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Prof. Dr. Amani S. Awaad
Professor of PharmacognosyPharmacognosy Department,
College of Pharmacy Salman Bin Abdulaziz
University,
Al-Kharj. KSA.
Email: [email protected]
Pharmacognosy- 1
PHG 222
*To recognize what are phenolic compounds
*To memorize Classes of phenolic compounds
*To know Tannins as one of phenolic *To know
memorize types of tannins and their reactions
*To know some types of Tannins containing drugs
Phenolic compounds
They can be:
Simple, low molecular weight,
single aromatic ringed
compounds TO-Large and
complex-polyphenols
1-Non-Soluble (condensed tannis,
lignins, cell-wall bound
hydroxycinammic acid
2-Soluble (Flavonoids, quinones,
phenolic acids and phenylpronanoids
Can be classified into two groups:
Phenolic compounds
Composed of a hydroxyl group attached to
an aromatic ring
A broad range of compounds - found in all
components of plants: leaves, flowers, fruit etc
Can be exist as free or combined form
Free= Aglycone
combined =Glycone or glycoside
Chemistry of phenolic compounds
Classes of phenolic compounds
Flavonoids
Coumarins
Xanthine
Anthraquinons
& anthracine
Lignins
Tannins
Simple phenolic
1-Flavonoids
For plants:Variety of functions: Protection from animalBreading photosynthesis
Most widely studied group of phenolic: over 4000 described
Seem to have major health benefits for humans :
Such as Antioxidant and other medicinal use
flavonoidsChemistry of
consisting of 2 benzene rings linked via a heterocyclic
pyrone or pyran ring They are benzo--pyrone derivatives .
plant pigments or co-pigments.
C6-C3-C6
The basic flavonoid skeleton can have a large number
of substitutions of Hydroxyl groups or Sugars on it.
Sugars and hydroxyl
groups increase the
water solubility of
flavonoids
Rhamnose
Glucose
Tests & identefecations of flavonoids
The extracts were dissolved in ethanol, filtered and
subjected to following tests.
(a) Shinoda test: The dried extracts were dissolved in
95% ethanol (5ml) and few drops of concentrated
hydrochloric acid (HCL) were added. Then the
magnesium turnings were put into the solution and
observed for appearance of pink color.
Color test
Spectroscopic methods of identify Flavanoids.
Identification
1-Ultraviolet spectrophotometry (UV)
range of 240-285 nm (band Ⅱ) and the other in the range of 300-400 nm (band Ⅰ).
O
O-
R
+ O
O
R
O
O-R
+
benzoyl( band ¢ò, 240~285nm)
flavone (R=H)flavonol (R=OH)
cinnamoyl( band ¢ñ,300~400nm)
2-Nuclear Magnetic Resonance (PMR, 13C-NMR)
OHO
OH O
A6
8
ORB
2' 3'
3-Mass Spectrometry (MS)
From normal mass spectra, information can be
obtained about:
Flavone
O
O
A C
B
+.
Pathway IIPathway I
O
C
O
C
C O BO+
+
+
B1+ B2
+O
+
Isolation Techniques
★High performance liquid chromatography (HPLC)
★ Thin-layer chromatography (TLC
★ Column chromatography
★ Paper chromatography (PC)
★Gas liquid chromatography (GLC)
Coumarins are derivatives of benzo-α-pyrone, or lactones of o-hydroxycinnamic acid (o-coumaric acid).
2-Coummarins
OO
Coumarine
OO
2-pyrone
The name coumarine is derived from the Caribbean word
“Coumarou” for the Tonka tree from which coumarine
with the characteristic aroma of new- mown hay was
isolated.
Un-substituted coumarine is widespread in nature.
Tonka tree .
Based on the substitution in benzene and pyrone rings.
Ⅱ. Classification
Most of these naturally occurring lactones are oxygenated at C-7 position
I) 7-hydroxy coumarine
O ORO
R= H Umbelliferone
R= Glc Skimin
O O
Coumarin
2II)Furanocoumarine
O
O
O
OCH3
OCH3
III)Pyranocoumarins
O OO
OMe
IV) Bis- and Tri-coumarins
O O
OH
O
OH
O
OHO O
O OO
O OO
Rhamnosyl-L-α
Edgeworoside
Extraction & Isolation
2-Sublimation
1-Solvent extraction
With alcohol and using suitable extraction
methods
B-Isolation
Using chromatography
(PC, CC)
A-Extraction
Coumarin and its derivatives exhibit
sublimation on heating to 100˚C
Identification of coumarins
A-Chemical tests Identification
Sublimation test: In a test tube one g of plant sample
was placed and covered with filter paper moistened with
dil. NaOH, then heated on water bath for a few minutes.
