FLAVANOIDS

Dr. Md. Rageeb Md. Usman

Associate Professor
Department of Pharmacognosy

Smt. Sharadchandrika Suresh Patil

College of Pharmacy,
Chopda, Maharashtra, India
 Contents

Chapter 1 The chemistry of Flavonoids

Chapter 2 Isolation and Identification of
Flavonoids
Chapter 3 Structure Elucidation
Flavanoids are the largest group of naturally
occurring phenolic compounds, which occur in
different plant parts both in free state and as
glycosides.

Flavanoids are also known as plant pigments

or co-pigments.
- responsible for the various colors and combination of
colors exhibited by bark, leaves, flowers, fruits and seeds of
plants.
- exhibit various biological activities in mammals, the most
important one being the antioxidant activity.
 Chapter 1
The chemistry of Flavonoids
1.1 occurrence

Flavonoids are widely distributed in nature and are

especially common in higher plants belonging families
Leguminoseae, Rutacee, Primulaceae, Polygonaceae,
Salicaceaem Pinaceae, Rosaceae, Asteraceae,
Lamiaceae, Bignoniaceae, Moraceae, Betulaceae,
Rubiaceae, and Mytaceae.

Flavonoids are found to occur in different parts of

the plants ( roots, bark, heartwood, leaves, flowers,
fruits and seeds).
1.2 biological activities

Flavonoids are found to have many biological activities

including:
★ estrogenic,
★ protein kinase inhibition,
★ estrogen receptor binding,
★ mitochondrial -adhesion inhibition,
★ prostaglandin-synthesis inhibition,
★ antiulcer, anticancer, antiarthritic, antimicrobial, antiangiogenic,
★ DNA synthesis/cell cycle arrest and topoisomerase inhibition.
1.2 biological activities

Proanthocyanidins
Proanthocyanidins extracts demonstrate a wide
range of pharmacological activity.

Their effects include increasing intracellular vitamin

C levels, decreasing capillary permeability and
fragility, scavenging oxidants and free radicals, and
inhibiting destruction of collagen, the most abundant
protein in the body.
1.2 biological activities

Green Tea
• potent antioxidant compounds that have demonstrated
greater antioxidant protection than vitamins C and E,

• increase the activity of antioxidant enzymes,

• inhibit cancer by blocking the formation of cancer-

causing compounds and suppressing the activation of
carcinogens. The major polyphenols in green tea are
flavonoids (catechin, epicatechin and so on).
1.3 NOMENCLATURE

The term “flavonoid” is generally used to describe a

broad collection of natural products that include a
C6-C3-C6 carbon framework, or more specifically a
phenylbenzopyran functionality.

B
O

A C

O
1.3 NOMENCLATURE

Depending on the position of the linkage of the aromatic

ring to the benzopyrano (chromano) moiety, this group of
natural products may be divided into three classes:

the flavonoids (2-phenylbenzopyrans) 1,

isoflavonoids (3-benzopyrans) 2,
the neoflavonoids (4-benzopyrans) 3.

These groups usually share a common chalcone

precursor, and therefore are biogenetically and
structurally related.
1.3 NOMENCLATURE
5'
O
6' 4'
B
3
8 3'
O 2
7 2'
A C
6 3
5 4 2 the isoflavonoids
(3-benzopyrans)
1 the flavonoids
(2-phenylbenzopyrans) O

4
3 the neoflavonoids
(4-benzopyrans)
1.3.1 2-Phenylbenzopyrans (C6-C3-C6 Backbone)

Based on the degree of oxidation and saturation present

in the heterocyclic C-ring, the flavonoids may be divided
into the following groups:
O O

O O

flavan flavanone flavone

O O
O

OH OH
OH
O O
dihydroflavonol flavan-3-ol
flavonol
1.3.2 Isoflavonoids (3-benzopyrans)

Isoflavonoids possess a 3-phenylchroman skeleton that

is biogenetically derived by 1,2-aryl migration in a
2-phenylchroman precursor.
8 O O
O
2
2'
6
4
O O

isoflavan isoflavone isoflavanone

O O

OH

isoflav-3-ene isoflavanol
1.3.2 Isoflavonoids (3-benzopyrans)

4 5 7
8 6 O
O O O
O 6a
3 9 O5
6a 7 D C B
2 10
11a 8 12a 4
1 11 12
A
11 O 9 1 3

coumestane 10 rotenoid 2 3-arylcoumarin

O O O
O O
A B
6a

11a
C D
O O

coumaronochromene coumaronochromone coumaronochromone

1.3.3. Neoflavonoids (4-benzopyrans)

The neoflavonoids are structurally and biogenetically

closely related to the flavonoids and the isoflavonoids.

