Formulation and Anti Bacterial Activity Test of Liquid Soap Extract

from Cocoa Beans (Theobroma cacao L.) against Staphylococcus

Aureus and Escherichia Coli Bacteria

Dian Ika Perbina Meliala

, Vera Estefania Kaban

, Christica Ilsanna Surbakti

, Wahyudi

, Parhan

Faculty of Pharmacy, Deli Husada Deli Tua Health Institute, Deli Serdang, Indonesia

Email:{dianikaperbinameliala, erastkaban.20, christicailsannas,

Keywords: Extract, Cocoa beans, Natural antibacterial, Cosmetics, Liquid soap

Abstract: Cosmetics are ingredients or materials mixtures that have functions to clean, maintain, add attraction or

change appearance. Antibacterial liquid soap is used to cleanse and soften skin. Many people are not aware

of making antibacterial liquid soap which can be made traditionally by using cocoa beans (Theobroma cacao

L.). This study aimed to formulate liquid soap extracts from cocoa bean extracts which are used as basis for

making liquid soap and tested the antibacterial activity against Staphylococcus aureus and Escherichia coli.

The research method was an experimental method; Simplisia was extracted by using maceration method with

80% ethanol solvent. Testing antibacterial activity used disk diffusion method and statistical analysis used

one way ANOVA test method. The evaluation results of soap formulation quality testing showed that pH and

froth ability had good results and fulfills SNI standard 06-3532-1996. The results showed that formulation III

with concentration of 20 g cocoa bean extract had great antibacterial activity in each bacterium with values

of 12.93 mm and 10.90 mm. In positive control, antibacterial activity showed values of 15.70 mm and 15.26

mm. The statistical analysis results used one way anova test method showed significant results p <0.05, with

significant value of 0.015 found in Staphylococcus aureus bacteria so that the results showed that it affected

the antibacterial activity test. Antibacterial liquid soap containing cocoa bean extract has antibacterial

potential but not as much as antibacterial potential such as Dettol bath soap.

1 INTRODUCTION

Cosmetics are materials or mixtures that rubbed,

glued, poured, sprinkled or sprayed on, put in, it used

on the body or human body parts to clean, maintain

and add the attraction or change the shape, protecting

to keep it in good condition repairing body odor but

it is not intended to treat or cure an illness (Mu and

Sprando., 2010).

Cosmetics have been widely used by the public

and the main purpose of their usage is to beautify

themselves, increase attractiveness, self-confidence,

and protect hair skin from UV damage, pollution and

other environmental factors, prevent aging and help

someone enjoy and appreciate life more (Mu and

Sprando, 2010).

There are many types of cosmetics that used to

protect the skin such as creams, gels, lotions,

ointments, powders and one of them is soap.

Currently in the market there are still many soaps that

use synthetic ingredients such as (diethanolamine

(DEA) and triclosan), but not all usage of active

ingredients in cosmetics are suitable for every skin

condition, which can cause skin irritation (Górnaś and

Rudzińska, 2010).

Today, there are many people do not realize

soap making can also be made traditionally. Cocoa

beans is one of the traditional ingredients used

(Tamarkin et al., 2018). Cocoa plant which has Latin

name Theobroma cacao L. It is tree plantations that

grow in areas that have soil and tropical climate that

are suitable for cultivating cocoa plants (Arzhavitina

and Steckel, 2010). The contents of secondary

metabolites found in cocoa beans are flavonoids,

saponins, and tannins which are chemical compounds

that have potential as antibacterial (Santana et al.,

2016).

Most people know that cocoa beans are only used

in food / beverage processing, but currently cocoa

beans can also be used in the pharmaceutical

(medicine) and cosmetics industries which one of

them in making liquid soap (Brito-vega, 2018).

Meliala, D., Kaban, V., Surbakti, C., Wahyudi, . and Parhan, .

Formulation and Anti Bacterial Activity Test of Liquid Soap Extract from Cocoa Beans (Theobroma cacao L.) against Staphylococcus Aureus and Escherichia Coli Bacteria.

