TEST EFFECTIVENESS ANTIMICROBA EXTRACT ETHANOL SEEDS CORN

(Caesalpinia bondus (L.) Roxb) ON GROWTH OF BACTERIA Staphylococcus aureus

Khairunnisa, Heti Rais Khasanah, Heru Laksono

Politeknik Kesehatan Kementerian Kesehatan Bengkulu

Prodi DIII Analis Kesehatan

ABSTRACT

Background: Staphylococcus aureus is a 1 μm diameter cocci-shaped bacteria that in Gram-

positive staining, when viewed under a microscope shaped like a grape. Staphylococcus is immobile
(nonmotil), does not form spores, and is a positive catalase. The bacteria is heat resistant up to 50 o
C, high salinity, and drought resistant. Many strains of this bacterial colony form an ivory or orange
yellow pigment. S.aures are widespread in nature and some live as normal flora in humans present
in the axilla, the inguinal and perineal regions, and the anterior nares (nares). Approximately 20-
30% of humans carry S.aureus in the nasal cavity and skin. Kebiul seeds contain saponins,
flavonoids and steroids that can inhibit growth in Staphylococcus aureus bacteria.
Purpose: This study aims to determine the effectiveness of antimicrobials contained in the extract
of seed kebiul (Caesalpinia bondus (L.) Roxb) on the growth of Staphylococcus aureus bacteria,
using Quasy Laboratory Experiments The data were tested by using univariate data analysis that is
to see the description average inhibitory zone diameter from the treatment of each concentration on
the seed of the kebiul.
Method: The method used is disc diffusion method by using ethanol solvent.
Results: This study showed that concentrations that could inhibit the growth of Staphylococcus
aureus bacteria were at concentrations of 20% that were categorized as moderate and 100%
concentrations were of moderate categorization.
Conclusions: The discovery of inhibitory zones in the variation of ethanol extract concentration of
seeds of kebiul from 20%, 40%, 60%, 80% and 100% resulted in average inhibition zones 6.87 mm,
7.81 mm, 8.25 mm, 8.87 mm dan 9.87 mm. at a concentration of 20% found the most effective
minimum inhibition against Stahylococcus aureus bacteria.
Keywords: Caesalpinia bondus (L.) Roxb, Staphylococcus aureus, and Effectiveness

PRELIMINARY
Infection is a disease that causes damage in the body or tissues caused by organisms such as
bacteria, viruses, and fungi. Infection occurs because the environment is too stretched with bacteria.
Based on data from the Provincial Health Office of Bengkulu in 2015 cases of skin infections that
occurred as much as 5.611 in Bengkulu City (Dinkes, 2015).
Liver damage can occur due to exposure of liver cells to toxic substances at a certain dose and
time One cause of skin infections are Staphylococcus aureus bacteria. Staphylococcus aureus is
generally found to be more than 20-30% in the nose and skin of adults. Staphylococcus aureus
bacteria can infect skin such as ulcers, acne, and wound infections. Ulcers or abscesses, acne and
ulcers, from the Staphylococcus aureus lipase enzyme will release fatty acids from lipids and cause
tissue irritation (Putri, 2013).
Treatment of infectious diseases that is by means of antibiotics, antibiotics act as an inhibitor
or killing infecting microbes. Antibiotics have been found nowadays some of them are ineffective
because of the large number of antibiotic resistant microbes (Akram, 2016). To prevent the
occurrence of bacterial resistance to antibiotics should be developed research in the discovery of
new drugs derived from nature (Brook, 2014).
The results support that medicinal plants do contain substances or compounds that are
clinically proven beneficial to the health of one type of plant used as a drug by the community is the
seed kebiul (local language) (Putra, 2013). Herb plants are found in South Bengkulu Regency. This
plant grows wild in the smallholder plantation bordering the protected forest. The herbaceous plant
is a single-seeded plant, its trunk is propagating and the entire surface of barbed stems (Son, 2013).
These seeds of kebiul are traditionally used by the community as a cure for malaria, diabetes
(diabetes mellitus), high blood pressure, and kidney stones. According to the experience of
medicinal society using seeds kebiul this has a good healing effect (Kusrahman, 2012). Kebiul
seeds contain compounds of flavonoids, alkaloids, saponins, triterpenoids (Nazeerullah, 2012).
Based on the test of phytochemical testing of phytochemical laboratory at Biology University of
Bengkulu, there are 3 types of active compounds namely flavonoid, saponin and streoid
Seeing the problem above, it is important to do research on antimicrobials that are more
effective and safe against Staphylococcus aureus bacteria such as kiul seed extract. . So researchers
are interested in reviewing more about the effectiveness of seed kebiul against Staphylococcus
aureus bacteria.

