Antibacterial and Characterization of Secondary Metabolite

Compound from Ethyl Acetate and Ethanol Fraction of Leaves

Moringa oliefera L

Siti Rofida

Pharmacy Departement, Faculty of Health Science, University of Muhammadiyah Malang, Jalan Bendungan Sutami 188A,

Malang 65145, East Java Indonesia.

Keywords: Fractionation, Moringa oleifera leaves, Secondary metabolite compounds, Antibacterial.

Abstract: The microorganisms that cause infections can mutate due to excessive antibiotic exposure. One of the new

drug search strategies is through the exploration of active ingredients derived from plants that have been used

empirically by the community. Moringa oleifera L leaf is a plant that has been used and has been shown to

have antibacterial, antifungal, analgesic, and antihypertensive activities. Moringa oleifera L leaves contain

secondary metabolites such as alkaloids, tannins, saponins, flavonoids, and phenols. The purpose of this study

was to obtain active ingredients from the leaves of Moringa oleifera L which will be used as a Standardized

Herbal Medicine product in the treatment of infectious cases. In order to get the active ingredient as an

antibacterial, multilevel extraction is carried out using different polarity solvents, so that a fraction containing

nonpolar, semipolar and polar compounds will be obtained. Antimicrobial potential will be tested on each

fraction using the disk diffusion method. The results of identification of the compounds in the ethyl acetate

fraction show the class of compounds Flavonoids, Terpenoids, Polyphenols and Anthraquinone while in the

ethanol fraction Moringa oleifera L. leaves show the compounds of the compounds Alkaloids, Flavonoids,

Terpenoids, Polyphenols, and Saponin. Antimicrobial activity is shown in the ethyl acetate and ethanol

fraction in both Staphylococcus aureus and Escherichia coli.

1 INTRODUCTION

Infection is a pathological condition caused by

microorganisms such as bacteria, viruses, fungi, and

protozoa, and can occur in the community or in

hospitals. Patients who are being treated at the

hospital, have a greater risk of contracting the

infection than outside the hospital. This can occur as

a result of interactions between patients,

environments, and microbes showed that 10 rooms

out of 16 inpatient rooms in the "X" hospital in

Semarang City had airborne germ exceeding the total

threshold of germs in the inpatient room (Wikansari,

2012). Infections that occur in hospitals and attack

patients who are in the process of treatment are

known as nosocomial infections. The prevalence of

nosocomial infections in Indonesia is 7.1%

(Wikansari, 2012). Various attempts have been made

by the hospital in dealing with nosocomial infections

namely by washing hands before and after contact

with patients; use personal protective equipment such

as gloves, masks, and other personal protective

equipment; decontaminate equipment after use in

service; sharp tool management; medical and non-

medical waste management.

Handling in cases of infection is antibiotic

therapy. But now some antibiotics are no longer able

to deal with cases of infection because they are caused

by antibiotic resistance. This antibiotic resistance

occurs because of the use of antibiotics freely, so that

microorganisms become more resistant to antibiotics.

The resistance of microorganisms to antibiotics

occurs because they are too often exposed to

antibiotics so that microorganisms undergo mutations

to form a biofilms layer so that the cell walls are

thicker and cannot be penetrated by antibiotics. This

mechanism is a self-defense mechanism for

microorganisms to be able to survive (Brooks et al.,

2013). The highest resistance found in antibiotics

penicillin and cephalosporin first generation (Yacob

et al., 2011).

This antibiotic resistance if left untreated can

cause cases of infection to become uncontrollable.

Roﬁda, S.

Antibacterial and Characterization of Secondary Metabolite Compound from Ethyl Acetate and Ethanol Fraction of Leaves Moringa oliefera L.

DOI: 10.5220/0009141002330239

In Proceedings of the 2nd Health Science International Conference (HSIC 2019), pages 233-239

ISBN: 978-989-758-462-6

233

The increase in incidence will be very rapid because

infection is a contagious disease. Efforts made to

prevent antibiotic resistance are through the

establishment of antibiotic use policies only in

diseases which according to laboratory data have

indeed been proven to be due to microorganisms.

