th protective effect of green tea extract on ovary tissue...

* Corresponding author, Address: Iran, Jahrom,Jahrom Branch, Islamic Azad University, Young Researchers

and Elit Club,

Tel: 09171095411 Email: [email protected] 45

Protective effect of green tea extract on ovary tissue function in rats treated

by Malathionin secticide

Hoseini Shahrkhafri Marziyah Sadat٭1, Hemayatkhahjahromi Vahid

2, Samani Jahromi

Elaheh1

Received:4/19/2015 Revised:12/14/2015 Accepted:12/20/2015

1. Young Researchers and Elit Club, Jahrom Branch, Islamic Azad University, Jahrom, Iran

2. Department of Biology, Jahrom Branch, Islamic Azad Unversity, Jahrom, Iran

Pars Journal of Medical Sciences, Vol. 13, No.3, Fall 2015

Abstract

Introduction:

This study was conducted to investigate the protective effect of green tea extract on ovary tissue

function in rats treated by Malathion insecticide.

Materials and Methods:

A total of 72 mature Wistar rats aged 2 to 3 months (approximate weight of 200±15 g) were

experimented. Rats were divided into 9 groups of eight. Control group did not receive any medication.

Sham group was given 0.2 cc physiological serum and experimental groups 1, 2 and 3 received 100,

200 and 400 mg/KG bw green tea extract respectively, experimental 4 was given 40 mg/KG bw

Malathion and experimental groups 5, 6 and 7 received 100, 200 and 400 mg/KG bw green tea extract

respectively and 40 mg/KG bw Malathion. After 15 days, the serum levels of sex hormones were

measured, and ovaries were removed for counting the ovarian follicles.

Results:

Serum concentration of estrogen, progesterone, FSH and LH were significantly decreased in

experimental group 4 compared to control and other groups. The primary and Graafian follicles

decreased in experimental group 4 compared to control group. Primary and secondary follicles and

corpus luteum significantly decreased in experimental group 4 compared to control group. All

examined parameters except Graafian follicles and corpus luteum increased in the experimental

groups 5, 6 and 7 compared to experimental group 4. Follicular atresia increased in experimental

group 4 compared to other groups and significantly decreased in experimental groups 5, 6 and 7

compared to experimental group 4.

Conclusions:

Malathion had an adverse effect on secretion of sex hormones in female rats, as well as the process of

oogenesis, and green tea extract decreased those negative effects.

Keywords:Malathion, Green Tea, Oogenesis, Sex Hormones, Rat

Introduction

Green tea is currently considered a useful

source for human health with biological

and pharmacological activities. The health

benefits of tea extract and its polyphenolic

catechins have attracted most scientific

research in the prevention and treatment of

many diseases. Tea leaves, as the only

food product having Epigallocatechin -3-

Th

Par J Med Sci 2015;13(3):45-57

Protective effect of green tea extract Hoseini Shahrkhafri M.S et al

Pars Journal of Medical Sciences, Vol. 13, No. 3, Fall 2015

46

gallate (EGCG) have active compounds

with eight free OH-groups and high

antioxidant activity (1, 2). Tea is called

khatai tea in traditional medicine and Shay

in Arabic, and its shrub or bush is called

Theier in French and Teaplant in English.

This plant belongs to Temsnstroemiaceae,

Camelliaceae, and Theaceae families, and

Camellia sinensis and Camellia Theifer are

its scientific names. Tea is a big shrub with

a maximum height of one and a half

meters and glossy leaves and beautiful

white flowers. This plant contains

polyphenolic compounds, including

Epigallocatechin -3- gallate (EGCG),

Epigallocatechin -EGC, Epicatechin -3-

gallate-ECG, and Epicatechin –EC (3, 4, 5,

6). The most potent of these antioxidants,

Epigallocatechin -3- gallate (EGCG) has

been reported to be 100 times as potent as

vitamin C and 25 times as potent as

vitamin E (7). The protective effect of

green tea on body tissues may be due to its

antioxidant property (8). The most

important ingredient in green tea is

catechin (ECGC, the most abundant

catechin). This substance is an extremely

potent and effective antioxidant and many

beneficial effects of green tea are

attributed to it (9, 10, 11). Studies have

shown that a cup of green tea (2.5 g green

tea in 200 ml of water) may contain 90 mg

EGCG (12). Green tea has been introduced

as an anti-inflammatory, anti-cancer, anti-

cholesterol, anti-diabetic, anti-mutation,

anti-microbial, anti-stroke, and anti-

oxidative agent (13). In addition, bioactive

components of green tea (catechins and

caffeine) stimulate the sympathetic system

and increase heat production and fat

oxidation in the body and accordingly,

exert their anti-obesity effects (14, 15).

