ldi an Journal of Chemi stry
'01. 4 1B, April 2002, pp. 845- 853
The chemistry of fossils of Ginkgo and its ancestors
S Ghosal*, A V Muruganandam, K S Satyan & S Chauh an
R&D Centre, Indi an Herbs Ltd. , Sharda Nagar, Saharanpur 247 00 I, India
Received 21 Decelllber 2000; accepted 8 August 200 I
Chemical analysis of orga ni c solve nt extrac tives of tcn foss il-taxa (Gymnosperms) of the Paleozoic - Mesozoic eras
indicate the chemical preservation of five main classes of naturall y occurring compounds, viz. ginkgo lic ac ids, terpcnoidal
laclO nes (gink golides A & B, bilobalide), biO avo nes (amentoOavo ne, bilobetin ), procya nidins and anthocyani dins, in nine of
them. Thc contents of thcse compounds are highest in Gill kgo biloba-foss il. The survival feat of G.biloba is remarkabl e. It is
the las t survivi ng represc ntati ve of a race that, ages before the evolution of man, occupi ed a ccntral position among the
Ooras of the world. The preserva tion of the consortium of orga nic compounds and the longev ity of G. bi/oba must in part be
due to the poten t antiox ida nt fun ction of some of these compounds and the robust cage structures/molecul ar laminates of the
metal ion complexes of the terpenoida l lac tones ancl the lipophili c phenolic ac ids. A poss ible proge nitor-progeny
re latio nship betwee n Glossopteris/Dadoxyloll and Gillkgo was conside red on the basis of simil arities of their chemica l
constituents.
l inkgo biloba Linn. (Ginkgoaceae) , th e maiden hair
I'ee, fondly called by herbali sts as the 'living fo ss il'
s the last survivin g representative of a race that,
ges before th e evolution of man , occupied a central
losition in the floras of th e world I. In early
:eological times the family Ginkgoaceae, which
ncluded several genera and a large number of
pecies, was almost cosmopolitan and was
epresented in many parts of Gondwana. Also,
lossible Ginkgo - ancestors, e.g. Glossorpteris and
)adoxylon, occurred in many parts of the world
luring the Permian period (300 - 250 million-year'efore-present, mybp). As recently as the late
'ertiary period (ca. 1.6 mybp), relatives of Ginkgo
:xisted in Spitzbergen and other high northern
egions 2 . Quite in contrast to its very recent
:onfinement, in the wild state, to a small area in the
astern China, members of the Ginkgo ranged far
md wide over the surface of the earth. The greatest
lays in the past annals of Ginkgo and its projected
mmediate
relatives,
e.g.
Glossopteris
and
)adoxylon (Gymnosperms), seem to have transpired
luring the Mesozoic era (250 - 60 mybp). Leaf
emains have turned up from geographically widely
epa rated localities where Triassic (220 mybp),
:specially Jurassic (180 mybp), and to a lesser extent
:halk age, rocks are found . In the Cretaceous period,
120 - 60 mybp) Ginkgo flourished in the subpol ar
egions which were, in all probability, much warmer
II1d more suited to comparatively luxuriant plant
~ rowth than at present2 .
The survival feat of G.biloba is indeed remarkable.
It has not only survived for hundreds of million years
of challenging geological and chemical onslaughts
including the ice age on the surface of the earth , it is
one of the very few living species that survived the
man's most destructive act, - the nuclear bombing at
Hiroshima. The tree of G.biloba, at Hiroshima, that
re-emerged after the devastation was normal and did
not show any chromosomal aberration due to th e
nuclear radiation. This could be due, at least partly, to
the strong antioxidant defence that G.biloba is known
to possess 3.4. Plants existing in Permo-Carboniferous
period (Ca. 300 - 250 mybp), when the percentage of
oxygen in the atmosphere was very high (>35 %) and
fluctuating 5 had, obviously, enhanced antioxidant
defence. It was, therefore, considered fascinating to
study the chemical constituents of the different parts
of fossil remains of Ginkgo and its possible ancestors,
such as those belonging to Glossopteridales 6 •
The plants included in the Glossopteridales are all
extinct representative of a flora that once dominated
the continent - Gondwana (which included the Indian
Peninsula) during the Permo-Carboniferous period
(Paleozoic era) . By far the most common element of
the flora is Glossopteris. There is evidence that the
leaves of Glossopteris were borne on long and short
shoots as in modern Ginkgo (Gymnosperm)6. The
stems of Glossopteris are typical of Gymnosperms
with abundant pycnoxylic wood and a pith surrounded
by a ring of primary bundles. Despite these
similarities,
some
other
morpho-taxonomic
INDIAN J. CHEM ., SEC B, APRIL 2002
846
considerations raised controversies? in respect of the
projected ancestor-progeny relationship of Glossopteris and Ginkgo. Chemotaxonomic analysis was,
therefore, warranted to settle this polemic. Again,
beds where Glossopteridales are dominant, yield a
great number of pycnoxylic stems with wood of the
Dadoxylon and Neol11ariopteris species (Gymnosperms)6.? These two fossils also constituted the
subject of the title study .
