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Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS


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An HPLC and GC study has been conducted on the aromatic oleoresins styrax and benzoin produced by several American, Mediterranean and East-Asian trees, and widely used in ancient civilisations for their therapeutic and odoriferous properties. Initial experiments were performed by HPLC-PAD-fluorimetry for the analysis of several aromatic components, and then completed by GC-MS for the characterisation of both aromatic and triterpenic derivatives. In this work, it was crucial to isolate from fresh natural exudates, and to characterise by two-dimensional NMR, some of the major constituents in order to extend the standard molecular pool prior to chromatographic identifications. This study reveals coniferyl benzoate as an excellent distinctive fluorescent biomarker of Siam benzoin substrate. It also confirms that fluorimetric-coupled detection is a powerful analytical tool for the identification of compounds in Hamamelidaceae extracts that are almost undetectable by UV. GC-MS was successfully applied to the determination of the botanical origin of Sumatra benzoin, and to the identification of lupeol [3beta-lup-20(29)-en-3-ol] for the first time in such balsam-type materials.
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Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 301–310 (2008)
DOI: 10.1002.pca
Phytochemical Analysis
Phytochem. Anal. 19: 301–310 (2008)
Published online 9 November 2007 in Wiley InterScience ( DOI: 10.1002/pca.1048
* Correspondence to: Catherine Vieillescazes, Université d’Avignon
et des Pays de Vaucluse, Laboratoire de Chimie Bioorganique et des
Systèmes Moléculaires Vectoriels (LCBSMV), Equipe ‘Chimie appliquée
à l’Art et à l’Archéologie’, 33, rue Louis Pasteur, 84000, Avignon, France.
Contract/grant sponsor: PACA Regional Council; Contract/grant spon-
sor: Amoroso-Waldeis Workshop of Conservation and Restoration of
Works of Art.
Styrax and benzoin balsams have been widely employed
since ancient times (Gianno et al., 1990; Modugno
et al., 2006) by Romans, Egyptians and Phoenicians
in order to treat chronic infections of the respiratory
tract, by virtue of their therapeutic and pharmaco-
logical properties including disinfectant, expectorant
and vulnerary activities (Hew, 1998). Moreover, their
characteristic odoriferous properties have been employed
in ritual ceremonies together with myrrh or olibanum
(Chermette and Goyon, 1996; Vieillescazes and Coen,
1993; Hamm et al., 2004). Nowadays, their use is
extended to perfumery, as fixative agents, whilst their
antioxidant and organoleptic properties are valued in
the cosmetic and food industries for conservation and
improvement of flavour (Fernandez et al., 2003, 2006;
Castel et al., 2006).
Benzoin resins represent pathological exudates that
are only produced after deep incisions have been made
into the bark of trees belonging to the genus Styrax
(Styracaceae family), which are endemic in numerous
East-Asian countries such as Indonesia, Sumatra, Java,
Laos, Thailand and Vietnam. Siam benzoin balsam
(Styrax tonkinensis C.) and Sumatra benzoin balsam
(S. paralleloneurum P. and S. benzoin D.) represent the
two main categories of balsams that are commercially
available. Such balsam-type materials are composed
of a volatile extract mainly consisting of benzoic and
cinnamic derivatives, and a resinous extract containing
oleanane triterpenes (Fig. 1). Thus, Siam benzoin resin is
typically composed of coniferyl, benzyl and p-coumaryl
benzoates, cinnamyl cinnamate (styracin), free benzoic
acid, a small amount of coniferyl alcohol (lubanol), and
traces of vanillin and siaresinolic (3
12-en-28-oic) acid (Reinitzer, 1914; Schroeder, 1968;
Saleh et al., 1980; Nitta et al., 1984; Wahbi et al., 1987;
Popravko et al., 1994; Castel et al., 2006; Fernandez
et al., 2006). Sumatra benzoin resin is reported to con-
tain traces of benzaldehyde, styrene and a larger pro-
portion of free cinnamic acid and cinnamate derivatives
than Siam species. The resinous part of Siam benzoin
contains only sumaresinolic (3
12-en-28-oic) acid, which is different from the resin of
Sumatra benzoin. Furthermore, the presence of diter-
penic compounds has also been described (Tchapla
et al., 1999). However, the aromatic composition of
these materials depends not only on the botanical and
geographical origins but also on the extraction process
employed prior to analysis. Indeed, after applying
Analytical investigation of styrax and benzoin balsams by
HPLC-PAD-fluorimetry and GC-MS
1Université d’Avignon et des Pays de Vaucluse, Laboratoire de Chimie Bioorganique et des Systèmes Moléculaires Vectoriels (LCBSMV), Equipe
‘Chimie appliquée à l’Art et à l’Archéologie’, 33, rue Louis Pasteur, 84000, Avignon, France
2Université du Sud Toulon Var, Laboratoire des Matériaux à Finalités Spécifiques (MFS), Equipe ‘Chimie des Produits Naturels Marins’, Avenue de
l’Université, BP 20132, 83957 La Garde, Cedex, France
Received 27 September 2007; accepted 27 September 2007
Abstract: An HPLC and GC study has been conducted on the aromatic oleoresins styrax and benzoin produced by several Ameri-
can, Mediterranean and East-Asian trees, and widely used in ancient civilisations for their therapeutic and odoriferous properties.
