SENYAWA FENOLIK TANAMAN

PENDAHULUAN

8000 Jenis senyawa fenol tanaman
Fenol sederhana dg 1 cincin aromatik,
rendah
Fenol komplek atau polifenol
BM

Umumnya ditemukan di alam dalam bentuk

berkonjugasi dengan gula atau asam organik
Biosynthesis of phenolics
Shikimic acid pathway is most common in plants. Converts simple
carbohydrates into aromatic amino acids. Not present in animals.

Most plant phenolics are derived from cinnamic acid
formed from phenylalanine by phenylalanine ammonia lyase (PAL) enzyme.
PAL activity is inducible: fungal infection, low nutrient levels, high light
PAL
KLASIFIKASI

SENYAWA FLAVONOID
SENYAWA NON FLAVONOID


1.

2.
TIPE UTAMA SENYAWA FENOLIK

1.

2.
3.
FENOL SEDERHANA

LIGNIN
- KUMARIN

FLAVONOID 2 cincin aromatik

anthocyanins, flavones/flavonols
TANIN TERKONDENSASI : polimer flavonoid
TANIN TERHIDROLISIS :
4.

5.
fenol

+ gula

BM lebih kecil dari tanin terkondensasi

1. simple phenolics
Fig. 13.10
Involved in
defense against
insect herbivores
and fungi, some may
have allelopathic
function.
Simple phenolics
Caffeic acid
& ferulic acid
implicated in
allelopathy.
Psoralen is one
of several
phototoxic
furanocoumarins,
(UV activated)
Fig. 13.11
Simple phenolics
(C
)
o
II

(H

Fig. 13.11
(OOH

HO
OH

O(H3

Vanilli
n
Salicyl
ic
aci
d
[@-
(1]
Benzoi
c
acid derivative
s
PLANT PHYSIOLOGY, Third Edition, Figure 13.11 (Part 2) 2002 Sinauer Associates, Inc.
2. Lignin
Second most abundant compound in plants.
Highly branched polymer of phenylpropanoid groups (benzene-C
3
)

- often found in vessel elements, tracheids,
and stems; confers structural support.
Primary structural
role!
- Secondary role as a herbivore deterrent by
reducing digestibility of plant matter
- also difficult for microbes to degrade; its
presence slows litter decomposition.
FLAVONOID

Flavonoid mrpk polifenol terbanyak
Senyawa dengan 15 atom C
C6 C3 C6




4500

Banyak terdapat di epidermis

buah
Potensial sebagai antioksidan
daun dan kulit


3. Flavonoids - basic structure is two aromatic
rings joined by a 3C bridge.
a)
b)
c)
d)
anthocyanins
flavones
flavonols
isoflavonoids
Fig. 13.10
From shikimi
c
acid
phenylalanin
e
pathwa
y
via
[(9-(3]
2'
From
acid
maloni
c
pathway
[(Q
]
2 \
f
I I
I I
,; 3 I
/\/
1
(
,
4
_
......
/
The three-
carbon
skeleton
bridg
e
Basic
flavonoid
PLANT PHYSIOLOGY, Third Edition, Figure 13.12 2002 Sinauer Associates, Inc.
Fig. 13.13
Flavonoids
a) Anthocyanidins and
anthocyanins are pigments
that give plant tissues
red, blue, and purple
color.
Pollinator attraction
Disperser attraction
FLAVONOID

Kerangka dasar flavonoid dpt mengalami
banyak substitusi pada gugus hidroksi
Terutama glikosilasi. Gula : glukosa, ramnosa,
galaktosa

Selain itu dapat mengalami : prenilasi,
metilasi.



KEGUNAAN FLAVONOID

Memberi warna pada bunga ----- penyerbukan
Mengeblok sinar UV
Meloloskan sinar biru hijau dan merah untuk
fotosintesis.
Bersifat antibakteri dan antifungi
Antioksidan, antimikroba, antikanker, cardio-
protective
Various classes of Flavoniods: Anthocyanins,
Flavonols, Flavones, IsoFlavones






FLAVONOID

Gula dan OH ------ kelarutan dalam air
Metil dan isopentil ------ flavonoid lipofilik