Ginkgophytes and Glossopterid ales have received
considerable attention from paleo botanical standpoint
but their chemistry has so far remained unexplored .
As a first step, we have studied the chemistry of ten
fossils of Ginkgo, Glossopteris, Dadoxylon and
Neol11ariopteris, of the early-Permian-JurassicCretaceous periods (260 - 60 mybp), collected from
different regions of India, England and USA
Cfable I).
Results and Discussion
The list of the test samples is given in Table I. The
chemical analysis was focussed on five main classes
of compounds, viz. ginkgolic acids, terpenoidal
lactones, biflavones, procyanidins and an thocyanidins, occurring in the extinct (fossils) and extant
(G.biloba) members of Ginkgo and related taxa.
Additionally, the important metal ions (Fe, Cu, Zn)
which constitute some crucial antioxidant defence and
other enzyme systems in the living G.biloba 8, were
analysed in the extinct and extant Ginkgo and in
related fossils (Table II). The comparative analytical
data of the chemical constituents of the fossils vis-avis living G.biloba are summarized in Table II.
Table I SI
No.
Fossil
Genus/Species
Ginkgo bi/oba
The ginkgolic acids are represented by the genel
structures 1a-f; the terpene lactones, viz. ginkgolid
A 2a, ginkgolide-B 2b and bilobalide 2c; tl
biflavones, amentoflavone 3a and bilobetin 3b; tl
procyanidins 4, stereochemistry undetermined) ; aJ
the anthocyanidins, viz cyanidin Sa and delphinid
by 5b (Chart 1). The separation and establishment
identities of these compounds were accomplished I
co lumn
chromatography,
prep.
TLC
aJ
comprehensive HPTLC, HPLC, GLC and GC-N
analyses using authentic markers (for detection aJ
for calibration of spectra for quantification).
The d ifferent parts of living G.biloba were rich
ginkgo lic acids. Among the fossil-taxa also,G.biloi
contained highest amount of these compound
Glossopters and Dadoxylon also contained readi
detectable amounts of ginkgolic acids, whi
Neol11ariopteris was devoid of these compounl
(Table II) .
The
known
natural
occurrence
ginkgolic/anacardic acids has been limited
members of only three primitive fami lies, vi
Anacardiaceae, Ginkgoaceae and Myristicaceae9. f<
several reasons, however, the present detection '
ginkgolic acids in the fossil-taxa should not be view(
as mere occurrence of phenolic lipids. Studies
biosynthesis of ginkgolic acids in G.biloba showed
that the salicylic acid moiety was synthesized by
po lyketide pathway via malonic acid. TI
alkyllalkenyl chains of ginkgolic acids were in
different state and sub-cellular site of activatio
Compounds and chains, e.g. malonic acid ar
palmitic acid, that were used for the biosynthesis I
I
List and details of the fossil samples analysed
Fossi l part
Period of natural existence in mybp
J urassic/200-180
Location of collection
Y
2
G. biloba (Sc.Td.36 I)
3
4
G. bi/oba
G. biloba
5
G.digitata
Cretace us/ 120-60
A
6
G.hullonii
Jurrassic
7
Glossopteris sp. (PPUCu)
8
Glossopteris sp.
st
Y
S
S
9
10
Dadoxylon sp.
w
S
Neomariopteris sp.
(Sc.Td. 131)
S
S
R
p
w
Permian/280-240
S
I, leaf; p, petiole; st, stem; w, wood
A, Alaska, USA; R, Rajasthan (National Fossil Park), India; S, Soharjuri Colliery, Bihar, India, Y, Yorkshire, England.
GHOSA L el al. : TH E CH EMI STRY O F FOS S ILS O F GINKGO AND ITS AN C ESTORS
847
Table 11- Comparat ive analys is of th e chem ica l co nstituents of livin g G.biloba, foss il Ginkgo and re lated fo ssil- taxa
Fossil S.No.
