FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J. 2003; 18: 207– 210

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1185

The essential oils of Rinorea subintegrifolia O. Ktze and

John Wiley & Sons, Ltd.

Drypetes gossweileri S. Moore occurring in Gabon#

Huguette Agnaniet,1,2 Hortense Mounzeo,1 Chantal Menut,2* Jean-Marie Bessiere3 and Marc Criton2
ESSENTIAL OILS OF RINOREA SUBINTEGRIFOLIA AND DRYPETES GOSSWEILERI

1
Université des Sciences et Techniques de Masuku, BP 911 Franceville, Gabon
2
Laboratoire de Chimie Biomoléculaire, Ecole Nationale Supérieure de Chimie, 8 Rue de l’Ecole Normale, 34296 Montpellier
Cedex 5, France
3
Laboratoire de Phytochimie, Ecole Nationale Supérieure de Chimie, 8 Rue de l’Ecole Normale, 34296 Montpellier Cedex 5,
France
Received 7 February 2002
Revised 26 June 2002
Accepted 26 June 2002

ABSTRACT: The essential oils from root bark of Rinorea subintegrifolia (Violaceae) and bark of Drypetes
gossweileri (Euphorbiaceae) from Gabon were analysed by GC–FID and GC–MS. The oils were characterized
by a high percentage of benzyl cyanide and a minor amount of terpenes (<1.5%). The antioxidant and the antiradical
activity of both samples were evaluated. Copyright © 2003 John Wiley & Sons, Ltd.

KEY WORDS: Violaceae; Rinorea subintegrifolia; Euphorbiaceae; Drypetes gossweileri; essential oils; benzyl
cyanide; antioxidant; antiradical

Introduction such as headaches or bronchopneumonia. Sometimes, it

is even used as an aphrodisiac.
Rinorea subintegrifolia (Violaceae) is a shrub of forest The oil isolated from the root bark of Rinorea sub-
clearings, growing to a height of 7 ft and found in the integrifolia collected in Gabon was analysed by GC
humid underwood forest area and savanna.1–4 and GC–MS and the results were compared to those
Drypetes gossweileri (Euphorbiaceae) is a tree of the obtained from bark oil of Drypetes gossweileri from
dense humid forest which can grow up to 42 m high, with the same country. The second species was investigated
a straight cylindrical trunk. Its bark is grey, rough to the previously8 in 1997, whereas to our knowledge there has
surface, with a pungent smell of horseradish.1–4 been no previous chemical investigation of the volatile
Traditionally, in Africa, the roots of Rinorea sub- constituents of Rinorea subintegrifolia.
integrifolia are used as aromatics which are offered to the The antioxidant and the antiradical activity of both
spirits and the manes of ancestors.1 They are also used samples were evaluated in the present study.
against eye diseases, when mixed with kaolin. The juice
of the leaves, added to water, is used for heart diseases.
A decoction of the leaves is used as an expectorant; it Experimental
also serves to cure feverish aching. As an infusion, the
root bark is good for constipation, stomach aches, rheum- Collection of Plant Material
atismal aches and œdema.5–7
Drypetes gossweileri bark and fruit are used to narcotize Samples of the roots of Rinorea subintegrifolia were
fish. A decoction of the fresh bark, mixed with Capsicum taken from 10 individual arborescent shrubs, whereas
fructescens, is used as an anthelmintic and against fevers bark samples of Drypetes gossweileri were originated
in young children. This same bark is used in the treat- from three different trees. All the samples were collected
ment of rheumatism and sinusitis. Used as a pomade in March 2000 in various localities of the Nkomo-
mixed with palm oil, it is said to act as a revulsive.5–7 In Moanda area (Gabon estuary).
the same way, the bark is used against various diseases,

#
Part XXXIX in Aromatic Plants of Tropical Central Africa. Isolation of Essential Oils
* Correspondence to: C. Menut, Laboratoire de Chimie Biomoléculaire,
Ecole Nationale Supérieure de Chimie, 8 Rue de l’Ecole Normale, 34296 Fractions of 300 g of both samples were subjected to
Montpellier Cedex 5, France.
E-mail: cmenut@univ-montp2.fr hydrodistillation for 6 h immediately after comminuting,
Contract/grant sponsor: Gabonese Republic. using a Clevenger-type apparatus. An additional experiment

Copyright © 2003 John Wiley & Sons, Ltd.

