Nuclear Magnetic Resonance

Analysis of Flavonoids

Tom J. Mabry, Jacques Kagan, Heinz Rosler

HO

OH 0

THE UNIVERSITY OF TEXAS PUBLICATION

AUSTIN, TEXAS

Nuclear Magnetic Resonance

Analysis of Flavonoids

Tom J. Mabry, Jacques Kagan, Heinz Rosler

Department of Botany and

Cell Research Institute

The University of Texas, Austin, Texas

THE UNIVERSITY OF TEXAS PUBLICATION

NUMBER 64I8 SEPTEMBER I5, Ig64

PUBLISHED TWICE A MONTH BY THE UNIVERSITY OF TEXAS,

UNIVERSITY STATION,

AUSTIN, TEXAS, 78712. SECOND-CLASS POSTAGE PAID AT AUSTIN, TEXAS .

Contents

PAGE

Acknowledgments 4

Introduction . 5

Materials and Methods 6

Interpretation of NMR Spectra of Trimethylsilyl Ethers of Flavonoids 7

Discussion 1 0

Literature Cited 10

NMR Spectra 1-51 12

Acknowledgments

This investigation was supported by Grant F-130 from the Robert A. Welch Foundation, The National Institutes of Health Grant GM"l 1111-02 and the sup· plemental grant NIH-GM-ll l 1 l-02S1. One of u~, J. K, thanks the Robert A. Welch Foundation for a Post-doctoral Fellowship, 1963-1965. The authots thank the Chemistry Departments of Rice University, The University of Texas and Texas Christian University for the use of Varian A-60 spectrometers.

Many of the flavonoids used in this investigation wern generously provided by Margaret Seikel, J. Herran, A. R. Kidwai1 H. Wagrtet, E. W. Underhill, E. M. Bickoff, R. Neu, F. De Eds, M. Hasegawa, Artnn Nilsson and J. Chopin.

The editorial assistance of Ursula Rosler, Myra Mabry and G. Knipfet is grate­fully acknowledged.

Finally, we thank the Graduate School of The Utiiversity of Texas for grant SRF-289 for publication support.

Nuclear Magnetic Resonance Analysis of Flavonoids

ToM J. MABRY, jACQUEs KAGAN, and HEINZ RosLER* Department of Botany and Cell Research Institute

The University of Texas, Austin

Introduction

Flavonoids constitute a large class of secondary compounds, widespread in the higher plants, which are especially useful for taxonomic purposes at the species level. In an extensive biochemical systematic investigation of the genus Baptisia (family Leguminosae), flavonoid patterns, as disclosed by two-dimensional paper chromatography, were used to validate natural hybridization and to study the structure of populations.1 Subsequently, this work was extended to include not only the isolation and chemical analysis of the Baptisia flavonoids but also those from selected genera of other families.

Although the isolation of natural products frequently requires elaborate pro­cedures, the tools of modern organic chemical analysis, gas chromatography and nuclear magnetic resonance ( NMR), infra-red, visible-ultraviolet, and mass spec­troscopy, often allow rapid structure analysis of pure substances without time­consuming chemical degradations and syntheses. One of the more recent tech­niques, NMR spectroscopy, has had limited application for naturally occurring flavonoids, most of which are glycosides, because of their low solubility in most organic solvents. 2·• Common derivatives, such as methoxy and acetyl, are generally not suitable for the NMR analysis of all flavonoids because the signal pattern of the natural product is often obscured, in part, by the signals of the additional groups.

Following the report of Sweeley and co-workers5 for preparing the trimethylsilyl ethers of carbohydrates, we investigated the potential of these derivatives for the NMR analysis of flavonoids. 2 Independently, and using a different procedure Waiss, Lundin and Stern3 reported NMR data for the trimethylsilyl ethers of several flavonoid aglycones. Subsequently, Batterham and Highet•b described an alternate method in which they used deuterated dimethylsulfoxide. Most flavonoids are soluble in this solvent and the identification of a large number of them was reported. In our work, we have analyzed, in particular, the NMR spectral patterns displayed by the sugar components of flavonoid glycosides. We have found the readily prepared carbon tetrachloride-soluble trimethylsilyl ethers satisfactory for the NMR analysis of all flavonoids thus far examined.

We believe the availability of the actual NMR spectra is of considerable value for structural studies since most tabular presentations of NMR data do not provide adequate descriptions of the spectra in all detail.

*Permanent address of H.R.: Institut fiir Pharmazeutische Arzneimittellehre der Universi­

tat Miinchen.

