Amazonian Vanilla species as potential

source of vanilla essence

Alvaro Zevallos, Cristhian Caari, Alex Nieva and Helena Maruenda*
Ponticia Universidad Catlica del Per, Departamento de Ciencias - Qumica
Lima 32, Per

Natural vanilla is a spice of recognized commercial value. It is derived from the fruits of Vanilla planifolia (Orchidaceae), V. tahitensis, and V. pompona. In order to quickly assess the potential of recently
[1]
1
discovered Amazonian Vanilla species as new sources of vanilla essence, H-NMR analyses of their leaves were undertaken. The accumulation of bis[4-(-D-glucopyranosyloxy)-benzyl]-2-isopropyl-tartrate
(glucoside A), bis[4-(-D-glucopyranosyloxy)-benzyl]-2-(2-butyl)-tartrate (glucoside B), and 4-hydroxybenzyl alcohol glucoside, in the leaves, stems, and pods was proved to be positively correlated with avor development
in vanilla pods. Hence, for the rst time, the presence of these metabolites is evaluated in the leaves of wild V. riberoi and V. palmarum, as well as in wild Peruvian V. pompona subsp. grandiora and commercially available
V. planifolia. Differences among species are addressed through multivariate data analyses.

Commercial vanilla (V. planifolia)

Biosynthetic pathway to vanillin

The development of commercial vanilla pods into the avorful and scented cured product takes between 13 and 17 months after hand-pollination
of the ower (A - C). Nine to eleven months later, the vanilla pods (D) are hand-picked to undergo a long curing process (5 to 8 months), after which the
avor and aroma are optimal (E).

(A)

(B)

(D)

(C)

Day 0

Day 1

(E)

Month 9

Month 17

Figure 1. Processes involved in natural vanilla production.

NMR proling of the leaf metabolome of Vanilla sp.

Aglycones reported in cured fruits

[2]

Figure 3. Proposed biosynthetic pathway to vanillin considering the expression of key phenylpropanoid genes during pod maturation[3].
This pathway is based on glucosides identied in extracts of green pods.

vanillin
vanillyl alcohol
vanillic acid
4-hydroxybenzyl alcohol
4-hydroxybenzaldehyde
4-hydroxybenzoic acid

V. planifolia

vanillin
vanillyl alcohol
vanillic acid
4-hydroxybenzyl alcohol
4-hydroxybenzaldehyde
4-hydroxybenzoic acid

V. pompona

Multivariate analysis
anisyl alcohol

An unsupervised principal component analysis (PCA) allowed the distinction between Vanilla species based on their leaf
metabolome, while supervised orthogonal partial least square discriminant analysis (O-PLS-DA) pinpointed the
metabolites responsible for such discrimination.

V. riberoi

Not studied yet

V. palmarum

Figure 4. Score plot (PC1 vs PC2) of PCA

1
results obtained from H NMR spectra of
Vanilla sp. leaves.

MeOD

TSP
HDO

Not studied yet

Figure 2. 1H-NMR spectra of representative samples of the leaves of each Vanilla sp. in the range 0-10 ppm.

The identity of all reported compounds was conrmed through 1D and 2D NMR experiments. Critical regions in the spectrum
were unraveled through J-resolved spectroscopy. The metabolites found are shown Table 1.

glucoside B

glucoside A

Table 1. 1H chemical shifts (), coupling constants (Hz) of signals from Vanilla sp. leaf metabolites identied by
references and using 1D and 2D NMR spectra (methanol-d4/KH2PO4 in D2O, pH 6.0). Due to lack of space fructose,
glutamine and homocitric acid were not included in this table.
Compound
Sucrose

-Glucose

-Glucose

Tyrosine

Alanine
Asparagine
Acetic acid
Gallic acid
Formic acid
Malic acid

trans-Ferulic acid

4-Hydroxybenzyl alcohol

4-Hydroxybenzyl alcohol glucoside

Bis[4-(-D-glucopyranosyloxy)-benzyl]2-isopropyltartrate (glucoside A)

Bis[4-(-D-glucopyranosyloxy)-benzyl]2-(2-butyl)tartrate (glucoside B)

Not detected

Assigment
1-CH
15-CH2
2-CH
1-CH
3-CH
2-CH
1-CH
3-CH
2-CH
3,5-CH (ring)
2,6-CH (ring)
-CH
-CH2
-CH
-CH3
-CH
-CH
CH3
2,5-CH (ring)
CH
2-CH
3-CH
3-CH
10-CH
3-CH (ring)
5-CH (ring)
6-CH (ring)
9-CH
3,5-CH (ring)
2,6-CH (ring)
7-CH2
3,5-CH (ring)
2,6-CH (ring)
1-CH (4-O-glucoside)
7-CH2
2,6-CH (ring)
3,5,3,5-CH (ring)
1-CH (4-O-glucoside)
7-CH3
5-CH3
2,6-CH (ring)
3,5,3,3-CH (ring)
1-CH (4-O-glucoside)
7-CH2
8-CH3