O O O O O

4-arylcoumarin 3,4-dihydro-4-arylcoumarin neoflavene

1.3.4. Minor Flavonoids

Natural products such as chalcones and aurones also

contain a C6-C3-C6 backbone and are considered to be
minor flavonoids.
2' OH OH OH

O O O
2'-OH-chalcone 2'-OH-dihydrochalcone 2'-OH-retro-chalcone

A OH

O B
O

aurone auronols
1.3.5 Anthocyanins

Basically anthocyanins are 3,5,7-thihydroxy flacylium

salts and are classified based on the substitution pattern
in the B ring of the molecule. Generally 3-hydroxy and
5-hydroxy positions are glycosylated.
R4 R4
R4
R5 R3 O R5
R5
R3 O R6
+ R6
R3 O R2
R6
R1 O
R1
R9 R7
R1
R2 O
R2 - R8
Cl
Derivatives of flavones Derivatives of isoflavones
Anthocyanidin including glycosides including glycosides
R1=R2=R3=R4=R5=R6 R1=R2=R3=R4=R5=R6 R1=R2=R3=R4=R5=R6=R7=R8=R9
=H,OH or OMe or =H,OH or OMe or Alkylor =H,OH or OMe or Alkylor
glycoside/ Anthocyanin glycoside/ glycoside glycoside/ glycoside
1.3.6 Bioflavonoids

Bioflavonoids are the dimers of various flavonoids.

O OH

OH
O

O
OH

HO

Bioflavone
1.4 The Biosynthesis of Flavonoids

There is good evidence that secondary metabolic

systems are derived from primary metabolism, with a
variety of enzymes, including

◆ members of the cytochrome P450 hydroxylase,

◆ 2-oxoglutarate-dependent dioxygenase (2-ODDs),
◆ short-chain dehydrogenase/reductase (SDR),
◆ O-methyltransferase (OMT),
◆ glycosyltransferase (GT) families.

recruited into new functions during the rapid evolution

that accompanied the movement of plants onto land.
 Chapter 2
Isolation and Identification of
Flavonoids
2.1 INTRODUCTION

Flavonoids and their conjugates form a very large

group of natural products. They are found in many plant
tissues ( inside the cells or on the surfaces of different
plant organs).
2.1 Introduction

Due to:
* Different classes of flavonoids and their conjugates have
numerous functions

* Different biological activities of plant secondary metabolites, their

regular consumption may have serious consequences for health, both
positive and negative .

So:
methods for the efficient and reproducible analysis of
flavonoids play a crucial role in research conducted
in different fields of the biological and medical sciences.
2.1 Introduction

Analytical methods for the characterization of flavonoids:

A. nuclear magnetic resonance (NMR) analyses (1H and 13C)

- Unambiguous identification of unknown compounds

B. other instrumental methods (mass spectrometry, UV and IR

spectrophotometry)
- The identification of organic compounds ( cannot to
answer all the structural questions)

- Utilization of standards comparison of retention

times, spectral properties
2.2 Isolation
Isolation of Flavonoids and their Conjugates from
Biological meterials:
Initially, determine
⊙ whether the researchers are interested in the
identification of individual components present in a
mixture of target compounds

⊙ whether they would like to estimate the total amount of

phenolic compounds in the biological material
investigated.
- nutritional studies ( foods , fodders, mainly
of plant origin).
2.2 Isolation

Then

Consider the factors that influence the profile of the

qualitative and quantitative composition of flavonoids
and their derivatives in the obtained extracts.

In many cases additional cleaning based on solid-

phase extraction (SPE) of the extracted samples is
required.
2.2 Isolation
2.2.1 Preparation of Plant or Animal Tissue and
Foodstuffs for Flavonoid Analysis

The utilization of dried plant material for extraction

may cause a substantial decrease in the yield of
flavonoid conjugates.