6 Nuclear Magnetic Resonance Analysis of Flavonoids

Materials and Methods

The following conditions were typically employed for preparing the trimethyl­silyl ether derivatives of flavonoids (Figure 1). Fifty mg of substance was dissolved

rhafHOfilCCIH-0

OH

HESPERIDIN

BcH,),sD2"H

(Cff,)3SiCI pyndlne

OH~

\ llCll, lllulri:i.=::z:· ri 1-0

~ aqu.lleOlt

SILYLATED HESPERIDIN

Figure 1. Trimethylsilylation and hydrolysis procedure.

OSHCH,>J

OCH3

in 3 ml of pyridine and treated successively with about 0.5 ml of hexamethyldisila­zane and 0.5 ml of trimethylchlorosilane (Applied Sciences Products, State Col­lege, Pa.) . The solvent and excess reagents were immediately removed under high vacuum and the dry residue was extracted with carbon tetrachloride. The clear carbon tetrachloride solution obtained by filtering off the salts was ready for NMR analysis when concentrated to a suitable volume. These steps require about 20 minutes. The original substance could often be regenerated unaltered by allowing the trimethylsilyl ether to stand several hours or to reflux 30 minutes in 50 ml of 20% aqueous methanol usually with a drop of acetic acid. Frequently the flavo­noid crystallized directly, otherwise the hydrolyzed product was chromatographed over silica gel or polyamide. The same procedure was used for all phenolics and sugars investigated. However, it was observed that the total conversion of flavo­noids with sterically hindered hydroxyl groups to their trimethylsilyl ether often required a reaction time of more than one hour at room temperature. We subse­quently observed that the addition of a small amount of trimethylsilyl chloride and hexamethyldisilizane into the NMR tube prevented hydrolysis and insured com­plete trimethylsilylation. The NMR spectra were obtained on a Varian A-60 spec­trometer in carbon tetrachloride. In most instances, tetramethylsilane (TMS) was an internal standard, but similar results were observed when external TMS in carbon tetrachloride was used as reference.

Mabry, Kagan and Rosier

Interpretation of NMR Spectra of Trimethylsilyl Ethers of Flavonoids

7

All hydroxyl groups in both the aglycones and glycosides were converted to their trimethylsilyl ethers as shown by the integration of the NMR spectra. In contrast to methoxy and acetyl derivatives of flavonoids, the NMR signals arising from the trimethylsilyl groups occur out of the absorption region of protons in flavonoids, at about 2.0 ppm from tetramethylsilane. Most of the signals from the silyl groups occur downfield from TMS, however groups attached at C-3 in some flavonoids are found at about 0.1 ppm upfield with respect to TMS (spectra 32- 35). Occa­sionally the trimethylsilyl group at the 5-position was hydrolyzed when the deriva­tive was exposed to a moist atmosphere. The C-5 hydroxyl group was then readily detectable since the proton, which is hydrogen-bonded to the C-4 keto group, gives a singlet near 13 ppm (cf. spectrum 2). The hydroxyl substitution pattern of the flavonoid nucleus as well as the position of glycosidation and the nature of the sugar moiety in glycosides can be deduced from the spectra of these trimethyl­silylated derivatives.

8 0

2 4' 1

c 6 3

0

Figure 2. Numbering system for flavonoids.

A-ring Protons

The two A-ring protons of flavonoids with the usual 5,7-hydroxylation pattern give rise to two doublets (J meta = 2 .5 cps) between 6.0- 6. 7 ppm from tetramethyl­silane. There are however, small but predictable variations in the chemical shifts of the C-6 and C-8 proton signals depending on the 5- and 7- substituents. In the spectra of the four luteolin derivatives ( 1-4) which vary only in the C-5 and C-7 substituents, the B-ring protons display practically identical signal patterns. In con­trast, in the spectrum of 7 ,3', 4' -tri-trimethylsiloxy-luteolin ( 2), the C-3 proton signal is shifted downfield while the C-8 proton is shifted upfield, each about 10 cps from their positions in the spectrum of the totally trimethylsilylated luteolin ( 1).

8 Nuclear Magnetic Resonance Analysis of Flavonoids

The signal of the C-6 proton ( 6.18 ppm) does not depend on the presence of the trimethylsilyl group at C-5 (cf. spectra 1, 2, 3, and 4).

