Conjugation Reaction between Citronellal and L-Tyrosine and Its

Antimicrobial Properties against Bacteria and Fungi

Magister Program, Department of Chemistry, Universitas Syiah Kuala, Banda Aceh, Indonesia

Department of Chemistry, Universitas Syiah Kuala, Banda Aceh, Indonesia

Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia

Department of Cardiovascular, Dr. Zainoel Abidin Hospital, Banda Aceh, Indonesia

Department of Chemistry, Universitas Tadulako, Palu, Sulawesi Tengah, Indonesia

Herbal Research Centre, Universitas Yarsi, Jakarta, Indonesia

Department of Chemical Engineering, Universitas Syiah Kuala, Banda Aceh, Indonesia

PUI-Nilam Aceh-Atsiri Research Centre, Universitas Syiah Kuala, Banda Aceh, Indonesia

Departement of Pharmacy, Universitas Syiah Kuala, Banda Aceh, Indonesia

Pusat Riset Obat Herbal, Universitas Syiah Kuala, Banda Aceh, Indonesia

Pusat Riset Etnoscience, Universitas Syiah Kuala, Banda Aceh, Indonesia

Keywords: Conjugation Reaction, Citronellal, L-tyrosine, Staphylococcus aureus, Escherichia coli, Candida

albicans

Abstract: The study of citronellal with L-tyrosin conjugation for antimicrobial properties has been

conducted. The aim of this study to determine the relationship stucture between two compounds

citronellal and L-tyrosine on antimicrobial activity against Staphylococcus aureus, Escherichia

coli, and Candida albicans. The conjugation product obtained was yellow-white solid amorphous

with the Rf value was 0.84 and the percentage of yield was 71.12%. The FT-IR spectra peak at

3205.69 cm

-1

is represented the N-H stretching vibration from L-tyrosine, while the spectra appears

at 1460.11 - 1438.90 cm

-1

are represented the C=N which derived from imine or immonium from

shift base reaction between citronellal and L-tyrosine. The GC-MS analysis showed that the peak

15 observed at RT 10.27 min. be expected a conjugation product with the m/z 316 [M+H]

ion.

The antimicrobial activity were determined by well diffusion method and the results showed that

product of conjugation were have no antimicrobail activities at concentration tested.

1 INTRODUCTION

Citronellal is a monoterpene that has two optical

isomers with a molecular weight of 154.25 g / mol.

The reactivity of citronellal is resulting from carbonyl

group, double bond, and acidity of Hα. These groups

allowing citronellal to react with an acid or a base.

Some biological activities of citronellal including

insecticides (Griffith and Grentile, 1979), perfumery

(Anderson et al., 1993; Sangwan et al., 2001),

stimulants, antidepressants, analgesics, antipyretics,

and antimicrobials (Adhikari et al., 2015) L-tyrosine

or 4-hydroxyphenylalanine is a non-essential amino

acid, a primary amine, has a polar group. L-tyrosine

widely used in food industry and pharmaceutical

industry (Tina, 2007). The conjugation of natural

product with several constituents has attracted some

researchers due to their biological reactivity on

several microorganisms and cells (Martinez et al.,

2015; Hong et al., 2017). For examples, novobiocin,

serrulatane, xanthorhizol (Finland and Nichols, 1957;

Lewis and Klibanov, 2005; Rukayadi and Hwang,

2006), which contain prenil and aromatic hydroxy

groups are believed to play an important role in

antimicrobial activity.

2,4-Dimethyl-2,6-heptadiene-1-ol and 5-Amino-

2-methylphenol are two compounds produced from a

conjugation reaction are known to have antibacterial

activity against Staphylococcus epidermidis (Ys,

2015), Rusdin reported that product conjugation

between citronellal and L-tyrosine has antibacterial

Rila Suryani

, Nazaruddin

, Kartini Hasballah

, Muhammad Diah

3,4

, Hardi Yusuf

, Juniarti

Syaifullah Muhammad

7,8

, Khairan

8,9,10,11

Suryani, R., Nazaruddin, N., Hasballah, K., Diah, M., Yusuf, H., Juniarti, ., Muhammad, S. and Khairan, .

Conjugation Reaction between Citronellal and L-Tyrosine and Its Antimicrobial Properties against Bacteria and Fungi.

