International Journal of Social, Service and Research, 2(5) 441-452 441

https://ijssr.ridwaninstitute.co.id/index.php/ijssr/

INTERNATIONAL JOURNAL OF SOCIAL

SERVICE AND RESEARCH

ISOLATION AND IDENTIFICATION OF SECONDARY

METABOLITE COMPOUNDS OF LAOR WORM EXTRACT AS

ANTIBACTERIAL

Martha Kaihena

1*

, Mas’uth Pratomo MS

2

, Abdul M Ukratalo

3

Faculty of Mathematics and Natural Sciences, Universitas Pattimura Ambon, Indonesia

1

Faculty of Science and Technology, Universitas Islam Negeri Maulana Malik Ibrahim Malang,

Indonesia

2,3

Email: [email protected]*

Abstract

Laor worms (

Lysidice oela

) are worms that appear on the surface of the Maluku sea to reproduce

in March or April. Laor worms are usually consumed by most people because they contain 13.92%

protein, 81.51% water, 1.01% fat, and 2.41% ash and contain 9 types of essential amino acids.

Secondary metabolite compounds (bromophenol) from marine worms are antimicrobial. This

research aimed to determine the potential of secondary metabolites in inhibiting the growth of

E.

coli

bacteria and to determine the secondary metabolites contained in laor worms. Laor worm is

extracted maceration with ethanol, ethyl acetate and petroleum ether. The results of each extract

were tested for antibacterial activity using the disc diffusion method with variations in

concentrations of 25, 50, 75, 100, and 125 mg/mL against

E. coli

bacteria. The results of the

extract which had the highest antibacterial activity were then tested for phytochemicals, then

separated using TLC, and identified active compounds using UV-Vis spectrophotometers and their

functional groups using FT-IR spectrophotometers. The results showed that laor worm extract

could inhibit the growth of

E. coli

bacteria with an inhibition zone area of 13.4 mm for ethanol

extract; 14.8 for ethyl acetate extract and 12.6 mm for petroleum ether extract. The

phytochemical test showed that the ethyl acetate extract of la worms contained flavonoids,

saponins, steroids, triterpenoids, and alkaloids. Identification using a UV-Vis spectrophotometer

obtained λ

max

was 203 nm which showed steroid compounds. The identification of active

compounds using an FTIR spectrophotometer shows the functional groups O-H, C = O, C-C, C-

OH primary, and C-H which are thought to be steroid compounds.

Keywords: Laor worms; Secondary Metabolite; Antibacterial; Escherichia coli

Received April 22, 2022, Revised May 14, 2022, May 28, 2022

INTRODUCTION

Infection is a type of disease caused by

micro-organisms and affects most people in

developing countries, including Indonesia

(Ningsih & Zusfahair, 2016; Sari, 2012).

Escherichia coli is usually a resident of the

digestive tract. Several strains of E.coli are one

of the causes of diarrhea in both children and

adults, which can cause enterobacterial

infections, for example diarrhea to meningitis.

suffered by society.

To treat infectious diseases, antibiotics

are the target. The irrational use of

antibacterial has caused many pathogenic

microbes to adapt to their environment and

become resistant to these drugs (Candrasari et

al., 2011)

.

The problem of bacterial resistance

to existing drugs has prompted the importance

of extracting antibacterial compounds from

442 International Journal of Social, Service and Research, 2(5), 441-452

Martha Kaihena, Mas’uth Pratomo MS, Abdul M Ukratalo

natural ingredients that are more patent,

inexpensive, have fewer side effects and are

continuously available in large quantities

(Hartini, 2001).

Maluku is an area in Indonesia which

consists of a very wide archipelago with seas

that stretch around each island. This area has

marine biodiversity and provides opportunities

to utilize marine biota in the search for

secondary metabolites of new bioactive

compounds. Marine biota (marine organisms)

are a very rich source of natural materials with

unique biological activities (Handayani et al.,

2012). Polychaeta are invertebrate animals

that belong to the phylum Annelida. People in

Indonesia know polychaeta by the name of sea

worms, because most of their habitat is in the

sea.

