92 lampiran 1. hasil identifikasi poguntano (picria fel-terrae lour.)

92

Lampiran 1. Hasil identifikasi Poguntano (Picria fel-terrae Lour.)

Universitas Sumatera Utara

93

Lampiran 2. Gambar daun poguntano (Picria Fel-terrae Lour.)

a

b

Keterangan: a. Gambar daun poguntano poguntano b. Gambar simplisia daun poguntano


94

Lampiran 3. Hasil Pemeriksaan Mikroskopik Serbuk Simplisia Daun Poguntano

Keterangan :

Perbesaran 10 x 40 a. Trikoma b. Sel epidermis c. Kristal kalsium oksalat bentuk prisma d. Stomata tipe diasitik e. Stomata tipe anomositik f. Berkas pembuluh angkut bentuk spiral g. Sel tetangga

a b

d

g

e

c

f


95

Lampiran 4. Bagan skrining fitokimia, karakterisasi simplisia dan ekstrak

Dicuci Ditiriskan Dikeringkan (diangin-anginkan)

Dihaluskan

Daun poguntano

S i m p l i s i a

Serbuk Simplisia

Skrining Fitokimia

Pembuatan Ekstrak

Karakterisasi Simplisia

- Alkaloid - Flavonoid - Saponin - Tanin - Glikosida - Antrakuinon - Steroid/Triterpenoid

- Pemeriksaan mikroskopik - Pemeriksaan makroskopik - Penetapan kadar air - Penetapan kadar sari yang larut dalam etanol - Penetapan kadar sari yang larut dalam air - Penetapan kadar abu total - Penetapan kadar abu yang tidak larut dalam asam

Ekstrak


96

Lampiran 5. Bagan ekstraksi serbuk simplisia secara maserasi bertingkat

di maserasi dengan n-heksana

di maserasi dengan etilasetat

dipekatkan

di maserasi dengan etanol dipekatkan di

skrining

dipekatkan

di skrining

di skrining

Serbuk simplisia

Ampas Maserat

Ampas Maserat

Ekstrak kering n-heksana*

Ampas Maserat

Ekstrak kering etilasetat*

Hasil

Ekstrak kental etanol*

Hasil

Hasil


97

Keterangan : * = Uji Sitotoksik

Lampiran 6. Perhitungan kadar air simplisia daun poguntano (Picria Fel-terrae Lour.)

% Kadar air simplisia = 100%x(g)sampelberat

(ml)airvolume

No. Berat sampel (g) Volume awal (ml) Volume akhir (ml) 1. 5,022 1,90 2,20 2. 5,015 2,20 2,50 3. 5,018 2,50 2,80

% Kadar air = 100%xsampelberat

awalvolumeakhirvolume −

1. Kadar air = 5,97%100%x5,022

1,90-2,20=

2. Kadar air = 5,98%100%x5,015

2,20-2,50=

3. Kadar air = ,97%5100%x5,018

2,50-2,80=

% Rata-rata kadar air = 5,97%3

5,97%5,98%5,97%=

++


98

Lampiran 7. Perhitungan kadar sari larut air simplisia daun poguntano (Picria fel-terrae Lour.)

% Kadar sari larut dalam air = 100%x20

100x(g)sampelberat

(g)sariberat

No. Berat sampel (g) Berat sari (g) 1. 4,008 0,212 2. 4,002 0,208 3. 4,010 0,220

1. Kadar sari larut dalam air = 26,45%100%x20

100x4,0080,205

=


100x4,0020,208

=


100x4,0100,222

=

% Rata-rata kadar sari larut dalam air = %62,263

27,43%25,99%26,45%=

++


99

Lampiran 8. Perhitungan kadar sari larut etanol simplisia daun poguntano (Picria fel-terrae Lour.)

% Kadar sari larut dalam etanol = 100%x20

100x(g)sampelberat

(g)sariberat


1. Kadar sari larut dalam etanol = 19,75%100%x20

100x5,0120,198

=


100x5,0090,182

=


100x5,0170,205

=

% Rata-rata kadar sari larut dalam etanol = %45,193

18,17%20,43%19,75%=

++


100

Lampiran 9. Perhitungan kadar abu total simplisia daun poguntano (Picria

fel-terrae Lour.)

% Kadar abu total = 100%x(g)sampelberat

(g)abuberat

No. Berat sampel (g) Berat abu (g) 1. 2,0121 0,0780 2. 2,0118 0,0791 3. 2,0132 0,0812

1. Kadar abu total = 3,87%100%x2,01210,0780

=

2. Kadar abu total = 3,93%100%x2,01180,0791

=

3. Kadar abu total = 4,03%100%x2,01320,0812

=

% Rata-rata kadar abu total = %94,33

4,03%3,93%3,87%=

++


101

Lampiran 10. Perhitungan kadar abu tidak larut asam simplisia daun

poguntano (Picria fel-terrae Lour.)


(g)abuberat


1. Kadar abu total = 0,76%100%x1,99810,0151

=

2. Kadar abu total = 0,67%100%x1,99350,0135

=

3. Kadar abu total = 0,81%100%x2,00270,0163

=


0,81%0,67%0,76%=

++


102

Lampiran 11. Perhitungan kadar air ekstrak n-heksana daun poguntano (Picria fel-terrae Lour.)


