FLAVONOID

I NYOMAN ADI WINATA

JURUSAN KIMIA FMIPA UNIV. JEMBER

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 FLAVONOID
 Carbon Skeleton (C6-C3-C6)

4 4'
B B
9 O 2 9 O 2 9 O 2
4'  7 1' 7 7
1
A A C A C A C
3 1'
 3 3
1' 10 4 10 4 10 4
5 5 B 5
O O O 1'
4'

B
Chalcone Flavone Isoflavone 4'

Neoflavone

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FLAVONOID OH OH O

 Structure type O O O
calkon dihidrocalkon flavanon

O O O O O

OH OH OH OH
O O O
antosianidin flavanol flavonol flavon dihidroflavonol

O O O
O O

H
O O O O etc
auron isoflavon rotenoid pterokarpan

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FLAVONOID
 Sample preparation
• Flavonoids especially those in plants could be extracted from several
parts such as roots, barks, leaves, fruits, woods and flowers

• Dry, lyophilized, or frozen samples is advisable. Flavonoids

(particularly glycosides) on fresh or nondried samples can be
degraded by enzyme action.

• When dry plant material is used, it is generally ground into a powder.

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 FLAVONOID
 Flavonoid polarity and solvent extraction
Structures type Polarity Solvent Extraction
isoflavones, flavanones, methylated Less polar CHCl3, CH2Cl2, Et2O,
flavones, and flavonols relativelly EtOAc
flavonoid glycosides and more polar More polar MeOH, EtOH, or
aglycones (-O-Glc, - Glc) relativelly alcohol–water mixtures
OH
OH
HO OH
HO O
O O
OH
O
OH O OH
O quercetin-3-O-β-D-
OH O OH
Glucopyranoside
Artelastoheterol OH Zhu, et.al, Molecules 2013
OH OH
(Ko, et.al. J. Nat. Prod. 2005
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FLAVONOID
 Extraction Methods
a process in which one or more components are separated
selectively from a liquid or solid mixture

Polar solvent Crude polar flavonoid

Plant
material
Non Polar solvent Crude non polar
flavonoid

Maceration

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FLAVONOID
 Soxhlet extraction

n-hexane lipid

Flavonoids aglycones less

CHCl3, CH2Cl2 polar compound

Flavonoids glycosides , more

Alcohol , + water
polar compound

CRUDE EXTRACT
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 Bioactive Isolation
Plant
procedures material

Crude extract

Fraction I Fraction II Fraction III Fraction IV

Fraction IIa Fraction IIb Fraction IIc Fraction IId Fraction IIe Fraction IIf

Pure compound

Structure elucidation Bioactivity test

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Bioactive Isolation Don’t forget to record
Plant
procedures material
plant identity (name,
part, age, and
place/location)

Crude extract

1. Solvent extraction
1. Record physical properties (mass,
2. Concentrated by solvent
colour, and shape or state of
evaporation
matter) Shinoda Test
2. Bioactivity test (if bioassay giude (pink solution
isolation) for Flavonoid)

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Bioactive Isolation
Plant
procedures material 1. Solvent partition (n-hexane, CH2Cl2,
EtOAc, MeOH/H2O)
2. Chromatographic separation (Vacuum
Crude extract
Liquid Chromatography, Filtration
Chromatography, etc)

Fraction I Fraction II Fraction III Fraction IV

1. More than 4 fraction is possible

2. Bioactivity test for bioassay guide isolation
3. Fractionation will be continued to the most
prospective fraction

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 Bioactive Isolation
Plant
procedures material

Crude extract

Fraction I Fraction II Fraction III Fraction IV

Fraction IIa Fraction IIb Fraction IIc Fraction IId Fraction IIe Fraction IIf

Fractionation will be continued Pure compound

until pure compounds are obtained

Structure elucidation Bioactivity test

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FLAVONOID
 Structure Elucidation By UV-VIS Spectroscopy

