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Identification of Trans-Cinnamic Acid in Sinyo Nakal (Duranta repens) Fruits’
Methanol Extract
Article in International Research Journal of Pure and Applied Chemistry · January 2015
DOI: 10.9734/IRJPAC/2015/17616
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International Research Journal of Pure &
Applied Chemistry
8(2): 73-80, 2015, Article no.IRJPAC.2015.075
ISSN: 2231-3443
SCIENCEDOMAIN international
www.sciencedomain.org
Identification of Trans-Cinnamic Acid in Sinyo Nakal
(Duranta repens) Fruits’ Methanol Extract
Sigit Eko Januar1, Purwantiningsih Sugita1* and Budi Arifin1
1
Department of Chemistry, Bogor Agricultural University, Bogor, West Java, Indonesia.
Authors’ contributions
This work was carried out in collaboration between all authors. Authors PS and BA designed the study
and author SEJ wrote the protocol. Authors PS and BA managed the literature search and author SEJ
wrote the first draft of the manuscript with assistance from author PS. All authors read and approved
the final manuscript.
Article Information
DOI: 10.9734/IRJPAC/2015/17616
Editor(s):
(1) Edmond Dik-Lung Ma, Department of Chemistry, Hong Kong Baptist University, Hong Kong, China.
Reviewers:
(1) Anonymous, Cumhuriyet University, Turkey.
(2) Weiting Wang, Tianjin Institute of Pharmaceutical Research, China.
(3) Anonymous, Universidade Lusófona de Humanidades e Tecnologias, Portugal.
(4) Anonymous, Universidade Federal do Paraná, Brazil.
(5) Anonymous, Vittal Mallya Scientific Research Foundation, India.
Complete Peer review History: http://www.sciencedomain.org/review-history.php?iid=1050&id=7&aid=9302
th
Received 20 March 2015
Original Research Article Accepted 6th May 2015
th
Published 19 May 2015
ABSTRACT
Aims: To isolate and to characterize the chemical constituents in sinyo nakal (Duranta repens)
fruits’ methanol extract. The fruits were collected from Jombang, East Java, Indonesia.
Methodology: The fruits have been extracted with n-hexane in previous study to separate the
nonpolar components. Fractionation of the methanol extract was done by using liquid vacum
chromatography (LVC) with step gradient elution from n-hexane to ethyl acetate, followed by
methanol.
Results: Methanol fraction gave the highest yield. A yellowish white crystal was obtained in further
fractionation of this fraction. The compound was still impure and could not be purified and
characterized further because of limited amount of sample.
Conclusion: The compound has been identified as trans-cinnamic acid based on 1D and 2D-
nuclear magnetic resonance (NMR) spectra analysis.
_____________________________________________________________________________________________________
Januar et al.; IRJPAC, 8(2): 73-80, 2015; Article no.IRJPAC.2015.075
Keywords: Trans-cinnamic acid; Duranta repens; fruits methanol extract.
1. INTRODUCTION chromatography; and silica gel Merck 60 GF254
for TLC.
Duranta repens is a member of Duranta species
which is widely spread in tropical and subtropical 2.2 Instrumentation
regions [1]. Most of this plants are cultivated as
ornamental plants in West India, Pakistan, Basic instruments used were TLC plates (silica
Middle and South America. In Indonesia, D. gel 60 F254); liquid vacuum chromatography
repens is generally known as sinyo nakal. Its (LVC), gravitational column chromatography, and
fruits are traditionally used to treat malaria and radial chromatography apparatus; rotary
worms, while its leaves are used as abscess evaporator; and a set of common glassware. The
medicine, antipyretic, diuretic, and antimalarial nuclear magnetic resonance (NMR) spectra were
agents [2]. obtained with an Agilent spectrometer working at
a frequency of 500 MHz (1H) and 125 MHz (13C)
Several studies concerning the bioactivities of D. at Bandung Institute of Technology.