(See Table I)
1
Re lati ve abunda nce of co m~o und s (s tructures)"
2a
2b
2c
3a
3b
4
5
Fe
(% w/w)
I
1.9 1
87
108
+
102
222
+
I. II
128
202
++
2.80
146
266
+
0 .77
44
46
tr
0.86
53
58
Ir
1.66
74
90
1.02
98
124
2.07
104
68
++
+
++
++
++
+
+
+
+
++
++
++
+
4
+
tr
tr
+
+
+
++
5
+
tr
tr
+
++
++
+
6
+
tr
tr
+
++
++
tr
7
+
tr
tr
+
++
+
tr
8
tr
tr
tr
tr
++
+
+
+
9
tr
tr
tr
+
+
++
++
3
++
10
++
++
+
Zn
(ppm)
2 .03
+
+
++
b
+
+
2
Metal ions
Cli
(ppm)
+
+
++
+
nd
G.
+++
++
+++
++
+++
++
tr
tr
2.44
1I4
242
Biloba c.d
+++
++
+++
++
++
+
+
+
1.74
92
107
, Percen tage (w/w) in t he solvent extractives; b in the released fossil, aft er soni ca ti o n/ HF treatmen t,C fresh lea f extract,d fresh slem exraCI+++, indi ca tes >0.2 %, see also ref. 3,4; ++ , 0 .1 - 0.2;+ , '" 0. 1;lr, ~ 0.05;-, abse nt ; nd, not detected
;ommon lipids, were fo und to be very poor precursors
'or the side chain of ginkgolic acids 10. This significant
;omplex ity in the bi osy ntheti c path way of ginkgo lic
lcids does seem to prov ide metabo lic advantage to th e
)roducer organisms. The glycerol-3-phos ph ate
1ehydrogenase-inhibiting acti vity of ginkgo lic ac ids"
)rovides th e producer orga ni sms protecti on aga inst
ni crobi al in fes tati on. The stability and selective
onophori c pro perti es of metal ion (Cu , Zn):omp lexes of ginkgo li c ac ids l2 may ex pl ain their wide
;pec trum of bi ological activiti es I3 . 15.
Leaves and stems of liv ing G.bi/oba contained
ligher amounts of gin kgo lides th an bilobalide. By
:ontrast, the bil obali de contents of di fferent parts of
he foss ils we re hi gher than their gin kgo lides co ntent
Table I). The biosynthesis of the terpenoids
Jri ginall y in vo lved an enzy me co mplex with an
so prene carri er protein (lCP), simil ar to th at of the
·atty ac id sy nthetase. The latter catalyzes th e
)roducti on of C I 6 and C 1Hfa tty ac ids from acetyl -CoA
:traightaway with no smaller intermedi ates (e.g. C4 ,
=8 ac ids) being fo rmed l6. The evolutionaril y most
)fI mltlve terpenoid biosy ntheti c systems in
Jrokaryotes were thus directed to hi gher pulymers
tri-C3o , tetra-C40 , terpenes). Prokaryotes lack mono, esq ui - and diterpenes . These major terpene
Ji osy ntheti c ta rgets in prokaryotes were modi fied
luring the evoluti on of eukaryotes to release farn esy l
Jy rophosphate, from the ICP-complex, (leadin g to
esq ui terpe nes) and geranyl-ge ranyl pyrophos phate
(to produce diterpenes) 16. The sesqui- and diterpenes
thus produced interfered with the biosy nthesis of triand tetraterpenes in competitors and predators, and/or
detrimentally changed the permeabili ty of their cell
membranes. In other wo rds, th e sesqui - and diterpenes
were evolved in eukaryotes for their abi lity to act as
defensive agent. In addition to thi s, the presence of
terti ary butyl substituent and the poly lactone rings,
with cage - structures, are two unique fea tures of the
terpenoids of Ginkgo and related species. Also, the
extent and types of the oxygen fun cti ons in the
ginkgolides and bilobalide, fo und in the Ginkgo and
related fo ss il s, sugges t th at these spec ies in the remote
past developed th eir sesqui- and diterpene
bi osyntheti c path ways under a situat ion of hyperbaric
oxygen press ure. Indeed, durin g that peri od, th e
atmospheri c oxygen percentage was both hi gh (>
3.5%) and fluctu ating5.