RESEARCH PURPOSES
To determine the effectiveness of antimicrobial contained in the extract of seed kebiul
(Caesalpinia bondus (L.) Roxb on the growth of Staphylococcus aureus bacteria.

RESEARCH METHODS
This research uses Quasy Laboratory Experimental research. This research has been
conducted at this Research Laboratory conducted in Integrated Laboratory of Poltekkes Kemenkes
Bengkulu on December 2017 - June 2018. Samples in this research are seeds of cultivation plants
(Caesalpinia bondus (L.) Roxb) that have not been dried and cultured Staphylococcus aureus
sufficiently.
A. Tools and Materials
1. Tools
Erlenmeyer 250 mL (pyrex) ®, 500 mL beaker glass (pyrex) ®, petri dish, 100 mL
(pyrex) ® pumpkin, test tube (pyrex) ®, 50 mL (pyrex) ® measuring cup, 75 mL funnel
(pyrex ), Watch glass, vacump pump, 10 μL micropipet, tip, spatula, spray bottle, cotton
swab (sterile sticky cotton), ose needle, tweezers, scales (analytical balance), autoclave,
oven (LabTech LDO-030E) incubator (NUVE EN 120) ®, hotplate, bunsen, disc disc
blank and peanut paper.
2. Materials
Seeds, 96% ethanol, aquades, disc disks blank, Mueller Hinton Agar (MHA), BHIB,
pure Staphylococcus aureus culture.
B. Ways Of Working
1. Preparation Of Sample Extract Solution
Samples used in the form of seeds kebiul from the plant kebiul, seed kebiul done drying
in a way in sangria then done soaking. Maseration was performed 7 days using 96%
ethanol with a ratio of 1: 10 samples and ethanol. To separate the extract and sample is
done filtering. The concentration of ethanol extract sample of 96% of honey beads made
from 20%, 40%, 60%, 80%, and 100%. The extracts were then obtained as antibacterial
substances of the sample to be tested.
2. Tool Sterilization
All tools to be used are washed until clean and continued drying. The next step was dry
sterilizer using oven for 2 hours at 180o C.
3. Media Creation
Weighing 4.08 grams of Mueller Hinton Agar then inserted into 250 mL Erlenmeyer,
dissolved in 120 mL of aquades, heated in a hot plate until homogeneous, sterilized in an
autoclave with a temperature of 121o C for 15 minutes of 1 atm pressure, subsequently
poured into each sterile dish dish, then allow the media a few minutes to freeze, then
store in an upside-down on the cold cabinet.
 4. Bacterial Regeneration
Taking bacteria one eye ose from pure culture of bacteria to be used. Then inoculated
with BHIB media and cubed for 24 hours at 37o C.
5. Antibacterial Testing
Prepared medium Mueller Hinton Agar that has been cold and solidified. Planted with
Staphylococcus aureus bacteria, planted bacteria leveled to the entire surface of Mueller
Hinton Agar by using swab cotton is done in LAF (Laminar air flow). Taken discs put in
Mueller Hinton agar medium that has been planted with bacteria. Then the discs are
dropped with a concentration variation that has been prepared as much as 10 μL to
spread evenly across the disc surface. The incubation was performed for 1 x 24 h at 37°
C. The following day was observed and measured inhibitory zone diameter with dark or
dark background paper with direct eye and in contrast with the inhibitory zone of the
control.

