Food Chemistry 138 (2013) 161167

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Food Chemistry
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Exploration of Vanilla pompona from the Peruvian Amazon as a potential source

of vanilla essence: Quantication of phenolics by HPLC-DAD
Helena Maruenda a,, Maria del Lujan Vico a, J. Ethan Householder b, John P. Janovec b, Cristhian Caari a,
Angelica Naka a, Ana E. Gonzalez a
a
b

Ponticia Universidad Catolica del Peru, Departamento de Ciencias Quimica, Av. Universitaria 1801, Lima 32, Peru
Botanical Research Institute of Texas (BRIT), 1700 University Drive, Fort Worth, TX 76107-3400, USA

a r t i c l e

i n f o

Article history:
Received 7 April 2012
Received in revised form 27 August 2012
Accepted 1 October 2012
Available online 8 November 2012
Keywords:
Vanilla pompona
HPLC-DAD quantication
Glucovanillin
Phenolic prole
Peruvian wetlands

a b s t r a c t
This study provides the rst chemical investigation of wild-harvested fruits of Vanilla pompona ssp. grandiora (Lindl.) Soto-Arenas developed in their natural habitat in the Peruvian Amazon. Flowers were
hand-pollinated and the resulting fruits were analysed at different developmental stages using an
HPLC-DAD method validated for the quantication of glucovanillin and seven other compounds. The
method showed satisfactory linearity (r2 > 0.9969), precision (coefcient of variation <2%), recoveries
(70100%), limit of detection (0.0080.212 lg/ml), and limit of quantication (0.0270.707 lg/ml). The
evaluation of crude and enzyme-hydrolyzed Soxhlet-extracted samples conrmed the leading role of glucosides in fruit development. LCESI-MS studies corroborated the identities of four glucosides and seven
aglycones, among them vanillin (5.7/100 g), 4-hydroxybenzyl alcohol (3.6/100 g), and anisyl alcohol (7.1/
100 g) were found in high concentrations. The attractive avor/aroma prole exhibited by wild V. pompona fruits supports studies focused on the development of this species as a specialty crop.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Natural vanilla, a unique spice of great commercial value on the
international market, is borne from the fruit of Vanilla planifolia Andrews (syn. Vanilla fragans) (Orchidaceae), Vanilla tahitensis J.W.
Moore (commonly referred to as Java), or Vanilla pompona Schiede.
These vanilloid orchids produce large owers that are ephemeral
and, in commercial settings, require pollination by hand. Subsequent development of the fruit into a mature and harvestable fruit
takes about seven to nine months. Upon harvest, the fruits undergo
a long curing process (58 months) to nally become the avorful
and scented product. All of these laborious steps contribute to the
high price of vanilla, known to have reached about $500 per kg in
the last decade (Brownell, 2011). Each year approximately 2000
3000 tons are used commercially by the cosmetic, avoring, and
food industries. Madagascar, despite the crises experienced during
the last two decades, is still the main producer (Brownell, 2011),
while, France, Germany, Japan, and the United States are the main
consumers.
Vanilla fruits are chemically complex. More than 300 compounds have been identied from cured Vanilla fruit extracts (Toth,
Lee, Havkin-Frenkel, Belanger, & Hartman, 2011), and that number
continues to rise. Twenty-ve phenolic compounds with concen Corresponding author. Tel.: +51 1 6262000x4226; fax: +51 1 626 2853.
E-mail address: hmaruen@pucp.edu.pe (H. Maruenda).
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.10.037

trations higher than 1 ppm (Brodelius, 1994) have been pinpointed

as responsible for the characteristic aroma and avor of vanilla.
Among them, the most frequently discussed are vanillin (1),
vanillyl alcohol (2), vanillic acid (3), 4-hydroxybenzyl alcohol (4),
4-hydroxybenzaldehyde (5), 4-hydroxybenzoic acid (6), anisyl
alcohol (7), anisaldehyde (8), and anisic acid (9), whose structures
are illustrated in Fig. 1. These compounds tend to accumulate in
the fruits as odourless glucosides (Brodelius, 1994; Havkin-Frenkel
et al., 2004). Their hydrolysis carried out by b-glucosidases released during early stages of the curing process yield the key aromatic aglycones, a logical reason why the majority of prior
chemical studies on vanilla have been oriented to the cured fruit.
Because of the irrefutable contribution of vanillin to vanilla avor
and aroma, the hydrolysis of glucovanillin (GV, structure depicted
in Fig. 1) during the various stages of the curing process (Dignum,
Kerler, & Verpoorte, 2002; Havkin-Frenkel et al., 2004; Odoux,
2000; Sreedhar, Roohie, Venkatachalam, Narayan, & Bhagyalakshmi,
2007), as well as the biosynthetic pathway of glucovanillin
(Kanisawa, 1993; Negishi, Sugiura, & Negishi, 2009), have been
thoroughly discussed.
The presence of the aforementioned aromatic compounds 19
in cured fruits of V. planifolia and V. tahitensis has been conrmed
using various analytical approaches with different degrees of sensitivity (Cicchetti & Chaintreau, 2009; Da Costa & Pantini, 2006;
Sharma, Sharma, Gupta, Kumar, & Sinha, 2007; Sinha, Sharma, &
Sharma, 2008; Sinha, Verma, & Sharma, 2007). In general, the

