Testing Inhibitory Power of Avocado Seed Extract (Persea americana

Mill.) for the Growth of Staphylococcus aureus and Escherichia coli

Ni Wayan Sukma Antari

, Ida Ayu Manik Damayanti

, A. A. Istri Mas Padmiswari

Nadya Treesna Wulan Sari

, I Gusti Bagus Teguh Ananta

and Putu Rima Sityadewi

Faculty of Health, Institute of Technology and Health Bali, Bali, Indonesia

Keywords: Avocado Seeds, Staphylococcus aureus, Escherichia coli.

Abstract: Infections are commonly treated using traditional medicine, especially made of natural ingredients such as

plants. The use of medicinal plants for complementary or alternative traditional medicine, increases.

Medicinal plants are considered to have fewer side effects when compared to chemical-based drugs. Apart

from that, low price and accessibility are two aspects that medicinal plants contribute to be traditional

medicine. One of the medicinal plants is avocado seeds (P. americana) which has the potential to be an

alternative source of antibacterial ingredient. This study tested the inhibition power of avocado seed extract

for the growth of E. coli and S. aureus bacteria; furthermore, it determined levels of phytochemical

compounds (tannins and flavonoids) in avocado seed extracts. The experimental design used in this study was

Completely Randomized Design (CRD). This study involved four types of avocado seed extract

concentration: 10%, 15%, 20%, and control. Data analysis was carried out quantitatively using a computer

statistics program (SPSS 22.0 for Windows). To test the normality of the data, the Kolmogorov-Smirnov test

was carried out; if the data were not normal, the Kruskal Wallis test was carried out. To see the homogeneity

of the variance, the Leven's test was employed. The effect of the treatment was tested using the One-way

ANOVA test. If the test showed a significant result, the process was continued by the performing Duncan's

multiple range test with a confidence level of 5% (p < 0.05).

1 INTRODUCTION

Avocado is a popular fruit in Indonesia. Generally,

avocados have thick yellowish-green flesh and a

brownish seed in the middle. The fruit has widely

been used as traditional medicine to treat various

diseases. The avocado’s flesh can reduce pain and

treat canker sores. Avocado leaves are usually used to

treat nerve pain, stomach pain, reduce high blood

pressure, and treat kidney stones (Anggrella, 2014;

Widiyanti, Cacik, & Winata, 2017).

Currently, many infections are treated using

traditional medicine, especially those made of natural

ingredients such as plants. Natural ingredients are

commonly used for either complementary or

https://orcid.org/0000-0003-2198-4486

https://orcid.org/0000-0001-5863-8734

https://orcid.org/0000-0003-2399-7555

https://orcid.org/0000-0001-9375-0238

https://orcid.org/0000-0002-0750-8849

https://orcid.org/0000-0002-3917-0168

alternative traditional medicine. Medicinal plants are

considered to have fewer side effects when compared

to chemical-based drugs. Besides, they have

affordable price and accessibility (Jiménez-Arellanes

et al., 2013).

Persea americana Mill. is a generally edible fruit

known as avocados which grow in tropical countries.

However, not all parts of avocados are effectively

used. For example, avocado seeds are less utilized

than avocado flesh (Gunawan, Setiabudy, &

Nafrialdi, 2007).

Avocado seeds are known to give

therapeutic effects, including antibacterial, anti-

oxidant, anti-inflammatory, anti-fungal and analgesic

effects (Lianti, 2014; Malangngi, Sangi, & Paendong,

2012).

Antari, N., Damayanti, I., Padmiswari, A., Sari, N., Ananta, I. and Sityadewi, P.

Testing Inhibitory Power of Avocado Seed Extract (Persea americana Mill.) for the Growth of Staphylococcus aureus and Escherichia coli.

DOI: 10.5220/0011939000003576

In Proceedings of the 2nd Bali Biennial International Conference on Health Sciences (Bali BICHS 2022), pages 54-58

ISBN: 978-989-758-625-5

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

Pathogenic bacteria that often infect humans

include S. aureus and E. coli causing intestinal

inflammation and diarrhea (Hermawan, Hana, &

Wiwiek, 2007). The bacteria are distinguished based

on the composition of their cell walls and the nature

of their staining. They include Gram-negative and

Gram-positive bacteria that can cause diarrhea.

Diarrhea can cause the body to lose a lot of fluids. It

is commonly found and becomes a major contributor

to mortality in Indonesia.

The number of infectious diseases in Indonesia

increases every year due to several factors, e.g., poor

public awareness of hygiene, shortages of trained

health workers, increasingly dense population,

insufficient knowledge, and lack of guidelines

(Bontjura, 2015). Overuse dosage of antibiotics and

the lack of government policies may lead to bacteria

resistance (Omojate Godstime, Enwa Felix, Jewo

Augustina, & Eze Christopher, 2014).

Research conducted by Idris et al. on the effect of

3.25 g ethyl acetate avocado seed extract (Persea

americana Mill.) against Staphylococcus aureus and

Streptococcus pyogenes shows antibacterial activity

was found in 37-mm and 25-mm inhibition zones.

