Synthesis and antibacterial activity of 1-monolaurin

Article abstract of DOI:10.13005/ojc/340233

An improvement of method for synthesizing 1-monolaurin from lauric acid and glycerol has been done. The reaction was carried on mol ratio between lauric acid and glycerol 1:1 at 130 °C for 6 h with variation of pTSA catalyst of 2.5%, 5%, 7.5% (w/w of lauric acid). The purification of 1-monolaurin was conducted only by extracting with alcoholic solution. The product of 1-monolaurin was obtained as a white solid with 100% of purity from variation of 2.5% and 5% of pTSA catalyst with 43.54% and 27.89% yield, respectively. 1-Monolaurin could inhibit the growth of S. aureus and E. coli bacteria at 500 μg/mL of concentration.

Full text of DOI:10.13005/ojc/340233

ISSN: 0970-020 X
CODEN: OJCHEG
2018, Vol. 34, No.(2):
Pg. 863-867
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
www.orientjchem.org
Synthesis And Antibacterial Activity of 1-Monolaurin
FEBRI ODEL NITBANI1*, JUMINA2,, DWI SISWANTA2, ETI NURWENING SHOLIKHAH3
and DHINA FITRIASTUTI4
1Department of Chemistry, Faculty of Science and Engineering, Universitas Nusa Cendana, Kupang,
Indonesia.
2,4Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada,
Yogyakarta, Indonesia.
3Department of Pharmacology and Therapy, Faculty of Medicine, Universitas Gadjah Mada Yogyakarta,
Indonesia.
*Corresponding author E-mail : febri_nitbani@yahoo.com
http://dx.doi.org/10.13005/ojc/340233
(Received: January 04, 2018; Accepted: March 20, 2018)
ABSTRACT
An improvement of method for synthesizing 1-monolaurin from lauric acid and glycerol has
been done. The reaction was carried on mol ratio between lauric acid and glycerol 1:1 at 130 °C
for 6 h with variation of pTSA catalyst of 2.5%, 5%, 7.5% (w/w of lauric acid). The purification of
1-monolaurin was conducted only by extracting with alcoholic solution. The product of
1-monolaurin was obtained as a white solid with 100% of purity from variation of 2.5% and 5% of
pTSA catalyst with 43.54% and 27.89% yield, respectively. 1-Monolaurin could inhibit the growth
of S. aureus and E. coli bacteria at 500 µg/mL of concentration.
Keywords: Lauric acid, Glycerol, Monolaurin, Antibacteria.
INTRODUCTION
Coconut oil contained lauric acid with
relative percentage of methyl laurate of 52%2. Lauric
acid as white solid could be synthesized through
base hydrolysis of methyl laurate with 84% of yield.
The other product that can be derived from coconut
oil is monoacylglycerol of medium chain fatty acid
such as 2-monolaurin3, 1-monokaprin4 and
1-monomiristin5. Monolaurin compound is known
to have antimicrobial activity (antibacterial, antivirus
Indonesia is the biggest coconut
production country beside Malaysia and India but
diversification of coconut based product is still low1.
Currently, in Indonesia or other countries, coconut
oil mostly was developed as drug such as virgin
coconut oil (VCO). Thus, the diversification of
coconut based product is still necessary.
This is an
Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike
4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted
NonCommercial use, distribution and reproduction in any medium, provided the original work is properly cited.