DOI: 10.5220/0009488502930300

In Proceedings of the International Conference on Health Informatics and Medical Application Technology (ICHIMAT 2019), pages 293-300

ISBN: 978-989-758-460-2

293

Liquid soap is a liquid form that is generally made

by using oils or fats intended to clean the skin, made

with additives namely surfactants, preservatives,

foam stabilizers, deodorizers and dyes that are

allowed, and used for bathing without causing

irritation to the skin. Liquid soap has the ability to

emulsify oily dirt so that it can eliminate bacteria and

dirt that sticks to the skin (Foddai, Grant, and Dean.,

2016).

Based on research conducted by Baharium (2014)

states that ethanol extract of cocoa beans (Theobroma

cacao L.) has an antibacterial effect in inhibiting

Pseudomonas aeruginosa bacterium which is carried

out in vitro. Therefore, this research conducted on the

formulation and antibacterial activity test of liquid

soap extract from cocoa beans (Theobroma cacao L.)

against Staphylococcus aureus and Escherichia coli

bacteria. This research included extracts making from

cocoa beans by maceration and liquid soap

formulation that is tested on Staphylococcus aureus &

Escherichia coli bacteria to determine the inhibitory

power of bacteria to the formulation of liquid soap

preparations from cocoa bean extract with alkali

(KOH), and then testing soap evaluation namely

organoleptic test, pH, foam ability test and irritation

test.

2 MATERIALS AND METHODS

Ripe cocoa beans and fresh yellow color are taken

from Lake Rambai Village, Batang Gansal Sub

District, Gansal District, Inhu, Riau. Cocoa beans that

have been taken are sorted to get fresh seeds, then

washed with running water and dried by direct

exposure to sunlight.

2.1 Making Simplicia Powder

Cocoa beans that have been dried are mashed by

pounding them before blend to get more uniform size.

Then the powder is weighed as much as 500 grams,

and then put into a container for extraction purposes

by using the maceration method.

2.2 Research Tools and Materials

The tools used in this study include laboratory

glassware, aluminum foil, autoclaves, 65 mesh

sieves, stirring rods, blenders, bushes, petri dishes,

hotplates, incubators, calipers, ose needles (diameter

1, 78 dm), cotton, disc paper, filter paper, LAF

(Laminar Air Flow), magnetic stirrer, 20 μl

micropipette, microscope, oven, pH meter,

pycnometer, rotary evaporator, digital scales and

vortex. The materials used in this study include stearic

acid, aquadest, cocoa beans, Butyl Hydroxy Toluene

(BHT), 80% ethanol, ethyl acetate, FeCl

, glycerin,

, HCl 2N, hydroxypropyl methyl cellulose

(HPMC), Ethanol 80%, ethyl acetate, FeC

, glycerin,

, HCl 2N, hydroxypropyl methyl cellulose

(HPMC), indicators of phenolphthalein, potassium

hydroxide (KOH) 0.1 N, chloroform, 0.9% NaCl

solution, n-hexane solution, Mueller-Hinton Agar,

coconut oil, castor oil, olive oil, Nutrient agar,

anhydrous, NaOH 2 N , and Dragendorff

reagent, Lieberman-Bouchardat reagent, lead (II)

acetate 0.4 N. The bacteria used were pure cultures of

Escherichia coli and Staphylococcus aureus bacteria.

2.3 Phytochemical Screening Test

Phytochemical screening is carried out to analyze

bioactive content which is useful as an antibacterial

or as a treatment. The phytochemical screening test of

this cocoa bean powder, namely:

2.3.1 Flavonoids Test

10 g of simplicia powder were added with 100 ml of

hot water. The mixture is then boiled for about 5

minutes, and then filtered when it is hot. As much as

5 ml of filtrate was obtained, added 0.1 g of Mg

powder, 1 ml of concentrated HCL and 2 ml of amyl

alcohol, shaken, and allowed to separate. Flavonoids

are positive if there is red, yellow, or orange color in

the amyl alcohol layer (Cocan et al., 2018; Delazar,

Asgharian, and Asnaashari., 2017).

2.3.2 Tanin Test

0.5 g of simplicia powder sample was added with 10

ml of distilled water. The extraction result is filtered

then the filtrate obtained is diluted with distilled water

until it is colorless. The results of this dilution are

taken as much as 2 ml, and then added with 1-2 drops

of iron (III) chloride. A blue or blackish green color

indicates tannins (Cocan et al., 2018;, Delazar,

Asgharian, and Asnaashari., 2017).

