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 .