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The Effect of Beetroot (Beta vulgaris L.) Juice on Cholinesterase 

Activity In Farmers Exposed to Organophosphate Pesticides 
 

Devyana Dyah Wulandari1, Andreas Putro Ragil Santoso1, Devyani Diah 

Wulansari 2 
 

1Department of Medical Laboratory 

Technology, Faculty of Health, 

Nahdlatul Ulama University of 

Surabaya, Indonesia 

2Department of Pharmacy, Faculty 

of Pharmacy, University of 

Surabaya, Indonesia 

Correspondence: 

Devyana Dyah Wulandari,  

Jl. Jemursari No. 51-57, Surabaya, 

East Java, Indonesia 

Zip Code : 60237 

 

Email: devyanadyah@unusa.ac.id 

 

Received: June 20, 2019 

Revised: June 27, 2019 

Accepted: October 6, 2019 

 

 

 

 

 

 

 

 

 

 

 Abstract 

A decrease in cholinesterase (chE) enzyme activity is an 

indicator of pesticide poisoning, especially 

organophosphate pesticides. The chE activity reduction will 

result in an increase in the amount of acetylcholine and will 

bound to muscarinic and nicotinic receptors in the central 

and peripheral nervous system which can lead to the onset 

of Alzheimer's disease. However, antioxidant products can 

slow the progression of Alzheimer's disease by protecting 

neurons from oxidative stress. Red Beetroot (Beta vulgaris 

L.) contains betalains and phenolic compounds which act as 

an antioxidants that are capable of preventing such illness. 

This study aims to prove the concept that antioxidant 

compounds in red beetroot juice can increase the chE 

activity in subjects exposed to organophosphate pesticides. 

This research is an observational study with an experimental 

approach. This research used 25 farmers who were exposed 

to organophosphate pesticides as respondents. They were 

given 500 ml of beetroot juice twice a day for 2 consecutive 

months. Cholinesterase levels were measured before and 
after consuming red beet juice. Measurement of 

cholinesterase levels was carried out using a kinetic 

photometric test. This method was developed based on 

recommendations from the German Society of Clinical 

Chemistry (DGKC). The results showed that the average of 

chE (U/L) Level Before Treatment was 8.102 and 8.380 

after treatment with a p value 0,62. It can be concluded that 

there was an increase in the level of cholinesterase but it was 

not statistically significant. This may be caused by a 

different response to activity of cholinesterase after 

consuming beetroot juice in each subjects, age difference, 

smoking habits, and personal protective equipment. 

Keywords 

Beetroot, cholinesterase, organophophate, pesticide 

 

 

 

 



 

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INTRODUCTION 

Pesticides are poisons intentionally made 

by humans to kill plant-disturbing organisms 

and disease-spreading insects (1). The high 

usage of pesticides are detrimental to health, 

particularly for the workers who contact with 

pesticides and the impacted population. The 

health risk of pesticides exposure in the 

workers may due to improper handling of that 

poisoning substance. Besides, the population 

are also vulnerable to pesticides poisoning 

due to the contamination of such substance 

that enter the food chain, either because of 

ingestion and inhalation of pesticides residue 

or direct contact of pesticides through the 

skin.  

Every day, a thousands of farmers 

working on agriculture land are poisoned by 

pesticides. it is estimated that millions of 

people involved in agriculture sector suffer 

from pesticide poisoning every year. Based 

on data from the World Health Organization 

(WHO) and the United Nations Environment 

Program (UNEP), 1-5 million cases of 

poisoning occur in people working in the 

agricultural sector. In addition, the 

population around the agricultural sites are 

very at risk of being exposed to pesticides 

either through air, soil or contaminated water. 

Moreover, organophosphate pesticides are 

the most toxic insecticides that is usually 

used in the consumer goods so that the 

population will also be impacted. Pesticides 

exposure in a small amount can cause death, 

but it takes more than a few mg to cause death 

in adults. Organophosphate will inhibit the 

action of pseudokholinesterase in plasma and 

cholinesterase in red blood cells and in 

synapses. Based on previous research, 

exposure to pesticides can cause cancer, 

Alzheimer's disease and even birth defects. 

Pesticides also have the potential to damage 

the nervous system, reproductive system, and 

endocrine system (2). In a Nutrients Journal 

have reported that betalains has high 

antioxidant and anti-inflammatory abilities in 

vitro and in vivo tests. This has sparked 

interest in researchers to examine the role of 

beets in the field of clinical pathology in 

several diseases characterized by oxidative 

stress and chronic inflammation such as liver 

disease, arthritis and even cancer (3).   

