ArticlePDF Available

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

Authors:

Abstract and Figures

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.
Content may be subject to copyright.
ANALYTICAL INVESTIGATION OF STYRAX AND BENZOIN BALSAMS 301
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 (www.interscience.wiley.com) 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.
E-mail: cathy.vieillescazes@univ-avignon.fr
Contract/grant sponsor: PACA Regional Council; Contract/grant spon-
sor: Amoroso-Waldeis Workshop of Conservation and Restoration of
Works of Art.
Phytochemical
Analysis
INTRODUCTION
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
β
,19
α
-dihydroxyolean-
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
β
,6
β
-dihydroxyolean-
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
MICHAEL HOVANEISSIAN,1 PAUL ARCHIER,1 CAROLE MATHE,1 GÉRALD CULIOLI2 and CATHERINE VIEILLESCAZES1*
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
β
-lup-20(29)-en-3-ol]
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.
302 M. HOVANEISSIAN ET AL.
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
β
-hydroxyolean-12-en-28-oic) and
3-epi-oleanolic (3
α
-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.
EXPERIMENTAL
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
dichloromethane.
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 (
δ
) relative to tetramethyl-
silane and coupling constants in Hertz.
ANALYTICAL INVESTIGATION OF STYRAX AND BENZOIN BALSAMS 303
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 (
λ
ex/
λ
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
compounds.
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.
RESULTS AND DISCUSSION
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.
304 M. HOVANEISSIAN ET AL.
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
λ
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 (
λ
ex = 330 nm/
λ
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
ANALYTICAL INVESTIGATION OF STYRAX AND BENZOIN BALSAMS 305
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 (
λ
ex = 260 nm/
λ
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.
(Hamamelidaceae).
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.
306 M. HOVANEISSIAN ET AL.
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,
ANALYTICAL INVESTIGATION OF STYRAX AND BENZOIN BALSAMS 307
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.)
308 M. HOVANEISSIAN ET AL.
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
species.
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).
Acknowledgements
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).
ANALYTICAL INVESTIGATION OF STYRAX AND BENZOIN BALSAMS 309
Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 301–310 (2008)
DOI: 10.1002.pca
Figure 8 Mass spectra of the observed triterpenoids.
REFERENCES
Acar I, Anil H. 1991. Composition of natural oriental sweet gum
(Liquidambar orientalis M.) balsam determined by combined gas
chromatography–mass spectrometry. Doga Turk Kim Derg 15: 34–
38.
Budzikiewicz H, Wilson JM, Djerassi C. 1963. Mass spectrometry in
structural and stereochemical problems. XXXII. Pentacyclic
triterpenes. J Am Chem Soc 85: 3688– 3690.
Castel C, Fernandez X, Lizzani-Cuvelier L, Loiseau AM, Périchet C,
Delbecque C, Arnaudo JF. 2006. Volatile constituents of benzoin
gums: Siam and Sumatra, part 2. Study of headspace sampling
methods. Flav Fragr J 21: 59 – 67.
Chermette M, Goyon JC. 1996. Le catalogue raisonné des producteurs
de styrax et d’oliban d’Edfou et d’Athribis de Haute Egypte. SAK
23: 54– 59.
Fernandez X, Lizzani-Cuvelier L, Loiseau AM, Périchet C, Delbecque C.
2003. Volatile constituents of benzoin gums : Siam and Sumatra.
Part 1. Flav Frag J 18: 328– 333.
Fernandez X, Castel C, Lizzani-Cuvelier L, Delbecque C, Puech Venzal
S. 2006. Volatile constituents of benzoin gums : Siam and
Sumatra, part 3. Fast characterization with an electronic nose.
Flav Fragr J 21: 439–446.
Gianno R, Erhardt D, Endt PW, Hopwood W, Baker MT. 1990.
Archaeological resins from shipwrecks off the coasts of Sarpan
and Thailand. Masca Res Papers Sci Archaeol 7: 59– 67.
Gigante B, Barros AM, Teixeira A, Marcelo-Curto MJ. 1991. Separation
and simultaneous high-performance liquid chromatographic deter-
mination of benzocaine and benzyl benzoate in a pharmaceutical
preparation. J Chromatogr A 549: 217– 220.
