Ekstrak Moringa oleifera Sebagai Antiseptik Terhadap Escherichia Streptococcus Menggunakan Metode Percentage Kill Moringa oleifera Extract as An Antiseptic Towards Escherichia coli and Streptococcus pyogenes by Using Percentage Kill Method Conny Riana Tjampakasari1.
Ariyani Kiranasari1.
Alfia Chairunnisa2.
Fatindhiya Mahira Hilmy2 Department of Microbiology.
Faculty of Medicine.
Universitas Indonesia Medical Education.
Faculty of Medicine.
Universitas Indonesia *Korespodensi Penulis :
Conny Riana Tjampakasari Email : connyrianat@yahoo.
Abstrak Latar belakang: Kandungan kimia daun kelor (M.
menunjukan sifat antibakteri terhadap bakteri Gram positif dan Gram negatif dengan menghambat sintesis dan metabolisme DNA serta menghancurkan dinding sel.
Penelitian ini bertujuan untuk mengetahui signifikansi kemampuan ekstrak daun M.
oleifera sebagai antiseptik terhadap E.
coli dan S.
Metode: Uji percentage kill dilakukan untuk mengetahui persentase kematian bakteri setelah kontak dengan M.
oleifera pada menit ke 1, 2 dan 5.
Variabel yang digunakan yaitu kontrol dan perlakuan yang dilakukan secara bersamaan.
Uji dinyatakan memenuhi standar bila memberikan hasil Ou90% untuk setiap waktu kontak.
Hasil: Uji terhadap E.
coli pada menit ke 1, 2 dan 5 menunjukkan hasil masing-masing 93.
41%, 94.
14%, dan 96.
87%, sedangkan terhadap S.
pyogenes masing-masing 27%, 83.
15% dan 94.
Terhadap S.
pyogenes pada menit ke-5 memenuhi standar.
Kesimpulan: M.
oleifera efektif mengeliminasi E.
coli karena nilai percentage kill untuk semua waktu kontak Ou90% dengan nilai tertinggi pada menit ke-5 .
87%), sedangkan terhadap S.
pyogenes ekstrak daun M.
oleifera menunjukkan aktifitas mikroba yang baik pada waktu kontak 5 menit .
19%).
Kata kunci: M.
oleifera, ekstrak E.
pyogenes, percentage kill Abstract Background: Chemical constituents of Moringa oleifera (M.
leaves show antibacterial properties against Gram-positive and Gram-negative bacteria by inhibiting the DNA synthesis and metabolism extract as an antiseptic for E.
coli and S.
Methods: Using the Percentage Kill test to determine the percentage of bacterial death after contact with M.
oleifera extract at 1, 2 and 5 minutes.
There are two variables that will be used, namely control and treatment that are carried out at the same time.
The Percentage Kill test is considered meet the standards if the result is Ou90% for each contact time.
Results: The discovered that the Percentage Kill in minute 1, 2, and 5 to E.
coli are 93.
41%, 94.
14%, and 96.
87% respectively.
While to S.
pyogenes are 73.
27%, 83.
The result found in the fifth minute is categorized as good as it fulfilled the minimum criteria of Ou 90% for S.
Conclusions: M.
oleifera was effective to eliminate E.
because the percentage kill value for all contact times was above 90% with 5th minute as the highest kill percentage .
87%).
While to S.
pyogenes the M.
oleifera leaves extract presented a good antimicrobial activity in a contact time of 5 minute .
19%).
Keywords: M.
oleifera, extract.
pyogenes, percentage kill The Indonesian Jounal Of Infectious Disease | Volume 8 No.
Background high burden caused by GAS is Escherichia coli which particularly apparent in developing predominant facultative member of countries with minimal income and the intestinal microbiota in human can also be a major causative agent Antiseptic is defined by the of infections in extraintestinal organs WHO as a disinfectant which can such as sepsis (SEPEC or sepsis- damage or prevent the development associated E.
, bacteremia and of microorganisms present in the This occurs when the tissue without producing any harmful pathogenic bacteria adhere, invade effect on the body.
Antiseptics are and be followed by colonization of membranes with unbroken skin or increased number of Escherichia coli open wounds in order to prevent sepsis and bacteremia events in recent years from the contamination microorganisms within the area.