DOI: 10.5220/0009957000740079

In Proceedings of the 2nd International Conference of Essential Oils (ICEO 2019), pages 74-79

ISBN: 978-989-758-456-5

activity against Stapylococcus aureus (Al-Garawi et

al., 2012). The computational analysis result showed

the formation of imine conjugation bonds (imine fond

formation) between the citronellal (3,7-dimethyl-6-

octenal) with the amino acid L-tyrosine (Rusdin et al.,

2018). However, the research conducted by Rusdin

and Hardi has not been able to determine the type of

conjugate product. In this study, we interest to

determine product of conjugation obtained by GC-

MS spectroscopy and evaluate its activity against

Staphylococcus aureus, Escherichia coli, and

Candida albicans.

2 EXPERIMENTAL

2.1 Materials

Citronellal and L-tyrosine were received from

Department of Chemistry, Universitas Tadulako,

Palu, Sulawesi Tengah, Indonesia. Pentane hexane,

ethyl acetate, methanol, potassium hydroxide (KOH),

diethyl ether, 96% ethanol, distilled water,

physiological NaCl, and dimethyl sulfoxide (DMSO)

were obtained from Department of Pharmacy,

Universitas Syiah Kuala, Banda Aceh, Indonesia.

2.2 Conjugation Reaction Citronellal

and L-Tyrosine

The conjugation reaction between citronellal and L-

tyrosine was conducted by Al-Gharawi and Rusdin

methods (Al-Garawi et al., 2012; Rusdin et al., 2018).

Briefly, 0.18 grams of citronellal (1.2 mmol) in 10

mL of methanol was reacted with 0.18 grams of L-

tyrosine (1 mmol) in 15 mL of methanol, then 0.056

grams of KOH was added. The mixture then refluxed

for 8 hours at 60ºC. The conjugate (product) then

concentrated using rotary evaporator and washed

three times with pure ethanol. The product washed

again with diethyl ether and evaporated at room

temperature to obtain yellow-white solid amorphous.

2.3 Column Chromatography

Purification of conjugation product obtained was

conducted by column chromatography using silica

gel F

250

as stationary phase. As mobile phase, we used

a mixture of hexane: ethyl acetate (9:1). The collected

fractions were submitted into thin-layer

chromatography (TLC), using mobile phase a mixture

of hexane: ethyl acetate (9:1), and the chromatogram

was observed using UV-lamp at 250 nm. The major

fraction obtained then analysis by GC-MS

spectroscopy.

2.4 FT-IR Analysis

The FT-IR analysis spectrophotometer was

performed at wave numbers 4000-500 cm-1 using

Schimadzu model.

2.5 GC-MS Analysis

The conjugation product and collected fraction were

analysed by gas chromatography-mass spectroscopy

6890 equipped with capillary column Agilent HP 5

MS (60 x 0.25 x 0.25). The operating condition of the

gas chromatography were 1.0 ml/min (He), with

volume injection was 0.5µl. Oven temperature 300ºC

for 40 min.

2.6 Antimicrobial Activity

The antifungal activity was determined by Kirby-

Bauer method. The sterile Sabouraud's Dextrose Agar

(SDA) media was poured into petri dish and allowed

to solidify. The strains of Candida albicans was

spread out on the solidified media of SDA by using

the sterile cotton bud. The paper disc was laid out on

the surface of the agar medium. To each of disc 12 µl

of negative control (solvent), positive control

(nystatine), and tested compound and was loaded and

subsequently incubated at 37ºC for 48 hours. In the

same procedure, the antibacterial activity of the tested

compounds against Staphylococcus aureus and

Escherichia coli were performed using Mueller

Hinton Agar (MHA) media and subsequently

incubated at 37ºC for 24 and 48 hours. In the

antibacterial assay we used ciprofloxacin and

gentamycin as positive controls for Staphylococcus

aureus and Escherichia coli respectively. Then, the

inhibition effect of the tested compunds were

determined. The antimicrobial activities were

performed in triplicates.

3 RESULTS AND DISCUSSION

3.1 Conjugation Reaction Citronellal

and L-Tyrosine

Conjugation reaction between citronellal and L-

tyrosine were used potassium hydroxide (KOH) as a

catalyst. Ritter mentioned that carbonyl group, double

bond, and Hα atom from citronellal allows to react

with an acid or base. L-tyrosine is a primary amine, a

Conjugation Reaction between Citronellal and L-Tyrosine and Its Antimicrobial Properties against Bacteria and Fungi

base, is readily react to citronellal (an aldehyde)

(Griffith and Grentile, 1979). Murray mentioned that

the reaction between an aldehyde or a ketone with a

primary amine will produce an imine through a shift

base mechanism. L-tyrosine act as a nucleophilic, and

a citronellal act as electrophilic to form an immonium

salt, through the mechanism of base shift reaction,

this readily to form an imine compound (R

C=NR)

(Murray, 2010). The mechanism reaction of imine

formation from primary amine and an aldehyde

shown in Figure 1 below.

