42 LAMPIRAN
Lampiran 1. Metode analisa antioksidan
No Metode analisa In vitro/
in vivo
1. DPPH scavenging activity In vitro
2. Hydrogen peroxide scavenging (H2O2) assay In vitro
3. Nitric oxide scavenging activity In vitro
4. Peroxynitrite radical scavenging activity In vitro
5. Trolox equivalent antioxidant capacity (TEAC) method / ABTS radical cation decolorization assay
In vitro
6. Total radical-trapping antioxidant parameter (TRAP) method In vitro
7. Ferric reducing-antioxidant power (FRAP) assay In vitro
8. Superoxide radical scavenging activity (SOD) In vitro
9. Hydroxyl radical scavenging activity In vitro
10. Hydroxyl radical averting capacity (HORAC) method In vitro
11. Oxygen radical absorbance capacity (ORAC) Method In vitro
12. Reducing power method (RP) In vitro
13. Phosphomolybdenum method In vitro
14. Ferric thiocyanate (FTC) method In vitro
15. Thiobarbituric acid (TBA) method In vitro
16. DMPD (N,N-dimethyl-p-phenylene diamine dihydrochloride) method In vitro
17. b-carotene linoleic acid method/conjugated diene assay In vitro
18. Xanthine oxidase method In vitro
19. Cupric ion reducing antioxidant capacity (CUPRAC) method In vitro
20. Metal chelating activity In vitro
21. Ferric reducing ability of plasma In vivo
22. Reduced glutathione (GSH) estimation In vivo
23. Glutathione peroxidase (GSHPx) estimation In vivo
24. Glutathione-S-transferase (GSt) In vivo
25. Superoxide dismutase (SOD) method In vivo
26. Catalase (CAT) In vivo
27. c-Glutamyl transpeptidase activity (GGT) assay In vivo
28. Glutathione reductase (GR) assay In vivo
29. Lipid peroxidation (LPO) assay In vivo
30. LDL assay In vivo
Lampiran 2. Analisa statistik kadar air bahan baku
Kadar air berat basah (wet basis)
Descriptives KAdb
N Mean Std. Deviation Std. Error
95% Confidence Interval for
Mean Minimum Maximum
Lower Bound Upper Bound
1.00 6 18.9857 .75990 .31023 18.1882 19.7831 17.87 19.81
2.00 6 15.4145 2.09444 .85505 13.2165 17.6125 12.44 17.89
3.00 6 16.9992 2.50303 1.02186 14.3724 19.6259 15.41 22.03
Total 18 17.1331 2.35856 .55592 15.9602 18.3060 12.44 22.03
Test of Homogeneity of Variances KAdb Levene Statistic df1 df2 Sig. 1.509 2 15 .253 ANOVA KAdb Sum of
Squares df Mean Square F Sig.
Between Groups 38.421 2 19.211 5.132 .020 Within Groups 56.146 15 3.743 Total 94.568 17 KAdb Duncan uji N
Subset for alpha = .05
1 2
2.00 6 15.4145
3.00 6 16.9992 16.9992
1.00 6 18.9857
Sig. .176 .096
Means for groups in homogeneous subsets are displayed. a Uses Harmonic Mean Sample Size = 6.000.
Keterangan:
Kode 1.00= kadar air berat basah C.longa 2.00= kadar air berat basah C.zedoaria 3.00= kadar air berat basah C.mangga
44
Kadar air berat kering (dry basis)
Descriptives KAdb
N Mean Std. Deviation Std. Error
95% Confidence Interval for
Mean Minimum Maximum
Lower Bound Upper Bound
1.00 6 18.9857 .75990 .31023 18.1882 19.7831 17.87 19.81
2.00 6 15.4145 2.09444 .85505 13.2165 17.6125 12.44 17.89
3.00 6 16.9992 2.50303 1.02186 14.3724 19.6259 15.41 22.03
Total 18 17.1331 2.35856 .55592 15.9602 18.3060 12.44 22.03
Test of Homogeneity of Variances KAdb Levene Statistic df1 df2 Sig. 1.509 2 15 .253 KAdb Duncan uji N
Subset for alpha = .05
1 2
2.00 6 15.4145
3.00 6 16.9992 16.9992
1.00 6 18.9857
Sig. .176 .096
Means for groups in homogeneous subsets are displayed. a Uses Harmonic Mean Sample Size = 6.000.
