The Effect of Ethanol Extract of Okra (Abelmoschus esculentus L.)

Moench) on Tumor Growth in Breast Cancer Rats Model Induced by

Benzo-a-Pyrene

Syarifah Riska Mela Putri

, Salomo Hutahaean

and Syafruddin Ilyas

, Widya Syahfitri

and Fitri

Elizabeth

Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia

Keywords: Okra (Abelmoschus esculentus), BAP-induced Breast Cancer, Angiogenic Effect.

Abstract: Okra plant (Abelmoschus esculentus L. Moench)) is known as a medicinal plant that is traditionally used in

treating various diseases. This study aims to investigate the effect of ethanol extract of okra seed pods on the

growth of tumors in the rat breast cancer model. The experiment was carried out using a completely

randomized design, with 5 treatments and 6 replications. These treatments were: 2 control groups

(K- = normal group; K+ = breast cancer rat model due to benzo-a-pyrene or BAP injection), and 3 extract

groups (breast cancer model rats were given okra extract at dose of 150, 300, or 450 mg/kg BW (body

weight), respectively). The results showed that tumor growth occurred slowly in the first 3 months, but after

that, the growth accelerated marked by an increase in tumor weight and tumor diameter. The average tumor

weight due to BAP induction was 0.6 g, administration of the extracts of doses of 150 and 300 mg/kg BW

gave no significant effect (P>0.05), but unexpectedly in doses of 450 mg/kg BW the average weight was 4.5

g (P<0.05). There is an indication that high-dose extracts stimulate tumor growth. The number of

blood vessels has the same pattern as tumor growth. In the group of rats which were induced by BAP

the number of blood vessels was 6 per visual area, whereas in the extract group the number increased by

9 to 10 vessels. The result suggest that A. esculentus extract may have an angiogenic effect at high doses.

1 INTRODUCTION

Breast cancer is one type of cancer causes a high

mortality rate, especially against women. Breast

cancer ranks second after cervical cancer in

Indonesia. In the United States, breast cancer is the

second leading cause of death in women (after lung

cancer) (Price & Lorraine, 2006). Breast cancer is an

important public health problem, because of its high

mortality and morbidity. Based on research results in

the Jakarta Breast Cancer in 2001 to 2003, of the

2,834 people who had breast lumps examined, 2,229

of them (78%) were benign tumors, 368 people

(13%) were diagnosed with breast cancer and the

rest were infections and breast congenital

abnormalities (Djoerban Z, 2003).

The use of plants for medicinal purposes is

common practice. Okra has been widely used in

various traditional treatments. Even today, many

communities use plants as the main source of

treatment. Okra (Abelmoschus esculentus) is one of

the most widely used plants for treatment. This plant

began to be used to treat various diseases such as

cancer, microbial infections, hypoglycemia,

constipation, urinary retention and inflammation

(Kumar, Patil, Patil, Patil, & Paschapur, 2009;

Tomoda, Shimizu, Gonda, Kanari, & Yamada,

1989).

Okra, Abelmoschus esculentus L. (Moench)

commonly known as "lady’s finger" is cultivated as

an important vegetable crop in tropical, subtropical

and warm temperatures throughout the world.

(Benchasri & Benchasri, 2012; S. Kumar et al.,

2010; Lamont, n.d.; Ndunguru & Rajabu, 2004;

Oyelade et al., 2003). Okra is rich in phenolic

compounds and has high antioxidant activity. Okra

has the potential to prevent several deadly diseases

such as cardiovascular disease, type 2 diabetes,

digestive diseases and some types of cancer

(Gemede et al., 2015). Based on the latest research

on lectins isolated from okra (Abelmoschus

esculentus) tested in human breast cancer and

502

Putri, S., Hutahaean, S., Ilyas, S., Syahﬁtri, W. and Elizabeth, F.

The Effect of Ethanol Extract of Okra (Abelmoschus esculentus L.) Moench) on Tumor Growth in Breast Cancer Rats Model Induced by Benzo-a-Pyrene.

DOI: 10.5220/0010208200002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 502-508

ISBN: 978-989-758-556-2

fibroblast cells in the skin, okra has potential as an

anti-tumor (Monte et al., 2014) and dried okra seeds

have the potential to reduce tumor necrosis rates

(Okada et al., 2010). This shows that okra has

potential as an antioxidant contributor and promising

chemopreventive agent for the treatment of diseases

in human

Based on the description above, the research that

will be carried out aims to find the effect of okra as

an anti-tumor by looking at its effect on the growth

of cancer cells by using the right dosage so that it

can be a source of information for those who need to

be developed into an alternative cancer treatment,

especially cancer breast with raw materials derived

from plants.