Catechin, as a natural antioxidant, affects

the reproductive system and stimulates

testosterone production by the Leydig cells

and decreases the content of total

cholesterol, triglycerides and

phospholipids in testicular tissue. It also

prevents the mutagenicity of mutagenic

chemicals on chromosomes (8). The

ability of sperm viability, mobility, and

fertilization highly depends on its

antioxidant capacity expressed in seminal

plasma. Being rich in polyphenols, as

potent antioxidants, green tea inhibits

reactive oxygen and nitrogen species, and

thus improves sperm quality (9).

Malathion (S-(1,2-dicarboethoxyethyl) O,

O-dimethyl phosphorodithioate) is one of

the most widely used insecticides in Iran.

Malathion is a yellowish dark brown

concentrated and oily liquid that is used as

a broad-spectrum pesticide in agriculture

(16). This substance is a derivative of D-

tonic phosphoric acid and one of the oldest

P-group insecticides introduced in 1950

(17). Organophosphates (OPs) are a class

of pesticides widely used in agriculture,

veterinary medicine, industry, residential

areas, and as nerve agents in wars. These

compounds cause the most accidental

poisonings with acute or chronic

complications (18, 19).

Studies have shown that toxic effects of

some OPs are not limited to cholinesterase

inhibition, but as a result of cholinergic

crisis (increased acetylcholine), changes

such as cell membrane damage, free

radical production, and the antioxidant

system malfunction are also observed (20,

21). By conducting a study on the

genotoxic effects of Malathion, Giri et al.

(2002) found that this toxin affects sperm

count and causes cell mutation (22).

Weisburger (2002) reported that tea

polyphenols have potent antioxidant

activity and can reduce LDL oxidation and

oxidized DNA metabolism, and

subsequently decrease the risk of heart

diseases and cancer (13). Crespy and

Williamson (2004) reported that green tea

extract has antioxidant properties and

eliminates free radicals (23). Since green

tea is high in catechins, it is considered a

potent antioxidant. Azarnia et al. (2013)

conducted a study on the impact of

hydroalcoholic extract of green tea on rats

with polycystic ovary syndrome (PCOS)

and reported reduced serum concentrations

of LH, CRP, IL-6, insulin, and glucose in



47

groups treated with green tea extract.

Histomorphometric studies also indicate

an increase in the number of follicles and

changes in the thickness of the follicular

theca cells for improving PCOS. Green tea

can be a potential medication for the

treatment of PCOS and type 2diabetes

(24).

The results of the study of Sanaei et al.

(2014) also showed that administration of

green tea extract for 42 days significantly

increased the number of primary, growing,

and antral follicles (25). Shariatzadeh and

Mohammadi (2014) reported that the use

of green tea can significantly compensate

damage caused by the use of sodium

arsenite, sperm count, motility, viability,

and normal morphology, as well as the

diameter of the lumen of the seminiferous

tubules, germinal epithelial thickness, and

the levels of MDA (P<0.001) (26).

The present study aimed to investigate the

protective effect of green tea extract on

ovary tissue function in rats treated by

Malathion insecticide.

Materials and Methods

Animals used: A total of 72 mature

female Wistar rats aged 2 to 3 months

(approximate weight of 200±15 g) were

purchased from Animal House of School

of Veterinary Medicine, Shiraz, Iran. Rats

were transferred to the Animal House of

Jahrom University of Medical Sciences

and were housed in special sterilized

containers at a temperature of 22-20 °C

and moisture content of 60-40 percent for

two weeks to be adapted to the

environment. During the experiment, they

were given foods provided from Fars

Livestock and Poultry Company and tap

water in special drinkers.