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H. Maruenda et al. / Food Chemistry 138 (2013) 161167

In this paper, the aromatic chemical composition of wild V.

pompona ssp. grandiora fruits, over three and a half to nine
months of development, is reported. The quantication was based
on High-Performance Liquid Chromatography with Diode Array
Detection (HPLC-DAD) methodology. Liquid chromatography electrospray ionisation mass spectrometry (LCESI-MS) was used to
assist in the identication of all compounds. This is the rst comprehensive chemical investigation of V. pompona fruits at different
stages of development as well as the rst analytical study dedicated to a biologically characterised wild Amazonian Peruvian Vanilla species.

2. Materials and methods

2.1. Plant material and chemicals

Fig. 1. Chemical structures of GV and phenolic compounds (19).

results indicate that vanillyl (13) and benzyl (56) derivatives are
key components of cured V. planifolia fruits. Vanillin is found in
higher concentrations (between 2 and 2.5/100 g dry weight) than
the other aglycones 26 (Brodelius, 1994; Cicchetti & Chaintreau,
2009; Gassenmeier, Riesen, & Magyar, 2008; Odoux, 2000; Odoux,
2011; Sinha et al., 2007; Toth et al., 2011). The importance of compounds 1, 3, 5 and 6 in dening the avor and aroma of vanilla essence is apparent from the role they play as markers in the
authenticity and quality control of natural V. planifolia products
(Gassenmeier et al., 2008; Jhon & Jamin, 2004). In contrast to the
chemical prole exhibited by V. planifolia fruits, in cured fruits of
V. tahitensis, the sweet sensory impact of compounds 13 is
blended with oral notes exerted by the anisyl derivatives (79;
Ranadive, 2011). Three compounds are found in considerable
amounts: vanillin (1.31.5/100 g dry weight), anisyl alcohol (1.4/
100 g dry weight), and anisic acid (0.8/100 g dry weight)
(Leper-Andrzejewski, Brunschwig, Collard, & Dron, 2011).
While the composition of the fruits of V. planifolia and
V. tahitensis is well documented, the chemical prole of V. pompona
fruits has received scant attention. This could be due to the inaccessibility of V. pompona fruits or to the fact that they have been
described as carrying signicantly less vanillin than the fruits of
the other two species (Ehlers & Pster, 1997; Ranadive, 2011; Toth
et al., 2011). However, most of the information available is based
on cured fruits of V. pompona of unknown origin.
Wild V. pompona ssp. grandiora has recently been reported as
the most abundant of six Vanilla species present in wetland ecosystems of Madre de Dios, a region within the southern Peruvian Amazon (Householder et al., 2010). Early eld observations established
that the large fruits of wild populations of V. pompona ssp. grandiora exhibited strong aromatic and avorful properties. This suggested the hypothesis that V. pompona ssp. grandiora of the
Peruvian Amazon could represent a new potential source of vanilla
essence. The complete lack of knowledge regarding the chemistry
of Amazonian Vanilla species in comparison with those from other
regions of the world was surprising. As a consequence, wild populations of V. pompona ssp. grandiora were selected from their natural wetland habitat to serve as sources of properly identied fruits
ideally suited for chemical analysis. The objective at this early
stage of the program was to determine if these wild fruits had
any value in terms of aromatic chemical content, and if so, to identify the month at which the chemical prole yield was optimal.

