International Journal of Health and Pharmaceutical

Bioautographic Analysis And Antibacterial Activity Test Of Mengkudu Leaf

(Morinda Citrifolia L.) Ethanol Extract On The Bacteria
Propionibacterium Acnes

Rati Satri Situmorang1, Haris Munandar Nasution2*, Minda Sari Lubis3, Yayuk Putri Rahayu4
1,2,3,4
Department of Pharmacy, Universitas Muslim Nusantara Al Washliyah., Jalan Garu II Medan Amplas 20147,
North Sumatera, Indonesia
*Corresponding author:
Email : harismunandar@umnaw.ac.id

Abstract.
Noni contains alkaloids, flavonoids, tripertinoid, protein, lime, iron, carotene, glycosides antarkiron. The content of
anthraquinin, scaloptin, flavonoid and adaptogen compounds are active compounds that have antifungal and
antimicrobial effects. The antibiotics commonly used are antibiotics such as tetracycline, clindamycin and others.
These chemical-based antibacterials have many negative side effects, therefore other alternatives are needed as
antibacterials, one of which is by using noni. This study aims to analyze bioautography and determine the
antibacterial activity of noni leaf ethanol extract against Propionibacterium acnes bacteria.This research was
conducted experimentally using noni leaf ethanol extract with concentrations of 30%, 40%, 50% as independent
variables and bioautographic analysis and antibacterial activity as dependent variables. The bioautography analysis
was performed using TLC and the antibacterial activity test was performed using paper discs.The results of
antibacterial testing of noni leaf ethanol extract with a concentration of 30% had an inhibition zone of 8.36 mm with
a medium category, a concentration of 40% had an inhibition zone of 10.3 mm with a strong category and a
concentration of 50% had an inhibition zone of 11.1 mm in a strong category. positive control tetracycline 30µg had
an inhibition zone of 20.8 mm which was categorized as very strong. The results of the bioautography test using
contact bioautography using the mobile phase of chloroform: n-hexane (7:3). The inhibition zone was obtained at an
Rf value of 0.17, and the characteristics of the chromatogram spots were carried out by spraying 10% FeCl3
suspected that the spots were flavonoid compounds.

Keywords: morinda citrifolia L, antibacterial, propionibacterium acnes, bioautography test.

I. INTRODUCTION
Noni leaves contain protein, lime, iron, carotene, and ascorbin. Meanwhile, noni flowers contain
inter-chiron glycosides. The content of anthraquinin, scaloptin, and adaptogyn compounds are active
compounds that have antifungal and anti-microbial effects (Hartanti, 2020). Propionibacterium acnes is a
Gram positive bacterium which is a normal floral part of the skin and causes opportinistic infections that
produce lipase as a contributor to acne formation. Propionibacterium acnes plays a role in the pathogenesis
of acne by producing lipases that break down free fatty acids from skin lipids (Afifi, 2018).The population of
P acnes bacteria can be reduced by giving antibiotics such as erythromycin, clindamycin and tetracycline.
Tetracycline class antibiotics show a broad antibacterial spectrum, especially in affecting microorganisms
that are dividing. Tetracyclines are widely used for the treatment of infections caused by several types of
gram-positive and gram-negative bacteria.
Although the use of antibiotics is quite effective in treating acne, the use of antibiotics as the main
choice for healing acne must be reviewed to limit the development of bacterial resistance to antibiotics.
Meanwhile, excessive use of antibiotics can cause bacteria that were originally sensitive to become resistant.
Therefore, it is necessary to search for natural antibacterial compounds that do not have a negative impact on
humans, namely by utilizing active bacteria-killing substances contained in plants (Afifi, 2018).The active
compounds found in plants as antibacterial are alkaloids, flavonoids, and tannins. Flavonoids function as
bacteriostatic and their mechanism of action is to form complex compounds with proteins and dissolve so
that they can damage bacterial cell walls. Tannins have activity as an antibacterial. The mechanism of action
is by shrinking the cell wall itself, the cell cannot carry out life activities so that growth is inhibited or even
dies. Alkaloid compounds have an inhibitory mechanism by interfering with the peptidoglycan constituent
components in bacterial cells, so that the cell wall layer is not fully formed and causes cell death (Budianti,
2018), (Susanti, 2020).