Fl avonoids are characteri stic of land plants and
presumably evolved mainl y durin g th e Devon ian
period (350 mybp)16. The elaborati on of the most
primitive flavo noids, vi z. th e biflavones, in land
pl ants, seemed to fo llow the malonyl-coenzyme A
pathways (leading to phenolic acids and lipids)1 7. It is
argued th at the initi al fun cti on of biflavo nes (and
equi valents) was th at of a filter aga inst UV irrad iati on
and as intern al reg ul atory agen t, e.g. in redox
reacti ons 17. Fl avo noids often occ ur in higher
concentrati ons and in greater structural diversi ty in
the leaves (and stems) of pl ants th an in th e oth er parts
INDIAN J. CHEM., SEC B, APR IL 2002
848
a: R
=C 1JH27
- H
= C 1Jli2S
R = C 'SHJ'
b : R
C :
,.... C(CH Jb
H -
d:R=C, sH 29
o ;
e : R = C 17HJJ
f :R
H
= C 17H31
1: Silyl Ginkgolates
. £<l: R
~ b : R
H
=H
=OH
OH
;! a : Amentoflavonc , R
;! b : Bilobe t in R CH J
=
=H
H
R
OH
H
HO
OH
OH
HO
HO
~
: Procyanidins
(Stereochemistry at C 2,3,2", 3" undetermincd)
~:l :
~
=
Cyanidin, R H
b : Delphinidin, R OH
=
Chart I -Structures of main che mi cal const ituents of Ginkgo & related fossils
(fl owers, wood ). Obviously , their role in the leaves
must be different th an in other parts of prod ucer
organisms. Consistent with thi s postul ate, the hi gher
ab und ance of the bifl avo noids in the leaves and stems
of th e fossi ls (Tab le II) would seem to suggest their
essentiall y protective ro le. The C - C linkages (3a,b,
Chart 1) are a hallmark of almost all ' primiti ve'
f1avo noids. The ' target' compound fo r the fl avonoid
biosy nthesis in Ginkgo and in related foss il taxa was
apigenin (the monomer of amentoflavone, 3a). The
C3" Cw· -linkage of two ap igenin mo ieties produced
amentoflavone (3a), wh ich on partial 0 - meth ylati on
prod uced bilobetin (3b). Surprisi ngly, the con'es-
ponding monomer (apigenin or its C 4· - 0 - metl'
analogce) was absent in th e fossil-taxa, analysed
this study. The preservation of bi f1avon es in t
fo ssil s supports th e postul ate about the robustness
the biflavo nes. These compounds have ~ tood t
of
geological
and
chemic
onslaughts
transformations fo r hundreds of million yea rs '
earth .
The woods and stems of Ginkgo, GLossopTeris a
Dadoxylon fossils co ntained hi ghes t concentrations
the procyanidins (4) and anthocyani dins (Sa,
Cfable I). NeolllariopTeris also showed similarity
the other fossil in respect of th e occurrence
GHOSAL el al. : THE CHEM ISTRY OF FOSSILS OF GINKGO AND ITS ANCESTORS
849
procyanidins and the anthocyan idins, but in lower
concentrations. Procyanidins sparsely occurring in
gym nosperms are believed to act as a constitutive
defence factor I7 .18 . They are reported to be
potent antioxidants, anti-mutagen ic and radiation
protectors 18.
routinely used as developers for the three major
classes of compounds, viz. ginkgol ic ac ids,
terpenoidal lactones, and biflavonoids, respectivel y.
For the separation of ginkgolic acids from other
common lipids by prep. TLC, chloroform - methanol 10 M NH 4 0H (65:30:5) was used .
The fossils (Table I) showed remarkable consistency in respect of their metal ion contents
(Table II) . These metal ions are the essential
constituents of a number of vital enzymes in
euk aryotic cells 5 .
HPLC - Waters Associate HPLC assembly
(Millennium 2.10) with a RP C-18 column, equipped
with a PDA (Model 996) detector was employed.
Two mobile phase, viz. methanol - water - aceti c
acid (85: IS :0.2) (so lvent-4) and acetonit ri Ie - water phosphoric acid (80:20:0.1) (solvent-S ), were used.
Additionally, for the analy sis of biflavonoids, in th e
fo ss il leaf extracts, a previously described meth od 2.3 ,
used for the detection and quantification of
biflavones in the leaf extracts of fresh G.biloba, was
followed. This method involved a LichroSorb - Di ol
col umn and a ternary eluti on system , - hexane chloroform (25:75) - tetrahydrofuran (l00) (solvent 6), the biflavones were detected at 330 nm.