Another effort undertaken is to explore new

medicines with plant sources that have been proven

empirically efficacious as antimicrobials. Secondary

metabolite compounds that have been shown to have

antimicrobial properties are a group of phenolic

compounds such as simple phenols, phenolic acids,

quinones, flavones, flavonoids, tannins, coumarin;

terpenoid compounds and essential oils; and alkaloids

(Luqman et al., 2012; Akinyeye, Solanke and

Adebiyi, 2014; Gyawali and Ibrahim, 2014; Minaiyan

et al., 2014).

One of the plants that has been empirically

proven as an antibacterial is Moringa oleifera L. This

plant contains alkaloids, tannins, saponins,

flavonoids, and phenols (Oluduro, 2012). Moringa

oleifera leaf extract has antimicrobial activity against

bacteria Klebsiella pneumonia, Escherichia coli,

Staphylococcus aureus, and Streptococcus

pneumonia (Kalpana, Moorthi and Kumara, 2013).

The study was conducted using agar diffusion

methods and extracts used 200-800mg / ml. The

solvent used is chloroform, petrolatum ether, ethanol,

and water. From these studies, the results showed that

all extracts showed antimicrobial activity. At a

concentration of 800mg the average inhibition zone

showed Klebsiella pneumonia 9.3 ± 0.46, Escherichia

coli 11.0 ± 0.00, Staphylococcus aureus 13.0 ± 0.00,

and Streptococcus pneumonia 7.0 ± 0.81. The ethanol

extract showed that the maximum inhibition zone was

S. aureus and the smallest inhibitory zone water

extract was in Streptococcus pneumonia. For this

study, the positive control used was tetracycline 10

mcg.

Based on the results of previous studies show that

the leaves of Moringa oleifera L. have the potential

as raw materials for standardized herbal medicines as

antimicrobials. Raw materials for standardized herbal

medicines can be in the form of extracts or fractions

resulting from the separation of plant secondary

metabolite compounds (Sarker, Latif and Gray,

2006). Based on research conducted by Rofida, et al

(2017) that the fractionation process of Garcinia

mangostan Linn extract can increase its cytotoxicity

effect on T47D cell culture. This shows that there is

an accumulation of active compounds in the

extraction fraction.

In order to obtain active ingredients from Moringa

oleifera L. leaves, multilevel extraction will be

carried out using multilevel extraction techniques

using solvents that have different polarities so that

non-polar compounds, semipolar compounds, and

polar compounds will be obtained. Multilevel

extraction using different polarity solvents will

separate secondary metabolites based on their

solubility. Groups of base form alkaloids, free

flavonoids, and free terpenoids will be more easily

extracted with nonpolar to semipolar solvents.

Whereas the alkaloids in the form of salts, flavonoids

glycosides, terpenoid glycosides, and polyphenols are

more easily extracted in polar solvents (Sarker et al.,

2006). The fraction of the results of the separation

will be tested for antimicrobial potential, especially in

gram-positive bacteria (Staphylococcus aureus) and

gram-negative (Escherichia Coli) in vitro by disc

diffusion method. Furthermore, the active ingredient

of Moringa oleifera L. leaves which has potential as

an antimicrobial will be characterized by secondary

metabolite compounds by the TLC method.

2 METHODS

Material

Moringa oleifera leaves L., Staphylococcus aureus

Escherichia Coli, Silica Gel TLC Plate GF 254, Ethyl

acetate, Ethanol, Mueller Hinton Agar medium.

Fractionation

Moringa oleifera L. leaf powder was extracted

stratified with hexane, ethyl acetate, and ethanol as

solvent respectively with maceration technique. The

ingredients are soaked in hexane solvent for 24 hours,

then filtered and separated the filtrate. The residue is

given the same treatment. This treatment is repeated

until the filtrate does not show stains on the silica

plate. The filtrate obtained was concentrated using a

rotary evaporator at 50°C until a thick extract was

obtained. Each viscous fraction obtained was dried in

an oven at 40°C. Each thick fraction ready for use is

stored in a refrigerator at 5-8°C.