Figure 1: The mechanism reaction of imine formation from

citronellal and L-tyrosine.

The obtained product was white-yellow in colour and

amorphous in shape. The fragrant of the product was

lighter than citronellal. The percentage yield of

product was 71.12% or 0.51 gram. The TLC result

showed that the product has four bands with the Rf

values were 0.17; 0.28; 0.44; and 0.84 (Figure 2A).

Figure 2A: TLC result of the conjugation product: 1.

Citronellal; 2. L-tyrosine; 3. Citronellal + L-tyrosine; and 4.

Conjugation product: B. TLC result of the conjugation

product from column chromatography using mobile phase

a mixture of hexane: ethyl acetate (9:1), and the

chromatogram of the fractions was observed using UV-

lamp at 250 nm.

3.2 FT-IR Analysis

The FT-IR analysis of citronellal, L-tyrosine, and

conjugation product shown below. Figure 3A showed

that the spectra at 1729 cm

-1

indicated the presence of

carbonyl groups (-C=O), while the absorption at 2913

-1

and 1423 cm

-1

were represented of functional

group of C-H and C=N. All these functional group are

typical for citronellal. Fig. 3B pointed that the

absorption at 1589 cm

-1

, 3363 cm

-1

and 1242 cm

-1

were indicated the presence of aromatic functional

group of C=C, N-H, and C-N respectively. These

functional groups are typical for L-tyrosine.

Figure 3: The FT-IR analysis of A. Citronellal; B. L-

tyrosine; C. Conjugation product.

The FT-IR analysis of the conjugation product

showed strong absorption of N-H groups at 3205.69

-1

(Figure 3C). Murray and Silverstain mentioned

that the absorption of C=O carbonyl appears at

1759.08 cm

-1

with low intensity. The absorption of

C=C alkenes appears at 1669.64 cm

-1

and the

spectrum of C=C aromatic absorb at 1512.19 cm

-1

ICEO 2019 - 2nd International Conference of Essential Oil Indonesia

While the C=N spectrum appears at 1460.11-1438.90

-1

(Murray, 2010; Silverstein et al., 2005).

3.3 GC-MS Analysis

The major fraction (fraction 2, Figure 2B) obtained

from column chromatography with the Rf value of

0.86 that to be expected conjugation product was

analysis by GC-MS. The GC spectral of fraction 2 is

presented in Figure 4.

Figure 4: The GC analysis of fraction 2 isolated from

conjugation product.

The composition of fraction 2 isolated from

conjugation product is shown in Table 2. The profile

of fraction 2 from conjugation product contains 20

compounds. The main compound of the fraction 2 is

phenolic compound. The major of phenolic

compounds in fraction 2 was p-cresol (13.20%) and

L-Tyrosine and Octanal,7-hydroxy-3,7-dimethyl

(12.92%). The other phenolic compounds in

moderate percentage were assigned to 3-propyl-

phenol (2.3%), 3-ethyl-phenol (2.49%), 2,4-

dimethylphenol (4.14%), 4-ethyl-2-methyl-phenol

(4.53%), 4-ethylphenol (5.99%), and Octanal,7-

hydroxy-3,7-dimethyl (0.53%).

The Ritter stated that L-tyrosine (a primary

amine) is readily react to citronellal (an aldehyde) to

form an imine compound (conjugation product)

through a shift base mechanism (Figure 1) (Griffith

and Grentile, 1979). The characterization of the

product conducted by GC-MS, to identify the

conjugation product between citronellal and L-

tyrosine. The MS characterization of the product is

shown in Figure 5, and be expected observed at RT

10.27 with the percentage area of the peak of 12.92%

(peak 15).

Table 1: The GC-MS analysis of fraction 2 isolated from

conjugation product.