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II. RESEARCH METHOD

2.1 Phytochemical Screening
2.1.1 Alkaloids Examination
The simplicia powder and ethanol extract of noni leaf were weighed as much as 0.5 g, then added 1
ml of 2 N hydrochloric acid and 9 ml of distilled water, heated on a water bath for 2 minutes, cooled and
filtered. The filtrate used for the alkaloid test is as follows:
1. 3 drops of filtrate is added with 2 drops of Mayer reagent, a positive reaction is indicated by the
formation of a white or yellow lumpy precipitate.
2. 3 drops of filtrate was added with 2 drops of Bouchardat reagent, a positive reaction was indicated by the
formation of a brown to blackish precipitate.
3. 3 drops of filtrate is added with 2 drops of Dragendorff's reagent, a positive reaction is indicated by the
formation of a red or orange color precipitate.
Alkaloids are considered positive if there is a precipitate or turbidity in at least 2 reactions from the 3
experiments above (Depkes RI, 1989), (Emelda, 2021).
2.1.2 Flavonoid Examination
The simplicia powder and ethanol extract were weighed as much as 10 g and then added 100 ml of
hot water, boiled for 5 minutes and filtered in hot conditions. To 5 ml of the filtrate was added magnesium
powder, 1 ml of concentrated hydrochloric acid and 2 ml of amyl alcohol, shaken vigorously and allowed to
separate. The presence of flavonoids is indicated by the appearance of a red, yellow or orange color on the
amyl alcohol layer (Susanti, 2019).
2.1.3 Saponin Examination
The simplicia powder and ethanol extract were weighed as much as 0.5 g were put into a test tube,
added 10 ml of hot water, cooled and shaken for 10 seconds. If foam is formed as high as 1-10 cm which is
stable for no less than 10 minutes, and does not disappear with the addition of 1 drop of 2 N hydrochloric
acid, it indicates the presence of saponins (Depkes RI, 1989).
2.1.4 Tannin Examination
The simplicia powder and ethanol extract were weighed as much as 0.5 g and added with 10 ml of
distilled water and then filtered. The filtrate was diluted with water until it was colorless. 2 ml of the solution
was taken and 1-2 drops of 1% FeCL3 reagent were added. If a blue or blackish green color occurs, it
indicates the presence of tannins (Depkes RI, 1989).
2.1.5 Glycoside Examination
The simplicia powder and ethanol extract were weighed as much as 5 g, dissolved in 96% ethanol
solvent, evaporated on a water bath, dissolved the remainder in 5 ml of anhydrous acetic acid, and added 10
drops of sulfuric acid P. Blue or green color formed indicates the presence of glycosides ( Susanti, 2019).
2.1.6 Steroid/Triterpenoid Examination
Simplicia powder and ethanol extract were weighed as much as 5 g, each was macerated in 20 ml of
ether for 2 hours and then filtered. 5 ml of filtrate was evaporated in an evaporating dish to dryness. To the
residue was added 20 drops of anhydrous acetic acid and 1 drop of concentrated sulfuric acid (Lieberman-
Bouchard reagent). The formation of purple or red color that changes to blue-green indicates the presence of
steroids/triterpenoids (Harbone, 1987).
2.2 Sterilization of Tools and Materials
Growth media were sterilized by autoclaving at 1210C for 15 minutes and glassware sterilized in an
oven at 160-1700C for 1-2 hours, except for materials made of rubber which were sterilized by immersing
them in 70% alcohol and using a needle on a Bunsen flame. until it turns red (Zulvia,2017)
2.3 Preparation of Mueller Hinton Agar (MHA) Media
A total of 38 g of MHA media was dissolved into sterile distilled water little by little, then the
volume was made up to 1 L and heated until completely dissolved. The media was sterilized in an autoclave
at a temperature of 121 0C for 15 minutes.

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2.4 Bioautography Test

a. Preparation of the Mobile Phase (Eluent). Before elution, the eluents used were chloroform and n-
hexane in a ratio of (5:5), (6:4), (7:3), (8:2), (9:1), (10:0) The eluent in one vessel is saturated first, each
mixture of the mobile phase is inserted into the chamber and then closed tightly and saturation is carried out.
This saturation is done to equalize the vapor pressure in the entire vessel.
b. Faction Sample Highlighting. The sample is spotted using a capillary tube on the TLC plate in
each comparison eluent, the TLC plate is inserted into the chamber and then dried by aerating.
c. Bioautographic bacterial activity test. The bioautography test was carried out to detect active
compounds that have antibacterial activity. A total of 1 ml of the suspension was put into a sterile petri dish.
Pour 20 ml of MHA medium. Furthermore, the cup is shaken on a flat table surface, so that the media and
bacterial suspension are mixed evenly and let stand until solid. The chromatograms resulting from the
separation of compounds by TLC were placed on the solidified medium. It was allowed to stand for 30
minutes, the chromatogram plate was removed and removed from the medium. Then incubated for 1 x 24
hours at a temperature of 370C. sprayed with 10% FeCl3 reagent if it produces a black/grey color, it
indicates a positive flavonoid compound (Andarini, 2019).
2.5 McFarland Standard Solution 0.5
Composition : 1% sulfuric acid solution 9.95 ml
Barium chloride solution 1.75% 0.05 ml
Ways of making :
9.95 ml of 1% sulfuric acid solution was pipetted and 1.75% barium chloride solution was pipetted
as much as 0.05 ml, put into a test tube and shaken homogeneously. Until a standard Mc Farland solution of
0.5 was obtained (Andarini, 2019).
2.6 Preparation of Bacterial Suspension
To make a suspension of Propionibacterium acnes bacteria, by culturing Propionibacterium acnes
taken with sterile wire, then suspended into a test tube containing 10 ml of 0.9% NaCl. Furthermore, it is
compared with the standard turbidity of the McFarland solution of 0.5 (Andarini, 2019), (Sari,2015).
2.7 Activity Test of Noni Leaf Ethanol Extract Against Propionibacterium acnes
The inhibition test used the agar diffusion method, namely disc. A total of 1 ml of the suspension
was put into a sterile petri dish. Pour 20 ml of MHA medium. Furthermore, the cup is shaken on a flat table
surface, so that the media and bacterial suspension are evenly mixed. Let stand until solid, then take the
paper disc using tweezers which was previously heated over a Bunsen fire. Disc paper was inserted into the
noni leaf extract with a predetermined concentration of 30%, 40%, 50%, DMSO as a negative control and
Tetracyline as a positive control. After that it is placed on the surface of the media so that it is carefully used
with tweezers and marked each location of each concentration. Then the media was incubated in an incubator
at 37°C for 24 hours. After that, the inhibition zone formed around the disc was measured using a digital
caliper. Marked by a clear zone around the disc (Retno, 2020), (Nasution, 2022).