When glycosidation occurs in the A-ring ( c.f. spectra 3, 4, 6, 12, 16, 18, 39, 41, 43, and 45), the patterns for the signals of the A-ring protons differ slightly with respect to the aglycones, while the B- and C-ring proton signals for these glycosides compare closely with the corresponding signals in their aglycones. The NMR pat­terns of the A-ring protons of ftavonoids that have sugars attached at either C-3 or C-4' (spectra 22, 23, 24, 29, and 31) are usually similar to those observed for their respective aglycones.

If a hydrogen has replaced a hydroxyl group at C-5, the C-4 keto group dia­magnetic-anisotropically deshields this C-5 proton which appears near 8.0 ppm (spectra 26, 35, and 36 ) . Flavonoids frequently have substituents at C-6 or C-8 and their assignment by conventional methods is often difficult. Furthermore, the well known Wessely-Moser reaction tends, in effect, to equilibrate a substituent between these two positions by opening of the ring C and closure in the alternate position. However, the NMR signals for the C-8 proton generally occur downfield with respect to the C-6 proton, thus providing a method for ascertaining the position of the substituent. This technique allowed Hand and Horowitz6 to propose structures for the isomeric carbon glycosides vitexin and saponaretin ( c.f. spectra 9 and 10).

8-ring Protons

All B-ring protons absorb around 6.7-7.7 ppm, a region separate from the usual A-ring protons. Protons are normally present on adjacent carbon atoms in the B-ring and the expected ortho coupling of about 8.5 cps is observed. Generally, the C-3' and C-5' proton signals occur upfield with respect to those of the C-2' and C-6' protons.

C-ring Protons

Flavonoids are classified according to the oxidation level and substitution in the C-ring. Considerable variation is generally found for the chemical shifts of the C-ring protons among the several ftavonoid classes. For example, the C-3 proton in ftavones gives a sharp singlet near 6.3 ppm ( 1-10). The C-2 proton of iso­ftavones is normally observed at about 7. 7 ppm ( 11- 18), while the C-2 proton in ftavanones, which have a saturated carbon-carbon bond between C2 and Ca, is split by the C-3 protons into a quartet (J e l s = 5 cps, J trans = 11 cps) and occurs near 5.2 ppm (36-46). The two C-3 protons occur as two quartets (JH-aa,H-ab = 17 cps) near 2. 7 ppm ( 3 7). However they often appear as two doublets since two signals of each quartet are of low intensity. The C-2 proton in dihydroftavonols appears near 4.9 ppm as a doublet (J = 11 cps) coupled to the C-3 proton which comes at about 4.2 ppm ( 32- 35). A detailed analysis of the spectra of ftavans

Mabry, Kagan and Rosier 9

allowed Clark-Lewis4d and others to make stereochemical assignments in the C­

ring. The chalcone protons which are equivalent to the C-ring protons of other flavo­

noids are designated as the a- and ,8-protons and occur as doublets (J = 17 cps) at about 7.15 and 7.55 ppm (47).

Sugar Protons

The C-1 protons of a-glucose and a-rhamnose have axial-equatorial and equa­torial-equatorial coupling, respectively, with the C-2 proton and a small coupling constant ( J = 2-3 cps) is observed ( 50- 51 ) . Glucose commonly forms a ,8-linkage in glycosides and the C-1 proton has, therefore, an axial-axial coupling. The broad signal near 5.0 ppm (J ca. 7 cps) is characteristic for glucose ,8-linked to the 7-position in ( flavonoids 3, 4, 6, 12, 16, 18, 39, 43 and 45 ). If, on the other hand, glucose or galactose is attached to the 3-position, as in some flavonols, the C-1 proton of the sugar appears as a sharp doublet near 5.7 ppm. For example, in hyperin ( 23), the galactose C-1 proton appears as a doublet at 5 .63 ppm (J = 7 cps) and in isorhamnetin 3-glucoside ( 29 ), the glucose C-1 proton is found at 5.73 ppm (J = 7 cps). The remaining protons of glucose occur between 3.3 and 3.9 ppm.

Rhamnosides and rhamnoglucosides of flavonoids occur naturally with an a-L­rhamnose moiety in which the rhamnose C-1 proton has an equatorial-equatorial coupling. This C-1 proton is observed as a doublet (J = 2 cps) at 5.25 ppm when the rhamnose is at the 7-position as in robinin (spectrum not shown) and at 5 .OS ppm when it is at the 3-position as in quercitrin ( 22). So far as is known the di­saccharide of rhamnoglucosyl-flavonoids is either rutinose or neohesperidose, 6-and 2-0-a-L-rhamnopyranosyl-D-glucopyranose, respectively.

