BAHAN BAKU OBAT

STRATEGI
OBAT OT
• ISOLASI • BIOASSAY GUIDED
• SINTESIS FRACTIONATION
• SEMI SINTESIS
Bioassay guided isolation
MUTU
• KEMURNIAN BAHAN AKTIF • STANDARISASI BAHAN AKTIF
• CEMARAN • CEMARAN

FARMAKOPE FARMAKOPE HERBAL

Single compound Multi compounds

???
STANDARISASI BAHAN AKTIF ???
 Standarisasi Bahan Baku Obat Herbal

Aspek Parameter Spesifik Aspek Parameter Non

Fokus pada senyawa atau Spesifik
golongan senyawa yang Fokus pada aspek kimia,
bertanggung jawab mikrobiologi dan fisis
terhadap efek yang mempengaruhi
farmakologinya keamanan konsumen dan
Analisis kimia melibatkan stabilitas (kadar logam
analis kualitatif dan berat, aflatoksin, kadar
kuantitatif air)
Parameter Spesifik
• Aspek Profil KLT
• Aspek Penetapan Kadar bahan aktif (multi compounds)
• Aspek Penetapan Kadar Total Golongan Metabolit
• Aspek Kelarutan Ekstrak dalam etanol dan air
Parameter Non Spesifik
• Aspek Penetapan Sisa Air
• Aspek Penetapan Sisa Pelarut Organik (Etanol)
• Aspek Penetapan Kadar Abu
• Aspek Cemaran Mikroba
• Penetapan Keberadaan Aspergills flavus
• Penetapan Cemaran Aflatoksin
• Penetapan Residu Pestisida untuk Fosfor dan Klor
Organik
• Cemaran Logam Berat
Aspek Penentapan Kadar Marker

Tujuan: Untuk menunjukkan secara kuantitatif kadar senyawa marker.

Parameter: terbaca senyawa target pada kadar tertentu
Problem:
1. Senyawa target tidak berwarna  derivatisasi
2. Senyawa marker tersedia dalam jumlah terbatas
3. Bentuk peak pada pembacaan densitometer atau HPLC tidak
simetris
4. Linearitas dan reprodusibilitas rendah  seri konsentrasi yang lebar
pada larutan baku
MARKER
• Curcumae xanthorrizae Rhizoma : Xantorizol
• Andrographidis paniculatae Herba : adrografolida
• Boesenbergiae Rhizoma: pinostrobin
 Powder pericarp of G. mangostana (5.5 Kg)

Extraction by Ethanol 96%

Cytotoxicity assay on T47D breast
cancer cell lines by MTT assay method

Ethanol extract (1.387 Kg; IC50=8.964 ppm)

Residue
a b c d

Identification of ethanol extract on TLC Densitometr

a b
.

TLC profile extract G. mangostana, stationary phase: silica gel

F254; mobile phase: hexan:chloroform:methanol=5:5:1; detection
spray: H2SO4 10% (a:UV254;b:UV366; c:Spray H2SO4 10%,UV366;
d:VIS a.) G. mangostana Extract Chromatogram at 254nm; b.) Spectrum UV
Cytotoxicity assay of extract ethanol pericarp G. mangostana

The cytotoxicity effect of extract ethanol

pericarp G. mangostana
120

100

% Cell viability T47D

80

60

40

20

0
-50 0 50 100 150 200 250 300 350 400 450 500
Concentrations (µg/mL)

Average % cell viability T47D

IC50
No Extract at test concentration (µg/mL)
(µg/mL)
500 100 20 4 0.8
Ethanol Extract G.
1 7.36 8.51 10.53 82.83 107.36 8.964
mangostana
 Ethanol extract
•Separation by chromatography vacuum column, stationary phase: silica
gel G 60; mobile phase: gradient n-hexan:ethyl acetate (100% n-hexan
until 100% ethyl acetate ); Chloroform:MeOH (1:1); MeOH (100%); EtOH
(100%); fractions with similar TLC profiles was merged
•Cytotoxicity assay on T47D breast cancer cell lines by MTT assay method