repens have been reported. The fruits and stem
show antifungal and antibacterial activities, 2.3 Preparation and Extraction
cytotoxicity, and larvicidal activity against Culex
quinquefascitatus [3,4]. The leaves show Milled dried sample (2.28 kg) containing 12.0%
antibacterial [2], antioxidant [5], and larvicidal of water was macerated in n-hexane for 24
activities against C. quinquefascitatus [6]. The hours. The filtrate was then saponified by liquid-
whole plant also shows antioxidant, antiviral [7], liquid extraction with several portions of 0.5 N
and insecticidal activities against Aedes aegypti NaOH. The unsaponified n-hexane extract (the
and Attagenus piceus [8], and inhibits alpha upper phase) was collected and evaporated
glycosidase enzyme [9]. under reduced pressure. The sample residue,
which is now free of lipid and other non-polar
Many active compounds have been isolated from components, was re-macerated in methanol (6 L)
the leaves and the whole plants of D. repens, for 24 hours. This maceration was done in three
including terpenoids and steroids [1,3,4,7,10–14], replications. Phytochemical tests were conducted
flavonoids [1,7,9,12], iridoid glycosides [10], on the n-hexane and methanol extracts based on
coumarins and coumarinolignoids [8,13], and standard procedures [15].
phenylethanoid glycosides [1,2]. Still few studies
on D. repens fruits have been conducted.
2.4 Secondary Metabolites Compounds
Moreover, different growing place can influence
the amount and type of secondary metabolites Isolation from Methanol Extract
produced by a plant. Therefore, this study aims
to isolate and to characterize the chemical The first fractionation step was tannin removal.
constituents of D. repens fruits from Indonesia. Tannin was precipitated by pouring acetone (100
The isolation and characterization are focused on mL) into the methanol extract, and the precipitate
the polar fraction of the methanol extract. was separated by filtration. This step was
repeated until visually no more precipitate was
2. MATERIALS AND METHODS formed. The tannin-free methanol extract was
then concentrated under reduced pressure.
2.1 Materials
Brief fractionation was done by using LVC with
step gradient elution of two eluents. The
Materials used include D. repens (collected from nonpolar eluent retarded the spots near the start
Jombang, East Java); twice distilled technical line of the TLC plate, whereas the more polar
grade methanol, ethyl acetate, n-hexane, and one ran the spots near to the finish line. The
dichloromethane; Wagner, Mayer, Dragendorf, separation pattern of the eluates was checked by
and Liebermann-Burchard reagents; 10% NaOH using the best eluent, which was a mixture of two
(technical); 1% FeCl3 (technical); H2SO4 (p.a); solvents giving the most well-separated spots on
silica gel Merck 60 G for thin layer the TLC plate. Eluates with the same separation
chromatography (TLC); silica gel Merck 60 (0.2– pattern were combined into fractions.
0.5 mm and 0.063–0.200 mm) for column
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Januar et al.; IRJPAC, 8(2): 73-80, 2015; Article no.IRJPAC.2015.075
Fraction with the highest yield was fractionated Tannin removal from 49.1 g of the methanol
further by using gravitational column extract left 28.1 g of tannin-free methanol extract.
chromatography with the same step gradient This extract was soluble in acetone. The best
elution system. Fractions which produced single eluent composition for this extract was n-hexane-
spot on the TLC plate were elucidated by using ethyl acetate (4:6). The step gradient elution in
NMR spectrometer. Fractions which still LVC apparatus was carried out on this extract
contained several components were purified (20.0 g) by using n-hexane (1×), n-hexane-ethyl
further by using radial chromatography, before acetate 8:2 (2×), 6:4 (2×), 5:5 (4×), 4:6 (2×), 3:7
being elucidated. The best eluent was (2×), ethyl acetate (2×), and methanol (10×),
determined again, and used as the mobile phase respectively. Nine fractions were collected based
in an isocratic elution system. on TLC checking using the best eluent (Table 1).