One of the worms in Worms Laor

(Lysidice oela). This wormis one type that

belongs to the family

Eunicidae

which is usually

consumed by most people in Maluku

(Pamungkas, 2009).These worms usually

come to the surface of the water in March or

April while moving his body or dancing. The

spiral movement of the worms to the surface

of the water allows these two worms of

different sexes to meet, which eventually

breaks off and sperm or egg cells come out.

The meeting between the egg and sperm will

become a zygote that will go to the seabed. It

was at that time that the people caught the

sea worms and until now it has become a

tradition for the people of Maluku.

The abundant number of laor worms

during the timbah laor activity is an indication

that the laor worms have the ability to protect

themselves from other creatures in the sea

(Jekti et al., 2008). The ability to take care of

itself is possible because the laor worm has

active ingredients (natural products) that can

kill or inhibit the growth of other living things.

The ability of the Laor worm to inhibit the

growth of benthos bacteria is related to the

place where the Laor worm lives, namely in

coral. The chemical components of sea worms

encourage the development of isolating and

identifying secondary metabolites found in sea

worms. Secondary metabolites have the ability

as bioactive compounds so they are very

promising as lead compounds for materials

that have pharmacological activity.

Identification of secondary metabolite

content is an important initial step in research

to search for new bioactive compounds from

natural ingredients that can be precursors for

the synthesis of new drugs or prototypes of

certain active drugs (Rasyidi, 2016). The group

of secondary metabolites is very abundant and

commonly found in organisms including

alkaloids, flavonoids, phenols, steroids, and

terpenoids (Marliana, 2007). This study aims

to determine the potential of secondary

metabolites from the Laor worm (Lysidice oela)

in inhibiting the growth of Escherichia coli

bacteria andTo find out the secondary

metabolite compounds contained in Laor

worms.

METHOD

Material

The materials used were Laor worm samples,

96% ethanol, 50% methanol, Mg metal, ethyl

acetate, petroleum ether, 1 N HCl,

concentrated HCl, 2% HCl, chloroform, DMSO,

chloramphenicol, acetic acid anhydrous,

concentrated H2SO4, Aquades, 1% FeCl3,

Reagent solution (dragendorff, mayer), KBr,

Medium NA, Medium NB, Aluminum foil,

Escherichia coli, disc paper and cotton.

Method

The research method used is descriptive

qualitative and quantitative research through

two stages of experimental testing in the

laboratory, the first stage aims to determine

the effect of the type of solvent on the

antibacterial activity of laor worm extract. Laor

worms were extracted with a variety of

solvents consisting of ethanol, ethyl acetate

and petroleum ether. Extraction of active

compounds was carried out using the

maceration method, the extraction results

International Journal of Social, Service and Research, 2(5) 441-452 443

Isolation and Identification of Secondary Metabolite Compounds of Laor Worm Extract as

Antibacterial

were tested for antibacterial using the agar

diffusion method. The antibacterial test was

carried out in duplicate and repeated 3 times.

In the second stage, the best antibacterial

activity of the extract will be tested for

phytochemicals, then the active compounds

will be separated using TLC. Identification of

chemical compounds using a UV-Vis

spectrometer while determining their

functional groups using FTIR.

Work procedures

Sampling

Sampling of laor worms in Latuhalat Village,

Ambon Island, Maluku Province.

Sample Preparation

Sampling results are included in the coolbox.

Then soaked in cold water 14 ° C for 24 hours.

Aerated at room temperature covered with

aluminum foil. Next, the samples were ground

using a blender. The milled products were

dried in an oven at 50 °C for 12 hours and

sieved (90 mesh) to obtain flour extract

(Aninda, 2016).