(ml)airvolume




1. Kadar air = 4,98%100%x2,005

1,90-2,00=

2. Kadar air = 4,99%100%x2,001

2,00-2,10=

3. Kadar air = ,00%5100%x2,002

2,10-2,20=


5,00%4,99%4,98%=

++


103

Lampiran 12. Perhitungan kadar sari larut air ekstrak n-heksana daun



100x(g)sampelberat

(g)sariberat



100x2,0110,004

=


100x2,1050,002

=


100x2,0250,005

=


1,23%1,42%0,99%=

++


104

Lampiran 13. Perhitungan kadar sari larut etanol ekstrak n-heksana daun poguntano (Picria fel-terrae Lour.)


100x(g)sampelberat

(g)sariberat



100x2,0110,012

=


100x2,0230,013

=


100x2,0080,009

=


2,24%3,21%2,98%=

++


105

Lampiran 14. Perhitungan kadar abu total ekstrak n-heksana daun



(g)abuberat


4. Kadar abu total = 0,41%100%x2,00110,0083

=

5. Kadar abu total = 0,45%100%x2,00520,0091

=

6. Kadar abu total = 0,44%100%x2,00180,0088

=


0,44%0,45%0,41%=

++


106

Lampiran 15. Perhitungan kadar abu tidak larut asam ekstrak n-heksana

daun poguntano (Picria fel-terrae Lour.)

% Kadar abu tidak larut dalam asam = 100%x(g)sampelberat

(g)abuberat


1. Kadar abu tidak larut dalam asam = %10,0100%x2,00950,0021

=


=


=

% Rata-rata kadar abu tidak larut dalam asam = 3

%05,0%08,0%10,0 ++

= 0,08%


107

Lampiran 16. Perhitungan kadar air ekstrak etilasetat daun poguntano

(Picria fel-terrae Lour.)


(ml)airvolume




1. Kadar air = 4,97%100%x2,010

1,90-2,00=

2. Kadar air = 4,97%100%x2,012

2,00-2,10=

3. Kadar air = ,98%4100%x2,008

2,10-2,20=


4,98%4,97%4,97%=

++


108

Lampiran 17. Perhitungan kadar sari larut air ekstrak etilasetat daun poguntano (Picria fel-terrae Lour.)


100x(g)sampelberat

(g)sariberat



100x2,0150,053

=


100x2,0100,048

=


100x1,9800,050

=


12,62%11,94%13,15%=

++


109

Lampiran 18. Perhitungan kadar sari larut etanol ekstrak etilasetat daun poguntano (Picria fel-terrae Lour.)


100x(g)sampelberat

(g)sariberat



100x2,0180,242

=


100x2,0060,231

=

3. Kadar sari larut dalam etanol = ,60%61100%x20

100x2,0210,249

=


61,60%57,5859,96%=

++


110

Lampiran 19. Perhitungan kadar abu total ekstrak etilasetat daun poguntano (Picria fel-terrae Lour.)


(g)abuberat


1. Kadar abu total = 0,52%100%x2,01020,0104

=

2. Kadar abu total = 0,48%100%x2,00940,0097

=

3. Kadar abu total = 0,43%100%x2,00830,0086

=


0,43%0,48%0,52%=

++


111

Lampiran 20. Perhitungan kadar abu tidak larut asam ekstrak etilasetat daun poguntano (Picria fel-terrae Lour.)

% Kadar abu tidak larut dalam asam = 100%x(g)sampelberat

(g)abuberat



=


=


=

% Rata-rata kadar abu tidak larut dalam asam = 3

%10,0%05,0%08,0 ++

= 0,08%


112

Lampiran 21. Perhitungan kadar air ekstrak etanol daun poguntano (Picria fel-terrae Lour.)


(ml)airvolume




1. Kadar air = 8,00%100%x5,014

1,90-2,30=

2. Kadar air = 7,98%100%x5,010

2,30-2,70=

3. Kadar air = ,98%7100%x5,012

2,70-3,10=


7,98%7,98%8,00%=

++


113

Lampiran 22. Perhitungan kadar sari larut air ekstrak etanol daun poguntano (Picria fel-terrae Lour.)


100x(g)sampelberat

(g)sariberat



100x2,0080,307

=


100x2,0060,281

=


100x2,0370,319

=


78,30%70,03%76,44%=

++


114

Lampiran 23. Perhitungan kadar sari larut etanol ekstrak etanol daun poguntano (Picria fel-terrae Lour.)


100x(g)sampelberat

(g)sariberat



100x2,0720,348

=


100x2,0560,339

=


100x2,0440,328

=


80,2382,44%83,98%=

++


115

Lampiran 24. Perhitungan kadar abu total ekstrak etanol daun poguntano (Picria fel-terrae Lour.)


(g)abuberat


1. Kadar abu total = 1,64%100%x1,97750,0325

=

2. Kadar abu total = 1,86%100%x1,99500,0372

=

3. Kadar abu total = 95%,1100%x2,00580,0391

=


1,95%1,86%1,64%=

++


116

Lampiran 25. Perhitungan kadar abu tidak larut asam ekstrak etanol daun poguntano (Picria fel-terrae Lour.)