Initial experiments were performed by HPLC-PAD-fluorimetry for the analysis of several aromatic components, and then completed
by GC-MS for the characterisation of both aromatic and triterpenic derivatives. In this work, it was crucial to isolate from fresh
natural exudates, and to characterise by two-dimensional NMR, some of the major constituents in order to extend the standard
molecular pool prior to chromatographic identifications. This study reveals coniferyl benzoate as an excellent distinctive
fluorescent biomarker of Siam benzoin substrate. It also confirms that fluorimetric-coupled detection is a powerful analytical tool
for the identification of compounds in Hamamelidaceae extracts that are almost undetectable by UV. GC-MS was successfully
applied to the determination of the botanical origin of Sumatra benzoin, and to the identification of lupeol [3
for the first time in such balsam-type materials. Copyright © 2007 John Wiley & Sons, Ltd.
Keywords: High-performance liquid chromatography; gas chromatography; photodiode array–fluorimetric detection; mass
spectrometry; styrax; benzoin.
Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 301–310 (2008)
DOI: 10.1002.pca
Figure 1 Main aromatic and triterpenic constituents of styrax and benzoin balsams.
solvent extraction, some authors (Pastorova et al.,
1997) have reported that coniferyl benzoate is the main
constituent of the volatile fraction of Siam benzoin,
whereas, when hydrodistillation has been employed,
other researchers (Fernandez et al., 2003) have claimed
that benzyl benzoate is the major aromatic compound
in both Siam and Sumatra exudates.
Some resinous materials produced by trees of the
genus Styrax are not called benzoin but styrax. They
derive exclusively from a Mediterranean tree of the
species S. officinalis L. (Tayoub et al., 2006). In fact,
genuine styrax (sometimes named storax) substances
are produced by Liquidambar spp. (Hamamelidaceae
family) from Turkey (L. orientalis M.), Java, China and
Australia (L. altingiana B.) or Guatemala, Honduras,
Mexico and North-East America (L. styraciflua L.).
The volatile extract (Hafizoglu et al., 1996; Pastorova
et al., 1998) contains styrene, free cinnamic acid and
cinnamate derivatives (cinnamyl, ethyl, benzyl and
phenylpropyl cinnamates), but no benzoic compounds
have been reported (Acar and Anil, 1991). Free or
esterified oleanolic (3
-hydroxyolean-12-en-28-oic) and
3-epi-oleanolic (3
-hydroxyolean-12-en-28-oic) acids
mainly constitute the major part of the triterpenic
fraction (Huneck, 1963).
Such a complex and ambiguous botanical classifica-
tion, combined with various improper common desig-
nations, gives rise to difficulties in correlating with
precision the botanical species and chemical com-
positions of the exudates. In previous work (Hovaneis-
sian et al., 2006), we demonstrated the possibility of
adulteration of these resinous substrates by dammar,
a triterpenic resin produced by Indonesian trees be-
longing to the family Dipterocarpaceae. This present
paper describes a better characterisation of these
plant exudates via the identification of both aromatic
and triterpenic compounds by HPLC and GC analysis.
Indeed, although these resins are widely used as
flavours and fragrances, there have been only a few
studies on their global chemical composition. Further-
more, whilst several analytical methods involving
chromatography and spectrophotometry have been
applied to the investigation of these resinous materials,
no studies have been made using fluorimetric detection.
Solvents and reagents.
Solvents and reagents were all
of analytical grade and obtained from Merck (Darmstadt,
Germany). Styrene and lubanol were purchased from
Acros Organics (Morris Plains, NJ, USA), benzoic acid
and ethyl cinnamate were from Fluka AG (Buchs,
Switzerland), cinnamic acid and vanillin from Merck,
and oleanolic acid and lupeol from Extrasynthese
(Genay, France). Oleanonic (3-oxoolean-12-en-28-oic)
acid was synthesised by the classical oxidation of
oleanolic acid with pyridinium chlorochromate in
Reference resinous substances.
Siam benzoin (S.
tonkinensis), and Turkey (L. orientalis) and Honduras
styrax (L. styraciflua) resins were purchased from
Les Encens du Monde, Asie Concept (Castelnau-le-Lez,
France). Sumatra benzoin (S. benzoin or S. paralle-
loneurum) balsam was obtained from Okhra
(Roussillon, France).
NMR analysis.
1H-, 13C-, gCOSY 1H-1H-, gHSQC- and
gHMBC-NMR experiments were recorded on a Bruker
(Wissenbourg, France) Avance 400 MHz spectrometer.
Samples were dissolved in deutero-chloroform; chemical
shifts are quoted in ppm (
) relative to tetramethyl-
silane and coupling constants in Hertz.
Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 301–310 (2008)
DOI: 10.1002.pca
HPLC-PAD-fluorimetry analysis.
Analyses were performed
with a chromatographic system consisting of a Spectra-
Physics (San Jose, CA, USA) SP-8800 ternary gradient
pump, a Rheodyne 7125 injector equipped with a 20 μL
loop, a LiChroCART Superspher (Merck) 100 RP-18e
reverse-phase column (250 × 4 mm i.d.; 5 μm) and
a Waters (Milford, Massachusetts, USA) model 996
PAD and a Waters model 470 fluorimetric detector, all
controlled by Millennium32 version 3.05.01 software.