2.3.3 Test of Saponins

As much as 0.5 g of simplicia powder was put into a

test tube and 10 ml of hot aquadest was added, chilled,

then shaken vigorously for no less than 10 minutes to

as high as 1-10 cm of the froth obtained. Furthermore,

with the addition of 2N hydrochloric acid, if the foam

does not disappear, the results obtained indicate the

presence of saponins contained in a simplicia (Cocan

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

294

et al., 2018; Delazar, Asgharian, and Asnaashari.,

2017).

2.3.4 Alkaloide Test

The simplicia powder of cocoa beans was weighed

0.5 g then added 1 ml HCL 2 N and 9 ml aquadest,

heated over water bath for two minutes, cooled and

filtered. The resulting filtrate is used for testing. 10

drops of filtrate are taken into test tube with 2 drops

of Meyer reagent added and white / yellow precipitate

is formed. Next 10 drops of filtrate were added into

test tube and 2 drops of bouchardate reagent were

added to form a brown to black precipitate. Then 10

drops of filtrate were put into test tube and then added

2 drops of dragendrof tuning and an orange to red

brown formed. If at least 2 out of 3 reagents produce

the same precipitate then it positively contains

alkaloids (Cocan et al., 2018; Delazar, Asgharian, and

Asnaashari., 2017).

2.3.5 Steroid / Triterpenoida Test

A total of 1 g of simplicia powder was macerated with

20 ml of n-hexane for 2 hours, and then filtered. The

filtrate is evaporated in a vaporizer cup. To remaining

2 drops of anhydrous acetic acid and 1 drop of

concentrated sulfuric acid are added. The purple or

red color then turns green blue indicating the presence

of steroids / triterpenoids (Cocan et al., 2018; Delazar,

Asgharian, and Asnaashari., 2017).

2.3.6 Glycoside Test

Simplisia powder was weighed as much as 3 grams,

then mixed with 30 ml mixture of 7 parts by 80%

ethanol volume and 3 parts by volume of distilled

water (7: 3), refluxed for 10 minutes, chilled and

filtered. To 20 ml filtrate, 25 ml of lead (II) acetate

0.4 N were added, shaken, allowed to stand for 5

minutes and then filtered. The filtrate was extracted 3

times, each time with 20 ml mixture of 3 parts by

volume of chloroform (p) and 2 parts by volume of

isopropanolol (p). To the chloroform layer, sodium

sulfate anhydrous (p) is added to taste, filtered and

evaporated at temperature of no more than 50

Dissolve the rest with 2 ml of methanol, then take 0.1

ml of the experimental solution put into test tube,

evaporated on a water bath. To remaining 2 ml of

water added and 5 drops of molish reagent, carefully

added 2 ml of sulfuric acid formed purple ring at

boundary of both liquids indicating the presence of

sugar bonds (Cocan et al., 2018; Delazar, Asgharian,

and Asnaashari., 2017).

2.3.7 Test of Anthraquinic Glycosides

A total of 0.2 g cocoa bean simplex powder was

weighed, and then 5 ml of 2 N sulfuric acid was

added, heated briefly, after being cooled, added 10 ml

of benzene, shaken and allowed to stand. The benzene

layer is separated and filtered, shaken the benzene

layer with 2 ml of NaOH 2N, allowed to stand. The

red water layer and the colorless benzene layer show

anthraquinone (Cocan et al., 2018; Delazar,

Asgharian, and Asnaashari., 2017).

2.4 Making Cocoa Bean Extract

The method of making cocoa bean extract is done by

using cold extraction method, namely maceration.

Simplisia which has been in the form of powder

weighed 500 grams, then macerated by soaking 10

parts of simplicia, then put in a vessel and poured 75

parts of the liquid, then covered and allowed to stand

for 3-5 days in place which is protected from sun

light. Shaken repeatedly, filtered then squeezed. The

pulp from maceration is washed by using 25 parts of

liquid until the juice is obtained. The vessel is closed

and left for 2 days in a cool place and protected from

sunlight, then separated the precipitate obtained. The

filtrate that has been produced is then evaporated by

using rotary evaporator until it thickens slightly, and

then evaporates over water bath to form a thick

extract (Baharium, 2014).