There Moringa oleifera (M.
is one of the most common species of Streptococcus the family Moringaceae and can be Group A streptococci (GAS) is one easily found around Africa and Asia.
of the major Gram-positive bacterial oleifera contains a wide range of pathogen exclusive to humans.
The bioactive components.
These active interaction of GAS and humans phenolic acids, flavonoids, tannins, healthy individuals, manifestation of GAS is usually limited to mild purulent infections of the skin and While benefits as well as antibacterial existing conditions GAS may present purposes that are beneficial.
The severe systemic manifestations.
WHO
countries, 80% of the populations manifestations include streptococcal opt more towards the use of herbals as the main therapy in favor of that in necrotizing fasciitis which produce Making high morbidity and mortality rate.
The Indonesian Jounal Of Infectious Disease | Volume 8 No.
antibacterial or antiseptic use, which which was then used as extract in is also supported by the extensive safety margin of M.
Pharmacy, oleifera for humans and animals.
Department Faculty Medical Medicine.
Universitas Indonesia.
specifically the leaves have very Extract weight 120.
8495 g which high active contents are proven to provide great advantages in chronic By subtracting 100% out of conditions, such as diabetes, high the water content, it will results in 70% and it accounts for the purity of the extract, later it will be divided concentration of the extract, resulting Based on the risk of developing in 847 mg/ml.
This number further infection due to sepsis.
coli and defines that in each 1 mL volume Streptococcus pyogenes, the usage taken from the sample consists of of antiseptic becomes crucial in 847 mg concentrated M.
This research will target of manifestation due to the infection.
using an 800 mg concentration and Along with the high burden that this Carboxymethyl countries and the preferred usage of cellulose (CMC) Na 1%.
CMC is herbals as therapy.
oleifera which prepared firstly by mixing CMC into the disc and combine it with 50 ml becomes an appropriate option.
warm sterile water until the color is order to prove the efficacy of M.
clear and then add on another 50 ml oleifera as an antiseptic against E.
of warm water.
7 After the CMC is coli and S.
pyogenes, it will be ready, a simple dilution chemistry evaluated using the percentage kill
4,5,6
concentration volume of extract from the sample should be, as shown Methods The below V1M1 = V2M2.
Bacterial strain culture of E.
oleifera Lamk were obtained from and S.
pyogenes were obtained from Tropical Study Center of Biopharmaceutical Department Microbiology.
IPB University The Indonesian Jounal Of Infectious Disease | Volume 8 No.
Faculty Medicine Universitas Percentage killed is measure by the following equation :8,9,10 Indonesia.
Bacteria optimization Bacteria is then incubated for Percentage Kill = (CAeX)/C x 100% 18-24 hour and the turbidity is C : Total colony of control equalized to 0.
5 McFarland.
One X :Total colony of M.
oleifera mixture inoculating loop of bacteria is added Significant to 10 ml of Tryptic Soy Broth (TSB) achieved with > 90% .
(Oxoi.
Afterward, a ten-fold serial dilution is made.
Each were poured to petri dish, added with another 15 mL of of molten Plate Count Agar (PCA) (Ikapharmind.
Once solid, it is incubated at 35AC for 24-48 hours in inverted Electronic colony counter is used to identify the statistically viable plates containing between 100 to 200 colonies.
This concentration of bacteria is used for percentage kill Percentage Kill test Mix 0.
5 mL Results Based on the preliminary result, we find the optimal concentration of bacteria was obtained in 105 both coli and S.
Diluting the colony of E.
coli and M.
oleifera with 1, 2 and 5 minutes contact time using the Percentage Kill methods will produce the result as shown in the Table 1.
The results showed the number of colonies of E.
coli in control with three times repetition with a contact time of 1 minute, respectively 183, 166 and 152 colonies.
For a contact .
oncentration 105 in TSB) in 4.
5 mL time of 2 minutes, the number of oleifera extract (X) and 0.
5 mL colonies is 147, 141 and 139.
While bacteria .
oncentration 10 in TSB) for a contact time of 5 minutes, the 5 mL sterile water (C).
Pour to number of colonies was 112, 109 petri dish after 1, 3, and 5 minutes On the contrary, the number .
n triplicat.
Mix it gently with 15 mL of colonies of E.
coli in treatment of of molten PCA and incubate at which is given M.
oleifera extract 35oC for 24-48 hours once its with three times repetition of a 1 Both are held at the same minute contact time is 12, 11 and 10.