■ Acylated flavonoid glycosides are especially labile

at elevated temperatures and are frequently
thermally degraded during the process of drying
plant tissues.
2.2 Isolation
2.2.1.A isolation of the solvent systems

a. Free flavonoid aglycones exuded by plant tissues

(leaf or root)
- Nonpolar solvents:
methylene chloride, ethyl ether, or ethyl acetate.
b. more polar glycosidic conjugates
- Polar solvents:
methanol and ethanol.
c. flavonoids and their conjugates from solid biological
material (plant or animal tissues and different food
products).
- Mixtures of alcohol and water in different ratios
2.2 Isolation
2.2.1.B The methods of enhance the extraction efficiency

* application of ultrasonication,

* pressurized liquid extraction (PLE),

- A procedure performed at elevated temperature ranging
from 60℃to 200℃.

* Supercritical fluid extraction with carbon dioxide CO2

- Careful the thermal degradation of the flavonoid
derivatives.
2.2 Isolation
2.2.1C Further purification and/or preconcentration of
the target compound fraction
commonly used: liquid–liquid extraction (LLE) , SPE

a. Estimation of the extraction yield

- Spike biological materials with proper internal
standards
Methods: Compounds labeled with stable isotopes
(2H or 13C) MS
b. The extraction of flavonoids from biological materials
- Need to adddifferent classes of phenolic compounds.
2.2 Isolation
2.2.1C Further purification and/or preconcentration of
the target compound fraction

On the other hand, quantitative analysis of consecutive

components of the analyzed flavonoid mixture needs
reference standard compounds necessary for
preparation of calibration curves essential for a precise
quantification.
2.2 Isolation
2.2.1D The extraction procedure for obtaining flavonoid
conjugates from biological material

Attention !

- Depends on the goals of the conducted research.

- Avoid the chemical and/or enzymatic degradation
of the metabolites.
2.2 Isolation
2.2.1D The extraction procedure for obtaining flavonoid
conjugates from biological material

On the other hand, in the phytochemical analysis of

plant species or phytopharmaceutical studies of plant
material, the repeatable isolation of all biologically
active flavonoid aglycones with a good yield is more
important.

- Need more drastic extraction conditions.

2.2 Isolation
2.2.1F Isolation of individual components

a. Robust multistep chromatographic methods

- Isolation of individual components from plant extracts
containing new uncharacterized compounds.

The sequence and kind of separation methods

used depends on the composition of the sample
and the experience of the researcher.

However, minor Flavonoids components are

difficult to obtain as pure compounds.
2.2 Isolation
2.2.1F Isolation of individual components
b. Chromatographic systems with multiple detector systems
▲ Analysis of samples containing a number of compounds
present in small amounts, the application of an analytical
chromatographic systems enhanced by proper detectors
(UV, NMR, and/or MS)
- Gives spectrometric information sufficient for
establishing the structure of minor target components.

▲ Liquid chromatography with multiple detector systems

(UV diode array detector, mass spectrometers, and
nuclear magnetic resonance spectrometer).
- It is possible to achieve complete structural information
about isomeric flavonoids and their conjugates in this way.
2.2 Isolation
2.2.2 Preparation of Body Fluids
Isolation of flavonoids and their derivatives from
liquid samples like beverages (wine or fruit juice) and
physiological fluids (blood or urine).
Two different approaches :
a. liquid–liquid extraction
b. solid-phase extraction
- mainly on RP C-18 silica gel cartridges.

Body fluids need special procedures：

- Should to avoid degradation of target compounds
( due to the activity of different enzymes present).
2.3 Isolation Techniques

★ Thin-layer chromatography (TLC)

★ High performance liquid chromatography (HPLC)

★ Gas liquid chromatography (GLC)

★ Paper chromatography (PC)

★ Column chromatography
2.3 Isolation Techniques
2.3.1 Thin-layer chromatography (TLC)
Thin layer chromatography continues to be one of
the versatile tools employed for the analysis of
flavonoids.