RESEARCH RESULT
Result of analysis of analysis of antimicrobial effectivity test of ethanol extract of seed of
kebiul (Caesalpinia bondus (L.) Roxb) to Staphylococcus aureus bacteria presented in table 4.1
below.
Table 4. 1 Univariate test results of antimicrobial effectiveness of ethanol extract of seed kebiul
(Caesalpinia bondus (L.) Roxb) against Staphylococcus aureus bacteria

Univariate analysis aims to analyze one variable. From the results of the research in Table 4.1
it was found that the effectiveness of antimicrobial extract of ethanol seed kebiul (Caesalpinia
bondus (L.) Roxb) against Staphylococcus aureus bacteria given the treatment of concentration
variation from the smallest concentration of 20% to the largest concentration of 100% showed an
increase that was directly proportional to each other. Where the concentration of 20% obtained an
average of 6.75 mm while at a concentration of 100% obtained average inhibition zone 9.75 mm.

DISCUSSION
Quasy Laboratory Experimental Research aimed to find out antimicrobial ability of ethanol
extract of seed kebiul (Caesalpinia bondus (L.) Roxb) to Staphylococcus aureus. The method used
in this research is agar diffusion. The disc containing the antimicrobial agent is placed on the agar
medium which has been planted with microorganisms that will diffuse on the agar agar medium.
Clear areas identify the presence of microorganisms barriers by antimicrobial agents on the surface
of the agar medium (Pratiwi, 2010). In this study the inhibitory zone is used as an indication of
obstruction in Staphylococcus aureus bacteria measured using a ruler (ruler) expressed in units of
millimeter (mm).
Inhibitory zone diameter with concentration variation in Staphylococcus aureus bacteria was
compared with the inhibitory zone around the disc containing positive control (tetracycline) and
negative control (aquades). If the drag zone produced by the ethanol extract of the seed of kebiul is
greater than the positive control, the ethanol extract of the seed of kebiul is more effective as
antibacterial than the positive control, whereas if the drag zone produced by the ethanol extract of
the seed of kebiul is smaller or nonexistent than the positive control the ethanol extract of the seed
of kebiul is not as effective as antibacterial. The use of negative controls aims to ensure that there is
no antibacterial effect of the solvent. If the negative control has inhibition zone, the antibacterial
effect on the ethanol extract of the seed of kebiul will decrease its validity. From the result of the
research that has been done with the variation of ethanol extract concentration of seed of kebiul, it is
found that it has antibacterial effect, inhibition zone that formed at each variation of extract
concentration 20%, 40%, 60%, 80%, and 100% 6.75mm, 7.75mm, 8.25mm, 8.75mm and 9.75mm.
While the positive control produces an average drag zone of 19.50 mm.
This antibacterial effect comes from compounds contained in the seeds kebiul flavonoids,
steroids and saponins. Fertilizer Flavonoid is a group of phenol compounds with the largest and has
a working mechanism in inhibiting bacterial growth and the way of inactivation of proteins
(enzymes) in the cell membranes that result in the structure of proteins to be damaged (Rinawati,
2010). Steroids are triterpenoid compounds that are suspected of working mechanisms of bacterial
inhibition by damaging cell membranes resulting in cell leakage (Taroreh, Rumampuk and Siagian,
2016). Saponin is a polar-shaped surfacta that will break the fat layer on the cell membrane that
ultimately lead to disruption of cell membrane permeability, it causes the diffusion process of
materials or substances required by the fungus can be disrupted, eventually the cell swells and
breaks (Sugianitri, 2011 ).

CONCLUSION
Based on the result of antimicrobial effectivity test of ethanol extract of seed kebiul
(caesalpinia bondus (L.) Roxb) on the growth of staphylococcus aureus bacteria can be concluded
as follows:
1. The discovery of drag zones on the concentration variation of ethanol extract of horn seeds
from 20%, 40%, 60%, 80% and 100% resulted in average inhibition zones 6.75 mm, 7.75
mm, 8.25 mm, 8.75 mm and 9.75 mm.
2. Found at a concentration of 20% already has the most effective minimum inhibition
against Stahylococcus aureus bacteria.

SUGGESTION
For Educational Institutions as inputs and reading materials for students in Poltekkes
Kemenkes Bengkulu as a reference comparison for further research.
For the Community it is advisable for people to use the powder of kebiul seed as an
alternative medicine. Because, the seed api extract has been effective to inhibit the growth of
Staphylococcus aureus bacteria.
For other researchers it can be used as a reference to perform research in advanced stages
such as using solvents other than ethanol 96% eg ethyl acetate and ethanol 70%, can replace the
maceration method using infusa method, using bacteria other than Staphylococcus aureus bacteria,
and can decrease or increase concentration used.

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