12 Fraction

F1 F3 F5 F7 F9 F11
IC50=6.023 ppm IC50=3.428 ppm IC50=7.442 ppm IC50=21.191 ppm IC50=542.550 ppm IC50=1005.625 ppm
(357.2mg) ( 1093.4mg ( 298.5mg) ( 181.6mg) (393.8 mg) (10 mg)

F2 F4 F6 F8 F10 F12
IC50=1.799 ppm IC50=6.094 ppm IC50=10.441ppm IC50= 411.517 ppm IC50=5746.048 ppm IC50=360.807 ppm
(2639.7mg) (477.2mg) (214.1mg) (1005.5mg) (25.9mg) (43 mg)

TLC profile F1-12, stationary phase: Silica gel F254;

mobile phase: Hexan:Choloform:Methanol=5:5:1;
Detection spray: H2SO4 10% (a:UV254;b:UV366;
c:Spray H2SO4 10%,UV366; d:VIS; E: Extract;
S:standard: α-mangostin; 1-12: Fraction 1-12
Cytotoxicity assay of Fraction 1-12

The cytotoxicity effect of Fraction 1-12

150
F1

% Cell viability T47D

F2
100 F3
F4
50 F5
F6
0 F7
0 50 100 150 F8
F9
Concentration (µg/mL) F10

Average % cell viability T47D

IC50
No Fraction at test concentration (µg/mL)
(µg/mL)
100 16 2.56 0.41 0.07
1 Fraction 1 1.86 31.69 77.35 93.37 95.48 6.023
2 Fraction 2 0.6 5.29 66.78 77.64 85.92 1.799
3 Fraction 3 2.10 6.10 82.44 85.91 94.85 3.428
4 Fraction 4 2.09 3.44 88.79 108.37 101.05 6.094
5 Fraction 5 2.12 36.00 87.98 93.10 93.85 7.442
6 Fraction 6 2.19 44.88 90.85 95.97 96.11 10.441
7 Fraction 7 0.41 82.58 92.42 93.37 96.45 21.191
8 Fraction 8 61.41 93.92 94.40 96.99 99.93 411.517
9 Fraction 9 64.21 94.19 95.70 96.65 108.13 542.550
10 Fraction 10 67.31 70.42 84.74 85.27 88.26 5746.048
11 Fraction 11 57.51 71.24 83.27 85.27 85.80 1005.625
12 Fraction 12 51.00 80.11 81.28 85.80 92.31 360.807
 F2 = 2 g

•Separation by chromatography open column, stationary phase: silica gel 60; mobile phase:
gradient solvent [n-Hexan:Ethyl acetate (6:4; 2:8); Ethyl acetate: Chloroform 9:1;
Chloroform :Methanol 1:1; methanol]. fractions with similar TLC profiles was merged
•Cytotoxicity assay on T47D breast cancer cell lines by MTT assay method

7 Subfraction

F2.1 F2.2 F2.3 F2.4 F2.5 F2.6 F2.7

IC50= 7.591 ppm IC50=1.755ppm IC50=3.579ppm IC50=4.461ppm IC550=8.067ppm IC50=23.636ppm IC50=31.753ppm
(237.6mg) (736.5 mg) (278.6mg) (567.3mg) (45.3mg) ( 9.4mg) ( 148.1mg)

TLC profile SF2.1-2.7, Stationary phase: Silica gel F254; mobile phase:
Hexan:Chloroform:Methanol=5:5:1; Detection spray: H2SO4 10%
(a:UV254;b:UV366; c:Spray H2SO4 10%,UV366; d:VIS; F2: Fraction 2; S:
standard: α-mangostin; 1-7 : subfraction 1-2
 Cytotoxicity assay of Subfraction 2.1-2.7

The cytotoxicity effect of Subfraction 2.1-2.7

120

100
SF2.1
80

% Cell viability T47D

SF2.2
60 SF2.3
SF2.4
40
SF2.5
20
SF2.6
0 SF2.7
0 20 40 60 80 100 120
-20
Concentration (µg/mL)