The NMR spectra of rutinosides and neohesperidosides are characteristically different. The rhamnose C-1 proton in trimethylsilylated rutinosides displays a peak at 4.25- 4.35 ppm and a broad methyl peak at 0.8-0.95 ppm ( 4, 24, 39) while the rhamnose C-1 proton signal in neohesperidosides occurs at 4.85 ppm and a doublet (J = 7 cps) at 1.2 ppm is observed for the methyl group ( 43, 45).

Coumarins

The silylated derivatives of the coumarins, aesculetin and its 6-glucoside, aesculin, gave spectra with sharp signals ( 48 and 49). The C-3 and C-4 protons, a and ,8 to the lactone carbonyl, appear as doublets (J = 9.5 cps) at 6.1 and 7.5 ppm, respec­tively. Two singlets around 6. 7 and 7 ppm are assigned to the C-8 and C-5 protons.

10 Nuclear Magnetic Resonance Analysis of Flavonoids

Discussion

The procedure herein described for the NMR analysis of ftavonoids offers sev­eral advantages over alternative methods now available. It avoids the use of ex­pensive deuterated solvents, which would have to be of high isotopic purity for a detailed analysis of ftavonoid glycosides; all trimethylsilyl ethers thus far en­countered were soluble in carbon tetrachloride. The signals for the trimethylsilyl groups occur well out of the absorption region of the protons of the ftavonoid nucleus and the glycoside moiety in contrast to the peaks observed for the sub­stituents in the more common ftavonoid derivatives such as methyl ethers and acetates. The method does not require long or elaborate procedures since a typical ftavonoid or sugar can be converted to its trimethylsilyl ether and prepared for NMR analysis in a few minutes. Furthermore, the trimethylsilyl groups are hydro­lyzed quantitatively under mild conditions.

Our results, combined with the data published elsewhere demonstrate that the structures of ftavonoids can be assigned almost solely on the basis of their NMR spectra, therefore limiting the need for time consuming chemical degradations. In practice, when a naturally occuring ftavonoid is encountered, confirmation of the NMR results is often obtained from other sources. For instance, the ultraviolet spectra of the unknown sample alone and in the presence of standard reagents re­veals the presence of hydroxyls at the positions 3, 5, 7, 3' and 4' in most ftavonoids. 1

It also reveals the positions of glycosylation by comparison with the spectra of the hydrolyzed material. The nature of the sugar can be confirmed by paper or gas chromatographic analysis. We have developed a method for the routine gas chro­matographic determination of sugars obtained from ftavonoid glycosides.8 It in­volves the acid hydrolysis of the glycoside, separation of the aglycone by adsorption on polyamide and conversion of the sugars into their volatile trimethylsilyl ethers.

The trimethylsilylation is a convenient procedure to obtain derivatives which are soluble in common nonpolar organic solvents and which are considerably more volatile than other derivatives. It should be applicable to other classes of highly hydroxylated natural products.

In NMR as well as in ultraviolet or infrared spectroscopy many subtle details of the shape of the curve are not included in tabulated expressions of the spectra. We feel that the actual spectra of reference compounds are required for detailed struc­tural studies. We hope that this collection of spectra will be of value to workers in­terested in ftavonoid chemistry and in NMR spectroscopy.

Mabry, Kagan and Rosier 11

Literature Cited

1. (a) R. E. Alston and B. L. Turner, Proc. Natl. Acad. Sci., U.S., 48, 130 (1962); (b) R. E. Alston and B. L. Turner, Am. J. Bot., 50, 159 ( 1963); ( c) R . E. Alston, T. J. Mabry, and B. L. Turner, Sci., 142, 545 ( 1963).

2. (a) T . J . Mabry, J. Kagan, and H. Rosier, Phytochem., 4, 177, ( 1965); (b) T. J. Mabry, J. Kagan, and H. Rosier, Phytochem, 4, in press, ( 1965).

3. A. C. Waiss, Jr., R . E. Lundin, and D. J. Stern, Tetrahedron Letters, No. 10, 513, (1964).

4. Leading References: (a) J. Massicot, J.P. Marthe, and S. Heitz, Bull. Soc. Chim. Fr., 2712 ( 1963); (b) T. J. Batterham, and R. J. Highet, Aust. J. Chem., 17, 428 (1964); ( c) C. A. Henrick and P. R. Jefferies, Aust. J. Chem., 17, 934 ( 1964) ; (d) J. W. Clark-Lewis, L. M. Jackman, and T. M. Spotswood, Aust. J. Chem., 17,

632 (1964).

