Synthesis of Antimicrobial and Emulsifier Compunds through

Hydroxyl Group Esterification of Oxidized Ricinoleic Acid

Rifqah Azzahra Naulidia, Sri Handayani, Siswati Setiasih and Sumi Hudiyono

Department of Chemistry, Universitas Indonesia, Depok, Indonesia

Keywords: Ricinoleic Acid, Oxidation, Esterification, Emulsifier, Antimicrobial Agents

Abstract: In this study, ester synthesis from commercial oxidized ricinoleic acid using various carboxylic acid was

conducted. The double bond in ricinoleic acid was oxidized using KMnO

in alkaline condition to form two

hydroxyl groups. Oxidized ricinoleic acid was then esterified chemically using palmitic acid, decanoic acid,

and butyric acid by ZnCl

as catalyst with the molar ratio of oxidized ricinoleic acid to carboxylic acid was

3:1. Esters produced were characterized using FTIR examined as emulsifier and the antimicrobial activity.

The results showed that each ester product gave absorption band C=O ester at the range of 1600 cm

-1

-1720

-1

. The highest conversion percentage of esterification was obtained by oxidized ricinoleic acid-palmitic

acid esters with the value of 75%. Simple emulsifier test was performed for each ester and the result showed

that esters were able to maintain an emulsion form approximately 24 hours with water-in-oil emulsion (w/o)

type. The antimicrobial activity test of esters gave positive results in the presence of inhibition zone to the

growth of Propionibacterium acnes and Staphylococcus epidermidis. The highest antimicrobial activity

against P. acnes and S. epidermidis was produced by oxidized ricinoleic acid-decanoic acid esters.

1 INTRODUCTION

Ricinus communis L. is one of industrial plant that

has bright prospects to be developed in

Indonesia. Ricinus communis L. seeds contain 46%

oil, which are known as castor oil. Castor oil content

is dominated by ricinoleic acid (89,5% of total fatty

acids). Ricinoleic acid (C18) is an unsaturated fatty

acid with one double bond at carbon-9 (C

) and

hydroxyl group in the side chain (R) position of

carbon-12 (C

). Other than double bonds and

hydroxyl groups, ricinoleic acid has carboxylic

groups which make this fatty acid can be reacted

through various reactions to get its derivative

product. One form of its derivative product is

oxidized ricinoleic acid esters (Kajikawa et al.,

2016).

The double bond in ricinoleic acid can be

oxidized by using a strong oxidizer to produce diol.

The addition of diol groups can increase polarity of

ricinoleic acid which makes this compound to

reduce surface tension, because it can combine polar

and nonpolar phase. The hydroxyl groups of

oxidized ricinoleic acid can be esterified by

carboxylic acids to form oxidized ricinoleic acid

esters which can be used as an emulsifier due to its

properties.

Previous study showed that derivatives of fatty

acid in the form of long chain esters could be used to

be skin barrier (Pérez et al., 2016). In this study,

oxidized commercial ricinoleic acid esters were

synthesized by esterification reaction with palmitic

acid, decanoic acid, and butyric acid using ZnCl

catalyst. Esterification products were then

characterized using FT-IR and examined its ability

as emulsifier and its antimicrobial activity against

Propionibacterium acnes and Staphylococcus

epidermidis.

2 EXPERIMENT

2.1 Materials

Materials used in this study were commercial

ricinoleic acid, sodium hydroxide, potassium

permanganate, chloroform, Wijs solution, potassium

iodide, sodium thiosulphate, starch, palmitic acid,

decanoic acid, butyric acid, zinc chloride, n-hexane,

eosin, phenolphthalein, methanol, antibiotic

clindamycin, nutrient broth, nutrient agar,

Azzahra Naulidia, R., Handayani, S., Setiasih, S. and Hudiyono, S.

Synthesis of Antimicrobial and Emulsiﬁer Compunds through Hydroxyl Group Esteriﬁcation of Oxidized Ricinoleic Acid.

DOI: 10.5220/0010133000002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 42-46

ISBN: 978-989-758-556-2

Propionibacterium acnes and Staphylococcus

epidermidis from Universitas Indonesia.

2.2 Methods

2.2.1 Oxidation of Commercial Ricinoleic

Acid

Oxidation was carried out by mixing commercial

ricinoleic acid, NaOH 2 M, and KMnO

0.5 M. This

mixture was stirred using magnetic stirrer at

temperature 25

C-27

C for 30 minutes. After 30

minutes, there would be two phases. Organic phase

was then separated and filtered.

2.2.2 Determination of Iodine Value

Oxidized ricinoleic acid was mixed with chloroform

and Wijs solution, then left for 30 minutes in dark

place. After 30 minutes, KI 15% and distilled water

were added. The mixture was then titrated using

sodium thiosulphate 0.1 N until the colour of

mixture turn to yellowish. Starch was added and the

mixture was titrated again by sodium thiosulfate

until the colour turned clear (Goud et al., 2006).