The growing colonies counted While on the second minutes, the using an electronic colony counter.
data shows 12, 7 and 6 respectively.
Then, on the fifth minutes, it shows The Indonesian Jounal Of Infectious Disease | Volume 8 No.
5, 3 and 2 colony growth.
The bar and 5 minutes and a three time chart represents the average E.
repetition for each contact time is colony growth within the first, second presented in Table 2.
Based on the and fifth minutes in both control and data, the average growth of S.
A significant difference pyogenes colony which is given M.
olefera and control in 1, 2, and 5 minutes, thus, we could state that M.
oleifera leaves extract hinder the control, the average colony growth in growth of E.
coli even from the first the first minute is 173.
33, in the minute, since it results in more than second minute the obtained mean is 90% (Figure .
33, while the average growth of Results of the percentage kill For colony in the fifth minute is 143.
procedure with contact time of 1, 2.
Table 1.
Average Amount of E.
coli colony growth in control and M.
Time
Colony Count
(C)
i 1 Minute 2 Minutes 5 Minutes Average Colony Growth
(C)
Colony Count
(X)
Average Colony Growth
(X)
i I : First Repetition.
II : Second Repetition, i : Third Repetition C : Total colony of control X :Total colony of M.
oleifera mixture Table 2.
Average amount of S.
pyogenes colony growth in control and M.
Time
Colony Count
(C)
i 1 Minute 2 Minutes 5 Minutes Average Colony Growth
(C)
Colony Count
(X)
Average Colony Growth
(X)
i I : First Repetition.
II : Second Repetition, i : Third Repetition I C : Total colony of control X :Total colony of M.
oleifera mixture The Indonesian Jounal Of Infectious Disease | Volume 8 No.
(A)
(B)
Figure 1.
(A) Average E.
coli bacterial colony growth.
(B) Average S.
pyogenes bacterial colony C : Total colony of control.
X :Total colony of M.
oleifera mixture In order to obtain the final outcome requirement of >90%.
Therefore, only of the Percentage Kill method, the results of the fifth minute is able to meet the specified standard.
Percentage Kill formula.
Based on the calculations, the final results of the Percentage Kill method during minutes 1, 2, and 5 respectively are 93.
14% and 96.
for E.
coli and 27%, 83.
15%, 94.
188% respectively for S.
According to these results.
It can be stated that the higher percentage kill results, thus showing the eradication level of E.
coli is bigger.
The Percentage Kill results can be exceeded Ou 90%.
From the first minute, percentage level .
bove 90%), hence we can state that it has already given benefit in terms of hinder the growth of Meanwhile for S.
findings in the first and second minute Discussion This experiment presence of antimicrobial efficacy of M.
oleifera to suppress the growth of E.
and S.
Supported by the smooth growth of colony which is given treatment in comparison to the higher growth of colony found in control.
From the average colony number of these repetitions, it can be stated that the extract of M.
oleifera can eradicate E.
The hypothesis of the above mentioned notion is due to the antiseptic properties of M.
extract that can slow the growth rate of It can be explained by several possible mechanisms.
The first one is that the phytoconstituents from the M.
oleifera itself such as Isothiocyanate and it derivates, for instance Allyl isothiocyanate (AITC) as it will provide The Indonesian Jounal Of Infectious Disease | Volume 8 No.
effects towards the bacterial membrane a result, the metabolism of bacteria is 12,13 bacteria by inhibiting the activity of Tannin thioredoxin which is a key role enzyme for its metabolism.
activity, its action is dependent on viral Moreover, aqueous extract shows activity and MIC results that are effective towards capacity to assault human cells being Gram negative bacteria such as E.
due to the active components of mechanisms lead to the decrease of E.
Tannins.
Phenol Alkaloids.
All Polyphenols that existed accumulate on contributing to the probability of M.
bacteriaAos increased hydrophobicity and elimination of Escherichia coli and even eventually an increase in bacterial may acts as an antiviral, indicated by growth inhibition percentage.
On the the significant difference on the colony results based on time contact and Tannins' efficacy is explained by their capacity to repetition in comparison to control.
14,15
penetrate through the bacterial cell wall Previous research by Dima LR et up to the interior membrane, causing interference with the cell's metabolism Manado, in 2016 stated that different and, as a result, its death.