Medications and materials used: Green

tea plant (medicinal herb shops in the

city), Malathion (Moshkfamfars factory),

the FSH hormone kit (Iran Pishtaz Teb,

serial number Catnom BYEK1370), the

LH hormone kit (Iran Pishtaz Teb, serial

number Catnom BYEK1370), the

progesterone hormone kit (REF DKO 006,

Germany), the estrogen hormone kit (REF

DKO 003, Germany).

Green tea extraction method: To prepare

the extract, green tea was first milled and

soaked in 80% ethanol for 24 hours. The

solution was then filtered and the extract

was obtained through vacuum

concentration method. The extract will be

soluble in distilled water (27, 2).

Methods: This experimental study was

conducted in Animal House of Islamic

Azad University, Jahrom Branch, May

2013. All ethical considerations were

observed in relation to animal care and

working with laboratory animals during

the study and the topic was approved by

the Ethic Committee of Islamic Azad

University, Jahrom Branch (D/P/2990

dated February 28, 2013). According to

previous articles, rats were divided into 9

groups of eight as follows:

Control group did not receive any

medication. Sham group was given only

physiological serum. Experimental groups

1, 2, and 3 received 100, 200, and 400

mg/kg bw green tea extract respectively,

experimental 4 was given 40 mg/kg bw

Malathion, and experimental groups 5, 6,

and 7 received 100, 200, and 400 mg/kg

bw green tea extract respectively and 40

mg/kg bw Malathion. Rats were injected

intraperitoneally with green tea extract and

Malathion for 14 days.

Determination of serum levels of

hormones: On the 15th

day, rats were

anesthetized with diethyl ether and blood

samples were taken from their hearts.

Blood samples were placed in a steam bath

at 37 °C to be clotted. After centrifugation

at 3000 rpm for 10 minutes, the serum was

separated. LH, FSH, progesterone, and

estrogen levels were measured using the

ELISA method. After the sampling, the

abdominal region of the rats was incised

with a scalpel. Left and right ovaries were

removed with a scalpel and forceps from

the surrounding fat tissues and the

fallopian tube and after washing with

physiological saline, they were placed in

separate containers of 3% formalin



48

solution. Tissue sections were then

prepared from the samples. Prepared slides

of left and right ovaries of rats were

studied separately by a light microscope.

In each slide, the number of primary,

secondary, Graafian, and atretic follicles,

and corpus luteum were counted with 40x

and 100x magnification.

Statistical analysis method: One-way

analysis of variance (ANOVA) was used

for statistical analysis. Since the data were

normally distributed according to the

Kolmogorov-Smirnov test results,

parametric tests were used. Duncan’s test

was applied to find the difference between

the means when there was a significant

difference between different groups.

Statistical analysis was performed using

SPSS-16 and the significance level was

considered P<0.05. The results are

presented as Mean±SEM in the results

section. Figures were drawn by Excel

software.

Results

Changes in plasma concentration of LH:

The results of changes in plasma

concentrations of LH showed that LH

levels reduced significantly in

experimental group 4 (Malathion group)

compared to control group and other

experimental groups at P<0.05. A

significant increase was observed in

experimental groups 5, 6, and 7 (groups

receiving minimum, average, and

maximum Malathion and tea) compared to

Malathion group at P<0.05 (Figure 1).

Figure 1: Comparison of LH concentrations between the groups studied

The values are shown as Mean±S.E.

According to Duncan’s test, if columns have at least one common letter, it indicates there is no

significant difference between columns.

Changes in plasma concentration of

FSH:


concentrations of FSH among the groups

studied showed that FSH levels reduced

significantly in experimental group 4

(Malathion group) compared to control

group and other experimental groups at

P<0.05. A significant increase was

observed in experimental groups 5, 6, and

7 (groups receiving minimum, average,

and maximum Malathion and tea)

compared to Malathion group at P<0.05

(Figure 2).

b

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b

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b

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rmo

ne

(LH

(IU

/L)

control shahed Tea mini. Tea average Tea max. malathion malt & T.min malt&T.aver malt&T.max



49

Figure 2: Comparison of FSH concentrations between the groups studied

Changes in plasma concentration of

estrogen:


concentrations of estrogen among the

groups studied showed that estrogen levels

significantly increased in experimental

group 2 (group receiving average dose of

tea) and significantly decreased in


compared to control group at P<0.05. A

significant increase was also observed in

experimental groups 5, 6, and 7 (group



Malation group at P<0.05 (Figure 3).