2.2.3 Esterification

Esterification was carried out by reacting oxidized

ricinoleic acid in n-hexane as solvent with palmitic

acid on reflux system at temperature 60

C for 6

hours using ZnCl

as catalyst. ZnCl

used in this

reaction used in this reaction was 0.3 % of the total

mass substrate (w/w substrate). The mol ratio of

oxidized ricinoleic acid to palmitic acid used in this

reaction was 3:1 (mol/mol) (Gonçalves et al., 2011).

The same steps were performed using decanoic acid

and butyric acid. Ester products were then purified

by extraction using n-hexane and methanol.

2.2.4 Determination of Conversion

Percentage

The conversion percentage was determined by

titrating the organic phase that has been obtained

from extraction in n-hexane using 0.1 N NaOH and

phenolphthalein as indicator (Handayani et al.,

2012).

2.2.5 FT-IR Analysis

Commercial ricinoleic acid, oxidized commercial

ricinoleic acid, and all esters were characterized

using FTIR.

2.2.6 Simple Emulsifier Test and

Determination of Emulsion Type

Emulsifier test was performed by mixing 0.1 g

esters, oil, and water according to Table 1. The

mixtures were shaken, and the emulsion stability

was observed up to 24 hours. The emulsion type

determination was observed under microscope using

eosin as indicator.

Table 1: Variation of Oil and Water Composition in the

Making of Emulsion.

Oil in Wate

Water in Oil

Tube

Numbe

1 2 3 4 5 Tube

Numbe

1 2 3 4 5

Water

(ml)

2 2 2 2 2 Water

(drops)

2 4 6 8 10

Oil

(drops)

2 4 6 8 10 Oil (ml) 2 2 2 2 2

2.2.7 Antimicrobial Activity Assay

Disc diffusion method was performed to determine

antimicrobial activity. An aliquot of 200 µL bacteria

suspension with density 1x10

cells/mL was

aseptically mixed by 20 mL nutrient agar in a sterile

petri dish. The sterile paper disc (± 6 mm in

diameter) was placed on top of the medium and

dropped by 4 μL sample to be tested. The medium

was then incubated at 37°C for 24 hours. The clear

zone around the paper discs were measured. The

bacteria that used in this research were

Staphylococcus epidermidis and Propionibacterium

acnes.

3 RESULT AND DISCUSSIONS

3.1 Oxidation of Commercial

Ricinoleic Acid

The double bond on commercial ricinoleic acid were

oxidized through dihydroxylation reaction, which

would produce two hydroxyl groups. In this study,

oxidation was performed in an alkaline condition. At

cold temperatures with low concentrations of

oxidizing agents, alkenes tend to form glycols. At

the end of reaction, MnO

was produced. The MnO

could be removed by filtering oxidation product.

3.2 Determination of Iodine Value

Oxidation product was identified through

determination of iodine value to testify that

oxidation has successfully carried out. The iodine

Synthesis of Antimicrobial and Emulsiﬁer Compunds through Hydroxyl Group Esteriﬁcation of Oxidized Ricinoleic Acid

value of oxidized ricinoleic acid before oxidation

was 45.30 mg/g and after oxidation it decreased to

17.29 mg/g. The decreased in iodine number proved

that ricinoleic acid was successfully oxidized.

3.3 Esterification

In this study, esterification reaction was carried out

on the OH group of oxidized ricinoleic acid with

palmitic acid, decanoic acid, and butyric acid using

ZnCl

catalyst. The ester products expected to be

produced were monoesters. The mole of oxidized

ricinoleic acid is higher than fatty acid. This referred

to Le Chatelier’s principle. The increased of

reactants amount will produce more products.

3.4 Determination of Conversion

Percentage

Esters that formed were purified by extraction using

n-hexane and methanol. The n-hexane layer was at

the top. This layer consisted of non-oxidized and

non-esterified compounds, while methanol layer is at

the bottom consisted of esters. The conversion

percentage is shown in Figure 1. The highest

conversion percentage was obtained by oxidized

ricinoleic acid-palmitic acid esters with the value of

75%. At the beginning of reaction, conversion

percentage of ester products will decrease along with

the chain lengthening of fatty acid used. This is

because ester products from short-chain fatty acid

are more soluble in organic solvents, such as n-

hexane. However, when the reaction is completed,

conversion percentage will remain the same

(Macierzanka & Szela̧ g, 2004).

Figure 1: The Curve of Fatty Acid Variation vs

Conversion Percentage.

3.5 FT-IR Analysis

The formed product was characterized using FTIR to

determine the success of the hydrolysis, oxidation,

and esterification reaction. IR spectrum of ricinoleic

acid, oxidation products, and ester products are

shown in Figure 2.

The FTIR spectrum of ricinoleic acid showed the

absorption peak of group C=O carboxylic acid at

wave number 1710.93 cm

-1

. In addition, there was

also the absorption peak of the -OH at wave

numbers 3408.36 cm

-1

Oxidized ricinoleic acid which was successfully

oxidized has the addition of two -OH groups to the

fatty acid carbon chain. The -OH group’s intensity

increased indicated the oxidation reaction has

succeeded. Oxidation products had an absorption

peak of –OH at wave number 3426.69 cm

-1

. In this

study, there was still C=C group with a small

intensity which indicated that not all double bonds

were oxidized.