2.5 Sterilization of Tools and Materials

The tools used for antibacterial testing are first

sterilized by heating process carried out to kill all

forms of organisms. Non-glass tools are sterilized by

using an autoclave at 121

C for 15 minutes. Whereas

glassware is sterilized using an oven at 170

C for 1-

2 hours (Widyaningsih, et al., 2018).

2.6 Soap Formulation

According to Saryanti and Setiawan (2018),

formulations used in making liquid soap preparations

can be seen in Table 1.

Formulation and Anti Bacterial Activity Test of Liquid Soap Extract from Cocoa Beans (Theobroma cacao L.) against Staphylococcus

Aureus and Escherichia Coli Bacteria

295

Table 1: Formulations of Liquid Soap

Ingredient FI (g) F

II (g)

III

(g)

FIV

(Comparis

on)

Cocoa

bean

extract

5 10 20

Conventio

nal

Castor Oil 10 10 10

Olive Oil 15 15 15

Coconut

Oil

10 10 10

KOH 8 8 8

HPMC 3 3 3

Stearic

acid

2 2 2

Glycerin 18,75 18,75 18,75

BHT 0,02 0,02 0,02

Aquadest ad

100

2.6.1 Soap Making

Castor oil is first mixed with olive oil and coconut oil,

then stir slowly until homogeneous. Then KOH

solution is added little by little to the oil mixture at

temperature 60-70

C to form a paste. Then added

stearic acid and BHT which had previously been

melted, then put into the mixture and stirred until

homogeneous, then put HPMC which has been

developed in hot aquadest into mixture. Furthermore,

glycerin and cocoa bean extract are added and stirred

until homogeneous. Then add up to 100 ml of distilled

water and then stir until homogeneous and put into a

container (Saryanti and Setiawan., 2018).

2.7 Evaluate of Liquid Soap

Preparations

2.7.1 pH Test

Liquid soap pH requirements based on SNI meet the

requirements namely 8 - 11. pH Conditions that are

too low can cause irritation to the skin (Widyaningsih,

et al.,2018)

2.7.2 Irritation Test

The irritation test technique is an open sample test

(Patch Test) on the back of the ear against 20

panelists. A positive irritation reaction is

characterized by redness, itching, or swelling in the

skin area of the inner ear that is treated. The irritation

test parameters include swelling, itching, redness,

peeling and feeling dry with numerical assessment (1)

irritation, (2) slight irritation, (3) absence of irritation.

2.7.3 Determination of Antibacterial

Activity

Antibacterial activity test used disc diffusion method,

as much as 15 ml media Nutrient agar (NA) is poured

into the cup until evenly distributed, then

homogenized and allowed to stand at temperature

room until the media is solidified. Then as much as

0.1 ml of Staphylococcus aureus bacterial inoculum

suspension is inserted into sterile petri dish, then put

disc paper into petri dish, then dropped 3 drops of

cocoa bean extract test sample soap with various

formulations (FI, FII, FIII). In this study also used

Aquadest as negative control and Dettol liquid bath

soap as positive control. The cup is left at temperature

room for 10-15 minutes, and then incubated at 37° C

for 24-48 hours, then the diameter of the inhibition

zone around the disc is measured by using calipers.

Tests carried out with three repetitions (triplo). The

same treatment was carried out on Escherichia coli

bacterial inoculums (Ditjen POM, 1995).

3 RESULTS AND DISCUSSIONS

3.1 Phytochemical Screening

Phytochemical screening results of cocoa beans are

carried out to determine the class of secondary

metabolite compounds contained in it. The

examination of secondary metabolites is carried out

on groups of flavonoid compounds, tannins,

saponins, alkaloids, steroids / triterpenoids,

glycosides and anthraquinone glycosides. The results

of phytochemical screening of cocoa beans can be

seen in Table 2.