Figure 3: Comparison of estrogen hormone between the groups studied

Changes in plasma concentrations of

progesterone:


concentrations of progesterone among the

groups studied showed that progesterone

levels significantly decreased in

experimental groups 4 (Malathion group)

and 5 (the group receiving Malathion and

the minimum tea) and significantly

increased in experimental group 2 (group

receiving average dose of tea) compared to

control group (P<0.05). A significant

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50

increase was also observed in progesterone

levels in experimental groups 5, 6, and 7

(group receiving minimum, average, and


Malation group at P<0.05. A significant

increase was also observed in plasma

concentrations of progesterone in

experimental groups 6 and 7 compared to

experimental group 5 (Figure 4).

Figure 4: Comparison of progesterone between the groups studied

The number of primordial follicles:

The results of the number of primordial

follicles among the groups studied showed

that the number of primordial follicles

increased in experimental groups 2 and 3

(the group receiving average and

maximum doses of tea) and significantly

decreased in experimental group 4


group (P<0.05). A significant increase was

also observed in experimental 5, 6, and 7

(the group receiving minimum, average,


compared to Malathion group (P<0.05)

(Figure 5).

Figure 5: Comparison of the number of primordial follicles between the groups studied

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51

Changes in the number of primary

follicles:

The results of the number of primary


that the number of primary follicles

significantly decreased in experimental

group 4 (Malathion group) compared to

control group and other groups (P<0.05).

A significant increase was also observed in

experimental 5, 6, and 7 (the group



Malathion group (P<0.05) (Figure 6).

Figure 6: Comparison of the number of primary follicles between the groups studied

Changes in the number of secondary

follicles:

The results of the number of secondary


that the number of secondary follicles


(Malathion group) and significantly

increased in experimental group 2 (average

dose of tea) compared to control group

(P<0.05). A significant increase was also

observed in experimental 5, 6, and 7 (the

group receiving minimum, average, and



Figure 7: Comparison of the number of secondary follicles between the groups studied

c c

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(%)


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52

Changes in the number of Graafian

follicles:

The results of the number of Graafian


that the number of Graafian follicles

increased in experimental groups 2 and 3

(the group receiving average and

maximum doses of tea) and significantly



group (P<0.05). A significant increase was

also observed in experimental 5, 6, and 7

(the group receiving minimum, average,


compared to Malathion group (P<0.05)

(Figure 8).

Figure 8: Comparison of the number of Graafian follicles between the groups studied

Changes in the number of corpus

luteum:

The results of the number of corpus luteum

among the groups studied showed that the

number of corpus luteum increased in

experimental group 2 (average dose of tea)

and significantly decreased in

experimental groups 3 (maximum dose of

tea) and 4 (Malathion group) compared to


A significant increase was also observed in

experimental 5, 6, and 7 (the group




Figure 9: Comparison of the number of corpus luteum between the groups studied

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)




53

Changes in the number of atretic

follicles:

The results of the number of atretic


that the number of atretic follicles


group 4 (Malathion group) compared to


A significant decrease was also observed

in experimental 5, 6, and 7 (the group




Figure 10: Comparison of the number of atretic follicles between the groups studied

Discussion

The results showed that progesterone,

estrogen, FSH, and LH levels significantly

reduced in experimental group 4 compared

to control group and other groups, and


groups 5, 6, and 7 (the group receiving

minimum, average, and maximum

Malathion and tea) compared to Malathion

group (P<0.05). Increased serum levels of

sex hormones have been reported useful

for the evaluation of human and animal

fertility (28). In general, a significant

reduction in concentration of sex

hormones in fertility activities leads to

fertility disorders in people who are

exposed to chemicals (29).

Exposure to chemicals led to ovarian

tissue destruction and significant changes

in progesterone and estrogen in rats which

it was also confirmed in previous studies.