Authentic markers were used for setting the
calibration curves and establi shin g th e identity of the
compounds .
Conclusion
Only a few studies were reported in the past four
decades on the chemistry of preserved remnants of
organic compounds in fossil-taxaI 9. 22 . It is too soon to
comment what th e practical significance of the earlier
studies could be in tracing the role of the detected
chemical in the stability and metabolism of the extant
species. The present study, on the other hand , has
demonstrated that fossils have been found in which
the degrad ative processes have not been too severe
and/or their robust chemical compounds have been
competent to withstand the onslaughts of nature for
hundreds of million years. The comparative chemical
profiles of Ginkgo and related taxa (extinct and extant
species) have establi shed thi s point. Thi s study has
shown, for the first time, striking simil arities between
Ginkgo and Glossopteris/Dadoxylon fossi ls, in respect
of their chemical constituents, and thereby indicated
possibility of their progeny-progenitor relationship, a
subj ect which hitherto remained polemical.
Experimental Section
Test samples
The details of th e test samples, their genus/species,
fossil parts analysed, their age, and location of
collection, are summari zed in Table I.
Techniques
HPTLC - CAMAG TLC (Plate material, si lica gel
60 F254 ) evaluat ion assembl y (CATS integration,
V4.0S) was employed. The detection of spots was
done by both quenching and fluorescence modes
using authentic markers. Prior heating of the
developed plates, at 110°C for 20 min , was necessary
to detect th e ginkgo lides and bilobalide spots . Three
solvent systems, viz. chloroform-methanol (90: 10)
(solvent-I) , tol uene - ethy I acetate - acetone - hexane
(4:3:2 : I) (so lvent-2), and ethyl acetate - formic acid acetic ac id - water ( 100: 11 : 11:27) (solvent-3 ), were
GLC - A Hewlett-Packard (HP-S890) instrument,
equipped with a flame-ioni zati on detector (FID),
coupled to a microprocessor controli ed integrator
(HP-3394A) was employed. Gl ass column ( 1.8 m x
0.2 cm i.d) packed with a 3% SE - 30 non-pol ar liquid
phase coated on chromosorb - W (HP) (80 - 120
mesh) was used for the analyses of the sil ylated
terpenoidal lactones. The conditions employed were oven 210°C (2 min -hold), 6°C/min increase upto
320°C (15 min hold), FID, 380°C; carrier gas, N2 , 30
mLimin. To substantiate the findin gs, a previously
described GC-FID method 24 for the analysis of
ginkgolides and bilobalide in the extracts, was also
applied.
GC-MS - Gas chromatographic separati on was
performed on a DB-S (30 m x 0.2Smm i.d. ) ca pillary
colu mn, oven temperature at 80°C (2 min hold) to
250°C (30 min hold) at 10°C/min rise; inj ection
temperature was 250°C. MS data were obtained from
a Varian 3800/Saturn 2000 model instrument,
operating at an ionization potenti al of 20 eV.
Electron Probe Micro-Analyzer (EPMA) - The
analys is of metal ions in the fossil-wood marc
(Scheme II) was performed on a Shimadzu EPMA 8705 instrument. The conditions were ACC.V (kV)
20.0; S.c. (micro -) , 0.20; beam size, 30 - 159 )..tm .
INDIAN J. CI-IEM ., SEC B, APRIL 2002
850
Marker samples
Ginkgolides, bilobalide and gi nk golic acid mi xture
were obtained from Or.Willmar Schwabe GmbH ,
Karl sruh e, Germ any; the biflavonoids were isolated
from G.bi/aba (fresh leaves) followi ng a published
procedure-73 .
Treatment of fossil samples - The ge neral
proced ure followed for th e separation of the fragile
parts (leaf, stem. Petiole) of foss il s, from mineral
matrix, and iso lation of their chemical constituents is
depicted in Scheme I. A different procedure was
followed for the ex tracti on and separation of chemical
constituents from the fossi l- wood(s) (Scheme II).
The first step was to disentang le the specimen, e.g.
the whole or pan of the frag il e organ (leaf, stem,
petiole) from the mineral matrix. This was carefull y
carried out by physical/mechan ical, rather th an
chemica l, means in order to avoid possible chemical
alterati on of the secondary metabolites (Scheme I ).