The separation was performed at 35°C with a binary
elution mixture composed of acetonitrile (A) and double-
distilled water (B) containing 0.01% trifluoroacetic
acid. Chromatography was carried out in 80 min at a
continuous flow-rate of 1 mL/min with the following
solvent sequence: (i) linear increase in 50 min from 10
to 60% of A; (ii) second linear increase in 10 min from
60 to 100% of A; and (iii) an isocratic period of 20 min
with 100% of A, followed by a return to the initial
conditions in 10 min after analysis. All chromatograms
were scanned at 275 nm. The identification of different
peaks was carried out by the comparison of retention
times and UV–vis data with those of pure standards.
Fluorimetric measurements were carried out directly
using the Waters 470 fluorimetric detector in the
scanning mode. The appropriate excitation and emis-
sion wavelength couple (
em) was established
manually using the maximum absorption wavelength,
previously determined from the UV spectra, as the
initial excitation wavelength. After recording the
fluorescence spectrum, the emission wavelength was
obtained. Then, the opposite process was carried out in
order to optimise the excitation wavelength, and this
process was repeated until the final optimum wavelen-
gth couple was obtained. Samples (10 mg) were dis-
solved in 2 mL of analytical grade methanol. Solutions
were then maintained at 25°C for 15 min in an ultra-
sonic bath. After centrifugation, the supernatant was
filtered in a 0.2 μm DynaGuard™ cartridge before
injection into the HPLC in triplicate.
HPLC purification and NMR identification of reference
In order to extend the standard molecular
pool, other chemical markers were isolated directly
from methanolic extracts of resinous materials and
purified by HPLC-PAD. For this purpose, 2 g each of
Siam benzoin, Sumatra benzoin and Turkey styrax
were separately dissolved in 10 mL of methanol.
After filtration, reference compounds were purified
from these solutions by sequential injections onto a
Nucleosil (Interchim, Montluçon, France) C18 reverse-
phase column (250 × 3.2 mm i.d.; 5 μm) employing
isocratic elution with acetonitrile:water (60:40, v/v).
These conditions of elution were used in order to permit
a reduction in retention time of the major compounds
in each resin. Then, each structure was characterised
on the basis of chemical and spectral evidence in-
cluding two-dimensional NMR experiments. Methanolic
extracts from resinous material gave coniferyl benzoate
(from Siam benzoin at tR = 9.7 min), coumaryl cinna-
mate (from Sumatra benzoin at t
R = 10.1 min), benzyl
benzoate and benzyl cinnamate (from Turkey styrax at,
respectively, tR = 13.3 and 15.9 min; Gigante et al.,
1991; Hausen et al., 1995; Villa et al., 2007).
GC-MS analysis.
Analyses were performed on a Varian
(Walnut Creek, CA, USA) Saturn 3900 gas chromato-
graph with a model 1177 injector coupled with a 2100 T
ion trap mass spectrometer. The gas chromatograph
was equipped with a Varian fused-silica CP-Sil 8 CB
low bleed/MS capillary column (30 m × 0.25 mm i.d.)
coated with a 0.25 μm film of poly (5% phenyl, 95%
dimethyl) siloxane. The MS electron multiplier voltage
was set at 1400 V and the ionisation time was 25000 μs;
the transfer line, ion trap and manifold temperatures
were set, respectively, at 300, 200 and 50°C. MS spectra
were measured in the electron impact (EI) mode with
an ionising voltage of 70 eV; the scan range was set to
40–650 m/z. Samples were injected (1 μL) with a split
ratio of 1:20, and the injector temperature was set to
250°C. A continuous flow-rate of 1 mL/min of chromato-
graphic grade helium was employed. The column oven
was initially at 50°C and was held for 2 min after
injection, followed by temperature ramping at 8°C/min
up to 250°C, and then at 3°C/min up to 350°C. No hold
time was performed at the upper limit. Before injection,
each sample (5 mg of commercial resin or standard)
was trimethylsilylated with a solution containing
0.45 mL of hexamethyldisilazane and 0.3 mL of trime-
thylsilyl chloride in 0.5 mL of anhydrous pyridine. The
reaction was carried out at room temperature over
30 min and the solvents removed under a stream of
nitrogen. The dried residue was immediately dissolved
in 0.6 mL of dichloromethane, and the solution was
filtered on 0.2 μm DynaGuard™ cartridge prior to
analysis. Identifications were made by comparison of
retention times and mass spectral data with those
of pure isolated and/or commercial standards. The
chemical structure determinations of some unknown
compounds were performed using the NIST’98 mass
spectral database.
HPLC-PAD-fluorimetric investigation
Reference chemical compounds used in this section
(Table 1) corresponded to commercial (1–5) and/or
isolated (6–9) aromatic constituents. Experimental
conditions employed were optimised for the separation
and identification of only the benzoate and cinnamate
derivatives. The triterpenoids were preferentially inves-
tigated by GC-MS (see following section) owing to their
spectrophotometric properties.
Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 301–310 (2008)
DOI: 10.1002.pca
Table 1 High-performance liquid chromatographic and spectroscopic data for the reference compounds 1–9
Solvent composition,
Compound tR
a (min) UV
max (nm) % (A/B)b (v/v)
1Lubanol 11.7 212, 263, 300 21.7/78.3
2Vanillin 14.3 228, 277, 308 24.3/75.7
3Benzoic acid 14.8 228, 272 24.8/75.2
4Cinnamic acid 25.5 214, 219, 275 35.5/64.5
5Ethyl cinnamate 43.7 215, 220, 276 53.7/46.3
6p-Coumaryl cinnamate 45.9 206, 273 55.9/44.1
7Coniferyl benzoate 46.1 223, 267, 305 56.1/43.9
8Benzyl benzoate 50.3 229, 267 60.2/39.8
9Benzyl cinnamate 54.1 205, 278 62.1/37.9
atR, retention time. bMobile phase was composed of acetonitrile (A) and double-distilled water (B) containing
0.01% trifluoroacetic acid (pH = 3).
Figure 2 HPLC-PAD and HPLC-fluorimetry chromatograms of commercial Siam and Sumatra benzoin resins. Peak numbers refer
to compounds in Table 1. (For analytical conditions see Experimental section.)
Appropriate analytical conditions allowed the charac-
terisation of several aromatic derivatives as p-coumaryl
cinnamate (6) in Sumatra benzoin, coniferyl benzoate
(7) in Siam benzoin, benzyl benzoate (8) and benzyl
cinnamate (9) in Turkey styrax, but no free benzoic
and cinnamic acids were detected (Figs 2 and 3).
However, taking into account the similarity in UV
absorption of such aromatic derivatives, it would be too
restrictive to rely on an analytical investigation with
UV detection only. Therefore, selective and sensitive
additional fluorimetric detection was employed. The
choice of such spectroscopic technique was determined
according to previous results obtained using small
amounts of archaeological resinous materials (Martin
et al., 2001).
Under optimum excitation and emission wavelength
conditions (
ex = 330 nm/
em = 365 nm), coniferyl
benzoate represents an excellent fluorescence marker
of Siam benzoin resin, whereas neither p-coumaryl
cinnamate nor any other aromatic compounds present
in Sumatra benzoin resin exhibited such fluorescence
(Fig. 2). This interesting characteristic provides a better
differentiation of those aromatic esters for which reten-
tion times and spectroscopic data are not sufficiently
distinct for an unambiguous identification. In particu-
lar, it excludes the presence of traces of coniferyl
Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 301–310 (2008)
DOI: 10.1002.pca
Figure 3 HPLC-PAD and HPLC-fluorimetry chromatograms of commercial Turkey and Honduras styrax resins. Peak numbers
refer to compounds in Table 1. (For analytical conditions see Experimental section.)
benzoate in the Sumatra benzoin sample. It can be
noted that this compound has a similar retention time,
in our analytical conditions, to p-coumaryl cinnamate,
and this component is present in Sumatra benzoin and
has a strong UV absorption. A similar strongly fluores-
cent compound (
ex = 260 nm/
em = 340 nm) is eluted
at tR = 21.7 min in both Turkey and Honduras styrax
chromatograms (Fig. 3). In such cases, the fluorimetric-
coupling technique provides visualisation of compounds
for which UV detection is very difficult. Furthermore,
this unknown fluorescent constituent, whose structure
is currently being investigated, could provide a plausi-
ble fluorescence marker of styrax balsams and more
precisely of plant exudates from Liquidambar spp.
GC-MS investigation.
Standard chemical compounds used
in this section (Table 2) corresponded with those previously
Table 2 Gas chromatographic and mass spectrometric data of reference compounds 1–16
Compound tR
b (min) m/z values of main characteristics fragments (relative intensity) (%)
1Styrene 5.8 77 (25), 104 (100) [M+]
2TMSa benzoic acid 12.7 77 (29), 105 (44), 135 (51), 179 (100), 194 (4) [M+]
3TMS vanillin 16.1 73 (10), 195 (100), 209 (55), 224 (27) [M+]
4Ethyl cinnamate 16.7 77 (1), 103 (16), 131 (100), 148 (1), 176 (85) [M+]
5TMS isovanillin 17.5 73 (10), 195 (100), 209 (55), 224 (27) [M+]
6TMS cinnamic acid 17.7 77 (15), 103 (26), 131 (34), 161 (44), 205 (100), 220 (19) [M+]
7Benzyl benzoate 21.3 77 (24), 91 (38), 105 (100), 194 (29), 212 (14) [M+]
8TMS lubanol 23.4 73 (71), 131 (23), 204 (64), 235 (33), 293 (97), 324 (100) [M+]
9Benzyl cinnamate 25.5 91 (78), 103 (56), 131 (100), 193 (72), 238 (15) [M+]
10TMS p-coumaryl benzoate 29.6 77 (39), 105 (100), 135 (23), 179 (43), 221 (60), 326 (25) [M+]
11TMS coniferyl benzoate 31.8 77 (23), 105 (100), 180 (29), 204 (33), 235 (84), 251 (44), 356 (64) [M+]
12TMS p-coumaryl cinnamate 34.7 103 (13), 131 (34), 161 (1), 205 (100), 307 (18), 352 (19) [M+]
13TMS lupeol 43.3 73 (19), 189 (100), 203 (59), 218 (30), 408 (13), 483 (10), 498 (16) [M+]
14TMS oleanonic acid 47.1 55 (15), 73 (44), 189 (73), 203 (100), 218 (6), 408 (38), 526 (27) [M+]
15TMS oleanolic acid 47.4 73 (43), 189 (47), 203 (100), 218 (10), 320 (34), 483 (31), 600 (1) [M+]
16Unidentified triterpene 51.4 73 (55), 189 (19), 203 (100), 218 (12), 426 (29), 501 (34), 602 (4) [M+]
aTMS, trimethylsilylated. btR, retention time.
Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 301–310 (2008)
DOI: 10.1002.pca
Figure 4 TIC chromatogram of a trimethylsilylated sample of commercial Siam benzoin resin. Peak numbers refer to compounds
in Table 2. (For analytical conditions see Experimental section.)
employed in the HPLC section of the present paper
(2–4, 6–9 and 11 and 12), together with styrene
(1), some commercial (13 and 15) or hemi-synthetic
(14) pentacyclic triterpenes, and other NIST’98 mass
spectral database reference structures (5 and 10).
Experimental conditions employed were optimised for
the simultaneous separation and identification
of both aromatic volatile and triterpenic non-volatile
compounds. Furthermore, trimethylsilylation was pre-
ferred to methylation for the preliminary derivatisation
in order to distinguish unambiguously the natural
methyl ethers and/or esters from the corresponding
synthetic compounds.
As a complement to the HPLC investigation, it was
shown that Siam benzoin is not exclusively composed
of coniferyl benzoate (11). Indeed, inspection of its
corresponding total ion current (TIC) chromatogram
(Fig. 4) revealed the presence of other aromatic deriva-
tives such as benzoic acid (2), isovanillin (5), lubanol
(8) and p-coumaryl benzoate (10). Moreover, these re-
sults confirm the absence of free cinnamic acid, as had
been previously described (Reinitzer, 1914; Schroeder,
1968; Saleh et al., 1980; Nitta et al., 1984; Wahbi
et al., 1987; Popravko et al., 1994; Castel et al., 2006;
Fernandez et al., 2006). In contrast to Siam benzoin,
Sumatra resin is composed of styrene (1), isovanillin
(5), a large quantity of free cinnamic acid (6), p-
coumaryl cinnamate (12), and traces of both free
benzoic acid (2) and p-coumaryl benzoate (10) (Fig. 5).
As was previously mentioned, Sumatra benzoin balsam
can be produced from S. benzoin and/or S. paralle-
loneurum. However, the relative proportion of p-
coumaryl benzoate (10) to p-coumaryl cinnamate (12)
may be employed in the determination of the botanical
origin. In fact, S. paralleloneurum balsam contains more
cinnamates than S. benzoin, and among these deriva-
tives p-coumaryl cinnamate (12) is predominant
(Pastorova et al., 1997). This clearly specifies the identi-
fication of this resinous substrate as Styrax paralleloneurum
P. exudation product. Moreover, Turkey styrax balsam
is composed of benzoic acid (2), isovanillin (5), cinnamic
acid (6), a large amount of benzyl benzoate (7) and
traces of benzyl cinnamate (9) (Fig. 6). It is obvious
that Turkey styrax exudate is mainly composed of benzyl
benzoate whereas this compound is also described as
a major constituent of some solvent extracts of Siam
and Sumatra benzoins. In consequence, this ambiguity
implies that benzyl benzoate (7) cannot be considered
as a characteristic marker, unlike coniferyl benzoate
(11), which is exclusively observed in Siam benzoin. Thus,
it is possible to characterise coniferyl benzoate as an
excellent fluorescence marker of Siam benzoin resin,
Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 301–310 (2008)
DOI: 10.1002.pca
Figure 6 TIC chromatogram of a trimethylsilylated sample of commercial Turkey styrax resin. Peak numbers refer to compounds
in Table 2. (For analytical conditions see Experimental section.)
Figure 5 TIC chromatogram of a trimethylsilylated sample of commercial Sumatra benzoin resin. Peak numbers refer to com-
pounds in Table 2. (For analytical conditions see Experimental section.)
Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 301–310 (2008)
DOI: 10.1002.pca
Figure 7 TIC chromatogram of the triterpenic region (retention time 40 –55 min) of a trimethylsilylated sample of commercial
Siam benzoin resin. Peak numbers refer to compounds in Table 2. (For analytical conditions see Experimental section.)
which increases its distinction in comparison with res-
inous materials produced by other Styracaceae plant
Styrax and benzoin balsams are generally reported
to contain terpenic compounds, and more precisely,
oleanane triterpenic compounds (Wang et al., 2006).
Nevertheless, among all resinous samples studied here,
only Siam benzoin exudates appeared to contain such
derivatives (as indicated by tR values in the range 40–
60 min). This observation could be explained by con-
sidering the modes of preparation of these commercial
products. Indeed, they often consist in collecting
boiling decoction extracts (Hew, 1998), which would
explain the exclusive occurrence of previously observed
volatile chemical compounds. The Siam benzoin sam-
ple was composed of oleanonic acid (14), oleanolic acid
(15) and lupeol (13) (Fig. 7). Although the lupane
triterpene is generally described in the chemical com-
position of birch bark and some plant resins (Lawrie
et al., 1970; Masaoud et al., 1995), it has never previ-
ously been characterised in a balsam-type product.
Another triterpenoid (16) also observed in the sample
showed a molecular ion at m/z 602, which excludes
its identification as siaresinolic acid, a compound gen-
erally described in this resin, but its characteristic
mass spectral elements (see Table 2; Fig. 8) seem to
indicate an oleanane skeleton. (Budzikiewicz et al.,
1963; Karliner and Djerassi, 1965; Mathe et al., 2004).