(g)abuberat


1. Kadar abu total = 0,34%100%x1,89900,0065

=

2. Kadar abu total = 0,36%100%x1,99230,0071

=

3. Kadar abu total = 0,23%100%x1,82550,0042

=


0,23%0,36%0,34%=

++


117

Lampiran 26. Perhitungan persen sel hidup sel MCF-7

a. Kontrol sel dan kontrol media

Absorbansi kontrol sel

Rata-rata absorbansi kontrol sel

Absorbansi kontrol media

Rata-rata absorbansi

kontrol media

Absorbansi kontrol sel dikurangi kontrol media

1.232 1,061

0,150

0,154 0,907 1.02 0,147

0.931 0,164

b. Ekstrak n-heksana daun poguntano

Kadar (µg/ml) Absorbansi % sel hidup % sel hidup

rata-rata 31.25 1.002 0.911 0.879 93.497 83.468 79.941 85.636

62.5 0.947 0.929 0.705 87.436 85.452 60.764 77.884

125.0 0.682 0.655 0.602 58.229 55.253 49.412 54.298

250.0 0.363 0.342 0.345 23.071 20.757 21.087 21.639

500.0 0.201 0.215 0.241 5.217 6.760 9.625 7.201

c. Ekstrak etilasetat daun poguntano

Kadar (µg/ml)

Absorbansi % sel hidup % sel hidup rata-rata

31.25 0.923 1.01 0.9 84.791 94.379 82.256 87.142

62.5 0.9 0.904 0.759 82.256 82.697 66.716 77.223

125.0 0.557 0.639 0.59 44.453 53.490 48.090 48.677

250.0 0.309 0.341 0.397 17.120 20.647 26.819 21.528

500.0 0.232 0.248 0.243 8.633 10.397 9.846 9.625


118

d. Ekstrak etanol daun poguntano

Kadar (µg/ml) Absorbansi % sel hidup

% sel hidup rata-rata

31.25 1.069 0.958 0.976 100.882 88.648 90.632 93.387

62.5 0.986 1.074 0.866 91.734 101.433 78.508 90.558

125.0 0.929 0.905 0.788 85.452 82.807 69.912 79.390

250.0 0.688 0.724 0.744 58.891 62.858 65.062 62.270

500.0 0.514 0.376 0.359 39.713 24.504 22.630 28.949

e. Doxorubicin





kontrol media


0.865

0.853

0.159

0,159 0,694 0.851 0.161

0.843 0.157


rata-rata

25 0.836 0.749 0.982 97.550 85.014 118.588 100.384

50 0.706 0.809 0.782 78.818 93.660 89.769 87.416

100 0.767 0.789 0.719 87.608 90.778 80.692 86.359

200 0.694 0.728 0.723 77.089 81.988 81.268 80.115

400 0.404 0.425 0.43 35.303 38.329 39.049 37.560


119

Lampiran 27. Perhitungan persen sel hidup sel T47D






kontrol media


1.059

1.061

0.13

0.126 0.934 1.078 0.115

1.045 0.134


Kadar (µg/ml) Absorbansi Absorbansi rata-rata % sel hidup

rata-rata 31.25 0.899 0.974 0.978 0.950 88.191

62.5 0.785 0.888 0.898 0.857 78.202

125.0 0.751 0.804 0.916 0.824 74.634

250.0 0.681 0.714 0.712 0.702 61.648

c. Ekstrak etilasetat daun poguntano


rata-rata 31.25 0.917 0.959 0.932 84.624 89.119 86.229 86.657

62.5 0.762 0.618 0.572 68.034 52.622 47.699 56.118

125.0 0.505 0.486 0.451 40.528 38.494 34.748 37.924

250.0 0.354 0.326 0.369 24.367 21.370 25.972 23.903

500.0 0.294 0.247 0.296 17.945 12.915 18.159 16.340


120

d. Ekstrak etanol daun poguntano

Kadar (µg/ml) Absorbansi % sel hidup

% sel hidup rata-rata

31.25 1.02 1.087 1.091 95.648 102.818 103.247 100.571

62.5 1.021 0.997 1.037 95.755 93.186 97.467 95.469

125.0 0.938 0.978 0.966 86.871 91.152 89.868 89.297

250.0 0.761 0.825 0.795 67.927 74.777 71.566 71.423

500.0 0.316 0.277 0.307 20.300 16.126 19.336 18.587


121

Lampiran 28. Perhitungan persen sel hidup sel Vero






kontrol media


0.479

0.502

0.071

0.065 0.437 0.498 0.063

0.528 0.06


Kadar (µg/ml) Absorbansi Absorbansi rata-rata % sel hidup

rata-rata 31.25 0.487 0.51 0.482 0.493 98.017

62.5 0.486 0.498 0.498 0.494 98.246

125.0 0.475 0.449 0.478 0.467 92.143

250.0 0.108 0.096 0.122 0.109 10.069

500.0 0.077 0.079 0.083 0.080 3.432


122

Lampiran 29. Bagan pembuatan media DMEM

Dimasukkan kedalam erlenmeyer

Ditambahkan 800 ml aquabidest steril

Dihomogenkan dengan menggunakan stirer magnet

Diatur pH 7,2 – 7,4 (HCl 1N atau NaOH 1N)

Ditambahkan aquabidest steril samapai 1 l

Dilakukan sterilisasi dengan penyaringan

Ditampung dalam botol steril

Diberi identitas pada botol media

Disimpan pada suhu 2 – 80 C

DMEM Sachet 2 g Hepes 2 g NaHCO3

Media DMEM

Larutan media


123

Lampiran 30. Bagan pembuatan media kultur lengkap (MK) DMEM

Dicampur

Diberi identitas pada botol MK

Disimpan pada suhu 2 – 80C

Fetal Bovine Serum (FBS)

(10%)

Penisilin- Streptomisin

(2%)

Fungizone (amphotericin

B) (0,5%)

DMEM ad 100%

Media Kultur Lengkap (MK)