Table 2: Results of phytochemical screenings

Comp

ound

Class

Reacto

Result Conclus

ion

Refere

nce

(Indira

et al.,

2014)

dan

(Dedy,

2016)

Flavo

noid

Mixtur

e 0.1 g

of Mg

Ring is

formed

(+)

Flavono

(+)

Flavo

noid

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

296

powder

, 1 mL

HCl

(p), 2

Amil

alcohol

Colour

ed Red

in the

amyl

layer

alcohol

Tanin

FeCl3

(blue

blackis

green)

Blue

color is

formed

(+)

Tanin

(+)

Tanin

Sapon

Aquad

est is

hot

and

whippe

strong

Foam

formed

(+)

Saponin

(+)

Sapon

With

the

additio

n of 1-

2 drops

of HCl

Froth /

foam is

not lost

Alkal

oids

Meyer

reagent

(sedim

ent

yellow

white)

Was

formed

precipi

tate

yellow

/ white

(+)

Alkaloid

(+)

Alkalo

Reacto

Bouch

ardat

(sedim

ent

brown-

black)

Reacto

Bouch

ardat

(sedim

ent

brown-

black)

Steroi

(p)

(purple

and

red,

and or

change

green /

blue)

First of

all

purple,

then

change

green

(+)

Steroid

(+)

Steroi

Glyco

sides

Lead

(II)

acetate

0.4 N

purple

ring

forms

at the

second

fluid

bounda

(+)

Glikosid

(+)

Glikos

ida

Anthr

acino

Glyco

sides

2 N

sulfuri

c acid,

benzen

e, 2N

NaOH

No red

water

layer

formed

(-)

Anthraci

none

glycosid

(-)

Anthr

acinon

glycos

ides

3.2 Soap Quality

Soap testing is carried out to determine the physical

appearance and quality of cocoa bean liquid soap

preparations, organoleptic test examinations which

include the shape, odor and color of the preparation,

pH test, foam ability test and irritation test on

volunteers.

3.2.1 pH Test and Froth Capability

pH testing of liquid soap is done because the high and

low pH values of the soap preparations can affect the

absorption rate of the skin. Testing the ability of the

foam is done by fast shaking using a test tube, aiming

to determine the stability of the soap scum caused.

The testing results of pH analysis can be seen in Table

Table 3: pH value and foam stability

NO. Preparation pH Froth

Ability

(cm)

1. Formula I 10,32 6,8

2. Formula II 10,14 7,2

3. Formula III 9,5 7,2

pH testing of liquid soap is done because the high and

low pH values of the soap preparations can affect the

absorption rate of the skin. According to SNI, good

pH of soap is used which is alkaline in the range of 8-

11 to be able to clean the dirt on the skin. The pH

testing result on each formulation of cocoa beans

extract liquid soap has values ranging from 9.5 -

10.25. The addition of strong alkaline KOH can

increase pH value and the addition of high cocoa bean

extract to the formula results in decrease the pH

value. When compared with the comparator Dettol

Formulation and Anti Bacterial Activity Test of Liquid Soap Extract from Cocoa Beans (Theobroma cacao L.) against Staphylococcus

Aureus and Escherichia Coli Bacteria

297

soap which has pH 9 available on the market, the pH

of liquid soap with cocoa bean extract additive is also

close to the commercial standard.

The observations showed that each formula

produced relatively similar foam because it had value

of 6.4 cm while Dettol soap had value of 8.2 cm. The

difference occurred due to the surfactant used by

Dettol soap, namely sodium lauryl sulfate (SLS), so

that the resulting bubble foam was more numerous.

The bubbles shape produced in the liquid soap extract

of cocoa beans is very thin so that the rate of decline

in the foam very quickly disappears within ± 180

seconds. Measurement of foam height after 5 minutes

decreases with each formulation. It is due to the small

amount of surfactant that is added to the liquid soap

preparation so that there is no repulsion between

bubbles resulting in lower voltage and the bubbles

break easily (Politova, et al., 2018).

3.2.2 Irritation Test

The questionnaire results found that liquid soap

extract of cocoa beans showed no irritation. The

results of irritation testing data on 15 volunteers can

be seen in Table 4.

