the plant was first defatted with petroleum ether...
TRANSCRIPT
3
General discussion of experimental procedure:
The methods of extraction and purification of natural
1—h. 5—nproducts, described in reviews and research publications ' ,
are employed ror the isolation of particular class of
compounds. The general procedure for extraction and purification
are substantially documented and proved helpful in working out
methods of extraction and subsequent work up of the plants
undertaken during the course of the present study..
The plant was first defatted with petroleum ether and
the petroleum exhausted material was then percolated either 1
directly with ethanol or a prior extraction with benzene, if it
appeared advantageous, was interposed. The petroleum ether
extract occasionally deposited crystalline material on
concentration and cooling but usually contained o ily and fatty
constituents which were not investigated further.
The alcohol extract was concentrated to a manageable
volume, cooled and filtered from any separated material and
finally taken to dryness. The alcohol insoluble constituents
obtained in this way were subjected to TLC examination and
purified by a suitable procedure. The residue left on complete
evaporation of alcohol was taken up with water and continuously
extracted with ethyl acetate to isolate glycosidic material.
Though this procedure for isolation of glycosides normally
4
gives good results, presence of significant amount of ethyl
acetate soluble impurities v e ry often makes it necessary to
Apurify the glycosidic material by treatment with lead acetate *
The amount of plant material extracted varied with the
nature of the constituents and the quantity of plant available
was often a limiting factor, so that in some cases ir itial
studies could not be persued to completion.
Coumarins are usually found to be readily extraetable
with petroleum ether and benzene. Flavones and their glycosides
are mostly isolated from alcoholic extract whereas triterpenes
are present both in the alcoholic and petroleum ether extracts.
Purification :
A multiplicity of methods has been resorted to, in
recent years to effect tedious separations of intractable
mixtures. Column chromatographic methods of separation have
10been refined by the introduction of prepared columns of nylon
or other such materials, which allow the separated zones to be
.cut out and then processed according to the nature of the
compound involved. The method is roughly a duplication of TLC
on a column. The use of GLC has been extended to phenolic
products with the introduction of the silylation method1 1 .
Along with these methods of separation the homogeneity of
5
isolated product3 is now routinely checked by TLC. This,
though occasionally misleading, as with the mixture of
1 °coumarins reported by Sharma e_t al " , is usually a reliable
indicator of the purity of isolated m aterials. Almost all the
adsoroants that nave been used for coluirji chromatography have
also been employed for TLC. Comparatively recent innovations
1 3include the use of polyamide powder , either as such or in
mixture with cellulose formamide impregnated s ilica g e l * \ ar.d
silica gel exposed to moisture by steaming . Polyvinyl
16pyrrolidone has also been used with improved results as substi
tute for polyamide. Most of these methods have been applied to
phenolic glycosides where conventional adsorbants are unsuitable
17 18owing to their strong retentive powers. Preparative TLC *
has been extensively used to effect separation in cases where
column chromatography fa ils to give good results. This generally
happens i f a prior TLC check up shows that components of the
mixture have only marginal separation on chromatostrips. It has
been observed that substances which flouresce under UV light
are readily separated by this method, but poor results are
obtained where a sprayed plate has to be used as reference
since Rf values are seldom reproducible.
In an interesting variation of normal methods of
developing the spots on chromatostrips zinc dust 2% is added
6
after elution with 6 normal HCl . The procedure has been
applied to flavonols which are indicated by the production of
red spots.
Identification :
Colour reactions: The compounds isolated can be related
to known classes of organic compounds by specific colour
reactions. Triterpenes are identified by Libermann Burchard
20reaction with acetic anhydride-sulphuric acid and by N oller 's
21reaction with thionylchloride and tin which gives a whole
range of colours on standing. Flavones are detected by the
22 2 5colouration produced with Mg/HCl and ferric chloride J and
pi i OKspecific colour reactions such as Dimroth and Wilson’ s
boric aci*d tests are also used to infer the presence of
hydroxyl groups at specific positions-. Hydroxy isoflavones and
rotenoids give the normal coiour reactions of phenols and can
be distinguished from the isomeric flavones by a negative Mg/HCl
and a positive sodium amalgan/HCl test. Isoflavones and
rotenoid both give a positive Durham test. There is no such
diagnostic, colour reaction for coumarins but the deep yellow
26colour produced by these compounds in sodium hydroxide
solutions is useful in their characterisation. The presence of
steroids in the extracts is detected by Libermann Burchard
reaction and of alkaloids by Dragendorff’ s and Meyer’ s test.
1 9
7
Investigations of the structure of plant constituents
nowadays rely heavily on spectroscopic evidence. The importance
of different spectroscopic techniques to problems of the type
encountered in the present work is , therefore, b riefly discussed
below:
Ultra Violet Spectroscopy:
• Ultra violet spectroscopy was employed during the course
of the present investigation and was extremely helpful,
specially in characterisation of the phenolic plant constituents*
Coupled with'diagnostic colour reaction, it provides at the
outset a clear distinction between coumarins, flavones and
isoflavones. A number of reviews on the ultra violet absorption
27 23spectroscopy of flavonoids have appeared J and on the basis of
comparison’of the a b s o ~ r p t T o r T l a r g e number of flavones and
isoflavones in neutral and basic medTta , the effect of
substitution at various position has beeitx^etermined.
The UV spectra of flavones show two regions of maximum
absorption which have been definitely correlated to the
existence of the benzoyl and cinnamoyl chromophoric. systems (l)
and (2) with contributions ^lso from structures (3) and (U ) .
The benzoyl chromophore is responsible for the maximum in the
region 21+0-270 nm (band II) whereas the cinnamoyl chromophore
8
absorbs at 320-350 nm (band I ) . In isoflavones only the benzoyl
chromophore is present and consequently the high wave length/
absorption is either totally absent or present only as an
inflection. Substitution in ring B specially at U ’ stabilises
the cinnamoyl chromophore resulting in a bathochromic shift of
band I whereas substitution in ring A has a similar effect on
the position of band I I . This makes it d ifficu lt to distinguish
between flavanones and isoflavanones. The localisation of
positive charge at 7-position in the benzoyl chromophore has
the effect of making this hydroxyl group more acidic than
hydroxyls at other positions and consequently its ionization is
brought about even by a weak base, such as sodium acetate,
9
resulting in a bathochroraie shift of 8-10 run. Though this
provides a sufficient indication of the presence of hydroxyl
29group at the 7-position it has been found that lucidin ,
acerocin^0 and scaposin^] all having the 7-OH, did not respond
to this test. A free hydroxyl at C-5 differs in its reactivity ,
considerably from those at other positions in the flavone
molecule and can be identified by the bathochromic shift
produced on addition of AlCl^ and orange to red colouration in
presence of H ^B O y /A ^O . Though no longer as prominent as before
such techniques are still useful and sometimes help in locating
the substituents.