Flowers of V. pompona ssp. grandiora (Lindl.) Soto-Arenas were

hand pollinated in October 2008 at selected wetland ecosystems of
Madre de Dios, Peru. For a description of eld research methods
and the habitats, see Householder et al. (2010). Fruits were harvested during months three and half to nine after pollination. Herbarium voucher specimens (MC366, MC684, MC599, MC660,
MC296, PM865 and JJ2671) were collected in duplicate sets and
deposited at the San Marcos Herbarium in Lima, Peru, and at the
BRIT Herbarium in Fort Worth, Texas. Specimens were identied
by the late neotropical orchid experts E.A. Christenson and Miguel
Soto-Arenas (Householder et al., 2010). Details of these collections
are posted in the Atrium Biodiversity Information System, programmed and managed at BRIT (http://atrium.andesamazon.org).
The standard compounds vanillin, vanillyl alcohol, vanillic acid,
4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, and anisyl alcohol, as well as Viscozyme (V2010, lot #
085K1587) and Celluclast (C2730, lot # 074K1156), were purchased from SigmaAldrich. Glucovanillin was obtained from
Chromadex. Methanol (HPLC-grade) and ethanol (99.9%) were purchased from J.T. Baker, Co. The water used was Milli-Q-puried.
The studies were performed with three individual fruits per month,
except for month nine, in which six fruits were considered.
2.2. Humidity determination
Upon arrival to the laboratory, no more than two days after harvest, the fruits were frozen in liquid nitrogen and immediately
lyophilized (LABCONCO, Freeze Dry System/Freezone 4.5) for 3
5 days. Humidity is expressed as percentages [(wwet  wdry/
wwet)  100].
2.3. Vanilla sample preparation
Individual lyophilized fruits were ground to a ne powder using
an electric grinder (SigmaAldrich, model Z278181). Two grams of
the ground material was Soxhlet-extracted with 48% aqueous EtOH
(200 ml) for 24 h (Voisine, Carmichael, Chalier, Cormier, & Morin,
1992). The ethanol was removed by vacuum evaporation
(150 mBar, 40 C) and the aqueous, turbid solution was taken to
50 ml in a volumetric ask. Hydrolysis of the glucosides was
achieved by incubating 10 ml of the extract with 150 ll of Viscozyme and 300 ll of Celluclast at 45 C for 8 h (Inkubator 1000/Unimax 1010, Heildolph Instr.). Ethanol (4.5 ml) was added and the
process continued for 1 h to yield the hydrolyzed fraction. The
non-hydrolyzed portion followed the same procedure except for
the enzyme addition. The volume of both samples was adjusted
to 15 ml prior to HPLC analysis. The experiments were performed
in duplicate. The vanilla extract was passed through a 25 mm PTFE

H. Maruenda et al. / Food Chemistry 138 (2013) 161167

0.45 lm syringe lter prior to injection (10 ll) into the HPLC system. Each extract was evaluated in triplicate.
2.4. HPLC analysis
The samples were analysed on an Agilent 1200 series DAD
equipped with a binary pump unit (G1312A), UVVIS detector
(G1315D), degasser (G13798), column oven (G1316A), a 20 ll loop
manual injector (G1328B) controlled by Chemstation software
LC3D (Agilent Technologies, Inc.). The column used was a Cromolith RP 18e (100  4.6 mm, Merck-Darmstadt), and the mobile
phase a mixture of two solvents: A (1  103 M K PO4, pH 3.1)
and B (MeOH). Elution was achieved at 30 C with a gradient of
37% B in 2 min (1.0 ml/min), 79% B in 10 min (1.02.0 ml/min),
and 919% B in 7 min (2.0 ml/min). The compounds were monitored at: 230 nm for GV, vanillin, and alcohols 2, 4, and 7;
254 nm for vanillic acid and 4-hydroxybenzoic acid; and 280 nm
for 4-hydroxybenzaldehyde. The injection volume was 10 ll, and
all samples were ltered through a PTFE 0.45 lm syringe lter
(Millipore, Germany) prior to analysis. The LCESI-MS experiments
were performed on a Bruker Daltonics Esquire 6000 controlled by
Compass 1.3 for Esquire/HCT software (Bruker Daltonik GmbH).
The HPLC system was as described for the HPLC-DAD. The gradient
separation was carried out at 35 C using a volatile mobile phase
composed of solvents A (4.2  103 M Formic acid, pH 3.1) and B
(MeOH), following the same gradient as above but at a ow rate
of 1 ml/min. After reaching 19% B it was held isocratic for
16 min. The injection volume was 7 ll. The ESI-MS operating
conditions were: positive ionisation mode, drying gas (N2) ow,
12 l/min; nebulizer pressure, 65 psi; gas drying temperature,
350 C; capillary voltage 4000 V; scan mode m/z, 90800. ESI-MS
and UV-DAD spectra of authentic standards, 17 and GV, were recorded and used to corroborate identity. The semipreparative
HPLC purication was conducted with a Cromolith RP 18e
(100  10 mm, Merck Darmstadt) using the conditions established for the LCESI-MS run, but with a ow rate of 2.5 ml/min.
The lyophilized extract (75 mg), dissolved in 1.5 ml of 30% aqueous
ethanol, was injected through a 2 ml loop. Fraction collection was
guided by retention time and UV spectra of the eluate.
2.5. Method validation
The HPLC method was validated according to ICH requirements
(ICH, 1996). The external calibration curves were prepared with
solutions containing compounds 17 in concentrations between
0.167 and 992.8 lg/ml. In the case of GV the range was 0.864
1035 lg/ml. The linearity was checked by regression analysis of
at least six concentrations of each compound. Repeatability was
conrmed by performing injections on the same day (intra-day
precision, n = 5) at a given concentration of each compound. The
intermediate precision was calculated over ve different days
(inter-day) at an established concentration per substance. Highly
diluted solutions of each compound were used to achieve signalto-noise (S/N) ratios of 3 (limit of detection, LOD) and 10 (limit
of quantication, LOQ). The recovery of the complete analytical
protocol was evaluated by the enrichment of vanilla samples
(2 g) with 50 mg of compounds 1, 4, and 7, and 10 mg of 2, 3, 5,
and 6. The recovery was expressed as the percentage of the total
amount recovered. The process was repeated in triplicate. The
fruits used for this study were nine-month old.
2.6. Statistical analysis
Statistical analysis of the data was carried out using IBM SPSS
Statistics Version 19.0 (SPSS, Inc. Chicago, IL) and the multivariate
analysis was performed using MATLAB software Version 7.11