208 H. AGNANIET ET AL.
was performed with Drypetes gossweileri, by recovery of
the volatile constituents obtained with variable distillation
times (30 min, 1 h, 2 h, 6 h). The oils were separated from
water by decantation and dried by filtration over anhydrous
sodium sulphate. The oil yields were 0.19% for Drypetes
gossweileri and 0.06% for Rinorea subintegrifolia.
Analysis
Gas Chromatography
The oils were analysed on a Varian GC 3380 with flame
ionization detectors fitted with two fused silica capillary
columns (25 m × 0.25 mm i.d., coated with OV-101,
film thickness 0.25 µm; 25 m × 0.22 mm, coated with
Carbowax 20 M, film thickness 0.25 µm); temperature
programme 50–200 °C at 5 °C/min; injector temperature,
220 °C; detector temperature, 250 °C; carrier gas, N2 at a
rate of 0.8 ml/min. The linear retention indices of the com-
ponents were determined relative to the retention times
of a series of n-alkanes and the percentage compositions
were obtained from electronic integration measurements
without taking into account relative response factors.
Gas Chromatography–Mass Spectrometry
GC–MS analyses were performed using a Hewlett-Packard
apparatus equipped with a DB-5 fused silica column (30 m
× 0.20 mm; film thickness 0.15 µm) and interfaced with a
quadrupole detector. Column temperature was programmed
to 50–200 °C at 3 °C/min; injector temperature, 220 °C;
helium flow rate, 1 ml/min; ionization energy, 70 eV.
Identification of Components
The identification of the constituents was assigned on the
basis of comparison of retention data and mass spectra
with data banks.9
Antioxidant and Antiradical Activities
Evaluation of the Antioxidant Activity
The antioxidant activity of both essential oils was evalu-
ated using a β-carotene/linoleate model system.10–15
A solution of β-carotene from Fluka (Catalogue No.
7235-40-7) was prepared by dissolving 2.0 mg β-carotene
in 10 ml chloroform; 1 ml of this solution was pipetted into
a round-bottomed flask which contained 20 µl purified
linoleic acid (Avocado, Catalogue No. 60-33-3) and 200 mg
of Tween 40 emulsifier (Aldrich, Catalogue No. 27,435-6).
After chloroform was removed under vacuum using
a rotary evaporator at 40 °C, 50 ml aerated, distillated
water was added to the flask with vigorous shaking.
Copyright © 2003 John Wiley & Sons, Ltd.
The antioxidant activity was evaluated by measuring,
at 470 nm, the kinetics of decolouration of β-carotene in
the absence (control) or in the presence of the antioxid-
ant solution containing different concentrations (10 ML
methanolic solutions containing different concentrations
of essential oils or BHT for comparative purposes) at
50 °C. The blank is a solution prepared in the same con-
ditions as above, but without β-carotene.
All the kinetics obtained are of first order. Their
computer treatment (curve expert), on the basis of the
equation: A = A0e−kt + C in which A0 represents the
absorbance at time zero and C the absorbance at infinite
time, allows rate constants (mn−1) to be obtained with
the correlation coefficients, r, always >0.999. The inhibi-
tion percentage is obtained through the relation:
 k − k
Ip =  o  × 100
 ko 
where ko and k represent the degradation rate constants
of β-carotene in the absence and in the presence of
inhibitor. The outline of the inhibition percentage as a
function of the inhibitor concentration allows evaluation
of the IC50 of the tested antioxidants.
Evaluation of the Antiradical Activity
The antiradical activities were determined using 2,2-
diphenyl-1-picrylhydrazyl (DPPH), using the Mellors
and Tappel method.16 –19
DPPH·, a free stable radical scavenger, was dissolved
in ethanol to give a 100 µm solution. To 2.0 ml ethanolic
solution of DPPH was added 100 µl methanolic solution
of the antioxidant reference (BHT, ascorbic acid or
tocopherol) at different concentrations (10–50 µm). The
essential oils were tested respectively by the same
method. The control, without antioxidant, is represented
by the DPPH ethanolic solution containing 100 µl
methanol. The decrease in absorption was measured at
517 nm after 30 min at room temperature. The actual
decrease in absorption induced by the test compound
was calculated by substracting that of the control.
Measurements were performed in triplicate and the
concentration required for 50% reduction (50% scaveng-
ing concentration, SC50) was determined graphically.
All the spectrophotometric measures were performed
with a SAFAS UV mc2 spectrophotometer, equipped
with a multicells/multikinetics measure system and with
a thermostated cells case.
Results and Discussion
The oils obtained from Drypetes gossweileri and Rinorea
subintegrifolia both present an herbaceous-green odour
with a powerful pungent aspect in spite of sweet and
heavy floral undertones.
Flavour Fragr. J. 2003; 18: 207–210
 ESSENTIAL OILS OF RINOREA SUBINTEGRIFOLIA AND DRYPETES GOSSWEILERI 209

Table 1. Comparative percentage composition of the oils of Rinorea subintegrifolia and Drypetes gossweileri
from Gabon

RI (OV101) RI (CW20M) Compounds Distillation time (h)