The filter paper was examined under UV light, blue to
yellow fluorescence indicates the presence of coumarins
B-Spectroscopic methods of Identification
•i) UV spectroscopy
Coumarin absorb ultra-violet light at~300 nm
-An unsubstituented coumarin displays absorption bands at 274 nm and 311 nm
7-hydroxycoumarin shows strong bands at ~217 nm and 315-330 nm
-furanocoumarins absorb at 4 wavelengths namely 205-225 nm, 240-55 nm.
ii) Infrared spectroscopypyrone carbonyl(C=0) ------1700-1750 cm-1
C=C skeletal vibrations1-------600-1660 cm-1
aromatic ring band at 1500 cm-1.
iii) 1H-Nuclear magnetic resonance spectroscopy
In the NMR spectra of coumarins the H-3 and H-4 protons exhibit characteristic chemical shifts which enable the substituted coumarins to distinguish from one another.
iv) Mass spectroscopystrong molecular ion peak (M+) at m/e 146(76%)
base peak at m/z 118(100%)
O O
-COO
++
-CO
C7H6
m/e 146 (M. 76%) Coumarin
m/e 118 (100%) m/e 90 (43%)
3-Xanthones
Xanthones composed of tricyclic aromatic systeme
with a variety of phenolic, methoxy, or isoprene
substituents giving rise to numerous derivatives.
Xanthones have been reported to have
antioxidant, antibacterial, antimicrobial,
antifungial,
solid polycyclic aromatic hydrocarbon consisting of three fused
benzene rings. It is a component of coal tar.
Oxidation occurs readily, giving anthraquinone
(i)Anthracene
4-Anthracene&Anthraqu.
(ii) Anthraquinones
are colored substances and many of them are used technically as dyes
7
6
5
10a
8a
8
10 4a
9a9
4
3
2
1
O
O
OHOH OH
CH3
OOH OH
CH3
They are isomeric and one may be partially converted to the other in solution.
These reduced anthraquinone derivatives occur either free or combined as glycosides
(iii) Anthranol , anthrone derivatives
(iii) Oxanthrone
OH O OH
H OH
Intermediate products between
anthraquinones and anthranols
(iii) DianthroneOO OH
COOH
OO OH
COOH
HH
glc
glc
Derived from two anthrone
molecules, which may be identical
or different
Extraction and isolation(i) Extraction Commonly method:
(1) back streaming extraction with ethanol, then remove the solvent by
depressed evaporation to get the crude extract.
(2) extracting with ammonia or 0.1% - 0.5% sodium hydroxide solution at
room temperature.
(3) remove the fat-soluble impurities from alkaline extraction with ether.
(4) acidifying the alkaline extraction.
(5) extract the anthraquinones with ether and retrieve solvent.
used absorbent are silica gel,
polyamide, in general aluminium oxide
is not applied in order to avoid
irreversible chemical absorption.
Sephadex and reverse silica gel
chromatography often have good effect.
2. Seperation
using silica gel plate and different
solvent systems
TLC
Column chromatography
Identificationi) UV spectroscopy
The peak position of the peak I
(230nm ),peak II (310 ~ 335nm)
the more the hydroxyl replacements
is, the longer the wavelength of
absorption.
ii) Infrared
spectroscopy
position of C=O
absorption peak , the
peak number, α-
hydroxy number and
location can be
estimated.
O
O
182.5132.9
126.6
134.3
O
O
187.9113.8
161.3
123.7
181.5132.6
118.8
136.3
OH
iii)NMR spectroscopy
NMR spectrum analysis is the most
powerful tool in the the structure
determination of anthraquinones..
iv ) Mass spectroscopy
To MS spectrum, anthraquinone compounds’
molecular ion peak always is the base peak and it
often lost 1-2 CO peak of debris
O
+ .