163

(Matworks, Inc., Natick, MA). The PCA analysis was carried out
using standardized variables.
3. Results and discussion
3.1. Humidity
The average humidity of intact V. pompona ssp. grandiora
fruits, three and a half to nine months old, is presented in Table 1.
The observed increment of dry weight through fruit maturation
has been noted earlier with V. planifolia fruits (Brodelius, 1994).
The size of the 24 fruits used to calculate humidity ranged from
10 to 25 cm in length and 24 cm in width.
3.2. HPLC-DAD validation
The HPLC method developed for this study achieved adequate
separation among the eight standards, GV and 17, in 19 min,
Fig. 2A. The linear regression parameters obtained for all eight calibration curves were optimal in the concentration range established for each compound (Table 2). The same could be argued
for the sensitivity of the method, with LOD values in the range of
0.0080.212 lg/ml and LOQ of 0.0270.707 lg/ml. In terms of linear range, sensitivity, and HPLC run-time, the results are comparable to those achieved by two other validated HPLC methods
available in the literature for the quantication of compounds 1
6 (Cicchetti & Chaintreau, 2009; Sinha et al., 2007). However, this
is the rst validated HPLC method to assess GV and anisyl alcohol
in the presence of aglycones 16. A good linearity was achieved for
both of these compounds, GV and compound 7, over a wide concentration range, 11000 lg/ml, Table 2. Precision (retention time
and peak area) was addressed through intra-day and inter-day
repetitive analysis (n = 5) at four different concentrations per compound. In general, the results were satisfactory, displaying coefcient of variation (CV) below 2%, as shown in Table 3 for a
particular concentration of each standard.
Accuracy of the proposed method was tested only for the free
aglycones 17, since the extraction protocol followed has already
been reported as optimal for the isolation of GV (Voisine et al.,
1992). The recoveries for the experiment were high (>97%) for almost all aglycones and, acceptable (>70%), for compounds 2 and
4 (Table 4), suggesting that, overall, the validated method is suitable for quantication of vanillin and related phenolic compounds.
3.3. Quantitative determination of GV and aglycones 17 present in
vanillons at different stages of development
The presence of GV and phenolic compounds 17 in extracts of
V. pompona ssp. grandiora was demonstrated through comparisons of their HPLC-retention times, UV-DAD proles, and ESI-MS
spectra against that of authentic samples. The alcohols were identied through the ion [M+HH2O]+, whereas all others showed the
[M+H]+ signal. In the case of GV, its structure was veried through
the presence of m/z 353, 337 and 153, corresponding to the ions
[M+K]+, [M+Na]+ and [M+1162]+, respectively. The latter ion fragment corresponds to loss of glucose.
The HPLC-quantitative study was performed on twenty-four
green intact fruits at seven different stages of maturation. The
quantities of GV and free and total aglycones 17, were calculated
based on the amount present on enzyme-treated and non-treated
extracts. The results, plotted against time after pollination, are
shown in Fig. 3. The data suggest that vanillin and the other six
phenolic compounds accumulate in the fruit as both glucosides
and free forms throughout maturation. This result coincides
with previous observations (Brodelius, 1994). In the case of

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H. Maruenda et al. / Food Chemistry 138 (2013) 161167

Table 1
Average humidity (H) of Vanilla pompona ssp. grandiora fruits at different stages of development.
Month
a

H(%) SD
a
b
c

91.3 0.5

86.3 1.6

83.6 1.3

82.8 0.8

79.4 1.6

80.6 0.4

76.7 2.0c

Humidity is expressed as percentages [(wwet  wdry/wwet)  100]. The values are average of three fruits unless otherwise indicated.
SD, standard deviation.
The value is an average of six fruits.

Fig. 2. Representative HPLC chromatogram at 230 nm of (A) standard compounds

GV and 17, (B) crude vanilla extract (B) and (C) enzyme-treated vanilla extract. The
numbers 30 , 40 and 70 represent the glucosides of the respective aglycones.