Preparation Sample

The test solution to be used was made by weighing

each fraction of Moringa oleifera L. as much as 50

mg, 25 mg, 12.5 mg dissolved in 0.1 ml of 1% DMSO

then added with sterile aquadest to 1 ml to obtain the

concentration of the test solution in the amount of 50

ml / mL; 25 ml / mL; 12.5 mg / mL

Antibacterial Activity by Disk Diffusion Method

The test was carried out by filling sterilized Petri

dishes with nutrient media to as much as 20 mL and

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waiting for it to harden. Then the bacteria is applied,

evenly with the streak plate method. The media used

for bacterial culture is sterile aquadest that has been

standardized with McFarland's standard (106 CFU /

ml) (McFarland, 1907). The hexane, ethyl acetate and

ethanol fractions of M. oleifera leaves were weighed

50mg each dissolved in 1 ml of solvent (according to

the solvent's flavor) and then bottled on the TLC plate

as much as 5 µl. The hexane fraction was eluted with

n-hexane: ethyl acetate: formic acid (6.5: 3.5: 3

drops) mobile phase, the ethyl acetate fraction used

the n-hexane: ethyl acetate (4: 6) mobile phase system

and ethanol fraction uses a mobile phase system of n-

hexane: ethyl acetate: methanol (0.5: 4: 0.5) plus 1

drop of formic acid. The spot stains that are covered

are cut and sterilized for 30 minutes using UV light in

LAF (Laminar Air Flow). The TLC plate is then

planted in a petri dish that contains bacterial cultures.

Petri dishes are incubated for 24 hours at 37oC. Then

the inhibition zone is formed. Antibacterial testing

was replicated three times. As a positive control,

erythromycin 15 µg / disk was used. As a negative

control, TLC plates were used which were incubated

without any test material spills.

Characterization of Secondary Metabolite

Compounds with Thin Layer Chromatography

Methods

Each extract produced was carried out by thin-layer

chromatography test using silica gel F254 stationary

phase and various eluent mobile phases. The TLC

profile was observed with UV lamps 254 and 365. To

find out the class of compounds, the TLC results were

derivatized with dragendorf solvent, anisaldehyde-

sulfuric acid, FeCl3, KOH, and 10% H2SO4.

3 RESULTS AND DISCUSSION

From the concentration process, it was obtained that

the ethyl acetate fraction of M. oleifera leaves was as

thick as 7.83 g yield of the fraction produced was

3.132%. The yield of ethanol fraction was 42.65 g,

the yield of the fraction extracted from M. oleifera

leaves was 17.06%. Antibacterial activity of M.

oleifera L. leaves

fractionation against Staphylococcus aureus and

Escherichia Coli bacteria can be seen in Table 1. The

identification test carried out on M. oleifera leaf ethyl

acetate fraction using thin-layer chromatography

(TLC) method showed that there were no alkaloid

compounds. The results of the TLC test can be seen

in Figure 1. Based on table 1, the ethl acetate fraction

and ethanol fraction showed that the compound with

Rf 0.9 showed the highest antibacterial activity

against Staphylococcus aureus Escherichia Coli.

Gram-negative bacteria have a way to protect their

cell membranes from penetrating antibacterial agents,

because they have a unique outer membrane,

relatively thinner peptidoglycan walls, and

periplasmic space between the cell wall and

membrane. This outer membrane structure contains

Lipopolysaccharides (LPS) or endotoxins, a complex

structure consisting of Lipid A, short chains of sugar

and long chains of carbohydrates called O-antigens.

O antigens and polysaccharides contained in bacterial

outer membranes play a role in preventing the

penetration of hydrophobic compounds , such as

anthraquinone compounds, into the cell membrane,

while the penetration of hydrophilic compounds, such

as phenol and tannin compounds, into the cell

membrane is prevented by the lipid properties they

have (Brooks et al., 2013).

The results of identification by TLC method

against the ethyl acetate fraction of M.oleifera leaf

showed that there was a class of terpenoids

compounds (Figure 1), flavonoids compounds

(Figure 2), polyphenol compounds (Figure 3),

anthraquinone compounds (Figure 4). The results of

identification of compounds by TLC technique on

spot color observation both visually and irradiated by

UV 254 nm and 365 nm, obtained Rf value presented

in Table 2. In previous studies it was found that the

ethyl acetate fraction of M. oleifera leaves contained

chemical compounds such as alkaloids, flavonoids,

saponins, tannins, terpenoids (Moyo, Masika and

Muchenje, 2012; Kalpana, Moorthi and Kumara,

2013; Abdallah, 2016).