The UV spec'tra of coumarins resemble the three banded
spectrum of 2 .U-dlh.vdrox.v trans cinnamic acid which has maxima
at 216, 290 and 330 nm. This is only to be expected as most
coumarins are derivatives of umbelliferone, the cyclization
product of 2 , b-dihydroxy cinnamic acid . However, the situation
here is not as consistent as in the case of flavonoids and
32-35comparison ^ of the UV spectra of a large number of coumarins
makes it quite evident that a definite correlation-between
substitution pattern and UV absorption does not exist. The
absorption bands of a number of coumarins are listed in Table I ,
which shows that 7-hydroxy coumarin has only one maximum at
325 nnr , in direct contradiction to the parallelism irawn
1 0
earlier with 2 ,Li.-dihydroxy cinnamic
TABLE 1
acid.
S .No . Name Max. (nm)
1 . Coumarin 275,312
2. 5-hydroxy coumarin 2U5,300
3 . 5-methoxy coumarin 2U5,301
h . 6-hydroxy coumarin 22U ,276,3U8
5 . 6-methoxy coumarin 225 ,275 ,335
6 . 7 -hydroxy coumarin 325
7 . 7-methoxy coumarin 213,318
8 . 8-hydroxy coumarin 210 ,25U ,289
9 . 8-methoxy coumarin 251,286
On the basis of resonance structure (5) and (6) and by-
analogy with flavones, presence of hydroxyls at 5 and 7 could be
(5) (6 )
11
expected to lead to a red shift compared to the parent
37compound in substituted coumarins . The absence of this effect
is partly due to the reduced basic character of the lactone
carbonyl compared to the pyrone carbonyl which makes
contributions of structure such as (5) and (6) less pronounced
than those of the benzoyl and cinnamoylchromophores in flavones.
According to Mangini and P a s s e r in i^ the absorption maximum in
the region 300-333 nm’ is due to the combination of a ll the
resonating structure of the coumarin and the maximum in the
region 270-290 nm is due to antisymmetric combination of the
polar structure only.
Spectroscopic data on a large number of naturally
occurring coumarin have been compiled by several w o rk ers^-^«
but attempts, chiefly by .Bohme _et a l , to find a definite corre
lation between substitution pattern and UV absorption have not
been successful. Coumarins, specially when they give rise to a
quinoidal system on ring, opening, are unstable towards alkali
and consequently UV absorption of alkaline solutions of
coumarins were seen to vary with time. However, according to
Bohme the lactone ring is sufficiently stable towards alcoholic
sodium methoxide to allow measurement of the spectrum in this
medium which is useful in establishing the presence of phenolic
hydroxyls, ferric colouration not being very clear with
12
hydroxy coumarins.
Coumarina having the phloroglucinol substitution
pattern are liable to undergo isomerisation in alkaline solutions
which may be associated with changes in UV absorption as for-7 O
instance in the conversion of mammein to isomammein . Spectral
measurements in alkaline solution should therefore be carried
out as quickly as, possible.
39Work by Mendez and Lojo has shown that a free OH on
the benzene ring causes bathochromic shift of the longer
wavelength maximum in tlie presence of KOH. While successful use
has been made in the flavone fie ld of the shifts produced by the
addition of specific reagents, such as NaOAc»H^BO^ and AlCl-^,
they do not seem to have any appreciable effect in the case of
icoumarins. Comparative UV absorption study of furanocoumarins
by Lee & Soine^0 has revealed that linear and angular furano-
coumarins show distinctly different spectra. The X max at
2U2-2U5 nm and above 260 nm are characteristic of th® former
and are absent in the latter . C^ and Cg monosubstituted linear
furanocoumarins have X. max at about 260 nm and X min at 276 nm,
where as C^ or C^ disubstituted ones have characteristic X®ax
at 273 and 286 nm. The C^ monosubstituted linear furanocoumarins
have X max at 273 and 286 nm. The C^ monosubstituted compounds
having X max at about 268 and 308 nm and X min at 25k can be
13
readily differentiated from Cp substituted compounds, which
have X* max at 301 nm, The nature of substituents
_0-cn2-CH=C(CH3 ) 2 ,-0-CH2-CH-^-{CH3 ) 2 or-0CH3 has little
influence, since almost identical spectra are obtained with them.
Infra Red Spectroscopy:
The application of IR spectroscopy has been overshadowed
in recent years by NMR spectroscopy as many of the structural
features brought out by the IR spectrum are more clearly
1 1 3discernible in the H and spectra. Inspite of this the IR
spectrum, in practice, plays an important role and offers the
first clue to the nature of the compounds. Thus in flavonoids
IR measurements are helpful in providing evidence for the
presence of (a) pyrone ring (b) chelated hydroxyl groups and*
(c) the gem dimethyl grouping of substituents i f present.
The substitution pattern of the benzene ring can hardly be
inferred from bands in the 690-800 cm” ”* region. Such evidence
is helpful in distinguishing between flavonoids and coumarins
but otherwise offers little information of structural value.
IR spectroscopy has mostly been used in recent years to adduce
corroborative evidence. The -C=0 region of the IR spectra of
flavones shows a number of bands the most prominent of which is
usually assigned to -C=0 stretching. This region has also been
k1the subject of detailed studies by Murray and Me Cabe in
14
related, systems such as chromones.
Attempts to correlate carbonyl absorption frequencies
with the substitution pattern have been made by various
w orkers^- 4’ and have found the existence of both
intramolecular and intermolecular hydrogen bonding in hydroxy
flavones. In contrast to the behaviour of flavanones and ortho
hydroxy acetophenones intramolecular hydrogen bonding was,
however, found to have little effect on carbonyl frequencies
of flavones and 5-6'ydroxy flavones and further more1methylation
of the 5-hydroxyl did not lead to any appreciable hypsochromic
sh ift .
Another interesting feature of the IR spectra of
flavones is- that the carbonyl frequency is independent of the
substitution pattern in ring A and B and is effected only by
the introduction of a hydroxyl at 3-position. Looker and
Hannenan^4- attributed this to the predominant contribution of
mesomeric structures ( 7 ) , (8) and (9)
15
The existence of chelation is , however, clearly
demonstrated by the absence of the hydroxyl bands at the usual
position in 5-hydroxy compounds. Apparently it comes to lie in
the—OH stretching region and is thus obliterated.
As noted earlier IR spectroscopy is helpful in
distinguishing between flavones and coumarins. The -C=0 band
of the former occurring at higher wavelengths owing to the
reduced basicity of the lactone carbonyl. Aromatic absorption
b5bands are found in the usual regions. Bukreeva and Pigulivskii
have noted that 5-substituted furanocoumarins have bands at
16i6-2^,1601-8 and 1577-81 cm”1 the strongest being at I616-I62U
cm , whereas those substituted at 8-position have bands at
1621-25, 1583-85, 1559-61 and 15U5 cm~\ the strongest absorp-
-1tion being at 1583-5 cm . Coumarins with substituents at 5 and
8-positions have bands at 162U-27, 1607-1k, 1588-1595 and
—1 —115U6-57 cm , the strongest being at 1588-1595 cm
Furanocoumarins also show very strong absorptions at 7^0-780
_-icm , due to the C-H m plane deformation vibra-tion. In other
coumarins this band is weak or absent.