Red Beetroot (Beta vulgaris L.) is 

preferred as a rich source of betacyanin, 

including a group of fruits that have reddish 

to purple betalains pigments consisting 

mainly of betanin and isobetanins. Betalains 

and phenolic compounds present in red 

beetroot have been reported to increase 

resistance in low density lipoprotein (LDL) to 

oxidize and to prevent cancer and 

cardiovascular disease by reducing the 

oxidative effects of free radicals on lipids (4). 

Free radicals or reactive oxygen species 

(ROS) play many important roles in 

physiology and pathological processes. 

Oxidative stress is a biological phenomenon 

that results from a biochemical imbalance 



 

 

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between the formation and cleansing / 

buffering of free radicals (5). 

Workers in the agrochemicals and 

biological fertilizers company are the most 

often exposed to pesticides, especially 

organophosphate pesticides. Based on the 

results of the situation analysis, many 

complaints arose from the operators who 

indicated that there were symptoms of 

poisoning. While the temporary solution that 

has been done is to move positions in other 

parts for a while and there is no way to 

minimize the symptoms of poisoning that 

arise. Whereas, from the results of previous 

studies, (5) explained that natural antioxidant 

products can slow the progression of 

Alzheimer's disease by protecting neurons 

from oxidative stress. Over the past few 

years, oxidative stress has been described as 

a co-lethal factor in cases of poisoning caused 

by organophosphate pesticides. Many studies 

emphasize the role oxidative stress in cases of 

organophosphate poisoning. Previous studies 

have shown that the administration of date 

palm extract is able to restore atrazine 

poisoning, a herbicides that cause oxidative 

damage to sperm parameters, reduce 

testicular antioxidant enzymes, GSH, SOD, 

CAT, and GPx levels in mice (6). Another 

studies found the presence of oxidative stress 

and inhibition of acetylcholinesterase on 

farmers exposed to organophosphate 

pesticides (7). Antioxidants have been 

suggested as organophosphate antidotes. 

Meanwhile, Nurulain et al. did not find that 

giving glutathione (GSH) and NAC do not 

provide a survival effect on acute exposure 

induced paraoxon (a very dangerous type of 

OP pesticide) in Wistar mice. This shows that 

antioxidants have no role in the toxicity of 

acute phase II pesticides (8).  

Several studies have shown that the 

administration of antioxidant activity 

compounds can minimize pesticide 

poisoning. Thus the researchers want to 

examine whether red beetroot juice (Beta 

vulgaris L.) that is proven to have antioxidant 

activity is able to minimize pesticide 

poisoning through increased cholinesterase 

activity. 

 

MATERIALS AND METHODS  

The equipments that used in this research 

were Terumo volume 3 ml syringe, EDTA 

vacutainer tube, torniquet, cotton, Centrifuge 

Brand: TOMY, Type: MX-105, GENESYS 

™ 10S UV-Vis spectrophotometer - Thermo 

Fisher Scientific, Thermometer, Dropper, 

Dropper measure, ice box, ice pack. 

Meanwhile, the materials used were 96% 

alcohol, distilled water, deionized aquades, 

phosphate buffer pH 7.4, thiol 5.5'-dithio-bis 

(2-nitro benzoic acid) {DTNB} 0.33 mM, 

acetylcholine 5 mM. 

Red beetroot juice was made by mixing 

150 mg of red beetroot in 1 L of water. 

Afterward, it was blended using a blender 

(servings per person), divided into 2 servings 



 

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to drink twice a day (500 ml/serving), at the 

morning and at the evening (9). They 

consume this juice every day for 2 months. 

Blood sampling was done by damming 

with tourniquet. Alcoholic cotton was 

applied in a circular manner as an antiseptic 

and was allowed to dry. The syringe needle 

was inserted in positions 30-45°C until a sign 

of blood comes out of the needle then the 

syringe was drawn to the limit of 2 with a 

blood volume of 2 mL. The needle was taken 

from the vein and the blood was inserted into 

the vacuum tube containing the EDTA and 

was homogenized (10). 

Separation of Erythrocytes and Plasma 

was done by inserting the blood that has been 

taken into the vacuum container and it was 

centrifuged using Centrifuge Brand: TOMY, 

Type: MX-105 for 10 minutes at 4000 rpm. 

