The Physicochemical Characteristic and Inhibition Zone of

Eschericia coli in Ketepeng (Cassia alata L.) Leaf Extract

Transparent Soap

Encik Eko Rifkowaty

and Adha Panca Wardanu

Department of Plantation Product Management, Politeknik Negeri Ketapang, Ketapang, Indonesia

Keywords: Inhibition Zone of Eschericia coli, Ketepeng Leaf Extract, Physicochemical Characteristic, Transparent Soap.

Abstract: Ketepeng (Cassia alata) is a wild plant that is traditionally recognized as a medication for skin diseases. The

leaf of Ketepeng has a phytochemical content that plays a role to inhibit the growth of Eschericia coli. A

transparent soap with an addition of leaf extract is one of the innovations to increase the economic value of

the Ketepeng plant. This research aims to identify the effect of adding leaf extract on the quality of transparent

soap based on the Indonesian National Standards (SNI). The experiment design that used is Completely

Randomized Design (RAL) with a three-time repetition. It comprised three different treatments consist of the

addition of leaf extract as much as 2, 4, and 6 grams in 50-gram coconut oil as the basic material of soap. The

result of this research indicated that increasing the concentration of leaf extract might increase saponification

value, hardness, pH, inhibition zone of Eschericia coli ATCC 25922. It was also reported to increase the

attribute texture of the transparent soap, as well as to decrease water content, foam stability, free alkali, and

soap transparency. The best treatment in this study was the transparent soap which was added by 6 grams of

ketepeng leaf extract.

1 INTRODUCTION

The COVID-19 (coronavirus) pandemic requires

people to live clean. Washing hands with antiseptic

soap is one way to prevent COVID-19. Transparent

soap with the addition of ketepeng left extract is an

alternative to antiseptic soap. According Widiarto,

Janiarta, Intan and Hajiriah (2018) antiseptic soap is

a soap that contains chemical compounds that are

used to kill or inhibit the growth of microorganisms

on living tissues such as the surface of the skin and

mucous membranes. The use of antiseptics is highly

recommended when there is an epidermal disease

because it can slow the spread of the disease the

spread of the disease.

Ketepeng leaf extract has secondary metabolite

compounds for example alkaloid, flavonoid, tannin,

anthraquinone, and saponin have antibacterial

activities against Escherichia coli, Staphylococcus

aureus and Pseudomonas aeruginosa (Ekwenye and

Okorie, 2010; Lumbessya, Abidjulua, and Paendonga

https://orcid.org/0000-0003-2984-6977

https://orcid.org/0000-0002-4970-248X

2013). Ecoli is a gram-negative bacteria that can

cause skin diseases. However, it can be prevented by

the use of antibacterial soap (Stevens, Nicholas, and

David 2003). Generally, secondary metabolite

compounds have antibacterial. Thus, they can be used

as active materials in a soap-making process.

Meanwhile, saponin contains several properties like

the foaming agent, easily soluble, surfactant

compounds, and antibacterial (Tebogo, 2004).

Indonesia is home of tremendous natural

resources. One of the natural resources is Ketepeng

plant which is known as a cure and anti-bacteria for

skin problems such as ringworm and itching

(Kusmardi, Kumala, and Triana, 2007). Traditionally,

Ketepeng plant is applied by rubbing or sticking its

leaves to the affected skin. Esimone, Nworu, Ekong,

and Okereke (2007) discuss that this plant has an

excellent wound-healing characteristic. The results of

herbal soap which adding ketepeng leaves extract

with ethanol extract (95%) indicate has antibacteria

activities covering S. aureus, B. subtilis, E. coli, P.

786

Rifkowaty, E. and Wardanu, A.

The Physicochemical Characteristic and Inhibition Zone of Eschericia coli in Ketepeng (Cassia alata L.) Leaf Extract Transparent Soap.

DOI: 10.5220/0010953600003260

In Proceedings of the 4th International Conference on Applied Science and Technology on Engineering Science (iCAST-ES 2021), pages 786-793

ISBN: 978-989-758-615-6; ISSN: 2975-8246

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

aeruginosa, and C. Albicans. The soap with adding of

ketepeng extract on the market.

Ecoli is microorganism which can serve as an

indicator towards the possible existence of pathogen.