4-hydroxybenzyl alcohol, 4-hydroxybenzoic acid, and anisyl

alcohol, it is clear, based on the ratio of free/total aglycone, that
these compounds exist mainly as glucosides throughout fruit
development. For all other compounds, the glucoside form begins
to predominate towards the end of maturation. These ndings support unsubstantiated data, recently referred to by Lapeyre-Montes,
Conejero, Verdeil, and Odoux (2011), which were associated with
the evolution of vanillin/glucovanillin during V. planifolia fruit
development. Only three glucosides, in addition to GV, were recognised by comparing the HPLC-DAD chromatograms of crude and
enzyme-treated extracts (Fig. 2B and C). These were glucosides of
3, 4, and 7 that appeared as broad peaks at retention times of
6.26.4 min (30 ), 3.94.3 min (40 ), and 16.917.4 min (70 ). These
glucosides have been reported earlier in V. planifolia extracts
(Dignum, van der Heijden, Kerler, Winkel, & Verpoorte, 2004;
Kanisawa, 1993; Odoux, 2011) and in order to unequivocally
conrm their presence in extracts of V. pompona ssp. grandiora,
an LCMS study was conducted. The ions m/z [M+K]+, [M+Na]+,
and [M+H-162]+ in the MS spectra corroborated their glucosidic
nature (see Supplementary data). In addition, and due to the
unusual behaviour exhibited by 70 glucoside eluting after the
aglycone this compound was isolated by semipreparative HPLC.
The NMR data (1H NMR, 13C NMR, COSY and HSQC) agree with that
reported in the literature (Itokawa, Oshida, Ikuta, Inatomi, &
Adachi, 1982; Kitajima et al., 1998). Glucosides of the other minor
constituents, 2, 5 and 6, were not identied possibly because of
signal loss due to broadening or overlapping.
The ndings also indicate that 4-hydroxybenzyl alcohol is the
most prominent compound present in three and a half month old
fruits of V. pompona spp. grandiora (311 lmol/g dry weight,
Fig. 3). Its concentration decreased over time, while the amount
of the other vanillyl compounds gradually increased. A similar variation pattern, including the minor increment noted in month six
for 4-hydroxybenzyl alcohol was demonstrated previously in
developing fruits of V. planifolia using HPLC (Kanisawa, 1993) and
through an NMR study (Palama et al., 2009). As opposed to the
behaviour observed in V. planifolia (Kanisawa, 1993; Palama
et al., 2009), the production of 4-hydroxybenzyl alcohol in
V. pompona ssp. grandiora fruits increased again after month seven, reaching values as high as 3.6/100 g dry weight in the fully
matured fruit. The leading role of the glucoside of 4-hydroxybenzyl
alcohol in the biosynthesis of GV has been noted earlier (Kanisawa,
1993). Based on the results shown in Fig. 3, this role is conrmed in
V. pompona spp. grandiora fruits.
It is also clear from Fig. 3 that production of glucovanillin in the
fruit starts as early as the third month and continues throughout
the ninth. The maximum amount quantied for GV was
315 lmol/g dry weight, a value that corresponds to 9.9/100 g dry
weight, similar to the levels achieved by V. planifolia fruits (Palama
et al., 2009). An interesting observation derived from these curves
is that vanillin production in V. pompona spp. grandiora fruits occurs at a slower pace during the period 3.58 months (20 lmol/g
per month) compared to the almost constant rate observed in V.
planifolia fruits (68 lmol/g per month). It is only during the ninth
month that the concentration of vanillin in V. pompona spp. grandiora fruits increases sharply from 100 to 375 lmol/g dry weight,

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H. Maruenda et al. / Food Chemistry 138 (2013) 161167

Table 2
Calibration curves of analytes in study and sensitivity of the HPLC-DAD method.
Concentration range (lg/ml)

Compound

4-Hydroxybenzyl alcohol
4-Hydroxybenzoic acid
4-Hydroxybenzaldehyde
Glucovanillin
Vanillin
Vanillyl alcohol
Vanillic acid
Anisyl alcohol
a
b
c

Linear regression

1.20287
0.17126
0.34255
0.861035
0.26501
0.56416
0.23176
1.06993

LODb (lg/ml)

LOQc (lg/ml)

0.212
0.008
0.020
0.196
0.048
0.157
0.073
0.053

0.707
0.027
0.065
0.654
0.159
0.524
0.244
0.177

2a

y = mx + b

2158.5x  3.7
5372.5x  10.6
4981.8x  25.9
3511.9x  120.5
3636.4x  8.3
2334.4x  8.5
2899.4x  12.8
2186.7x  3.3

0.9999
0.9999
0.9999
0.9969
1.0000
0.9999
0.9998
0.9998

Correlation coefcient.
LOD = limit of detection (S/N = 3).
LOQ = limit of quantication (S/N = 10).