Table 4: Result of irritation test

Formu

Volunteer Skin Condition

Swellin

Red

dish

Itc

hin

F I I 1 1 1

II 1 1 1

III 1 1 1

IV 1 1 1

V 1 1 1

VI 1 1 1

VII 1 1 1

VIII 1 1 1

IX 1 1 1

X 1 1 1

XI 1 1 1

XII 1 1 1

XIII 1 1 1

XIV 1 1 1

XV 1 1 1

FII I 1 1 1

II 1 1 1

III 1 1 1

IV 1 1 1

V 1 1 1

VI 1 1 1

VII 1 1 1

VIII 1 1 1

IX 1 1 1

X 1 1 1

XI 1 1 1

XII 1 1 1

XIII 1 1 1

XIV 1 1 1

XV 1 1 1

FIII I 1 1 1

II 1 1 1

III 1 1 1

IV 1 1 1

V 1 1 1

VI 1 1 1

VII 1 1 1

VIII 1 1 1

IX 1 1 1

X 1 1 1

XI 1 1 1

XII 1 1 1

XIII 1 1 1

XIV 1 1 1

XV 1 1 1

Irritation testing is done to determine safety when

using antibacterial liquid soap extract of cocoa beans.

The irritation test on liquid soap is done by applying

preparations to the volunteers' inner arms. The inner

arm skin is more sensitive because the horny layer is

thin enough so that the absorption of soap

preparations by the skin is faster (Widyaningsih,

Chasani, and Diastut., 2018). The results of observing

the irritation test by spreading the questionnaire to 15

volunteers gave choice number 1 which indicated that

liquid soap extract of cocoa beans did not cause

irritation in the form of redness, swelling and itching.

It can be attributed to the fact that the liquid soap

extract of cocoa beans is formulated with natural

ingredients that are not harmful so it is safe to use.

3.2.3 Antibacterial Activity Test for Liquid

Soap Cocoa (Theobroma cacao L.)

Extract

The results of antibacterial activity testing on liquid

soap of cocoa bean extracts were carried out by using

three different formulations namely FI 5 g, FII 10 g,

and FIII 20 g. The results of inhibitory zones of liquid

soap extract of cocoa beans on the bacteria

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

298

Staphylococcus aureus and Escherichia coli can be

seen in Figures 1, 2, and Table 5.

Figure 1: Area of circle of inhibition zone of

Staphylococcus aureus

Figure 2: Area of circle of inhibition zone of

Escherichia coli

Table 5: Inhibition Zone of Theobroma Kakao L

Extraction

No. Treatment

Average diameter of

resistance (mm)

Staphylococcus

aureus

Escherichia coli

Aquadest - -

Dettol 15,70 15,26

FI 10,33 9,23

FII 11,70 10,53

FIII 12,93 10,90

The antibacterial activity results of liquid soap

preparations from cocoa bean extracts showed that

diameter of inhibitory zones in Staphylococcus

aureus and Escherichia coli bacteria were categorized

as having strong activity in inhibiting bacterial

growth. Inhibitory zones on positive control of Dettol

soap are 15.70 in Staphylococcus aureus bacteria and

15.26 in Escherichia coli bacteria and are categorized

as having strong inhibitory zone activity and the

results of aquadest as negative control do not form

inhibitory zones because aquadest does not have the

ability to inhibit bacterial growth.

The antibacterial activity of liquid soap extract of

cocoa beans showed that Gram positive testing of

Staphylococcus aureus bacteria had a greater

inhibition zone than Gram negative testing of

Escherichia coli bacteria from several variations of

liquid soap preparation formulas. According to Volk

(1992), it is due to differences in both of bacteria

testing where they have different cell wall

compositions and structures so that gram-positive

bacteria are more susceptible to chemical compounds

than gram-negative.

A Gram-positive bacterium has a simpler cell wall

structure, which is single layer that has low lipid

content (1-4%) so that bioactive materials more easily

enter the cell. While gram-negative bacteria have

more complex cell wall structure, which is three

layers consist of the outer layer of lipoprotein, the

middle layer of lipopolysaccharide that functions as a

barrier to the entry of antibacterial bioactive material,

and the inner layer of peptidoglycan which has high

lipid content (11-12%). In addition, according to

(Arafat and Rahman., 2017) certain antibacterial

compounds can increase their activity from

bacteriostatic to bacteriocidal if a concentration of the

compound in the preparation is increased. The greater

the concentration contained in an antibacterial agent,

the stronger the work activity.

4 CONCLUSIONS

Liquid soap extract contains cocoa beans that have

antibacterial activity but it is not greater than

antibacterial activity of Dettol soap.

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