Yarbe et al. (2009) reported that reduced

serum levels of progesterone and estrogen

occur due to ovarian damage which is

consistent with the results of this study. At

the time of fertility and ovulation, estrogen

and progesterone are normally affected by

the pituitary gland producing LH and FSH

(30). Thus, it can be said that ovarian

tissue damage and impaired secretion of

estrogen and progesterone which can occur

increasingly or decreasingly justify a

significant reduction in estrogen and

progesterone in experimental group 4

compared to control group. LH and FSH

changes may also be due to reduced GnRH

which decreased the serum levels of LH

and FSH. The hypothalamic-pituitary-

gonadal axis regulates reproductive

activities, a multifactorial process, by

genetic and hormonal control. In this

study, the results of histological evaluation

showed that treatment with Malathion

leads to ovarian cell damages and induces

extensive histopathological changes in

ovarian follicles. Malathion can cause

toxic effects on the reproductive system

through oxidative stress (31), which can be

reduced with concurrent administration of

antioxidants. This result is consistent with

our results. By conducting a study on the

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a a

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nu

mb

er o

f at

reti

c fo

llicl

es(%

)




54

protective effect of green tea extract on

ovary function in mice treated by

Paclitaxel drug, Mohseni Kouchesfehani

and Asoudeh (2014) reported that green

tea extract improves ovarian parameters

treated by Paclitaxel. Green tea extract

with its antioxidant properties has

protective effects on ovarian tissue

parameters in mice and LH and FSH levels

after treatment with Paclitaxel (32). This

protective effect of green tea on the

parameters of ovarian tissue is consistent

with our results. According to a study

conducted by Ghafuriyan et al. on the

effect of green tea extract on the histology

of the ovary in polycystic ovary syndrome,

it was found that green tea affects oocyte

maturation and follicular development in

polycystic women. Green tea extract

reduces the theca layer thickness in rats

and may increase lipolysis and reduce the

hypertrophy of this layer. Accordingly, the

production of androgens and steroids also

decreases by the mentioned layer (33). It

seems that the difference in the type of

animal, developmental stage, treatment

duration, and the type of field is the main

reason for these differences. The results of

the study of Allahdadian et al. showed that

green tea consumption has a significant

effect on reducing free testosterone in

patients with polycystic ovarian disease

(34). According to a laboratory study

conducted by Figueiroa et al. on the effect

of green tea compounds on testosterone

production in rabbit Leydig cells, it was

found that green tea compounds inhibit

basic and stimulated production of

testosterone (35). According to a study

conducted by Wu et al. on the effect of

two months of green tea consumption on

testosterone level in healthy

postmenopausal women, it was reported

that green tea supplements caused no

constant changes in the testosterone levels

(36). According to a study conducted by

Shayeghi et al. on the effect of Malathion

insecticides on the inhibition of

cholinesterase enzyme among the

agricultural sprayers, it was found that

Malathion, as a phosphate insecticide, has

gastrointestinal toxicity and fumigant

property. They also found that decreased

cholinesterase activity will be less if health

regulations are observed when using

Malathion (37).

Fortunato et al. (2006) reported that

Malathion induces the production of free

radicals and oxidative stress in the brain

and increases the activity of antioxidant

enzymes (31). Oxidative stress is caused

by free radicals and mitochondria are

identified the main region of the

production of free radicals (38). By

evaluating the effect of Malathion-induced

stress on fat metabolism, scientists found

that the lethal and sub-lethal doses of this

toxin increase fatty acids, glycerol and

lipase activity in tissues studied and can

have a negative impact on reproductive

activity. Malathion also has a negative

impact on reproductive activity and

decreases the amphibian population which

is consistent with the present study (39).