124

Lampiran 31. Bagan penumbuhan sel

Diambil diambil dari freezer

Diambil beberapa tetes

Dimasukkan kedalam konikel yg berisi DMEM

Disentrifuge 6000 rpm selama 5 menit

Dibuang supernatan

Ditambahkan 4 ml MK DMEM

Di resuspensi hingga homogen

Dimasukkan ke dalam flask

Ditambahkan 5 ml MK kedalam setiap flask

Dihomogenkan

Diamati kondisi sel dengan mikroskop inverted

Diberi identitas pada flask

Disimpan dalam inkubator CO2

Sel dalam freezer

Sel

Konikel

Flask


125

Lampiran 32. Bagan panen sel

Dipersiapkan dan dikondisikan

Diamati apakah sel telah konfluen 80%

Dibuang MK dari flask dengan mikropipet

Dicuci sel 2x dengan PBS

Ditambahkan 400 µl trypsine-EDTA 0,25%

Diinkubasi dalam inkubator CO2 selama 5 menit

Ditambahkan 4 ml MK

Di resuspensi dengan mikropipet

Diamati sel dibawah mikroskop inverted

Di resuspensi kembali jika masih ada sel yang menggerombol

Ditransfer sel kedalam tabung konikel

Sel, alat, dan bahan

Panen Sel

Sel yang melekat


126

Lampiran 33. Bagan penghitungan sel

Diambil 10µl panenan sel

Dipipetkan kedalam hemositometer

Dihitung jumlah sel dibawah mikroskop

Kultur Sel

Jumlah Sel


127

Lampiran 34. Bagan pembuatan larutan uji

Ditimbang sebanyak 50 mg

Dimasukkan kedalam polytube

Dilarutkan dalam 1.000 µl DMSO

Di vortex

Dibuat pengenceran sampai diperoleh konsentrasi 500 µg/ml, 250 µg/ml, 125 µg/ml, 62,5 µg/ml, dan 31,25 µg/ml

Ekstrak n-heksan

Ekstrak Etilasetat

Ekstrak Etanol

Larutan Uji


128

Lampiran 35. Bagan pengujian sitotoksik

Ditanam pada microplate 96 sumuran dengan kepadatan 1 x 104 Diinkubasi selama 24 jam

Dibuang medium

Ditambahkan medium baru

Ditambahkan larutan uji

Diinkubasi selama 24 jam

Dibuang media dan larutan uji setelah 24 jam

Dicuci dengan PBS

Ditambahkan 100 µl MK dan 10 µl MTT (5 mg/ml)

Diinkubasi selama 4-6 jam

Ditambahkan SDS (sebagai stopper)

Dibungkus dengan aluminium foil

Dibiarkan selama 1 malam

Dibaca serapan dengan ELISA reader pada λ 595 nm

Dihitung % sel hidup

Dihitung IC50 dengan analisa probit menggunakan SPSS 17

Sel

Absorbansi

IC50


129

Lampiran 36. Sel MCF-7 dan T47D di bawah mikroskop

Keterangan: a. Sel MCF-7 sebelum diberi larutan uji (perbesaran 10 x 10) b. Sel MCF-7 setelah diberi larutan uji (sel mengalami perubahan bentuk morfologi) (perbesaran 10 x 10)

Keterangan: a. Sel T47D sebelum diberi larutan uji (perbesaran 10 x 10) b. Sel T47D setelah diberi larutan uji (sel mengalami perubahan bentuk morfologi) (perbesaran 10 x 10)

b a


130

Lampiran 37. Sel Vero di bawah mikroskop

Keterangan: a. Sel Vero sebelum diberi larutan uji (perbesaran 10 x 10) b. Sel Vero setelah diberi larutan uji (sel mengalami perubahan bentuk morfologi) (perbesaran 10 x 10)

b a


131

Lampiran 38. Microplate -96 sumuran dan kristal formazan

Keterangan: microplate-96 sumuran yang berisi sel dan larutan uji

Keterangan: a. Kristal Formazan (perbesaran 10 x 10)

ekstrak n-heksana

Kontrol media

Kontrol sel

500 µg/ml

250 µg/ml

125 µg/ml

62,5 µg/ml

31,25 µg/ml

a

ekstrak etilasetat

ekstrak etanol


132

Lampiran 39. Hasil penentuan IC50 sel MCF7 dengan analisa probit SPSS 17 a. Ekstrak n-heksana daun poguntano