5. C. C. Sweeley, R. Bentley, M. Makita, and W. W. Wells, J. Amer. Chem. Soc., 85, 2497 ( 1963) .

6. E. S. Hand and R. M. Horowitz, J . Am. Chem. Soc., 86, 2084 ( 1964).

7. L. Jurd, In "The Chemistry of Flavonoid Compounds," T . A. Geissman, ed. Chap. 5, The MacMillan Co., New York, (1962).

8. J . Kagan and T. J. Mabry, Anal. Chem., 37, 288 ( 1965) .

12 Nuclear Magnetic Resonance Analysis of Flavonoids

NMR Spectra

Spectra 1- 51 were obtained with the totally trimethylsilyated ethers of the com­pounds listed below except spectrum 2 which represents 7 ,3' ,4'-tri-trimethylsiloxy­luteolin. All the signal assignments are recorded using the numbering scheme shown for flavone (Figure 2). The spectra were obtained in CCI. on a Varian A-60 spec­trometer with tetramethylsilane as reference, usually internal. The region 0-1 ppm was frequently recorded at a reduced spectrum amplitude.

Flavones

1. Luteolin 2. Luteolin as 7,3',4'-tri-trimethylsiloxy-

Luteolin 3. Luteolin-7-,8-o-glucoside 4. Luteolin-7-( rhamno-,8-o-glucoside) 5. Apigenin 6. Apigenin-7-,8-o-glucoside 7. Tectochrysin 8. 5,7-Dihydroxy-3',4'-dimethoxyflavone 9. Vitexin

10. Saponaretin

lsoflavones

11. Orobol 12. Orobol-7-,8-o-glucoside 13. Genistein 5-methyl ether 14. Irisolidone 15. Biochanin A 16. Tectoridin 17. Irigenin 18. Iridin 30. Penduletin 31. Pendulin

Flavonols

19. Kaempferol 20. Myricetin 21. Quercetin 22. Quercitrin 23. Hyperin 24. Rutin 25. Morin 26. Robinetin 27. Rhamnetin

28. Isorhamnetin 29. Isorhamnetin-3-,8-o-glucoside

Dihydroflavonols

32. Dihydrokaempferol 33. Dihydroquercetin 34. Dihydrofisetin 35. Dihydrorobinetin

Flavanones

36. Liquiritigenin 37. Homoeriodictyol 38. Hesperetin 39. Hesperidin 40. Sakuranetin 41. Sakuranin 42. 5,7-Dihydroxy-3',4' -

dimethoxyflavanone 43. Neohesperidin 44. Naringenin 45. Naringin 46. 6-Hydroxyflavanone

Chalcones

47. 2',4'-Dihydroxy-3,4-dimethoxychalcone

Coumarins

48. Aesculetin 49. Aesculin

Sugars

50. a-o-Glucose 51. a-L-Rhamnose

1

2

1! ,k ,J. ~

1! ,k ,J. ~

H-6' H-2'

H-3

H-~ ! H~-6 __ _

OH

y0yo~ .. HO w 'L_ff

OH 0

Luteolin

H-5'

H-6' H-2'

1.0

~,..,.,.,~

.. '

HOWoO OH free OH-5 / / ~ j ""

offset 400 c.p.s. ~

L OHO

uteolin-( 7,3',4' -Tri-Trimethylsilyl) Ether

~ . H-SH-6

H-3

~J0~~-~

+ ... (I )

o~~

1MS

.---rll ...._ (JI

o7i?

TMS

3 ! .~ .J. !

4 'I .~ .J. !

H-6' H-2'

H-6' H-2'

H-5'

H-3 H-6

H-3 H-6

glucose H-1

OH ,., Wo 0 OH '''"''-o : I I ~ #

OH 0

Luteolin-7-glucoside

OH

yyo~OH ,, ... ,.,,," .. -0 YV 'L1'

OH 0

Luteolin-7-rhamnoglucoside

rhamnoglucose

rhamnose H-1

10 proto'.:'.:n~s~------- rhamnose CH,

plus spinning sidebands

+fWlll Cl l

0 >-;!'

TMS

• ~=-o ~lJ>

TMS

6 r

'i i 'j

!