Average % cell viability T47D

No Subfraction at test concentration (µg/mL) IC50 (µg/mL)
100 16 2.56 0.41 0.07
1 Subfraction 2.1 10.95 22.39 78.98 95.87 110.70 7.591
2 Subfraction 2.2 0.65 9.92 68.90 70.42 84.74 1.755
3 Subfraction 2.3 0.71 1.69 73.41 94.55 103.44 3.579
4 Subfraction 2.4 0.45 7.20 88.20 94.49 96.24 4.461
5 Subfraction 2.5 1.04 28.02 91.63 93.90 101.75 8.067
6 Subfraction 2.6 5.12 78.15 89.95 99.55 106.55 23.636
7 Subfraction 2.7 18.09 72.89 94.88 99.87 101.69 31.753
 F2.2 = 1 g

•Separation by chromatography vacuum column, stationary phase: silica gel G

60; mobile phase: gradient solvent n-Hexan:Dichlormethane (100% n-Hexan -
100% Dichlormethane, gradient decrease in 5%; gradient solvent
Dichlormethane:Ethyl acetate (100% Dichlormethane – 100 % ethyl acetate,
gradient decrease in 5%) ; methanol, fractions with similar TLC profiles was
merged.
•Cytotoxicity assay on T47D breast cancer cell lines by MTT assay method

8 Subfraction

F2.2.1 F2.2.2 F2.2.3 F2.2.4 F2.2.5 F2.2.6 F2.2.7 F2.2.8

IC50=2.189 ppm IC50=3.138ppm IC50=5.787ppm IC50=1.117ppm IC50=2.543ppm IC50=4.719ppm IC50=3.127ppm IC50=10.858ppm
(10.7 mg) (29.5 mg) (8.0mg) (283.0 mg) (454.4 mg) (30.0 mg) (21.0 mg) (4.6 mg)

TLC profile SF2.2.1-2.2.8, Stationary phase: Silica gel

F254; mobile phase:
Hexan:Chlooroform:Methanol=5:5:1; Detection spray:
H2SO4 10% (a:UV254;b:UV366; c:Spray H2SO4
10%,UV366; d:VIS; 1-8 : Subfraction 1-8; F22:
Subfraction 2.2
 Cytotoxicity assay of Subfraction 2.2.1-2.2.8
The cytotoxicity effect of Subfraction 2.2.1-2.2.8
120

100 SF 2.2.1

% Cell Viability T47D

SF 2.2.2
80
SF 2.2.3
60
SF 2.2.4
40
SF 2.2.5
20 SF 2.2.6
0 SF 2.2.7
0 10 20 30 40 50 SF 2.2.8
-20
Concentration (µg/mL)

Average % cell viability T47D

IC50
No Subfraction at test concentration (µg/mL)
(µg/mL)
40 20 10 5 0.5 0.25 0.02
1 Subfraction 2.2.1 0.88 3.00 16.93 61.20 84.30 108.73 85.89 5.328
2 Subfraction 2.2.2 0.97 0.53 5.38 50.26 58.11 97.35 102.56 3.138
3 Subfraction2.2.3 0.62 1.23 24.51 60.49 94.80 97.97 100.79 5.787
4 Subfraction2.2.4 1.41 1.15 1.59 3.70 70.63 99.74 100.88 1.117
5 Subfraction 2.2.5 1.04 0.87 1.57 3.92 85.01 99.83 102.09 2.543
6 Subfraction 2.2.6 0.35 1.04 3.05 52.31 82.68 103.31 110.44 4.719
7 Subfraction 2.2.7 0.61 1.57 1.83 5.48 79.37 110.10 110.97 3.127
8 Subfraction 2.2.8 1.39 2.61 65.27 98.09 99.48 113.93 106.01 10.858
 F2.2.4=100 mg

•Separation by chromatography open column, stationary phase: silica gel 60; mobile
phase: gradient solvent n-Hexan:Dichlormethane 1:3; Dichlormethane:Methanol 1:1;
Methanol 100%, fractions with similar TLC profiles was merged.
•Cytotoxicity assay on T47D breast cancer cell lines by MTT assay method

