Flavonoids

O
O
By : Drs.Syarifuddin K.A.,M.Si
Jurusan Farmasi Universitas Pancasakti Makassar
Pendahuluan
Senyawa Flavonoid memberikan kontribusai keindahan dan
warna pada bunga dan buah-buahan di alam, Flavin
memberikan warna kuning atau jingga, antosianin
memberikan warna merah, ungu atau biru kecuali warna hijau.
Sejumlah flavonoid mempunyai rasa pahit hingga dapat
bersifat menolak jenis ulat tertentu.
Flavonoid ditemukan juga pada bagian-bagian tanaman

seperti akar, kulit kayu, kayu batang, daun, bunga, buah dan
biji.
Flavonoid merupakan senyawa antioksidan, anti-inflamasi,

anti-hepatoksik, anti-tumor, anti-mikroba dan anti-virus.
Tata Nama Senyawa Flavonoid
Flavonoid adalah senyawa yang mengandung C15

(C6-C3-C6) terdiri atas dua inti fenolat (Benzena) yang
dihubungkan dengan tiga atom karbon atau turunan dari
1,3 difenilpropan Cincin A memiliki karakteristik bentuk
hidroksilasi phloroglusinol (resorsinol) dan Cincin B
mempunyai 3,4,5-terhidroksilasi.
O
Tergantung pada posisi keterkaitan cincin aromatik ke
benzopyrano (chromano) menghasilkan 3 bagian.
Kelompok flavonoid dapat dibagi menjadi tiga kelas :

1.Flavonoid(2-phenylbenzopyrans),
2.Isoflavonoid(3-benzopyrans),
3.Para-Neoflavonoids(4-benzopyrans).
Kelompok-kelompok ini biasanya berbagi prekursor

chalcone umum, dan oleh karena itu sifat biogenetik dan
struktural sangat berpengaruh.
5'
6' 4'
O
B
8
O
3' 3
2
7 2'
A C
6 3
5 4
2. Isoflavonoids
(3-benzopyrans)
1. Flavonoids
(2-phenylbenzopyrans) O
4
3. p-Neoflavonoids
(4-benzopyrans)
Flavonoid 2-Phenylbenzopyrans (C6-C3-C6)
Berdasarkan derajat oksidasi dan saturasi yg terdapat

dalam heterosiklik C-ring, flavonoid dapat dibagi ke
dalam kelompok berikut:
O O
O O
flavan flavanone flavone
OH
O O
O
OH OH
OH
O O
flavan-3-ol
flavonol dihydroflavonol OH
Isoflavonoids (3-benzopyrans)
Isoflavonoid memiliki kerangka 3-phenylchroman yang
biogenetically diperoleh migrasi 1,2-aril dalam
Prekursor 2-phenylchroman.
8 O O
O
2
2'
6
4
O O
isoflavan isoflavone isoflavanone
O O
OH
isoflav-3-ene isoflavanol
Isoflavonoids (3-benzopyrans)
4 5
8 7 6 O
O O O
O 6a
3 9 O5
6a 7 D C B
2 10
8 12a 4
1 11a 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

Neoflavonoids (4-benzopyrans)
Para-neoflavonoids secara struktural dan biogenetically

erat terkait dengan flavonoid dan isoflavonoid misalnya :
O O O O O
4-arylcoumarin 3,4-dihydro-4-arylcoumarin neoflavene

Minor Flavonoids
Produk alami seperti chalcones dan aurones juga

mengandung tulang punggung C6-C3-C6 dan dianggap
minor flavonoid, misalnya :
2' OH OH OH
O O O
2'-OH-chalcone 2'-OH-dihydrochalcone 2'-OH-retro-chalcone
A OH
O B
O
aurone auronols
Senyawa Anthocyanin.
Antosianin merupakan bentuk dasar pigmen warna
merah,ungu dan biru pada tanaman, terutama sebagai
bahan pewarna bunga dan buah. Antosianin adalah glikosida
antosianin dalam bentuk garam polihidroksiflavilium
(2-arilbenzopirilium) misalnya :
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 Cl - R8
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
Bioflavonoid
Bioflavonoid merupakan dimer (gabungan) dari berbagai

Flavonoid misalnya :
O OH
OH
O
O
OH
HO
Bioflavone
1.2. Aktivitas Biologis
Flavonoids mempunyai aktifvitas biologis adalah :