8.0

2.0

1.0

H-2' H-6'

7.0

3.0

H-3 ' H-5'

7.0

H-3 H-6

6.0

4.0

6.0

5.0 Pl'M (I) 4.0

s'.o PPM ( T ) 6.0

s.o WM~

3.0

7.0

2.0

8.0

1.0

9 .0

yyo~OH 1tucost- yy 'L....!I

OH 0

Apigenin-7 -glucoside

glucose 6 protons

3.0 2.0 1.0

tb

H C> 0"'

TMS

- -ts (J) ~ . ts (J)

~ . :;s

l~ t :;s t

· + ~ + ~ E-< >::: 1 0

>::: :> co ·r"<

~ <fl

' >, ' 0 0 ;... ' x ,..s::: 0 u ,..s::: 0 +-' +-'

"' <J.) i5 u

~ s <J.) r-- ----------- ---------- -- ;.a ~ I

~~ NI

I >, x

i5 0 ;...

'"O 0 >,

i % ,..s::: ·r-<

Cl I

"'-"-

N;~ \.(") ' i :ii u uu 0 00

. ~

~ ~~~~oo:==~~~~~~==: :!:

~-§-+-~-·------,..--------~~~ ~-§-~~-·---~--------~~~

00

I ts VJ -, i ts VJ -+ ::s

I 0-+ ~ ----------t-<

i!i i!i Q) ~ ~ "d .....

<Fl 0

....... u 0 ::s

...c ....... bl)

0 I ... .,.... 0 I

i!i ....... i!i 0 U) ...c CIJ ~

0 U) 0

~ ... O+-' uo 0 ;:s .... '"So°"'

<D

' ~

CIJ

"'-0 ' ~ - ~

g p:: bo

l-1-+·-f-•-------------------.J I-I-!-§-•------------------''-'

2.0 3.0 4.0

13 ' I : I I 'j i ""' 'i !

H-2 H-2' H-3' H-6 H-6' H-5' H-8

I !~

14 ! ,~ J, ~

1.0 7.0

H-2' H-3' H-6' H-5'

H-2

6.0

H-8

: s:o PPM !Tl 6.0

I :

I I ""'

OCH3-5 11

" ,,....-

5.0 PPM {OJ 4.0

OCH,-6 OCH3-4'

: ~

~

7.0 1.0 9 .0 10 I I I : I

'"" 07:

HO'O?o ""'-I I f_, OH

OCH1 0 TMS

Genistein 5-methyl ether

3.0 2.0 1.0

+- Cl) o ~~

HOX)O-o-1 0 I f ' oc.., ""'- -CH30

OH 0

Irisolidone TMS

' :

"' 0

~ 1-====-==;o==i ===::::::::""

~i'o

u 0

C'l ~'1:::~:L....>...-====~ ~

l-i-~-§-•----~---------L---1

'9 i u 0

<!)

"' o ..-. <..) ' ;:l~

'"5'.o

IX!

rn>n ~

~~

~i'o ~~

C'l

~

~-i-1!-§-•----~-----------

17

18

I .L .J. !

'i i 'i Ir

2.0

H-8

H-2 H-2' I H-6'

OCH3-6 OCH3-3' OCH3-4'

I

.f.HM !ll

o>-;.,~

Hoyy"') r{cH, CH,o~ocH,

OH 0 OH

Irigenin

~ i

I ~-1i l

3.0 4 .0 s'.o ""M (T ) 6.0

OCH3-6 OCH3-3' OCH3-4'

H -8 H -2' H-6'

H-2

7.0 8.0 9 .0 10

,_,'> '"'

-- CH30

3 11um1-o~O H

OCH1

0 OH

Iridin glucose 6 protons

TMS

2.0 1.0 0

-1 i [/)

• -+ I "'•

~ l u

-+

~ ~

6 ~ ~ ...... 0 ... CJ)

'+-< A. s CJ) co

i!i ::.:::

0 %

ts

0 N

0 %

i!i

i!i

[/)

~

'f

j i::: ·.;::i

CJ) u ·c

I >.

:;E

~ I i ts 0-+ ~--==~

i!i ~ :

i::: ...... <ll +'

Cl.> <J)

u 0 ;... >:: Cl.> a ;:3 = = "' O' ...<::

i!i ....

0 %

f i!i

i!i i::: ...... ;... +' ......

- : : - : u ;... Cl.> ;:3

O' i!i

(CJ ~ (CJ

:i:: :i:: 00

00 :i:: :i:: -=i l{) l{)

:i:: :i:: Ci

<o ~ ' Ci :i:: '9 :i:: ::r:

1-i-~1-• 1-141-•

'"""' N N N

2.0 3.0 I :

4.0 s'.o

23 + I I ' PPM ( T l 6.0

I ; ' I 7.0 8.0

'j

I : I 9.0 1b

""' ""' ""' .OH

0 >-;:t

'j 'i HO

i ~ OH --r

/ Hyperin TMS

..