7 Subfraction

F2.2.4.1 F2.2.4.2 F2.2.4.3 F2.2.4.4 F2.2.4.5 F2.2.4.6 F2.2.4.7

IC50=694.947 ppm IC50=198.970 ppm IC50=206.185 ppm IC50=6.932 ppm IC50=7.973 ppm IC50=98.874 ppm IC50=324.618ppm
(15.4 mg) (3.5 mg) (13.2 mg) (9.4 mg) (24 mg) (16.2 mg) (11.3 mg)

TLC profile SF2.2.4.1-2.2.4.7, stationary phase:

Silica gel F254; mobile phase:
Hexan:DCM:Methanol=5:15:1; Detection spray:
H2SO4 10% (a:UV254;b:UV366; c:Spray H2SO4
10%,UV366; d:Vis; F: Fraction 224; S:
Standard; 1: 1-7: SF2.2.4.1- SF2.2.4.7
 Cytotoxicity assay of Subfraction 2.2.4.1-2.2.4.7

The cytotoxicity effect of Subfraction 2.2.4.1-2.2.4.7

120

100

% Cell Viabily T47D

SF 2.2.4.1
80 SF 2.2.4.2
60 SF 2.2.4.3

40 SF 2.2.4.4
SF 2.2.4.5
20
SF 2.2.4.6
0
SF 2.2.4.7
0 10 20 30 40 50 60 70
Concentration (µg/mL)

Average % cell viability T47D

at test concentration (µg/mL)
No Subfraction IC50 (µg/mL)
60 45 30 15 7.5 3.75
20(*) 10(*) 5(*) 2.5(*) 0.25(*)
1 Subfraction 2.2.4.1 91.28 91.10 95.83 100.78 104.21 103.69 694.947
2 Subfraction 2.2.4.2 86.72 88.59 94.10 101.82 105.99 103.72 324.618
3 Subfraction 2.2.4.3 70.70 83.85 89.54 95.53 97.05 98.52 190.296
4 Subfraction 2.2.4.4 1.02 10.99 82.50 103.93 103.28 - 6.932
5 Subfraction 2.2.4.5 1.72 25.42 89.18 99.52 101.08 - 7.973
6 Subfraction 2.2.4.6 50.78 73.44 78.08 79.82 88.11 96.05 98.874
7 Subfraction 2.2.4.7 92.36 97.22 98.70 103.47 100.69 104.56 198.970
 SF2.2.4.4 (9.4 mg) disolved in 1 ml acetonitrile SF2.2.4.5 (24 mg) disolved in 1 ml acetonitrile

SF2.2.4.4 Isolation by HPLC Shimadzu LC-05, Diode Array

Detector SPD-M20A, shimpack column 4.6x 250mm, flowrate
0.6 ml/min, mobile phase: A= ACN-; B=Water, gradient
program : 40% A(10 minute), 40-60% A (5 minute), 60-70% A
(15 minute), 70-80% A (15 minute), 80% A(30 minute).
Isolat
SF 2.2.4.5 Isolation by HPLC Shimadzu LC-05, Diode Array
Detector SPD-M20A, shimpack column 4.6x 250mm, flowrate 0.8
ml/min, mobile phase: ACN:methanol:water,=65:28:7

Purification by HPLC Shimadzu LC-05, Diode Array Detector SPD-

M20A, shimpack column 4.6x 250mm, flowrate 0.8ml/min,mobile
phase: ACN:methanol:water,=65:28:7
Identification by:
• TLC Densitometer Isolat GM-1
• HPLC IC50=4,291ppm
• FTIR Spectrometer (1.5 mg)
• NMR Spectrometer

Chromatogram profile isolation of SF2.2.4.4 (A) and SF2.2.4.5 (B)

A. B.
TUGAS
• Merekap Nama senyawa marker sesuai FHI (Nama ilmiah, nama
daerah, nama senyawa marker, golongan senyawa, prosedur analisis
kualitatif dan kuatitatif senyawa marker)  KELAS
• Membuat prosedur analsis standarisasi berdasarkan Keputusan
Menteri Kesehatan RI No: 55/MENKES/SK/I/2000 tentang Parameter
Standar Umum Ekstrak Tumbuhan Obat dari salah satu simplisia yang
terdapat pada FHI.  INDIVIDU