16
Nuclear Magnetic Resonance Spectroscopy:
The application of the NMR spectroscopy to flavonoids
was hampered initially by the poor solubility of polyhydroxy
compounds in chloroform. Recourse to silylation, already an
established technique in the carbohydrate fie ld , overcame the
difficulty but direct measurement in very dilute solution
through the use of CAT or in other deuterated solvents eg.
DMSO-dg is the more common practice these days.
Flavones and isoflavones are distinguished by the UV
spectra but, since isoflavones also occasionally show high wave
length absorption, structural assignments had to be supported by
colour test and finally confirmed, either by correlation to a
known compound or degradation. NMR evidence in this regard is ,
however, conclusive the singlet of the 0-2 proton appearing
substantially downfield at 8 and that the C-3 proton at 6 .3 .
In flavonols the 3-OH gives rise to a signal at 9.b and it can
therefore be easily differentiated from the more strongly chela
ted 5-OH at 13«0 and other phenolic functions. Another Important
feature Of the spectra of flavonols is that.the presence of the
3-OH results in reduced deshielding of the 5-OH which now
absorbs at 1 2 .5 . Similarly flavanones, 3 hydroxy flavanones and
isoflavanones can be distinguished by the position, multipli
cities and coupling constants of the heterocyclic ring
17
protons (Table I I ) .
Table II
H-2 (ppm) H-3 (•ppm)
Flavanones 5 .0 - 5 .5q near 2 .8qq
Dihydroflavonol (3-0-glycosyl) 5 .0- 5.6d U.3-U.6d
Dihydroflavonols U.8-5.0d lu1-/+.3d
Isoflavanone i+,51 ,dd , U .07q ,J=1l+,5.5 Hz J = 5 .5 Hz
The situation is , however, more complex in compounds
in which the heterocyclic ring is saturated for the signals in
the 3-5 8 region can also arise from other structural types e.g.,
substituted pyrans, dihydrobenzofurans, C and O-glycosides. One
has also to bear in mind that the heterocyclic ring in such
compounds may adopt different conformations leading to changes
in coupling constants of the methylene and methine protons.
Even slight alteration of the bond angles may lead to
significant changes in the NMR spectrum.
The substitution pattern is nowadays determined by the
N!® and mass spectroscopy. In flavones carbons 5 , 7 , 2 ' , 14.’ , 6 ' ,
and in isoflavones and flavanones 5 ,7 are electron deficient
and therefore signals of the protons at these positions occur
Chemical shifts are reported in 8 values throughout the
thesis.
18
downfield from the standard value 7 .3 0 ppm for the benzenoid
protons. This deshielding by the carbonyl group is , however,
counter balanced by -OH and -OMe groups present in the molecule
and the NMR spectrum of any given flavone or isoflavone has to
be analysed in terms of these factors. ThU3 according to
Ballantine and Pillinger^6 the effect of introduction of oxygen
on benzenoid protons is additive and is given in the following
table I I I .
Substituents shielding values measured in ppm from
benzene (10% solution of benzene in CDGl^) absorbs at 7 *30 .
Table I I I
Substituents S ortho S meta S para
-OH 9.55 9 .9 0 9 .6 0
-0 Alkyl 9 .55 9 .9 0 9o60
-0 COR 9 .80 10 .10 9 .8 0
In flavonoids specifically such effects are responsible
for the high fie ld positions of the doublets of C-6 and C-8
protons in 5 ,7-oxygenated flavones and deshielding 2' ,h '
protons compared to 3 ' » 5 f protons. The simplest spectra are
those of 5 >7 ,U ’-trisubstituted compounds in which, owing to
19
symmetrical substitution ring B protons appear as superimposed
doublets corresponding to an AgBg system ana ring A protons as
AB doublets. In other casds interpretation is not so simple ow
ing to superimposition of signals and appearance of complex
multiplets of protons of an. ABX or ABC system. Acetylation is
also helpful here as it identifies protons ortho or para to the
-OH group which are deshielded by the introduction of acetyl
U7group
Solvent induced shifts have also been used for assigning
the positions of methoxyls in the structure analysis of methoxy
flavones, flavoiols and isoflavones. By measuring the NhCR
spectra first in CDCl^ and then in CgHg, Wilson4"® et ajl found
that the size of the benzene induced shift ( A ) of certain
methoxyl signal was to some extent indicative of the position
of the methoxyl group on the flavone nucleus (Table IV ) . Large
shifts were noted for methoxyls at positions conjugated to the
carbonyl and it was noted that these shifts were diminished in
the presence of ortho oxygenation (-0H or -OMe). Pelter and
[iQAmenechi concluded that the benzene - induced shift for
signals of methoxyl groups (in either A or B ring) ortho to an
aromatic hydrogen is greater than 0 .3 ppm; whereas signals of
methoxyl groups lacking such a proton move by only 0 .0- 0 .2 ppm.
20
A Values ( CDCl^-C^Hg ppm) for flavone methoxyl signals in
the absence of ortho substituents.
Methoxyl at C-3 - 0 .07 to + 0 .3U ppm
C-5 + 0 ,k3 to + 0 . 5 8 ppm
C-7 + 0.5U to + 0e76 ppm
C-2' + 0.!+6 to + 0«53 ppm
C-k' + 0.5U to + 0.71 ppm
Table IV
A more recent innovation in this f ie ld is that of
50lanthanide induced shift . The technique is extremely helpful
in establishing the internuclear junction of biflavonoids and
also for the distinction of A and B ring methoxyl signals.
In a recent article on the structure determination of
an 8-isoprenyl-3»7,V-trimethyl quercetin, Pinhey and Southwell^"*
used the nuclear overhouser effect (NOE) to substantiate the B
and C ring substitution pattern. Irradiation of the methoxyl
group resonances was found to produce a 13$ enhancement of the
integral of the H-2' signal, presumably due to a NOE between
the 3-methoxy and the C-2' proton.
21
Cournarins:
The N'v.R spec Lr > of each class of cour::arins are 30
characteristic that one could assign the -structure 30lely from
NMR data. The chemical shift for the protons in the 3 and U
position of coumarin C10) are almost the same as those observed
for ethylenic protons in o-coumaric aaid . The coupling constant
J= 9 .8 Hz confirms that the protons at the 3 and U position are
cis to each other as expected. The chemical shifts of all the
52protons of coumarins and furanocoumarins published , are
reproduced in table V.