IR spectra of esterification products showed

absorption peak of C-O ester group in the range of

wave numbers of 1300-1000 cm

-1

which indicated

the ester has formed. The -OH group’s intensity

decreased since esterification has occurred.

(a)

(b)

(c)

Figure 2: FTIR Spectra (a) Commercial Ricinoleic Acid,

(b) Oxidized Ricinoleic Acid, (c) Esters.

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

3.6 Simple Emulsifier Test and

Determination of Emulsion Type

The simple emulsifier test showed that commercial

ricinoleic acid, oxidized ricinoleic acid, and esters

can act as emulsifier up to 24 hours. All products

had ability to form water in oil (w/o) type of

emulsion (Figure 3). Figure 3 showed that clear zone

surrounded red droplets. Red droplets formed were

water phase with eosin dye which dissolved because

they have same polarity, while the surrounding clear

solution was oil.

The ability of a compound as an emulsifier is

determined based on the presence of a hydrophobic

group and a hydrophilic group. The hydrophilic

group will bind to water, while the hydrophobic

group will bind to oil. If the ratio of water and oil

composition is balanced, the emulsion formed will

be stable.

Figure 3: Emulsion Types (a) Commercial Ricinoleic

Acid, (b) Oxidized Ricinoleic Acid, (c) Oxidized

ricinoleic acid-palmitic acid ester, (d) Oxidized ricinoleic

acid-decanoic acid ester, (e) Oxidized ricinoleic acid-

butyric acid ester.

3.7 Antimicrobial Activity Assay

In this study, bacteria that used were Staphylococcus

epidermidis and Propionibacterium acnes. These

two bacteria are acne causing bacteria. Table 2

shows the effectiveness classification of

antimicrobial substances (Greenwood, 1995), while

Table 3 shows antimicrobial activity of all products.

The positive control used in this study was

clindamycin 0.5%. Clindamycin is an antibiotic

commonly used for acne-causing bacteria. The

negative controls used were n-hexane and methanol

as they were used as solvents for dilution. The

purpose of using negative control was to compare

that the solvent used did not affect antimicrobial

activity (Natheer et al., 2012).

Based on Table 3, it was known that all esters

showed antimicrobial activity, while the positive

control showed no antimicrobial activity. Oxidized

ricinoleic acid-decanoic acid esters showed the most

effective antimicrobial activity against

Propionibacterium acnes and Staphylococcus

epidermidis.

Table 2: Classification of effectiveness of antimicrobial

substances (Greenwood, 1995).

Inhibit Zone

Diameter

Response of growth

barriers

> 20mm Strong

16-20 mm Medium

10-15 mm Weak

< 10 mm Not effective

Table 3: Diameter of the Inhibitory Zone of Various

Compunds against P. acnes and S. epidermidis

Sample Inhibitory

Zone (mm)

Effectiveness

acnes

S.epide

rmidis

P. acnes S.epider

midis

Commercial

Ricinoleic

Acid

11 7 Weak Not

effective

Oxidized

Ricinoleic

Acid

20 22 Medium Strong

Oxidized

Ricinoleic

Acid-Palmitic

Acid Ester

13 12 Weak Weak

Oxidized

Ricinoleic

Acid-

Decanoic

Acid Ester

22 29 Strong Strong

Oxidized

Ricinoleic

Acid-Butyric

Acid Ester

15 20 Weak Medium

Palmitic Acid - - Not

effective

Not

effective

Decanoic

Acid

10 15 Weak Weak

Butyric Acid 27 25 Strong Strong

Clyndamycin 10 10 Weak Weak

n-hexane - - Not

effective

Not

effective

Methanol - - Not

effective

Not

effective

Antimicrobial activity is influenced by the form

of esters. Monoesters are more active as

antimicrobial agents than diesters and triesters

because the longer carbon chain the more nonpolar

Synthesis of Antimicrobial and Emulsiﬁer Compunds through Hydroxyl Group Esteriﬁcation of Oxidized Ricinoleic Acid

molecule. So that its solubility in water is low

(Kabara, 1984).

4 CONCLUSIONS

Synthesis of oxidized ricinoleic acid esters using the

ZnCl

as catalyst have been successfully performed

by showing the characteristics of the C-O ester

group at FTIR spectra. Esters could act as

emulsifiers with a type of water-in-oil emulsion and

have antimicrobial activity against

Propionibacterium acnes and Staphylococcus

epidermidis. Oxidized ricinoleic acid-decanoic acid

esters showed the highest inhibition zone against

both bacteria.

ACKNOWLEDGEMENTS

This work was funded by Hibah Publikasi

Internasional Terindeks 9 (PIT 9) Universitas

Indonesia No.

NKB.0031/UN2.R3.1/HKP.05.00/2019.

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