No. Name

(min)

Area

m/z

1. Carboxylic acid 3.70 16.47 44.0

2. Phenol 5.74 25.35 94.1

3. Hydroxytoluene 6.31 5.34 108.14

4. p-cresol 6.49 13.20 108.14

5. 2,3-dimetyl-phenol 6.87 1.44 122.16

6. 3-ethyl-phenol 7.03 2.49 122.16

7. 2,4-dimethylphenol 7.15 4.14 122.16

8. 4-ethylphenol 7.30 5.99 122.16

9. 3-propyl-Phenol 7.50 2.30 136.19

10. 2-propyl-Phenol 7.73 1.19 139.19

11. 2-ethyl-4-methyl-

Phenol

7.86 2.27 137.01

12. 4-ethyl-2-methyl-

Phenol

7.97 4.53 137.01

13.

2,3-Dimethyl-N-

phenylalanine

8.50 0.49 197.00

14. Octanal,7-hydroxy-

3,7-dimethyl

8.63 0.57 154.00

15. L-Tyrosine and

Octanal,7-hydroxy-

3,7-dimethyl

10.27 12.92 316.00

16. Benzophenone 12.54 0.24 182.00

17. Phenol, 4-(2-

aminoethyl)

13.05 0.18 137.00

18. 2,2-

methylenediphenol

14.21 0.57 200.23

19.

4,4'-

methylenediacetami

14.76 0.16 282.33

20. Tyramine 14.85 0.15 181.00

Total 100.00

From the result, the product (peak 15), lose a

common molecule of C=O (-28 Da) forming the

conjugation product with the m/z 316 [M+H]

ion.

Figure 5, also showed that the fragment of m/z 154

[M+H]

and m/z 181 [M+H]

were characteristic for

citronellal (C

O) and L-tyrosine (C

)

respectively. However, these results need to be

further analysed to ensure that the product formed is

a true conjugate product (imine compound).

2. 00 4. 00 6.00 8. 00 10. 00 12.00 14. 00 16.00 18. 00 20.00 22. 00

2000000

4000000

6000000

8000000

1e+07

1. 2e+07

1. 4e+07

1. 6e+07

1. 8e+07

2e+07

2. 2e+07

2. 4e+07

2. 6e+07

2. 8e+07

Ti me-->

bundance

TI C: SAMPEL 2. D

3. 70

5.74

6. 31

6.49

6. 87

7. 03

7. 16

7.30

7. 50

7.73

7. 86

7.98

8.50

8. 64

10. 27

12.54

13. 05

14.22

14. 75 14.84

Conjugation Reaction between Citronellal and L-Tyrosine and Its Antimicrobial Properties against Bacteria and Fungi

Figure 5: The GC-MS analysis of fraction 2 isolated from

conjugation product (peak 15) at RT 10.27.

3.4 Antimicrobial Activity

The antifungal activity of the conjugation product

against Candida albicans is presented in Figure 6C.

The results showed that the conjugation product at

concentration of 10 and 50 mg/ml has no activity

against Candida albicans. While, the positive control

(nystatin) showed antifungal activity with the

diameter of inhibition zone was 10.70 nm (Table 2).

Figure 6: Antimicrobial activity of the conjugation product.

A. against S. aureus; B. E. coli; and C. C. albicans.

Table 2: The inhibition effect of the conjugation product

against Staphylococcus aureus, Escherichia coli, and

Candida albicans.

Sample

Diameter of inhibition

zone (mm)

aureus

coli

albicans

Solvent nie nie nie

Nystatin nd

nd 10.70

Ciprofloxacin 21.78 nd nd

Gentamycin nd 29.60 nd

Citronellal 8.51 nie nie

Product 10 mg/ml nie nie nie

50 mg/ml nie nie nie

Note: C

: negative control; C

: positive control; nie: no

inhibition effect; nd: not determined.

The antibacterial activity of the conjugation

product also tested, and the results exhibited that the

product was has no antibacterial activities on

Staphylococcus aureus and Escherichia coli at

concentrations used. In this assay, we used

ciprofloxacin and gentamycin as positive controls for

Staphylococcus aureus and Escherichia coli

respectively (Table 2).

The results also showed that, ciprofloxacin,

gentamycin and citronellal (as precursor for synthesis

of conjugation product) have antibacterial activities

with the diameter inhibition zone were 21.78; 29.60;

and 8.51 mm respectively (Figure 6B and 6C). In the

conclusion, the antimicrobial assays showed that the

conjugation product have no inhibition effect against

Staphylococcus aureus, Escherichia coli, and

Candida albicans at concentration tested.