Erythrocytes that have been separated from 

plasma were taken and placed in test tubes 

(11). 

Measurement of chE was done by taking 

erythrocytes that have been separated from 

plasma and it was then added with deionized 

water to the same as the initial volume of 

blood. Afterwards, it was diluted 60 times 

using a buffer (phosphate buffer 0.1 M, pH = 

7.4), and it was frozen for hemolysis. The 

mixture was thawed again and diluted with 

buffer 9 times. After that, it was added with 

thiol reagent 5.5'-dithio-bis (2-nitro benzoic 

acid) {DTNB} (DTNB concentration 0.33 

mM) and was allowed to stand for 10 

minutes. Then the mixture was added to the 

ATCh substrate (ATCh concentration of 1.0 

mM) and was read with absorbance of 412 

nm using a UV-Vis GENESYS ™ 10S 

spectrophotometer - Thermo Fisher 

Scientific by using a blank containing 

erythrocyte hemolysis in a buffer (12). 

This research is an observational study 

with an experimental approach since the data 

was taken through laboratory tests. 

Additionally, the participant was interviewed 

by using a questionnaire which ask data on 

age, length of work and symptoms felt. The 

population and sample in this study were 

male farmers in the Mojokerto region who 

were exposed to organophosphate pesticides. 

The inclusion criteria were 30 years old and 

above, and has been working for at least 1 

year. 

The data was analyzed by a paired t-test 

using SPSS 16 software. Statistical tests were 

performed at 95% confidence level and a 

significant difference if p <0.05. The 

participants were given an informed consent 

and the number of Ethical Clearance 

Certificate is 091/KE/VII/2019. 

  

RESULTS 

The study population (respondents) were 

farmers exposed to organophosphate 

pesticides. All of the respondents were male 

as many as 25 people with different working 

periods as shown in table 1. Respondents 

were exposed to organophosphate pesticides 



 

 

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daily because they work as farmers on an 

agricultural land. 

The parameters of the examination 

carried out were the levels of cholinesterase.  

Cholinesterase levels were measured before 

and after administration of red beet juice. The 

results of the examination of cholinesterase 

levels can be seen in table 1. The normal 

range of cholinesterase levels ranged from 

4,620 - 11,500 U / L. The higher levels of 

cholinesterase the better because 

cholinesterase has a function to convert 

acetylcholine into acetate and choline that 

can prevent the accumulation of 

acetylcholine. Conversely, if the level of 

cholinesterase is lower than 4,620 U / L, it 

will show the symptoms of poisoning due to 

the accumulation of acetylcholine. 

 

 

Table 1. Results of Cholinesterase Level Before and After Treatment 

Patient 

Code 

Age  

(years old) 

chE (U/L) Level 

Before Treatment 

chE (U/L) Level 

After Treatment 

Normal 

Value of 

chE (U/L) 

p Value 

1 50 11.340 9.405 

4.620 – 

11.500 
0.620 

2 46 3.967 10.479 

3 62  5.670 6.436 

4 54  5.666 6.830 

5 54  9.371 8.276 

6 50  8.759 7.759 

7 53  7.302 9.915 

8 57  9.775 5.770 

9 63  8.718 4.547 

10 50  8.441 7.357 

11 60  7.446 9.047 

12 41  6.655 9.342 

13 38  4.351 7.380 

14 63  11.284 9.685 

15 60  9.927 9.611 

16 60  7.133 9.954 

17 64  6.673 8.013 

18 32  8.198 7.995 

19 64  11.086 6.007 

20 52  6.840 10.999 

21 65  9.410 10.035 

22 35  10.583 7.428 

23 60  6.542 8.723 

24 61  6.121 7.989 

25 50  11.287 10.516 

Average 8.102 8.380   
*U/L = Unit per Liters  

 

 



 

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The statistical analysis of the effect of red 

beet juice consumption on cholinesterase 

levels can be seen in table 1. The results show 

that consumption of red beet juice on 

cholinesterase levels was not statistically 

significant with a P value 0.620. It means that 

consumption beetroot juice was not able to 

increase cholinesterase levels significantly.

 

DISCUSSION 

Farmers are one of the subjects who are 

vulnerable to pesticides. In a plantation area 

in Mojokerto, east Java, the farmers use 

organophosphate pesticide. 