A. Silica gel with the solvent systems

B. Polyamide with the solvent systems
C. Cellulose with solvent systems
2.3 Isolation Techniques
2.3.1 Thin-layer chromatography (TLC)

A. Silica gel with the solvent systems:

a. Flavone aglycones
benzene: pyridines: ammonia (80:20:1 drop)
enzene: pyridine: formic acid (72:18:10)

b. Flavanone aglycones
benzene: acetic acid (45:4)
methylene chloride: acetic acid: water (3:1:1)
2.3 Isolation Techniques
2.3.1 Thin-layer chromatography (TLC)
A. Silica gel with the solvent systems:
c. Flavone-O-glyconsides, flavone-C-glycosides and
flavonol-O- glycosides .
butanol: acetic acid: water (3:1:1),
formic acid: ethyl acetate: water (9:1:1),
chloroform: ethyl acetate: acetone (5:1:4),
ethanol: pyridine: water: methanol (80:12:10:5),
chloroform: methanol: water (65:45:12).

d. Flavanone-O-glycosides
chloroform: acetic acid: methanol,
chloroform: acetic acid (100:4),
benzene; acetic acid (100:4).
2.3 Isolation Techniques
2.3.1 Thin-layer chromatography (TLC)

B. Polyamide with the solvent systems:

a. Flavonoids aglycones
benzene: methyl ethyl keton: methanol (4:3:3),
benzene: hexane: methyl ethyl ketone: methanol (60:26:7:7).

b. Flavonoid glycosides
water: ethanol: methyl ethyl ketone: acetyl acetone (65:15:15:5),
water: butanol: acetone: acetic acid (16:2:2:1),
methanol: water: acetic acid (95:5:5).
2.3 Isolation Techniques
2.3.1 Thin-layer chromatography (TLC)

Distinguish between flavones and flavonoids on

polyamide TLC plates:
• toluene: petrol: methyl ethyl ketone and methanol and
toluene: dioxan: methanol
• toluene: methyl ethyl ketone: methanol and acetic acid:
dioxan: dimethyl formamide: water.
Best spraying agents:
β-aminodiethyl ester of dephenylboric acid (5%
solution in MeOH) ,produces various colours due to
different substitution patterns.
2.3 Isolation Techniques
2.3.1 Thin-layer chromatography (TLC)

C. Cellulose with solvent systems:

a. Flavonoid glycosides
water: acetic acid (25:75),
butanol: acetic acid: water (4:1:5).

b. Flavone aglycones
benzene: acetic acid: water
(12.5:7.2:0.3),
chloroform: acetic acid: water (10:9:1).
2.3 Isolation Techniques
2.3.2 High performance liquid chromatography (HPLC)

HPLC is one of the most versatile tools amongst the

modern techniques for the separation of flavonoids.
Sometimes different buffers are also used. HPLC -MS
has an added advantage for characterizing the various
flavonoids.

Columns:
Normal phase: Lichrosorb RP-8,
Reverse Phase: μ-Bondapak C18, Lichrosorb RP-18
For instance:
Stationary phases: zorbax ODS ,
Mobile phases: methanol: acetic acid: water, acetonitrile: water,
ethanol: ethyl acetate .
2.3 Isolation Techniques
2.3.3 Gas liquid chromatography (GLC)

The non-volatility of hydroxylated flavonoid aglycones

and glycodise has a disadvantage for the application
by GLC. However analysis of various flavonoids has
been carried out by dramatization as silyl ethers.

Column: SE-30, carbowax , Apizon

Detectors : TCD or FID.

2.3 Isolation Techniques
2.3.4 Paper chromatography (PC)
Since flavonoids are coloured compounds paper
chromatography has been an important tool for their
analysis and isolation in micro quantities. Paper
chromatographic analysis can be used in a one-
dimensional or two-dimensional system for effecting
better separation

Normally employed:
Whatman No.1 filter paper

Common solvent system:

n-butanol: acetic acid: water( in different
ratios).
2.3 Isolation Techniques
2.3.5 Column chromatography

As with many natural products column

chromatography has been a very useful tool for the
sepatation of flavonoids.

Common stationary phases include silica gel,

sephadex, polyamide and cellulose, while mobile
phases commonly employed are both non-polar and
polar organic solvents either singly or in combination
and often using the method of elution gradient.
 Chapter 3
Structure Elucidation
All physicochemical methods applied in the field of organic
chemistry are useful for structural characterization or
identification of individual flavonoids and their conjugates.

Spectroscopic methods of identify Flavanoids.

★ Ultraviolet spectrophotometry (UV)

★ Nuclear Magnetic Resonance (PMR, 13C-NMR)

★ Mass spectrometry (MS)

3.1 Ultraviolet spectrophotometry (UV)

UV spectroscopy has been of immense use for the

characterization and identification of different flavonoids.
UV1 visible spectra of these plant pigments differ due to
the presence of different chromophores, which in turn
are responsible for the different colours.

