THE IDENTIFICATION OF PETAL FLAVONOL

GLYCOSIDES FROM CALTHA PALUSTRIS,

LOTUS CORNICULATUS, AND

TRAGOPOOON PRATENSIS

A Thesis

Presented to

The School of Graduate Studies

Drake University

Tn Partial FulfilDnent

of the Requirements for the Degree

r-laster of Arts

by

riary Owen Slaybangh

Au~ust 1969

J 41C-) c / l,? ./

GLYCOSIDES r~VJSTRIS,

LOTUS COFmIcuI",rc:..~rUs, AND

TFUiGOPOGON PlQtrENSIS

Owen Slaybaur·:h

~-/~~~/ / /

) /

').::'Z //, . <~ /- >~~fl:~~..1-~b{7'</

IT>fTHODUC'I' ION' 1

OF'

..

))13 ION

1.

of tJ:1e fla~vonoJ~ 081(1138 1)[l]~llst11is • ~-------~.,;.

e t; of the flavono18 of .L,.

~Qlr0:E}lJ:!:~~::J? e

of T.J,<-.;-' e

I. Ethanolic s c~rum of the fo:c querce'tin i.J1 (j ~3 i cle

1

nr2110DUCTIOH

Relationships based upon external, morphological

characteristics have been the IJajor botanical approach to

}Jlant clcLssification for ml3.ny years Hecently a number ofe

other methods, including cytology, pollen morphology, and

chcBotaxonomy, have provided additional evidence of these

relationships. Concerning chemotaxonomy, Ercltman (1963)

ethat 1 t ,'Jas simple in principle 'rhe chemotaxonom­

ie '-,lethod essentially consisted lIof the investigation of

the distribution of chemical compounds, or groups of

biosynthetically related compounds, in series of related or

SUj)posedly related, plants." Tech..Yliques such as tvJO

dimensional paper chromatography, thin layer chromatoGra­

:3}}8C troscopy have ?:lade t chemotaxonoI:li c

C11e:-1ot axoI10rr:~r

valuable, because t eCl1.11iq118 S

invo illd.e '-''-;j.h~,-,.ut of t older descri ive

, lCl<?'J.:..../U..} It

Ollt tl·ltJ.t tl'18 ication of chemotaxonOl~T been

to clicl vCilidi ty of t older

c1escrintlve me s of c slfico.tion. so ctcd,

lon~ time to come cheI:licnl 'e

fOT' t 118to c:,:lst

011 111\le~ti '~(_ltc;(l

2

established that flavon,olds v7ere excellent

COiJlpounds for such studies, because of their cJ.1emical

stabillty, ease of precise iclentifi.catlon, lac]e of use

cell !!wtivitles other than cell 1'T9.11 formation, lULlvex-sal

presence in plant tissues in sizable quantities, and

ospEJcies sPEJci.fic' varianCE; in detailed st"'"1''''''' 8.rn0 11.EC

one or more relet or unrelated speciese diversity

flavonoids is due in part to the wide of

tern of substituted c , S11Cl1 as

su0ar molecules att to t 1c

e COiTI.f)

flavonoid glycosides, are easily c eel

to 1 SlS 02'- lIse of

10 ;:­ is

I of b

(2 )

covor~ot(I

oc cal 1 crre tl011shi93 01

(1)t\rotl0l

1

1J It

C o.f' t

3

las of ots

to e for ,'In (;)volu"tl

1:1110 to

JCJ:C

of

't1t.r' t (j

01 (]~ [:1,11(1ot

\I • entification of the

Cl

11 e tiOYi of

tt

i

J 1

1 '

4­

Spectral analysis iias also employed d.urinG thls

nariod and some aglycones could be identified by spectral

cu.rves. Aronoff (19 l !-0) revie1'!ed the l:tterature fJnd

tcrnrrt to correlate absorption spectra with aglycone

f'l8..vonols "t'TClr:; due to the f)enzo'.=>yrone lJortion of the nucleus

[11'1(1 tri.:::tt the B 1.... il1?; clicl not 1)[1sical13T altel~ t11is Cllrvcs

I of 3 8h01'J8 the structure of flavone.