Organophosphate pesticides are the most 

toxic pesticides than the other groups. This 

pesticide is able to inhibit the performance of 

the cholinesterase enzyme. The enzyme 

normally hydrolyzes acetylcholine into 

acetate and cholin. When the enzyme is 

inhibited, it causes the amount of 

acetylcholine to increase and binds to the 

muscarinic and nicotinic receptors of the 

central and peripheral nervous system. 

Therefore, this causes symptoms of 

poisoning that affect all parts of the body. 

There are some people who have shown the 

indications of poisoning symptoms marked 

by a decrease level of the enzyme 

cholinesterase, characterized by complaints 

such as dizziness and nausea. It was also 

found in the farmers working on agricultural 

land in Mojokerto. However, symptoms of 

organophosphate poisoning vary greatly. 

Each symptom is very dependent on the 

presence of persistent acetylcholine or 

depression stimulation followed by central or 

peripheral nerve stimulation. Early 

symptoms such as salivation, lacrimation, 

urination and diarrhea (SLUD) occur in acute 

organophosphate poisoning due to 

stimulation of muscarinic receptors so that 

the acetyl kholin content in the blood 

increases in the eyes and smooth muscle (13). 

Organophosphate is a type of pesticide 

that is most widely used in agriculture. 

Cholinesterase levels are known as 

biomarkers in patients exposed to 

organophosphate pesticides. In the results of 

the study in table 1 showed that some 

cholinesterase levels are still in the normal 

range, but there are some others that have chE 

levels close to the lower limit (symptoms of 

poisoning), as in patient coded by number 2 

and number 13. Based on the results of 

statistical calculations obtained, the p value 

was 0.62 which indicates that consumption of 

red beet juice did not significantly influence 

the levels of cholinesterase. It may be caused 

by a different habits and personal protective 

equipment. It can be seen in patient coded by 

number 2, he was experienced a very 

significant increase in cholinesterase levels 

with an increase of 6512 chE (U/L). 

Furthermore, the patient coded by number 11 

increase in cholinesterase levels by 2613 chE 

(U/L), the patient coded by the number 12 



 

 

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increase in cholinesterase levels by 2687 chE 

(U/L), the patient coded by the number 13 

with an increase 3029 chE (U/L). Lastly, the 

patient coded by the number 20 increase in 

cholinesterase levels by 4159 chE (U/L). 

From this data, there was an increase in the 

level of cholinesterase but it was not 

statistically significant.  

One study suggested that the nitrate 

content was not the only compound contained 

in red beetroot that were proposed to have 

beneficial effects on health and disease. 

Beetroot is a rich source of phytochemical 

compounds including ascorbic acid, 

carotenoids, phenolic acids and flavonoids. 

Beets are also one of the few vegetables that 

contains a group of highly bioactive pigments 

known as betalains. Members of the betalains 

family are categorized as red-purple 

betacyanin pigments or yellow-orange 

betaxanthin pigments. Based on other 

studies, it mentioned that phenolic 

compounds can react with residues of active 

amino acids found on the active side of ChE 

through hydrogen bonds, hydrophobic 

interactions and π-π interactions. The results 

show that hydroxyl groups in the structure of 

phenolic compounds are believed to increase 

cholinesterase activity (14). This is the 

underlying mechanism why antioxidant in 

beetroot can increase cholinesterase level. 

This supports the results of this study on 

several patient codes that indicate an increase 

in cholinesterase activity. 

However, there are several other factors 

that can influence the level of cholinesterase, 

including nutritional status, health 

conditions, age, sex, ambient temperature, 

smoking habits, and habits of using personal 

protective equipment (PPE) (15). In the 

results of this study, it was note that each 

respondent has a different response after 

consuming red beet juice on cholinesterase 

activity. This can be due to the influence of 

age, smoking habits and habits of using PPE 

that are different from each respondent. 

Unfortunately, this research did not examine 

the effect of age, smoking habits and habits 

on cholinesterase levels, but it could be that 

these factors are the cause of not being 

statistically significant results. This research 

is preliminary data that can be used as a basis 

for further research to determine the efficacy 

of red beet juice in increasing levels of 

cholinesterase by involving age and habits. 

 

CONCLUSIONS 

Based on the results of the study 

concluded that there was an increase in the 

level of cholinesterase but it was not 

statistically significant. The increasing of 

cholinesterase levels can be caused by 

antioxidant properties of red beetroot juice 

(Beta vulgaris L.) so it can be minimize the 

pesticide poisoning through increased 

cholinesterase activity. In further studies it is 

recommended to increase the dose, duration 



 

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of treatment and involve factors of age and 

habits. 

