Problem Herbal medicine with following combination: R/ Andrographidis herbs 1 g Curcuma domesticae 1 g Curcuma xanthorrhiza 1 g Sappan lignum 1g Ginseng 1g 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 • Cinchona calisaya Atropine • Atropa belladona • Atropa accuminata • Hyoscyamus niger • Hyoscyamus muticus

all contain quinine

all contain hyosiamine

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 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, verodoxin inactive, its combination reduce side effect. Neem extract 300 mg

Rauwolvia radix containg ¼ mg 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: Combinaion without X

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

B

C

D

E

A: Baeckea leaves B: Jamu+Baeckea (40%) C: Jamu+Baeckea (5%) D: Jamu+Baeckea (15%) E: Jamu+Baeckea (15%) F: Jamu-Baeckea G: 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) & 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, pereaksichemical 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

Sovent Percolate volume No (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 μm – Higher resolution – Short elution distant. – Migration time shorter.

Comparison of TLC and HPTLC Parameter Plate dimension

TLC 20 x 20 cm

HPTLC 10 x 10 cm

Adsorbent Initial spot Sample volume Spot diameter Migration distance Elution time

Silica gel 3 cm 1-5 μL 3-6 mm 10 -15 cm 30 – 200 menit > 10 μm

Silica gel 1,2 cm 0,1-0,2 μL 1 – 1,5 mm 3 – 6 cm 3 – 20 menit

Particle size

5 – 9 μm

Narrower particle distribution • Small particle Æ 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 = kt zf = migration distance, k= velocity constant.

Optimum speed and resolution Slow speed Poor resolution

HPTLC : • Small particles were removed to increase k • Large particles were removed to reduce H

% 60 HPTLC 30

Conventional TLC

0 4

12

20

30

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 12% 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: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. 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