3. RESULTS AND DISCUSSION Fraction-H had the highest yield (62.4%). This
fraction was eluted in methanol. Tannins were
From 2.28 kg of D. repens fruits, 45.2 g of n- still detected in this fraction and removed by
hexane extract was obtained, 10.0 g of which precipitation in acetone to avoid spot tailing
was unsaponifiable. Maceration of the residue in during further fractionation. From 12.5 g of
methanol resulted in 466 g of methanol extract, fraction-H, 6.45 g of tannin-free fraction-H was
or 23.2% of yield based on dried weight (12.0% obtained, and 3.95 g of which was fractionated
of water content). Based on phytochemical by using a gravitational column chromatography.
studies, the unsaponifiable n-hexane extract only Step gradient elution system of n-hexane-ethyl
contained steroids, whereas the methanol extract acetate gave 15 fractions. Two fractions (H6 and
also contained tannins, saponins, alkaloids, and H9) produced only one spot each in the TLC test,
flavonoids. Steroids were evidenced by green with Rf value of 0.83 and 0.65, respectively,
colour formation in Liebermann-Burchard test. using the best eluent. Fraction H6 was obtained
Tannins produced blue colour with 1% FeCl3. only in small amounts (1.7 mg), so that only
Saponins formed stable foam after being shaked. fraction H9 (13.3 mg) was elucidated with 1H
Alkaloids were proved by orange, yellowish NMR. However, its spectrum still showed mixture
white, and brown precipitate with Dragendorf, of compounds and could not yet be determined.
Mayer, and Wagner reagents, respectively.
Flavonoids produced red colour with 10% NaOH The most abundant fraction, H7 (51.05 mg),
and pink colour with concentrated H2SO4. produced only two components in TLC test with
Jayalaksmi et al. [2] reported that D. repens Rf value of 0.83 and 0.60. These components
leaves’ methanol extract contains tannins, were then purified by using radial
saponins, flavonoids, steroids, and terpenoids. chromatography. The best eluent composition
Serena et al. [6] reported tannins, saponins, was dichloromethane-ethyl acetate (3:7), The
alkaloids, flavonoids, and terpenoids in the same first fraction, H71 (11.4 mg), was a yellowish white
extract. These results showed that both D. crystal with the same Rf value as fraction H6. Its
repens leaves and fruits contained approximately chemical structure was then elucidated from the
the same group of compounds, except NMR spectra.
terpenoids, which was only found in D. repens
leaves.
Table 1. LVC fractions from tannin-free methanol extract of D. repens fruit
Fraction Eluent Weight (g) Yield (%)
A n-Hexane + n-hexane-ethyl acetate 8:2 (elution 1) 0.0105 0.05
n-Hexane-ethyl acetate:
B 8:2 (elution 2) + 6:4 (elution 1) 0.113 0.57
C 6:4 (elution 2) + 5:5 (elution 1) 0.194 0.97
D 5:5 (elution 2–4) 0.289 1.44
E 4:6 0.089 0.44
F 3:7 0.115 0.57
G Ethyl acetate 0.224 1.12
H Methanol (elution 1–2) 12.5 62.4
I Methanol (elution 3–10) 4.82 24.1
75
Januar et al.; IRJPAC, 8(2): 73-80, 2015; Article no.IRJPAC.2015.075
13
The C NMR spectrum (Fig. 1) showed 7 carbon When the proton in 6.4 ppm was irradiated, only
signals from 9 carbon atoms in 117.2, 128.3, peak in 7.8 ppm appeared. It suggested a direct
128.9, 130.7, 134.0, 146.9, and 171.7 ppm. correlation between the two adjacent protons.