1H (multiplicity)
5.40 (d, 3.6 Hz)
3.67 (s)
3.51 (dd, 8.5, 3.6 Hz)
5.18 (d, 3.7 Hz)
3.82 (dd, 9.8, 3.2 Hz)
3.46 (dd, 9.8, 3.7 Hz)
4.57 (d, 7.9 Hz)
3.44 (dd, 9.8, 4.0 Hz)
3.21 (dd, 9.8, 7.9 Hz)
7.16 (d, 8.8 Hz)
6.85 (d, 8.8 Hz)
3.81 (d, 7.5 Hz)
3.13 (dd, 7.5, 3.3 Hz)
3.77 (m)
1.47 (d, 7.2 Hz)
2.96 (dd, 17.0, 8.1 Hz)
2.82 (dd, 17.0, 4.1 Hz)
1.97 - 1.98 (s)
7.04 (s)
8.45 (s)
4.31 (dd, 7.8, 4.8 Hz)
2.79 (dd, 16.0, 4.8 Hz)
2.40 (dd, 16.0, 7.8 Hz)
7.50 (d, 16.0 Hz)
7.34 (d, 2.1 Hz)
7.10 (dd, 7.3, 2.1 Hz)
6.87 (d, 7.3 Hz)
6.36 (d, 16.0 Hz)
7.12 (d, 8.6 Hz)
6.77 (d, 8.6 Hz)
4.33 (s)
7.21 (d, 8.6 Hz)
6.85 (d, 8.6 Hz)
5.01 (d, 7.8 Hz)
4.51 (s)
7.29 (d, 9.5 Hz)
7.11 (d, 9.5 Hz)
5.03 (d, 7.8 Hz)
0.94 (d, 7.0 Hz)
0.86 (d, 7.0 Hz)
7.29 (d, 9.5 Hz)
7.11 (d, 9.5 Hz)
5.03 (d, 7.8 Hz)
1.40 (m), 1.10 (m)
.79 (t, 7.5 Hz)

trans-ferulic 4-hydroxybenzyl
acid
alcohol

4-hydroxybenzyl
alcohol glucoside

glucoside A

ESI-MS (m/z)
[M+Na]+= 365.1

Figure 5. Loading plots of O-PLS-DA results obtained from

1
the H NMR spectra of Vanilla leaves. O-PLS coefcients were
color coded to contain correlation.

[M+Na]+= 203.2

glucoside B
glucoside B

[M+Na]+= 203.2

The aromatic region is clearly responsible for remarkable

differences among the Vanilla species.

glucoside A
glucoside A

[M+H]+= 182.1

[M-H]-= 88
[M+H]+= 133.1
NDa
[M-H]-= 169.1
ND
[M-H]-= 132.9

Conclusions
- The leaf metabolomes of four different Vanilla species (V. planifolia, V. pompona, V. riberoi, and V. palmarum) have been
studied by NMR, and were clearly distinguished through multivariate analysis.aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
- 1D and 2D NMR spectroscopy allowed the identication of 18 compounds (amino acids, organic acids, sugars, and
phenolics), among them, known vanillin precursors were found in all four species studied.aaaaaaaaaaaaaaaaaaaaaa

ND

- NMR analyses of these wild Vanilla orchids suggest that V. riberoi and V. palmarum, whose fruits have not been studied,
could potentially be considered as new sources of natural vanilla essence.

[M+H]+= 125.1

Acknowledgements
[M+1-162]+= 125.1

[M+Na]+= 751.2

We thank EE.GG.LL from Ponticia Universidad Catlica del Per for providing an environment in their greenhouse.
We also thank Professor Emeritus Dr. Geoffrey A. Cordell for reviewing this work.aaaaaaaa aaaaaaaaaaaaaaaaaaa

Referencesaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
1. Householder, J.E.; Janovec, J.P; Balarezo, A; Huinga, J.; Wells, J.; Valega, R.; Maruenda, H.; Christeson, E. Diversity, Natural History, and
Conservation of Vanilla (Orchidaceae) in Amazonian Wetlands of Madre de Dios, Peru. Journal of the Botanical Research Institute of Texas
2010, 4 (1), 227-243.
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[M+Na]+=

765.2

2. Maruenda, H.; Vico, M.; Householder, J.E.; Janovec, J.P.; Caari, C.; Naka, A.; Gonzalez, A.E. Exploration of Vanilla pompona from the
Peruvian Amazon as a potential source of vanilla essence: quantication of phenolics by HPLC-DAD. Food Chemistry 2013, 138, 161-167.
3. Fock-Bastide, I.; Palama, T.; Bory, S.; Lcolier, A.; Noroit, M; Jot, T. Expression proles of key phenylpropanoid genes during Vanilla planifolia
pod development reveal a positive correlation between PAL gene expression and vanillin biosynthesis. Plant Physiology and Biochemistry 2014,
74, 304-314.