Pathogen is gram negative bacteria that can cause

skin diseases. However, it can be prevented by the use

of antibacterial soap (Stevens et al. 2003).

antibacterial properties such as saponin, tanin,

terpenoid (Ugbabe, Ezeunala, Edmond, Apev and

Salawu., 2010), alkaloid (Jasim, Hussein, Hameed

and Kareen 2015), flavonoid, tripenoid (Cushnie and

Lamb, 2005). Thus, they can be used as active

materials in a soap-making process. Meanwhile,

saponin contains several properties like foaming

agent, easily soluble, surfactant compounds, and

antibacterial (Widyasanti and Hasna, 2017 ; Tebogo,

2004).

This research uses coconut oil as a basic

ingredient in making soap. Coconut oil contains

saturated fatty acids which play a role in the foaming

properties of the soap. In particular, Lauric acid

(C12:0) in coconut oil produces soap with high

solubility and soft foam characteristics (Anggraini,

Ismanto, and Dahlia, 2015; Habib, Kumar, Sorowar,

Karmoker, Khatun and Al-Reza, 2016). This

becomes the basis of the research because it is

assumed that the addition of ketepeng leaf extract

with different concentrations can affect the

physicochemical characteristics of the resulted soap

and increase the inhibition zone of Escherichia coli.

This research could be antiseptic soap alternative in

the pandemic situation.

2 MATERIAL AND METHODS

2.1 Raw Material

This research was conducted in two phases. It started

by preparing sorting ketepeng leaves, extraction, and

a transparent soap making process. The ketepeng leaf

were taken into 5 parts from left to right of the stem

base of the Ketepeng. Prepare 50 g coconut oil. After

that, the soap involved chemical materials by using

NaOH (merck) 5 g, NaCl (merck) 0.2 g, citric acid 0.2

g, glycerin (brataco) 6.5 g, aquadest 5 ml, sugar

(brand: Gulaku) 7.5 g and ethanol (merck) 70% 7.5 g.

Pure inoculant of ATCC 25922 Escherichia coli.

2.1.1 Ketepeng Leaf Extraction

The process of ketepeng leaf extraction was

performed by refining 20 g ketepeng leaves which

had been sorted and dissolved with a 200 ml alcohol

solvent. The ratio of ingredients and solvents was

1:10. Having filtered the basic ingredients, then the

filtrate was evaporated by using a rotary evaporator

(RV-10, IKA-WERKE, Germany) at a 70⁰C

temperature, a speed of 160 rpm, and a pressure of

2.5. The process took place for 9 hours in order to

obtain thick extracts (Hernani, Bunasor and Fitriarti,

2010).

2.1.2 Making of Transparent Soap

The transparent soap was made by heating 50 g

coconut oil at 70⁰C temperature. After that, 5 g NaOH

was dissolved to 5 ml of water and stirred until

dissolved. Put the solution in the oil and then add 7.5

g of ethanol and 7.5 g of sugar. Prior to that, the

ethanol and sugar should be dissolved. Finally, add

glycerin by 6.5 g. All materials were stirred until the

stock of soap was formed. It was followed by adding

0.2 g of NaCl, 0.2 g of citric acid, and extract of

ketepeng leaf by 2, 4, 6 g until all the mixture was

immunized. After that, the mixture was poured into a

mold and it was cooled until the soap hardens. The

resulted soap was then examined in several tests like

free alkali test, water content test, hardness, pH test,

saponification value, foam stability, inhibition zone

test, and organoleptic test of color, texture,

transparency.

2.2 Statistical Analysis

This research used a completely randomized design

using 3 (three) repititions. After gathering the data, if

F table was smaller than F count, further test was

conducted namely the least significant different

(LSD) with 0.01 rate. The observation parameter

covered:

2.3 Parameter Analysis

2.3.1 Water Content

This research counted 5 gram of transparent soap in a

petri dish and put in the oven at 105⁰C temperature

for 4 hours. Then, the soap was cooled, desiccated,

weighted, and heated for 1 hour to obtain a constant

weight. After that, the water content was calculated

by using the following formula:

2.3.2 pH

The soap was weighted 1 g and dissolved in 10 ml

aquadest. When necessary, the mixture could be

heated to accelerate the dissolution process. After

that, a pH meter was dipped in the solution. Acidity

The Physicochemical Characteristic and Inhibition Zone of Eschericia coli in Ketepeng (Cassia alata L.) Leaf Extract Transparent Soap

787

degree (pH) was observed to obtain and take note the

results.