Hafizoglu H, Reunanen M, Istek A. 1996. Chemical constituents of
balsam from Liquidambar orientalis. Holzforschung 50: 116–117.
Hamm S, Bleton J, Tchapla A. 2004. Headspace solid phase
microextraction for screening for the presence of resins in Egyptian
archaeological samples. J Sep Sci 27: 235– 243.
Hausen BM, Simatupang T, Bruhn G, Evers P, Koenig WA. 1995.
Identification of new allergenic constituents and proof of evidence
for coniferyl benzoate in Balsam of Peru. Am J Contact Dermatitis
6: 4199– 208
Hew Kian Chong H (1998). Benjoins, styrax et storax. PhD Thesis.
University of Montpellier.
Hovaneissian M, Archier P, Mathe C, Vieillescazes C. 2006. Contribution
de la chimie analytique à l’étude des exsudats végétaux styrax,
storax et benjoin. CR Chimie 9: 1192–1202.
Huneck S. 1963. Triterpene IV. Die triterpensäuren des balsams von
Liquidambar orientalis M. Tetrahedron 19: 479 –482.
Karliner J, Djerassi C. 1965. Terpenoids. LVII. Mass spectral and
nuclear magnetic resonance studies of pentacyclic triterpenes
hydrocarbons. J Org Chem 31: 1945 –1956.
Lawrie W, McLean J, Olubajo OOO. 1970. Triterpenes from balata
resin. Phytochemistry 9: 1669 –1670.
Martin P, Archier P, Vieillescazes C, Pistre MS. 2001. HPLC coupled
with fluorimetric detection for the identification of natural resins in
archaeological materials. Chromatographia 53: 380– 384.
Masaoud M, Schmidt J, Adam G. 1995. Sterols and triterpenoids from
Dracaena cinnabari. Phytochemistry 38: 795– 796.
Mathe C, Culioli G, Archier P, Vieillescazes C. 2004. Characterization
310 M. HOVANEISSIAN ET AL.
Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 301–310 (2008)
DOI: 10.1002.pca
of archaeological frankincense by gas chromatography–mass
spectrometry. J Chromatogr A 1023: 277– 285.
Modugno F, Ribechini E, Colombini MP. 2006. Aromatic resin charac-
terisation by gas chromatography–mass spectrometry: raw and
archaeological materials. J Chromatogr A 1134: 298– 304.
Nitta A, Tani S, Sakamaki E, Saito Y. 1984. On the source and evalua-
tion of benzoin. Yakugaku Zasshi 104: 592– 600.
Pastorova I, De Koster CG, Boon JJ. 1997. Analytical study of free and
ester bound benzoic and cinnamic acids of gum benzoin resins by
GC-MS and HPLC-frit FAB-MS. Phytochem Anal 8: 63–73.
Pastorova I, Weeding T, Boon JJ. 1998. 3-Phenylpropylcinnamate,
a copolymer unit in Siegburgite fossil resin: a proposed marker for
the Hamamelidaceae. J Org Geochem 29: 1381–1393.
Popravko SA, Sokolov IV, Torgov IV. 1994. Derivatives of unsaturated
aromatic alcohols in propolis and Styrax benzoin. Khim Prir Soedin
152–160.
Reinitzer F. 1914. Untersuchungen uber Siambenzoe. I. Verfahren
zur Darstellung eines neuen krystallisierten Bestanteils der Siam-
benzoe. Arch Pharm 252: 341– 349.
Saleh MRI, Habib AAM, El-Shaer N. 1980. Spectrometric estimation of
cinnamic and benzoic acids in Tolu balsam and benzoin. J Assoc
Off Anal Chem 63: 1195–1199.
Schroeder HA. 1968. The p-hydroxycinnamyl compounds of Siam
benzoin gum. Phytochemistry 7: 57– 61.
Tayoub G, Schwob I, Masotti V, Rabier J, Ruzzier M, Viano J. 2006.
Contribution de la microscopie électronique à balayage et photoni-
que à la connaissance de l’anatomie et de la morphologie de Styrax
officinalis L. CR Biologies 329: 712–718.
Tchapla A, Bleton J, Goursaud S, Méjanelle P. 1999. Contribution à la
connaissance des substances organiques utilisées en Egypte
ancienne. L’apport de techniques physico-chimiques d’analyse. In
Encyclopédie religieuse de l’Univers végétal. Croyances phytor-
eligieuses de l’Egypte ancienne, Vol I> Orientalia Monspeliensia X:
Montpellier; 445– 487.