Biogenesis berasal dari kombinasi antara jalur
shikimat dan jalur asetat-mevalonat.
Merupakan senyawa fenol terbanyak ditemukan di
alam.
Merupakan zat warna merah, ungu, biru, dan
sebagian zat warna kuning.
Kerangka dasar terdiri atas 15 atom karbon yang
membentuk susunan C
6
-C
3
-C
6
.
3
A
3
2
A
B
2
1
3
1
B A
2
B
1
1,3-diarilpropana
flavonoid
1,1-diarilpropana
neoflavonoid
1,2-diarilpropana
isoflavonoid
Istilah flavonoid berasal dari kata flavon yang
merupakan salah satu jenis flavonoid yang terbanyak
dan lazim ditemukan (selain flavonol, antosianidin).
Flavon mempunyai kerangka 2-fenilkroman.
Berdasarkan tingkat oksidasinya, flavan adalah yang
terendah dan digunakan sebagai induk tatanama flavon.
O 1
O
C
B
O
2
3
A
OH
4
katecin
(flavan-3-ol)
flavan
2-fenilkroman
O
OH
O
O
OH
leukoantosianidin
(flavan-3,4-diol)
calkon
dihidrocalkon
O
O O O
OH
OH
O
O
flavon
O
flavonol
O
flavanon
flavanonol
+

O
+

O
O
C
H
OH
O
auron
antosianidin garam
flavilium




Flavan = 2 fenilkroman
Flavanol = 3 hidroksi flavan = 3 hidroksi 2 fenilkroman
Flavanon = 2,3 dihidro 2 fenilkroman 4 on (Hesperitin)
Flavanonol = 3 hidroksiflavanon = 2,3 dihidroflavonol = 3
hidroksi 2,3 dihidro 2 fenilkroman (Taxifolin)
Flavon = 2 fenilkroman 4 on (Luteolin)
Flavonol = 3 hidroksi flavon = 3 hidroksi 2 fenilkroman 4 on
(Quercetin)


Isoflavonoid dan neoflavonoid hanya ditemukan
dalam beberapa jenis
Ragam isoflavonoid:
isoflavon
tumbuhan.
pterokarpan
MeO O HO O
O R O
OH
O
R = H
R = OH
daidzein
genistein
pterokarpin
rotenoid
kumestan
R
O O
R2 O O
O
R3
R O
R1 O
OMe
OH
OMe
R1=R3=H R2=OH kumestrol
R1=R3=OH R2=OMe medelolakton
R = H rotenon
R = OH amorfigenin
Anthocyanins





Warna bervariasi

dari merah --- ungu dan biru

Komponen utama dari warna bunga pada tanaman

Penting untuk menarik serangga pada penyerbukan
Interaksi mutualistik
ANTOSIANIDIN

Flavonol and Flavone

Umumnya berupa pigmen berwarna dalam

daun
Mengontrol sinar yg mengenai daun :
photosynthesis and UV protection
Menyebabkan perubahan warna daun



:

bila klorofil menurun -

flavonol akan

diubah

mjd anthocyanins

IsoFlavones

Found mostly in Legumes (Fabaceae)
Allelopathy herbivory defense, pathogen
defense!



Tannins

Tanin terkondensasi

-polimer dari flavonoid
-umumnya terdapat tan.
Tanin terhidrolisis
Berkayu

-

-
-
terdiri dari fenol: asam galat & gula

BM << BM tanin kondensasi
lebih mudah terhidrolisis dan terdegradasi
Tannin : menghambat pertumbuhan herbivore

Juga bersifat antioksidan
Foods contains tannins : tea

Tannins juga menyebabkan protein lebih
sulit dicerna

Animals can sense high levels of tannins in
their food and opt for another food
resource (e.g. mule deer, beavers).
High levels of tannins in diet can actually

some animals.

kill

Condensed tannins are
polymerized
flavonoids.
Hydrolyzable tannins are made of phenolics and sugars.
(B) Hydrolyzable
tannin
OH

OH

o
II
0-(
OH

o
II~
HO

OH

HO

(
H 0
I
(==0
HO

Fig. 13.15
0
I
(==0
Galli
c
aci
d
HO

OH

OH

HO

OH

OH

PLANT PHYSIOLOGY, Third Edition, Figure 13.15 (Part 2) 2002 Sinauer Associates,
Inc.
The term tannin is derived from the tanning
process in which raw animal hides are preserved by

rubbing tannins on them. The tannins help to

complex the proteins and keep them from

degrading.

This protein-binding property of tannins lends them
their toxicity to herbivores.
tannins can bind digestion enzymes in the gut of
herbivores.

tannins also form complex polymers when bound
to proteins which are difficult to digest, thus
decreasing the nutritional value of the plant
material.
Tannins can reduce nutritional

value

them
of tissues

by

binding

to

proteins,

making

less

digestible.