Treatment of fractions
Fractions f1/-f5 - Gin kgolic ac ids (GA ), occurri
in these two fract ions, (f/fs), were se parated from t
common lipids by prep. TLC on silica gel
(E.Merck). Samples of 10 mg, in chloroform, W(
streaked on 20 x 20 cm pl ates (l mrn thi ckness) a
developed in chlorofo rm - methanol - 10 M NH 4C
(See techniques). The band of GA appearing arou
Rr 0.65 was detected by the blue fluoresc ence unc
UV-B light. TLC scrapings of th e GA - band we
eluted with diethyl eth er, saturated with conc.H<
The extract was washed with water to neutrality, al
the solvent was removed under red uced pressure.
portion of the residue was subj ected to HPTL
(solvent-I ), Rr 0.28 ; Amax, reflectance 243 , 302 nr
HPLC (solvent-5), tR 2.12 min (mai n peak); Am;
POA 240, 290nm. Authentic gin kgo lic ac id mixtu
was used to substantiate the identity of th e compoun
The remaining portion of the mixture of gin kgol
Plant fossil on mineral matrix
(lea f, stem or petiole)
sonicated, ex tracted with
acetone-water (80:20)
l
Marc
Supernatant
I so lve nt
... evapd.
Foss il released
hot methanol
... ex tracti on / 8 h
Res idue
I suspe nded in
... water
I
Methanol ex tract
solvent
evapd .
Aq. suspens ion
extracd. wi th
i) II-hexane,
ii)methy lene ch loride
II-hexane &
methy lene chl oride ex trac t
I so lvent
... evapd.
I-1F (48%)
Aq .mother liq.
l
residue, Fraction - f4,
(Procyanidins anthocyanidins)
column chromoSephadex
LI-I-20/ I-1 20 -MeOI-l
Frac tion-f l
(processed for ginkgolic
acids)
Aq .el uates, residue,
Frac ti on - 1"3
(procyanidin s)
Methanol eluates
resi due,
Fraction - f2
(!Jiflavonoids & terpenoidal
lactones)
Scheme I-S eparation of fossils and extrac ti on of their chemica l constituents
GHOSAL et al. : THE CHEM ISTRY OF FOSS ILS OF GINKGO AND ITS ANCESTORS
851
G.biloba fossil wood -powder (110 g)
ex tracti on/Soxhlet,
methanol, 30 h.
Meth anol extract
Marc
tested for metal ions
(EPMA)
t evapd./red. press.
Res idue (534 mg)
t suspended in water
Ag. suspension
I
J
kept at ordinary
temperature for
2 days
t Solve nt evapd.
gu mmy residue (52 mg), Fraction -f5
(processed for ginkgoli c acids)
Ag. mother lig .
ppt. (4 1 mg),
Fraction -f6
(m ixture of terpene lac tones)
co lumn
chromat.,
celi te,
chl oro form ethanol (traces)
ginkgolide-A ( 10 mg), gi nkgolide-B.
(7 mg), bilobalide ( 18 mg), identities
establ ished by HPTLC, GLC and
GC-MS (usin g markers)
l
Ag . concentrate
Benzene extract
ex tracted with
chl oroform
Chloroform
extract
~ evapd
Resi due
(68 mg) .
Frac ti on -f7
(bi fla vo noids)
(HPLC)
Ag. layer
tevaPd .
Residue,
fraction-fR
procyanidins
cyan idin &
delphinidin
(HPTLC prep.
TLC, UV )
Scheme II-Extraction of chem ica l constituents fro m foss il-wood
lC ids was silylated . Th e mixture of gink go li c acids
:GA) (0.5 mg), was di ssolved in pyridine (0.2 mL)
ll1d treated with hexameth y l di slane-trimethyl
:hl oros ilane (2 : I, 0.1 mL) to form the corres pondin g
;il ylg inkgo latcs (OTMS derivatives). GC-MS anal ysis
)f the OTMS derivatives was carried out, unde r the
:onditi o ns ment io ned before (See techniques) and the
·esults arc s umm arized in Table III. Th e OTMS
Jer;vatives pre pared fro m the fraction-f l o f
VealIIariopteris (leaf fossil) did no t show the presence
)f ginkgolates .
The relative percentage composition of the C 6 a lkyl/alk eny l c ha ins of g ink go lic acids in the fo ssil
and in fresh G.bi/aba (leat) ex tracts was determined
after cataly ti c hydrogenat io n of the mix ture of
g ink golic ac id s. Thi s was done by usin g Pd-C (10 %),
in methanol , at 35-lbs pressure of hyd rogen. The
product with different alkyl side c ha ins was analysed
by HPLC (so lvent-4 ) and Gc. The res ults are
recorded in Table IV .