- CO
+ .
m/z 208 m/z 180
- CO
+ .
m/z 152
5-Phenanthrens & Stelbines
CH3
OH
CH3
HO
OH
CH3
CH3
HO
O
Juncusol Juncunone
Phenanthrene is a polycyclic
aromatic hydrocarbon composed
of three fused benzene rings
are secondary products of heartwood
formation in trees that can act as phytoalexins
. In chemical terms, they are hydroxylated
derivatives of stilbene
Stilbenoids
heartwood
Resveratrol
Antiviral effects
Anti-inflammatory effects
Veratrum album
Involved in defense against insect
herbivores and fungi,
6-Simple phenolic
The simple phenols consist of a singly substituted phenolic ring
with either alcoholic, aldehydic or carboxylic acid groups
1-Hydroxybenzoates or C6-C1 (Benzoic Acid) Derivatives
i-Arbutin ii-Salicin
2-Phenylpropanoids or C3-C6 Derivatives
i-Coniferinii-Ferulic acid
6-Simple phenolic
7-Lignans
Podophyllum Rhizomes
Plant products of low molecular weight formed
primarily from oxidative coupling of two p-propylphenol
moieties at their beta carbon atoms; products with units
coupled in other ways are neolignans
*derived from the Latin word lignum, meaning wood
*Lignin fills the spaces in the cell wall between
cellulose, hemicellulose, and pectin components
*Lignin is a cross-linked racemic macromolecule with
molecular masses in excess of 10,000 u.
Uses: can be used as starting for
anticancer drugsFig. : A small piece of lignin polymer
Lignin in beech has 100:70:7 of coniferyl: sinapyl: para-coumaryl
8-TanninTannins are "phenolic natural products that precipitate proteins
from their aqueous solutions".
1. Tannins are considered the source of energy through
their oxygen content.
2. They serve as a protective to the plant (plant
antiseptics).
3. They may have function in respiratory activity, i.e. in
the mechanisms of hydrogen transfer in plant cells.
4. Tannins play an important part in the acceptance of
many foods and beverages by consumers e.g. tea,
cocoa.
Tannins has capability to combine with macromolecules
and this explains why they precipitate cellulose,
pectins, and proteins;
it also explains their characteristic astringency
by precipitating the glycoproteins contained in saliva,
tannins make the latter lose its lubricating power.
1. They form colloidal solutions with
water.
2. Non crystalline substances.
3. Their aqueous solution is acidic.
4. They have sharp puckering taste.
5. They have astringent property.
6. Its Solutions precipitated
alkaloids, gelatin, salts of heavy
metals, proteins (enzymes) and
glycosi.
Physicochemical properties of tannins 8-Tannin cont….
Qualitative determination of tannin:
3-NaoH → yellow to red precipitate
1-With FeClз :
(a) H.T(hydrolysable tannin) → dark blue (gallitannins and
ellagitannins )
(b) Non H.T (condensed tannins eg catechol) → dark green
2-Vanillin_HCl reagent → red to pink color.
4-Goldbeater's skin testsoak a small piece of Gold beater's skin
membrane in 2% HCl, rinse with H2O, place
the piece for 5 minutes in the tannin solution
to be tested, wash with H2O, transfer to 1%
FeSO4 solution, a brown colour is formed
8-Tannin cont….
5-Gelatin test
Solutions of tannins (about 0.5-1 %)
precipitate a 1% solution of gelatin
containing 10% sodium chloride.
Gallic acid and other pseudotannins also
precipitate gelatin if the solutions are
sufficiently concentrated.
6-Phenazone test
7-Test for catechin
8-Test for chlorogenic acid
Qualitative determination of tannin:8-Tannin cont….
1- Hide powder method:
The difference in the dry weight of the extract before and after treatment with hide powder is taken as a measure for tannin content.
2- Copper acetate method:
Gravimetric method using copper acetate as precipitant.
Quantitative determination of tannin:8-Tannin cont….
True Tannins(High molecular weight compounds)
Hydrolysable Tannins(Pyrogallol)
Condensed Tannins(Catechol)
PseudotanninsLow molecular weight compounds
e.g. Gallic acid, Flavan-3,4-diol
Classification of tannins
As general Some of them have low
molecular weight but, most of them have
very large molecular weight
8-Tannin cont….
Classification of tannins cont..
flavan-3-ol units linked
together by carbon-carbon
bonds, most often
48(catechin)
I-True tannins
i-Condensed tannins
these are not readily hydrolyzed to simpler molecules by
acids or enzymes and they do not contain a sugar moiety
catechin
flavan-4-ol : units linked
46, which result from
coupling (leucocyanidin)
are polymeric flavans. They consist :
In higher plants, two groups of tannins are generally distinguished, which
differ by their structure, as well as their biosynthetic origin: condensed
tannins and hydrolysable tannins
8-Tannin cont….