Keterangan:
Kode 1.00= kadar air berat kering C.longa 2.00= kadar air berat kering C.zedoaria 3.00= kadar air berat kering C.mangga
Lampiran 3. Kurva standar curcuminoid
Kurva standar, atau disebut juga kurva kalibrasi, dibutuhkan apabila kita ingin mengetahui konsentrasi suatu zat dalam sampel yang mana konsentrasi zat tersebut tidak diketahui. Absis berupa konsentrasi senyawa standar, sedangkan ordinat berupa nilai absorbansi. Persamaan linear yang didapatkan adalah y = 0,3776 x + 0,0033 dengan R2 sebesar 0,9928.
Nilai kandungan curcuminoid dalam sampel didapatkan dengan memasukkan nilai absorbansi sampel ke dalam persamaan sebagai y sehingga didapatkan besarnya konsentrasi sampel (x).
y = 0.3776x + 0.0033 y = 0.3776x + 0.0033 R² = 0.9928 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 1 2 3 4 5 Konsentrasi μg/ml absorbansi
Lampiran 4. Perhitungan curcuminoid
absorbansi curcuminoid basis KA Curcuminoid
batch 1 batch 2 (y)
&
pengenceran
dlm 100 mg sampel rimpang b1.1 b1.2 b1.3 rata2b1 b2.1 b2.2 b2.3 rata2b.2 rata2
y = 0.3776x
+ 0.0033
CL 0.616 0.654 0.671 0.647 0.575 0.604 0.645 0.608 0.628 1.653 0.278 13.892 CZ 0.007 0.013 0.012 0.011 0.008 0.014 0.009 0.010 0.011 0.019 0.017 0.837 CM 0.015 0.012 0.019 0.015 0.017 0.017 0.015 0.016 0.016 0.033 0.028 1.414 absorbansi curcuminoid basis KA Curcuminoid
batch 1 batch 2 (x)
&
pengenceran
dlm 100 mg sampel ekstrak b1.1 b1.2 b1.3 rata2b1 b2.1 b2.2 b2.3 rata2b.2 rata2
y = 0.3776x
+ 0.0033
CL 1.724 1.732 1.723 1.726 1.717 1.723 1.719 1.720 1.723 4.554 0.765 38.270 CZ 0.000 0.073 0.038 0.056 0.011 0.013 0.016 0.013 0.035 0.083 0.072 3.582 CM 0.085 0.074 0.084 0.081 0.089 0.096 0.099 0.095 0.088 0.224 0.191 9.570
absorbansi curcuminoid Curcuminoid prosentase
batch 1 batch 2 (x)
dlm 100 mg sampel fraksi b1.1 b1.2 b1.3 rata2b1 b2.1 b2.2 b2.3 rata2b.2 rata2
y = 0.3776x + 0.0033 Curcumin 0.213 0.271 0.263 0.249 0.153 0.244 0.124 0.174 0.551 1.450 42.227 42.227 BDMC 0.135 0.127 0.135 0.133 0.138 0.107 0.128 0.124 0.331 0.868 25.279 25.279 Seny.19 0.147 0.109 0.151 0.136 0.197 0.193 0.185 0.192 0.425 1.116 32.494 32.494 Jml 3.435 100.000 100.000
Contoh perhitungan kandungan curcuminoid pada rimpang C.mangga
Persamaan kurva standar curcuminoid : y = 0,3776 x + 0,0033
Absorbansi rata-rata C.mangga = 0,016 plot-kan pada persamaan kurva standar sebagai y
y = 0,3776 x + 0,0033
0,016 = 0,3776 x + 0,0033 0,3776 x = 0,016 – 0,0033 0,3776 x = 0,0127