Fractions fJfJ f7- T he mixture- of terpenoid al
lac tones (TL,) and b·flavo noid s (BFJ, co ntai ned in
INDIAN J. CHEM., SEC B, APRIL 2002
852
Table 111- Mass spectral fragmentation patterns of si lyl-ginkgolates", isolated from Ginkgo and related fossils
a
Fragment ion peaks m/z (reI. abu ndance %)
Structure
Mol. ion peak
a
464 (2)
449( 100),374 (7), 359 (9), 293 (5), 219 (42), 205 (8),73 (70) .
b
462 (7)
447 (100), 372 (20), 357 ( 14) , 293 (4), 219 (36), 205 (4).
c
492 (3)
477 (100),402 ( 12),387 (9), 2 19 (38), 205 (J 1),91 (22), 73 (22).
d
490 (5)
475 ( 100),400 ( 12),385 (8), 293 (5), 219 (34), 205 (5), 91 (20), 75 (7) .
e
51 8 (5)
503 (100),428 (12), 4 13 (6), 293 (5) ,2 19 (35),205 (4), 93 (7).
f
516 (3)
50 I ( 100),426 ( 18),4 11 ( 11),293 (5), 205 (5), 90 (7), 75 (8).
see Chart 1
fraction-f2, was separated by column chromatography
over celite using chloroform and chloroform - ethano l
(traces) as th e eluents. The later fractions of el uates
were evaporated to give a mi xture of TLs (HPTLC,
see below)). The identities of the compo unds were
established by Gc. A portion of the residue (0.5 mg)
was di ssolved in pyridine (0.2 mL) and treated with a
mixture of trimethyichlorosilane (TMCS, 1%) and
N, O-bis-trimethyl silyltrifluoroacetamide (BSTFA,
0.1 mL). The mixture was warmed at 60°C for 30
min, cooled to room temperature and th en injected to
GC column . An internal stand ard of squ alene, in
chl oroform-meth anol ( 1: 1), was used for th e
calibration. Authentic markers were used, under
similar conditions, to substantiate the identification.
The respective peaks appeared at : 29.2 (bilobalide),
42.1 (gin kgo lide A), 43.5 min (ginkgolide B), and
35.5 min. (Squalene) .
The ea rly fractions from the column chromatographic run contained biflavonoids. The mixture was
subjected to HPLC using solvent-6 as th e eluent.
Amentoflavone (3a), tR 45.4, and bilobetin (3b), tR
42.6 min were detected and qu an ti fied in all th e fossil
samples (including Neal11ariapferis). Additionally,
G.bi/aba (leat)-fossils (2 samples) showed the
presence of isoginkgetin (str.3, Cr OCH3), tR 36.0
min. Fraction-f2 fro m fresh G.bi/aba (leaf) showed the
presence of all the four earlier reported 23 biflavonoids,
viz. Sciadopitysin , gi nkgetin , iso-gi nk getin, bilobetin,
and also amentoflavone (3a), Li kew ise, treatment of
fractions -f7 on HPLC and HPTLC showed the
presence of these biflavonoids.
HPTLC (solvent-3 )-Amen toflavone, Rr 0.26;
Amax, reflectance 270, 338 nm; bilobetin, Rr 0.32;
270, 334 nm ; isog inkgetin , 0.40; 271, 330 nm .
The resid ue from fraction-f6 (Scheme II) on
HPTLC and GLC (as above) showed the prese nce and
quantities of TLs (Table II).
Table IV - Composition of hydrogenated ginkgolic acids (G f
in the leaves of G.biloba (li ving) and in the fossil s leaves"·b
C6 -Side chain
Relative % of GA by
GC
HPLC
11-1 4
(tR 15.2 min)
C I3 H27
10-1 3
CIS HJI
6 1-72
60-70
(tR23.2 min)
C 17 H))
29-15
29- 16
(tR 32.5 min)
" G.biloba (3 sampl es), G.digitara, G.hUf/Oll ii, Glossopteris;
b Dadoxyloll (leaf) con tained on ly traces of GA;
Neol/lariopteris was free from GA.
Note - The inherent similarities in the compositi on of the C6
-
alkylaled ginkgolic acids preserved in the extant and ext inct
Gillkgo spec ies and in the possible ancestor (Glossopteris) .
