phenolic compounds in plants

1. A presentation
On
Phytochemical assays of phenolic compounds
Presented by – Gaurav
Reg. No. – 16TMM2363
In guidance of Dr. IP Singh
Department of Natural Products
NATIONAL INSTITUTE OF PHARMACEUTICAL EDUCATION AND
RESEARCH
S.A.S. NAGAR

2. Phytochemical Study of Phenolic Compounds
Phenolic Compounds - The term phenolic compound embraces a wide
range of plant substances which possess in common an aromatic ring
bearing one or more hydroxyl substituents. Phenolic substances tend to be
water-soluble, since they most frequently occur combined with sugar as
glycosides.
Among the natural phenolic compounds, of which over a thousand
structures are known, the flavonoids form the largest group but simple
monocyclic phenols, phenylpropanoids and phenolic Quinone's all exist in
considerable numbers.

3. The classic procedure for detecting simple phenols is by means of the intense
green, purple, blue or black color many of them give in solution when 1 %
aqueous or alcoholic ferric chloride is added. This procedure, modified by using
a fresh aqueous mixture of 1 % ferric chloride and 1 % potassium ferricyanide, is
still used as a general means of detecting phenolic compounds. Where as these
phenolic compounds which mainly include
1. Phenols and phenolic acids
2. Phenylpropanoids
3. Flavonoid pigments
4. Anthocyanins
5. Flavonols and flavones
6. Minor flavonoids, xanthones and stilbenes
7. Tannins
8. Quinone pigments

4. Phenols and phenolic acids
Acid hydrolysis of plant tissues releases a number of ether soluble phenolic acids,
some of which are universal in their distribution. These acids are either associated
with lignin combined as ester groups or alcohol-soluble fraction bound as simple
glycosides . phenols are relatively rare in plants. Hydroquinone is probably the most
widely distributed; others such as Gallic acid , catechol, orcinol, phloroglucinol and
pyrogallol, have been reported from only a few sources.
And for detection of phenols and phenolic acid , usually Thin layer chromatography
method follows on silica gel of simple plant phenols. Key: solvent 1, 10% acetic acid
in chloroform; solvent 2, 45% ethyl acetate in benzene .
Exg . phenols and phenolic acids

5. 1. Gallic acid
2. 3,4-dihydroxybenzoic acid
3. Hydroquinone
(FCR) or Folin's phenol reagent or
Folin–Denis reagent,(sodium 1,2-naphthoquinone-4-sulfonate )
1. 2,5-dihydroxybenzoic acid
2. Rhododendrol , orcinol,
3. p-hydroxybenzoic acid
4. Syringic acid
5. Vanillic acid
6. Salicylic acid
(FCR) or Folin's phenol reagent or
Folin–Denis reagent,(sodium 1,2-naphthoquinone-4-sulfonate)
Give
blue
color
Give blue
color after
fuming
with
ammonia
Detection of phenols and phenolic acid -

6. 1. Orcinol Reac
t
vanillin-HCl.
Give pink
color
PHENYLPROPANOIOS
Phenylpropanoids are naturally occurring phenolic compounds which have an
aromatic ring to which a three-carbon side-chain is attached. They are derived
biosynthetically from the aromatic protein amino acid phenylalanine and they
may contain one or more C6-e3 residues.
Thin layer chromatography separation on microcrystalline cellulose of
cinnamic acids. Solvent 1 = benzene-acetic acid-water (6: 7: 3). Solvent 2 = 15%
acetic acid in water.

7. In phenylpropanoids compounds like
1. Caffeic acid
2. Pcoumaric acid
3. o-coumaric acid
4. Sinapic acid
5. Ferulic acid.
can be detected as different shades of blue under UV light and fuming
with ammonia.
And where these two other
1. Gallic acid
2. Cinnamic acid
can be detected as different shades of blue under UV light and fuming
with ammonia.

8. Anthocyanins
The anthocyanins are the most important and widespread group of colouring
matters in plants. These intensely coloured water-soluble pigments are
responsible for nearly all the pink, scarlet, red, mauve, violet and blue colours
in the petals, leaves and fruits of higher plants. The anthocyanins are all based
chemically on a single aromatic structure, that of cyanidin, and all are derived
from this pigment by addition or subtraction of hydroxyl groups or by
methylation or by glycosylation . Examples of Anthocyanins
1 2
3

9. Chemical test for determination of Anthocyanins :
Sodium Hydroxide Test/ Alkaline reagent test :
About 0.2 gm of plant extract was weighed in separate test tube, 1ml of 2N
Sodium hydroxide was added, and heated for 5 minutes at 100 ± 2°C and
observed for the formation of bluish green color which indicates the
presence of anthocyanin.
Other tests for distinguishing anthocyanin
Procedure Anthocyanin response
1. Heat with 2M HCI
for 5min at 100°C
Color stable (can be
extracted into amyl
alcohol)
2. Add 2M NaOH
dropwise
Changes to blue-green
and slowly fades
3. Chromatography in
1% aqueous HCI
Low to intermediate RF
4.Visible spectrum
in Methanol-HCI
Maximum in the range
505-535nm
Test
(extra
ct)