2.3.3 Hardness

To measure the level of hardness towards the resulted

soap, the research employed a penetrometer tool.

2.3.4 Foam Stability

1 g of sample was dissolved in 9 ml water and then

poured in a reaction tube. Then, the mixture was

shaken by using vortex for 30 seconds. This process

resulted foam and the height of the foam was

measured. The sample foam was left for 1 hour and

the foam was measured again. If the sample number

was more than one, the dimensions of all tubes must

be similar. To measure, the foam stability, the

following formula could be performed (1).

   ()

   ()

x 100 % (1)

2.3.5 Saponification Value

The soap was weighted 1.5 g in 250 ml of

Erlenmeyer. Add 50 ml NaOH solution in alcohol.

After that, the solution was covered by a condenser

and boiled for 30 seconds. Then, add several drops of

phenolphthalein indicators and titrate with 0.5 N HCl

until its color changed from pink to transparent.

Saponification measure following formula (2).

(



)

   ,

,  

𝑥 100% (2)

Description:

= The volume of the sample titration

= The volume Blanco titration

N = Normality 0,5 HCl

0,04 = Atom Weight NaOH

0,258 = The average saponification value of

coconut oil

W = Sample weight

2.3.6 The Number of Free Alkali

The transparent soap was weighted 10 g and

dissolved in 50 ml of hot alcohol. After that, drop

indicators pp 2-3 drops, refluxed for about 30

minutes. Then the soap was cooled and titrated by

using KOH 0.1% until it turned to pink. Take note the

titration volume and calculate the free alkali (3).

 

  

𝑥 100% (3)

Description:

V = Titration volume

BM = molecule weight

W = Sample weight

2.3.7 Bacteria Inhibition Zone Test

1) Microbe Rejuvenation Test

NA Media was weighted 5 g and dissolved by

aquadest by 250 ml. Sterilization was performed until

the temperature reached 121⁰C. Keep it still for 15

minutes then the media was put into reaction tubes

respectively to form titled NA. After the solid titled

NA was obtained from the Eschericia coli ATCC

25922 breed as much as one inoculating loop. Then,

they were inoculated in the titled NA media. The

tested bacteria were incubated for 24 hours at 37⁰C

and it could be used as tested bacteria.

2) Making test bacterial suspension

Test bacteria resulted from rejuvenation process went

through suspension process by using NaCl 0.9%

solution which was put into respective reaction tubes.

After that, it was mixed with a sterilized NA media

and then immunized.

3) Bacteria inhibition zone test

NA media was turned to a solid form in a petri dish.

After being solid, prepare a bacteria suspension test.

Dip a stick with cotton at the end on the bacteria

suspension test than swab vertically and horizontally

on the NA surface which had been solidified until all

surfaces were covered. Leave it until all surfaces

dried. Meanwhile, each transparent soap sample was

melted on a hot plate. Soak disc paper for 1 hour in

the soap sample which was melted and then dry the

disc paper.

After all NA surfaces dried, the disc paper which

was soaked in the transparent soap was put on the NA

surface by using pin set. It was then incubated at 37⁰C

for 24-48 hours. The petri dish which was incubated

for 24-48 hours then was observed and measured to

obtain its inhibition diameter.

2.3.8 Organoleptic Test

Organoleptic test was performed to score the attribute

of transparency and texture. Panelists in this research

were 25 semi-trained panelists. The assessment score

was based on the level of quality where 1 (one)

indicated the lowest score and 7 (seven) meant the

highest.

iCAST-ES 2021 - International Conference on Applied Science and Technology on Engineering Science

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3 RESULT AND DISCUSSION

3.1 Water Content

High water content in the soap may cause a hydraulic

reaction between water and fat which is not

sapofinied to form free fat acid and glycerol. This

may cause the decrease of quality during storage

(Vivian, Nathan, Osano, Mesopirr and Omwoyo,

2014). The high water content may lead the soap to

easily dissolve in water, shrink, and have a soft

texture (Hambali, Bunasor, Suryani and Kusumah

2005). In addition, it may cause a short span of the

soap storage (Habib, et.al., 2016). Table 1 shows that

the water content kept decreasing as the addition of

ketepeng leaf extract. The water content of soap

which was added by ketepeng leaf extract by 2, 4,

and 6 gram decreased by 18.15, 12.09, and 10.78%.