4 CONCLUSION

The conjugated product between citronellal and L-

tyrosine produces a powdery and yellowish-white

product, with percentage of yield was 71.12%. The

TLC analysis showed that a band with the Rf value of

0.85 was thought to be the conjugation product. The

GC-MS analysis showed that the fragment ion at m/z

316 [M+H]

, lose a common molecule of C=O (-28

Da), was expected to be conjugation product. The

antimicrobial assays showed that the conjugation

product at the concentrations of 10 and 50 mg/ml

have no inhibition effect on Staphylococcus aureus,

Escherichia coli, and Candida albicans.

ACKNOWLEDGEMENTS

The authors thank to Atsiri Research Center (ARC)

and Herbal Medicine Research Center (ProHerbal) of

Universitas Syiah Kuala for their support of this

study.

REFERENCES

Adhikari, S., Saha, S., Bandyopadhyay, T, K., Ghosh, P.,

2015. Efficiency of ISSR Marker for Characterization

of Cymbopogon Germplasms and Their Suitability in

Molecular Barcoding, Plant Systematics and Evolution,

301(1), 439–450.

Al-Garawi, Z, S., Tomi, I, H., Al-Daraji, A, H., 2012.

Synthesis and Characterization of New Amino Acid-

Schiff Bases and Studies Their Effects on the Activity

of ACP, PAP and NPA Enzymes (In Vitro), E-journal

Chem, 9(2), 962–969.

Anderson, J, A., Churchill, G, A., Autrique, J, E., Tanksley,

S, D., Sorrells, M, E., 1993. Optimizng Parental

Selection for Genetic Linkage Maps, Genome, 36(1),

181–186.

ICEO 2019 - 2nd International Conference of Essential Oil Indonesia

Finland M, Nichols R 1957 Practitioner 179:84.

Griffith, R, C., Gentile, R, J., Davidson, T, A., Scott, F, L.,

1979. Convenient One-Step Synthesis of N-

Substituted. Alpha.-Methylphenethylamines Via

Aminomercuration-Demercuration, Jurnal of Organic

Chemistry, 44(20), 3580-3583.

Hong Y, Junkai L, Shengtao X, Zheying Z, Jinyi X 2017

Exp. Opin. Drug Disc. 12 121–140.

Lewis, K., Klibanov, A, M., 2005. Surpassing Nature:

Rational Design of Sterile-Surface Materials, Trends in

Biotechnology, 23(7), 343-348.

Martinez, D, M., Barcellos, A., Casaril, A, M., Perin, G.,

Schiesser, C, H., Callaghan, K, L., Lenardão, E, J.,

Savegnago, L., 2015. Twice Acting Antioxidants:

Synthesis and Antioxidant Properties of Selenium and

Sulfur Containing Zingerone Derivatives, Tetrahedron

Lett, 56, 2243–2246.

Murray, J, M., 2010. Fundamental of organic chemistry,

seventh edition; Cornell University. Brooks/Cole 20

Davis Drive: Belmont CA 94002-3098 USA.

Rukayadi, Y., Hwang, J, K., 2006. Effect of Coating the

Wells of A Polystyrene Microtiter Plate with

Xanthorrhizol on the Biofilm Formation of

Streptococcus Mutans, Journal of Basic Microbiology,

46(5), 410-415.

Rusdin F, Hardi Y, Syamsuddin 2018 Kovalen 1

Sangwan, N., Yadav, U., Sangwan, R., 2001. Molecular

Analysis of Genetic Diversity in Elite Indian Cultivars

of Essential Oil Trade Types of Aromatic Grasses

(Cymbopogon species), Plant Cell Reports, 20, 437–

444.

Silverstein, R, M., Webster, F, X., Kiemle, D, J., 2005.

Spectrometric identification of organic compounds,

New York, 7

eddition.

Tina Lütke-Eversloh 2007) Applied microbiology and

biotechnology 77(4) 10-31.

Ys H 2015 J. Nat. Sci. 4 111–118.

Ys, H., Rusdin, F., Syamsuddin, Rahim, E, A., 2019.

Comparison Analysis Between Experiment and

Computational Chemistry Data on Citronellal and

Tyrosine Conjugation, IOP Conf. Series: Journal of

Physics: Conf. Series, 1242.

Conjugation Reaction between Citronellal and L-Tyrosine and Its Antimicrobial Properties against Bacteria and Fungi