• Biosynthetically, flavonoids are derived from
acetate and shikimate pathways.
• Condensed tannins occur due to polymerization
(condensation) reactions between flavonoids.
• The polymers may include up to 50 monomer units.
• On treatment with acids or enzymes condensed
tannins are converted into red insoluble compounds
known as phlobaphenes.
• Phlobaphenes give the characteristic red colour to
many drugs such as red cinnamon bark.
• Examples of drugs containing Condensed tannins:
Some drugs (e.g. tea, hamamelis leaves and
hamamelis bark) contain both hydrolysable and
condensed tannins. The following are rich in
condensed tannins.
i-Condensed tannins
I-True tannins Classification of tannins cont..8-Tannin cont….
I-True tannins
ii-Hydrolysable tannins
1- They are esters of a sugar (β-D-glucose) with one or more
trihydroxybenzene carboxylic acid.
2-The tannins which derived from Gallic acid called Gallo-tannin
(Clove buds)
Hydrolyzed by acids or enzymes (Tannase).
were formerly known as pyrogallol tannins, because on dry distillation
gallic acid and similar components are converted into pyrogallol
Examples of drugs from Gallitannins: rhubarb, cloves, Chinese
galls, Turkish galls, hamamelis, chestnut and maple.
Classification of tannins cont..8-Tannin cont….
I-True tannins
ii-Hydrolysable tannins
Tannis which derived from Ellagic acid called
Ellagitannin like in Pomegranate roots bark.
Ellagic acid can arise by lactonization of hexahydroxydiphenic
acid (= HHDP) during chemical hydrolysis of the tannin.
Biosynthetically, gallic acid (= 3,4,5-trihydroxybenzoic acid)
arises from the metabolism of shikimic acid.
Examples of drugs containing Hydrolysable tannins:
Ellagitannins: pomegranate rind, pomegranate bark, eucalyptus
leaves, and oak bark.
Classification of tannins cont..8-Tannin cont….
II-Pseudo tannins
They are compounds of lower molecular
weight than true tannins and they do not
respond to the goldbeater's skin test.
they occur:
as Gallic acid like in Rhubarb rhizomes.
as catechin like in Cacao, Acacia.
as chlorogenic acid like in coffee.
Classification of tannins cont..
Gallic acid
COOH
OH
OH
HO
Catechin
OHO
OH
OH
OH
OH
Chlorogenic acid
O
O
OH
HO
OHHOOC
OH
OH
8-Tannin cont….
Medicinal and biological properties
The applications of tannin-
containing drugs are limited, and
result from their affinity for proteins.
• Tannin-containing drugs will
precipitate protein and have been
used traditionally as styptics and
internally for the protection of
inflamed surfaces of mouth and
throat.
• They act as antidiarrhoeals and have
been employed as antidotes in
poisoning by heavy metals, alkaloids
and glycosides.
8-Tannin cont….
Industrially; in leather tanning, ink manufacture.
Medicinally; astringent, haemostatic, antidiarrheal, antidote in alkaloid and heavy metals toxicity.
In labs; reagent for detection of proteins, alkaloids and heavy metals because of their precipitating properties.
8-Tannin cont….Uses of Tannins
Origin: dried excrescence (pathological outgrowth) resulting from the deposition of the eggs of Cynips gallae tinctoria F. Cynipidae on the young twigs of Quercus infectoria F. Fagaceae. Collected before the escape of the insect and known as blue galls.
Morphology: subspherical, 12-20 mm in diameter, external surface is bluish green to olive green in colour, showing blunt short projections. Internally it is buff in colour.
Tannin containing drugs
1-Galls (Blue Galls, Aleppo Galls, Turkish Galls)
8-Tannin cont….
Active constituents: 50-70% pyrogallol
tannin: gallotannic acid, gallic acid and
ellagic acid.
Uses: Medicinally: astringent, haemostatic
used in treatment of hemorrhoids in
form of supp. or oint.
Industrially:
1- in manufacture of ink and paints
2- in tanning of leather.
Chemical test: FeCl3 → bluish black
colour.
Tannin containing drugs
1-Galls (Blue Galls, Aleppo Galls, Turkish Galls)
8-Tannin cont….
Synonyms Kattha, Cutch, Khadir-catechu, Catechu
Biological Source It consists of dried aqueous extract
prepared from the heart- wood of Acacia catechu Wild
and Acacia chundra Willd,
family Leguminosae.
