Characterization of Volatile Constituents from Heterotrophic Cell
Suspension Cultures of Ruta graveolens
M ichael Jo rd a n , C laus H . R olfs, and W olfgang Barz
Lehrstuhl für B iochem ie der Pflanzen, W estfälische W ilhelm s-U niversität, Hindenburgplatz 55,
D -4400 M ünster, Bundesrepublik Deutschland
R a lf G . B erg er, H u b e rt K ollm annsberger, and F riedrich D raw ert
Institut für L ebensm itteltechnologie und Analytische C hem ie, T U M ünchen,
D -8050 F reising-W eihenstephan, Bundesrepublik Deutschland
Z. Naturforsch. 41c, 8 0 9 —812 (1986); received July 15, 1986
H eterotrophic Cell C ultures, Ruta graveolens, Volatile O ils, Terpenoid H ydrocarbons, Aliphatic
K etones and A lcohols
H eterotrophic cell suspension cultures o f Ruta graveolens were established by reversion of
photom ixotrophic cultures without any change in the chemical com position o f the growth
m edium . The heterotrophic cultures were qualitatively and quantitatively analyzed by gaschrom atography and m ass spectroscopy for volatile com pounds. The terpenoid hydrocarbons
geijerene and pregeijerene, C 6 -C 8 ketones, acetic acid n-butylester and a series o f aliphatic C 4 -C 9
primary and secondary alcohols were found as main constituents. Two isom eric sabinene hydrates
were also isolated as new constituents of rue cells. The data are discussed in com parison to results
obtained with photom ixotrophic cell suspension cultures.
Introduction
C o m p arativ e studies on secondary constitutents in
p hoto sy nthetically active an d h etero tro p h ic cell cul­
tu res o f hig h er plants have show n th at the m ode of
carbon supply and the p a tte rn o f cellular differen tia­
tion g reatly determ in es th e spectrum of accum ulating
com pounds [1—6]. R eversion of p h o to a u to tro p h ic or
p h o to m ix o tro p h ic cell suspension cultures to h e te ro ­
tro p h ic cells and vice versa rep resen ts a m eans to
d eterm in e w h eth er the p rese n ce or absence of func­
tioning chloroplasts co n trib u tes to secondary product
fo rm atio n [4, 5, 7].
In co n tin u atio n of o u r analyses of volatile con­
stitu en ts from p h o to m ix o tro p h ic cell suspension cul­
tu res of R u ta g raveolen s [8 ] we have now reversed
these cells to h etero tro p h ic cell cultures and here
rep o rt a d etailed investigation of the volatiles from
non -g reen rue cell cultures. T hese studies again
prove th e d ifferent p o te n tia l for secondary m e tab o ­
lism of h etero tro p h ic and photosynthetically active
cell cu ltures because th e high yields of aliphatic es-
ters ch aracteristic for th e p h o to m ix o tro p h ic cultures
have n o t b een fo u n d in h etero tro p h ic cells.
Materials and M ethods
C ell cultures
P h o to m ix o tro p h ic cell suspension cultures as
grown for previous investigations [8] w ere reversed
to h etero tro p h ic cell cultures by in cubation in co m ­
plete d ark n ess w ith o u t any alteratio n in th e n u trie n t
m edium . T he suspensions w ere k ep t in 200 ml E rlenm eyer flasks w ith 40 ml m edium and 2,4-D as p h y to ­
h o rm o n e. A ll o th e r details of th e cu ltu re p ro ced u re
and th e collection of cells w ere essentially as p rev i­
ously described [8]. T h e reversion process was fol­
low ed by m easuring th e d ecrease of th e chlorophyll
co n ten t [8 ].
E xtraction p ro c e d u re
O ur established m eth o d s for the ex tractio n of fro ­
zen ( —40 °C) cells (250 g) and for th e fractio n atio n
of cellular co n stitu en ts w ere as described earlier
[8 - 10],
A bbreviations: 2 ,4 -D , 2,4-dichlorophenoxyacetic acid; MS,
mass spectroscopy; G C, gaschromatography; RT, reten­
tion time.
Reprint requests to Prof. Dr. W . Barz.
