Problem

Herbal medicine with following combination:

R/ Andrographidis herbs 1 g
Curcuma domesticae 1 g
Curcuma xanthorrhiza 1 g
Sappan lignum 1 g
Ginseng 1 g
How will you assure that the jamu mixture
containing crude drugs as described in the
above composition.
Qualitative and Quantitative
Analysis of Herbal Medicine
Analytical Pharmacognosy
1. Introduction
Most monographs describe identification
or specification of crude drugs.
Indonesian Herbal Medicines contain
mixture of crude drugs, some of them
reach 60 components. .
2. Active component
Caffein
Coffea arabica
Coffea robusta all contain caffein
Camelia sinensis
Quinine
Cinchona ledgeriana
Cinchona succirubra all contain quinine
Cinchona calisaya
Atropine
Atropa belladona
Atropa accuminata all contain hyosiamine
Hyoscyamus niger
Hyoscyamus muticus
Active principle is not always applicable to trace the crude drug.
3. Classical Approach
Classical approach suitable for conventional
medicine. Such as: quinine tablet,
reserpine tablet, vincristine and vinblastine
injection.
This approach is not suitable for herbal
medicine.
4. Reverse Approach
The selected method must be suitable for
most J amu producer:
Simple,
Not too expensive,
Rapid
Accurate.
Conventional Medicine using high dose with
higher side effects
Modern Traditional
Quinine causing hearing
disorder.
Reserpine 1 mg/tablet
Digitoxin active, verodoxin
inactive, its combination
reduce side effect.
Neemextract 300 mg
Rauwolvia radix containg
mg reserpine gives the same
effect.
Verodoxin present in
Digitalis leaf
People use 7 pieces of
neemleaves
Determination of Marker
Marker:
Compound that can be used to identify a
crude drug.
C. domestica : 4-OH,3-OCH3-dicinamoyl
methane
C. xanthorhiza : xanthorizol
TLC using the best system.
A: 6 spots of X, B: Combination containing X; C: Combinaion
without X
A: Sapan 6%
B: J amu + sapan (20%)
C: J amu + sapan (10%)
D: J amu Sapan
E: J amu - Sapan
A B C D E
A: Baeckea leaves
B: J amu+Baeckea (40%)
C: J amu+Baeckea (5%)
D: J amu+Baeckea (15%)
E: J amu+Baeckea (15%)
F: J amu-Baeckea
G: J amu-Baeckea
A B C D E F G
TLC
A. Spectrophotodensitometry
1. At least 3 concentrations.
2. Sample (jamu combination) & reference jamu is
spotted quantitatively
3. Spots must not destroyed by spraying agent.
4. Spot of marker from sample and reference mixture is
measured at the same wave length.
5. Results: - Area under curve
- Absorbed energy (absorbance)
6. Quantity is measured using calibration curve
UV Spectrophotometry
1. Reference jamu at least 3 level.
2. Sample (jamu combination) & reference
3. Applied on TLC plates quantitatively.
4. Sample solution is better applied as band
rather than spot.
5. Scraped the developed band and dissolved
in solvent and filtered.
6. Measure the absorbance at the same wave
length.
7. Calculate using calibration curve.
Step I:
Find the best TLC system.
Identify marker.
Spray reagent could be the same or different,
such as: Vis, UV, UV254, UV365, pereaksi-
chemical reagent)
Extraction process for quantitative
analysis
Case in analysis of andrographolide in A
paniculata:
One gram of powder in 5 mL solvent
(dichlormethane-hexane= 1:1) for 24 hours,
Percolate step wise for 5 times with 3 mL solvent
Percolate obtained is determined its bitterness.
Amount of bitter compound extracted during
maceration and percolation of A paniculata herbs
Percolate
No
Sovent
volume
(mL)
Accumulated
solvent (mL)
Bitterness of
extract
(unit)
Cumulative
bitterness
value (unit)
Bitter
compound
extracted (%)
1 5 5 3125 3125 44.41
2 3 8 2000 5125 72.84
3 3 11 1000 6125 87.05
4 3 14 500 6625 94.16
5 3 17 200 6825 97.00
6* 3 20 100 6925 98.42
7* 3 23 50 7025 99.84
8* 3 26 10 7035 99.99
9* 3 29 1 7036 100.00
*Data obtained fromextrapolation
Step II: Quantification
Spectrophotodensitometry
Spectrophotometry
HPTLC (High Performance Thin
Layer Chromatography)
Initial publication 1968-1973
First employed 1973
Adsorbent:
Fraction with diameter 5 m
Higher resolution
Short elution distant.
Migration time shorter.
Comparison of TLC and HPTLC
Parameter TLC HPTLC
Plate dimension 20 x 20 cm 10 x 10 cm
Adsorbent Silica gel Silica gel
Initial spot 3 cm 1,2 cm
Sample volume 1-5 L 0,1-0,2 L
Migration distance 10 -15 cm 3 6 cm
Elution time 30 200
menit
3 20 menit
Spot diameter 3-6 mm 1 1,5 mm
Particle size > 10 m 5 9 m
Narrower particle distribution
Small particle contribution of molecule
diffusion on zone broadening is high.
Elution higher than 5 cm is not recommended.
Distance > 5 cm the efficiency of HPTLC
become smaller.
Distribution of particle size affect solvent migration.
Ideally k high without increasing H.
Zf
2
= kt
zf = migration distance, k= velocity constant.
HPTLC :
Small particles
were removed to
increase k
Large particles
were removed to
reduce H
%
60
30
0
4 12 20 30
Slow speed
Optimum speed
and resolution
Poor
resolution
HPTLC
Conventional TLC
Smaller particle with narrow range can give excellent
resolution
Adsorbent as Thin Film
Thickness 10-15 m
Diameter 1-2 m
Elution distance 2 3 cm
Without binder or with 1-
2% gypsum
Silica amount 0,8-1,2
mg/cm2 (1 x < TLC)
Support: object glass for
microscopy
Ex 12 steroid was well
separated in 2D-TLC of
1,5x1,5 cm
TLC of steroid on Silica 1,5x1,5 cm,
J .Liq.Chromatogr. 5:1573 (1982)
Variation of performace among
product
a. Initial spot
b. Chromatogram of pigment
c. Separation pattern: red:
application as spot, green:as
spray
d. Application as a strip by spotting
and spray
Higher the position, the velocity become
slower
Derivatization
Exstract Hypericum, a. without, a2. reagent, a3. reagent + PEG
a. Room temperature 3 min, b. 105
o
C, 5 min, c. 105
o
C 30 min, b2 reagent,
b3 reagent 30 min, b.4 and b5 reagent/PEG after 30 min.
Pustaka
Sutrisno, R.B. (1993), Reverse Approach,
Fakultas Farmasi Universitas Pancasila