Confidence Limits

Probability

95% Confidence Limits for konsentrasi ekstrak

95% Confidence Limits for log(konsentrasi ekstrak)b

Estimate

Lower Bound

Upper Bound

Estimate

Lower Bound

Upper Bound

PROBITa

.010 9.001 .012 29.397 .954 -1.905 1.468

.020 12.192 .036 35.579 1.086 -1.449 1.551

.030 14.780 .069 40.240 1.170 -1.160 1.605

.040 17.083 .114 44.204 1.233 -.943 1.645

.050 19.219 .171 47.763 1.284 -.767 1.679

.060 21.246 .241 51.062 1.327 -.618 1.708

.070 23.198 .326 54.182 1.365 -.487 1.734

.080 25.098 .426 57.178 1.400 -.371 1.757

.090 26.961 .544 60.085 1.431 -.265 1.779

.100 28.797 .680 62.930 1.459 -.168 1.799

.150 37.830 1.701 76.840 1.578 .231 1.886

.200 46.990 3.481 91.282 1.672 .542 1.960

.250 56.598 6.334 107.446 1.753 .802 2.031

.300 66.888 10.646 126.718 1.825 1.027 2.103

.350 78.087 16.824 151.148 1.893 1.226 2.179

.400 90.443 25.209 184.086 1.956 1.402 2.265

.450 104.255 35.923 231.186 2.018 1.555 2.364

.500 119.906 48.755 302.040 2.079 1.688 2.480

.550 137.907 63.232 412.953 2.140 1.801 2.616

.600 158.968 78.898 592.288 2.201 1.897 2.773

.650 184.121 95.595 892.096 2.265 1.980 2.950

.700 214.948 113.580 1415.331 2.332 2.055 3.151

.750 254.031 133.568 2385.435 2.405 2.126 3.378

.800 305.968 156.889 4350.330 2.486 2.196 3.639

.850 380.056 186.082 8913.542 2.580 2.270 3.950

.900 499.271 226.935 22339.662 2.698 2.356 4.349

.910 533.279 237.630 27942.929 2.727 2.376 4.446

.920 572.855 249.660 35656.796 2.758 2.397 4.552

.930 619.769 263.410 46650.018 2.792 2.421 4.669


133

.940 676.721 279.454 63026.633 2.830 2.446 4.800

.950 748.090 298.696 88904.222 2.874 2.475 4.949

.960 841.613 322.684 133313.514

2.925 2.509 5.125

.970 972.760 354.395 219643.651

2.988 2.549 5.342

.980 1179.264

400.736 427288.056

3.072 2.603 5.631

.990 1597.289

484.898 1223353.197

3.203 2.686 6.088

a. A heterogeneity factor is used. b. Logarithm base = 10. b. Ekstrak etilasetat daun poguntano

Confidence Limits

Probability


95% Confidence Limits for log (konsentrasi ekstrak)a

Estimate

Lower Bound

Upper Bound Estimate

Lower Bound

Upper Bound

PROBIT

.010 9.748 5.989 14.006 .989 .777 1.146

.020 13.082 8.460 18.119 1.117 .927 1.258

.030 15.766 10.528 21.342 1.198 1.022 1.329

.040 18.143 12.409 24.146 1.259 1.094 1.383

.050 20.338 14.180 26.701 1.308 1.152 1.427

.060 22.414 15.884 29.092 1.351 1.201 1.464

.070 24.408 17.542 31.369 1.388 1.244 1.496

.080 26.343 19.172 33.562 1.421 1.283 1.526

.090 28.237 20.782 35.693 1.451 1.318 1.553

.100 30.099 22.382 37.778 1.479 1.350 1.577

.150 39.213 30.384 47.851 1.593 1.483 1.680

.200 48.387 38.662 57.857 1.685 1.587 1.762

.250 57.950 47.442 68.233 1.763 1.676 1.834

.300 68.138 56.888 79.303 1.833 1.755 1.899

.350 79.171 67.147 91.381 1.899 1.827 1.961

.400 91.289 78.373 104.825 1.960 1.894 2.020

.450 104.775 90.745 120.072 2.020 1.958 2.079


134

.500 119.990 104.487 137.684 2.079 2.019 2.139

.550 137.416 119.909 158.410 2.138 2.079 2.200

.600 157.716 137.452 183.279 2.198 2.138 2.263

.650 181.854 157.780 213.777 2.260 2.198 2.330

.700 211.302 181.918 252.181 2.325 2.260 2.402

.750 248.451 211.536 302.240 2.395 2.325 2.480

.800 297.554 249.575 370.723 2.474 2.397 2.569

.850 367.167 301.865 471.561 2.565 2.480 2.674

.900 478.339 382.467 639.987 2.680 2.583 2.806

.910 509.896 404.832 689.207 2.707 2.607 2.838

.920 546.541 430.562 747.066 2.738 2.634 2.873

.930 589.882 460.687 816.411 2.771 2.663 2.912

.940 642.362 496.760 901.622 2.808 2.696 2.955

.950 707.938 541.274 1009.875 2.850 2.733 3.004

.960 793.583 598.581 1153.997 2.900 2.777 3.062

.970 913.201 677.250 1359.989 2.961 2.831 3.134

.980 1100.578

797.787 1692.410 3.042 2.902 3.229

.990 1476.972

1032.146 2390.336 3.169 3.014 3.378

a. Logarithm base = 10.