Plants used for preparation of catechu are grown in India
and Myanmar. History Possibly, the use of black catechu
could be traced back in history from the time of chewing
betel leaf, in which it has been used as adjuvent. In old
days, it was used by women as a colouring agent for the
feet. Since 15thi century, this natural material has, been
exported to Europe. The old information about Catechu
is by a Portuguese writer Garcia de Orta in 1574. Dr.
Wrath first used the scientific process to extract catechu,
and showed that catechu consists of two parts, viz.
kattha and cutch.
2-Black Catechu
Tannin containing drugs8-Tannin cont….
Synonyms Kattha, Cutch, Khadir-catechu, Catechu
Biological Source It consists of dried aqueous extract
prepared from the heart- wood of Acacia catechu Wild
and Acacia chundra Willd,
family Leguminosae.
Plants used for preparation of catechu are grown in India
and Myanmar. History Possibly, the use of black catechu
could be traced back in history from the time of chewing
betel leaf, in which it has been used as adjuvent. In old
days, it was used by women as a colouring agent for the
feet. Since 15thi century, this natural material has, been
exported to Europe. The old information about Catechu
is by a Portuguese writer Garcia de Orta in 1574. Dr.
Wrath first used the scientific process to extract catechu,
and showed that catechu consists of two parts, viz.
kattha and cutch.
2-Black Catechu
Tannin containing drugs8-Tannin cont….
Synonyms Kattha, Cutch, Khadir-catechu, Catechu
Biological Source It consists of dried aqueous extract
prepared from the heart- wood of Acacia catechu Wild
and Acacia chundra Willd,
family Leguminosae.
Plants used for preparation of catechu are grown in India
and Myanmar. History Possibly, the use of black catechu
could be traced back in history from the time of chewing
betel leaf, in which it has been used as adjuvent. In old
days, it was used by women as a colouring agent for the
feet. Since 15thi century, this natural material has, been
exported to Europe. The old information about Catechu
is by a Portuguese writer Garcia de Orta in 1574. Dr.
Wrath first used the scientific process to extract catechu,
and showed that catechu consists of two parts, viz.
kattha and cutch.
2-Black Catechu
Tannin containing drugs8-Tannin cont….
Traditional method: the separated heart wood is boiled
in earthen vessels, Fired by sap wood, till all the soluble
portion is extracted from it. It is cooled naturally till it is
converted to semisolid mass. On cooling less soluble
fraction separates out. The latter is removed as kattha and
semi solid mass as cutch. It is transferred to rectangular
pits, at the bottom of which sand and clay are placed. It is
kept for several days, till the cutch part is absorbed by
clay and solid mass, called kattha, is taken out and
mouldéd into blocks.
Modern methods: The red heart wood obtained by
felling the tree and separating the bark and sapwood, is
cut into chips mechanically and put into extractors. The
steath is passed through the drug for maximum extraction.
The extract is concentrated under vacuum and is cooled
by refrigeration. It is then centrifuged to isolate the cake
of kattha. The cake is moulded in desired sizes and dried
in proper condition. By this way, a good quality of kattha,
ready for market is obtained. The mother liquor, left
behind during centrifugation is concentrated, which on
cooling gives cutch.
Manufacture
2-Black Catechu 8-Tannin cont….
Description Colour:
Light brown to black Odour: None Taste: Very astringent
Size: About 2.5 to 5 cm Shape: Cube or irregular fragments
of broken cubes or brick shaped pieces. Extra Feature The
cubes as well as brick shaped pieces of catechu show the
presence of vegetable debris break with a short fracture The
broken pieces are angular with pale cinnamon-brown colour
it is friable and porous
Chemical Constituents: 10% of acacatechin It is
distereoisomer of 5, 7, 3’, 4’ tetrahydroxy flavan-3-ols
Acacatechin undergoes oxidation to catechutannic acid in
presence of water The other contents of black catechu are
catechu red, quercetin, gum and quercitrin. Black catechu
does not contain chlorophyll
Uses: Kattha is used as an astringent externally for boils, skin
eruptions and ulcers. It is also used in cough and diarrhoea. Kattha
has cooling and digestive properties. Cutch is not much used
medicinally, but for other purposes like dyeing and colouring,
water softening, reducing the viscosity of drill mud protective
agent for fishing nets and in the manufacture of ion-exchange
resins.
2-Black Catechu
8-Tannin cont….
8-Tannin cont….