?lavonol has the same structure except for a hydroxy group

in the 3 posl tion. He further Y'laintainecl that the curve

Has caused by the presence of the oxygen on the phenone,

~p at t e 1"'11 •

c

i1

in 300 to 0 1:1!-t

1, and t

,...~ "\:-"IJr-'!'" Y (....• ..L '~_c-.2..II.

cttrvc iil

rescarcl1 of Carts I "1 \ -'-

Otlt t

oul.

1­t \..' ;;olven

1 \ro i

(;

of research. PartridGe (19Lf.6) o.9plied clescending chroma­

to[;!'D.ph;yr to the analysis of reduciY'..f\ SUF';$l.rs • Pate-Smith

(19 L:.[3) used descending chro:matorsraphy and the solvent syf3­

tem of n-butanol: acetic acid: water (4:1:5) to separate

anthocyanl,n::;, outein, and a flavone glycoside, and

employed acid h;,,rdrolysis before he chromatographed the

sU,:';ars. He introduced the method of direct lication of

Ll ]jlo.nt rewt to chro1:1Ettoc~raphic iOJ:l

t e-Smi th (19)+9) tabulat ed Flp data for flayones and

f1c,vono18 and concluded that flavonoid structure can be

values.

Wender and Gase (1949a) applied the descend

chromat raphic method to flavonoid microquanti tati ve ,wrk

R~ values of nine flavones aUQ flavonols and

t color ions to five chemical

f'lavOl101s 1.1

tion tiol!. of altlnirunn chlorid.e, t it

vIaS 5vrain (195lq who first indicated that the shift Has due

to t t i :rl:~ c f fee t 0 f

11'[ r'~ncc in beh[lvlor 1Jetwcen

venal J~C2(:11CC of tl

SElf fo

j.k ..>'\or.lt~.iC:Il

6

with or without a free 5-hydroxy group,

flavone or 5-~fdroxYflavonol With a J position protected

81).I'ar or a methoxy group undergoes a +20 to +1..)-5 illJ.L shift

for l~nd Ie A bathochromic shift refers to any shift in

tern ochromic refers to a shift toward the shorter

1'J8.ve lenGths upon the addition of rEm":ents. R:'lthochronic

values are denoted by a + and hypsochromic values aTe

the vEtlue of dilute sodium ethoxide (l,aVL;Jv) solutions for

the location of monohydroxy

is. In the Dresence of

osi tiOYlfj 1.111CleT'1'Tcnt cliLl.. r~D"cteristic s11ifts of ~:"50 to

(1953) 1.1scd t11is

lfication of five f vone lycosi s fran

scri 1)8c1 ,[1 2118 tiOll t c;--:l.l lie

fOI~ c2tlon o-r su~nr att

thnt f vonols and flnvone

1.11 v 9 1'>0~:' it 1011

,--" :'~ '1 to ",-, ,..; .J£.• ......' i to

"'\ .... r'. r, ~, ~- " (~ 0 r olic lJOI'lc ci

l"bCC ()

~..i er of t 58 r~rou

to

OYl

o

i "

to

~.JCJ~e :)1'"'otcct o:c raet:

C.Yi

101

o rClY; e

or~

1 o it.l '7

"c-.o ~~ .. '1rIO ~ ~. ~

J } ct ;1U

" .

I

r;

t:t t /... ,\r{)t18

,~" ,.....e ,.-,l./."'\..,.I

10 f-l,

1:JJ) t ,'J~ t 10110

r (1961) est eel t ~sl bility of thin

('T''T'r<\ .... ,t. .L:'J ) for

o erol~ cet ltl COYlcluclod thCtt

/'~ (':of­t '_," ..1.• 0 v i cd

....-'f'.'\ 1'1 '! I'" ,L-l.~ r ,j_ L L_'" ~

{- ~-"\ ,­~.' i 1. -~jt

;~"£...... --;'V -.:,.

~'''co:n 1 y--'--- ""-' -'- :cc t:~ ~

-:'~ ("1 -~~ ': ,~ • "", i....... ~ '; ';-'. ~" \.,/ -" ',-- >j

f-,--.,-\C 1.tl0Yl- ~ L .1."';. j

I 1 \ ­

.~.. -;1 ;; to

-,, ' (; i

o /

(1961b) erxrployed molecular e:;:t lnctlon coefficients for

cletermining; aglycono-sugar molar ratios.