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and antifungi)6,7,8,9. Monolaurin also has antivirus
activity i.e. active to dissolve lipid and phospholipid
in the outer layer of virus so that the virus will be
degraded5.
ethanol and distilled water. All chemicals, except
distilled water which was obtained from Laboratory
of Fundamental Chemistry Universitas Gadjah
Mada, were purchased from E. Merck with high
grade and used without any further purification.
Monolaurin has a promising antimicrobial
activity but the compound is not easy to be
synthesized and relatively expensive. The attempts
to synthesize monolaurin has been done by Pareira
et al.,10 and Langone et al.,11 by esterification
reaction of lauric acid and glycerol using Lipozyme
IM 20 catalyst but the yield is still low (26.8%). Review
article written by Nitbani et al., (2015)12 stated that,
the successful application of MCM-41 hybrid
materials as the catalysts in the esterification of
glycerol with either lauric acid or oleic acid. By
maintaining the ratio between methyl groups to the
sulphonic acid and applying the optimum conditions
of the catalyst, monolaurin and monoolein
compounds can be generated with a yield of 63%
and 45% respectively.
Instrumentation
Equipment used in this research were
laboratory glassware and analytical mass balance
(Mettler AT200). Infra-red spectra were obtained
using Shimadzu-Prestige 21 spectrometer. 1H-NMR
spectra were recorded at 400 MHz with a JEOL
JNM-MY spectrometer using TMS as an internal
reference. Chromatogram and mass spectra were
measured on a Mariner LC-MS using C-18 column
(15 x 1 mm) and injected volume of 2 µL. The
mobile phase was methanol with flow rate of
0.1 mL/minutes.
Procedure
Synthesis of 1-monolaurin
Synthesis of 1-monolaurin through
esterification reaction of lauric acid and glycerol
using sulfuric acid catalyst and solvent-free has
been done by Widiyarti et al., (2009)7. Purification
of 1-monolaurin compound was done by using
column chromatography with silica gel as stationary
phase and a mixed solvent of n-hexane and ethyl
acetate as mobile phase. The reaction was carried
out using lauric acid: glycerol (1:1), 5% of catalyst
at 130 °C for 6 h and solvent-free. The synthesized
1-monolaurin has 31.05% of yield and 91% of
purity.
Glycerol (0.01 mol, MW = 92.09 g/mol;
0.92 g), lauric acid (0.01 mol, MW= 200.03 g/mol;
2.003 g) and p-toluene sulfonic acid (pTSA) catalyst
(2.5% w/w of lauric acid) were added in
three-necked flask. The mixture was heated with
continuous stirring at 130 °C for 6 h in oil bath. After
that, crude product was cooled and dissolved in
ethyl acetate. The solution was washed with distilled
water until neutral pH. The organic layer was dried
with Na2SO4 anhydrous and evaporated with rotary
evaporator. The purification of 1-monolaurin from
crude product (ethyl acetate layer) was conducted
by using extraction with hydroalcoholic solution.
The amount of hydroalcoholic solution
(ethanol : water = 8 : 2) is 10 times of crude product
(ethyl acetate layer) volume. The mixture in
hydroalcoholic solution and the extracted 3 times
with an amount of n-heksane. The optimization of
reaction was carried out by using variation of pTSA
catalyst of 5% and 7.5% w/w of lauric acid. FTIR
(KBr) vmax: 3487 (OH), 1736 (C=O), 1180 (C-O),
2924-2854 (Csp3-H), 1400 (-CH2-), 1300 (CH3) cm-1.
1H-NMR (400 MHz, CDCl3) δ: 0.86 (3H, t, CH3 of
C12), 1.237-1.269 (16H, m, CH2 of C4-11), 1.573-1.646
(2H, m, CH2 of C3), 2.334 (2H, t, CH2 of C2), 3.590
In this current paper, research of synthesis
and purification method of 1-monolaurin was done.
The reaction used pTSA catalyst rather than H2SO4
and purification of the product employed
liquid-liquid extraction. This synthesis innovation is
expected to increase the yield and purity of the
1-monolaurin compound. The product will also be
tested for its antibacterial activity against S. aureus
and E. coli bacteria.
EXPERIMENTAL
Materials
Chemicals used in this experiment were
lauric acid2, glycerol, pTSA, n-hexane, ethyl acetate,