- Table V
H-3' 6 .11- 6.39
U-k 7.58-8.1,5
H-5 6.78-7 c5U
H-6 6.37- 7.38
H-7 7.U 2
H-8 6.26-7.U1
H-2 ’ 7.5U- 7.70
H-3’9
6.78- 7*12, J = 2 1 , 3 ’ =2Hz
Solvent induced shift in coumarins have been studied
53 ' 5L.by Crigg, Knight and Roffay and Nakayama and the method has
22
(lO)
(II)
found extensive application in determining the position of
substituents. Their conclusion are summarised in the formula (1 3 ) .
0.32-0.52
0.25
0.53-0.570.76-0.79
0.15-0.29
(13)
Methoxyl substituents at C-U» C-5 and C-7 exhibited large
(0 . 52-0 . 7 7 ppm) solvent shifts, while the methoxyl groups at
C-3 and C-8 gave small solvent sh ifts .
(
23
Another technique which has been utilised in recent years
for providing structural information about coumarins is "Nuclear
overhouser effect" (NOE). It was first applied in 1 9 6 5 ^ . The
structure of n e ish o uto l^ (lU) was confirmed by determining the
peri relationship of the C-U proton which caused a 12% increase
in the integrated intensity of the C-5 methoxy protons, when
compared with the intensity on irradiating at approximately
50 Hz upfield from the G-k proton signal. Conversely, a 11%
increase in the integrated intensity of C-U- proton resulted
from the saturation of C-5 methoxyl signal. NOE has also been
used for establishing the stereochemistry of double bond in thejr “7
side chain of murralogin (15)
OMc
(14)
24
Though NOE is no doubt a powerful technique, the
requirement of sophisticated instrumentation has, restricted
its wide applicability and indirectly contributed to the rapid
development, of lanthanide shift reagents. Recently Eu(fod)^
has been employed for differentiating between the possible
CQisomers of avicenol5 ( 1 6 ) .
25
13q NMR spectro3copy:
Recently ^JC NNiR spectroscopy has been used in natural
product chemistry in variety of ways at various stages of the
structure determination. 1 NMR spectral data furnishes key
information such as the number of carbon atoms and.establishes
if they are primary, secondary, tertiary, aromatic, olefir.ic or
part of functional groups.
13The C NMR spectra of flavones and couinarir.s are of
some interest in the context of compounds isolated|during the
course of thi3 work. The spectra can be analysed by reference
to those of simple compounds such as acetophenones and cinnamic
acids which possess structural features characteristic of
flavonoids and coumarins.<
It is worthwhile to see how introduction of oxygen at
various positions of these effects the chemical sh ifts . In
hydroxy acetophenone ( 1 8 ) the nuclear carbons linked directly
to oxygen of hydroxyl groups give rise to singlets at 161 .5 ppm
and the two adjacent carbons to the two singlets at 118 .0 ppm.
The carbons para to the carbonyl is the most deshielded and its
singlet appears at 135 .5 ppm. In 2,6-dihydroxy acetophenone (19)
the carbons bonded directly to oxygen give rise to singlets at
161.U ppm and the two adjacent carbons produce singlets at 106«5pp
i65. r104.0 32.10
202.9
(19) (20)
The meta carbon which is para to the acetyl group is deshielded
and its singlet appears at 13U.0 ppm.
Thus chemical shifts correlate to those for protons on
these carbons, the protons ortho and para to hydroxyls being
shielded more than the ones at meta positions and protons para
and ortho to carbonyl being the ones most exposed to the
deshielding influence of the carbonyl group. In 2,U,6-trihydroxy
acetophenone (20) the oxygenated nuclear carbons show singlets
at "iSSolO ppm while in dihydroxy acetophenone it is 1 6 1 . ppm.
This slight deshielding of 3 »60 ppm can be attributed to the
27
hydroxy ait meta position i .e the U-hydroxy in (2 0 ) . The
unsubstituted aarbons 3 and 5 are shielded due to enhanced
mesomeric effect and their singlets appear at 9k»5 ppm. These
effects can be assumed to be general and are relied upon in
making assignments in flavonoid spectra. The other structural
13unit of flavonoids is akin to cinnamic acid and the -'c chemical
shifts of cinnamic acid derivative are therefore, of interest.
The chemical shifts of the parent compound, mono, d i , trisubsti
tuted cinnamic acids are indicated in the structures (21 —2U-) •
12 8.5___[29.0
I28.5
(21)
HO
131.70 II7.26
146.68 / J6°,33h
131.70II726
(22)
OMe
II2.28
^739 7-----OH149.78
124.23116.89 HO
V o ’
OMe
io 7.Q-/|49,17
147.23 / / \126.30 138.79
_ J49.I7 1070
59 'OMe
•OH
(23) (24)
28
The 3 , U type of substitution is the one most commonly
encountered in flavones and chemical skifts of carbon 3 and U
of 3-methoxy, U-hydroxy cinnamic acid (23) 1U9.11 and 1^9.78 ppm
respectively are substantially different from those of carbons
under oxygen in acetophenone. This makes it possible to ,
distinguish between oxygenated ring A and ring B carbons of
flavones. The carbons ortho and para to phenolic hydroxyls are
shielded compared to unsubst'ituted benzene and appear at 112.28
and 116 .89 ppm, the cinnamic acid double bond causing a further
shift of C-2 resonance, Carbbn-1 adjacent to the olefinic
double bond of cinnamic acid , is almost at the same value as in
substituted benzene but different in unsubstituted benzene. The
oC—carbon appears aft 1 1 7 . 5 ppm and the carbon at 1U7.1 ppm.
In trisubstituted benzene (2U ), the carbons under oxygen are
further shielded and in 3»5-dimethoxy, U-hydroxy cinnamic acid
appear at 1U 9 .17 , 138 .79 ppm respectively.
The carbon under hydroxyl is shielded to a greater
extent because of resonance contribution from the flanking
methoxyl groups. The same type of resonance effect is
responsible for the shielding of 2 and 6 carbons.
The chemical shifts of flavones, substituted flavones
59and isoflavones are reproduced in the table.
Chemical shift ( in pprr. dcwnfield from T .M .3 . )29
Table VI
Carbon^r
Flavone 7—methoxy f lavone
5— hydrqx.y l'l.avon e
5 t7 , 5 ’ ,U ' , to tra —hydroxy flavone.
7-methoxyisoflavone
163 .2 162 .6 104.07 16 5 0 07 152J4
3 107 .6 1 07 . 2 105.61 1 03 .9/4 1 25.1
178.U 1 7 7 .k 182.90 182.63 175.3
5 125 .2 126.7 1 5 5 .p-5 1 5 8 .2U 127.6
6 1 25 .2 11 h . 1 107.22 9U.9 11U.6
7 1 3 3.7 163 .7 135.61 16U.3U ' 163 .8 .
P 11^.1 100.2 110.83 99.91 1 0 0 . 0
9 156 .3 157.7 159.82 16 1 .56 157.7
10 12i+.0 117 .6 110.13 10U .82 11^.3
1 ' 131 .8 131 .6 130 .5U 123.06 127 .9
2 * 126 .3 1 25 .8 126 .39 11/4.38 128 .2
3 ’ 129 .0 128.7 128.91 1U5.95 128.8
U ’ 131 .6 1 31 .1 131 .97 1U9.8U 131 .8
5 ’ 129 .0 - 128.91 117 .05 -
6» 1 26 .3 — 126.39 1 20.1 U
!