Ethanol extract of avocado seeds becomes an

alternative agent that can fight against Enterococcus

faecalis. Avocado seed extract amounting to 10%

plays a role in the antibacterial activity with an

inhibition zone diameter of 2.3 ± 0.12 mm.

Back-to-nature lifestyle becomes quite popular at

this time; hence, people start utilizing various natural

materials, including medicinal plants for treatment

(Dalimartha, 2008; Malangngi et al., 2012).

Medicinal plants have more economic values and

smaller side effects compared to synthetic drugs

(Somchit et al., 2011). One of the medicinal plants is

avocado seed (P. americana) which can provide an

antibacterial effect. This study aimed to test the

inhibition power of avocado seed extract for the

growth of E. coli and S. aureus bacteria.

2 MATERIALS AND METHODS

This study was conducted at the Agricultural

Laboratory and the Laboratory of Biosciences of

Udayana University from January to April 2020.

Completely Randomized Design (CRD) was used as

the research design. This study involved four groups,

namely:

1 Control group

2 Experiment group with 10% of avocado seed

extract concentration

3 Experiment group with 15% of avocado seed

extract concentration

4 Experiment group with 20% of avocado seed

extract concentration

All groups were given different avocado seed

extract concentrations to test the inhibition

power of S. aureus and E. coli.

1. Making avocado seed extract

Preparation of avocado seed powder was done by

washing the seeds first to remove dirt and then

draining them. The seeds were then cut into small

pieces using a knife. They were then dried using

traditional drying methods under sunlight for six

hours with occasional stirring. The dry weight was

determined by subtracting the initial leaf weight from

the final dry leaf weight multiplied by 100% until the

total moisture content reached 13%. Seeds that were

completely dry were then grinded using a blender.

The dry powder was then sifted through a coffee filter

and stored in a plastic container.

2. Avocado seed extraction

In extraction process, 100 grams of avocado seed

powder was macerated in 1,000 ml of each solvent

(ethanol, n-hexane and ethyl acetate) (1:10 ratio

(w/v)) for 24 hours at 25

C. The powder sample was

then filtered with a filter paper. The residue was

macerated again in the same way, up to two times (for

two days). The macerated extract or filtrate was then

collected together and evaporated to separate the

solvent. Evaporation was carried out using a rotary

evaporator at a temperature of 60

C for n-hexane

solvents and 70

C for ethanol and ethyl acetate

solvents, until the solvent evaporated completely.

This process was done to gather a thick extract of

avocado seeds.

3. Bacterial inhibitory test

The test was preceded by making Nutrient Agar (NA)

media. To make the media, 100 μl of each suspension

of E. coli and S. aureus were taken from the bacterial

decay and then mixed in each medium and vortex.

Once becoming homogeneous, the medium was

poured into a petri dish to solidify. In the media with

the bacteria, seven holes were made per petri dish

using a sterile aluminum pipe with a diameter of 0.5

cm. Then, the extract solutions at various

concentrations were inserted into the holes; the

solvent used as the negative control group and

chloramphenicol solution as the positive control

group amounting to 100 μl each were poured into the

other holes. The petri dishes were left for 10 minutes

during the diffusion process and then incubated for 24

hours at 36 – 37

C. After 24 hours, the area of

bacterial growth and the hole diameter in millimeters

were measured using a caliper at least three times.

Testing Inhibitory Power of Avocado Seed Extract (Persea americana Mill.) for the Growth of Staphylococcus aureus and Escherichia coli

4. Determination of minimum inhibitory

concentration

The media containing the test solution, solidified

positive control and negative control groups were

observed visually. The growth of microbial colonies

was slow down perhaps due to the turbidity of the

media; the smallest concentration showed inhibition

power for microbial growth after the diameter of the

inhibition zone was measured by using a caliper.

Observation was made after optimum incubation of

microorganisms for one time. The inhibition power of

the ethanol extract of avocado seed for the growth of

E. coli and S. aureus was from the clear zone which

was measured with calipers and then subtracted by

the 0.5-cm diameter of the well.

3 STATISTICS

Data analysis was carried out quantitatively using a

computer statistics program (SPSS 22.0 for

Windows). To test the normality of the data, the

Kolmogorov-Smirnov test was carried out; if the data

were not normal, the Kruskal Wallis test was carried

out. To see the homogeneity of the variance, the

Leven's test was carried out. Furthermore, the One-

Way Anova test was employed to see the effect of the

treatment with avocado seed extract. If the test

resulted in a significant result, the Duncan's multiple

range test was utilized with a confidence level of 5%

(p < 0.05).

4 RESULTS

This study was an experimental test to see whether

there is inhibition of avocado leaf extract (Persea

americana Mill) for the growth of E. coli and S.

aureus bacteria. Four treatment groups consist of a

control group and three different dose treatments,

namely avocado leaf doses of 10%, 15% and 20%.

Table 1 shows the results of antibacterial test after the

administration of avocado seed extract.

Table 1: Antibacterial test results of avocado leaf extract

(Persea americana Mill) for the growth of S. aureus

bacteria.