Verlag der Zeitschrift für Naturforschung, D -7400 Tübingen
0 3 4 1 -0 3 8 2 /8 6 /0 9 0 0 -0 8 0 9 $ 0 1 .3 0 /0
G a sch ro m a to g ra p h y a n d m a ss sp e c tro sc o p y
T he p ro ced u res for g asch ro m ato g rap h y o f volatile
constituents and th e ir identification by m ass spec­
troscopy have b een re p o rte d [8 , 11]. T he q u an tita-
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810
M. Jordan et al. • Volatile Constituents from Cultures o f Ruta graveolens
tive d eterm in a tio n of all com pounds using undecanecarboxylic acid m eth y lester as reference com pound
has also been outlined in earlier rep o rts [8 , 11].
R eference co m p o u n d s
(E )- and (Z )-sab in en e hydrate w ere synthesized
from sabinene according to [16]. All o th e r referen ce
com pounds w ere from the collection of the W eihenstephan institute.
Results
W ithout change of th e culture m edium the p hotom ixotrophic R u ta g ra veo len s cell suspension cultures
as used in previous experim ents [8] w ere tu rn ed into
h etero tro p h ic [4] cell suspensions by incubation in
darkness. T he grow th yield was hereby som ew hat
reduced though these suspensions finally yielded
5 ± 0 .2 g fresh w eight from 1 g inoculum in 14 days.
A fter 4 —5 passages th e chlorophyll con ten t had
d ro p p ed below the level of d etection. Cells for d e te r­
m ination o f volatiles w ere h arvested in early sta tio n ­
ary phase.
Isolation a n d stru ctu ral elu cidation o f volatile
com pounds
T he to ta l m eth an o l extract [9] of 250 g h e te ro ­
trophic rue cells was fra ctio n ated into 3 fractions and
analyzed by gaschrom atography-m ass-spectroscopy
[8 ]. T he identification o f com pounds and th eir q u a n ­
titative d eterm in a tio n w ere carried o u t essentially as
in o u r ea rlier experim ents [8 —11].
F raction 1 (T able I) co n tain ed a series of h y d ro ca r­
bons which could only partially be identified. G eijeren e and p reg eijeren e w ere again found as th e
m ost p ro m in en t constituents in this fraction. T he
com plete structural elucidation o f th e various isogeije re n e com pounds w arrants fu rth e r investigations
though these pro d u cts ap p ear to be artifacts fo rm ed
during the isolation pro ced u re.
F raction 2 (T able II) m ostly co n tain ed a series of
k etones an d , as the m ain co n stitu e n t, acetic acid nb utylester in addition to sm all am o u n ts of various
esters of aliphatic and arom atic acids. In co n trast to
the ph o to m ix o tro p h ic rue cultures [8 ] aliphatic a ld e ­
hydes and prim ary alcohols could n o t be d etec ted in
this fraction.
In fraction 3 (T able III) low am o u n ts o f n u m ero us
prim ary and secondary alcohols could be identified
which have also not b een fo u n d in free form in the
green cell cultures of R. g ra veo len s. Q uite su rp risin g ­
ly the tw o isom eric sabinene h y d rate s w ere found as
h ith erto u n rep o rted co n stitu en ts o f rue cells or
plants. O th e r terp en o id s previously re p o rte d to
occur in R. g ra veo len s such as m yrcene and linalool
as well as lim onene, 1 , 8-cineol an d pinene [ 12] w ere
absent from th e cell extracts. A ccording to M aarse
et al. [13] such com pounds m ight be artifacts o rig i­
nating from im p ro p er isolation p ro ced u res such a
prolonged steam distillation o f volatiles.
T he q u an titativ e d ata in T ab les I —III on th e c
currence o f volatile co n stitu en ts in R . g ra veo len s
h etero tro p h ic cell cultures d o cu m en t th at m ost o f the
com pounds occur in very sm all am o u n ts only. E x cep t
for p reg eijeren e all com pounds o ccur in m uch low er
Table I. G C —MS analysis and quantity o f hydrocarbons (fraction 1) obtained from
heterotrophic cell suspension cultures o f Ruta graveolens. The retention index (R,)
of each com pound was determ ined on a carbowax 20-M column using a defined
temperature gradient [9]. The mass fragments (m/e) are arranged in decreasing
intensity with M" indicating the largest mass fragment observed.
R.
C om pound
Mass fragments
(m /e)
M+
Quantity
(Hg/kg fr.w.)