Earborne (1965a) l)oint ed. out the v"t1ue of 'spec'tes

S oif1c' flavonoid. Clycosides in describing a method by

l'[11io11 oxyr;en-linked. and carbon-linlred C;lycosides could be

disting:ulshecl by acid and enzyrae hydrolysis. He found

that the length of time, type of enzyme used, and

resistance to a,cid hydrolysis characteristically rovc:::llod

the sitiol1 of glycoside attachment and the nature of the

bond. Hedin et ala (1968) identified 10 new glycosides

from the petals of Hibiscus e~c?lentus us chromato­

~raphic, hydrolytic, and spectral methods.

the occurrence of as-flavonol Slycoside for

t

J~llC t \11'-'0..1 ell out on f vonol

the

t 1-1S, 't)l) t 11.0

r (1959) '--' ted.

erolcone in c.

n}~c erit to [;1. i CGt iC~l

L:c:i 1 (I I'L~tecl t ~·)1~eSe11CC

ClllC):'C et 1. \1

of the nillC neclef~~

:cti 1 ':"j

;",;',,,,:"\;;i l!::;.....:lh,

c: r~l C,-i I

,}\ (o I r"l r-~ r: n '001'",,1 r"'1

() i.:3 o C'; f""\ f"C.) () o 'or'! () •.-1 >',~':: ·t..) -~ ).. ,-j

'r~i ("'" ()~",") f'o>'c", j,-' -I')t"'I'

i-l) o ~"jC} r

-4.:1 ('"'I r-~ Gi () ~"1 ·,1,,) (''',.,.. o ";':":

1-".~. o 0\ r-~! :;:: C". i (,., rc::: r--i o ..-i u r" r:; () f] {> cJ "".

;:~ ~,".~'j .;:::> r~::; ..$.) "r,,1"I'! [-.] (io S-"L~ () ;=.,~: t > ,p r-·j (' \ 0,1'''' ~ rt>' (D ;::­ _c.\c'~ r;i: re,:; f·i c- () ()

~1 (,D (', LJ <,-I ~r~l r-)(" ()t,;, () r,

{:,' ",< ;~~ r~J "0) to (,-! re' o ~'> o " ~--, , ' -ot",,'-;;_.,­ () ·,-l ()l-')

~: Cl r:'; c ~'}-) ,q'~') o .,.,; () CJ (j (" j

{..'~ J:":; r-} $--1 .p .rl A'C'~ (j ..j -4.j \,!.: r..: () -~,,) I (.

~,

-{J t'2 .s::: ~'~ .r~ () 1..".-,""j

~r-'J ..r"~"0 ...ri +:> or'l4·~)

c ~

o r')('lOJ re'c~ w")C) c.\ .'r-!:> () .!:: ~'''I

r~" C c,-~ r->,_J {) [ ;4'4 C) -l,) ,-,,:1 .. /,,-it-~ oG " ov-~')

() 'i"e'+)r-c; o(, ~,: rJ C,'o o j>".}c cS

C"C'.. t"l o (j -!-.,) bo rr:;'

"1"",j C I o~r-~.r! ~r~'1o -0-) o 4-)

~,

rrj r-'~--,",(il ()(L C) .r'"

~,,) o ..r-.j ~~,,! r'.-..... ..c:; .j..,)(':,) CD ()

§ V.;, -4.-'>

() r"''': ·r·b !;;: o L) is~ .,-i (J r(~:, ' -p

(') ~r-'J ("\

4-) C'\..ri:

,.....')Q) () 7"""""',rl , ' r\' t..:' (iJ C) C ......'--- ..... '()

~, I",L~: t--\ o (' '!Or"

11 ~i .~.) C) r' ~t-j y'i r

....--f -rl: ~...) :='i' r~-'j f,"i """') /"\

(J) ~"

':./~) I )

..ri .-i (' ~::1 C·~"J ~rl ~r~1I'"

:r ,~C,"'", () ... ) o r'" ./",,; <or",j~> r"~; () f:) o QO' t~.' ~~,~~r1 '·

C) r:;:,~ $-; ·,.1 ( j " r-'- c; ·d C' ~~_"I ~ .,

1--:,",!"m1

;,-~ c ~.-I {')

,--' ('() t:1 r': r

C."" }:-:: o ~"'" r'~ i~r-~ ('.•,1 , \.,'

C~ i"i

~\ o r'l r'

() C,.: ::::i 1'-', c .