No Treatment Diameter of Inhibition

Zone against S. aureus

(mm)

Length

(mm)

Average

(mm)

II III

Extrac

10% 3.83 5.65 4.43 13.91 4.63

Ekstrak 15% 4.62 4.46 5.84 14.92 4.97

Ekstrak 20% 6,89 6,77 6,54 20,2 6,73

ontrol 21,44 21,44 21,44 64,32 21,44

Table 1. Shows the diameter of the inhibition zone

at concentrations of 10%, 15% and 20% has an

average value of: 4.63, 4.97, 6.73 and the control

group, 21.44

Table 2: Antibacterial Activity Test Results of avocado leaf

extract (Persea americana Mill) on the growth of E. coli

bacteria.

No Perlakuan Hasil Diameter Zona

Hambat Terhadap Bakteri

S. aureus

(mm)

Jumlah

(mm)

Rata-rata

(mm)

II III

Ekstrak 10% 3,63 5,23 4,33 13,19 4,39

Ekstrak 15% 5,77 4,23 5,67 15,67 5,22

Ekstrak 20% 7,34 6,34 6,67 20,35 6,78

ontrol 21,44 21,44 21,44 64,32 21,44

Table 2. Shows the diameter of the inhibition zone at

a concentration of 10%, 15% and 20% has an average

value of: 4.39, 5.22, 6.78 and the control group, 21.44

Table 3: Categories of Inhibition Zone Diameter.

Inhibition Zone Diameter Category

5 mm or less weak

5 - 10 mm Currently

10 - 20 mm strong

20 or more Very Strong

Based on the results of the normality test that was

carried out using the Shapiro-Wilk Test statistic using

SPSS version 2.2 software, it showed a significance

number > 0.05 which indicated that the sample in this

study was normal. The average diameter of the

inhibition zone of avocado leaf extract in inhibiting

the growth of Staphlococcus aureus and Escherichia

coli is shown in Table 4 below.

Table 4: Mean Diameter of Inhibition Zone of Avocado

Leaf Extract Against S. aureus and E. coli.

Extract

concentration

S. aureus E. coli

Ekstrak 10%

4,63± 0,45c 4,39± 0,33b

Extract 15%

4.97± 0.49d 5.22± 0.38c

Extract 20%

6.73 ± 0.76f 6.78± 0.48d

Control

21.44± 0.43g 21.44± 0.43g

Information ** = mean ± standard deviation is the

average of six repetitions. Different letters in both

columns and rows show significantly different results

(p < 0.05) based on the test (ANOVA).

Bali BICHS 2022 - The Bali Biennial International Conference on Health Sciences

5 DISCUSSION

Based on Tables 1 and 2, concentration of 20% is the

highest concentration with moderate inhibition for the

growth of S. aureus and E. coli; as a reference from

Table 3, the diameter of the inhibition zone 5-10 is in

the moderate category.

Avocado leaves are plants that are widely used for

treatment because they have an antibacterial effect.

Avocado leaf extract is able to inhibit the growth of

S. aureus and E. coli. The leaves consist of active

compounds resulting in antibacterial properties such

as flavonoids, saponins, alkaloids, tannins, and

polyphenols.

Flavonoids have antibacterial effects by inhibiting

bacterial DNA function. In this process, some errors

might be found in bacterial replication and

translation. Inhibition occurs when the bacterial

cytoplasmic membrane which consists of lipids and

amino acids removes the alcohol group in the

flavonoid compound. This process will damage the

cell walls, and thus the compound can enter the

nucleus of the bacterial cells (Pisoschi & Pop, 2015).

Saponin compounds in avocado seeds can also

have antibacterial properties which have the same

mechanism of action as flavonoid compounds.

Tannins are active compounds in avocado seeds. The

way tannin produces an antibacterial effect is by

precipitating bacteria and their proteins. The effect of

the antibacterial content in tannin will disrupt the

function of genetic materials and inactivate enzymes,

causing bacterial cells not to form (Dwi, Tanjung,

Chairani, Bikarindrasari, & Dewi, 2021).

The mechanism of alkaloids in inhibiting bacterial

growth works by damaging the constituent

components of peptidoglycan in bacterial cells; as a

result, the cell wall layer is not formed completely

and causes cell deaths. In addition, alkaloids have an

inhibitory mechanism by binding to DNA

presumably because the alkaloids have a base group

that contains nitrogen. This base group will react to

acidic compounds in bacteria such as DNA which is

the main constituent of the cell nucleus. With the

disruption of DNA, the synthesis of proteins and

nucleic acids in cells will be disrupted. As a result,

cell metabolism is distracted, and bacterial growth is

inhibited or stopped (Isnarianti, Wahyudi, & Puspita,

2013).

6 CONCLUSIONS

Avocado leaf extract is effective in inhibiting the

growth of S. aureus and E. coli with an average

diameter of the inhibition zone that varies depending

on the concentration of each extract. The content of

metabolite compounds found in avocado leaves has

the potential to inhibit the growth of these bacteria.

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Bali BICHS 2022 - The Bali Biennial International Conference on Health Sciences