Laor Worm Extraction

As much as 25 g of Laor worm powder was put

into a 100 mL Erlenmeyer flask. Then each

solvent was added, namely 96% ethanol, ethyl

acetate and 50 mL petroleum ether. Shaking

was carried out with a shaker for 3 hours at a

speed of 120 rpm (rotation per minute) and

macerated for 24 hours. Then filtered the

maceration residue with a Buchner funnel and

redissolved using the same solvent until it is

clear. The filtrate obtained was combined in an

extract storage container (Nurhayati &

Purwaningsih, 2017). The ethanol, ethyl

acetate and petroleum ether extracts obtained

were concentrated using a rotary evaporator

at a temperature of 30-40 ºC, and flowed with

N2 gas until the solvent completely

evaporated.

Antibacterial Activity Test

The NA (Nutrien Agar) media is heated until it

melts, cooled to a temperature of 40˚C. The

NA solution was poured into sterile petri

dishes, mixed with 0.1 mL of E. coli bacteria

solution, then homogenized and allowed to

solidify.Laor worm extract was prepared in

various concentrations (25, 50, 75, 100 and

125 mg/mL). A total of 0.01 gram of Laor

worm extract was dissolved in 10 mL of

ethanol, ethyl acetate and petroleum ether as

the main solution and then divided into several

predetermined concentrations (Elayaraja et

al., 2010). Paper discs with a diameter of 5 mm

were soaked in the resulting laor worm extract

and controls (positive controls were given

chloramphenicol and negative were given

DMSO solution). The disc paper is placed on

the surface of the media using sterile tweezers

and pressed slightly. Incubated at 37 ˚C for 24

hours until the inhibition area appears (Mulyadi

et al., 2017). The antibacterial activity test for

each solvent was carried out in duplicate and

repeated 3 times. The inhibition zone was

measured using a ruler to determine bacterial

activity.

Phytochemical Test of Active Compounds

in Laor Worms

The extract which has the widest inhibition

zone will be identified for its chemical content,

including flavonoids, terpenoids, saponins,

tannins, phenolics and alkaloids (Kristianti et

al., 2008).

Classification of Compounds with

Analytical TLC

Extracts that showed antibacterial activity

were then tested chemically by Thin Layer

Chromatography (TLC) which was carried out

using silica gel GF254 plates as the stationary

phase. Silica gel plates are made with a width

of 1x10 cm2 at the top and bottom ends with

a limit of 1 cm. 0.001 gram of the extract to be

tested was dissolved in 2.5 mL of the solvent

(4,000 ppm) used, then spotted 10 times ± 1

cm from the bottom of the plate. Developers

(mobile phase) used for each compound were:

steroids (n-hexane 1 : 1 ethyl acetate),

alkaloids (chloroform 4 : 1 methanol),

saponins (chloroform 13 : 7 methanol : 2

water), triterpenoids (n- butanol 4 : 1

444 International Journal of Social, Service and Research, 2(5), 441-452

Martha Kaihena, Mas’uth Pratomo MS, Abdul M Ukratalo

Ammonia), flavonoids (ethyl acetate 4.5 : 0.5

methanol) and phenolics (Toluene 3 : 3 ethyl

acetate : 0.2 formic acid). Then it was dried

and eluted with eluent in a TLC flask which had

been saturated and tightly closed. After the

eluent reaches the top line, the plate is

removed and dried (Jekti et al., 2008).The

spots were observed visually with a UV lamp

at a wavelength of 245 nm and 366 nm and

using a universal spray reagent to reveal the

spots with the Liebermann-Burchard reagent.

Then the Rf value is calculated and the spot

shape is observed in various eluents.

Identification of Compounds Using a UV-

Vis Spectrophotometer

Laor worm extract which had been dissolved

based on the solvent with a concentration of

100 ppm was then put into a cuvette and

analyzed using a UV-Vis spectrophotometer at

a wavelength range of 200-800 nm (Maharani

et al., 2016).

Functional Group Identification Using

FTIR

The characterization in this study used FTIR

(Fourier Transform Infra Red) spectroscopy.