According to Indonesian National Standard (SNI),

the maximum water content of soap is 15%.

Table 1: Water content of Transparent Soap.

Ketepeng Leaf

Extract Treatmen

Water Content

(%)

18.15

±0.23

12.09

±0.41

10.78

±0.28

The decrease of water content in the soap which

was added by ketepeng leaf extract was resulted from

active compound of saponin contained in the plant.

This compound possessed a characteristic to absorb

water which could decrease the percentage of water

in the soap. Widyasanti and Hasna (2017) in their

study on transparent soap with white tea extract claim

that the more white tea extract added to the soap, the

more water will be absorbed by sugar so that the

water in the soap decreases. Saponin is a glycoside

compound which will produce glycone (sugar) and

glycone (non-sugar) if hydrolyzed. Sugar has

hygroscopic properties and causes the soap to

solidify.

3.2 Saponification Value

The saponification value gives information about the

solubility in water and soap formation (Ohimain,

Izah, and Fawari, 2013). The degree of saponification

depends on the type of ion, its ionic strength, the

temperature of the solution, and above all on the pH

of the solution (Briscoe, Evans, and Tabor, 1976).

Saponification value indicates the neutralization

process between fatty acid and base in the process of

soap stock. If the saponification is not perfect, it may

increase the percentage of free alkali or unsaponified

fat acid. As a result, it can decline the quality of the

soap.

The addition of leaf extract increased significantly

the value of saponification (Tabel 2). The increase of

this value is a result of alkaloid content in the leaf

extract. Alkaloid constitutes a hydrogen base so that

the addition of leaf extract will affect the solution's

basicity. Cotte, Checroun, Susini, Dumas, Tchoreloff,

Besnard and Walter (2006) explain that the process of

triolein saponification with various types of lead salts

on pH base can increase the percentage of soap

amount. Sears and Schulman (1964) argue that pH 13

saponification is essentially complete. At a lower pH

8.5 the degree of association between the cation and

the fatty acid could be less than complete.

Table 2: Saponification Value of Transparent Soap.

Ketepeng Leaf

Extract Treatmen

Saponification Value

/NaOH)

155.5

± 0.81

164.8

± 0.18

178.2

± 0.46

3.3 Hardness

Level of hardness test aims at understanding the

efficiency of the soap when used. Hard soap is

considered higher resistance towards damage or form

changing as a result of physical disturbances.

Table 3: Hardness of Transparent Soap.

Ketepeng Leaf

Extrac

Treatmen

Hardness

0.5

±0.04

1.1

±0.05

1.4

±0.02

Level of hardness on the soap correlated with the

percentage of water content and saponification value.

The water content decreases when the leaf extract is

added and the level of hardness increases too.

Similarly, the increase also occurs to saponification

value that can affect the hardness of the soap.

Saponifaction value provides information about the

soap forming process (Ohimain et al. 2013). A high

value of saponifation that has a lot of greased fat

indicates a high quality of the soap (Ketaren, 1986

cited in Kusumaningsih and Hastuti, 2014). Soap

texture can be affected by the length of hydrocarbon

chain and oil doubled bond.

The Physicochemical Characteristic and Inhibition Zone of Eschericia coli in Ketepeng (Cassia alata L.) Leaf Extract Transparent Soap

789

3.4 Foam Stability

Foam stability is measured by esthetic value of a soap

product. Customers perceive that good soap is those

which produce much foam. In fact, the amount of

foam is not necessarily correlated positively with its

ability to clean dirt. There is no requirement regarding

the minimum or maximum of foam height for soap.

The results of this research revealed that the

addition of leaf extract in the soap would make the

foam stability low. Based on LSD (0.01) soap that

was added 4 and 6 grams of the leaf extract was

significantly different from a 2-gram addition. The

concentration of leaf extract increases significantly

decrease the stability of the foam.

Table 4: Foam Stability of Transparent Soap.

Ketepeng Leaf

Extract Treatmen

Foam Stability

55.5

±1.08

38.4

±0.79

35.7

±0.92

Ketepeng’s leaf which is extracted by using

ethanol contains alkaloid, saponin, flavonoid, tannin,

and antraquinon compounds (Lumbessya et al. 2013).