Drypetes gossweileri Rinorea
subintegrifolia
0.5 1 2 6 1 2
925 1523 Benzaldehyde 0.2 0.3 0.3 0.8 — —
1004 1862 Benzyl alcohol 1.7 3.7 3.6 10.8 0.8 2.5
1044 1673 Benzyl formate 0.2 0.7 0.7 0.6 — —
1070 1463 trans-Linalool oxide (THF) 0.2 — — — — —
1081 1549 Linalool 0.9 0.5 0.4 0.6 — —
1095 1950 Benzyl cyanide 19.4 37.0 58.0 73.7 64.0 87.7
1130 1714 Benzyl acetate 0.5 0.3 0.5 0.7 — —
1210 1990 p-Methoxybenzaldehyde — — — — 0.9 1.3
1249 2202 p-Methoxybenzyl alcohol — — — — 0.4 0.5
1317 2131 Benzyl isothiocyanate 72.0 53.0 31.4 5.2 29.0 1.4
1336 2305 p-Methoxybenzyl cyanide — — — — 1.5 2.7
1402 1607 β-Caryophyllene 0.1 0.2 0.3 0.3 0.2 0.3
1460 2482 p-Methoxybenzyl isothiocyanate — — — — 0.6 0.8
1520 1995 Caryophyllene oxide 0.2 0.2 0.1 0.3 — —
1782 2571 Benzyl benzoate 0.2 0.2 0.1 0.2 — —
1816 2715 Benzyl salicylate 0.9 1.0 1.0 1.1 — —
Total 96.5 97.1 96.4 94.3 97.4 97.2

Eight components for Rinorea subintegrifolia and
eleven components for Drypetes gossweileri were identi-
fied, which comprised respectively 97.2% and 94.3% of
the oils analysed after 6 h distillation time (Table 1).
Rinorea and Drypetes essential oils are mainly con-
stituted by the decomposition products of glucosinolate
derived from phenylalanine. This hydrolysis classically
gives isothiocyanate, but it is often accompanied by the
formation of nitrile and other benzylic metabolites. It is
observed that the composition of essential oils varies
greatly during the preparation. The study, mainly per-
formed on Drypetes barks, which are easier to obtain,
showed that at the beginning of the distillation the essen-
tial oil is very rich in benzyl isothiocyanate, and that at
the end of the operation benzyl cyanide is formed, with
a final rate of 73.7% after 6 h distillation.
Benzyl cyanide is probably produced by a reaction
between cyanide ions and benzyl isothiocyanate. We
have checked that this reaction could occur in our experi-
mental conditions and it was effectively evidenced
that cyanide ions were released by the barks in boiling
water; these ions are not present in a cold maceration, but
they are formed as 0.4% weight of the barks after 2 h
ebullition.
Furthermore, it is worth noting that the total percent-
age of benzyl cyanide, benzyl isothiocyanate and benzyl
alcohol together remains close to 90%, so this may
indicate that benzyl alcohol also arises from benzylis-
othiocyanate, and not from glucosinolate hydrolysis as
frequently supposed.
These results should be compared to those obtained
from samples of different origins: barks gathered in
Central Africa gave an essential oil very rich in benzyl
isothiocyanate (after 10 h hydrodistillation), while the
essential oil issued from a Gabonese bark (from a differ-
ent source than that studied here) contained equivalent
amounts of isothiocyanate and nitrile.8 This discrepancy
in the results is probably due to the variable content of
cyanogenic compounds for the analysed samples.
The phenomenon of chemical transformation, which
is observed in Drypetes barks, also occurs during the
treatment of Rinorea barks. The essential oil obtained
after only 1 h of ebullition contains 29% benzyl isothi-
ocyanate and this content becomes lower than 2% at the
end of the distillation; concurrently, the benzyl cyanide
percentage rises from 64% to 87.7%. Rinorea essential
oil also contains p-methoxybenzyl derivates, probably
formed from p-methoxyphenyl alanine glucosinolate.
Finally, terpenes are very minor products in the essen-
tial oil of both barks, each being 1.5%.
The isothiocyanate derivatives are commonly found in
the family Cruciferae;20 they are present, although less
systematically, in other botanical families. Their presence,
more particulary in the family Violaceae, is reported
here for the first time.
The results relating to the evaluation of the antioxidant
properties of essential oils and BHT samples, according
to the β-carotene/linoleate method,10 –15 are given in
Table 2.
The outline of the inhibition percentage as a function
of the antioxidant concentration leads to the follow-
ing results: IC50 (BHT) = 82 µg/l and IC50 (Rinorea) =
700 µg/l.
The Drypetes essential oil was not sufficiently active
to allow the calculation of IC50, since for a concentration
of 666 µg/l the inhibition percentage was only 26%.

Copyright © 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2003; 18: 207–210
210 H. AGNANIET ET AL.

Table 2. Rate constants (min−1) of β-carotene decoloura- Acknowledgements— We thank the Gabonese Republic for its contribu-
tion in the absence (control) or presence of inhibitor tion and IRET (Gabon) Director P. Posso and his staff for their great
and inhibition percentage (Ip) help for collecting and identifying the Gabonese botanical samples.

Inhibitors [Inhibitors] µg/l k (10−3)* min−1 Ip

Control 0 52.3 — References
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Copyright © 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2003; 18: 207–210