Studies conducted in recent years

emphasize the role of reactive oxygen

species as a contributing factor in some

tissue damage. An imbalance in the

production of reactive oxygen species and

antioxidant enzyme activities leads to

tissue damage and it can occur through

either increased production of reactive

oxygen species or decreased antioxidant

enzyme activities, or both, suggesting the

existence of oxidative stress conditions

(40). Reactive oxygen species can severely

endanger cell life by weakening the

structure and function of plasma

membrane and intracellular membranes

(41). By examining ovarian tissues and

counting the follicles using a light

microscope, a decrease was observed in

the number of primordial and Graafian

follicles in experimental group 4

(Malathion group) and a significant

increase was found in experimental groups

2 and 3 (the average and maximum doses

of green tea) compared to control group

(P<0.05). The number of Graafian follicles

also increased in experimental group 6



55

compared to Malathion group. The number

of primordial follicles increased in

experimental groups 5, 6, and 7 (the group



Malathion group (P<0.05). The number of

atretic follicles significantly increased in


compared to other groups (P<0.05). Also,

the number of atretic follicles in

experimental groups 5, 6, and 7 (the group


maximum Malathion and tea) significantly

reduced compared to Malathion group

(P<0.05).

The number of primary and secondary

follicles and corpus luteum significantly

reduced in experimental group 4 compared

to control group (P<0.05). The number of

primary and secondary follicles


groups 5, 6, and 7 (the group receiving

minimum, average, and maximum

Malathion and tea) compared to Malathion

group (P<0.05).

Organophosphate insecticides have

alkylating properties and thus can affect

the cell nuclear DNA (42). These

chemicals have also electrophilic property.

Therefore, they can affect cellular proteins

and lead oocytes and follicular cells to lose

their efficiency. In this study, the

destructive impact of Malathion was also

confirmed (43). Malathion increases

malondialdehyde in ovarian tissue that

may be caused by released radicals or lipid

and body metabolism. With increasing

doses, Malathion reduces the number of

healthy follicles, increases the number of

atretic follicles, and causes changes in the

corpus luteum which is consistent with the

results of the present study (44). Salvadori

et al. (1988) found that Malathion induces

chromosomal aberrations in somatic cells

(bone marrow) and primordial germ cells

(primary spermatocytes) (45). Studies have

also indicated that oxidative stress plays an

important role in pathogenesis of various

diseases, including cancer, diabetes,

cardiovascular diseases, Parkinson's

disease, schizophrenia, atherosclerosis,

pulmonary diseases, and cataracts (46).

Oxidative stress is caused by free radicals

and mitochondria are identified in the

main region of the production of free

radicals (38). When cell properties are

changed, oocytes and follicular cells lose

their normal efficiency. According to what

was mentioned and the fact that the

production of primordial follicles in

mammals in embryonic period is a local

phenomenon caused by ovarian factors and

germ cells, and considering the fact that

the continued growth of the follicles is

influenced by the hypothalamic and

pituitary hormones, a significant decrease

in primordial, primary, and secondary

follicles can thus be justified. In this study,

significant changes in the pituitary

hormones were also observed. As

mentioned, cellular damage caused by

pesticides can be caused by various

reasons, but the impact of Malathion on

the structure of DNA and cellular proteins

can change cell function. Considering the

possible effect of Malathion on changes in

natural concentrations of gonadotropins at

the time of injection, it can be concluded

that with abnormal development of

follicular cells, reduced thickness of

granulosa layer and follicular sheath,

estrogen production decreases which is

consistent with our results. Development

of follicular sheath is directly affected by

factors secreted from the granulosa layer

and reduced granulosa layer can thus affect

the growth of follicular sheath (38).

Studies have shown that green tea has

antioxidant properties and eliminates free

radicals (23) through the inhibition of

cytochrome P450 expression (47, 48). It

has also been reported that green tea

extract can reduce oxidative damage

caused by cyclosporine A (27). Studies

have shown that green tea catechins

prevent lipid peroxidation caused by

chemicals in the liver and kidneys of

animals (49).



56

Conclusion

According to the results of histological

studies and ovarian hormones, it can be

concluded that Malathion insecticide

destroys ovarian tissue and reduces the

activity of the pituitary-gonadal hormones.

These effects can be generalized to

humans to a less extent. However, the

manner of use and extent of exposure to

this insecticide is very important.

Therefore, care should be taken in

generalizing these results to humans. Due

to its antioxidant property, green tea

extract can reduce the negative effects of

Malathion insecticide, so the use of green

tea extract is recommended to reduce the

harmful effects of Malathion.

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