PlantCell
Reports
Plant Cell Reports (1986) 3 : 231-233
© Springer-Verlag 1986
Phytochemical constituents of cultured cells
of Eucalyptus tereticornis SM.
R. Venkateswara, S. Sankara Rao, and C. S. Vaidyanathan
Enzymology and Plant Biochemistry Laboratory, Department of Biochemistry, Indian Institute of Science, B angalore 560 012, India
Received August 23, 1985 / Revised version received April 10, 1986 - Communicated by F. Constabel
ABSTRACT
Callus induced Irom shoot explants ol Eucalyptus
t e r e t i c o r n i s was maintained for eight months on a
defined MS medium. The lipid composition of t h e callus of E. t e r e t i c o r n i s were [3-sitosterol, s t i g m a s t e r o l and
cholesterol. In addition, we report the p r e s e n c e of a
Ilavanoidal glycoside, aglycon identified as Kaempferol.
F u r t h e r , t h e p r e s e n c e of 2,3-dihydroxybenzaldehyde and
3,4-dihydroxyphenyl a c e t i c acid was established from t h e
m e t h a n o l fraction.
List ol Abbreviations" 2,t~-D = 2,4-Dichlorophenoxyacetic
acid, Kn = Kinetin, NAA = l - N a p h t h a l e n e a c e t i c acid,
IAA = Indole-3-acetic acid, BA = 6-Benzylaminopurine.
INTRODUCTION
Eucalyptus is a large genus of subtropical and
w a r m t e m p e r a t e e v e r g r e e n a r o m a t i c t r e e s indigenous to
Australia, T a s m a n i a and New Guir~ea. About 100 species
have been tried in India for valuable t i m b e r , pulp and
paper production.
Most of the species are popularly known as Gum
t r e e s although the exudation from t h e m is not a gum but
an a s t r i n g e n t , t a n n i f e r o u s polyphenol called Kino. Bark
of Eucalyptus species contains varying proportions of
polyphenols. They c o n s t i t u t e an i m p o r t a n t source of
tanning m a t e r i a l s (Linchan, 1981).
E. tereticornis.
MATERIAL AND METHODS
Nodal and internodal s e g m e n t s from t e n d e r portions
of a c t i v e l y growing b r a n c h e s of E. t e r e t i c o r n i s plants
raised in the Indian I n s t i t u t e of Science nursery were
c o l l e c t e d . T h e y were surface sterilized with 0,1% m e r c u r i c
chloride solution for 15 minutes and rinsed thoroughly
with sterile distilled water. The m a t e r i a l was f u r t h e r cut
into pieces of suitable size (10mm) and implanted onto
c u l t u r e media.
Culture media composed of mineral salts and v i t a mins of Murashige and Skoog (1962) with 2% sucrose and
800 m g / L Polyvinylpyrrolidone (PVP). Only agar solidified
media (0.7%) were used.
A l l chemicals used were of analytical grade
(British Drug House, E. Merck, Sigma and Difco). The
following combinations of growth supplements were used
for callus i n i t i a t i o n .
1.
2.
3.
MS Basal medium + 2,4-D (5 mg/L) + Kn (0.5 mg/L)
MS Basal medium + 2,~-D {2 rag/L)
MS Basal medium + NAA(1 m g / L ) + IAA(0.2 mg/L)
+ BA (1 mg/L).
Callus was successfully maintained for 8 months with a
passage period of 3 weeks on MS(2) medium.
Culture media in tubes were autoclaved at 15
Ibs/cm 2 for 15 minutes. Cultures were incubated at
25 _+ 2°C, exposed to an a r t i f i c i a l neon light of 2001Jm/m2/
sec with dark light cycles of 8/16 hours.
Investigations of the phenolics of Eucalyptus have
revealed t h e p r e s e n c e of eucalyptin, betulinic acid and
urosolic acid from E.citriodora Wild. (Dayal Rameshwar,
1982), euglobal Ill ~-r0m E._.L.globulus (Sawad, 1980), robusteol-A (Qin Cuo-We, 1981) and l a t e r 7 - 0 - m e t h y l a r o m a dendin, a r o m a d e n d r i n and fustin from E . c i t r i o d o r a (Dayal
R a m e s h w a r , 1981). Morolic acid was c h a r a c t e r i s e d from
E__L. grossa by Cannon (1981). Earlier Wollenweber (1981)
reported flavanoids from t h e c u t i c u l a r layer of t h e leaf
of E. o v a t a . Abd-Alla (1980) identified flavanoidal glycosides in two c h e m i c a l races of E. camaldulensis c o r r e s ponding to north and south populations of Australia.
Fresh callus (100 gm) at the end of six week period
was separated from the media and washed with double
distilled water and lyophilised. The lyophilised callus
powder was extracted exhaustively with n-hexane in
soxhJet apparatus. The hexane fraction was separated and
the marc was dried and extracted with ethyl acetate
followed by another e x t r a c t i o n with methanol.