The target compound in solid form was

crushed with KBr salt (2:98) and then pelleted

using a diameter of 7 mm. The pellet was then

placed in the sample holder and its absorption

was measured using an FTIR spectrometer in

the area of 4000-400 cm-1. The sample to be

analyzed must be ensured that it is dry and

free from impurities around the container. The

data from the analysis results were then

processed using Ms. Excel 2010 (Sigee et al.,

2002).

Data analysis

The data obtained from the first stage of the

research were in the form of inhibition zones

from the results of the antibacterial activity

test of each variation of solvent, the best

solvent was indicated by the widest diameter

of the inhibition zone and the second stage of

research was the identification of chemical

compounds and functional groups from

extracts that had the widest inhibition zones.

Each data is presented in tabular form and

interpreted according to the results obtained

RESULTS AND DISCUSSION

Sample preparation

Wet laor worm samples 256 g were washed

thoroughly using running water. Then the

samples were dried by means of laor worm

samples placed on top of aluminum foil and

dried in the laboratory for ± 2 weeks. After

drying, a sample weight of 121.82 g was

obtained which was then refined using a 90

mesh sieve. Smoothing is done so that the

surface area of the sample is greater so that

the sample contact with the solvent is

maximized. In addition, the refining process

also allows the cells to rupture, thus facilitating

the uptake of active compounds by solvents.

Laor worms have a moisture content of

47.58%. Water content analysis aims to

determine the water content contained in laor

worm biomass.

Extraction of Laor worms by maceration

Sample extraction in this study was carried out

by maceration. Extraction was carried out

using 3 solvents namely ethanol, ethyl acetate

and petroleum ether. Laor worm powder was

added to each solvent in the ratio (1 : 3) and

macerated for 24 hours at room temperature.

The filtrate obtained is then concentrated

using a rotary evaporator and N2 gas, this

aims to remove the solvent. The results of

observations on the color of the filtrate, extract

color, measurement of crude extract weight

and extract yield extract yield can be seen in

Table 1.

Table 1. Yield of Laor Worm Extract from various solvents

Solvent type

Filtrate Color

Extract Color

Crude

Extract (g)

Yield (%)

Ethanol

Dark brown

6.9481

27,79

Ethyl acetate

Brown

2.8335

16.89

Petroleum ether

Brown

1.4081

5,63

International Journal of Social, Service and Research, 2(5) 441-452 445

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The results in Table 1 show that the

characteristics of the filtrate in ethanol are

dark brown, while ethyl acetate and petroleum

ether have the same color, namely brown. The

type of solvent also affects the extraction

results and yield. The highest extract value

was ethanol solvent of 6.9481 g with a yield

value of 27.79%. followed by 2.8335 g of ethyl

acetate extract with a yield of 16.89% and

petroleum ether extract with an extract value

of 1.4081 g with a yield of 5.63%.

These results also indicated that the ethanol

extract produced a higher yield when

compared to the ethyl acetate and petroleum

ether extracts. The high yield of extracts in

polar solvents is because the active

compounds are generally still in the form of

glycoside bonds, namely compounds

consisting of sugar compounds (glycones and

primary metabolites) and non-sugar

compounds (aglycones and secondary

metabolites) (Utami et al., 2013).

Antibacterial Activity Test

The results of measuring the diameter of the

inhibition zone showed that the Laor worm

extract had activity in inhibiting Escherichia coli

bacteria. The average inhibition zone resulting

from the results of the antibacterial activity

test can be seen in Table 2.

Table 2. The results of testing the antibacterial activity of laor worm extract against Escherichia

coli bacteria

Concentrat

ion

(mg/mL)

Average Inhibition

Zone(mm)

Growth Response

Ethano

l

Extract

Ethyl

Acetat

e

Extract

Petrole

um

Ether

Extract

Ethanol

Extract

Ethyl

Acetate

Extract

Petroleu

m Ether

25

6,6

7,1

5,6

Intermediate

Weak

50

9,2

8,8

9,2

Intermediate

75

10,7

12,1

10.5

Intermediate

Strong

Intermediate

100

12

13,8

11,8

Strong

Intermediate

125

13,4

14,8

12,6

Strong

Control (+)

34

35

Very strong

Control (-)

-

There isn't

any

There isn't

any

There isn't

any

Based on Table 2 it can be seen that the

ethyl acetate extract of the laor worm has a

higher antibacterial activity when compared to

the ethanol and petroleum ether extracts. This

is indicated by the size of the inhibition zone at

each concentration of each ethyl acetate

extract. The higher extract concentration

causes a higher inhibition effect so that the

clear zone around the paper disk becomes

wider.