O O

- Benzoyl - Cinnamoyl
- Pita II - Pita I

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FLAVONOID
 Structure Elucidation By UV-VIS Spectroscopy
The peak locations, shapes and strength of band I and II could be
Determined by structure types and oxygen-bearing substituent types

Pita II (nm) Pita I (nm) Jenis Flavonoid

250-280 310-350 Flavon
250-280 330-360 Flavonol (3-OR)
250-280 350-385 Flavonol (3-OH)
245-275 310-330 (bahu) Isoflavon
275-295 300-330 (bahu) Flavanon / dihidroflavonol
230-270 (lemah) 340-390 Calkon
230-270 (lemah) 380-430 Auron
270-280 465-560 Antosianidin / antosianin

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FLAVONOID
 Structure Elucidation By UV-VIS Spectroscopy
Band I and II locations and shapes are affected by substituents on rings A and B.
Increasing number of OH on B ring  red shift of band I increases
Increasing number of OH on A ring  red shift of band II increases
Blue shift (shifting to lower wavelength) will appear if methylation, glycolisation, and acetylation
of oxygen that attach to A or B rings increase.

The flavonoid type and its oxygenation pattern can identified by UV-shift reagents
Shifting Reagent Function / Interpretation
NaOMe OH at C-4’
NaOAc OH at C-7
NaOAc/H3BO3 orto di-OH
More information showed
AlCl3/HCl OH at C-5 or C-3 on flavonoid book

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FLAVONOID
 Structure Elucidation By UV-VIS Spectroscopy

OH
C-hexosyl

HO O

arabinosyl-C

OH O

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FLAVONOID
 Structure Elucidation By UV-VIS Spectroscopy

The UV‑vis spectra of the isolated compound showed maximum CH3

wavelengths at 347 (band I) and 296 (band II) nm. These HO O
findings showed that the compound was identified as flavonoid
with flavanone backbone. The presence of C‑3 hydroxyl group
in flavonoid and the absence of C2‑C3 double bond gave H3C
particular characteristic in UV spectra. The flavanones had their OH O
primary absorption peak (band II) in the range of 270–295 nm
with only a shoulder or low‑intensity band I peak. Flavonoid structure can be determined using
shifting reagent. After addition of NaOH and CH3COONa, band II moved to 340 and 339 nm,
respectively. The difference of 35 and 34–37 nm showed that compound X was
5,7‑dihydroxyflavanones, whereas there was no change in the wavelengths after addition of
CH3COONa/H3BO3 and AlCl3. Those revealed that the B‑ring had no conjugation with the dominant
chromophore.

MUHAMAD INSANU, et al.: Isolation of 5,7‑Dihydroxy, 6,8‑Dimethyl Flavanone with Its Activities

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FLAVONOID
 Structure Elucidation By Infrared Spectroscopy
The Main Functional Group Region absorption band (cm -1)
Hydroxyl Groups (-OH) 3200–3650
Carbonyl groups (C=O) 1660–1680
Benzene rings 1500, 1580, 1600

OH
Benzene Rings
OH

HO O
Hydroxyl Groups
OH
Hydroxyl Groups OH O

Carbonyl Groups

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FLAVONOID
 Structure Elucidation By Infrared Spectroscopy

C=O

OH C=C Quercetin

Sumber : https://sdbs.db.aist.go.jp/sdbs/cgi-bin/direct_frame_top.cgi
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FLAVONOID
 Structure Elucidation By Infrared Spectroscopy

C-H

C=O Rutinoside
OH

C=C
Sumber : https://sdbs.db.aist.go.jp/sdbs/cgi-bin/direct_frame_top.cgi
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FLAVONOID
 Structure Elucidation By Mass Spectroscopy
• Very helpful in the structure elucidation of flavonoids.
• To determination of the molecular weight (MW) for establishing the distribution of Substituents
between the A- and B-rings.