2 MATERIALS AND METHOD

This research has been carried out at the Laboratory

of Structure and Development of Animals, Faculty

of Mathematics and Natural Sciences, Laboratory of

Organic Chemistry and Natural Materials, Faculty of

Mathematics and Natural Sciences, Pathology

Laboratory of Anatomy, Faculty of Medicine,

University of North Sumatra.

2.1 Experimental Design

This study uses a completely randomized design

(CRD) method which consists of 3 treatments with

different concentrations and 2 treatments as controls.

Both control and treatment each consisted of 6

replications so that there were 30 rats used.

2.2 Making of Okra Fruit Extract

The okra fruit was obtained from the Growth Center

Laboratory of KOPWIL 1, North Sumatra. After

being collected from the field, the okra fruit that has

been washed clean is dried in an oven at 40oC until

it meets the requirements of general moisture

content. Simplisia that is dried and then made into

powder until smooth and sieved with a B30 sieve.

Making ethanol extract of okra fruit is done by

maceration, ie okra fruit powder is put into a brown

bottle and ethanol is added until submerged and then

stirred and left for 1 night. Take the filtrate and re-

soak the residue with ethanol until a clear filtrate is

obtained. The filtrate obtained was separated with a

rotary evaporator so that a thick extract was

obtained.

2.3 Acclimatization of Experimental

Animals

The experimental animals used were rats (Rattus sp.)

Strains of healthy and fertile female Wistar aged 8-

11 weeks with a weight of 200-250 g of 30 animals

obtained from the North Sumatra Animal Disease

Investigation Center Medan. Rats are kept in cages

that are kept clean and feed and drink are done every

day on an ad libitum basis. Handling of experimental

animals by the requirements of the applicable code

of ethics and before the research is conducted, an

application for an Ethical Clearance to the Health

Research Commission of the North Sumatra Region

of Medan is submitted.

2.4 Extract Administration

Carcinogenic induction is carried out by injecting a

solution of benzo (α) pyrene to the subcutaneous

tissue of the Wistar strain rat in the mammary gland.

Benzo (α) pyrene 50 mg/kg BW was dissolved in

olive oil and given a single dose subcutaneously

then observed the emergence of tumor mass in the

breast of the rat by palpation (± 4 months), then

continued with the test substance for 15 days.

The administration of treatment in this research

a. negative control: without treatment

b. positive control: administration of Benzo [a]

Pyrene (BAP) at a dose of 50 mg/kg body weight

which will induce the growth of cancer cells in

experimental animals

c. Treatment I: administration of Benzo [a] Pyrene

(BAP) 50 mg/kg BW + ethanol extract of okra

150 mg/kg BW

d. Treatment II: administration of Benzo [a] Pyrene

(BAP) 50 mg/kg BW + ethanol extract of okra

fruit 300 mg/kg BW

e. Treatment III: administration of Benzo [a] Pyrene

(BAP) 50 mg/kg BW + ethanol extract of okra

fruit 450 mg/kg BW

2.5 Making the Histological

Preparations

The histological preparation of the paraffin method

begins with fixation, washing, dehydration, clearing,

infiltration, embedding, slicing, attachment,

deparaffination, staining, closing and labeling

(Suntoro H, 1983).

The Effect of Ethanol Extract of Okra (Abelmoschus esculentus L.) Moench) on Tumor Growth in Breast Cancer Rats Model Induced by

Benzo-a-Pyrene

503

2.6 Hematoxylin Eosin Staining

Hematoxylin-eosin staining is a standard coloring to

determine the general structure of cells and tissues in

an organ. The hematoxylin-eosin staining process

starts from the deparaffination process followed by

the rehydration process using multilevel alcohol then

the preparations werw stained with haematoxylin

and rinsed with distilled water for a few moments.

The preparations are stained again with eosin and

then proceed with the dehydration process. The

preparations were clarified with xylol solution and

continued with the mounting process (Suntoro,

1983).

2.7 Test Parameter Analysis

2.7.1 Visual Morphological Analysis

The morphological observations in this study were

body weight, tumor weight, tumor diameter.

2.7.2 Histological Analysis of Mammary

Gland

The histological part of the mammary gland

observed in Hematoxylin-eosin staining is

vascularization of the mammary gland. Observations

were made by counting the number of blood vessels

formed in the rat breast organs using a microscope

with a magnification of 100x. Observations were

made as many as 5 fields of view.

3 RESULTS AND DISCUSSION

3.1 Body Weight

Based on research that has been done, the

administration of ethanol extract of okra fruit

(Abelmoschus esculentus (L.) Moench) to the

bodyweight of rats, the highest weight was the

negative control group in which the weight was

261.6 g at week 13 and the lowest body weight was

P2 group which was 201.3 g at week 6. The results

of observations of body weight of rats (Rattus sp.)

can be seen in Figure 1.