10. Flavonoids
Flavonoids have the general structure of a 15-carbon skeleton, which consists of
two phenyl rings (A and B) and heterocyclic ring (C). This carbon structure can be
abbreviated C6-C3-C6. According to the IUPAC nomenclature,[1][2] they can be
classified as.
Flavonols
Kaempferol , Quercetin, Myricetin, lsorhamnetin, Azaleatin.
Flavones
Apigenin, Luteolin, Chrysoeriol, Tricin.
Glycosylflavones
Vitexin, Isovitexin, Orientin, Iso-orientin.
Bifiavonyl
Kayaflavone.
Figure 1– General moiety of flavonoid

11. Chemical test for flavosnoid –
Shinoda test – crude extract was mixed with few fragment of magnesium ribbon
and concentrated HCL was added drop wise. Pink scarlet color appeared after
few minute which indicate presence of flavonoids .
Alkaline reagent test – crude extract was mixed with 2 ml of 2% oh NaOH .
An intense yellow color was formed and which turned colorless on addition of
few Drops of diluted acid .which indicate presence of flavonoids .
Lead acetate test
The extract (50 mg) is dissolved in of distilled water and to this 3 ml of 10%
lead acetate solution is added. A bulky white precipitate indicates the
presence of phenolic compounds /flavonoids

12. Other test for flavonoids - The ethanol extract (5 ml) was added to a
concentrated sulphuric acid (1 ml) and 0.5g of Mg. A pink or red coloration
that disappear on standing (3 min) indicates the presence of flavonoids.
Tannins
The word tannins was first applied by Seguin –means the substance
present in plant extracts which are able to combine with animal hides,
prevent their purification and convert in to lather.or Tannins are naturally
occurring complex organic compounds possessing nitrogen free
polyphenols of high molecular weight . . They also precipitate proteins and
alkaloids
Classification of tannins
1. Hydrolysable tannins
2. Condensed tannins
3. Pseudo tannins

13. Chemical tests: for tannins
1. Gelatin test:
To a solution of tannin, aqueous solution of gelatin and sodium chloride are
added. A white buff coloured precipitate is formed.
2. Goldbeater’s skin test: Soak of small piece of goldbeater’s skin in 2% HCL,
rinse with distilled water and place in a solution to be tested for 5 min . Wash with
D.water and transfer to 1% solution of ferrous sulphate .a brown or black color on
the skin denotes the presence of tannins .goldbeater’s skin is a membrane prepared
from the intestine of ox and behave to untanned hide
3. Phenazone test:
A mixture of aqueous extract of a drug and sodium acid phosphate is
heated and cooled and filtered. A solution of phenazone is added to the
filtrate. A bulky coloured precipitate is formed.
of phloroglucinol.

14. 4. Match stick test (Catechin test):
A match stick is dipped in aqueous plant extract, dried near burner
and moistened with concentrated hydrochloric acid. On warming
near flame, the matchstick wood turns pink or red due to formation
of phloroglucinol.
5. Chlorogenic acid test:
An extract of chlorogenic acid containing drug is treated with
aqueous ammonia. A green colour is formed on exposure to air.
6. Vanillin-hydrochloric acid test:
Sample solution and added vanillin- hydrochloric acid reagent
(Vanillin 1 gm, alcohol 10 ml, concentrated hydrochloric acid 10 ml).
A pink or red colour is formed due to formation .

15. Quinones
Quinones are coloured and contain the same basic chromophore, that of
benzoquinone itself, which consists of two carbonyl groups in conjugation
with two carbon-earbon double bonds. For their identification, quinones can
conveniently be divided into four groups: benzoquinones, naphthaquinones,
anthraquinones and isoprenoid quinones. The first three groups are generally
hydroxylated, with 'phenolic' properties, and may occur in vivo either in
combined form with sugar as glycoside or in a colourless, sometimes
dimeric, quinol form. Some examples quinons are

16. Hydroxyquinones may appear during paper chromatographic surveys as
yellow or orange pigments which move to the Phenolic compounds
front in butanolic solvents (e.g. BAW) and which are generally immobile
in aqueous solvents.
Characterization quinones were carried out according the reaction
Borntraeger. In a capsule, 2 mL of the plant extract are evaporated to
dryness. The residue was triturated in 5 mL of HCl diluted 1/5 and then
brought the solution to the boiling water bath for 30 min in a test tube.
After cooling under a stream of cold water, the hydrolyzate was
extracted with 20 mL of chloroform in a test tube. The chloroform layer
was then collected in another test tube and then, 0.5 mL of ammonia
diluted twice was added thereto. The appearance of a color ranging
from red to purple characterizes the presence of quinones.

17. References - J. B. HARBORNE ,Phytochemical Methods “A Guide to Modern
Techniques of Plant Analysis” 3ed Edition 1978 , page No. 33-80
2- W.C EVANS, ”Trace Pharmacognosy” , 15th Edition ,Page No. -214- 235.
3- RNS Yadav* and Munin Agarwala, “Phytochemical Screening and identification of
some compounds” Journal of Phytology 2011, 3(12): 10-14.
4- S.F. Zohra, B.Meriem, S.Samira, “Phytochemical Screening and identification of
some compounds” J. Nat. Prod. Plant Resour., 2012, 2 (4):512-516