Futher, confirmation of the position of the subsitituents

especially hydroxyl groups can be sought by using shift
reagents such as sodium methoxide (CH3ONa) and
aluminum chloride (AlCl3).
3.1 Ultraviolet spectrophotometry (UV)

Most of the flavonoids exhibit two major absorption

maxima, one in the range of 240-285 nm (band Ⅱ) and
the other in the range of 300-400 nm (band Ⅰ).

+
O O O

R R R
O- O O-
benzoyl flavone (R=H) cinnamoyl
( band ¢ò, 240~285nm) flavonol (R=OH) ( band ¢ñ,300~400nm)
3.1 Ultraviolet spectrophotometry (UV)
The characteristic bands of flavanoid compounds in UV spectra.

Band I Band II
R3

R2 O R4
240-285 304
Flavones R5

R1 O
R3

O R4
R2 240-285 352-
Flavonol R5
OH
R1 O
R3

R2 O R4
270-295 300-3
Flavanone R5

R1 O
R3
 R3
R2 O R4

R2 R4 270-295O 300-3
Flavanone R5
270-295 300-3
3.1 Flavanone
Ultraviolet spectrophotometry
O
R5 (UV)
R1
R1 O R3
R3 Band I Band II
R2 O R3 R4

R2 O R4 270-295 300-320
Dihydroflavonol
R2 O RR45
OH 270-295
240-285 300-320
304
Dihydroflavonol
Flavones O OH R5
R5
R1

R1R1 O
O R3
R3R3
R2 R4
R2 R2 O RR
4 220-270
240-285 340-390
Chalcones
4
R5 220-270 352-
340-390
Flavonol
Chalcones R5
R5
R1 O OH
R1 O
R1 O
R3

R2 O O R4
O CH -
270-295 370-430
300-3
Aurones
Flavanone CH
R5 - 370-430
Aurones O
R1 O
O R3
R3
R3
R2 O +
R4
R2 O R4
+ 270-295
270-280 300-320
465-550
Dihydroflavonol R2 O R5 R4
Anthocyanidin OH R5 270-280 465-550
Anthocyanidin R1 O R6 R5
R1 - R6
Cl R3
R1 Cl -
3.1 Ultraviolet spectrophotometry (UV)
3.1.1 Flavones and Flavonols

Difference: Band Ⅰ

a. Flavones
b. Flavonols
3.1 Ultraviolet spectrophotometry (UV)
3.1.2 Isoflavones、Dihydroflavones and dihydroflavonols

Band Ⅱ is main peak , the strength of the Band Ⅰ is

very weak, and always have a shoulder peak (at the
side of Band Ⅱ).

c. Dihydroflavonols
d. Isoflavones
3.1 Ultraviolet spectrophotometry (UV)
3.1.3 Chalcones and Aurones

The strength of Band Ⅰ is very strong ( main peak)，

while Band Ⅱ is soft weak(secondery strong peak).

Difference：Band Ⅰ

e. Chalcones
f. Aurones
3.1 Ultraviolet spectrophotometry (UV)
The mechanism of both bathochromic and ipsochromic shift
OH OH OH
OH - +
O Na O

O O O
NaOMe

OH O OH O OH O -Na +
Flavone

O Al3+ Al3+
O
O O

O AlCl3 O

OH O O O
3+
Al
Flavone
3.1 Ultraviolet spectrophotometry (UV)
The use of various shift reagents to determine the hydrozylation
patterns in flavonoid moitey
3.2 Nuclear Magnetic Resonance

The studies of flavonoid structures using PMR were

initiated in 1960s and along with 13C-NMR have became
the method of choice for the structure elucidation of
these compounds.

The chemical shifts and multiplicity of signals

corresponding to particular atoms and their coupling with
other atoms within the molecule allow for easy
identification of the aglycone structure, the pattern of
glycosylation, and the identity of the sugar moieties
present.
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)
PMR spectroscopy has been an important diagnostic
tool which enables the identification of different
flavonoids by their characteristic chemical shifts.

Solvents： CDCl3、DMSO-d6、C5H5N
Using DMSO-d6:
5-OH: δ12.40; 7-OH: δ 10.93; 3-OH: δ 9.70.