A t t e m p t s to p r o p a g a t e Eucalyptus through tissue
culture have been successful (De Fossard 1978, Gupta
et al 1978, 1982, 1983, Hartney and Barker 1980, Mehrapalta 1982, Bennet and McComb 1982, Durand-Cresswel
1982, Sankara Ran and V e n k a t e s w a r a 1985). The phytoc h e m i c a l i n v e s t i g a t i o n s of t h e callus have not been c a r ried out so far and we wish to report the g e n e r a t i o n o£
callus and subsequent p h y t o c h e m i c a l i n v e s t i g a t i o n s of
The hexane fraction was dried and to the residue
chloroform was added. The chloroform soluble fraction
was separated to which hexane was added and kept at
room temperature. A white residue (A) was obtained posit i v e to sterols. The chloroform insoluble hexane fraction
was a greasy y e l l o w semisolid (60 rag). The white residue
(A), (25 rag) was chromatographed on a silicagel column
( 1 . 5 x 2 0 c m ) . T h e column when eluted with hex:CHCl 3
Offprint requests to: R. Venkateswara
Phytochemica] Examination
232
Analysis of phenois in m e t h a n o l fractions: C h r o m a tographic analysis was done with paper on Whatman No.10
paper and TLC on silicagei. A u t h e n t i c samples tried
include, 2,3-dihydroxybenzaldehyde, 3,*4-dihydroxyphenyla c e t i c acid, DL-t+-hydroxy 3-methoxy mandelic acid~
3 , 5 - d i h y d r o x y p h e n y l a c e t i c acid~ p r o t o c a t e c h u i c
acid~
t - c i n n a m i c acid, p-coumaric acid. Spots were identified
by fluorescence and Rf values. Freshly diazotised pnitroaniline and sodium hydroxide solutions were used
as spray reagents.
(50:50), CHCI3 (100), CHCL3 :EtOAc (70:30) gave spots
corresponding to 2 phytosterots. A silicagel TLC of
the f r a c t i o n with h e x : C H C l a : E t O A c (70:20:10) indicated
homogeneity of the samples.
Ethyl a c e t a t e f r a c t i o n when partitioned with e t h e r
and c h r o m a t o g r a p h e d on silicagel column (1.5 x 2 0 cm)
gave f r a c t i o n s positive to phenolics. The ethe;e~EtOAc
(50:50) f r a c t i o n upon repeated c r y s t a l l i z a t i o n in a c e t o n e e t h e r mixture gave pale yellow crystals (8 rag) which
was identified as a flavanoidal glycoside by c h e m i c a l
d e g r a d a t i o n and s p e c t r a l analysis.
RESULTS AND DISCUSSION
Callus from the shoot s e g m e n t s of E . t e r e t i c o r n i s
were induced under light at 25 + 2°C. The callus was
obtained with all t h e t h r e e MS (1),(2) and (3) media
tried. Callus was white and friable in medium supplemented with 2,#-D with or without Kn whereas t h e medium
containing BA~NAA and IAA produced coloured c o m p a c t
slightly nodulated callus. The callus grew vigorously
in MS(2) media and required subculturing every t h r e e
weeks. During routine passage, portions of the callus
were seen to develop red pigment. The c h e m i c a l constit u e n t s of the callus were repeatedly e x t r a c t e d in nhexane, EtOAc and metbanoi. The hexane f r a c t i o n upon
partition with CHCIs and the subsequent silicagel column
c h r o m a t o g r a p h y of CHCIs
soluble f r a c t i o n afforded
pure f r a c t i o n s of a major and two minor sterols identified
by c h r o m a t o g r a p h i c behaviour on TLC. I R s p e c t r a of major
sterol - 3200 cm-q (-OH), 1800 cm - I ( C = C ) ,
800 cm -~
(-CHa-) was superimposable with a u t h e n t i c 8 - s i t o s t e r o l 3200 cm -1 , 1750 cm -1 , 900 c m - 1 . Q u a n t i t a t i v e l y the
components of pbytosterols in the callus were d i f f e r e n t
with B-sitosteroi as the main c o m p o n e n t (70%)~ s tigrnast e r o l and cholesterol present only in t r a c e amounts.
Methanol f r a c t i o n when c h r o m a t o g r a p h e d on silicagel column gave f r a c t i o n s positive to phenols. A TLC of
the f r a c t i o n s with a u t h e n t i c phenols was carried out
using the following systems. CHCI3 : m e t h a n o l (80:20),
E t O A c : m e t h a n o l : H A C (80:18:2) and 2% HCOOH.
P h y t o s t e r o l s were c h a r a c t e r i s e d by c h r o m a t o g r a phy and spectroscopy. Silicagel TLC of the samples with
a u t h e n t i c samples of cholesterol, cholesterol a c e t a t %
stigmasterol,
B-sitosterol was carried out. Cochromatography of a m i x t u r e of a u t h e n t i c sterols and sample
was carried out in solvent systems h e x : C H C I 3 : E t O A c
(70:20:10), CHCl3:hex (50:50), ButOH:HAC:H20 (50:'40:10).
The IR s p e c t r a were recorded on P e r k i n - E l m e r IR
s p e c t r o p h o t o m e t e r by the nujol method.