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(1H, dd, -CH2-(OH)CH-CH2-), 4.129 (1H, dd, -CH2-
(OH)CH-CH2-), 3.590 (1H, dd, -CH2-(OH)CH-CH2-),
3.687 and 4.191 (2H, dd, -(OH)CH-CH2-OCO-),
7.246 (1H, s, OH). 13C-NMR (400 MHz, CDCl3) δ:
14.217 (C12), 22.77 (C11), 24.991 (C10), 29.206
(C9), 29.330 (C8), 29.415 (C7), 29.673 (C8), 29.530
(C5), 29.673 (C4), 31.990 (C3), 34.220 (C2), 63.397
(-OH)CH2-(OH)CH-CH2-), 65.237 (-(OH)CH-CH2-
OCO-), 70.338 (-(OH)CH-CH2-OCO-), 174.494
(C=O). LC-MS (100%). MS relative intensity (m/z) :
275,49 (base peak), 572 (2M+Na).
could be synthesized through esterification reaction
of lauric acid and glycerol using acid catalyst.
Widiyarti et al., (2009) synthesized 1-monolaurin
through this reaction using H2SO4 catalyst with
31.05% yield of 1-monolaurin and 4.48% yield of
dilaurin. The H2SO4 catalyst was predicted to be
too strong and induced the formation of dilaurin so
that reduced the yield of monolaurin. This reaction
was conducted for 6 h at 130 °C with 5% (w/w)
catalyst of total reactant. The synthesized
monolaurin was also purified by column
chromatography.
Antibacterial Assay
In the current paper, the synthesis of
1-monolaurin was conducted by reacting lauric acid
and glycerol (1:1) with pTSA catalyst. The reaction
was carried out at 130 °C for 6 hours. The amount
of pTSA catalyst was varied for 2.5%, 5.0% and
7.5%. The product was not purified by using column
chromatography like the previous research. The
esterification product was dissolved in ethyl acetate
and extracted with base solution until the pH of
organic layer is neutral. This step needs to be done
because the pTSA catalyst is a heterogeneous
catalyst and was separated from products by using
base solution. The base solution will neutralized
the catalyst to form a salt which is dissolved in water
and easy to be separated. The ethyl acetate fraction
was then evaporated and was dissolved in ethanol-
water (80:20) solution or so-called hydroalcohol
solution. The product was washed with n-hexane
to afford a white solid. The yield of 1-monolaurin for
2.5%, 5.0% and 7.5% of pTSA variation was
60.34%, 43.54% and 27.89%, respectively. Thus,
it can be seen that the highest percentage of yield
of 1-monolaurin was the reaction with 2.5% of pTSA
catalyst. The product was analyzed by using
LC-MS and shown in Fig. 1. Chromatogram of
esterification product showed that 1-monolaurin
was detected at retention time (tR) of 3.1 minutes.
Single product was shown in the product of
esterification using 2.5% and 5% of pTSA catalyst.
Mass spectrum of product (Fig. 2) showed fragment
of M+H and 2M+Na with m/z of 275,49 and 572
respectively. The elucidation of product by using
FT-IR, MS, 1H-NMR and 13C-NMR was described in
the procedure section.
The composition of media for bacteria such
as Nutrient Agar consisting of 5 g of peptone, 3 g of
beef extract and 15 g of agar which were dissolved
in 1 L of distilled water and then sterilized at a
temperature of 121 °C for 15 min. at 15 psi. On the
surface of the petri dish, 50 mL of 1.8% agar solution
was poured. After solidified, the wells are made by
putting a ring with diameter of 1 cm and then 50 mL
of media for pathogenic bacteria was poured in the
surrounding that have been added with 1% culture
of pathogens such as Staphylococcus aureus
FNCC 0047 and Escherichia coli FNCC 0091.
After solidified, as much as 150 µL of sample of
1-monolaurin was added by concentration of 500;
1,000; 2,500; 5,000 and 10,000 ppm with DMSO
(99.9%) as negative control. The positive control of
antibacterial assay is tetracycline with concentration
of 500; 1,000; 2,500; 5,000 and 10,000 ppm while
the negative control is distilled water. The dish was
incubated for 24 h. and the inhibitory zone was
measured. A clear zone with size of more than
2 mm on the surrounding wells indicated as positive
inhibition. The diameter of inhibition zone formed
was measured by using a caliper to determine the
effectiveness of antibacteria. Inhibition zone
was measured by reducing the overall diameter
(disk + inhibition zone) with diameter of disk.
RESULTS AND DISCUSSION
Synthesis of 1-monolaurin through esterification
of lauric acid and glycerol
1-Monolaurin compound could be
synthesized from some pathways. This compound