Signal at 171.7 ppm showed a carbonyl group of One signal was more downfield (H 7.8 ppm)
a carboxylic acid. Two signals at 117.2 and 146.9 because of deshielding effect. The double bond
2
ppm were sp carbon signals with different resonated due to the electron-withdrawing effect
chemical environment. The existence of benzene of the carbonyl group leaving partial positive
aromatic ring was proved by 4 signals in 128.3, charge at the -carbon. The -carbon, which was
128.9, 130.7, and 134.0 ppm. Each of two not deshielded provided lower chemical shift.
signals in 128.3 and 128.9 ppm rose from two Aromatic protons of the monosubstituted
equivalent carbon atoms, based on their higher benzene were evidenced from the absorption
intensities than the two other signals. This peaks in 7.4 ppm (3H, t) and 7.6 ppm (2H, m).
chemical shift pattern matched with a However, the exchangeable proton of –COOH
monosubstituted benzene structural unit. did not appear in the 1H NMR spectrum. It is
1
possibly because of a very rapid proton
The H NMR spectrum (Fig. 2) showed 4 proton exchange between the acidic proton and protons
signals in 6.4, 7.4, 7.6, and 7.8 ppm. Signals in from water residue which was still left during the
1
6.4 ppm (1H, d) and 7.8 ppm (1H, d) showed a preparation of the sample. The summary of H
13
trans-alkene group with coupling constant of 16 and C NMR analysis and their comparison with
Hz. This result was supported by the total the literature [16] are shown in Tables 2 and 3.
correlation spectroscopy (TOCSY) spectrum.
1
Table 2. Comparison of H NMR chemical shift of trans-cinnamic acid
H atom This study (500 MHz, CDCl3) [16] (300 MHz, CDCl3)
H H (ppm) (multiplicity, J (Hz)) H H (ppm) (multiplicity, J (Hz))
OH - - - -
2 1 6.4 (d, 16) 1 6.48 (d, 15.9)
3 1 7.8 (d, 16) 1 7.82 (d, 15.9)
2’/6’ 2 7.6 (m) 2 7.65–7.54 (m)
3’/4’/5’ 3 7.4 (t) 3 7.5–7.36 (m)
13
Table 3. Comparison of C NMR chemical shift of trans-cinnamic acid
C atom C (ppm) (multiplicity, J (Hz))
This study (125 MHz, CDCl3) [16] (75 MHz, DMSO-d6)
1 171.7 167.66
2 117.2 119.35
3 146.9 144.01
1’ 134.0 134.32
2’/6’ 128.3 128.24
3’/5’ 128.9 128.96
4’ 130.7 130.26
76
Januar et al.; IRJPAC, 8(2): 73-80,, 2015; Article no.IRJPAC.2015.075
no.IRJPAC.2015.
Fig. 1. 13C NMR spectrum of H71 fraction (125 MHz, CDCl3)
1
Fig. 2. H NMR spectrum of H71 fraction (500 MHz, CDCl3)
77
Januar et al.; IRJPAC, 8(2): 73-80,, 2015; Article no.IRJPAC.2015.075
no.IRJPAC.2015.
The heteronuclear single quantum coherence that C-3 atom was
s directly attached to the
(HSQC) and heteronuclear multiple bond benzene unit.
coherence (HMBC) spectra gave g further
evidence of the trans-cinnamic
cinnamic acid structure. Overall correlations observed in the HMBC
The HSQC spectrum showed ed correlation spectrum are given in Table 4. An unpredicted
between proton and carbon through a single correlation wass still observed between C-2
C atom
bond. Signal correlations between C 117.2 ppm and the orto hydrogens. As shown in Figs. 1 and
and H 6.4 ppm and also C 146.9 ppm and H 2, fraction H71 and H6 still contain aliphatic
7.8 ppm proved the existence of a disubstituted impurities. An aromatic compound with similar
alkenes, RCH=CHR’ (Fig. 3). The evidence of a structure as trans-cinnamic
cinnamic acid, but with a C-2
monosubstituted benzene was s explained as atom of the aliphatic moieties directly attached to
follows. Two ortho protons in H 7.6 ppm were a monosubstituted benzene ring was wa predicted to
correlated with C 128.3 ppm, two meta protons be an impurity accounts for this cor correlation.