,\._;~r '\1~;.,~..L) ·jl r' "

11

7-:nethyl other (IV). Qv.ercetagetl11 has a hyclroxy

In the 7 nositlon. Among the other Legu1111nosae Ka1'1'e:r

(1958) and Harborne (1965a and 1965b) recorded the

occurrence of the aglycones lraempferol, isorharrnetin,

pratoletin (3, 5, 8, 4'-tetrahydroxyflavone), quercetin,

and querceta:.;;et;in. L.eguminosae Clycosidos typlca11;y

ibitod the J glycoside or J, 7-dlg1ycoside

eaxon01Tlic sir:nificance for this pattern of substitution

and iJrocUcted that 1t should be present in unrelated

lie::-; • He also said that rslYCOSlcllC stllclies of tiils

substitution at the reneric species lCVE~l

I; ) • itrltioYl

t teI\11S other than tl18 J~ 7-disubstitutlon

tIle COI1YJosl tae

l")rCCCnCQ of the aslycones quercet ,Quercctacet ,ana

r:::tt ulce In ( nerc 11'1 6

ill ? 1)051 tiOT1S III

V()J10 1.

t 1"'811(1 f'DI" tll1~(}C hOG ,).11

c

.~

:. '......'.:­

ch

.Ie tens

l1inat

I'8so1v8cl tll1s

tec1 9

~-1et

Elor"e COIn;·}

etlon,

1::10

oral VIes often tl1c

CEJ,llSC t

lvc

1

'70+ -:­L.1~ \.J v

111 t

uti ~ vanol

os

tl

!1'his v7L~,S

r

I-~\ ...,..... r::" ~.:.

, j >~ "'--.-­ ~~:

j .. et

e:r[~ctecl }J1Jtett

eet

c

tt

';"C'

1 "1

It:;8 Cl s o i

E;" cole

,~_ "l.~

'-..,1 l/ to t

t:1.011

()l~ to :In •

.'. ,~ Cl11ere in 1')8tttl

to of the mor'c speci ZE~d st

the production of 4'-mono­

iVGSe Since both 0.11. ct

s were slml1a~ly

tiOl'l

Clll'SOI"' to bot11, al'1cl t}18 ft.ril1ef?Y"[;"Y1G of

'),-" , l!:'

cot t 18 in the comlJosite lineo

t si

C- orl t ~c·() 11

-,L, I)lc O'S

( -; n1 1

tOll

tin and cl'uercet in early in biosynt sis 0118 ~,.~ c2uJ..secl Sr1l11'1t of precursors which ~rere

1.1se rl to fO!'Ifl I"ol" Irl 8.. seC011.cl rILl

there was decreased ssypetin production and a

GorI'c~s~Jondinr: increase in quercetin e1'01

is ill t

lJalustris net s were collected in 1 11earQ.~~,'. ­

, Ill. in

in t Itlttcr of

v'J8st tt11~1 for Ind~ stnte Route 2e

,~ 1'rere collecte~ in

Tll.J... -,._ ~~ • ..:\.11 dl'ted DO t:-o

from two to five

f ~lere sto enve

i

11 1 \ ; .

c in silica L vc tc

mo(le]~ IJ.='

ce t t r 1'TJ.t

C',

~I

1.5

r;1~)cctr1).m 13 recorded by synchronized strip e:raph paper

and ink pen. (1'118 shape of thls curve and. the Nave length

at en JIlB,xlmum a~1J30rption occurs are very precise \fmys

of identifyinn; the compound. By using various reD.gents

the point of n8.ximum absorpt ion IflD.y be shifted m){l these

chanc;es give defini to inforr.Jation al)out the strl1cture of

the conpound u.nder study.

g[lCh c:rlJ.de extract Has strealced Ntth 8. pinette on

5'7 cm No. 3 Whatman chromatosraphy paper, and

developed by mass descending chromatop:raphy overnir:~ht

us i ;",0~ Ive ~ o.d (~ b·le l' .L"'~!~. H ~ol~rp.'l~~V•• k __ dOvn1 the1:1[',' -nl- va··.· t~e-. _ V moves'0 _

paper the more soluble pigments in the mixture are carried

near the solvent front and the less soluble 1al:::: behlnd.

the visible 9icment

t'iltor ~, elut With absolute ethanol,

to 11el~ deter~ine

of t nurifi

to was streaJted on 22 22 CT:I ..,. )

1

.Purific tlon of L.

f~ '\fOYle) 1

). @The: ,1

t ('~ . t i 011 1-J~_l::~: L~ e111 C "'\IC

, .; 1..