Vieillescazes C, Coen S. 1993. Caractérisation de quelques résines
utilisées en Egypte ancienne. Stud Conserv 38: 255–264.
Villa C, Gambaro R, Mariani E, Dorato S. 2007. High-performance
liquid chromatographic method for the simultaneous determina-
tion of 24 fragrance allergens to study scented products. J Pharm
Biomed Anal 44:755–762
Wahbi AAM, Abounassif MA, Gad-Kariem ERA, Ibrahim ME. 1987.
Liquid chromatographic determination of cinnamic and benzoic
acids in benzoin preparations. J Assoc Off Anal Chem 70: 689–
691.
Wang F, Hua H, Pei Y, Chen D, Jing Y. 2006. Triterpenoids from
the resin of Styrax tonkinensis and their antiproliferative and
differentiation effects in human leukemia HL-60 cells. J Nat Prod
69: 807– 810.
... In 2017, Styrax sumatrana covered up to 23,068 ha with a total production of 8,332 tones [2]. The main product of kemenyan is benzoid resin, a non-timber forest product locally known as kemenyan resin with a high economical value [3], and is widely used in various ritual ceremonies, as well as being used for cosmetic and medicinal purposes [4,5]. ...
... The local Grade I of kemenyan resin obtained from a community forest in North Sumatera, Indonesia has the highest purity and balsamic acid content as well as lower ash content, melting point, and moisture content [7]. It also has been reported that the kemenyan resin from North Sumatera mainly comprises of cinnamic acid and ethyl cinnamate, which indicates a high quality of the resin as compared to the resin obtained from other places [5]. The kemenyan resin is commonly harvested from the trees by a tapping process. ...
Article
Full-text available
Styrax sumatrana or the kemenyan tree grows in North Sumatera, Indonesia, and its resin is commonly utilized by the local community. Other parts of kemenyan, such as barks and fruits contain valuable compounds that can be extracted to produce high-value bioproducts. This study examined the effect of different resin grades on the physical parameters and cinnamic acid content, delignification pre-treatment period of kemenyan barks with Phanerocahete chrysosporium on the amount of extracted saponin from the barks, and different fruit ripeness on the composition of the fruits. This study showed that grade IV, V and VI resin contain 21.78 to 24.89% cinnamic acid. The isolated cinnamic acid had a purity of 90.9 to 94.3%. Pre-treatments of kemenyan barks were able to degrade 15% of the lignin after 21 days of incubation with Phanerochaete chrysosporium. and increased the amount of extracted saponin up to 7.5-fold higher compared to the non-pre-treated barks. Ripe kemenyan fruits had a higher protein (4.27-10.23%) and crude fat (0.91-7.36%) content as compared to the unripe fruits. Fatty acid composition of the crude fat had been determined and primarily consists of linoleic acid (31.71-42.33%) and palmitic acid (30.44-30.82%) for both ripe and unripe fruits.
... There have been many studies on bioactive [2], therapeutic effect [3]. The chemical compositions of Benzoin resin are Benzoic acid, Cinnamic acid, Vanillin, Benzyl benzoate, Cinnamyl cinnamate, Benzyl cinnamate, Coniferilic alcohol, and Siaresinolic acid [4,5,6]. ...
Article
Full-text available
Today, fixatives from natural raw materials are gradually replacing fixatives synthesized from chemicals. The arm of this research is the study of same useful resin in Vietnam for fixative substance in blending fragrance. We obtained Benzoin resin from Styrax tonkenensis Pierre plant in Ha Giang province, Canarium resin from Canarium Album L. in Dak Nong district, Dak Lak province, and Agarwood pulp of Aquilaria crassna plant in Binh Thuan province Vietnam. The material used in the experiment for the natural fragrance was taken from the project of Vietnam essential oils and related natural products. The method of this process is resin extraction by volatile solvents. The resin is dissolved in alcohol 96% and the distilled alcohol is removed to obtain absolute. The method of assessment of product quality in this study is using the olfactory to assess the odour of samples over time. Benzoin resin, Canarium resin, and Agarwood resin of Vietnam are useful fixatives in blending fragrance. The fixative ability of Benzoin resin absolute is not equal to the ability of Agarwood resin absolute but better than the ability of Canarium resin absolute. Through research and experiment, we can see Benzoin resin, Canarium resin and Agarwood resin are precious. They can be used as a good fixative in aromatherapy. This is a natural resin, a kind of resource available in Vietnam. Therefore, it is recommended for further research, exploitation, and effective use of this resource.
