PHYTOCHEMISTRY PRACTICUM

TUTORIAL
Khairunnisa, S.Farm.,M.S.Farm

School of Pharmacy
 PHYTOCHEMISTRY PRACTICUM :
TUESDAY 08.00-10.30 5B
10.40-12.10 5A

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 EVALUATION

1 POST-TEST
2 WORK REPORT
3 UTS
4 UAS

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 SCHEDULE AND TOPIC
FKB 331 PHYTOCHEMISTRY PRACTICUM
SEMESTER I 2022/2023
NO DATE TOPIC NOTE
1 Sept 20 & 27 2022 Phytochemical Screening offline
2 Oct 4, 11 & 18 2022 Extraction offline
3 Oct 25 & 1 Nov 2022 Extract monitoring offline
4 Nov 8 2022 UTS offline
5 Nov 15 – 22 2022 Fractionation offline
6 Nov 29 & 6 Des 2022 Purity Test offline
7 Des 13 & 20 2022 Isolation of essential oil offline
8 Jan 3 2023 Identification of compound offline
Jan 10 2023 Practicum Final exam offline

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 Phytochemical screening Crude drug
Method of extraction

Extract
Extract monitoring
1st Fractionation Method

Fraction
Fraction monitioring

Selected fraction

2nd Fractionation Method

Subfractionation
Subfraction monitoring

Selected subfraction
Purification

Isolate
Characterization
and Identification

School of Pharmacy Isolate structure

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 SECONDARY METABOLITES

Quinone

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 OBJECTIVES OF
PHYTOCHEMICAL
SCREENING

Guidance of isolation
Guidance of activities

Characterization
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 SAMPLE OF PHYTOCHEMICAL
SCREENING

Crude drug

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 GENERAL SCHEME OF
PHYTOCHEMICAL SCREENING
SAMPLE

ether/n-hexane + ammonia
+ chloroform
STEROID/ ALKALOID
water
TRITERPENOID
FLAVONOID
TANNIN
QUINONE
SAPONIN
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 Sample (1 g)
+ water 20 mL, boiled 15’,
filtered
Filtrate (5 mL)
+ 100 mg Mg, + HCl conc 1 mL
+ 5 mL amyl alcohol, shake
Red, yellow, orange in amyl alcohol
Flavonoid +

Principle: Cyanidin Willstatter reaction

detection of  - benzopyrone
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 Flavonoid

 - benzopyrone
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 H+
Mg + HCl MgCl2 + H2
H-
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 Cyanidin structure
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 Flavonoid

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 Flavonoid

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 Sample (1 g)
+ water 20 mL, boiled 15’,
filtered
Filtrate (15 mL)
+ gelatine 10% + Stiasny
+ FeCl3 5%
heated
green, blue, black white precip. pink precipitation
Tannin + Tannin + Catechic Tannin +
filtered

Filtrate
+ Na.acetate 1M precipitate
+ FeCl3 5%
Blue ink
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Hydrolizable Tannin +
 Gallic Tannin
O OH

C OH
HO O O

OH
HO C
CH2
O OH
O O

OH O O C OH
O
C O
OH
C O

OH
OH
OH
OH
OH
OH
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 Glucose

Gallic acid

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 Condensed Tannin
OH

HO O
7 OH

3
5 OH

OH OH
HO O
7 OH

3 n
5 OH

OH OH
HO O
7 OH

3
5 OH

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 Flavan-3 ol

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 Phlobaphene
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 Sample (1 g)
+ water 20 mL, boiled 15’,
filtered
Filtrate (5 mL)
+ few drops NaOH 1 N
red
Quinone positive
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 Quinone

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 Quinoid structure

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 Sample (1 g)
+ water 20 mL, boiled 15’,
filtered
Filtrat (5 mL)
in tube reaction,
shake vertically 10”
Stable foam 10 ‘
Saponin +
+ 1 drop HCl 2 N
Stable foam
Saponin +
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 Sample (1 g)
+ 20 mL ether, filtered
Filtrate (5 mL)
vaporated
Residue
+ Liebermann- Burchard
green- blue steroid (+)
red- purple triterpenoid (+)

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 Triterpenoid

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 Triterpenoid

H+

dehydroxilation
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 Steroid

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 Steroid

H+

dehydroxilation

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 Triterpenoid

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 Triterpenoid
aglycone

Triterpenoid Glycoside
(saponin)
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 Steroid aglycone

Steroid glycoside
(Saponin)
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 Steroid aglycone

Steroid glycoside
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 Sample (2 g)
+ 10 mL HCl 2N, filtered
Filtrate
+ 5 mL ammonia 25%, + 20 mL CHCl3
in filter paper
water phase CHCl3 phase residue
LLE + 10 mL HCl 2N + Dragendorff
reagent
CHCl3 phase water phase orange
Alkaloid +
+ Dragendorff + Mayer
reagent reagent
orange white precipitate
Alkaloid + Alkaloid + School Of Pharmacy
 Alkaloid

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 Alkaloid

.HCl

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 False positive reaction:
• coumarin
• protein

False negative reaction:

alkaloid quarterner

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 Coumarin

Amino acid

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 Alkaloid
quarterner

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 Filter paper Sample (0.5 g)

put into NaOH 1N + 5 mL CHCl3

allow to dry Extract
NaOH filter paper drop the extract to
NaOH filter paper
allow to dry
see under UV  366 nm
yellow to green fluorescence
Coumarin +
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 References
Burke RW, Diamondstone B I, Velapoldi RA, Menis 0,
(1974), Mechanisms of the Liebermann-Burchard and
Zak color reactions for cholesterol, Clin Chem 20(7):
794-801.

Departemen Kesehatan RI, 1995, Materia medika

Indonesia, Jilid VI, Depkes RI, Jakarta.

Farnsworth NR, 1966, Biological and phytochemical

screening of plants, J Pharm Sci, 55(3): 243-268.

Saito N, Reily M, Yazaki Y, 2005, Chemical structures

of (+)-catechin-formaldehyde reaction products
(Stiasny precipitates) under strong acid conditions,
Holzforschung, 55(2): 205-213.
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