HPTLC (solvent-2) (after heating ,he platl
Ginkgolide-A, Rr 0.58 ; Amax refl ectance 220, 3
nm; ginkgo lide-B, Rr 0.50, 220, 304 nm; bilobalil
Rr 0.62; 218, 300 nm.
Fractio ns f3/f4/fs - The resid ues from the
fractions were redish-pink in colour. Each of th e
res idues
co ntained
both
procyanidins
a
anthocyani dins (Tabl e II). These were separatl
ana lysed.
HPLC,
using
reference standal
(pycnogenols from pinebark) was used to detect a
quantify th e mixt ure of procyanidins prese nt in I
fossil ex trac ts. Two so lvent systems (Solvent 4 and
were used as eluents. Four major peaks
procyanidins (produced from a -and/or ~-combinati
of catechin and epicatec hin ) appeared in the eluti
range: tR 25-30 min. Amax PDA 222-228, 278-2
nm; th e tR-patterns and the Amax positi ons w
closely similar to th ose of th e major procyanid
from th e stand ard pycnogenols: The HPTLC patte
Rr 0.43- 0.48; Amax, refl ectance, 222-224, 278- ~
nm; were also similar to those of pycnogenols. 1
anthocyanidins were detected by HPTLC (solvent
GHOSAL et al . : TH E CHEMISTRY OF FOSSILS OF GINKGO A ND ITS ANCESTORS
853
using cyanidin and delphinidin (chloride) as markers.
Cyanidin, Rr 0.26, Amax 277, 534nm; delphinidin, Rr
0.15 ; Amax 275 , 542 nm . The prep. TLC scrapings of
th e corresponding zones, on usual processing,
afforded the two compounds, whose identities were
confirmed by direct comparison with authentic
markers.
5 Halliwell B & Gutteridge J M C, Free Radicals in Biology &
Medicine, Chapte r I (Oxfo rd Uni versity Press, UK). 1999. I.
6 Taylor TN & Taylor E. Biology & Evaluation of Fossil Plants,
Chapte r 14 (McGraw Hill, London - New York), 1993, 55 8.
7 Meyen S V, Fundamentals of Paleobotany (Cambridge Press,
UK) , 1987, 159.
8 Duke M V & Salin M L. Arch Biochem Biophys, 243, 1985,
305.
9 Spence r G F, Tj ark s L W & Kl eiman R, J Nat Prod. 43. 1980.
Acknowledgements
We thank Professor M.Banerjee and Dr.S.Bera,
Paleobotany Di vision, Department of Botany,
Calcutta University, and the Director General,
Geological Survey of India, Calcutta, for the authentic
fossil samples. Living Ginkgo biloba plant parts were
obtained from Indian Herbs Ltd.(IH), Saharanpur.
Financial assistance from the IH is gratefully
acknowledged by the Senior author (SG).
10 Gellerman 1 L. Anderson W H & Schle nk H, Biochelll Biophys
Acta. 43 1.1976. 16.
11 1rie 1. Murata M & Ho mll1a S, Biosci Biotech Biochelll . 60.
1996,240.
12 Nagabhushana K S. Sho va S V & Ra vind ranath B. J Nat Prod..
58, 1995. 807.
13 Grazz ini R, Hesk D & Heininger E, Biochel1l Biophys Res
COl/llnwl. 176. 1991 .775 .
14 Satyan K S. laiswal A K. Ghosal S & Bhattacharya S K. Biogenic Al1lines, 13B, 1997, 143.
15 Satyan K S. Ghosal S & Bhattacharya S K, Psychophannacol.
136.1998. 148.
16 Swain T & Cooper D G . Paleobotany, Paleoecology & Evolulion. Chapter IV (Freeman Pub. SanFranci sco) 1980. 103.
17 Stafford H A. Plant Physiol. 96, 1991.680.
18 Bornman 1 F. J PllOtochem PhOIObiol, 4, 1989. 145.
19 Barghoonn E S & Schopf J W, Science. 152. 1966, 758.
20 Niklas K 1. Brittonia. 32. 1976.11 3.
21 Niklas K 1 & Ginnasi D E, Science. 196. 1977. 877.
22 Mo ldwan M. G em Brit. 8, 2000, 34.
23 Scheid F B, Guth A L & Anto n R, Plallta Med.. 49. 1983,204.
24 Lo ll a E. Pale tti A & Pe te rion go F. Filotempia, 69, 1998,513.
724.
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