This research was financially supported by the PACA
Regional Council and Amoroso-Waldeis workshop of con-
servation and restoration of works of art (Avignon, France).
Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 301–310 (2008)
DOI: 10.1002.pca
Figure 8 Mass spectra of the observed triterpenoids.
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... The trees belonging to Styrax have no ability to secrete resin unless their stems get injured. Consequently, benzoin resin is regarded as a pathologic exudation of plants in a stress response to external injuries [17]. Pinyopusarerk described the tapping of benzoin in Laos [18]. ...
... Differing from the content and infrared absorption bands of benzoic acid and cinnamic acid, Sumatra benzoin and Siam benzoin have subtle differences in 1000 cm −1 to 600 cm −1 , which could be a significant basis of benzoin species identification [60]. Hovaneissian et al. used HPLC-PAD-fluorimetry to separate Siam and Sumatra benzoin, considering coniferyl benzoate as a fluorescence marker of Siam benzoin, while no compounds in Sumatra benzoin showed the same fluorescence [17]. ...
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Benzoin is a pathologic exudation produced by plants of the family Styrax. It is secreted by traumatic resin ducts after injury, which are derived from parenchymal cells in secondary xylem by schizolysigeny. Some 63 chemical constituents have been isolated and identified from this resin, including balsamic acid esters, lignans and terpenoids. It has a long history of applications, including as incense along with olibanum, a flavor enhancer in the food industry, materials in the daily chemistry industry as well as therapeutic uses. Up to now, high-performance liquid chromatography (HPLC) and gas chromatography mass spectrometry (GC-MS) have been widely used in qualitative and quantitative analysis of benzoin. Other technologies, including near-infrared reflectance spectroscopy (NIR), proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) and Fourier-transform infrared spectroscopy (FT-IR), have also been used to distinguish different resins. Herein, this paper provides a comprehensive overview of the production process, phytochemistry, traditional uses and quality control of benzoin and looks to the future for promoting its further research and applications.
... 11 Several phytochemical investigations have been performed on Sumatra benzoin. They include gas chromatography (GC) analysis of volatile constituents, 12,13 the detection and quantification of phenolic constituents by highperformance liquid chromatography with ultraviolet-evaporative light scattering detection (HPLC-UV-ELSD) analysis, 8 the analysis of triterpenoids and benzyl and cinnamyl esters by HPLC coupled with photodiode array detection and fluorimetry (HPLC-PDA-fluorimetry), 14 and the analysis of free and ester bound benzoic and cinnamic acids by GC−MS and HPLC−frit-fast atom bombardment−MS (HPLC−frit FAB−MS). 15 Sumatra benzoin was also included in a large study comparing the chemical profiles of various balsams. ...
Sumatra benzoin, a resin produced by Styrax benzoin and Styrax paralleloneurum, is used as an aromatic agent and may have the potential to be developed as a new agricultural fungicide. In this context, we performed a comprehensive metabolite profiling of a commercial grade A resin by high-performance liquid chromatography coupled with photodiode array detection, evaporative light scattering detection, and mass spectrometry (HPLC-PDA-ELSD-MS) analysis in combination with 1H NMR. Thirteen compounds including a new cinnamic acid ester containing two p-coumaroyl residues were identified after preparative isolation. These compounds accounted for an estimated 90% of the crude resin according to 1H NMR analysis. The two major constituents, p-coumaryl cinnamate (5) and sumaresinolic acid (11), were quantified by HPLC analysis. In a next step, the chemical profiles and the content in p-coumaryl cinnamate were compared in a large set of resin samples of different quality grades that were obtained from various commercial suppliers in Sumatra. The qualitative profiles of the samples were very similar, but significant quantitative differences were observed between different quality grades and origins of the samples for the relative contents.
... These compounds are most likely related to the original components of the resin. The most probable botanical origin for this amber is in fact the liquidambar plant (Altingiaceae family), whose resin has a high content of cinnamic acid and its esters [45]. Upon pyrolysis, these oxygenated species generate cinnamic acid derivatives such as 3-phenylpropanylcinnamate, which has been proposed by Pastorova and co-workers as a marker to distinguish siegburgite from New Jersey class III amber and from synthetic polystyrene [41]. ...
... [11], using "Rhizoma Coptidis (Huanglian)", "Trichosanthes Kirilowii Maxim (Gualou)", "Carthami Flos (Honghua)", "Persicae Semen (Taoren)"and "Borneolum Syntheticum (Bingpian)" as keywords. Also, the references [12][13][14][15][16][17] were retrieved to supplement the active components of these 6 herbs. The targets of these components were collected from TCMSP, and predicted using SwissTargetPrediction ( [18] and SEA ( ...