H -8 ~-/ galacto;o~

H-6' H-Z' H -6 6 protons

J'l -- 1 1 galactose

H -1

~~

8.0 7.0 I I ; I

6.0 ' ; I : 5.0 PPM (0 4.0

I I I

3.0 2.0 1.0

'j° ; 1t •t 24 ,t r jo

i

; ' I° : y '·' f.,..,,_ Ul

'"

I '

""'

c I

""' I ,.,. H<~

' '~

:r HO I

i ~ OH

H -6' Ru tin ~TMS

H-Z' I

\ H-8 H-6

H -5' ,l I glucose rharnnose

rhamnose

H-1 CH a

H-1 ~ rhamnoglucose 10 protons

~

~-(

;:,., ~

V"'\ N

0-+

>::: ..... I-< 0

~

s s

>::: ..... +-' Q)

>::: . .... ..c 0

i:c<

0

"'

l "'• Cf)

~ tu

~ -+ E-t

t-.. ' 5 ~~===!:s~====~

0

~-§-+-§-a----.,---------~~

28 !

29

J, J, ~

'j i i !

H-8

H-6' H-6

H-2' H-5'

2.0 3.0 4.0

H-8 H-6 H-6'

H-2'

J:jj . 7.0 6.0

OCH,-3'

PPM (T ) 6.0

OCH3-3'

5.Q-------,pji 4.0

..,..,

"°Yy"r<i=OH Y0~

OH 0

Isorhamnetin

'~~

7.0 8.0

HO

glucose OH 6 protons ~

9 .0

OH

Isorhamnetin~3-glucoside

u 2.0 1.0

... _ti )

0 >-;t

TMS

~)

10

o~~

TMS

0

30

31

1! ,k .~ ~

'i i 'i !

H-2' H-6'

2.0

H-2' H-6'

a.o

3.0

H-8

H-3' H-5'

H-8

H-3' H-5'-

7.0

4.0

6.0

s'.o

OCH3-3 OCH3 -6 OCH3-7

PPM (T ) 6.0

OCH3-3 OCH3-6

7.0

OCH3-7 !1111 I

llllJI glucose

I VI 11 6 protons

s.o ~----..-.a 3.0

.f.,,lit ( i )

o>-;..,"-"

CH30 OH

CH30

OH

Penduletin TMS

l,,------

~)

8.0 9.0 1b

>+>

T' ii Ir\\ i>--o - clucose

CH30 I II OH 0 TMS

Pendulin

2.0 1.0

. :

0 %

c<")

~

~

~

s s c<")

~

~

:l - :; ~

U) U) ~ "'• ~ Jv

E-< I "'•

~ ~ 0 -+

Jv -+

I I

) ...... i!i ~ 0 •.-<

» ~ t) ~

QfJ • .-< "t:i

·.;::i ·.-< I-; 0

·c ·.-<

~ & 0 s . ~ i8 0

~-c ::r: ~ . 0 rO ~

~ " J~ . ('()

~ ~

i ~ ::r:M u 0

t ::.! - !'.l Cl

0;1 ~ ~ ::r: ~_

l <.O

~ .<>;' OCJ

~ <.O ::r: ~ ~ ;;.., u-,

~~ -...,.. ~0~ Ci<o ::r:::r:::r: ~~

\0

~

~-§-~~-· ~-§-~~-·

\0 "' ("{") ('(')

38

39

I ,L J. !

I ,L J. !

H-2' H-5' H-6'

•oyyo~ w0~

4'-0CH3

OH 0

Hesperetin

H-8 H-6

yyo~"' '""'""'"""-ow~

H-2' H-5' H-6'

OH 0

Hesperidin

glucose H-1

OCH,-4'

H-3tranR H-3cts

rhamnoglucose 10 protons ....--------

H-3lrans H-3cis

rhamnose CH3

f,,.. ui

0 >-;t

TM

.f..,.,.,, U)

07.:s:;,.

TMS

40

41

I ,L ,J, !

i i

±

2.0

a.o

H-2' H-6'

3.0

H-3' H-5'

H-8 H-6

4.0

H -2' H-3' H-6 H-6' H-5' H-8

7.0 6.0

OCH,-7

s'.o PPM {; ) 6.0

OCH,-7

s.o -....-

yy•r-LJ--.. CH,0 yy ~ OH 0

Sakuranetin ______/'--

H-3 trans H-3cie

~·

7.0 8.0 9 .0

yy•r-LJ--.. CH,O yy ~ a:lueost-0 O

Sakuranin glucose 6 protons

H-3 trans H-3cis

Il 2.0 1.0

·-U) ,..