OJ c;l

,'-', r ' ­ .,

16 1. C 801 S~fste::nE

Solvent nixture Vo 10

l:Kl acetic acid:water

ar sa ted with 'henol

ac 10 acid:vratcr 15: 5

d Iso~n'opanol:1;jat(~r

Cyclohexane:et11yl acetate

Chloroform:me"thanol

tCT'

i

h

Yl-­ t 0.110 1 ~ ene:

)-4-:1:2.2

f vonol o~i

r n~'itt t \ ,l. j e

c

c

to of e ])e

...--~ "'-~' , 2..L ,-..lIne t t :3ili

tI0 i \

Go '01

'1 nO "1

\,.,

I~

\ ) 0

17 ~- J

olutc

, . I ')

t(; f1cld ted. c

odiuD ~c8ttlte (Juri,

fo~C' tIro

(~'IecltYl et ..J ) , c ,

+- \~11 f~~ V\ V -;_~.u:,~_,"-J.

i ,c.: "')""'.'-- _'-.. e

10 \,-TD.,tCT' as 2 solvent, the to

;J ol.vc21t 18 t acl~!COlj.e tlO\Tec. \T(;1~2:' Ii tt.

t off 01 -cc

1~ Cll1::'lC lte t c tIle

OJ" t: v"orJD 1 c

..... ~ fo.,Ctl t ()1_~ '-' _1. <

CJle

10

i t

) r

c

os~rams were spotted and each sheet was then

18

i iflcation was

solYEmts

achieved

8" h, i, [:mel j.

(t~rc1I~olYE:;8~te

i

pJ..l"G

for 10

vo.11lGS

" at 1100

Hith

c.

lil1C-II­

values

t eli ase movo':;

sol

e.i ;~~ t i rl,C~'ll i

ter CElme l1GCeS to hydrolyze cO'-:l~:eroially

described._ The resultiTIg hydrolysate was spott OYl 1

sorbose, sucro~c,

t 0 ,~'--'~. r a.rn 1 asc .ttl

c: cct cltll" ti

sneot

L 4­,-11}Ytetloll itl1 Ej1J l' i u J.

1 1 c

lCl -/

v< #'''~

a; ex: f'""

Table 2.. Spectral maxima of the flavonol glycoaldes

and aglycones of Q. palustris

Fused

Maxima Sodium sodium Boric

absolute ethoxide acetate acid "" Compound ethanol AICl; (NaOEt) (NaOAc) NaOAc

Ire. ;64 400 416 379 386

360b 332

296 302b

257 259 274 268 263

373 437 415 (u)c 386 394

366b 330

302b

255 269 295 258 261

III 377 431 q;. 406b (u) 398 392

366b 327 337

305b 277b

255 267 298 254b 259

375 i}1J.4 408 (u) 387 394

332 275b

255 270 298 257 261

n The spectral data for each glycoside is

SU::lt.1nt"1::od after the Roman numeral follo\~ed by aglyoone

dnta undorneath.

b Dcno:es on inflect1on.

c Refers to the chem1cal breakdown of the

flavonol 1n the presenco of this chemical.

--~--------_........_---------------------- ­

21

OAe

263

rTftO.A.c

flavonoid8

-:;c;h 382-,' J

')(,0 265~',,~. /

VII 3L~8 390 381}

355

?f­,~V ')nIbjV

?C:tjb'-'..){ 27L~ 272

II JS6

2 t 331

'7, 2 b

1.JI>:

/ b

;)r;< (~~ { v

bl0 l~.

c;l,bsolute

0111'1(1 ethanol

etral maxim.!'), of the

of fa oratensisQ m.

AICI~ OEt ./

01'10Y10

~IIII 111 no COSl rOT'

Dxnd II

::n + V)

c ~

U

'ff U

+0... o

22

Band I

350300250

..---------------------------.

the aglycone forFlsuro 1. Ethnno1ic spectrum of

quercetin 3-rnfflnoslde (II).