Table 3
Precision of the retention time and peak area of analytes in the HPLC-DAD method used.
Compounda

Retention time (min)

4-Hydroxybenzyl alcohol
4-Hydroxybenzoic acid
4-Hydroxybenzaldehyde
Glucovanillin
Vanillin
Vanillyl alcohol
Vanillic acid
Anisyl alcohol
a

5.34
8.17
9.93
8.77
14.29
7.41
11.43
15.10

Intra-day precision (CV%, n = 5)

Inter-day precision (CV%, n = 5)

Retention time

Peak area

Retention time

Peak area

0.105
0.083
0.058
0.423
0.073
0.071
0.057
0.222

1.948
1.806
1.836
1.777
1.422
1.867
1.510
0.745

0.228
0.573
0.361
0.067
0.095
0.100
0.297
0.190

1.177
1.365
1.869
1.976
1.912
1.908
1.277
0.279

Concentration of GV was 1.65 mM, anisyl alcohol 0.76 mM; all other analytes in the range 0.140.44 mM.

Table 4
Recovery of the method used for the determination of compounds 17 in vanilla fruits
(n = 3).

Compound

Spike (mg)

Recovery (%)

RSDa (%)

4-Hydroxybenzyl alcohol
4-Hydroxybenzoic acid
4-Hydroxybenzaldehyde
Vanillin
Vanillyl alcohol
Vanillic acid
Anisyl alcohol

50
10
10
50
10
10
56

72.7
100.8
101.8
96.5
70.4
100.3
93.7

3.32
2.65
2.54
4.05
1.48
2.98
0.32

Relative Standard Deviation.

reaching levels reported for eight-month old V. planifolia fruits,

340 lmol/g dry weight (Palama et al., 2009). A similar steep rise,
nearly one order of magnitude lower, is exhibited by the other
two vanillyl derivatives, 2 and 3. Studies were not continued beyond nine months of development because, by that time, most
fruits in the wild habitat begin to dehisce and turn brown on the
vine. However, it is possible that fruits from vanilla cultivars, harvested after nine months, could contain greater quantities of these
signature compounds.
Vanillyl compounds 2 and 3 were found in higher concentrations when compared to the benzyl derivatives 5 and 6. All of
these, with the exception of vanillyl alcohol (2), are present in uncured V. planifolia fruits (Palama et al., 2009) in larger amounts.
The only anisyl derivative observed in the uncured fruits of V.
pompona spp. grandiora was 4-methoxybenzyl alcohol, commonly
known as anisyl alcohol (7). This compound has been detected in
amounts up to 516 lmol/g of dry weight (Fig. 3), a value two to
four times greater than that reported to be present in extracts of
uncured fruits of V. tahitensis (Leper-Andrzejewski et al., 2011).
Table 5 shows the concentration range observed for GV and the
seven key aromatic compounds detected in nine-month old

uncured fruits of V. pompona spp. grandiora compared with the

composition of uncured fruits of V. planifolia and V. tahitensis. It
is apparent from such data that V. pompona spp. grandiora fruits
may be related to V. planifolia fruits in terms of overall vanillyl
(responsible of the sweet notes of vanilla) and benzyl (associated
with perfumed oral notes) contents and to V. tahitensis fruits
through the contribution of anisyl alcohol.
Principal Component Analysis (PCA) was used to analyse the
HPLC-data obtained for the 24 enzyme-treated extracts studied,
as well as GV content and humidity of each fruit. The loadings
and scores along the rst two axes are displayed in the biplot
exhibited in Fig. 4. The primary axis contrasts the concentration
of GV and the total free content of vanillyl and anisyl derivatives
with humidity. Through these variables, the differentiation of the
developmental stages of the fruits becomes possible. The contribution of PC2 is strongly inuenced by the composition of the benzyl
derivatives. It is also evident from the PCA that months ve to eight
after pollination do not show a dramatic variation in composition,
whereas the transition from month eight to nine is characterised
by a noticeable change in vanillyl compounds 13 and in anisyl
alcohol. The PCA factors explain over 75% of the total variance.
In conclusion, a fast and new validated HPLC - DAD method was
developed in order to detect compounds 16 in the presence of GV
and anisyl alcohol in developing fruits of V. pompona ssp. grandiora from the Peruvian Amazon. The precision, recovery, linearity,
and sensitivity values obtained suggest that the method is suitable
for quantication. Aglycones and glucosides were present during
all of the developmental stages after pollination, an important fact
for an improved understanding of the biosynthetic pathway of vanillin in developing fruits. The most abundant compound found
after three months of fruit maturation was the glucoside of
4-hydroxybenzyl alcohol. On the other hand, it is apparent that
glucovanillin is produced massively in the developing fruit during
the last month of maturation. We hypothesise that the nal

166

H. Maruenda et al. / Food Chemistry 138 (2013) 161167

Fig. 3. Free () and total (-) aglycone content in fruits of V. pompona ssp. grandiora at different developmental stages. Error bars indicate standard error.