The results of identification by TLC method

against the ethanol fraction of M.oleifera leaf showed

that there was a class of polyphenol compounds

(Figure 5), alkaloids compounds (Figure 6),

flavonoids compounds (Figure 7), saponin

compounds (Figure 8), terpenoids compounds

(Figure 9). The results of identification of compounds

by TLC technique on spot color observation both

visually and irradiated by UV 254 nm and 365 nm,

obtained Rf value presented in Table 3.

Antibacterial and Characterization of Secondary Metabolite Compound from Ethyl Acetate and Ethanol Fraction of Leaves Moringa oliefera

235

Table 1: Antibacterial activity result of M.oleifera L. leaf fractionation against Staphylococcus aureus and Escherichia coli

Sample concentration Rf

Average diameter of inhibitory zone

Staphylococcus aureus Escherichia Coli

Ethyl Acetate Faction

50mg/mL

0.28 9.21±3.72 7.1±1.9

0.38 10.21±3.49 10.2±2.9

0.49 8.77 ±3.19 10.6±1.1

0.69 9.91 ±1.88 8.8±1.7

0.79 10.17±1.11 10.7±1.8

0.86 12.26±2.69 11.8±1.5

0.94 13.73±0.29 14.5±0.7

Ethanol Fraction

50mg/mL

0.18 5.46 ±2.53 10.13±2.68

0.33 8.34 ±3.05 9.39±2.08

0.38 9.70 ±3.22 9.30±1.30

0.75 7.67 ±5.20 10.06±2.54

0.92 14 ±2.53 10.86±2.91

Erythromycin 15µg/disk 38.7±1.41 -

Chloramphenicol 30µg/disk - 32.33±0.58



Figure 1: Identification of terpenoids compounds with TLC,

(a) UV 254 nm (b) UV 365 nm (c) derivatization, UV 365

nm (d) derivatization, visual.

Figure 2: Identification of flavonoids compounds with

TLC, (a) UV 254 nm (b) UV 365 nm (c) derivatization, UV

365 nm (d) derivatization, visual.

Figure 3: Identification of polyphenols compounds with

TLC (a) UV 254 nm (b) UV 365 nm (c) derivatization, UV

365 nm (d) derivatization, visual.

Figure 4: Identification of anthraquinone compounds with

TLC (a) UV 254 nm (b) UV 365 nm (c) derivatization, UV

365 nm (d) derivatization, visual.

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Table 2: TLC Results from the ethyl acetate fraction of M.oleifera leaves mobile phase N-Hexane : Ethyl Acetate (4: 6).

Flavonoids

(10% Sulfuric

Acid)

Terpenoid

(Anisaldehyde-Sulfuric

Acid)

Polyphenol

s ( FeCl

)

Anthraquinone

(KOH 10% in methanol)

0.2

- Purple red - -

0.3

- - - Red purple

0.4

- - - purple green

0.6

- Purple red -

0.7

Intensive yellow - - -

0.8

- Purple red - -

0.9

- - Black Brownish yellow

Figure 5: Identification of polyphenols compounds with

TLC (a) UV 254 nm (b) UV 365 nm (c) derivatization, UV

365 nm (d) derivatization, visual.

Figure 6: Identification of alkaloid compounds with TLC

(a) UV 254 nm (b) UV 365 nm (c) derivatization, UV 365

nm (d) derivatization, visual.

Figure 7: Identification of flavonoid compounds with TLC

(a) UV 254 nm (b) UV 365 nm (c) derivatization, UV 365

nm (d) derivatization, visual.

Figure 8: Identification of saponin compounds with TLC (a)

UV 254 nm (b) UV 365 nm (c) derivatization, UV 365 nm

(d) derivatization, visual.

Antibacterial and Characterization of Secondary Metabolite Compound from Ethyl Acetate and Ethanol Fraction of Leaves Moringa oliefera

237

Figure 9: Identification of terpenoid compounds with TLC (a) UV 254 nm (b) UV 365 nm (c) derivatization, UV 365 nm (d)

derivatization, visual.