Figure 1: Effect of Okra Fruit Ethanol Extract on Body

Weight of Rats (Rattus sp.) K- = Negative Control (mice

not trained); K + = Positive Control (BAP distribution);

P1, P2 and P3 = Treatment with ethanol extract

concentrations of 150 mg/kg okra fruit, 300 mg/kg, and

450 mg/kg; unit in grams (g).

Statistical analysis showed that at week 6 the

body weight of the P3 treatment group was

significantly different (P <0.05) to the body weight

of the treatment group P1 and P2 rats but not

significantly different to the positive and negative

control groups. At the 13th week during the

administration of okra fruit ethanol extract, an

increase in body weight of rats in which the negative

control group was significantly different (P <0.05)

with the treatment groups P1, P2, P3 and not

significantly different from the positive control

group. At the 14th week after administration of okra

fruit ethanol extract there was a decrease in body

weight in which the negative control group was

significantly different (P <0.05) with positive control

and treatment groups P1, P2 and P3.

Week 4 to week 10 there is an increase and

decrease in body weight that is volatile.

Inflammation in mice after BAP administration does

not affect the body weight of mice but the

physiological conditions of rats and other external

factors such as weather can affect the body weight of

mice. The size of mammary gland tumors in mice

affects the body weight of mice. It can be seen in

Figure 1 that there was a gain in weight at week 13

due to a significant increase in tumor size from the

previous weeks so that the tumor mass affected the

body weight of mice. In recent years, there have

been reports showing that mice became fat in

carcinogenicity studies and tumor growth in these

mice. Selection, disease control, improved diet, and

better control of environmental conditions led to an

increase in body weight and life span of mice used

in long-term toxicity studies over the past 2 decades

(Rao et al., 1990). There was a decrease in body

weight of rats in the 14th week after giving ethanol

extract of okra fruit due to the size of the tumor that

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began to shrink so that the tumor mass also

decreased which would affect the body weight of the

rat. Anorexia and weight loss are part of end-stage

cancer syndrome which is a major cause of

morbidity and mortality in cancer (Johnen et al.,

2007).

3.2 Tumor Weight of Mammary Gland

Based on research that has been done, the

administration of ethanol extract of okra fruit

(Abelmoschus esculentus (L.) Moench) on mammary

gland tumor weight (Rattus sp.), it can be seen that

the highest tumor weight was the P3 group in which

the weight was 4.51 g and the lowest was the P1

group which was 0.5 g. The results of observations

of mammary gland tumor weight (Rattus sp.) Can be

seen in Figure 2.

Figure 2: Effect of Okra Fruit Ethanol Extract on Tumor

Weight of Mammary Gland of Rats (Rattus sp.) K- =

Negative Control (mice not trained); K + = Positive

Control (BAP distribution); P1, P2 and P3 = Treatment

with ethanol extract concentrations of 150 mg/kg okra

fruit, 300 mg/kg, and 450 mg/kg; unit in grams (g).

The results of statistical analysis showed that the

tumor weight of the P3 group was significantly

different (P <0.05) to the weight of the positive

tumor group, the treatment groups of P1 and P2. The

increase in tumor weight is caused by the continued

division of cells. Proto-oncogenes are genes that

help cells grow normally. When proto-oncogenes

mutate (change) or there are too many copies, cells

grow out of control. This can cause cancer. When

this gene changes, it no longer suppresses the growth

of abnormal cells and cancer is more likely to

develop (American Cancer Society, 2018).

3.3 Tumor Diameter of Mammary

Gland

Based on research that has been done, the

administration of ethanol extract of okra fruit

(Abelmoschus esculentus (L.) Moench) on mammary

gland tumor diameter (Rattus sp.), it can be seen that

the highest tumor diameter was the P3 group at week

13 which was 1.63 cm and the lowest tumor

diameter was the P2 group at 13 weeks which a

length was 0.64 cm. The results of observations on

the diameter of mammary gland tumors can be seen

in Figure 3.

Figure 3: Effect of Okra Fruit Ethanol Extract on Tumor

Diameter of Mammary Gland of Rats (Rattus sp.) K- =

Negative Control (mice not trained); K+ = Positive

Control (BAP distribution); P1, P2 and P3 = Treatment

with ethanol extract concentrations of 150 mg/kg okra

fruit, 300 mg/kg, and 450 mg/kg; unit in centi meter (cm).

Statistical analysis showed that at week 5th the

diameter of the mammary gland tumor mice in the

positive control group was significantly different (P

<0.05) to the diameter of the mammary gland tumor

in the treatment group P2 and P3 but it was not

significantly different in the treatment group P1.