When mixing in D2O，the signal of hydroxide

radical (OH) disappear.
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)

A. Protons of A ring
a. 5,7-dihydroxy-flavonoids
8
HO O

A
6

OH O

Substitiution H-6 (δ) H-8 (δ)

Flavones, Flavonols, Isoflavones 6.00-6.20 (d) 6.30-6.50 (d)
7-Glycosides 6.20-6.40 (d) 6.50-6.90 (d)
Flavanones, Dihydroflavonols 5.75-5.95 (d) 5.90-6.10 (d)
7-Glycosides 5.90-6.10 (d) 6.10-6.40 (d)
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)
b. 7-hydroxy-flavonoids
8
HO O

A
6
5

H-6: J=2.5, 9.0Hz, dd 峰

H-8: J=2.5Hz, d 峰
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)
b. 7-hydroxy-flavonoids

8
HO O

A H-5> H-6,H-8
6
5

Substitiution H-5(δ) H-6 (δ) H-8 (δ)

Flavones, Flavonols, Isoflavones 7.90-8.20 (d) 6.70-7.10 (dd) 6.70- 7.00 (d)
Flavanones, Dihydroflavonols 7.70-7.90 (d) 6.40-6.50 (dd) 6.30-6.40 (d)
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)

B. Protons of B ring
a. 4’-oxygenated flavonoids
2' 3'

B OR

H-2', 6' ： 7.1~8.1 d J=8.5 Hz;

H-3', 5' ： 6.5~7.1 d J=8.5 Hz。
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)
b. 3’,4’-dioxygenated flavonoids

OR
2'

B OR

6' 5'

H-5' : d， J=8.5Hz , δ6.70 ~ 7.10,

H-2' ：d，J= 2.5Hz

δ 7.20 ~7.90
H-6' ：dd，J= 2.5, 8.5Hz
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)

C. Protons of C ring
a. Flavones

O 2
H-3: s, δ6.30
C
3 OMe
H
HO O
O
Flavones

Distinguish H-3 and H-6: H

★ Long distance coupling; OH O
★ Deuterated methyl-silylanization of 5-OH alternatively 。
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)

b. Isoflavones
O 2 H
C

Isoflavones

H-2： 8.50~8.70 ppm (DMSO-d6), s;

 7.60~7.88 ppm; 7.80~8.10 (CDCl3), s。
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)

c. Dihydroflavones
H
O 2

H
3
H
O

H-2： J 2, 3-cis =5.0Hz dd, center of the chemical

shift atδ5.20
J 2,3-trans =11.0Hz
3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)

d. Dihydroflavonols

H
O
O
H
OH H
O H
O H
( 2R, 3S) ( 2S, 3S)

Normally, H-2，H-3 is trans coupling (d, J=11.0Hz)

H-2： δ ~ 4.90; H-3： δ ~ 4.30

3.2 Nuclear Magnetic Resonance
3.2.1 Proton magnetic resonance (PMR)
f. Chalcones and Aurones
d，J=17.0Hz,
β δ 7.30 ~ 7.70
Chalcones α
d，J=17.0Hz，
O
δ 6.70 ~ 7.40
7
1
O
Aurones CH
2
4 3 Benzyl proton：s,
O
δ 6.50 ~ 6.70
δ 6.37- 6.94(DMSO-d6)
3.2 Nuclear Magnetic Resonance
3.2.2 13Carbon- NMR spectroscopy (13C-NMR)

13C-NMR spectroscopy has been a powerful technique

for the structure elucidation of flavonoids and serves as
complimentary to PMR spetroscopy.

The information provided by this important tool with

regard to the number of carbon atoms reveals the
monomer, dimer, and trimer nature of the biflavones,
proanthocyanidins and condensed tannis.
3.2 Nuclear Magnetic Resonance
3.2.2 13Carbon- NMR spectroscopy (13C-NMR)
3.3 Mass Spectrometry (MS)

MS is one of the complimentary tools, which is

used along with other spectroscopic techniques for
the structure elucidation of flavonoids.

Indeed, significant structural data can be obtained from

less than 1 mg of the analyzed compound when
different MS techniques are used in combination with
chemical derivatization of the characterized
compounds .