★ Estrogenik,
★ Enzim Inhibition,
★ Pengikat estrogen receptor.
★ Inhibition mitochondrial,
★ Penghambat Sintesis Prostaglandin,
★ Anti-oksidan, anti-inflamasi, anti-hepatoksik, anti-tumor,
anti-mikroba dan anti-virus.,
★ Sintesis DNA.
Green Tea (Teh Hijau)
Teh Hijau merupakan senyawa antioksidan kuat yang telah
menunjukkan perlindungan antioksidan lebih besar dari
vitamin C dan E,
meningkatkan aktivitas enzim antioksidan, menghambat
kanker dengan menghalangi pembentukan senyawa
penyebab kanker dan menekan aktivasi karsinogen.
Polifenol utama dalam teh hijau flavonoid adalah catechin,
epicatechin dan sebagainya).
Hisperidin
Hisperidin terdapat dalam biji jeruk (Citrus) merupakan
flavonoid yang dimasukkan dalam berbagai sediaan
suplemen dan mempunyai efek untuk pengobatan
pendarahan kapiler.
OMe
OH
O- O
R
Hesperidin
O
R = Glukosa
OH
Sintesis Flavonoid
Biosintesis flavonoid asam sikimat
OH
HO
OH
O
O O SCoA
SCoA
OH SCoA O O O
+
O OH
kondensasi A
B
SCoA
O O O O
A B
OH OH
HO
HO O HO OH
OH
stilben
OH O OH O
flavon calkon
C5
HO
[O]
HO OH HO OH
COOH O
HO O
OH arilbenzofuran
OH
OH O
HO O O
HO OH
O O
OH
OH
OH O
aril kumarin
OH
O OH O OH
HO O OH HO HO OH
santon benzofenon
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.
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
2.3.1 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
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)
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).
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).
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.
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).
2.3.2 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 .
2.3.3 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.

2.3.4 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).
2.3.5 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.
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).
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)
The characteristic bands of flavanoid compounds in UV spectra.
3.1.1 Flavones and Flavonols
Difference: Band Ⅰ
a. Flavones
b. Flavonols
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
.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
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
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.
3.2.1 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.
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)
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
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)
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 。
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
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 。
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 。
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
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

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
O
Benzyl proton ： s,
δ 6.50 ~ 6.70
δ 6.37- 6.94(DMSO-d6)
3.2.2 13Carbon- NMR spectroscopy (13C-NMR)
13
C-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.
3.2.2 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
- methylation Methods of ionization:
- methanolysis LSIMS, FAB, ESI or APCI
- 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.

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
3.3.1 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 +
+ B1+ 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
B. Flavanol .+
O B
OH
O
Flavonol
O
+
O B
C OH
+
A1 + H B2 +
C. Flavanone
C. Dihydroflavanol
O B
A
OH
O
Dihydroflavanol
O
+
R B H3C
C O +
+
A1 B R=OH B4
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).
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.
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]−.
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.
Fragmentation pattern and daughter ion nomenclature of

different glycosides of isoflavonoids.
(A) Genistein-7-O-glucosyl-glucoside. (B) Genistein-8-C-glucoside.

Flavonoids

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Flavonoid ditemukan juga pada bagian-bagian tanaman

Flavonoid merupakan senyawa antioksidan, anti-inflamasi,

Flavonoid adalah senyawa yang mengandung C15

Kelompok flavonoid dapat dibagi menjadi tiga kelas :

Kelompok-kelompok ini biasanya berbagi prekursor

Berdasarkan derajat oksidasi dan saturasi yg terdapat

flavan flavanone flavone

isoflavan isoflavone isoflavanone

coumestane 10 rotenoid 2 3-arylcoumarin

coumaronochromene coumaronochromone coumaronochromone

Para-neoflavonoids secara struktural dan biogenetically

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

Produk alami seperti chalcones dan aurones juga

Bioflavonoid merupakan dimer (gabungan) dari berbagai

Flavonoids mempunyai aktifvitas biologis adalah :

There is good evidence that secondary metabolic

◆ members of the cytochrome P450 hydroxylase,

recruited into new functions during the rapid evolution

Flavonoids and their conjugates form a very large

* Different biological activities of plant secondary metabolites, their

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

- Utilization of standards comparison of retention

⊙ whether they would like to estimate the total amount of

Consider the factors that influence the profile of the

In many cases additional cleaning based on solid-

The utilization of dried plant material for extraction

■ Acylated flavonoid glycosides are especially labile

a. Free flavonoid aglycones exuded by plant tissues

* pressurized liquid extraction (PLE),

* Supercritical fluid extraction with carbon dioxide CO2

On the other hand, quantitative analysis of consecutive

- Depends on the goals of the conducted research.

On the other hand, in the phytochemical analysis of

- Need more drastic extraction conditions.

a. Robust multistep chromatographic methods

The sequence and kind of separation methods

However, minor Flavonoids components are

▲ Liquid chromatography with multiple detector systems

Body fluids need special procedures ：

★ Thin-layer chromatography (TLC)

★ High performance liquid chromatography (HPLC)

★ Gas liquid chromatography (GLC)

★ Paper chromatography (PC)

A. Silica gel with the solvent systems

A. Silica gel with the solvent systems:

B. Polyamide with the solvent systems:

C. Cellulose with solvent systems:

HPLC is one of the most versatile tools amongst the

The non-volatility of hydroxylated flavonoid aglycones

Column: SE-30, carbowax , Apizon

Detectors : TCD or FID.

Common solvent system:

As with many natural products column

Common stationary phases include silica gel,

Spectroscopic methods of identify Flavanoids.

★ Ultraviolet spectrophotometry (UV)

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

★ Mass spectrometry (MS)

UV spectroscopy has been of immense use for the