30
Mass ^ectrometry :
Though less informative than NMR at the initial stage
of characterisation of structural groupings mass spectrometry
offers information on several features of interest ’which cannot
be deterained, as readily at least, by NMR.
In mass spectrometery the driving force for cleavage
of a particular bond is formation of stable ionic species. The
Presence or absence of charge stabilizing substituents in
certain positions is an important factor in determinig the
course of bond cleavage. As most naturally occurring flavonoids
are highly oxygenated, the molecular ion is stabilised by charge
distribution over several oxygen fuctions and breakdown by any
well defined pathway is minimal. This situation changes, however,
in compounds where aromaticity of the flavonoid, which is«
essential for charge distribution, is destroyed by saturation
of the heterocyclic ring. Spectra of such compounds in which
this ring is saturated, show abundant cleavage by pathways
designated as A,B and G by Pelter and co-work e r s ^ " " ^ shown in
scheme I •
The Retro Diels Alder reaction 'A* affords fragments
in which the charge is retained either by the ketene or by the
aromatic fragment depending on the site of the intial loss of
Aliama Jqba! Library
These*
Scheme I31
B
cIIo
electron. The relative intensitiee of the two species show that
charge retention by the aromatic portion is favoured. The
further breakdown of this fragment is in accordance with the
behaviour of phenols and phenol ethers and results, through
successive losses of methyl and carbon monoxide, in .the
formation of fragments as shown below.
CH:CH2•f
CH-CH-
c h 3 -C O
32
Apart from these modes of cleavage the molecular ion can
eliminate either the side phenyl nucleus or a hydrogen atom
to give stable, fully aromatic, ions,shown in scheme I I .
m/z 147 (7.0) m/z 253 (30.0)
Scheme- I I
Path C, elimination of carbon monoxide, is observed in
3-phenyl-U-hydroxy coumarins, where the breakdown pattern has
been rationalised on the basis of tautomeric structure sh«wn in
scheme I I I .
Scheme I I I 33
Another feature of mass spectroscopy which is of great
significance in this f ie ld is the ready identification of
biflavonoids which can he recognised from the molecular ion peak
at mass number corresponding to fusion of the two flavone units .
It is noteworthy that a number of compounds e .g . fu ku g etin^
which were initially believed to be simple flavonoids have since
been shown to be infact dimeric.
69-71The mass spectra of furocoumarins are characterised
by several peaks corresponding to elimination of carbon
monoxide. The source of this carbon monoxide are the oxygen
functions of the pyrone and furan rings as' well as methoxyl
substituents which may be present. The resulting species can be
34
formulated as shown in scheme IV,
O CH 3
m/z 216 (IOO°/o)
CO“ CO
CH-
m/z 188 (ll°/o)
-C O CgHgO - C O
m/z 09 (25% ) m/z 117 (4°/o)
m/z 173 (56°/o)
m/z 145 (2 0 % )
Carbon atoms directly attached to the benzene nucleus
are incorporated in the formation of the fam iliar tropylium ion,
The corresponding peak Is stronger in the snectrum of compounds
having saturated side chain than those in which the side chain
is unsaturated. The allyloxy side chain is eliminated with
35
rearrangement, the hydrogen atom being most probably
provided 13y one of the methyl groups*
The mass spectral pattern of various types of
U-phenyl coumarins have been studied in d etail . In
unsubstituted U-phenyl coumarins (2 5 ) , 3-phenyl benzofuran
ion (26) is produced by the lono of cnrbon monoxide which
give rise to the fluorenyl cation (27) at m/z 165 by the
loss of aldehydic group shown in scheme V.
Scheme V.
(25)
- CO
— CHO(26)
(27)
36
B io g e n e s is of F l a v o n o id s :
The o r ig in of the Cg-C^-Cg u n it of f la v o n o id s is now
w ell k n o w n ^ ^ and summarised in scheme ( V I ) . The involvement
of ace t ic a c id and substituted cinnamic a c id has been confirmed
through studies with l a b e l l e d compounds, notably by G r i s e b a c h ^
and Geissm an^k . Tsoflavones are b io y e n e t lc a l ly .r e la t e d , and
a r i s e , as shown by fe e d in g experiments with l a b e l l e d chalcones ,
~~J *7 “J Q
through phenyl migration at soii;e stage of f la v o n o id b io g e n e s is
COOH Scheme V I ;
Shikimic acid
A
Carbohydrate
37
The exact details of the transformation of chalcones,
the immediate products of condensation of the cinnamoyl and
polyketom'ethylene units, to other members of the class is still
a matter of speculation. Their conversion to the isomeric
7Qflavanones occurs readily in the laboratory ' and has been
POdemonstrated in vivo" . In the latter case enzyme catalysis
must be assumed to account for the optical activity of naturally
occurring flavanones. Of the two chalcones were in itia lly
favoured as the starting point of the biogenesis of other
P1flavonoids, including isoflavones since laboratory analogies
i
existed for most of the required transformations. Thus alkaline
pp 0 5^2^2 0xidati0n chalcones, the Algar-Flynn-Oyamada reaction" *
affords flavonols and aurones, 3-hydroxyflavancnes being
Ak p,5intermediates in flavonol formation * . The mechanism accepted
iart; the time visualised formation of chalcone epoxides in the
reaction followed by nucleophilic attack by the 2 ’-hydroxyl
which cleaves the oxirane ring either o£ or p to the carbonyl
leading to aurones and flavonols (or a mixture of b o th ). Which
of the two is formed in abundance in actual practice depends on
the substitution in ring A, presence of a 6 ’-methoxyl directing
the reaction, for the steric reasons, towards aurone
86-88formation
38
Flavonol Formation:
Aurone Formation:
Reduction of 3-hydroxyflavanones is assumed in the
biogenesis of fla-van 3>U-diols but other p ossibilities exist.
AQ—QOThus as shown by Clark-Lewis and coworkers , NaBH,
k
reduction of chalcones gives flavenen which can conceivably
also serve as precursors of leucoanthocyanidins, anthocyanidins
39
and catechm s91
Anthocyanidin Catechin
Proposals for flavone "biogenesis include oxidation of
the enolic form of flavanone (2 8 )^ 2” ^ hut Wong has shown
QR-Q 6chalcone to he a better precursor .
40
The stage#at which the linear c6-c3“ cg unit of flavonesI
is modified to the branched aarbon skeleton of isoflavones has
been a subject of much controversy. By analogy with the
97rearrangement of catechin tetramethyl ether with PClc ,
D
3-hydroxy flavanone (29 ) was first implicated.