c. Ekstrak etanol daun poguntano

Confidence Limits

Probability


95% Confidence Limits for log (konsentrasi ekstrak)b

Estimate

Lower Bound


Lower Bound

Upper Bound

PROBITa

.010 15.532 1.856 35.622 1.191 .269 1.552

.020 22.039 3.510 45.793 1.343 .545 1.661

.030 27.518 5.248 53.813 1.440 .720 1.731

.040 32.520 7.092 60.848 1.512 .851 1.784

.050 37.252 9.050 67.322 1.571 .957 1.828

.060 41.819 11.126 73.447 1.621 1.046 1.866

.070 46.281 13.322 79.347 1.665 1.125 1.900

.080 50.679 15.640 85.106 1.705 1.194 1.930


135

.090 55.041 18.081 90.782 1.741 1.257 1.958

.100 59.387 20.647 96.419 1.774 1.315 1.984

.150 81.347 35.365 125.134 1.910 1.549 2.097

.200 104.460 53.187 156.988 2.019 1.726 2.196

.250 129.458 73.834 194.970 2.112 1.868 2.290

.300 156.965 96.766 242.620 2.196 1.986 2.385

.350 187.646 121.385 304.322 2.273 2.084 2.483

.400 222.287 147.321 385.495 2.347 2.168 2.586

.450 261.878 174.606 493.111 2.418 2.242 2.693

.500 307.719 203.639 636.789 2.488 2.309 2.804

.550 361.586 235.122 830.650 2.558 2.371 2.919

.600 425.986 270.039 1096.489 2.629 2.431 3.040

.650 504.627 309.759 1469.585 2.703 2.491 3.167

.700 603.265 356.273 2010.395 2.781 2.552 3.303

.750 731.446 412.705 2830.436 2.864 2.616 3.452

.800 906.480 484.431 4157.286 2.957 2.685 3.619

.850 1164.034 581.981 6529.187 3.066 2.765 3.815

.900 1594.480 730.507 11562.985 3.203 2.864 4.063

.910 1720.382 771.390 13280.488 3.236 2.887 4.123

.920 1868.449 818.267 15439.124 3.271 2.913 4.189

.930 2046.008 872.953 18222.991 3.311 2.941 4.261

.940 2264.332 938.173 21933.847 3.355 2.972 4.341

.950 2541.907 1018.294 27103.093 3.405 3.008 4.433

.960 2911.797 1120.903 34762.692 3.464 3.050 4.541

.970 3441.090 1260.878 47223.194 3.537 3.101 4.674

.980 4296.536 1473.623 70995.912 3.633 3.168 4.851

.990 6096.662 1882.339 135127.393

3.785 3.275 5.131

a. A heterogeneity factor is used. b. Logarithm base = 10.

d. Doxorubicin

Confidence Limits Probab

ility 95% Confidence Limits for

konsentrasi ekstrak 95% Confidence Limits for log(konsentrasi ekstrak)b


136

Estimate

Lower Bound


Lower Bound

Upper Bound

PROBITa .010 8878.180

. . 3.948 . .

.020 6115.250

. . 3.786 . .

.030 4827.115

. . 3.684 . .

.040 4040.271

. . 3.606 . .

.050 3495.839

. . 3.544 . .

.060 3090.641

. . 3.490 . .

.070 2774.195

. . 3.443 . .

.080 2518.450

. . 3.401 . .

.090 2306.389

. . 3.363 . .

.100 2127.006

. . 3.328 . .

.150 1521.182

. . 3.182 . .

.200 1165.394

. . 3.066 . .

.250 927.268 . . 2.967 . .

.300 755.197 . . 2.878 . .

.350 624.387 . . 2.795 . .

.400 521.277 . . 2.717 . .

.450 437.753 . . 2.641 . .

.500 368.632 . . 2.567 . .

.550 310.425 . . 2.492 . .

.600 260.686 . . 2.416 . .

.650 217.637 . . 2.338 . .

.700 179.939 . . 2.255 . .

.750 146.548 . . 2.166 . .

.800 116.604 . . 2.067 . .

.850 89.332 . . 1.951 . .


137

.900 63.888 . . 1.805 . .

.910 58.919 . . 1.770 . .

.920 53.958 . . 1.732 . .

.930 48.983 . . 1.690 . .

.940 43.968 . . 1.643 . .

.950 38.872 . . 1.590 . .

.960 33.634 . . 1.527 . .

.970 28.151 . . 1.449 . .

.980 22.221 . . 1.347 . .

.990 15.306 . . 1.185 . . a. A heterogeneity factor is used. b. Logarithm base = 10.

Lampiran 40. Hasil penentuan IC50 sel T47D dengan analisa probit SPSS 17

a. Ekstrak n-heksan daun poguntano Confidence Limits

Probability


95% Confidence Limits for log(konsentrasi ekstrak)a

Estimate

Lower Bound


Lower Bound

Upper Bound

PROBIT

.010 125061.187

14273.985 2.918E7 5.097 4.155 7.465

.020 64746.770 8999.177 9161910.651

4.811 3.954 6.962

.030 42640.364 6714.397 4394429.539

4.630 3.827 6.643

.040 31143.173 5386.029 2528845.221

4.493 3.731 6.403

.050 24119.303 4501.454 1613455.491

4.382 3.653 6.208

.060 19403.852 3863.684 1100710.895

4.288 3.587 6.042

.070 16034.310 3379.066 787187.406 4.205 3.529 5.896

.080 13516.959 2996.792 583095.471 4.131 3.477 5.766

.090 11572.394 2686.674 443837.897 4.063 3.429 5.647

.100 10030.672 2429.540 345264.200 4.001 3.386 5.538

.150 5549.421 1600.943 122128.624 3.744 3.204 5.087


138

.200 3466.784 1148.188 53518.436 3.540 3.060 4.729

.250 2315.462 862.505 26393.848 3.365 2.936 4.422

.300 1611.472 666.394 14004.117 3.207 2.824 4.146

.350 1151.739 524.056 7793.605 3.061 2.719 3.892

.400 837.412 416.548 4476.293 2.923 2.620 3.651

.450 615.207 332.847 2623.428 2.789 2.522 3.419

.500 454.178 266.059 1555.572 2.657 2.425 3.192

.550 335.298 211.587 927.115 2.525 2.325 2.967

.600 246.328 166.137 552.949 2.392 2.220 2.743

.650 179.101 127.090 329.996 2.253 2.104 2.519

.700 128.006 92.119 199.250 2.107 1.964 2.299

.750 89.087 60.040 125.323 1.950 1.778 2.098

.800 59.501 33.462 83.304 1.775 1.525 1.921

.850 37.171 15.615 56.106 1.570 1.194 1.749

.900 20.565 5.715 35.732 1.313 .757 1.553

.910 17.825 4.468 32.152 1.251 .650 1.507

.920 15.261 3.416 28.694 1.184 .534 1.458

.930 12.865 2.541 25.342 1.109 .405 1.404

.940 10.631 1.824 22.077 1.027 .261 1.344

.950 8.552 1.249 18.880 .932 .097 1.276

.960 6.624 .800 15.724 .821 -.097 1.197

.970 4.838 .462 12.570 .685 -.336 1.099

.980 3.186 .222 9.346 .503 -.653 .971

.990 1.649 .070 5.869 .217 -1.154 .769 a. Logarithm base = 10.