III. RESULTS AND DISCUSSION

3.1 Phytochemical Screening Results
Based on the results of the phytochemical screening examination of noni leaf powder and extract, it
showed that noni leaves contain chemical compounds of alkaloids, saponins, tannins, flavonoids,
steroids/triterpenoids, and glycosides as evidenced by chemical tests.
3.2 Bioautography Test Results
This test was carried out using a TLC plate which had been marked with the lower and upper
boundary of the plate as a sign of the elution boundary. The elution distance made was 8.5 cm in the hope
that this distance was sufficient to separate the compounds to be eluted on the TLC plate.
Data Table of Thin Layer Chromatogram Results of Noni Leaf Ethanol Extract
No Mobile Phase Rf value
1 Kloroform : n-heksana = 6:4 0,17
0,23

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2 Kloroform : n-heksana = 7:3 0,17

0,23
0,32
0,38
The results of the bioautography test showed that the best mobile phase was the ratio of Chloroform:
n-hexane 7:3, which was 4 stains. A good eluent is characterized by the number of spots that appear, spots
that are tailless, and the distance between one spot and another is clear. Furthermore, bioautography testing is
carried out which is a follow-up test. In this study, the contact bioautography method was used, this method
was chosen because it was considered simple in operation, and the results were more clearly visible. In
contact bioautography, the process of transferring the active compound into the medium in order to produce
a zone of inhibition and the ability to distinguish between active compounds with the same Rf value. The
eluted plate was contacted on the surface of solid media containing a suspension of Propionibacterium acnes
bacteria for 30 minutes. The plate was then removed from the media and the media was incubated for 18-24
hours, in order to obtain a zone of inhibition.
3.3 Bacterial Growth Inhibition Examination Results
In this study, the antibacterial activity was tested in inhibiting the growth of Propionibacterium
acnes bacteria using the agar diffusion method, namely disc paper. The basis for choosing this method is
because it is fast, easy and simple in its operation and does not require special equipment.
Bacterial Growth Inhibitory Zone Results Table
Obstacles zone (mm) Average
Concentration
Test sample Replication inhibition zone
(%)
1 2 3 (mm)
Negative Control - 0 0 0 -
30% 9,4 8,2 7,5 8,36
Noni Leaf Ethanol Extract 40% 10,6 10,2 10,1 10,3
50% 11,2 11,0 11,1 11,1
Positive Control - 21,3 20,8 20,3 20,8
The results of the examination of bacterial growth inhibition showed that the ethanol extract of noni
leaves with concentrations of 30%, 40%, and 50% each had an inhibitory zone against the bacteria
Propionibacterium acnes. The average clear zone diameter formed around the paper disc at each
concentration was: 30% (8.36 mm), 40% (10.3 mm), 50% (11.1 mm). Each concentration, namely 50% and
40%, has a strong resistance response. While at a concentration of 30% has a moderate resistance response.
The largest inhibition zones were produced in extracts with concentrations of 50% and 40% and then the
inhibitory power decreased again at concentrations of 30%.

Positive And Negative Control concentration 50%

concentration 40% concentration 30%

Fig 1.Results of Antibacterial Activity of Noni Leaf Ethanol Extract (Morinda citrifolia L.) Against
Propionibacterium acnes.

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IV. CONCLUSION
1 Ethanol extract of noni leaf (Morinda citrifolia L.) has a class of antibacterial compounds, flavonoids
which are characterized by the appearance of a blackish ash color with 10% Fecl3 spraying.
2 The ethanolic extract of noni (Morinda citrifolia L.) leaves has antibacterial activity against
Propionibacterium acnes, with the highest bacterial inhibitory activity at 50% concentration with an
inhibition zone of 11.1 mm categorized as strong.

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