Fig. 13.16
Care for a spot of milk in your tea?

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 60to 200.
* Supercritical fluid extraction with carbon dioxide CO
2

- Careful the thermal degradation of the flavonoid
derivatives.
2.2

Isolation

2.2.1C Further purification and/or preconcentration of
the target compound fraction
liquidliquid extraction (LLE)
commonly used:
a. Estimation of the extraction yield
- Spike biological materials with proper internal
standards
Methods: Compounds labeled with stable isotopes
(
2
H or
13
C) MS
b. The extraction of flavonoids from biological materials
- Need to addifferent classes of phenolic compounds.
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)
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.
c.
Silica gel with the solvent systems:
Flavone-O-glyconsides 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
toluene: dioxan: methanol
toluene: methyl ethyl ketone: methanol and acetic
dioxan: dimethyl formamide: water.
Best spraying agents:
-aminodiethyl ester of dephenylboric acid (5%
and
acid:
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: -Bondapak C
18
, 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(
ratios).
in different
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.
2. SENYAWA FENOLIK
10 ml SARI ETER
Diuapkan ad
kering
Residu
+ K heksasianoferat (III)
+ FeCl3
+ bbrp tts amonia
+ 2-3 tts FeCl3
Sinar UV
Biru ad hitam
Hijau, ungu, biru
hitam
Fluoresensi
(fenol-fenol bebas)
FENIL PROPANOID
Meliputi: kumarin dan turunannya
3 ml SARI ETER
Diuapkan ad
kering
Residu
+ air panas ad larut
II
mlNH4OH ad alkalis
I
Pembanding + 0,5
Sinar UV
Sinar UV
Fluoresensi
Fluoresensi
lebih terang
Kumarin
dan turunannya
FLAVONOIDA
Diuapkan
ad kering
Residu
3 ml SARI ETER
+ 1-2 ml MeOH (50%)p
dipanaskan
+ logam Mg
+ 5 tts HCl pekat p
Merah / jingga
aglikon Flavonoid
3. GLIKOSIDA
+ 20 ml Sari EtOH-Air
+ 15 ml Hcl 10% LP
direfluks 30
Dinginkan
Disari 3x15 ml Eter
Sari Air Asam
Sari Eter
+ Na2SO4 anh
disaring
+ basa ad netral
Sari air netral
Sari eter
Aglikon Steroid,
Triterpenoid, Kumarin,
Flavonoid, Antrakinon dll
Gula
IDENTIFIKASI

Analisis kualitatif senyawa fenol secara umum dapat
menggunakan FeCl3, di mana akan dihasilkan larutan
berwarna merah, violet atau merah-ungu
Flavonoid mengandung sistem aromatic yang terkonjugasi
sehingga akan menunjukkan pita serapan yang kuat pada
sinar UV dan sinar tampak (Harborne, 1987)
Dengan pereaksi AlCl3, flavonoid akan tampak berupa
bercak berwarna kuning dan tergantung strukturnya,
flavonoid akan berfluoresensi kuning, biru, atau hijau di
bawah UV 365nm
Tanin dapat bereaksi dengan protein membentuk
kopolimer mantap yang tak larut dalam air.






Tanin dan senyawa fenolik lain

Fase Diam

Silika Gel F254

Fase Gerak

Etil asetat : Metanol : Air ( 100 :
13,5 : 10 )

Sampel

Daun Beluntas

Deteksi

FeCl
3


Pembanding

Asam galat

Keterangan

Dibawah sinar tampak senyawa
fenolik akan berwarna hijau
Keterangan

Flavonoid

Fase Diam Silika Gel F254

Fase Gerak BAW (4:1:5)

Sampel Daun Beluntas

Deteksi AlCl3

Pembanding Rutin

Di bawah UV 365 berpendar
kuning intensif







Termasuk golongan apakah Gambar senyawa diatas ?
Pelarut yang digunakan untuk menyarinya senyawa tsb?
Metode penyarian yang digunakan?
Proses selanjutnya untuk mendapat aglikon dari senyawa
Fraksinasi dilakukan dengan menambah pelarut?
Proses isolasi dilakukan dengan metode?
Detektor yang digunakan untuk mendeteksi keberadaan
senyawa B?
tsb?

Termasuk golongan senyawa apa ?
1. .
2. .
3. .