b. Ekstrak etilasetat daun poguntano

Confidence Limits

Probability



Estimate

Lower Bound


Lower Bound

Upper Bound

PROBITa

.010 2480.677 863.709 44772.484 3.395 2.936 4.651

.020 1698.688 662.987 21764.332 3.230 2.822 4.338

.030 1335.891 559.821 13790.129 3.126 2.748 4.140


139

.040 1115.009 492.491 9792.180 3.047 2.692 3.991

.050 962.566 443.429 7417.199 2.983 2.647 3.870

.060 849.349 405.296 5858.919 2.929 2.608 3.768

.070 761.091 374.368 4766.997 2.881 2.573 3.678

.080 689.877 348.514 3965.119 2.839 2.542 3.598

.090 630.914 326.403 3355.135 2.800 2.514 3.526

.100 581.103 307.157 2878.212 2.764 2.487 3.459

.150 413.405 237.424 1534.579 2.616 2.376 3.186

.200 315.389 191.566 940.235 2.499 2.282 2.973

.250 250.045 157.537 624.719 2.398 2.197 2.796

.300 202.988 130.337 438.813 2.307 2.115 2.642

.350 167.326 107.483 321.791 2.224 2.031 2.508

.400 139.298 87.688 244.740 2.144 1.943 2.389

.450 116.658 70.362 192.209 2.067 1.847 2.284

.500 97.972 55.328 155.169 1.991 1.743 2.191

.550 82.280 42.567 128.034 1.915 1.629 2.107

.600 68.907 32.014 107.282 1.838 1.505 2.031

.650 57.365 23.496 90.697 1.759 1.371 1.958

.700 47.287 16.762 76.883 1.675 1.224 1.886

.750 38.388 11.533 64.935 1.584 1.062 1.812

.800 30.434 7.546 54.227 1.483 .878 1.734

.850 23.218 4.570 44.262 1.366 .660 1.646

.900 16.518 2.415 34.522 1.218 .383 1.538

.910 15.214 2.068 32.538 1.182 .316 1.512

.920 13.913 1.748 30.520 1.143 .242 1.485

.930 12.612 1.451 28.454 1.101 .162 1.454

.940 11.301 1.179 26.321 1.053 .072 1.420

.950 9.972 .930 24.092 .999 -.031 1.382

.960 8.609 .704 21.723 .935 -.153 1.337

.970 7.185 .499 19.139 .856 -.302 1.282

.980 5.651 .316 16.186 .752 -.501 1.209

.990 3.869 .153 12.447 .588 -.815 1.095 a. A heterogeneity factor is used. b. Logarithm base = 10. c. Ekstrak etanol daun poguntano


140

Confidence Limits

Probability



Estimate

Lower Bound


Lower Bound

Upper Bound

PROBITa

.010 1741.777 790.001 27458.872 3.241 2.898 4.439

.020 1420.898 689.497 17132.511 3.153 2.839 4.234

.030 1248.698 631.963 12711.285 3.096 2.801 4.104

.040 1133.055 591.567 10160.007 3.054 2.772 4.007

.050 1046.935 560.407 8470.663 3.020 2.749 3.928

.060 978.810 535.015 7258.231 2.991 2.728 3.861

.070 922.735 513.563 6340.513 2.965 2.711 3.802

.080 875.257 494.969 5619.039 2.942 2.695 3.750

.090 834.203 478.542 5035.501 2.921 2.680 3.702

.100 798.117 463.811 4552.974 2.902 2.666 3.658

.150 664.587 406.492 3007.714 2.823 2.609 3.478

.200 574.587 364.669 2171.467 2.759 2.562 3.337

.250 507.153 330.992 1648.170 2.705 2.520 3.217

.300 453.368 302.175 1291.900 2.656 2.480 3.111

.350 408.634 276.429 1035.697 2.611 2.442 3.015

.400 370.274 252.635 844.372 2.569 2.402 2.927

.450 336.591 230.020 697.630 2.527 2.362 2.844

.500 306.435 208.013 582.936 2.486 2.318 2.766

.550 278.981 186.200 492.102 2.446 2.270 2.692

.600 253.603 164.320 419.476 2.404 2.216 2.623

.650 229.796 142.303 360.906 2.361 2.153 2.557

.700 207.122 120.294 313.107 2.316 2.080 2.496

.750 185.156 98.616 273.312 2.268 1.994 2.437

.800 163.426 77.666 239.080 2.213 1.890 2.379

.850 141.295 57.782 208.135 2.150 1.762 2.318

.900 117.655 39.124 177.968 2.071 1.592 2.250

.910 112.565 35.533 171.724 2.051 1.551 2.235

.920 107.286 31.981 165.307 2.031 1.505 2.218

.930 101.765 28.462 158.648 2.008 1.454 2.200

.940 95.935 24.968 151.653 1.982 1.397 2.181

.950 89.693 21.483 144.182 1.953 1.332 2.159


141

.960 82.875 17.986 136.018 1.918 1.255 2.134

.970 75.200 14.438 126.776 1.876 1.160 2.103

.980 66.087 10.762 115.662 1.820 1.032 2.063

.990 53.912 6.751 100.398 1.732 .829 2.002 a. A heterogeneity factor is used. b. Logarithm base = 10.