Antibacterial activity in ethanol extract

was identified at concentrations of 25 mg/mL

(6.6 mm), 50 mg/mL (9.2 mm), 75 mg/mL

(10.7 mm), 100 mg/mL (12 mm) and 125

mg/mL (13.4 mm). Ethyl acetate extract

concentrations of 25 mg/mL (7.1 mm), 50

mg/mL (8.8 mm), 75 mg/mL (12.1 mm), 100

mg/mL (13.8 mm) and 125 mg /mL (14.8mm).

Whereas in petroleum ether extract

concentrations of 25 mg/mL (5.6 mm), 50

mg/mL (9.2 mm), 75 mg/mL (10.5 mm), 100

mg/mL (11.8 mm) and 125 mg/mL (12.6 mm).

There was an increase in the diameter of the

inhibition zone along with the increase in the

concentration of the worm extract in all

solvents.

The results of this study are different

from the results of Nurhikmah's research

(Nurhayati & Purwaningsih, 2017), where the

ethanol and ethyl acetate extracts of the

freeze-dried sea worm Siphonosoma australe-

australe did not have antibacterial activity.

While in research Elayaraja et al. (2010)

,Perinereis marine worm extract

concentration

cultrifera

effective in inhibiting

the growth of gram negative

446 International Journal of Social, Service and Research, 2(5), 441-452

Martha Kaihena, Mas’uth Pratomo MS, Abdul M Ukratalo

bacteriaEscherichia coli bacterianamely the

concentration of 25 mg/mL (7 mm) while the

sea worm extract

Eunice siciliances

96%

ethanol extract can inhibit the growth of

bacteria such as E. coli bacteria (11 mm) at a

concentration of 100 µg/mL (Jekti et al.,

2008)

.

The high value of the inhibition zone of

the ethyl acetate extract in this study is

thought to be due to the active compound that

has the potential as an antibacterial from the

Laor worm, which tends to be distributed in

semipolar solvents. The results of observing

the inhibition zones of Laor worm extract with

various solvents against Escherichia coli

bacteria can be seen in Figure 1.

Figure 1. Zone of Inhibition of Laor worm

extract from various solvents against

Escherichia coli bacteria. (A) Ethanol, (B) Ethyl

acetate, (C) Petroleum ether and (D) Control.

(1) 25 mg/mL, (2) 50 mg/mL, (3) 75 mg/mL,

(4)100 mg/mL, (5)125 mg/mL, (K+) positive

control and (K-) negative control

The positive control used in this study

ischloramphenicol. The use of chloramphenicol

is due to:Chloramphenicol is a broad spectrum

antibiotic (Nuria, 2016). Chloramphenicol is

bacteriostatic or inhibits bacterial growth

(Cahyono & Indrayudha, 2013). The test

results show that the average inhibition zone

formed onchloramphenicol was larger when

compared to the zone of inhibition in ethanol,

ethyl acetate and petroleum ether extracts.

The large inhibition zone in the positive control

was due to the fact that the active ingredient

in chloramphenicol was pure, while the

ethanol, ethyl acetate and petroleum ether

extracts were not pure because they were

mixed with other compounds so that the

inhibition of bacteria was not effective.

The negative control used was DMSO.

According to Pratiwi, Dimethyl Sulfoxide

(DMSO) is a solvent that can dissolve both

polar and nonpolar compounds that dissolve in

organic solvents and water. The absence of an

inhibition zone in the negative control in this

study proved that the inhibition zone formed

was not influenced by the type of solvent but

due to the activity of the active compounds

present in the laor worm extract as

antibacterial.

