Problem

Herbal medicine with following combination:R/ Andrographidis herbs 1 g

Curcuma domesticae 1 gCurcuma xanthorrhiza 1 gSappan lignum 1 gGinseng 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 componentCaffein• 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 vinblastineinjection.

This approach is not suitable for herbal medicine.

4. Reverse Approach

The selected method must be suitable for most Jamu 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, verodoxininactive, its combination reduce side effect.Neem extract 300 mg

Rauwolvia radix containg ¼mg reserpine gives the same effect. Verodoxin present in Digitalis leaf

People use 7 pieces of neem leaves

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: Combinaionwithout X

A: Sapan 6%B: Jamu + sapan (20%)C: Jamu + sapan (10%)D: Jamu – SapanE: Jamu - Sapan

A B C D E

A: Baeckea leavesB: Jamu+Baeckea (40%)C: Jamu+Baeckea (5%)D: Jamu+Baeckea (15%)E: Jamu+Baeckea (15%)F: Jamu-BaeckeaG: Jamu-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) & reference3. 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 duringmaceration and percolation of A paniculata herbs

Percolate No

Soventvolume (mL)

Accumulated solvent (mL)

Bitterness of extract (unit)

Cumulative bitterness

value (unit)

Bitter compound

extracted (%)1 5 5 3125 3125 44.412 3 8 2000 5125 72.843 3 11 1000 6125 87.054 3 14 500 6625 94.165 3 17 200 6825 97.00

6* 3 20 100 6925 98.427* 3 23 50 7025 99.848* 3 26 10 7035 99.999* 3 29 1 7036 100.00

*Data obtained fromextrapolation

Step II: Quantification• Spectrophotodensitometry• Spectrophotometry

HPTLC (High Performance Thin Layer Chromatography)

Initial publication 1968-1973First employed 1973Adsorbent:

– Fraction with diameter 5 μm– Higher resolution– Short elution distant.– Migration time shorter.

Comparison of TLC and HPTLC

Parameter TLC HPTLCPlate dimension 20 x 20 cm 10 x 10 cm

Adsorbent Silica gel Silica gelInitial spot 3 cm 1,2 cmSample volume 1-5 μL 0,1-0,2 μL

Migration distance 10 -15 cm 3 – 6 cmElution time 30 – 200

menit3 – 20 menit

Spot diameter 3-6 mm 1 – 1,5 mm

Particle size > 10 μm 5 – 9 μm

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.

Zf2 = ktzf = migration distance, k= velocity constant.

HPTLC :• Small particles

were removed to increase k

• Large particles were removed to reduce H

%

60

30

04 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 μm• Diameter 1-2 μm• Elution distance 2 – 3 cm• Without binder or with 1-

2% gypsum• Silica amount 0,8-1,2

mg/cm2 (1 ½ 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:asspray

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. 105oC, 5 min, c. 105oC 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