Table 5
Contents of GV and compounds 19 expressed in g/100 g dry weight present in
uncured V. planifolia, V. tahitensis and Peruvian V.pompona ssp. grandiora fruits.
Compound

Vanilla planifoliaa

Vanilla tahintensisb

Vanilla pomponac

GV
4
5
6
1
2
3
7
8
9

8.51
0.87
0.40
0.11
5.17
0.17
0.30

Not reported

0.21
0.60
2.02
0.06
0.02
2.11
0.01
0.79

5.869.88
1.733.55
0.010.03
0.010.02
2.665.71
0.220.61
0.050.14
4.057.13
NDd
ND

Values converted from lmol/g dry weight (Palama et al., 2009).

Values converted from ppm (Leper-Andrzejewski et al., 2011).
c
Concentration range observed in the evaluation of six individual nine-monthold fruits.
d
Not detected.
b

development stage from month nine to ten could be characterised

by a much greater increase in this important compound. As a
whole, the chemical prole of wild uncured fruits of V. pompona
ssp. grandiora is markedly different from those of the other two
commercial Vanilla species, specically in the simultaneous high
content of vanillin, 4-hydrobenzyl alcohol, and anisyl alcohol.
The results suggest that wild V. pompona ssp. grandiora from
the wetlands of Madre de Dios are good candidates as gene donors
for the improvement of existing Vanilla cultivars or the adoption of
alternative ones. Propagation and plantation experiments focused
on V. pompona ssp. grandiora in ex-situ settings in the region are
currently being conducted.

Fig. 4. Principal component analysis using GV content, total free aglycone 17

composition, and humidity data of vanilla samples at different developmental
stages. 1: Van, 2: Van Alc, 3: Van Ac, 4: PhB Alc, 5: PhB, 6: PhB Ac, 7: Anis Alc, H:
humidity.

Acknowledgements
The authors thank Jason Wells, Javier Huinga, Angel Balarezo
and Manuel Huinga for assisting with the pollination of Vanilla
owers and harvest of the fruits. We owe much appreciation to
Renan Valega of BRIT Peru for support provided in the

H. Maruenda et al. / Food Chemistry 138 (2013) 161167

administration of the project and to Alex Nieva from PUCP for his
assistance in the LCMS study. The Peruvian Instituto Nacional
de Recursos Naturales (INRENA) issued permits for research and
collection activities of this project. This research was nanced by
Programa de Ciencia y Tecnologia FINCYT (co-nanced by BID)
grant number PIBAP-2007-005. Field and herbarium research were
supported in part by the US National Science Foundation (NSF)
grant # 0717453 to BRIT, the Gordon Betty Moore Foundation,
and the Clayton Fund of Houston, Texas.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2012.
10.037.
References
Brodelius, P. E. (1994). Phenylpropanoid metabolism in Vanilla planifolia Andr. (V)
High performance liquid chromatographic analysis of phenolic glycosides and
aglycones in developing fruits. Phytochemical Analysis, 5, 2731.
Brownell, R. J. Jr., (2011). Fair trade The future of vanilla? In D. Havkin-Frenkel & F.
Belanger (Eds.), Handbook of vanilla science and technology (pp. 107116). West
Sussex, UK: Blackwell Publishing Ltd.
Cicchetti, E., & Chaintreau, A. (2009). Quantitation of the main constituents of
vanilla by reverse phase HPLC and ultra-high-pressure-liquid-chromatography
with UV detection: Method validation and performance comparison. Journal of
Separation Science, 32, 30433052.
Da Costa, N. C., & Pantini, M. (2006). The analysis of volatiles in Tahitian vanilla
(Vanilla tahitensis) including novel compounds. Developments in Food Science, 43,
161164.
Dignum, M. J. W., Kerler, J., & Verpoorte, R. (2002). Vanilla curing under laboratory
conditions. Food Chemistry, 79, 165171.
Dignum, M. J. W., van der Heijden, R., Kerler, J., Winkel, C., & Verpoorte, R. (2004).
Identication of glucosides in green beans of Vanilla planifolia Andrews and
kinetics of vanilla b-glucosidase. Food Chemistry, 85, 199205.
Ehlers, D., & Pster, M. (1997). Compounds of vanillons (Vanilla pompona Schiede).
Journal of Essential Oil Research, 9, 427431.
Gassenmeier, K., Riesen, B., & Magyar, B. (2008). Commercial quality and analytical
parameters of cured vanilla beans (Vanilla planifolia) from different origins from
the 20062007 crop. Flavour and Fragance Journal, 23, 194201.
Havkin-Frenkel, D., French, J. C., Graft, N. M., Pak, F. E., Frenkel, Ch., & Joel, D. M.
(2004). Interrelation of curing and botany in vanilla (Vanilla planifolia) Bean.
Acta Horticulturae (ISHS), 629, 93102.
Householder, E., Janovec, J., Balarezo, A., Huinga, J., Wells, J., Valega, R., et al. (2010).
Diversity, natural history, and conservation of vanilla (Orchidaceae) in
Amazonian Wetlands of Madre de Dios, Peru. Journal of the Botanical Research
Institute of Texas, 4(1), 227243.
International Conference on Harmonization (ICH). (1996) Guideline on the
validation of analysis procedures: Methodology Q2B.