Jin-Young Park et al. (2005) argue that saponin has

several properties such as easy to dissolve in water,

surfactant, and the ability of form stable foam in

solution. For these reasons, saponin is often used as

an agent of foaming, emulsifier, and detergent in meal

and non-meal products. However, as the

concentration of leaf extract increases, the stability of

the foam decreases. This is in line with a study by

Widyasanti and Hasna (2017) in transparent soap

with white tea extract. Adding white tea extract would

make the foam stability low. This occurs because the

foam stability is influenced by ethanol content in the

white extract. Putri (2017) supports that methanol and

ethanol in the extracts play a role as antifoaming

agent in the soap.

Beside saponin, alkaloid also affects the stability

of foam in soap. Saunders (1935) widely claims that

the tension of soap surface is affected by pH. pH itself

is influenced by the amount of organic base like

alkaloid. Widyasanti and Hasna (2017) posit that

alkaloid has base properties causing it to increase the

degree of acidity in soap. According to Gwi-Taek

Jeong, Hwa-Won Ryu, Yung-Il Joe, Don-Hee Park

and Tanner (2002) base pH can potentially decrease

the foam stability. Hence, it is assumed that the

addition of leaf extract will increase alkaloid content

in the soap that it can decrease the foam stability.

Ketepeng extract contains pigment chlorophyll,

the pigment can arise in the solution can increase

surface tension, thereby reducing foam stability. The

presence of dissolved substances in the liquid will

increase viscosity which in turn will increase surface

tension and result in reduced foaming ability.

3.5 pH

Incomplete hydrolysis from saponification process

produced high pH values so that acidity degree (soap

pH) should be measured. Normal soap pH is around

9.0-10.8 (Gusviputri, Meliana, Aylianawati and

Indraswati 2013). pH 10 has base properties and is

good for skin. If the pH is <9, it might cause the skin

dry. Meanwhile, pH values which are >10.8 may

cause skin irritations. Table shows that the addition of

leaf extract by 2, 4, and 6 gram has acidity degree or

pH around 9.33-9.67. It indicates that pH of

transparent soap which is added by the leaf extract has

met SNI 06 3532 1994.

Table 5: pH of Transparent Soap.

Ketepeng Leaf

Extract Treatmen

9.33

±0.94

9.67

±0.47

9.67

±0.47

3.6 Free Alkali

Free Alkali in soap is caused by the presence of alkali

which does not react with fat acid during a

saponification process (Zulkifli and Estiasih, 2014).

The increase of concentration of leaf extract

significantly raises the saponification values. It leads

to the decline of free alkali because of the lack of

alkali which reacts with fatty acid. Based on SNI 06

3532 1994 regulations, the value of free alkali in soap

should be a maximum of 0.1%. In this research, the

LSD (0.01) test of free alkali towards the three

treatments was not significantly different. It shows us

that the tested soap is safe. High free alkali (>0.1%)

will lead to skin irritations (Hernani et al. 2010) and

dry skin (Widyasanti and Hasna, 2017).

Table 6: Free Alkali of Transparent Soap.

Ketepeng Leaf

Extract Treatmen

Free Alkali (%)

0.0060

±0.006

0.0056

±0.006

0.0026

±0.003

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790

3.7 Inhibition Zone

Escherichia coli is bacteria that cause diarrhea,

urinary tract infection, meningitis, peritonitis,

mastitis, septicemia, pneumonia, and food poisoning

(Brooks, Butel and Morse 2008). Inhibition zone is an

active substance’s or compound’s ability to inhibit the

growth of microbe. It is marked by the existence of

transparent zone on agar media. Based on LSD (1%)

test, the inhibition zone of transparent soap towards

the three treatments was significantly different. The

more the addition of leaf extract on the soap would be

able to inhibit the growth of Escherchia Coli ATCC

25922. The difference of the inhibition zone size from

each concentration stemmed from the difference of

active compound contents. It was shown by the size

of the inhibition zone which increasingly widespread.