2)

, , , , , \ \

\ \

.. ' , \, ,

I

\ \

1

:n + l/)

c. ~

U

t(J

U ;­0..

---"0 ,, ...

2 ,.--

/' ... ,/ ,

,/ \

/ /

/ I

I /

/ _.....

Figure 2. Spectra of 5, 7, 2', 3', 4'-pentahy­

droxY-J-methoxY-5,4'-diglycoside aglycone (VI) in

(1) E;thanol ---- , (2) aluminum chloride - - - - - - --­

01-1

~ j OH

o 0 118 'c H 0 6 105

(II I)

O·(C H 0 ) OH 0, ;='\ 6 10 5 3 MeoW?")~OMe

~ O~ (\1 eO g (C H

(v) 6

OH

HO~r II

VVOH HO IIo

(v II)° (VIII)

g (I X)

r·1o.j or petal flavonolds of

OH

/. O'(CH 0 )II 6 105 x

OH

~ )OH

c. ~)}lstr.ts,

24

~ 0). 0

10 5 x

25 c. ~~~~~s flavono1s.

~u~roetin )-.raff).,no,side (II). The +1-l-4 and +6L~ mpJ

Pane I :::1I1fts in AICI) indicated that there 1Ims a hydroxy

,) at the 5 pos1tion and a 3 posi tlon TIlasked

The hano11c reading 364 A~~x was consistent viith the

reported soohromic shift for a protected :3 position

(Jura, 1962). The band I shift in NaOAc revealed that

there was a hydroxy group at either the 3 or 4' position.

3and II shift 1n NaOAo shO't'iTed that there 1'JaS a hydroxyl

croup at position 7. The stable glycoside and unstable

a 0 1ycone established that either the 3 or 4· position l1as

protected by sUl3ar. The shift of Band I in NaOEt l'rithout

decreased intensity indicated that the 4' position was

e @ '1'11l1S 9 t was attached to position )0

ric ac SY11ft s t t an o-dihydroxyl

choice vJas 4' or 4', 5', because tIle

did not t118 Cll.oice of t

Uilu~"Jsis revealed the nresence of')r , r)O~Jitlo:n.

tt t ')Oil1.t S 111£1-;;' I1D.ve ~jted.

0:::1 cetin was conside possible, because

ino

c

26 .!- }-.

'c ~'. '.J t,- 1t LOYl ':' i.1O:C .L

111

this decision (Egger and Keil, 19650.).

·~o 1 lr.t

icc;~111 C

25/

r"" \ "',y-:!, ') 1 (\J'j I .) (' , for'

't"'i ~.~ -r- ,~;:\.

,.;... ', '.v ...." CllOico of quercetin.

tiorl

i

Cl11 o i

{ -~- \.. i ! I :\ ........... ~~ I e Ii':

(A reI , y

..,~~'" of·· ~ \~". ',-"

1

.,

o

1

27 sitions, or Coss YPGtln ectad 5, 8 hydroxy

8t~11D~}J.o11 c CG of

c 01' C11.ls:rc ii.'1 [;1)..t rIot tl'o.l

s fts in bo C o.ci cl.CT1011st l"'"D.t

? , h r O"~ h t C fJ ,. J..., j Gi ti011S, t '_ rell 'v lY} ,-. ssibility, since its J'

osi tior~ is ed.. 'The -j".5'-i­

sition,.

'.....:.. ~,/~c; v

~30I:1E: c111ce flJJ.::C t i orl El11.Q

:fr"ee .3 r

tlydroxy function ( tl1'l~il(l G. r' 1 c1.'__ Q ~ .. iI ............ ~ ......... _ --, ~• ./. ,/ I

tin (C-EJethoxyquercet bi t s a -+70 illJ..L fo:c e.

J.'O '::,

itioY) of tileJ

L

l " <-. .1_ "",,",~, C' ".,._ ~.'J V _c... ~L.. '" iCOL} 110t

4. 1- t.., --<_~ J.., v':' ~ ~

to

o C 0

.r." 1 L

1. 1c

L

AIel] I

e ll.;I(ll~()X~Y si tiO:ri"

coside instability in

.~ 1~('~ :r'Oll. '.~ v sitions. Ethanolic

but not the choice of

ass ~etin or nyrlcetin.