Flavonoid Type Structure Basic of MW (amu)

Flavones, isoflavone and aurone 222
Flavanones and chalcones 224
Flavonols 238
Dihydroflavonols 240

One Unit Substituent of Addition to Basic MW (amu)

- OH 16
- OCH3 30 Awouafack, Tane &
- O-glucopyranoside 178 Morita,
Book Chapter
- C-glucopyranoside 162 . etc
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 FLAVONOID
 Flavonoids A Rings Structure Elucidation By 1H NMR
OH
8 8 8 8
HO 9 O HO 9 O HO 9 O HO 9 O
7 7 7 7
A A A A
6 6 6
5 10 10 HO 6 5 10
5 10

OH O
5
O OH O OH O
Feng, Hao & Li,
Book Chapter
5,7-dihydroxyflavone 7-hydroxyflavone 5,6,7-trihydroxyflavone 5,7,8-trihydroxyflavone

Type H-5 H-6 H-8

5,7-dihydroxy Flavone, Flavonol, Isoflavone  6,0-6,4 d  6,3-6,5 d
7-O-Glu of above compounds  6,2-6,4 d  6,5-6,9 d
Flavanone, Flavanonol  5,8-6,0 d  6,0-6,1 d
7-O-Glu of above compounds  5,9-6,1 d  6,1-6,4 d
7-hydroxy Flavone, Flavonol, Isoflavone  7,9-8,2 d  6,7-7,1 d  6,7-7,0 d
Flavanone, Flavanonol  7,7-7,9 d  5,7-6,0 d  5,9-6,1 d
5,6,7-trihydroxy  6,9 s
5,7,8-trihydroxy  6,3 s
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 Feng, Hao & Li,
FLAVONOID Book Chapter

 Flavonoids B Rings Structure Elucidation By 1H NMR

OH OH
5' 5' 5' 5' 5'
OH OH OH 5'
OH OH
4' 6' 4' 4' 6' 4' 4'
B 6' 6' 4' 6' 4'
B B B B 6'
B B
3' 3'
3' 3' 3' 3'
1'
1' 2' 1'
2'
1'
2' 1'
2' OH 1'
2' OH 1'
2'
OH OH OH
no substituent 2'-oxygenated 4'-oxygenated 2',4'-dioxygenated 3',4'-dioxygenated 3',4',5'-trioxygenated 2',4',5'-trioxygenated

Substituent Mode H-2’ H-3’ H-4’ H-5’ H-6’

No substituent δ7.1-7.6 m δ7.9-8.2 m δ7.9-8.2 m δ7.9-8.2 m δ7.1-7.6 m
2’-oxygenated δ6.8-6.9 m/dd δ7.2 m/dd δ6.8-6.9 m/dd δ7.4-7.5 m/dd
4’-oxygenated δ7.1-8.1 (2H, d, δ6.5-7.1 (2H, d, δ6.5-7.1 (2H, d, δ7.1-8.1 (2H, d,
J≈8.0Hz) J≈8.0Hz) J≈8.0Hz) J≈8.0Hz)
2’,4’-dioxygenated δ6.0–6.6(1H, d, δ6.30-6.50 (1H, δ7.0–7.4(1H, d,
J≈2.0Hz) dd, J≈8.0,2.0Hz) J≈8.0Hz)
3’,4’-dioxygenated δ7.2-7.8(1H, d, δ6.7-7.1(1H, d, δ6.7-7.9(1H, dd,
J≈2.0Hz) J≈8.0Hz) J≈2.0, 8.0Hz)
3’,4’,5’-trioxygenated δ6.5-7.5 (2H, s, H-2’,6’) as identical oxygen-bearing substituents are connected to position 3’,4’
and 5’ .
2’,4’,5’-trioxygenated δ6.4-6.6(s) δ7.2-7.5(s)
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 Feng, Hao & Li,
FLAVONOID Book Chapter