Identification of Laor Worm Secondary

Metabolite Compounds

The results of the antibacterial activity test

showed that the ethyl acetate extract of the

laor worm had the highest antibacterial

activity, so the ethyl acetate extract was then

identified as a secondary metabolite. The

results of the secondary metabolic compounds

test for the ethyl acetate extract of the Laor

worm qualitatively can be seen in Table 3.

Table 3. Test results for the content of secondary metabolites of ethyl acetate extract of Laor

worms

No.

Compound Class

Formed color/precipitate

Results

1

Flavonoids

Yellow

+

2

Steroids

Bluish green

++

3

Triterpenoids

Browning Ring

++

4

Saponins

Foam turbid

++

5

tannins

Yellow

-

6

Phenol

Dark brown

+

7

Alkaloids

a. Mayer

b. Dragendroff

Yellowish white precipitate

Orange precipitate

+

Note: (+) There is a group of active compounds and (-) There is no group of active compounds.

B

A

C

D

International Journal of Social, Service and Research, 2(5) 441-452 447

Isolation and Identification of Secondary Metabolite Compounds of Laor Worm Extract as

Antibacterial

Based on Table 3, it can be seen that

the ethyl acetate extract of the laor worm

contains flavonoids, steroids, triterpenoids,

saponins, phenols and alkaloids. The

secondary metabolites found in this study were

not much different from those found by Erviani

and Arif (2017) where the results of the

phytochemical screening of sea worm extract

Perinereis aibuhitensis also contain alkaloids,

flavonoids, saponins, triterpenoids/steroids

and tannins. In addition, the Siphonosoma

austral sea worm contains saponins,

flavonoids, steroids and alkaloids (Aninda,

2016). The results of the phytochemical

screening of the sea worm extract of Eunice

siciliensis contain alkaloids, flavonoids,

saponins, triterpenoids/steroids and tannins

(Erviani et al., 2019).

Thin Layer Chromatography Profile

The ethyl acetate extract obtained was

then analyzed by Thin Layer Chromatography

(TLC). Tests were carried out with a

combination of ethyl acetate: methanol, n-

hexane: ethyl acetate, n-butanol: NH4OH, the

TLC test can be seen in Table 4 and Figure 2

Table 4. Color and Rf value of loar worm ethyl acetate extract elution results

Compound

Class

Mobile Phase

Stain

Rf value

Stain Color

Compound

Suspect

Alkaloids

Chloro: meta

(4 : 1)

1

0.91

Orange

Alkaloids

Saponins

Chloro : meta :

water

(13 : 7 : 2)

2

0.80

0.83

Dark Purple Red

Saponins

Triterpenoids

n-Butanol :

NH4OH

(4 : 1)

4

0.20

0.37

0.50

0.89

Greenish blue

Greenish blue.

Greenish blue

Violet

Flavonoids

Triterpenoids

Steroids

n-Hexane : EA

(1 : 1)

6

0.39

0.58

0.64

0.7

0.83

0.93

Blue

Red

Red bluish green

Red

Flavonoids

Triterpenoids

Steroids

Triterpenoids

Flavonoids

EA : Methanol

(4.5 : 0.5)

2

0.83

0.95

Dark purple

Red

Saponins

Triterpenoids

Phenol

Toluene : EA :

Formic acid

(3:3:0,2)

4

0.52

0.57

0.90

0.96

Red

Dark blue

Dark purple

Triterpenoids

Flavonoids

Saponins

Notes : EA (Ethyl Acetate), Meta (Methanol) and Chloro (Chloroform).

Figure 2. TLC profile of Laor worm ethyl acetate extract

448 International Journal of Social, Service and Research, 2(5), 441-452

Martha Kaihena, Mas’uth Pratomo MS, Abdul M Ukratalo

Based on the results of the TLC in Table

4, it was shown that the ethyl acetate extract

of the laor worm contained flavonoids,

steroids, triterpenoids, saponins and alkaloids.