~-. -l­ +­-..::)~ v v

i11

sclosod t ~ there were hydroxy 8roups at either t

sitlonSe Thus, the sucar attac

5, 6 or 3' position. sa 'tacl1­

~.ent po ts have not en reported in the literature.

ols Hi [lycosidation in the 5 lJosition are rare

i I •

ice of 2 raffinose

~-~ 1;--, r-o, 'V~2' t c11(1 lj L 1'< i i

c

:H'~ ''1 I e

r: :Co 1-:; r,~o S lllco 1

1 .\ +. -'.L

1 n .~) to

29 t

roxy V J. sit

ft ~'Tas Yl0t 1::110ti Trl, s 111.Ce t

110St 0 r flavonols is around

A1C1l..-/

curves were not typical e1 thel~ a~

(::, "'I ,,- ­

'-.J J 51 J

,,~ , i, t l'JfiE~ d. ed.ll cecl t both were free,

c() t of the necea 2ochromic shi s of

lIe ch sochromlc shifts 1

Cl~ C1lTVC {-Yere at leal, because,J

3 or

r' .fO ttl011

'O~~), ~he disn~Dearance of.L / .... , (., I.i1 _ ~, _

I(',ll1""\J8, t

(If r:clos

.ition 7_ s i11terprst3tion of a

of

;, f riO ()~(Jj, tlon t Ol~ J'

ltlo11

iti

tc' oltc

I

not 811. T~(~I)o:et 0112

c :t o:f

I) ,1962)e Also, a .9rotected

--, ox' ~~)os:ttion cts fOl~

l\lCl'") .J

--. r'" .,!­l~"J 'v "tc =

!­l.... .~ t

COl1clitioll :1

.~ -i- 1" J~ ',."

'c -;-::0

I

r' !- (' l j'

v .~-,

1 ~ \ I •

c

c: '- (~11

c-l:rcos:1dc (VI)._- ..- +68 l1lf-L inflection in AICl':l.--.:~-1_. -","-,~-,~"", sted

./

t the 3 )ositj.on was hydro}:yl.atedo

(see f:1~ure 2) indlcat

01--- o~ 8el 5- with a nothylated 3 positiOTIe

one spectrum as Q if the 3 position

tedc The lac~ o~ a definite s ill

the sence of one in the OXIC

Tthat the 5-0E l't8S os ted. J..

etl1nnolic -lycoside one

i,f" c~ l'(.....'"UeOYlc.lllS i 01'10 et 11 c a::lycone I

~~ of 01JerC in in ethanol is not( Tn 1

'"'j ~'""1 r:,. ~., ..L_ ~ .'. \.,

orl ():i."' L ct:l1 II llsnolic shift t:zs 110t

t cl'loie o sh..l f :Co:r111 i eJ. C X'(:

4-- ~* +­ nro ~ ;'jT i

(; I

J.. 0"!",,,~_~ ("'; .'"- .i.. ,_~ \. •

1 t lOXl I

! 1 , \ ,J.. / 5

t~ t

7

i t se:r.lce of all 0 ......

of ti011 11"1 :TCt

f DO ition wac Clycosi

e no 0­ fllI1Ct i011 OCellI"

'1, 1'"'~' -f"-­-" 1., lJ 8~ lit:r of t

lit of c i i o

S, -t o f11J:'lct i

1 1011 e

1

!. +- 4,L 0L1. 011. 0

-J,o. {,..... ~r'~ l-' J.. U~.:. 0 1

no ion

L<1 V ., <3,

11tJ'

ts vrt of 005

ect

l1c c 1).. j~'\Te S ,

,.uv L •

llYli ,-.'~_ tifi e~vca to be heavi

C 01r ,l G C 1'1'[ 0" t 1 H fo tl1.C

101T'" I' of

'J:-c"t (::11.3 1 S ti ~,.",._._.'.._~-~,... ~.'.'~....-..­

VIII were obtained fron se

c­

1 c

'­ .:; -,"";"---,+" ('"',-,"" ~ ...... '-, ~

,"1-''-J ~L. '3 _1._ Ie •.'. t

I::

110t

c t11rlT1olic A

r., : !

u III 10 Cl "7:,

1.L, " 1

(VII I) • AIC1~, sclosJ

v.l; sltion hydroxyl croupG

I sh.lft 1.~Ti tl101Jt

1tlon was protect ich i~~

01 cant et to t118 OUS cone .101"18. The

reason for t is contradi ion was

II 8 C:l"'Oll~:) .i1'l t11.e ? sition.