 Flavonoids C Rings Structure Elucidation By 1H NMR

Type 2-H 3-H Note
Flavone δ6.3–6.8 (s) The signals maybe overlapped by H-6 or H-8.
Flavonol None signal.
Isoflavone δ7.6–7.8 (s) The signal is at rather low field because of influence
of oxygen atom at position 1 and carbonyl at position
4.
Chalcone -H: δ6.7–7.4 (d, J≈17 β-H: δ7.3–7.7 (d, J≈17 C-2 and C-3 form a trans double bond
Hz) Hz)
Flavanone δ5.0–5.5 (1H, dd, J≈11, δ2.3–2.8 (2H) H-2 is coupled by two protons of position 3.
5 Hz) (dd, J≈17, 11 Hz); (dd,
J≈ 17, 5 Hz)
Flavanonol δ4.8–5.0 (d, J≈11 Hz) δ4.1–4.3 (d, J≈ 11 Hz)
Flavanone-3-O- δ5.0–5.6 δ4.3–4.6 After glycosidation of 3-OH, resonance signals of
glycoside both H-2 and H-3 shift to low field.
Aurone Exocyclic proton:
δ6.5–6.7 (s)

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FLAVONOID
Carbon Chemical Shift H δ in ppm 1
Hakim, et.al. Proc. ITB,Vol 30, No 1, 1998
Number H-NMR (CDCl3, 500 MHz)
1

Heteroflavanon A 1 Quercetin 2 2
Aisyah et.al., 2017, Nat.Prod.Sci, 23(2),139-145
1
H-NMR (CD3 OD, 500 MHz)
2 δ 5.98 (1H, dd, J = 14
and 3 Hz) H3CO OCH3
3 δ 3.92 (1H, dd,
J =17 and 14 Hz), H3CO O
δ 2.50 (1H, dd,
J = 17 and 3 Hz) OCH3
5 δ 12.23 (1H, s)
OH O
6 δ 6.03 (1H, d, J = 2 Hz) δ 6.20 (1H, d, J= 2.0 Hz)
7 δ 3.78 (3H, s, OCH3) Heteroflavanon A
8 δ 6.02 (1H, d, J = 2 Hz) δ 6.40 (1H, d,J= 2.0 Hz) OH

2’ δ 3.81 (6H, S, 2 OCH3) δ 7.65 (1H, d, J= 2.1 Hz) HO O

OH
3’ δ 6.16 (2H, s)
4’ δ 3.83 (3H, s, OCH3) OH
5’ δ 6.16 (2H, s) δ 6.85 (1H, d, J= 8.4 Hz OH O
6’ δ 3.81 (6H. S. 2 OCH3) δ 7.50 (1H, dd, J= 8.4
and 2.1 Hz) Quercetin

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FLAVONOID
 Structure Elucidation By 13C NMR Spectroscopy
 Identification of skeleton structures of flavonoids
the characteristic signals of carbons in ring C allowed the identification of
different types of flavonoids
Type C-2 C-3 C-4
Flavone δ160.0–165.5 δ104.0–112.0 δ175.0–184.0
Flavonol (3-OH is free) δ145.0–149.5 δ135.6–139.0 δ172.0–178.0
Isoflavone δ149.8–156.5 δ120.3–126.0 δ174.5–182.0
Flavanone δ 75.0 – 80.5 δ 42.0 – 44.6 δ189.5–197.2
Flavanonol δ 82.7 – 83.5 δ 71.2 – 73.0 δ188.0–197.0
Chalcone δ136.9–145.4 δ116.8–128.1 δ188.0–194.6
Dihydrochalcone δ 39.2– 45.5 δ 28.5– 31.2 δ199.6–205.5
Aurone δ146.1–147.7 δ111.0–113.3 δ180.5–182.7
Flavan-3-ol δ 77.5– 82.7 δ 65.1– 68.2 δ 27.5– 28.6
Flavan-3,4-diols δ 80.0– 82.5 δ 72.5– 74.4 δ 69.8– 72.0
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FLAVONOID
 Structure Elucidation By 13C NMR Spectroscopy
 Signal characteristics of Flavonoids on ring A and B without Substituents