1315
1325
1429
1473
1492
1536
1577
G eijerene derivative
G eijerene
Isogeijerene A
Isogeijerene B
Isogeijerene C
Isogeijerene D
Pregeijerene
79, 94, 41, 91, 77
79, 94, 41, 77, 91
91, 105, 41, 92, 77
91, 79, 147, 105, 41
91, 147, 41, 105, 79
147. 91, 41, 105, 162
79. 94, 41, 77, 91
147
162
162
162
162
162
162
n.m.
480
n.m.
n.m.
n.m.
n.m.
1040
n.m. — not measured.
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M. Jordan et al. • V olatile C onstituents from Cultures o f Ruta graveolens
Table II. G C —MS analysis and quantity o f ketones and
esters (fraction 2 ) obtained from heterotrophic cell suspen­
sions o f Ruta graveolens. M eans o f identification were com ­
parison o f mass spectra (M S) and o f G C-retention times
(R T ) with authentic reference com pounds.
Com pound
Identi­
fication
3-hexanone
2 -hexanone
3-heptanone
2 -heptanone
4-octanone
3-octanone
2 -octanone
2 -nonanone
2 -decanone
2 -undecanone
2 -tridecanone
iso-octanone (C 8 H 160 )
5-nonanone
p-m enthan-4-ol
acetic acid-n-butylester
-«-nonylester
-2 -undecylester
2 -methylbutyric acid-octyl ester
3-methylbutyric acid-octyl ester
benzoic acid methyl ester
phenylacetic acid methyl ester
M S,
M S,
M S,
MS,
MS
M S,
MS,
MS,
MS,
MS,
M S,
MS
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
RT
RT
RT
RT
Quantity
(Mg/kg fr.w .)
80
200
240
60
10
RT
RT
RT
RT
RT
RT
80
20
15
< 1 0
80
20
< 1 0
RT
RT
RT
RT
RT
RT
RT
RT
RT
20
25
1000
20
50
< 1 0
< 1 0
25
40
Table III. G C —MS analysis and quantity o f aldehydes and
alcohols (fraction 3) obtained from heterotrophic cell sus­
pensions o f Ruta graveolens. M eans o f identification as
m entioned in legend o f Table II.
Com pound
Identi­
fication
(£)-sab in en e hydrate
(Z )-sabinene hydrate
malonic acid dimethylester
3-pentene-2-one
2 -m ethyl- 2 -butenal
(E , £)-2,4-h exad ienal
decanal
benzaldehyde
2 -pentanone
2 -m ethylbutan- 2 -ol
butan- 2 -ol
pentan- 2 -ol
heptan-3-ol
octan-4-ol
octan-3-ol
nonan-5-ol
2 -m ethylpropanol
«-butanol
«-pentanol
rt-hexanol
Ai-octanol
M S,
MS,
MS,
M S,
MS
MS,
MS,
M S,
MS,
MS
M S,
M S,
MS,
M S,
M S,
MS,
M S,
MS,
M S,
M S,
M S,
RT
RT
RT
RT
RT
RT
RT
RT
Quantity
( Mg/kg fr.w.)
25
70
< 1 0
20
60
25
10
< 1 0
20
20
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
RT
100
20
20
< 1 0
< 1 0
< 1 0
200
100
40
20
10
811
q uantity th an m easu red for th e volatiles of green rue
cells [8].
Discussion
T hese and o u r previous studies [8 ] b ased on d e ­
tailed and highly sensitive analyses d o cu m en t th at
cell suspension cultures of R. g ra veo len s possess th e
potential to form a large n u m b e r of volatiles. In view
of the com paratively low yield of th e co n stitu en ts it
m ust be rem em b ered th a t no selection processes for
high yielding cell lines have b een co n d u cted so far.
T he stru ctu res of th e co m pounds listed in T ables I
to III also show th a t a variety of d ifferent secondary
biosynthetic pathw ays m ust be expressed in these
h etero tro p h ic rue cells. F o rm atio n o f the h y d ro car­
bons g eijerene and p reg eijeren e w hich are ro o t co n ­
stituents of R. g ra veo len s [15] is again found (T a­
ble I). E xpression o f this biosynthetic pathw ay in cell
cultures ap p ears to be largely in d e p en d e n d from the
type of cell culture because th e p h o to m ix o tro p h ic
rue cell suspensions [8 ] show ed the sam e p ro p erty
though in m uch sm aller am ounts.