 

ACKNOWLADGEMENTS 

Thank you to the Ministry of Higher 

Education Research for providing funding for 

this research, thank you to the Nahdlatul 

Ulama Surabaya University for providing 

easy access to research facilities and thanks 

to all respondents who have helped this 

Research. 

 

CONFLICT OF INTEREST 

There are no conflicts of interest. 

REFERENCES 

1. Soemirat Juli, 2003. Environmental Toxicology, 
Gadjah Mada University Press, Bandung. 

2. Sarwar, Muhammad. The Dangers of Pesticides 
Associated with Public Health and Preventing of 

the Risks. International Journal of Bioinformatics 

and Biomedical Engineering. 2015.Vol. 1, No. 2, 

pp. 130-136. 

3. Clifford, Tom. Glyn Howatson, Daniel J. West 
and Emma J. Stevenson. The Potential Benefits of 

Red Beetroot Supplementation in Health and 

Disease. Nutrients 7.2015, 2801-2822 

4. Guldiken Burcu, Gamze Toydemir, Kubra Nur 
Memis, Sena Okur, Dilek Boyacioglu 

And Esra Capanoglu. Home-Processed Red 

Beetroot ( Beta Vulgaris L.) Products: Changes In 

Antioxidant Properties And Bioaccessibility. Int. 

J. Mol. Sci. 2016, 17, 858. 

5. Pervin, Mehnaz., Md. Abul Hasnat, Yoon Mi Lee, 
Da Hye Kim, Jeong Eun Jo and Beong Ou Lim. 

Antioxidant Activity and Acetylcholinesterase 

Inhibition. of 

Grape Skin Anthocyanin (GSA). Molecules. 

2014, 19, 9403-9418 

6. Ganga, U.K, Hemalatha C., Kishori B. Protective 
Role Of Date Fruit Extract Against Chlorpyrifos-

Induced Reproductive Toxicity In Albino Male 

Rats. International Journal of Green Pharmacy. 

2018 ,12 (1). 

7. Bayrami M, Hashemi T, Malekirad AA, Ashayeri 
H, Faraji F, Abdollahi M. Electroencephalogram, 

cognitive state, psychological disorders, clinical 

symptom, and oxidative stress in horticulture 

farmers exposed to organophosphate pesticides. 

Toxicol Indl Health 2012;28:90-6 

8. Nurulain, S.M, Szegi, P., Tekes K., and Syed NH 
Naqvi. Antioxidants In Organophosphorus   

Compounds Poisoning. Arh Hig Rada Toksikol 

2013;64:169-177 

9. Maria, Rita A. Porto, Vivian S. Okina, Tatiana C. 
Pimentel and Sandra Helena Prudencio. 

Physicochemical Stability, Antioxidant Activity, 

and Acceptance of Beet and Orange Mixed Juice 

during Refrigerated Storage. Beverages,2017,3, 

36 

10. EFLM (European Federation of Clinical 
Chemistry and Laboratory Medicine) 

Recommendation for Venous Blood Sampling, 

2017 

11. Standard Operating Procedure for Collecting, 
Proccessing, and Storage of Blood Samples, 2017 

12. Jamshhidzade, Akram, Hossein Nicknahad, 
Mohammadi-Bardbori A, Talati M.. Comparative 

measurement of serum Acetyl Cholinesterase 

Enzyme using three different methods. Iranian 

Journal of Toxicology, 2009. Volume 2 , No.4 

13. Aroonvilairat, Soraya., Wannapa 
Kespichayawattana, Thiwaree Sornprachum, 

Papada Chaisuriya, Taweeratana Siwadune and 

Kavi Ratanabanangkoon.. Effect of Pesticide 

Exposure on Immunological, Hematological and 

Biochemical Parameters in Thai Orchid Farmers-

A Cross-Sectional Study. Int. J. Environ. Res. 

Public Health.2015, 12, 5846-5861 

14. Szwajgier. D. Anticholinesterase activity of 
selected phenolic acids and flavonoids – 

interaction testing in model solutions. Annals of 

Agricultural and Environmental Medicine 2015, 

Vol 22, No 4, 690–694. 

15. Purba, IG. Analysis of Factors Related to 
Cholesteresterase Levels in Fertile Age Women in 

Agricultural Areas. Universitas Diponegoro; 

2009. Tesis