From the characteristic MS fragmentation patterns it is

possible to establish the structures of different types of
flavonoids.
 Unmodified flavonoid
glycosides

Chemical derivatization Methods of ionization:

- methylation LSIMS, FAB, ESI or APCI
- methanolysis
- additional methylation or
acetylation
normal MS spectra
CID MS/MS
spectra
- molecular mass,
GC/MS with EI - size of aglycone and sugaring,
ionization - type of glycosidic bond on
aglycone C- or O-,
- information about acylation.

- information about interglycosidic bond,

-Identification of sugars,
- identification of aglycone,
- site of sugar substitution on aglycone,
- differentiation of C-6 and C-8 glycosides.
- interglycosieic linkages between sugars,
- identification of aglycone.

Structural information obtainable with different mass spectrometric methods.

3.3 Mass Spectrometry (MS)

From normal mass spectra, information can be

obtained about:

the molecular The size of the sugar the molecular

weight of the moieties attached weight of the
whole conjugate to the aglycone aglycone
3.3 Mass Spectrometry (MS)
3.3.1 MS of the Flavanes

A. Flavone .+
B
Pathway I O
Pathway II
A C

O
Flavone

O O
+
+ C O B
C O C O +
+ B 1+ B2 +

+. +.
Substitiution A1 B1
Flavones 120 102
5,7- dihydroxy-flavone 152 102
5,7,4'-trihydroxy-flavone 152 118
5,7-dihydroxy-4'-methoxyl flavone 152 132
3.3 Mass Spectrometry (MS)
3.3.1 MS of the Flavanes

B. Flavanol .+
O B

OH
O
Flavonol

O
+
O B
C OH
+
A1 + H B2 +
3.3 Mass Spectrometry (MS)
3.3.1 MS of the Flavanes

C. Flavanone
O B
A

O
Flavanone
H+ transfer

O O R
+ A HC B
C O H
C O
+
A1 + H B3+ R=H
3.3 Mass Spectrometry (MS)
3.3.1 MS of the Flavanes

C. Dihydroflavanol
O B
A
OH
O
Dihydroflavanol

O
+
R B H3C
C O +
+
A1 B R=OH B4
3.3 Mass Spectrometry (MS)
3.3.2 MS of the Flavanoids
Flavonoid glycosides are thermally labile compounds
and the evaporation without decomposition of the
analyte is impossible, even in the ion source of a MS,
where high vacuum exists (about 3 × 10-5torr).

In this situation, soft ionization methods need to be

applied for the analysis of this group of compounds,
and the analyte molecules are ionized without
evaporation in high vacuum (FAB or LSIMS, MALDI)
or under atmospheric pressure (ESI, APCI).
3.3 Mass Spectrometry (MS)
3.3.2 MS of the Flavanoids
With FAB or LSIMS ionization, the desorption of the
analyte molecule ions from the liquid matrix may be
improved when the interactions of the polar groups
of the analyte with the matrix decrease. Improved
efficiency of ion desorption may be further achieved
after the methylation of the analyzed compounds.

In addition, the methylation of a flavonoid glycoside

may help to elucidate the glycosylation pattern of the
aglycone hydroxyl groups.
3.3 Mass Spectrometry (MS)
3.3.2 MS of the Flavanoids
A. The O-glycosides of flavonoids:

a. positive ion mass spectra

- containing intense [M+H] + ions as well as
fragment ions created after the cleavage of
glycosidic bonds between sugar moieties or
sugar and aglycone.

b. negative ion mass spectra

- occurring much lower fragmentation of the
deprotonated molecule ions [M-H]−.
3.3 Mass Spectrometry (MS)
3.3.2 MS of the Flavanoids

B. The flavonoid O-glycosyl-glycosides:

The rearrangement of sugars may take place
during the fragmentation process, and the
sequence of sugar losses does not correspond to
the sequence of the sugar moieties in the intact
molecule.
C. The flavonoid C-glycosides:
Rupturring of the sugar ring takes place and creating
strong [M+H-120]+ ions. The cleavage of the sugar
moiety in all types of flavonoid C-glycosides is
observed in both positive and negative mode mass
spectra.
3.3 Mass Spectrometry (MS)
3.3.2 MS of the Flavanoids

Fragmentation pattern and daughter ion nomenclature of different

glycosides of isoflavonoids.

(A) Genistein-7-O-glucosyl-glucoside. (B) Genistein-8-C-glucoside.