In general ag reem en t w ith previous research ers [1,
2, 14] using green and h etero tro p h ic callus cultures
the chem ical com position of th e volatiles of lightgrown and dark-grow n R. g ra veo len s cells is striking­
ly different. O u r d etailed G C —MS d ata clearly prove
these differences (T ables II, III and [8]).
T hus, the ch aracteristic co n stitu en ts of p h o to ­
m ixotrophic cells are the C 9-C 13 m eth y lk eto n es and a
series of esters of 2-m ethylbutyric acid and 3-m ethylbutyric acid esterified w ith straight chain o r b ranched
C8-C n alcohols. E xcept for trace am o u n ts of 2-undecan o n e, 2 -trid ecan o n e and th e o cty lester of
2-m ethylbutyric acid and 3-m ethylbutyric acid these
com pounds are practically ab sen t from th e h e te ro ­
trophic cells. It is safe to p o stu late th a t th ese ch arac­
teristic constituents of p h o tosynthetically active cells
are the result of ch loroplast m etabolism .
O n the o th e r h an d , h etero tro p h ic rue cells p ro ­
duce a com plex m ixture of C 6-C 8 k eto n es, acetic acid
«-butylester and a series of C 4-C 9 prim ary and sec­
ondary alcohols which have n ot been found in the
green cell cultures. It m ust again be m en tio n ed th at
this p ro n o u n ced shift in th e p a tte rn of secondary
volatile co n stitu en ts is th e result of reversion of a
green to a no n -g reen culture w ithout any change in
the chem ical com position of th e n u trie n t m edium
and w ithout any exogenous alteratio n of th e phytohorm onal situation of th e cells.
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812
M. Jordan et al. • Volatile Constituents from Cultures o f Ruta graveolens
F u rth e r application of reversion experim ents of
g reen to non-green cells and vice versa obviously of­
fers a m eans to analyze how secondary biosynthesis
is effected by cellular carbon m etabolism and by the
p resence of functioning chloroplasts. F u rth e rm o re ,
com parative studies using these two types of plant
cell cultures obviously w idens the applicability of the
cell culture technique for investigations on leaf and/
or ro o t co n stituents of higher plants because the es­
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(1975).
[2] M. N agel, D octoral Thesis, U niv. Tübingen 1974.
[3] W. Barz, H . H erzbeck, W. H üsem ann, G. Schneiders,
and H. M angold, Planta M ed. 40, 137 (1980).
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ture 1982 (A . Fujiwara, e d .), pp. 245—248, Proc. 5th.
Intern. Congr. Plant Tissue and Cell Cultures, Tokyo
1982.
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and W. Barz, Z. Naturforsch. 3 9 c , 525 (1984).
tablishm ent of b e tte r p roducing system s seem s to be
m ore feasable.
A ck n o w le d g em e n ts
Financial su p p o rt by D eu tsch e F orschungsge­
m einschaft (F o rsch erg ru p p e S ek u n d äre N aturstoffe/
Z ellk u ltu ren ) and F onds d er C hem ischen Industrie
(to W. B arz) is gratefully acknow ledged.
[9] F. Drawert, W. H eim ann, R. Em berger, and R.
Tressl, Chromatographia 2, 57 (1969).
[10] P. Schreier, F. Drawert, A . Junker, and W. M ick, Z.
Lebensm. Unters. Forsch. 164, 188 (1977).
[11] F. Drawert, R. G. Berger, B. Bröker, and S. N itz,
Chem. M ikrobiol. Technol. Lebensm . 7, 179 (1982).
[12] N. Christoph, D octoral T hesis, Technical University
M unich, 1983.
[13] H. M aarse, L. M. N ijssen, and M. N ow ak, in:
Ätherische Öle (K. H. K ubeczka, e d .), Thiem e V er­
lag, Stuttgart 1982.
[14] E. Reinhard, G. Corduan, and O. H. V olk, Planta
Med. 16, 8 (1968).
[15] K. H. K ubeczka, Flora A 158, 519 (1967).
[16] H. C. Brown and P. G eogh egan , jr., J. A m er. Chem.
Soc. 89, 1522 (1967).
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