The TLC test with ethyl acetate: methanol (4.5

: 0.5) eluent produced two spot stains at a

wavelength of 366 nm, namely blue and purple

with an Rf value of 0.83; 0.95, blue color

indicates flavonoids and purple indicates

saponins. In the n-hexane eluent: ethyl

acetate (1:1) six spots were obtained at a

wavelength of 366 nm with an Rf value of

0.39; 0.58; 0.71; 0.64; 0.67; 0.83 and 0.93. It

is suspected that at Rf 0.67 it is a steroid

compound due to the presence of a bluish-

green stain after being sprayed with the

Liberman-Buchard reagent, the blue color

indicates flavoid compounds with Rf 0.39 while

the red color indicates triterpenoid compounds

with Rf 0.58; 0.64; 0.83 and 0.93.

In the n-butanol eluent: NH4OH (4:1) at

a wavelength of 366 nm produced four spots

with an Rf value of 0.20; 0.37; 0.50 and 0.89

with blue-green color indicates flavonoid

compounds respectively and light purple is a

triterpenoid. In testing with the mobile phase

of chloroform: methanol: water (13:7:2) it

produced two dark purple spots which

indicated saponin compounds with an Rf value

of 0.80 and the red color indicated

triterpenoids with Rf 0.83 at a wavelength of

366 nm.

Meanwhile, using a phenolic eluent

using toluene as mobile phase: ethyl acetate:

formic acid (3:3:0,2) produces 4 stain spots,

namely red (triterpenoid), red (triterpenoid),

dark blue (flavonoid) and purple (saponin). )

with Rf values of 0.51 each; 0.57; 0.90 and

0.96 at a wavelength of 366 nm. In the eluent

chloroform: methanol (4:1) produced a spot of

orange stain which is an alkaloid compound

with an Rf value of 0.91 at a wavelength of 366

nm after being sprayed with Dragendroff's

reagent.

The TLC profile obtained from Laor

worms identified a flavonoid compound from

Curcuma aeruginosa Roxb with an Rf of 0.8

which was close to that of the study, namely

0.9(Nugrahaningtyas et al., 2005). The

saponin compound from Smilax rotundifolia

leaf wrap has an Rf value of 0.79 which is close

to the TLC profile Rf value of 0.8(Firawati &

PRATAMA, 2018). In alkaloid compounds, an

Rf value of 0.91 was obtained which was in

accordance with Rahmawati's research (2015)

who carried out the separation of alkaloid

compounds from Alstonia scholaris L. with an

Rf value of 0.9. Identification of steroid

compounds from Eucheuma spinosum

obtained an Rf value of 0.694 exactly with the

above observation, namely 0.7 (Laili, 2016).

Analysis of Compounds with a UV-Vis

Spectrophotometer

UV-Vis spectrophotometer is an analysis

based on measuring the absorption of a

solution through which monochromatic

radiation passes. In a UV-Vis

spectrophotometer the interaction between

electromagnetic radiation and molecules

causes electronic transitions. The spectrum

can be seen in Figure 3.

Figure 3.UV-Vis results of Laor worm ethyl acetate extract

International Journal of Social, Service and Research, 2(5) 441-452 449

Isolation and Identification of Secondary Metabolite Compounds of Laor Worm Extract as

Antibacterial

Table 5. The results of qualitative analysis of the ethyl acetate extract of Laor worms

Wavelength (nm)