Tannic acid concentration of M.
oleifera in 5%, 10%, 20%, 40% and 80% have shown a surfaces since it acts as the inhibitor of potent antibacterial result, shown when NorA efflux pump which is considered at the highest concentration percentage which is 80% can results the highest antibacterial activity.
Thus, bacterial cell MIC zone diameter of 24.
00 mm in E.
death comes from a loss of adherence coli, and the smallest diameter can be to the surface.
Tannic acid also inhibits found in the lowest concentration which the absorption of sugar and amino is 12mm from 5% concentration.
The smallest concentration has even shown Tannins are multidentate a significant result as antibacterial.
ligands that may bind to proteins Combining the highest percentage and number of contact time might eradicate interactions and hydrogen bonding.
a higher number of E.
coli growth.
16,17
Sam Ratulangi University.
Based on their research we continued The Indonesian Jounal Of Infectious Disease | Volume 8 No.
with antiseptic potency test using the antimicrobial efficacy is associated with percentage kill method and the result longer duration of contact.
showed promising.
Furthermore.
On the comparison of contact time affected by the presence of active towards the growth of E.
coli, there is an average number of colony growth on contained in M.
oleifera which minute 1, 2 and 5 minutes which are extracted by certain solvents including 11, 8.
33 and 3.
33 respectively.
The aquadest and ethanol.
The extraction of extract of M.
oleifera has antibacterial properties from the Percentage Kill generate polyphenol and flavonoid, results on minute 1, 2 and 5 with while the involvement of aquadest in extraction produce tannin, saponin, minute contact time using extract of M.
Flavonoid takes part in generating oleifera has already given a prominent antimicrobial activity in M.
and effective results and five minutes of through the disruption of bacterial cell wall and cytoplasm which inhibit the cell highest eradication number of E.
division process.
Supported by the ability of flavonoid to form a complex Meanwhile the result derived from with extracellular proteins and bacterial the Percentage Kill method of S.
cell wall.
18,19 Another phytochemical in pyogenes showed that the influence of oleifera is alkaloid which possess oleifera extract can already be bacterial DNA disturbing characteristics identified within the first 1 minute.
Although the outcome of 73.
27% and functioning in directing nucleotide to 15% found in the first and second single strand DNA.
This interruption of minute respectively still does not fulfill interference of bacterial cell division effective antimicrobial which is >90%.
and consequently the normal structure However, a notable improvement of the of the bacteria along with suppression plantAos antimicrobial action is identified of protein synthesis which takes part in in the fifth minute demonstrated by a the metabolism and construction of result of 94.
This information bacterial cell wall.
In addition to the signify that the increase of M.
DNA
polyphenol substance called Tannin is The Indonesian Jounal Of Infectious Disease | Volume 8 No.
also membrane cell disturbance.
20,21
Another action towards Gram positive bacteria.
Furthermore, this research finding is also congruent with another study Esimone et al in 2006 also suggest that performed by Floorentina in 2015 which antimicrobial effects of the leaf extract reported the efficacy of M.
oleifera in is further enhanced by the activity of inhibiting the growth of S.
pyogenes by Beta-lactams evaluating the minimum inhibitory zone transpeptidation of the cell wall.
The created by the bacteria.
This particular research also noted the association between a higher concentration of comprised of two phases.
In the first extract and a lower growth of bacteria as there was also higher level of present on the surface exhibit negative antimicrobial properties.
23,24
charges which attracts cationic amino Next, patches interact with aliphatic fatty acids while positively charged peptides interact with the anionic components.
Resulting in membrane disruption along with changes in membrane potential.
Conclusion We conclude that M.
oleifera leaf extract has potential as an antiseptic against E.
coli and it is necessary to do a concentration increase examination of oleifera leaf extract against S.
changes in membrane permeability, disrupted lipid distribution, leakage of Acknowledgement anionic cell elements, and stimulation of autolytic enzymes which contributes to the death of bacteria.
Therefore it can be acknowledge that the proteins or Clinical Microbiology Laboratory,Department of Microbiology.
Faculty Medicine.
Universitas Indonesia for the facilities peptides pose a crucial role in the antimicrobial defense system of M.
The outcome of this research is consistent with the report of Oluduro in 2012 which noted that the aqueous
References