l~le ~~lci ft indicated a fI'ee 3', 4' o~dlhy~

ro~:rl 8~rstem& This was consistent ~;ith t

~rcone • The ~~ ~.;:.t e CO:tlt ELi 118 (1

Sllcr"'os!.:? e1 r arabinose or xylose. No sUC3r

r', ,,:,, ~,"! ..!­ '''':< .'\'-'t;--::. '. ••.f ,,~ _ \.J il'lt C.c<..-l.. '---"

tl~e et 1c

cst , 1

t loss of t

c e T)1Jrc i f .i CElt i 011 0

se

o C)llr~d i t lor:

t -: thf.:}:·c

i J. f r' ~:_, .

r\ ,. (l""! ._ L .:_ \.";

.!-. t...

fllnctio11,e

o:~i 81 f j~ t e1

()r ect or t

1 ill /

S8Y1Ce an

C, 255

c~ COllClllS i 011.

e{~"-Yl11.0t ned, if it is as

StIcl] 8, ssi 8)ci

COile 1tj2t3 ~1. f "j}"011e, l)tlt

earch 1 nee d to c choice of one.

ob:3

o 1,: ()

1 f ex ti

.~ ..L

11 f

c

J. s1 lC-SJ1t t> (~rrle 1)X"eSel1ee {)f [-ill 2tntl~10'-

of tIle ell! villi conceals L. cornicu­

serves as a pollinator

E:ct S (I or more of the flavono COll1c1

"l 'j--- '1 'f '. v ,__"v.~ ..

1s for evolutionary selection of flavonols

"1)11t f':ror~1 q.ueJ~cet iEj 1'JOuld o 111 110

to ot flavonol enel

1"01:1 (1 valid

ation for the creater vari

is

to the

c cot t

..-\ \ t~-~ j e

of c in.

, .k

811.C ,1.11

10 j

•J. +-l/ 1,,"__ i'1

t ci" -,1.

CY.lOif:l-C

·i ( f"1 CD.lit 9 te_I~ ; c

C" t t j y. tc O:C ho­

"f'( ['~ 'Yl (\ If '-",.;.".i.,. ...v"

') , i:_ r TtE:CUrSorj , S £l11(1

esc cLiri

ycllow-colorec: D.1 S o:f t 11 i s i i1V e i t i 01"1 ,

tillt d.i cl in t

~YY'~d~~~~l was forDed instead of

Cl Hercet fore, the ~resence of 0 ~:~I'Ol1. _ :-'.

Ct:ltes t the cnYotenoid association

fl Y.rC.:l' _t,~

'v '1 ._~ of v ltc

r. ""···V1 Jli ',r' ~i --C010l-'C' c-c~.. i

10 lrl<" i

i

flc)l'J(; .~.

c c:n t:t r"ll:t~Ett i all th,c ... J

i:o';

ti tlor:

lc

11 (;

1 t

r~·o o tl1c

t:O COl1 i

."•...\f;~.S-------------------­

1

to Ck'la:n.ce rJll)tCttiol1 seD not

e for reclassification. Hote t

('~ 1" -r",-'1 r.;:.. r'~ ',.) "''.. ..dL'"-,, LJ e~c corrtrlins

cee ')c;sltio11

( 1 c; of

t __

'-" ,;

o

., till C

arc p cted for3il'lC c

1

tirl

..,- " ~ ~­,-', _.Letlan is U8 to C()~ i t

ci

o

01 ~LC

"'"-i-='1'~II~••••• •••••••••••••••••••••••••_ .~ ~ .

'::. J. \.rc .tl P-l ~,-.... "'.. C.l of' flavonol

to ~-~

ct to veri:{' e"f,..rol~t..lti

c-.l e:~tracts vrer'e

.') ~,-;.-, -: .~. '-.' ~

:tc

t T'11C

o f 9

cto::i

L~' .- t. ri

to J.c.;tc'T~ri11

1

1f1c(1 •

r") """"",.p.p -i -'!­ , ..•·Y J • .0... -'~

1.

1 l~ ~.---, ;., ',.\..

1

<;;',,-£-----------------------­

.;~ ~i : '" l-, ...L i orl

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~--------_"."1_

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