142

Lampiran 41. Combination Index

I= (D)1/(Dx)1 + (D)2/(Dx)2

I = CI Dx = konsentrasi dari satu senyawa tunggal (IC50) (D)1 dan (D)2 = besarnya konsentrasi kedua senyawa untuk memberikan efek yang sama

absorbansi tunggal

ug/ml ENDP

uM Doxorubicin tunggal 15.0 0.731 0.825 0.783

25.0 0.836 0.749 0.982 kontrol sel

30.0 0.681 0.781 0.74

50.0 0.767 0.799 0.819 1.189 1.025 1.012 1.075333 45.0 0.663 0.728 0.708

100.0 0.694 0.728 0.723 kontrol media

60.0 0.559 0.588 0.592

200.0 0.625 0.702 0.617 0.14 0.15 0.159 0.150

cell viability tunggal ug/ml ENDP rata-rata uM Doxorubicin tunggal rata-rata

15.0 62.80 72.96 68.42 68.06 25.0 74.14 64.75 89.92 76.27 30.0 57.40 68.20 63.77 63.13 50.0 66.69 70.15 72.31 69.72 45.0 55.46 62.48 60.32 59.42 100.0 58.80 62.48 61.94 61.07 60.0 44.22 47.35 47.79 46.45 200.0 51.35 59.67 50.49 53.84


143

absorbansi kombinasi

doxo 25

doxo 50 doxo100 doxo 200

ENDP 15.0 0.918 0.88 0.885 0.769 0.856 0.857 0.726 0.807 0.826 0.706 0.733 0.744 ug/ml 30.0 0.834 0.855 0.857 0.765 0.824 0.826 0.757 0.728 0.779 0.701 0.754 0.665

45.0 0.807 0.759 0.739 0.803 0.788 0.767 0.75 0.723 0.738 0.632 0.632 0.662

60.0 0.708 0.735 0.747 0.739 0.772 0.757 0.69 0.652 0.715 0.443 0.473 0.443

cell viability kombinasi

dox 25 dox 50 dox 100 dox 200

ENDP 15.0 83.00 78.90 79.44 66.91 76.31 76.41 62.26 71.01 73.06 60.10 63.02 64.21 ug/ml 30.0 73.93 76.20 76.41 66.47 72.85 73.06 65.61 62.48 67.99 59.56 65.29 55.67

45.0 71.01 65.83 63.67 70.58 68.96 66.69 64.85 61.94 63.56 52.11 52.11 55.35

60.0 60.32 63.23 64.53 63.67 67.23 65.61 58.37 54.27 61.07 31.69 34.93 31.69

EADP

y = -0.1682x + 81.93

Doxorubicin y = -0.1524x + 101.99

R² = 0.8752

R² = 0.9393

IC 50 = 120 µg/ml

IC50 = 369 nM

Doxo

ENDP 0 25 50 100 200 0 100 76.27 69.72 61.07 53.84 15.0 68.06 80.45 73.21 68.78 62.44 30.0 63.13 75.51 70.80 65.36 60.17 45.0 59.42 66.83 68.74 63.45 53.19


144

60.0 46.45 62.69 65.50 57.90 32.77 doxo tunggal

y = -0.1524x + 101.99

25 50 100 200 15.0 141.361 188.855 217.918 259.505 30.0 173.733 204.686 240.365 274.391 45.0 230.678 218.155 252.889 320.230 60.0 257.851 239.420 289.277 454.205

ENDP

y = -0.1682x + 81.93 25 50 100 200

15.0 8.820 51.852 78.185 115.865 30.0 38.150 66.196 98.524 129.353 45.0 89.746 78.399 109.871 170.886 60.0 114.367 97.667 142.841 292.276

CI

Doxo ENDP 25 50 100 200 15.0 1.88 0.55 0.65 0.90 30.0 0.93 0.70 0.72 0.96 45.0 0.61 0.80 0.81 0.89 60.0 0.62 0.82 0.77 0.65


145

Lampiran 42. Hasil penentuan IC50 sel vero dengan analisa probit SPSS 17

Confidence Limits

Probability



Estimate

Lower Bound


Lower Bound

Upper Bound

PROBITa

.010 553.113 . . 2.743 . .

.020 484.041 . . 2.685 . .

.030 444.760 . . 2.648 . .

.040 417.326 . . 2.620 . .

.050 396.264 . . 2.598 . .

.060 379.176 . . 2.579 . .

.070 364.801 . . 2.562 . .

.080 352.392 . . 2.547 . .

.090 341.474 . . 2.533 . .

.100 331.723 . . 2.521 . .

.150 294.228 . . 2.469 . .

.200 267.475 . . 2.427 . .

.250 246.470 . . 2.392 . .

.300 229.017 . . 2.360 . .

.350 213.949 . . 2.330 . .

.400 200.569 . . 2.302 . .

.450 188.421 . . 2.275 . .

.500 177.184 . . 2.248 . .

.550 166.616 . . 2.222 . .

.600 156.525 . . 2.195 . .

.650 146.736 . . 2.167 . .

.700 137.081 . . 2.137 . .

.750 127.374 . . 2.105 . .

.800 117.372 . . 2.070 . .

.850 106.700 . . 2.028 . .

.900 94.639 . . 1.976 . .

.910 91.937 . . 1.963 . .

.920 89.088 . . 1.950 . .

.930 86.058 . . 1.935 . .


146

.940 82.795 . . 1.918 . .

.950 79.225 . . 1.899 . .

.960 75.227 . . 1.876 . .

.970 70.586 . . 1.849 . .

.980 64.858 . . 1.812 . .

.990 56.759 . . 1.754 . . a. A heterogeneity factor is used. b. Logarithm base = 10.

Lampiran 43. Mikroskop inverted, elisa reader dan inkubator CO2

Keterangan : Elisa reader

Keterangan : Mikroskop inverted


147

Keterangan : Inkubator CO2

Keterangan: Flowsitometri


148

Keterangan: Laminar Air Flow


92 lampiran 1. hasil identifikasi poguntano (picria fel-terrae lour.)

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