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Background: Atherosclerosis (AS) is one of the leading causes of cardiovascular diseases. The traditional China herb pairs such as Huanglian-Gualou, Honghua-Taoren, and Suhexiang-Bingpian showed therapeutic effects on AS by clearing heat and resolving phlegm, invigorating blood and removing blood stasis, as well as aromatic resuscitation, respectively. However, the common and specific mechanisms of these pairs against the same disease are elusive.Objective: This study aimed to explore the molecular mechanisms of 3 herb pairs treating AS by network pharmacology, molecular modeling and mechanism experiments.Methods: The components and their corresponding targets of 3 herb pairs, as well as AS-related targets, were collected from multiple databases and literature. Then the protein-protein interaction network was built to identify the key components and targets associated with AS. The pathway enrichment analysis using KEGG was carried out for analyzing the common mechanisms of 3 herb pairs against AS. Finally, the binding modes of the key components and targets were analyzed by molecular docking and molecular dynamic simulation.Results: The PPI network indicated that the common targets of 3 herb pairs focused on four pathways, including regulated vascular shear stress, TNF, ARE-RAGE, and IL-17 pathways. The molecular docking analysis indicated that the key component quercetin showed highest docking score with PTGS2 in comparison to other targets. Molecular dynamics simulations revealed that quercetin stably anchored to the active pocket of PTGS2 by forming hydrogen bonds with Thr175, Asn351, and Trp356.Conclusion: The molecular mechanism of Huanglian-Gualou, Honghua-Taoren, and Suhexiang-Bingpian against AS was preliminarily expounded, and we wish to provide a theoretical instruction for clinical treatment of AS.
... Fermented styrax leaves was found to have significant inhibition zone and Styrax Leaves in SmF at stationary phase has the most significant inhibition zone which was shown in Table 1. This activity was equally the same compared to antibacterial activity of Styrax Resin which was famously known and widely used in the world for the effectiveness to inhibit antibacterial [17]. ...
... Loban crystals are burned in places of worship throughout the Indian subcontinent to produce a holy, sanctified environment. S. benzoin generally contains cinnamic acid, benzoic acid, methyl cinnamate, coniferyl benzoate, cinnamaylcinnamate, phenylethylene, phenylpropylic alcohol, and vanillin (Popravko et al. 1984;Hovaneissian et al. 2008). S. benzoin resin is used in traditional herbal medicine to treat a number of skin ailments, skin ulcers, wounds, and bedsores. ...
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Styrax benzoin fumes have a spiritual aspect from ancient times, magical essence like a pleasant perfume, and are employed in religious ceremonies in India. This study aims to identify the volatile compounds in S. benzoin extract, their binding affinity to the bacterial target proteins, and study the antibacterial activity of the potential extract. The compounds obtained from GC-MS analysis of S. benzoin extract were subjected to molecular docking studies against DHFR of Staphylococcus aureus, tRNA synthetase of Escherichia coli, DHPS of Mycobacterium tuberculosis. Molecular docking studies revealed that seventeen compounds out of 20 compounds exhibited higher binding affinity than co-ligand (-7.00 kcal/mol) against the Staphylococcus aureus enzyme DHFR. Consequently, the crude extracts were evaluated for antibacterial activity against S. aureus, and the acetone extract showed promising findings. S. benzoin fumes might replace synthetic room fresheners, and promising compounds could be exploited in the cosmetics industry.
Introduction: Depending on their terpenoid and phenolic constituents plant resins can be classified as diterpenoid, triterpenoid or phenolic resins; thereby the profile of diterpenes and triterpenes is considered as genus- or even species-specific. Objectives: We aimed to develop a simple, rapid, inexpensive, sensitive and specific method for the identification of resin-specific triterpenoid and phenolic compounds in plant resins using (HP)TLC [(high-performance) thin-layer chromatography] combined with APCI-MS (atmospheric pressure chemical ionisation mass spectrometry) and post-chromatographic detection reactions. Methods: Twenty resin samples from different plant species were analysed. Different extraction procedures, post-chromatographic detection reagents as well as various sorbents and solvents for planar chromatography were tested. To evaluate the potential of the optimised (HP)TLC-APCI-MS methods, parameter such as limit of detection (LOD) was determined for selected marker compounds. Results: Our protocol enabled qualitative analyses of chemotaxonomic molecular markers in natural resins such as dammar, mastic, olibanum and benzoin. For the first time, the application of thionyl chloride-stannic chloride reagent for a specific post-chromatographic detection of triterpenes is reported, sometimes even allowing discrimination between isomers based on their characteristic colour sequences. For triterpene acids, triterpene alcohols and phenolic compounds, detection limits of 2-20 ng/TLC zone and a system precision with a relative standard deviation (RSD) in the range of 3.9%-7.0% were achieved by (HP)TLC-APCI-MS. The applicability of the method for the analysis of resin-based varnishes was successfully tested on a mastic-based varnish. Thus, the method we propose is a helpful tool for the discrimination of resins and resin-based varnishes with respect to their botanical origin.
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Phytochemical investigation of the ethyl acetate extract of Styrax annamensis leaves has led to the isolation and determination of a new lignan, 7R,8S,7′S,8′R-3,4,3′-trimethoxy-9,4′,7′-trihydroxy-8.8′,7.O.9′-lignan (1), together with a known compound, 4-ketopinoresinol (2). Compound 2 was isolated from the genus Styrax for the first time, and showed moderate activity to capture the DPPH radical with the IC 50 value of 56.74 ± 0.79 μg/mL.
In a previous work, a difference spectrophotometric method was developed for quantitating benzoic and cinnamic acids, in the presence of resin acids. In the present work, the technique was adapted for estimating the individual balsamic acids in balsam tolu and benzoin. A procedure for determining purity of the balsamic acids fraction, recovered after alternative purification steps, was studied. Thus we developed an assay procedure relevant to both balsams. Results by the proposed method, the BPC method, and a literature spectrophotometric method are compared.