Table 3. TLC results from the Ethole Acetate fraction of M.oleifera leaves mobile phase Ethyl acetate: n-Hexane: methanol

(4: 0.5: 0.5) added 1 drop of formic acid.

Polyphenol

s ( FeCl

)

Alkaloids

(Dragen-droff)

Saponin

(Anisaldehyde-

Sulfuric Acid)

Flavonoids

(10% Sulfuric

Acid)

Terpenoid

(Anisaldehyde-

Sulfuric Acid)

0,18 Black Orange - - -

0,33 - - - - -

0,38 - - purple - -

0,75 - - - Yellow Intensive -

0,92 - - - - purple

The secondary metabolite compounds detected in

the ethyl acetate and ethanol fraction have

antibacterial activity. The flavonoid antibacterial

mechanism works by inhibiting the synthesis of DNA

and RNA from bacteria. In Proteus vulgaris bacteria,

flavonoids show a process of inhibiting the formation

of strong bacterial DNA. Whereas in the process of

inhibiting the formation of bacterial RNA the

strongest results were found in S. aureus. In addition

to inhibiting flavonoid DNA and RNA synthesis it

also inhibits the formation of bacterial cytoplasmic

membranes. Examples of antibacterial flavonoids are

apigenin, quercetin, flavonone, isoflavones, luteolin,

and derivatives of epigalotekin (Patel et al., 2014).

The terpenoid compound as an antibacterial works

by inhibiting bacterial growth through destruction in

the bacterial cell membrane. In the polyphenol group,

antibacterial activity works by binding to proteins,

damaging cell membranes, and inhibiting the reverse

transcriptase enzyme so that bacterial cells cannot be

formed (Emmanuel et al., 2014).

In this study also found the antrakinon group. In

this group has a broad antibacterial activity.

Anthraconone works by forming complexes with

nucleophilic amino acids in proteins that can cause

proteins to lose their function. Quinone reacts with

cell hair adhesion proteins, cell wall polypeptides,

and echoenzymes released through membranes

(Putra, 2010).

Polyphenolic compounds can also have

antibacterial activity. The mechanism of action of

polyphenols as an antibacterial is by binding to

proteins, damaging bacterial cell membranes and

inhibiting enzyme expenditure. Inhibiting the reverse

transcriptase enzyme (the reverse transcription

process that is copying RNA into DNA) and DNA

topoisomerase (curling) so that bacterial cells cannot

be formed (Nuria, Faizatun and Sumantri, 2009).

Alkaloid compounds contain nitrogen groups and

are usually present in high amounts in certain plant

parts. This compound is usually found in seeds, fruit,

leaves, roots and on the bark. One of the functions of

alkaloids is as a poison to protect plants from animal

and insect attacks, but some are used as treatments

such as morphine and quinine. Alkaloid compounds

can interfere with the formation of cross bridges of

peptidoglycan compounds in bacterial cells so that the

cell wall layer is not formed intact which then results

in cell death (Patel et al., 2014).

The mechanism of action of saponin as an

antibacterial is that it can cause leakage of proteins

and enzymes from within the cell. Saponins can be

anti-bacterial because of their surface active

substances which reduce the surface tension of

bacterial cell walls and damage membrane

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permebiality. Damage to the cell membrane is very

disturbing survival of bacteria. Saponins diffuse

through the outer membrane and cell walls of the

vulnerable and then bind to the cytoplasmic

membrane so that it interferes with and reduces the

stability of the cell membrane (Kalpana, Moorthi and

Kumara, 2013).

4 CONCLUSIONS

The compounds in the ethyl acetate fraction show the

class of compounds Flavonoids, Terpenoids,

Polyphenols and Anthraquinone while in the ethanol

fraction Moringa oleifera L. leaves show the

compounds of the compounds Alkaloids, Flavonoids,

Terpenoids, Polyphenols, and Saponin while in the

ethanol fraction Moringa oleifera L. Antimicrobial

activity is shown in the ethyl acetate and ethanol

fraction in both Staphylococcus aureus and

Escherichia coli.

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