TMS

1b

,_",.. ' '~

TMS

42

43

2.0 3.0 4.0 s~o .. MT 6.0 7.0 1.0 9 .0

i T = "" yc;r0· i HO

! OCHi

OH 0

OCH.-3' /I 5,7-Dihydroxy-3',4'-

H-2' OCH,-4' dimethoxyflavanone H-5' H-6'

H-8 H-6

1.0 7.0 6.0 5.0 PPM (O) 4.0 3.0 2.0 1.0

2.0 3.0 ... 0 5 ~0 PPM (T ) 6.0 7.0 8.0 9 .0

I I : I : . 1 ' I : ' I : I .... .. . - .

+ = "" I '''"''"''""-OWoO OH I I ~#°""1 ~ ~

1.0

H-2' H-5' H-6'

H-8 H-6

7.0 6.0

OCH,-4'

glucose H-1 ,---__ ___,

5.0 ,,. 4.0

OH 0

N eohesperidin

rhamnoglucose 10 protons

H-3trane H-3cie

3.0 2.0

rhamnose CH,

plus sidebands

1.0

10

>-H~ 0"'

/ TMS

10

,_,~

0"'

TMS

0

44

45

. - - - . -'j i •M ""' ··w-o-~ 'i 'i " ~ H-2' H-3'

H-6' H-5' ~ 0

H-8 H-6 Naringenin -----

,,./ - -

---- H-3trans H -3cis

l<l/fl\t•t'\),il\' .. l''""' Vil ~

H-2 ~ ~r~J 'Mf'I

'i i :r ~ yy·1--LJ-~ , ...... , ..... _. yy "--ff

H-2' H-3' H-{}' H-5'

~ 0

Naringin

H-8 H-6

rhamnoglucose 10 protons

glucose H-1~---

H-3lrans

~ n H-3ci s

1"- tll - - - -·. - - . -- - - ~ --

>--11 _,,.

/I TMS

_ ___; ~ \,

rhamnose CH a

f""" ! l l

-t-~ ( l l

07.:s~

TMS

46

47

+- ''' Jo I 'j' ,, ... I '"' I ; I '·' '- I : ~ ·,· I " r - - ' ' I ! 16 pmton< J '"' 1 ! ~proton; 6-Hydroxyflavanone "'~ js:

I I

H-3crans H-3cis

~~ 1L __ -----~------------ __ ,__.·1

I ' il T l~i ,,...,,,_,_,.. ~ ~J vL~ ~-

I Ir I

I _______ )J\

).0

... l'PM (i)

0 >-;;! 'i I .l. !

2', 4' -Dihydroxy-3, 4-dimethoxychalcone

H -6'

n

H-2 H-6

H-3'

OCH3-3 OCH,-4

HOnOH i J=d<OCH3 .yyf\-f"'"'

0

TMS

2.0 3.0 4.0 s'.o PPM !T l 6.o 7.o a.o 'j° 11 I ; I I I ' I I ; I

48 .:., ,_,'> ,l - ,....., o CPS

I

'.r H-5 I ~ H-8 , ,

H0~3

"°~oA,o I I

r ll TMS -------' H-3 Aesculetin

H-4

1 ..,..__.,,v ·~

8.0 7.0 6.0 5.0 PPM (li ) 4 .0 3.0 2.0 1.0

2.0 3.0 4.0 s:o PPM (T ) 6.0 7.0 8.0 9 .0 1b

I -~ 4 ·= 9 500 ~oo 100 200 100 c c•s I 'j

'i ro·. so &lucost-0 / ~ ~:

H0 7 Q 0 O

H-5 ~ I I TMS H-8 f .

glucose H-4 I 11 H-3 ~~11 6protons

glucose H-1

WVJWll_.J\~·~"'~·) ~1

l.O 7.0 6.0 5.0 - PPM lo 4:0 3.0

Aesculin

2.0 1.0

"' "' ~ ~ 0 0 ..... ..... 0 0 I-< I-< 0.. 0..

<O +

-- ~ - ~ ~ ~ - ~

(I,) (I,) en en

0 0 s::: u

;j s ....... 0 co

I ~ ~ ~

I

~