Article
Full-text available
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.
Article
Full-text available
Non-Timber Forest Products (NTFPs) management can lead to various benefits for community livelihood and forest sustainability. However, such management has not been carried out optimally and sustainably in Indonesia, due to various limiting factors including ineffective policies, undeveloped cultivation technologies, and inadequate innovation in processing technologies. Further, the diversity of NTFPs species requires that policy-makers determine the priority species to be developed. Agarwood (Aquilaria spp. and Gyrinops spp.), benzoin (Styrax spp.), sandalwood (Santalum album L.), and cajuput (Melaleuca cajuputi Powell) are aromatic NTFPs species in Indonesia that forest-dwellers have utilized across generations. This paper reviews the current governance, cultivation systems, processing and valuation, and benefits and uses of these species. We also highlights the future challenges and prospects of these NTFPs species, which are expected to be useful in designing NTFPs governance, in order to maximize the associated benefits for the farmers and all related stakeholders.
Chapter
Solanum is one of the largest genera of the family Solanaceae comprising > 2000 species distributed mostly in the tropical and subtropical regions of Australia, Africa, and some parts of Asia, such as China, India, and Japan. The nutraceutical and pharmaceutical values of the Solanum species are due to the presence of bioactive phyto-constituents such as steroidal saponins, steroidal alkaloids, terpenes, flavonoids, lignans, sterols, phenolic compounds, coumarins, etc. Among them, the presence of steroidal alkaloids and glycoalkaloids serves as major chemical markers of this genus. Steroidal alkaloids and glycoalkaloids have a special status in traditional and modern systems of medicine possessing a wide range of bioactivities, viz., antimicrobial, analgesic, hepatoprotective, immunomodulatory, anticancer, neurogenetic, etc. Steroidal alkaloids (STAs) are the major class of secondary metabolites found not only in plants but also in higher animals as well as in some aquatic invertebrates. They have a steroidal (cyclopentanophenanthrene) backbone skeleton with a nitrogen atom. The biosynthesis of these alkaloids takes place from steroids or triterpenoid pathway, on the basis of which they are further divided into different classes and subclasses. The present review is focused on the occurrence and biosynthesis of steroidal alkaloids in Solanaceae family. These compounds are mainly triterpene-derived molecules that are involved in various defense responses participating also in formulations of a wide range of phyto-pharmaceuticals. The addition of sugar moieties to the base skeleton by glycosyltransferases resulted in the formation of steroidal glycoalkaloids (STGAs), possessing a wide range of pharmacological values. The accumulation of these bioactive metabolites has been shown to be highly influenced by environmental and geographical factors. Hence, their production via tissue culture always offers an attractive alternate production platform. The current trends and biotechnological tools recently developed for the sustainable production and up-scaling of these bioactive constituents are focused in the present review.
Chapter
Full-text available
Genus Styrax belongs to the family Styracaceae, which is widely distributed in tropical and subtropical regions and has been applied in pharmacological uses and food chemical products. Original research articles related to Styrax plants are now abundant, but there has not been an overview account to demonstrate the highlights in phytochemical and pharmacological aspects of Styrax constituents. This chapter compiles a full list of secondary metabolites from this genus, along with their pharmacological effects. Herein, 165 isolated compounds are summarised with diverse chemical structures, and lignans and triterpenoids can be seen as major components. Pharmacological studies have introduced the use of Styrax components in anticancer, antioxidant, anti-inflammation, antimycobacterial, antiaging, antivirus HIV, and antischistosomicidal activities, estrogenic biosynthesis, etc. Regarding food chemistry, several Styrax plants can also be good candidates to provide essential oils and nutrient content of protein, especially benzoin resins.
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
The isolation of oleanonic acid (I) as methylester and 3-epi-oleanolic acid (II) from the resin of Liquidambar orientalis Mill. is described.
Article
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.