Rings A Chemical shift Rings B Chemical shift

C-5 δ124–126 C-2’,6’ δ126–130
C-6 δ124–126 C-3’,5’ δ128–129
C-7 δ133–135 C-4’ δ128–132
C-8 δ116–119 C-1’ (flavone, flavonol, isoflavone) δ130–133
C-9 δ154–156 C-1’ (flavanone, flavonol) δ138–139
C-10 δ121–124

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 FLAVONOID
 Structure Elucidation By 13C NMR Spectroscopy
 Signal characteristics of carbons on ring A
the substituents are leads to the changes of chemical shifts of other carbons in ring A.

Substituent mode Type C-5 C-6 C-7 C-8 C-9 C-10

5-Oxygenated δ160 δ109–111 δ135–137 δ107–108 δ157–160 δ111

7-Oxygenated Flavone, δ126 δ114 δ162–164 δ100–102 δ157 δ114–116
flavonol
Flavanone, δ128 δ109–110 δ165–166 δ100 δ162–164 δ114–116
flavanonol
Isoflavone δ127 δ115 δ161–163 δ102–104 δ156–158 δ117–119
5,7-Dioxygenation Flavone, δ160–162 δ97–100 δ162–165 δ 93– 95 δ155–158 δ103–109
flavonol,
isoflavone
Flavanone, δ162–163 δ93–97 δ165–167 δ 93– 97 δ161–163 δ100–104
flavanonol

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 FLAVONOID
 Structure Elucidation By 13C NMR Spectroscopy
 Signal characteristics of carbons on ring B

Substituent mode Type C-1’ C-2’ C-3’ C-4’ C-5’ C-6’

4’-Oxygenated Flavone, δ121–123 δ130 δ115 δ157–161 δ115 δ130
flavonol,
isoflavone
Flavanone δ128–130
flavanonol
3’-OH,4’-OCH3 Flavone, δ121–125 δ113–114 δ145–147 δ149–151 δ112–116 δ118–122
(or 3’,4’-di OH) flavonol,
isoflavone
3’-OCH3, 4’-OH δ121–125 δ110–111 δ150–152 δ148 δ115–117 δ118–122
3’-OH,4’-OCH3 Flavanone δ128–129 δ111–115 δ144–147 δ146–148 δ112–116 δ118–120
(or 3’,4’-di OH) flavanonol
3’-OCH3,4’-OH δ131–132 δ111–115 δ147–149 δ146–147 δ112–116 δ118–120
2’,4’-Dioxygenation Flavonoids δ108–113 δ156–158 δ102–104 δ157–162 δ104–108 δ131–132
3’,4’,5’-Trioxygenation δ120–126 δ106–109 δ146–153 δ93–97 δ136–142 δ106–109
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 Flavonoids Structure Elucidation By 13C NMR
Carbon Chemical shifts δC C NMR (50.0 MHz) in DMSO-d6
13
Number (ppm)
2 164.2 Carbon Chemical shifts δC
Number (ppm)
3 103.6
1’’ 73.9
4 182.7
2’’ 71.5
5 161.5
3’’ 79.8
6 109.8
4’’ 71.1
7 164.4
5’’ 80.0
8 94.5
6’’ 62.3
9 157.1
OH
10 104.2
1’ 122.0 HO O
HO
2’ 129.2 O
HO
3’ 116.6 HO Isovitexin
4’ 162.4 OH
OH O
5’ 116.6
Delazar, et.al. 2006, DARU Volume 14, No. 3
6’ 129.2
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FLAVONOID
 Flavonoid Carbon Framework Drugs

Genestein
Antioxidant, antihelmintic Hesperidin
beneficial effects on blood vessel disorders

Hesperetin
antioxidant, anti-inflammatory,
anti-allergic, hypolipidemic, 3-Rutinosyl quercetin
vasoprotective, anticarcinogenic. decreasing capillary fragility

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