in H 7.4 ppm were correlated with C 128.9 ppm, However, from NMR analysis explained above,
and a para proton in 7.4 ppm was
wa correlated the chemical compound in fraction H71 and H6
could be predicted as crude trans--cinnamic acid,
with C 130.7 ppm. The carbon signal in C 134.0
but still needed further purification.
ppm was s not correlated with any signal in 1H
NMR spectrum, suggesting that it was a
trans-Cinnamic
Cinnamic acid and trans-p-
quaternary ipso carbon.
methoxycinnamic acid have been repor reported by
Kuo et al. [10] in the ethanol extract of D. repens
The HMBC spectrum showed ed correlation
leaves grown in Taiwan. Ester of cinnamic acid
between proton and carbon atoms separated by
with an iridoid glucoside moiety, namely
two or three bonds. Conjugation of the carbonyl
durantoside I, has also been isolated from D.
group with the double bond was s evidenced from
erecta leaves [17]. trans-Cinnamic
Cinnamic acid itself is a
correlation between carbonyl signal (( C 171.7
common chemical ical compound found in plants.
ppm) and 2 proton signals (H 6.4 and 7.8 ppm) Therefore, investigation on other chemical
(Fig. 4). The C-2 atom (C 117.2 ppm) was constituents in polar as well as nonpolar fractions
correlated with H-3 atom (7.8 ppm), and the C-3
C of the methanol extract, which is characteristic of
atom (C 146.9 ppm) was s correlated with H-2’/6’
H D. repens fruits in Indonesia, needs to be carried
signals (H 7.6 ppm). This results also proved
prove out in immediate study.
Fig. 3. HSQC spectrum of H71 fraction
78
Januar et al.; IRJPAC, 8(2): 73-80,, 2015; Article no.IRJPAC.2015.075
no.IRJPAC.2015.
Fig. 4. HMBC spectrum of H71 fraction
Table 4. Two (α) and three (β) bonds correlation between 1H and 13C NMR peak in the
HMBC spectrum
13
C NMR C-1 C-2 C-3 C-1’ C-2’/6’ C-3’/5’
3’/5’ C-4’
1
H NMR ppm 171.7 146.9 117.2 134.0 128.3 128.9 130.7
H-2 6.4 α DB* α **
H-3 7.8 β DB α
H-2’/6’ 7.6 β DB α β
H-3’/4’/5’ 7.4 β α/ β DB DB
*DB: directly bonded
**unpredicted correlation
4. CONCLUSION 2. Jayalakshmi B, Raveesha KA, Amruthesh
KN. Phytochemical investigations and
Phytochemical investigations on D. repens fruits’ antibacterial activity of some medicinal
methanol extract from Jombang, East Java plants againts pathogenic bacteria. J. Appl.
showed the existence of tannin, saponin, Pharm. Sci. 2011;1(5):124-128.
128.
alkaloid, flavonoid, and steroid. The trans- 3. Nikkon F, Hasan S,, Rahman MH, Hoque
cinnamic acid was
as isolated from acetone-soluble
acetone MA, Mosaddik MA,, Haque ME.
fraction of the extract as confirmed
rmed by 1D and Biochemical, hematological and
2D-NMR spectra analysis. histiphatological effects of Duranta repens
stem on rats. Asian J. Biochem. 2008 2008;2:
COMPETING INTERESTS 366-372.
4. Nikkon F, Saud ZA, Hossain K K, Parvin MS,
Authors have declared that no competing Haque ME. Larvicidal effects of stem and
interests exist. fruits of Duranta repens against the
mosquito Culex quinquefasciatatus.
quinquefasciatatus J.
Pharm. Tech. Res. 2009;4:17091709-1713.
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(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.
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