41
Later, Ollis and Grisebach put forward the idea that9 ft
in rotenoid biogenesis rearrangement occurred after elaboration
of the heterocyclic ring system (Scheme V I I ) . Their mechanism
invoke ether cleavage of (31) at the benzylic carbon followed
by aroyl migration and cyclisation. This could also be valid
for simple isoflavanones and isoflavones but an attempt was
made to accommodate isoflavone formation in the biogenetic
99scheme based on chalcone epoxides . It had been shown that
these were cleaved by BF^, or other Lewis acids, to give
isoflavones in good yi elds1 o Tt was presumed that phenyl
1 02—migration occurred in the reaction but studies by House al
showed that an aroyl migration was infact involved.v •
Scheme V I I .
H2C : 0
O O
(30
42
Feeding experiments with appropriately labelled7 7 7 0
chalcones . however, showed that the rearrangements in
nature proceeds through phenyl migration and cleavage to give
the thermodynamically less stable cation (32) would have to be
assumed if chalcones epoxides are intermediates.
(32) Ph« Phenyl
It may be noted here that the whole basis of this theory
was demolished by the investigations of Bean and Podimunag”* ^
on the mechanism of AFO reaction. They argue that whereas
2 ,-methoxy chalcones afford epoxides on treatment with HgOg*
epoxides haye never been isolated from compounds in which the
2*-OH is free . They relate this to coulombic repulsion between
the phenoxide and hydrogen peroxide anions and deactivation of
43
the carbonyl in the phenoxide anion (3 3 ) • The reaction i3
therefore believed to proceed not through epoxide formation
but by electrophil'ic■ httack by molecular hydrogen peroxide( 5k-51) .
(37)
The concept of phenol oxidation offers an alternative
to the intermediacy of chalcone epoxides. Put forward by
P e l t e r * ^ in 1968, it assuir.es that the chalcone isomerises to
flavanone, the enolic form of which is oxidized by hydrogen
44
abstraction from the V or 2 '-hydroxyl and the radical or
cation 30 generated Induces eye 11 sat ions depicted in .scheme (V I11)
Scheme V III
45
A similar scheme (IX ) can be written for oxidative
cyclisation of chalcones to aurones or flavones and i f
intervention of the -OH radical is accepted 3-hydroxy
flavanone and the products derivable from it"'^^.
Scheme IX .
46
Flavonol (U1) formation i . e . hydroxylation at C-3 can
result from further oxidation of (UO) whereas participation by
the phenyl ring in establishing the radical (39) would lead
107eventually to isoflavone (J+2)
(41)
extended to include U-hydroxy-3-phenylcoumarins (U3) which can
47
be derived from isoflavones as shown1 °'Q910
(43)
48
Biosynthesis of Coumaring:
The coumarins are typical metabolic products of
higher plants; the simple ones are'formed from the corresponding
substituted trans-cinnamic acid derivatives. Hydroxylation of
the o-position of the particular cinnamic acid in question
takes place f irs t and the resultant o-coumaric acid derivative
is subsequently glucosylated. It is then rearranged in a
spontaneous light-dependent reaction to the corresponding
coumarinic acid glucoside, which is structurally derived from
cis-cinnamic acid . By enzymic elimination of glucose, free
coumarinic acid is formed which cyclizes spontaneously to»
coumarin. The biosynthesis of herniarin and umbelliferone takes
place through such glucosidised intermediates1^ . The glycosides,
aesculin and scopolin, on the contrary, are not intermediates
in the biosynthesis of aesculetin and scopoletin, but originate
from these compounds by subsequent glycosylation.
In more complex compounds, wherein coumarin ring is
substituted by a furan ring, the additional carbon atoms of the
furanocoumarins correspond to carbon atoms 1 and 2 of isopen-
tenyl pyrophosphate. The origin of the furano-carbon atoms in
111these coumarins has been explored extensively . using Thamnosma
montaria which synthesizes umbel lip renin, alloimperatorin
methylether and isoimperatorin, essentially confirming the
49
correctness of the earlier results that mevalonate is the
source of these two carbons, and 2-1^C-acetate is not a more
efficient precursor'. '
3-Phenylcoumarins are related closely to isoflavones
and flavans (2-phenylchroman) and may be synthesized from
isoflavones, formed in the plant by the shift of a phenyl group
from G—2 to C-3 of the flavans ring system. Since flavones are
good precursors of isoflavones, the shift probably takes place
at ttiis stage of the biosynthetic pathway; the exact mechanism
is , however, unknown. Thus,daidzein (M-i-)# which occurs together
with coumestrol (U5) in a lfa lfa , is a precursor of the latter
11 ^compound and a pathway is proposed for this conversion .
112
(44) (45)
(46) (47) (48)
50
Out of the two possibilities of origin of ^-phenyl
coumarins (neoflavonoids) , v iz , by means of a double shift of.
the phenyl group from flavones or by direct condensation, the'
latter seems more plausible . The incorporation of phenylalanine-
3- ^ C (U6 ) gave calophyllolide (hi) which was specifically
labelled at C-k. Thus a novel condensation is observed between
phenyl propane uiiits and acetate in this case.
The biosynthesis of dicoumarol (U8 ) , the toxic bicoumarin
of decaying sweet clover (Melilotus o f f ic in a l is ) , may tftke place
by bacteria in dead plants containing coumarins. Its direct
precursor was U-hydroxycoumarin which is formed from ,<2.-coumaric
acid viaphydroxymelilotic acid . The carbon of the methylene
bridge appeared to originate from formaldehyde formed during
decomposition of the plants. Biological one-carbon donors
(e .g . methionine, serine and choline) were not effective
115precursors
Trapping experiments using 7 - ^ C . demethyl sub ero sin and
1 hC-umbelliferone in Gonium maculatum, Heracleum lanatum, and
Ruta graveolens ~ have demonstrated that 7-demethylsuberosin
(U9) is a precursor of linear furanocoumarins, e .g . marmesin
(50 ) as osthenol (5 1 ) is of the angular furanocoumarins, e .g . ,
columbianetin (5 2 ) •
(51) (52)
Ri53, R| = R2=H
54, R,= OMe, R2= H
55, R, = H, R2=OMe
56, R, = R2 —» OMe
These observations were confirmed by carrying out the
same feeding experiments with cell cultures of Ruta graveolens.
In additions, it was found that both (U9) and (50) were
excellently incorporated into four coumarins with degraded
isoprenoid side-chains, i . e . , psoralen (53) xanthotoxin (5U ),
bergapten (55) and isopimpinellin ( 56 ) . Also , psoralen was found
52
to be a good precursor of the above three raethoxylated coumarins.
Evidence that the same basic pathway to furanocoumarins operated
in Ficus carica was obtained when feeding of tritiated marmesin
to the leaves for 72 hr caused incorporation into psoralen and
119bergapten . There has been some speculation about the mechanism
by which marines in is converted to psoralen ; it has been
postulated that the first step is the elimination of a 3-carbon
fragment by a yet unknown mechanism, leading to 2 ' , 3 f-dihydro
psoralen as an intermediate. Some evidence towards its
confirmation has been obtained in F . carica by feeding tritiated
2 ' f > '-dihydropsoralen and 2 , 3 ’-dihydrobergapten which were
converted to psoralen and bergapten respectively, although
dihydrobergapten is so far not known to occur as a natural
product .