Des substances résineuses provenant de tombes de la XIIe Dynastie Egyptienne ont été analysées par chromatographie liquide à haute performance (CLHP) et détection par barrette de photodiodes. Les identifications de deux prélèvements avec des résines standard de myrrhe et de mastic (Pistacia lentiscus) sont décrites dans cet article; ces caractérisations résultent de la comparaison de leurs analyses chromatographiques, des spectres UV des principaux constituants et de données infrarouge. /// Resinous substances from Egyptian XIIth Dynasty tombs were analyzed by high-performance liquid chromatography (HPLC) and diode array detection. The unambiguous identification of two samples with actual standards of myrrh and mastic (Pistacia lentiscus) is described. They are characterized by comparison of their chromatographic patterns, the UV spectra of the main constituents and infrared data. /// Harzige Stoffe, die aus Gräbern der zwölften ägyptischen Dynastie abstammen, sind durch Hochdruckflüssigkeitschromatographie (HPLC) mit Diodenarraydetektion analysiert worden. Die Identifizierung von zwei Entnahmen mit genormten Myrrhe und Mastix (Pistacia lentiscus) Harzen sind beschrieben. Diese Charakterisierungen leiten von dem Vergleich ihrer chromatographischen Analysen, der UV Spektren ihrer Hauptbestandteile, und der IR angeben ab.
Sumatra benzoin resins originating from two species of Styrax were studied using modern analytical techniques. Analysis of these types of resins usually involves several steps including alkaline hydrolysis, derivatization of the polar groups and chromatography. Two different resins, and three different qualities from each resin, were investigated using a fast analytical method which omits the hydrolysis step and makes it possible to identify all of the individual components of the resin in one gas-chromatography–mass spectrometry measurement. Resins from both Styrax benzoin Dryand and S. paralleloneurum Perk contain free cinnamic and benzoic acids and their corresponding esters with p-coumaryl and coniferyl alcohols, although in different relative amounts. Pinoresinol and some higher molecular weight esters of cinnamic and benzoic acids were also found. The structure of these compounds was verified by high performance liquid chromatography–continuous flow fast atom bombardment mass spectrometry. For quantitation, gas chromatography with flame ionization detection was performed. Molecular response factors of the identified compounds were calculated as correction factors of the peak areas. © 1997 by John Wiley & Sons, Ltd.
Siegburgite, an unusual Tertiary aromatic resin found near Bitterfeld (Germany), has been characterised earlier by FTIR spectrometry as polystyrene-like material. Here the molecular weight distribution of the soluble part is determined by size-exclusion chromatography (SEC) to be bomodal with maxima at 400 and 30,000 Da. Using combined gas chromatographic-mass spectrometric techniques (GC–MS) traces of oleanonic and 3-epi-oleanolic acid are found in the low MW fraction of Siegburgite. 3-Phenylpropanylcinnamate is detected in the high MW fraction by pyrolysis (Py)–GC–MS. Although also present in the soluble part of the high MW fraction, this compound is present especially in the insoluble residue, indicating that the ester is incorporated in the polymer chain. Oleanonic acid, 3-epi-oleanolic acid and 3-phenylpropanylcinnamate are constituents of commercially available Gum Storax from modern Liquidambar orientalis Mills. The proposed paleobotanical origin of Siegburgite from the Hammamelidacae family is thus chemically confirmed. In addition, a comparable polystyrene-like resin originating from North America (Squankum New Jersey) was compared to Siegburgite. The polystyrene matrix of this resin is comparable to that of Siegburgite, but no molecular markers confirming its origin could be found.
Balata resin from the “balata tree” (Mimusops globosa) is shown to contain squalene, β-amyrin, lupeol, cycloartenol, 24-methylenecycloactanol and the ketones corresponding to the aforementioned alcohols.
Cholest-4-en-3-one, 4α-methylcholest-7-en-3β-ol, 4α,14α-dimethylcholest-8-en-3β-ol, 31-norcycloartanol, lanost-7-en-3β-ol, cholesterol, campesterol, stigmasterol, sitosterol, stigmastanol, stigmast-22-en-3β-ol, cycloartanol, 24-methylenecycloartanol, lupeol and betulin have been isolated from resin (dragon's blood) or roots of Dracaena cinnabari and identified by capillary GC and GC-mass spectrometry.
The benzoate of trans-coniferyl alcohol and the benzoate of trans-p-coumaryl alcohol have been obtained from propolis and the styrax benzoin, this being the first time that the latter has been described.
The isolation of oleanonic acid (I) as methylester and 3-epi-oleanolic acid (II) from the resin of Liquidambar orientalis Mill. is described.
Siam Benzoin gum and coniferyl benzoate isolated from the gum were reduced with lithium aluminium hydride. In addition to the anticipated coniferyl alcohol, p-coumaryl alcohol was isolated from the phenolic fraction and it was found that the benzoate in Siam Benzoin gum contains 15 per cent p-coumaryl alcohol; sinapyl and caffeyl alcohol were shown to be absent. By use of a lithium aluminum hydride reduction coniferyl alcohol can be obtained directly from Siam Benzoin gum, whereas attempted saponification only results in polymerization of the benzoate ester.