The flavanoidal glycoside (5 rag) was dissolved in
20% HCL (6 ml) and boiled for 10 minutes to hydrolyse
it. The solution was c o o l e d a f t e r addition of w a t e r
(100 ml), then it was washed t h r i c e with benzene and
dried in vacuum. The yield of aglycon was 3.2 rag. The
aglycon when t r e a t e d with Shinoda's reagent gave
a bright red colour which was identical with t h a t of
a u t h e n t i c kaempferol. C h r o m a t o g r a p h i c analysis was done
on Whatman No.10 paper. Solvent systems used were
i s o p r o p : N H a : H 2 0 (80:10:10), 2% HCOOH, benzene:HAC:
H20 (.40:.40:20).
The ultra violet absorption s p e c t r a of the aglycon
measured in m e t h a n o l containing 0.01% HCI had an absorption maxima of carbonyl group a t 315 nm (kaempferol
I/
I I
|1
V
/
I .... ./
x
.....
.......
!
"ii
(b)
r ..............
~,l
f
r4
....
,~
r
~ j
~;
I
i
,I
I
,
I
I
I
I
I°I
3500
3000
I
I
I
I
I
2500
2000
1800
1600
1400
WAVENUMBER
I
1200
(cm -1)
Fig.1. IR spectra of the flavanoidal aglycon from cultured cells of E.tereticornis
Continuous line: Kaempferol, broken line: sample.
I
1000
233
320 nm) and aromatic peak at 280 nm (kaempferol 280 nm).
The IR spectra of the sample - 3tt20cm -1 ,3380cm -I,
3220cm -1, 3180 Cm-l(multiple OH peaks), strong peak at
1700cm -1 (carbonyl), 1600 c m - l ( A r o m a t i c ) and kaempferol
_ 3t+30cm -I ,3350cm -1 ,3300cm -1 ,3150cm-1 ,1700cm -1,
1600cm -1 was superimposable. We identified the aglycon
moiety by hydrolysing t h e g l y c o s i d e . Q u a l i t a t i v e analysis,
c h r o m a t o g r a p h i c and s p e c t r a l d a t a proved t h a t aglycon
was k a e m p f e r o l . The Fehlings r e a c t i o n was positive
indicating t h e g l y c o s i d i c linkage (Fig.l) Table 1).
The phenolics of the m e t h a n o l f r a c t i o n upon
silicagel c h r o m a t o g r a p h y afforded f r a c t i o n s positive to
phenolics identified by spot t e s t s with freshly diazotised
p-nitroaniline and NaOH solutions. We could identify
only two phenolics Pl and P2 by c h r o m a t o g r a p h y with
a u t h e n t i c samples. The IR s p e c t r a of P - 3350 crn-q
(-OH), 1650 cm-1
(carbonyl), 1660 cm-1
(Aromatic)
and 2~3-dihydroxybenzaldehyde - 3300 cm -1, 1650 cm -1,
1600 c m - 1 and P2 - split peak a t 3#00 cm -1, 3350 c m - 1
(-OH groups)) 1700 cm -1 (carbonyl), 1590 cm -1 (aromatic)
and 3,#-dihydroxyphenylacetic acid - 3#~0 cm -1 , 3320
c m - 1 , 1700 c m - 1 ) 1600 cm-1 , 900 cm -1 were superimposable.
Table 1
P r o p e r t i e s of the aglycon from the cultured cells of
E. t e r e t i c o r n i s
Rf values (X 100)
Spectral D a t a
!soprcp :
NH "H O
Benzenc:
HAC.H20
9%
'-'
HCOOH
Sample from
cultured
cells
#5
30
70
315
280
Authentic
kaempferol
#5
28
65
320
280
8071010
t max (nm)
During the tissue culture of E. tereticornis the
accumulation of phenolics in the absence of PVP in the
culture media was a routine problem which lead us to
investigate the chemical composition of the aromatic
metabolites~ in cultured cells. To our knowledge, this is
t h e first report on t h e p h y t o c h e m i s t r y of t h e callus of
Eucalyptus although report of flavanoidal glycosides
from t h e leaves of E. citriodora, E. o v a t a , E. c a m a l d u lensis are available (Dayal R a m e s h w a r 1980, 1981,
Wollenweber 1981, Abd-Alla 1980). Abd-Alla (1980)
identified
kaempferol-3-glycoside
from t h e leaf of
E. camaldulensis. We report for the first t i m e isolation
of k a e m p f e r o l glycoside from cultured cells of E . t e r e t i cornis. It appears, t h a t a major hydroxylation system
is a c t i v e as seen from the n a t u r e of aromatic products
in the callus of E. tereticornis. We believe it is possible
to alter t h e biosynthetic profile of phenolics in the
callus by changing cultural conditions like light and
hormonal manipulation.
ACKNOWLEDGEMENT
We wish to acknowledge Dr. Dayal Rameshwar~
Forest Research Institute, Dehradun for a u t h e n t i c samples
and Prof. Y. Yamada for the e n c o u r a g e m e n t to first
author during his r e c e n t visit to Kyoto, 3apan.
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