There was a decrease in the diameter of the

mammary gland in the second week after BAP

administration because at this stage the swollen

mammary glands due to inflammation that formed

after injection had begun to shrink. Inflammation

can cause tumor growth and development through its

influence on cell proliferation, tumor survival and

metastasis (Buckland et al., 2014).

In the following weeks, tumors began to form

marked by the presence of thickening in the skin of

the mammary gland of mice. The primary sign of a

tumor in the mammary gland is the irregular border

of the tumor due to the infiltration process into the

surrounding tissue or unclear boundary (comet sign)

and also changes in tumor mass both in size,

consistency, and shape. The only way to diagnose

gold (gold standard) in breast cancer is by

histopathological examination, with this type of

histology known (type), sub-type and cellular

grading and core grading (Ramli, 2015). One of the

histological examination of tumors is the

determination of Ag-Nor grains in which the

The Effect of Ethanol Extract of Okra (Abelmoschus esculentus L.) Moench) on Tumor Growth in Breast Cancer Rats Model Induced by

Benzo-a-Pyrene

505

Nucleolar Organizing Region (NOR) is a place of

ribosomal biogenesis in the cell nucleus whose

numbers increase with the increase in the activity of

cell protein biosynthesis (Hutahaean et al., 2009).

At week 12 there was a very significant change in

tumor size and metastasis that was marked by the

growth of tumors in other organs both in other

mammary glands and in the abdomen of mice. At

the 13th week during the administration of okra fruit

ethanol extract, a decrease in the diameter of the

tumor in mammary glands occurred. Lectins

contained in okra fruit can inhibit the growth of

mammary gland tumor cells in vitro (Monte et al.,

2014).

3.4 Tumor Vascularisation of

Mammary Gland

Based on research that has been done, the

administration of ethanol extract of okra fruit

(Abelmoschus esculentus (L.) Moench) against

vascularisation in mammary gland tumor can be

seen that the highest number of blood vessels is

found in the P3 group which amounted to 10.1 and

the lowest blood vessel is the negative control group

which amounted to 6.5. The results can be seen in

Figure 4.

Figure 4: Effect of Okra Fruit Ethanol Extract on Tumor

Vascularisation of Mammary Gland of Rats (Rattus sp.)

K- = Negative Control (mice not trained); K + = Positive

Control (BAP distribution); P1, P2 and P3 = Treatment

with ethanol extract concentrations of 150 mg/kg okra

fruit, 300 mg/kg, and 450 mg/kg.

Figure 5: Effect of Okra Fruit Ethanol Extract on Tumor

Vascularisation of Mammary Gland of Rats (Rattus sp.)

K- = Negative Control (mice not trained); K + = Positive

Control (BAP distribution); P1, P2 and P3 = Treatment

with ethanol extract concentrations of 150 mg/kg okra

fruit, 300 mg/kg, and 450 mg/kg. Vascularisation is

indicated by a red circle.

The analysis showed that the number of blood

vessels formed in the negative control group was

significantly different (P <0.05) for all treatment

groups. This is in line with the tumor mass of

mammary glands, where the greater the tumor mass,

the more blood vessels are formed. One of the

secondary features of the presence of a tumor in the

mammary gland is characterized by an increase in

blood vessels (Ramli, 2015). Vascularization or also

called angiogenesis is the formation of new blood

vessels originating from existing blood vessels.

Under pathological conditions, angiogenesis is

needed in the growth process of solid tumors and the

process of metastasis (Hicklin & Ellis, 2005; Lee S.

Rosen, 2002). Tumors require angiogenesis to grow

above 1-2 mm

in size (Lee S. Rosen, 2002).

Angiogenesis is needed for oxygen supply, nutrients,

growth factors and hormones, proteolytic enzymes,

influencing hemostatic factors that control

coagulation and fibrinolytic systems and the spread

of tumor cells to distant sites (Hicklin & Ellis,

2005).

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4 CONCLUSIONS

The results showed that tumor growth occurred

slowly in the first 3 months, but after that, the

growth accelerated marked by an increase in tumor

weight and tumor diameter. The average tumor

weight due to BAP induction was 0.6 g,

administration of the extracts of doses of 150 and

300 mg/kg BW gave no significant effect (P>0.05),

but unexpectedly in doses of 450 mg/kg BW, the

average weight was 4.5 g (P<0.05). There is an

indication that high-dose extracts stimulate

tumor growth. The number of blood vessels has

the same pattern as tumor growth. In the group

of rats which were induced by BAP the number of

blood vessels was 6 per visual area, whereas in the

extract group the number increased by 9 to 10

vessels. The result suggest that A. esculentus extract

may have an angiogenic effect at high doses.

ACKNOWLEDGEMENTS

We would like to acknowledge the support of

DRPM Ministry of Research and Technology and

the Higher Education Republic of Indonesia which

has provided funding for this research.

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