167

Itokawa, H., Oshida, Y., Ikuta, A., Inatomi, H., & Adachi, T. (1982). Phenolic plant
growth inhibitors from the owers of Cucurbita pepo. Phytochemistry, 21,
19351937.
Jhon, T. V., & Jamin, E. (2004). Chemical investigation and authenticity of Indian
vanilla beans. Journal of Agricultural and Food Chemistry, 52, 76447650.
Kanisawa, T. (1993). Flavor development in Vanilla beans. Kouryou, 180(5),
113123.
Kitajima, J., Ishikawa, T., Tanaka, Y., Ono, M., Ito, Y., & Nohara, T. (1998). Watersoluble constituents of fennel. V. Glycosides of aromatic compounds. Chemical &
Pharmaceutical Bulletin, 46(10), 15871590.
Lapeyre-Montes, F., Conejero, G., Verdeil, J.-L., & Odoux, E. (2011). Anatomy and
biochemistry of vanilla bean development (Vanilla planifolia G. Jackson). In E.
Odoux & M. Grisoni (Eds.). Medicinal and aromatic plants-industrial proles:
Vanilla (Vol. 47). Boca Raton, FL: CRC Press, pp 164.
Leper-Andrzejewski, S., Brunschwig, Ch., Collard, F.-X., & Dron, M. (2011).
Morphological, chemical, sensory, and genetic specicities of tahitian vanilla.
In E. Odoux & M. Grisoni (Eds.). Medicinal and aromatic plants-industrial proles:
Vanilla (Vol. 47, pp. 205228). Boca Raton, FL: CRC Press.
Negishi, O., Sugiura, K., & Negishi, Y. (2009). Biosynthesis of vanillin via ferulic acid
in Vanilla planifolia. Journal of Agricultural and Food Chemistry, 57, 99569961.
Odoux, E. (2000). Changes in vanillin and glucovanillin concentrations during the
various stages of the process traditionally used for curing Vanilla fragans beans
in Reunion. Fruits, 55, 119125.
Odoux, E. (2011). Developing the aromatic quality of cured vanilla beans (Vanilla
planifolia G. Jackson). In E. Odoux & M. Grisoni (Eds.). Medicinal and aromatic
plants-industrial proles: Vanilla (Vol. 47). Boca Raton, FL: CRC Press, pp 189
204.
Palama, T. L., Khatib, A., Choi, Y. H., Payet, B., Fock, I., Verpoorte, R., et al. (2009).
Metabolic changes in different developmental stages of Vanilla planifolia pods.
Journal of Agricultural and Food Chemistry, 57, 76517658.
Ranadive, A. S. (2011). Quality control of vanilla beans and extracts. In D. HavkinFrenkel & F. Belanger (Eds.), Handbook of vanilla science and technology
(pp. 141160). West Sussex, UK: Blackwell Publishing Ltd.
Sharma, U. K., Sharma, N., Gupta, A. P., Kumar, V., & Sinha, A. K. (2007). RP HPTLC
densitometric determination and validation of vanillin and related phenolic
compounds in accelerated solvent extract of Vanilla planifolia. Journal of
Separation Science, 30, 31743180.
Sinha, A. K., Sharma, U. K., & Sharma, N. (2008). A comprehensive review on vanilla
avor: Extraction, isolation and quantication of vanillin and others
constituents. International Journal of Food Sciences and Nutrition, 59, 299
326.
Sinha, A. K., Verma, S. C., & Sharma, U. K. (2007). Development and validation of an
RP HPLC method for quantitative determination of vanillin and related
phenolic compounds in Vanilla planifolia. Journal of Separation Science, 30,
1520.
Sreedhar, R. V., Roohie, K., Venkatachalam, L., Narayan, M. S., & Bhagyalakshmi, N.
(2007). Specic treatments reduce curing period of vanilla (Vanilla planifolia)
beans. Journal of Agricultural and Food Chemistry, 55, 29472955.
Toth, S., Lee, K. J., Havkin-Frenkel, D., Belanger, F. C., & Hartman, G. (2011). Volatile
compounds in vanilla. In D. Havkin-Frenkel & F. Belanger (Eds.), Handbook of
vanilla science and technology (pp. 183218). West Sussex, UK: Blackwell
Publishing Ltd.
Voisine, R., Carmichael, L., Chalier, P., Cormier, F., & Morin, A. (1992). Determination
of glucovanillin and vanillin in cured vanilla pods. Journal of Agricultural and
Food Chemistry, 43, 26582661.