absorbance

609.0

0.024

260,1

0.518

203.0

3,043

Based on the results of UV-Vis spectrum

measurements, there is a maximum

absorption at a wavelength of 203 nm. This

absorption peak is a typical absorption for

steroid compounds indicating the presence of

π π* electronic transitions caused by

unconjugated double bonds (C=C) in the

isolated compound. The resulting spectrum

pattern is similar to Baderos's

research(2017)who identified steroid

compounds from the sponge Xestospongia sp

de L. and the red algae Eucheuma cottonii

which produced a UV-Vis spectrum at a

wavelength of 203 nm indicating the presence

of steroid compounds that have hydroxyl

groups. At a wavelength of 260 nm. According

to Maulidiyah(2011)absorption at a

wavelength of 260 nm comes from the C=O

chromophore group withtype of transition n

π*. Meanwhile, at a wavelength of 609 nm, it

shows flavonoid compounds that are in

accordance with Suarsa's research (2011) who

identified flavonoid compounds from Musa

paradiasiaca L. Cv kapok and Musa

paradiasiaca L. Cv Susu. The transition at a

wavelength of 609 nm shows a bathochromic

shift, namely a shift in absorption towards

longer wavelengths, due to substituents or the

influence of solventsSastrohamidjojo (1996).

Results of Identification of Compounds

by FTIR Spectroscopy

Ethyl acetate marine worm extract

containing alkaloids, flavonoids, saponins,

steroids and triterpenoids in KLTA was then

followed by identification with FTIR

instruments. The results of the FTIR spectrum

are shown in Figure 4.

Figure 4. FTIR results of Laor worm ethyl acetate extract

Figure 6 shows the FTIR spectrum in the form

of transmittance data from wave numbers

4000–400 cm-1. A relatively high

transmittance value indicates that the signal

from the sample is mostly transmitted, while a

relatively low transmittance value indicates

that the signal from the sample is mostly

absorbed. The results of the spectrum

functional group analysis can be seen in Table

5.

450 International Journal of Social, Service and Research, 2(5), 441-452

Martha Kaihena, Mas’uth Pratomo MS, Abdul M Ukratalo

Table 5. The functional group of the FTIR spectrum of ethyl acetate extract of sea worms

No

Wavenumber

(cm-1)

Range

(cm-1)

Intensity

vibration

1

3445,377

4000-3200

Intermediate

-OH Stretch

2

3019,660

3100-3000

Intermediate

CH Aromatic Stretch

3

2924,766

3000-2800

Strong

-CH3 Stretch ash

4

2853,846

3000-2800

Strong

-CH2- Stretch sym

5

1711,306

1870-1550

Strong

C=O Stretch

6

1638.0

1680-1600

Intermediate

C=C Stretch

7

1541,025

1600-1450

Weak

C=C Stretch

8

1460,495

1467-1420

Intermediate

-CH2- bend (scissoring)

9

1273,214

1275-1225

Intermediate

CO Stretch

10

1243,214

1275-1225

Intermediate

CO Stretch

11

1048.0

1085-1030

Intermediate

Primary alcohol CO

12

942,773

995-675

Intermediate

CH Stretch

The results of the IR characterization of

the crude extract of the Laor worm showed

that there were specific absorptions for several

functional groups, including in the wave

number region of 3445.377 cm-1 indicating a

weak absorption as vibrational alcohol (OH)

which often appeared very weak and gave rise

to a band and supported by the presence of

primary alcohol (CO) Stretch vibrations at

wave number 1048 cm-1. In addition, there is

absorption at wave number 3019.66 cm-1

which indicates an aromatic Stretch CH

vibration which is supported by the

appearance of absorption at wave number

942.773 cm-1 with moderate intensity. The

presence of aliphatic CH groups at wave

numbers 2924.766 cm-1 and 2853.846 cm-1

indicates the asymmetric Stretch vibration of

CH3 (methyl) and the Stretch symmetry

vibration of CH2 (methylene).

CONCLUSION

Based on the IR spectroscopy data, it

can be seen that the compounds contained in

the crude ethyl acetate extract of Laor worms

are thought to be steroid compounds. The

FTIR spectra in this study are similar to Dewi's

research(2008)who identified steroids in

percolation extraction of sand sea cucumber

(Holuthuria scabra) where at a wavelength of

3400.97 cm-1 1 showed absorption as

vibrational alcohol (OH) which was supported

by the presence of stretching vibrations of

secondary alcohol (CO) at wave number

1097.03 cm -1. Then at wave numbers

1637.45 cm-1 and 1711.06 cm-1 there are

C=C and C=O carbonyl functional groups.

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