Juliansyah and Paotonan (2017) support that the

higher the concentration of the extract, the more

active compounds which potentially serve as

antibacterial. The ketepeng leaf extract contains

secondary metabolite compounds like alkaloid,

flavonoid, anthraquinone, and saponin. These

compounds constitute as phenolic compounds which

has a phenol group. Hernani et al. (2010) argue that

the phenol group has bacteriacide properties because

it may cause protein coagulation and bacteria cell

membranes may become lysis.

Table 7: Inhibition Zone of Transparent Soap.

Ketepeng Leaf

Extract Treatmen

Inhibition Zone

(mm)

1.8

±0.24

4.0

±0.47

6.0

±0

In this research, the inhibition zone of transparent

soap which was added leaf extract by 2 gram (1.8

mm) was categorized weak. The addition of the

extract by 4 gram (4 mm) and 6 gram (6 mm) was

grouped medium. Magdalena and Kusnadi (2015)

explain the level of resistance of negative Gram

bacteria (Escherichia coli ATCC 25922) was high

One of the reason was because the structure of

bacteria cell walls of the negative gram bacteria was

complex. It had three layers, outside layers

lipoprotein, middle layer lipopolysaccharide which

served to block antibacterial bioactive materials, and

inside layer peptidoglycan which had high lipid

contents (11-12%). This made the active compound

of leaf extract difficult to penetrate the nonpolar lipid

layer on Escherichia coli ATCC 25922.

3.8 Organoleptic Test

An organoleptic test for transparent soap with leaf

extract covered attribute of transparency and texture

ranging from 1 – 7. Soap texture assessment is

performed by pressing samples using fingers. In the

other side, transparency test is done by using the

senses of vision by observing samples with the help

of light (Meilgaard, civele and Carr, 1999).

Table 8 show that the texture attributes of the

three treatments were not significantly different. It

can be seen from the results of the panelist's

assessment which pointed out that the three

treatments had the same texture (from neutral to

rather hard). Meanwhile, the attributes of

transparency in the three different treatments were

very significant (neutral to transparent).

Table 8: Organoleptic Test of Transparent Soap.

Ketepeng Leaf

Extract

Treatmen

Organoleptic Test

Texture Transparency

4,32 5,48

4,40 5,36

4,72 4,20

The increased concentration of leaf extract could

improve the texture of the soap. This result is

correlated with the decrease of the soap water content.

Low water content in soap increases the hardness of

the soap (Tabel 1). Based on the organoleptic test,

The texture attributes of the three treatments were not

significantly different. It indicates that the addition of

leaf extract does not affect consumer perceptions

towards texture attributes.

Meanwhile, adding 2 and 4 grams of ketepeng leaf

extracts was significantly different from 6 grams on

the attribute of transparency (Tabel 8). It shows that

the addition of ketepeng extract affects consumer

perceptions of the attributes of transparency. The

more addition of leaf extract to transparent soap, the

browner and less transparent soap is. According to

Anggraini et al. (2015) a heating process causes

chlorophyll (green) to become a compound of

pheophytin (olive green). Therefore, if the soap is

added by more leaf extract, the soap will turn to

dominantly brown. The flavonoid content contained

in the extract also affects transparency. According to

Ahmad, Hasan, Muhamad, Bilal, Yusof and Idris

(2018) the brightness level of transparency decreases

with the increased content of flavonoids and phenol

compounds.

Based on the Indonesian National Standards

(SNI) for solid bath soap, the treatment of the addition

The Physicochemical Characteristic and Inhibition Zone of Eschericia coli in Ketepeng (Cassia alata L.) Leaf Extract Transparent Soap

791

of 6-gram ketepeng leaf extract had a better quality

soap than the two treatments. The treatment also had

the largest ATCC 25922 Escherichia coli inhibition

zone as much as 6 mm despite having low foam

stability by 35.7. Finally, the organoleptic score of

texture attribute was 4.72 (rather hard) and the

transparency value was 4.20 (from neutral to rather

transparent).

4 CONCLUSION

In conclusion, increasing the concentration of leaf

extract could increase saponification, hardness, pH,

Escherichia coli inhibition zone ATCC 25922, and

the texture attributes of transparent soap. In addition,

it is capable of reducing water content, foam stability,

free alkali and soap transparency.

The best treatment in this study was transparent

soap which was added by leaf extract as much as 6

grams, although it produced soap which its

transparency attributes ranged from neutral to rather

transparent.

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