Significant developments in this area have taken place
at the enzymic level with the discovery of two key enzymes, the
first of these is required for the ,o.-hycLroxylation of cinnamic
acid . This activity was detected in Melilotus alba seedling!^®,
principally through the use of chloroplast preparations as the
1Usource of this phenolase which converted trans 3- C-cinnamic
acid . The pH optimum was 7 .0 and the enzyme activity increased
U-fold when glucose-6-phosphate was added as a source of
reducing power. The o-hydrox.ylase appeared to be bound to the
53
lamellar membrane of the chloroplast; sonications of the crude
preparation gave 50°' increase in the activity .
The second new enzyme was discovered in suspension,
1 21cultures of young leaves of Ruta graveolens , it catalyzed
the reaction between umbelliferone and dimethylallyl pyrophos
phate to give demethylsuberosin, the f ir s t key intermediate in
the biosynthesis of linear furanocoumarins. The enzyme, which
has a requirement for Mn , showed a well defined specificity .
It failed to use 7- methoxycoumairin (herniarin) as substrate
and produced substitution at the C-6 position only and not at
C-8. The fact that the enzyme was particle-boumd in the
particulate fraction may explain earlier failures to find this
important biosynthetic enzyme.
54
PHYTOCHEMISTRY OF IRIS SPECIES
Iris is a well known genus of rhizomatous or bulbous
herbs belonging to the family Iridaceae distributed in the
1 22north temperate regions of the world. Nearly 150 Iris
species are known to occur in nature.
The plants are perennials and are mostly spring and
early summer bloomers. The genus is characterised by a simple
or branched erect stem bearing one or* more flowers at its top;
the leaves are mostly radical and cauline, linear to sword shape,
f la t and many nerved lengthwise; the segments of flowers are
generally united into a long or short tube (the perianth tube),
the outer three segments are hanging or refluxed and narrowed
towards the base, the inner three segments are usually erect
and often arched. Style is three branched, the branches are
coloured, petal lik e , expanded, spreading outwardly and covering
the three stamens.
In India the Ir is genus is represented by about a dozen
species and a few are cultivated for ornamental purposes. In
Kashmir valley the genus is represented by the following
species:
1 . Ir is ensaitg Thumb
2 . Iris corcea jacq. ex. R .C . Foster
55
3. Iris reticulata M. Bieb
h . Iris germanica linn .
5 . Iris flavescens Dc.
6 . Iris xiphoides Shsh.
7 . Iris kashmiriana Baker
8 . Iris aurea
9 . Iris kumaonensis Wall, ex
10 . Iris nepalensis W all.
11 . Iris florentina Linn.
1 2 . Iris spuria.
13 . Iris hookeriana Poster
All these species are growing wild on meadows and by
noadsides. Ir is germanica and Ir is florentiaa have considerable
economic importance and are cultivated for their rhizomes which
constitute the orris of commerce. Rhizomes of Ir is have been
used in perfumery from Greek and Roman times. The peeled and
dried rhizomes of Ir is germanica, Ir is florentina and Ir is
p allids are called. Orris, Orris root or Irdis Rhizoma. The
rhizones of Ir is pseudocorus and Ir is foetidissima have also
been used in Europe as Orris root. Ir is germanica is cultivated
on commercial scale in Italy and Morocco. I . pallida is a native
of eastern mediterranean countries. The rhizomes of these two
species constitute what is known as Verona O rris . The true
56
florentine orris is the variety florentina Dykes of I . germanica.
Its rhizomes are generally most fragrant although it is less
12^cultivated than the other two species . The natural perfume
is isolated from the aged and dried rhizomes either by
extraction with volatile solvents which yields the so called»
"Resenoids of Orris” or by steam d istillatio n which produces
the so called "Concrete of Orris" or Orris butter. The charac-i
teristic violet like odour is chiefly due to the presence of
Irones in the essential o i l . Removal of fatty acids from Orris
oil give'f Absolute of Orris , a most valuable and expensive
used in high class soaps, cosmetics, dentrifices and as a
f ixat ive1^ .
Medicinal importance:
»Roots and rhizomes of Ir is species have been used in
indigenous system of medicine as alterative , aperient, stimulant,
cathartic and diuretic , gall bladder diseases, liver complaints,
dropsy, purification of blood, venereal infections, fever,
ringworms, bilious infections and variety of heart diseases.
Extracts of leaves are employed for the treatment of frozen
fe e t . Externally root in powder or poultice is used as an
1 25application to sores and pimples . Crude alcoholic extract of
Ir is germanica has been shown to possess hypotensive and anti
inflammatory action. An aqueous solution of Ir is germanica has
57
been shown to suppress smooth muscle activity in vivo and has
9
a musculotropic spasmolytic effect on the duodenum and Oddi’ s
sphincter in vivo and in v itro » It has also been 3hown to
126stimulate respiration . Some antifungal activity has been
127reported from diseased Blue Ribbon Iris bulbs . Extracts of
iris rhizomes are employed in meat curing pickle solutions to
128prevent food poisoning , . Ir is powder is used as an ingredient
in formulating creams, lotions, shampoos and dentrifice.
129compounds . Dry leaves of I . ensata constitute an important
•fodder for cattle during winter months in Kashmir. It is a,lso
used for making ropes*
Chemical Constituents:
A variety of compounds of different carbon skeletons
«
have been isolated and characterised from various Ir is species.
The chemical components of Ir is may be c lassified as
follows:
1• Flavonoids
2 . Steroids/triterpenes.
3 . Amino acids
U . Patty acids/phenolic carfcoxylic acids
5 . Quinones
6 . Poly saccharides
58
7 . Irones
8 . .Uncharacterised alkaloids
9* Miscellaneous compounds
Flavonoids form the major group of compounds isolated
from Iris species. All the four members namely anthocyanins,
xanthones, flavones and isoflavones are represented. The main
pigment is delphinidin-3f5-diglycoside and 3-(p-coumaryl
. rutinoside)-5-glucoside. A partly characterised malvidine
derivative has been reported from I . ensata, I . chrysographes
and I . delavayl. Delphinin in pseudo base form is reported in ,
some white petalled varieties of the gardeA ir is . Mangiferin is
the main xanthone isolated from ir is species. Recently Ir is
xanthone, a-C-glycosyl xanthone, has been isolated from rhizomes
of I . florentina together with 1-hydroxy 3 , 5 ,6-trimethoxy.
xanthone-2-glucoside, isomangiferin and mangiferin.