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This purification technique is simpler and more
effective compared to column chromatography
technique from the previous research. The
extraction technique using hydro alcohol solution
could separate the 1-monolaurin until 100% of purity
from the product of esterification using 2.5% and
5% of pTSA catalyst. The advantages of
1-monolaurin purification using liquid-liquid
extraction are more effective in time and cost so
that 1-monolaurin could be easily produced in a
large scale.
(a)
Antibacterial assay of 1-monolaurin
1-monolaurin has two hydroxyl and one
lauryl groups that bound in glycerol scaffold. Lauryl
groups as a non-polar group is bound at C1 of
glycerol so that it called as 1-monolaurin (Fig. 3).
This compound was tested as antibacteria against
S. aureus and E. coli. This assay used five of variation
of sample concentration i.e. 500; 1,000; 2,500;
5,000; and 10,000 µg/mL and positive control of
tetracycline with concentration of 500 µg/mL. The
antibacterial assay used the same method as that
of Widiyarti et al., (2009). The result of antibacterial
assay was shown in Table 1.
(b)
(c)
Fig. 1. Chromatogram of esterification product
using (a). 2.5%; (b) 5%; (c) 7.5% (w/w) of pTSA
catalyst.
Fig. 2. Structure of 1-monolaurin
The result showed that 1-monolaurin could
inhibit the growth of S. aureus and E. coli in various
concentration even at the lowest concentration of
1-monolaurin (500 µg/mL) although could not
exceed the inhibition zone of tetracycline as positive
control at the same concentration. The data showed
that the higher concentration of 1-monolaurin will
have the larger inhibition zone. Inhibition zone of
synthesized 1-monolaurin against S. aureus and
E. coli was larger compared to those of
1-monolaurin standard and 1-monolaurin
synthesized by Widiyarti et al., (2009). Thus, it can
be concluded that the synthesized product have a
good antibacterial activity for Gram-negative and
Gram-positive bacteria.
Fig. 2. Mass spectrum of peak with Rt = 3,1
minutes
The purification of 1-monolaurin used the
liquid-liquid extraction with hydro alcohol solution
and n-hexane. Aqueous alcoholic solution which
contains of ethanol: water (80:20) could separate
1-monolaurin from the other non-polar impurities
such as dilaurin, trilaurin and lauric acid residue.

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Table 1: Diameter of inhibition zone of 1-monolaurin against
S. aureus and E. coli
Diameter of inhibition zone (mm)
Bacteria
500
1000
2500
5000
10000 Tetracycline
µg/mL 500 µg/mL
µg/mL µg/mL µg/mL µg/mL
S. aureus
E. coli
S. aureus *
S. aureus**
7,5
10.55
9.50
9.50
15.75
12.70
9.00
17.75
13.25
8.50
19.00
13.75
8.50
20,75
17.00
7.00
18.55
19.60
8.50
8.50
7.00
7.00
Negative control (DMSO): not inhibit the bacteria in all concentration
*Antibacterial activity of 1-monolaurin synthesized by Widiyarti et al., (2009)
** Antibacterial activity of 1-monolaurin standard (Widiyarti et al., (2009)
CONCLUSION
inhibit the growth of S. aureus and E. coli bacteria
at 500 µg/mL of concentration.
1-monolaurin was successfully synthesized
by reacting lauric acid and glycerol (1:1) at 130 °C
for 6 h using pTSA catalyst of 2.5%, 5% and 7.5%
with 60.34%, 43.54% and 27.89% of yield,
respectively. The synthesized 1-monolaurin could
ACKNOWLEDGEMENT
Authors showed gratitude to Biosciences
Laboratory at Universitas Nusa Cendana
(Indonesia) for the equipment support to this
research.
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