FLAVOUR AND FRAGRANCE JOURNAL, VOL.

1, 111-113 (1986)

The Volatile Oil of Ziziphora hispanica L.

A. Velasco Negueruela
Departamento de Boihica y Fisiologfa Vegetal, Facultad de Biolog(a, Universidad Complutense, Madrid, 28040, Spain

M. Mata Rico
Departamento de Farmacognosia y Farmacodinamia, Facultad de Farmacia, Universidad Complutense, Madrid, 28040, Spain

Analysis of the steam-volatile leaf and flower oil of Ziziphora hispanica L. by gas-chromatography (GC)
confirmed that pulegone and piperitenone are the main monoterpenes. Lesser amounts of limonene, 1,%cineole,
isopulegol, trans-isopiperitenol and thymol are present. 8-Cadinene is the main sesquiterpene together with
caryophyllene oxide and cis-nerolidol. The pulegone content of the oil tended to increase before flowering and
decrease once flowering had commenced.

KEY WORDS Essential oil Ziziphora hispunicu L. Lamiaceae Pulegone Piperitenone

INTRODUCTION in the author's laboratory.293We have also used

Previous chemical investigations of the con-
stituents of the foliage and flowers of Ziziphora
hispanica L., an endemic of the central region of
Spain, indicated the presence of pulegone and
piperitenone.' It was decided to reinvestigate the
steam volatile oil using GC to identify the further
components present in the oil.
EXPERIMENTAL
infra red spectroscopy (IR) and l3C-nuclear
magnetic resonance (I3C-NMR) analysis for
further identification.
Pulegone, piperitenone and cis-piperitone
oxide were also identified by ultraviolet spectro-
scopy (UV) and by analytical thin-layer
chromatography (TLC).
Component concentrations were calculated
from GC peak areas without using a correction
factor.

Three samples of Ziziphora hispanica were har- TL C

vested, one prior to flowering (ZHNF) and the
other two during flowering (ZHF1 and ZHF2)
near Arganda, Madrid, in the central part of
Spain. Dry leaves, stems and flowers were
hydro-distilled in a Clevenger apparatus. The
mean yield of the oil was 1.38% based on dry
weight of sample.
Voucher specimens were deposited in the Her-
barium of the Facultad de Biologia, Universidad
Complutense de Madrid (M.A.C.).
Identification of Components
Most of the individual components were tenta-
tively identified by comparing their retention
times (R,) with those of pure standards available
0882-5734/86/030111-03SO5.00
0 1986 by John Wiley & Sons, Ltd.
This was performed on pre-coated silica gel
plates4 using MeC6HS:EtOAc (93:7) as solvent.
Detection with vanillin-sulphuric acid gave the
following results: piperitenone (R, 0.26),
piperitone (R, 0.31), isomenthone (R, 0.65),
pulegone (R, 0.50) and menthofuran (R, 0.95).
Preparative Thin-layer Chromatography
This was performed on silica gel 60 PFz54(E.
Merck no. 7747), layer thickness 2mm, using
1 ml of sample. After detection by UV
(254-360 nm), pulegone, piperitenone,
piperitone and cis-piperitone oxide were isolated
using chloroform.
Received 29 April 1986
Accepted 19 August 1986
I12 A. VELASCO NEGUERUELA AND M. MATA RICO

GC
Analytical G C was carried out on a Perkin-Elmer
3920 gas chromatograph fitted with either ( 1 )
stainless steel column 2 m x 3.18 mm, packed
with UCON LB 550 X or (2) stainless steel
column 2 m x 3.18 mm, packed with APIEZON
L Detector used dual FID, carrier gas He, flow
rate 30 ml/min, temperature programmed
95"C-180°C/min, injector temperature 200°C,
detector temperature 2S0°C, injection volume for
all samples 0.04 pl. The quantitative composition
(relative percentages) was obtained in a comput-
ing integrator Varian SP 4270.
uv
The spectra were performed on a Perkin-Elmer
Hitachi 200 spectrophotometer, recorded in
E,OH. A,, found were consistent with those in the
IR
Spectra were performed on a Pye-Unicam SP 200
and were run as liquid films, NaCl cell
(0.025 mm). For further identification the 1R
spectra were performed directly on the essential
oil sample as in reference 11.
W-N M R
Analysis was performed directly on the essential
oil sample as in reference 12, on a Varian XL 300
spectrophotometer, solvent used ChDhr fre-
quency 300 MHz, internal standard TMS. The
values for the individual components of the oil
were consistent with those reported."
RESULTS AND DISCUSSION
The percentage composition of the oil and the
identification methods are given in Table 1, Com-

Table 1. Volatile components of Ziziphora hispanica L.

Percentage composition

Component ZHFl ZHF2 ZHNF Identification method

a-Pinene 0.30 0.30 0.35 "C-NMR. RT

Camphene 0.01 RT
P-Pinene 0.32 0.31 0.45 RT
Myrcene 0.1 1 RT
6-Car-3-ene 0.02 0.02 RT
Limonene + 1 ,8-Cineole 1.90 2.55 3.58 "C-NMR. RT
pCymene 0.80 0.04 I3C-NMR, RT
y-Terpinene 0.68 0.01 I3C-NMR, RT
Terpinolene 0.33 0.02 "C-NMR, RT
Octan-3-01 0.34 0.01 0.01 13C-NMR, RT
Oct- 1-en-3-01 0.44 0.18 0.13 I3C-NMR, RT
cis-allo-Ocimene 0.94 0.04 0.03 RT
a-Thujone 0.57 0.06 13C-NMR, RT
rrans-Thujan-4-01 0.10 0.08 0.01 RT
Menthofuran 0.66 0.39 0.28 "C-NMR,RT, TLC
Isopulegol 1.33 0.74 0.98 "C-NMR, RT
!runs-Dihydrocarvone 0.15 0.04 RT
Menthol 0.42 0.28 0.12 "C-NMR, RT
Pulegone 64.57 62.69 76.66 1R. UV, I3C-NMR, RT, TLC
Piperitone 0.10 0.04 0.01 IR, UV, "C-NMR, RT, TLC
cis-Piperitone oxide 1.02 0.60 0.25 IR, UV, RT
Nerol 0.27 0.44 0.20 I3C-NMR, RT
rrans-Isopiperitenol 2.71 1.47 0.42 RT
Piperitenone 15.64 16.67 11.65 IR, UV, RT, TLC
Thymol 0.97 0.97 0.31 I3C-NMR, RT
cis-Nerolidol 1.31 0.22 RT
Caryophyllene oxide 1.46 0.50 RT
Humulene oxide 1.13 0.52 RT
6-Cadinene 2.03 1.79 0.25 I3C-NMR, RT
 VOLATILE OIL OF ZIZIPHORA HlSPANlCA L.
ponents were arranged in order of GC elution
(UCON).
From these results we may note that
piperitenone, the biogenetic precursor of
p ~ l e g o n e , ' ~increases during flowering. This
might well mean that the stereospecific reduction
of piperitenone to pulegone is blocked during
flowering (substrate inhibition of the enzyme?)
This oil is of interest for the high content of
pulegone. Our results are in good agreement
with that p r o p o ~ a lthat' ~ piperitenone is the bio-
genetic precursor of pulegone.
Acknowledgement-We wish to express our very grateful
appreciation to Dr M. P. Pardo and Dr M. T. Ortega for
collecting and providing sample ZHl and for valuable per-
sonal communications.
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