2 and 4 hours incubation.

Average PI ±SD

Methanol Water

2 hours incubation

Fresh Boiled Fresh Boiled

C1 0.949 ±0.10aA 0.97 ±0.08aA 0.973 ±0.06aA 1.019

±0.02aA

C2 1.001 ±0.12aA 0.931 ±0.12aA 0.972 ±0.08aA 0.996

±0.01aA

C3 0.668 ±0.47aA 0.844 ±0.08aA 0.975 ±0.06bA 0.018

±0.03bA

4 hours incubation

C1 4.509±0.19aA ***bA ***bA ***bA

C2 2.104±0.02aA ***bA ***bA ***bA

C3 1.559±0.11aA ***bA ***bA ***bA

Means with different superscript capital letter in the same column were significantly different

(p≤0.05). Means in different superscript small letter in the same row were significantly

different (p≤0.05).

C1 – concentration of pepper extract: 0.375 mg/ml

C2 – concentration of pepper extract: 0.750 mg/ml

C3 – concentration of pepper extract: 1.250 mg/ml

*** - no PI value, because living cells were not detected

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4. CONCLUSIONS

The results of the toxicity test performed on human red blood cells show that the green

chili pepper has high protection properties. It can contribute to increasing the quality of health

and protecting our body against diseases. Statistically significant is the type of raw material

processing used but not the thermal treatment. Rather than the amount of extract acting on

blood cells. Fresh material showed the highest protection properties, which were

demonstrated even after 4 hours of incubation (RBC+E+BA for both solvent). The obtained

data showed that fresh green peppers had the same bioactivity for every concentration (C1,

C2, C3) of water extract. In the highest concentration (C3) significant differences of used

solvent were detected for both kind of vegetable (fresh and boiled). Human red blood cells

(RBC) were total degraded after 4 hours of incubation, except for fresh pepper samples in

methanol (RBC+BA). It can be assumed that fresh vegetables have the highest protecting

influence for cells, because of bioactive compounds contained. Bioactivity of green chili

pepper should be subjected to further research by various methods in order to determine the

antioxidant activity on the human blood cells.

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