Flavone and f lava none glycosides occur widely in ‘Iris
plants. They include embinin, Orientin , iso-orientin homo— *
orientin; saporarietin, ^xy lo sy lsw ertisin , swertia ja p o n in ,'
flavoayamenin, kaenrpferol, quercetin, v itexin , leucinin , vicenin ,
kanzakiflavones.
Isoflavones are well known in Iris and seem to be of
erratic distribution in the genus. Ir is is also a rich source
of C-glycosides* particularly from the isoflavonoid group of
59
natural products. Irigenin was the first isoflavone reported
from Ir is in 19th century. This was followed "by tectorigenin
and tectoridin from I . tectorum; irisolone, irisolidone and
irid in from I . nepalensis, other isoflavonoids isolated are
ir iflo sid e , irisflorentin , iris tecto rig en in _B , iris tectoridin*-
A , iristectoridin-B, 5 , 2 '-dimethoxyT-6 ,7-methylenedioxy
isoflavone; 5 , 3 ' ,U ’-trimethoxy,6 ,7 ^methylenedioxy isoflavone;
5 , 7 , 2 *—,trihydroxy> 6 ,UL,dimethoxy isoflavone; 5 ,Ui-jdihydroxy,
677—methylenedioxy isoflavone; 5 »UV“-dimethoxy>6,7—methylene-
dioxy isoflavone; 5 »U’—dimethoxy^3 1-hydroxy.>6 , 7-jnethylenedioxy
isoflavone. Steroids and terpenoids are not a permanent feature
in I r is . Only a few namely ft -sitosterol and its glucoside,
stigmasterol, campesterol and octacosanol,c< -amyrin and
P -amyrin have been isolated so fa r .
Quinones like irisquinone with plastaquinones have been
reported recently. Benzophenone derivatives have also been
isolated and characterised.
A number of aminoacids are also present in the genus
and the oil extracted from Iris contains the methyl esters of
several fatty acids. The essential oil is a mixture of ©c , p and
rtf irones. Other odouriferous compounds present are acetovanill-
one and tectoruside.
Fhenolip carboxylic acids and polysaccharides have
also been reported from this genus.
Ir is species are good source of ascorbic acid .
Dihydrothiamine has been reported from I . tectorum. Some
uncharacterised alkaloids have been reported from I . drepanophyla•
A review of literature of various Ir is species is given
in Table I . '
TABLE - VII
Botanical source , __________________ Substances isolated____________
1 "50Ir is germanica Homotectoridin, tectoridin }5»3 ' >*+* »5-L
tetramethoxy*6,7—methylenedioxy isoflavone,
5 , 3 ' tk'—trimethoxy*6,7-methylenedioxy
i soflavoneJ 5 »7 ,3 trihydroxy-»6 ,U dimethoxy
isoflavone; 5 » 7 ,U ’—trihydroxy>6, 3 '-.di
methoxy isoflavone; acetovajjillone;
irisolidone, irigenin , irisolone,
tectorigenin, dihydroquercetin-7,3 I— dimethyl
ether1 ^ 1 ; delphinidin glycosides1
mangif erin1-^, starch1^ , caprylic, capric,
1 *5lauric , oleic and linoleic acids ,
embinin1-^, ascorbic acid1 " ^ , L-hydroxy
1 1 39proline D ' , irones , p -sitosterol,
61
Ir is florentina
I . Kashmiriana
I . japonic a
©<-amyrin and y5-amyrin^^.
Iriflogenin , ir iflo sid e , irisolone,
irisflorentin , iristectorigenin—B,
1U1irigenin , iriflophenone and iridin ,
i-somangiferin, mangiferin, iris xanthone,
1-hydroxy—3 ,5 ,6 —trimethoxy xanthone-2-
g lu c o s id e ^ 2 . -sitosterol and its
glucoside , starch, ascorbic acid,
irilone-i;’-glucoside; irisolone-i±f-
b io sid e^^4-.
Irigenin , irisolone”* i r i s o l i d o n e ^ ^ ,
11±7irilone ; 2,U,6,Ui-tetrahydroxy
A I. Qbenzophenone .
Embinin and s w e r t is in ^ ^ . - a la n in ^^ ,
150m-carboxy. -L-phenyl glycine , v itexin ,
isoorientin, swertia japonin, swertisin,
JD-xylosylswertisin and delphinin^Lin-?-«
1 S1(p-coumaroyl rutionside)-5-glucoside 5 ,
152-amino isobutyric acid , acetovanillone,
irisflorentin , irisolidone, irigenin ,
5 , 3 ’ ,Ul—trimethoxy-6,7-methylenedioxy
isoflavone; 5 »U’—dimethoxy-3'-hydroxy-6,>-
methylene d io xy isoflav o n e^^ , tecftoridin^^,
62
I . tectorum
I . p a llid *
I . nepalensis
I . hollandica
I . pumila
I . tanex
I . chrysophylla
I . pseudocorus
I . nertshinskia
I , drapanophylla
I . kumaonensis
I . pallasi
Iristectoridin-A. and androsin, iristecto-
i p c • j cZT
ridini-B, tectoruside J , dihydro thiamine ,
157 embinin ,
Irones, ascorbic acid ,starch .
I r i g e n i n * i r i s o l o n e ^ ^ , ir iso lid o n e^® ,
^3-sitosterol, stignasterol, campesterol,
16*1 162pctacosanol , plastaquinone , isopre-
1 63 'noid quinone, tocopherols
Starch, ascorbic acid .
Homo-orient in , saponarietiij. t
Kaempferol and quercetin glycosides,
orient in , v itexin , leucinin <4 vicenin
glycosides1^ .
Shikimic acid , maleic acid and quinic
acid^ 5 , dihydrothiamine1^^ , irisquinone1^ •
Luteoayamenin, f lavoayamenin,. swertisin
1 67and swertia japonin .
166Uncharacterised alkaloids , glucose,
galactose, arabinose, rhamnose. xylose,
169mannose and uronic acid .
170 171Irid in , irigenin , iriskumaonin .
172Irisquinone , pallasone~B and
palla so ne*C ^ .
63
I . unguicularis poir
I . ensata
I . elegantissma
I . aphylla
I . sambucina
I . variegata
I . heigo
I . f oetidissima
I . ruthenica
I . stolenifera
I . warleynsis
Kanzakiflavone-I, irigenin , iri3tectori-
g e n in l ^ , kanzakiflavone-II, irid in ,
1 75mangiferin and isomangiferin ,
Perulic, p-coumaric, vanillic and
p-hydroxy benzoic acid1^ , k ' , 7 ,—dimethoxy
apigenin-6-c-D-gluco pyranosyl-O-L-
177rhamnose .
Cystine, cysteine, ornithin , lysine,
h istidine , asparagine, asparatic acid ,
serine, glycine,glutamic acid , alanine,»
*
proline, ^ -alanine, tyrosine, methionine,
valine, nor-valine, phenyl alanine,
leucine and nor-leucine1
Ascorbic acid
178
Glucose, galactose, arabinose, rhamnose,
169xylose, mannose and uronic acid .