x = 0,033 mg / 2 mg sampel kandungan curcuminoid
massa bahan baku 2 g kadar air C.mangga = 16,99
Kandungan curcuminoid C.mangga
= nilai x * kadar air massa bahan baku
= 0,033 * 16,9992 2
= 0,028 mg / g sampel = 0,028 * 100
= 2,8 mg / 100 mg sampel
Cara perhitungan yang sama digunakan pada rimpang C.longa dan C.zedoaria, serta ekstrak
48
Contoh perhitungan kandungan curcuminoid pada fraksi C.mangga
Persamaan kurva standar curcuminoid : y = 0,3776 x + 0,0033
Absorbansi rata-rata senyawa 19 = 0,425 plot-kan pada persamaan kurva standar sebagai y
y = 0,3776 x + 0,0033 0,425 = 0,3776 x + 0,0033 0,3776 x = 0,425 – 0,0033 0,3776 x = 0,4217 x = 1,116 mg x curcumin = 1,450 mg x bis-demethoxycurcumin = 0,868 mg x senyawa 19 = 1,116 mg Total = 3,335 mg Sehingga x senyawa 19 = 1,450 mg / 3,435 mg
Kandungan curcuminoid senyawa 19 dalam 100 mg ekstrak
= nilai x * 100
= 1,450 mg * 100 3,435 mg
= 32,494 mg / 100 mg
49 Lampiran 5. Perhitungan aktivitas antioksidan
batch 1 batch 2 %SA
rimpang DPPH b1.1 b1.2 b1.3 rata2b1 %SA DPPH b2.1 b2.2 b2.3 rata2b.2 %SA rata2 CL 1.063 0.776 0.776 0.776 0.776 27.011 1.079 0.760 0.746 0.776 0.761 29.478 28.244 CZ 1.063 0.863 0.876 0.901 0.880 17.238 1.079 0.863 0.876 0.901 0.880 18.432 17.835 CM 1.063 0.856 0.996 0.728 0.860 19.093 1.079 0.858 0.863 0.858 0.860 20.307 19.700
batch 1 batch 2 %SA
ekstrak DPPH b1.1 b1.2 b1.3 rata2b1 %SA DPPH b2.1 b2.2 b2.3 rata2b.2 %SA rata2 CL 1.063 0.356 0.330 0.289 0.325 69.428 1.079 0.298 0.337 0.363 0.333 56.271 62.849 CZ 1.063 0.714 0.125 0.156 0.332 68.816 1.079 0.060 0.068 0.077 0.069 92.200 80.508 CM 1.063 0.125 0.112 0.084 0.107 89.951 1.079 0.116 0.121 0.115 0.117 86.386 88.169
batch 1 batch 2 %SA
fraksi DPPH b1.1 b1.2 b1.3 rata2b1 %SA DPPH b2.1 b2.2 b2.3 rata2b.2 %SA rata2 C 1.063 0.213 0.271 0.263 0.249 67.921 1.079 0.153 0.244 0.124 0.174 77.158 72.540 BDMC 1.063 0.135 0.127 0.135 0.133 84.938 1.079 0.138 0.107 0.128 0.124 85.889 85.413 seny.19 1.063 0.147 0.109 0.151 0.136 84.228 1.079 0.197 0.193 0.185 0.192 77.701 80.965
Contoh perhitungan aktivitas antioksidan pada rimpang C.mangga
Batch 1
Absorbansi rata-rata DPPH = 1,063 Absorbansi rimpang C.mangga = 0,860
%SA = (absorbansi DPPH – absorbansi rimpang C.mangga) * 100 Absorbansi DPPH = (1,063 – 0,860) * 100 1,063 = 19,092 Batch 2 Absorbansi rata-rata DPPH = 1,078 Absorbansi rimpang C.mangga = 0,859
%SA = (absorbansi DPPH – absorbansi rimpang C.mangga) * 100 Absorbansi DPPH
= (1,078 – 0,859) * 100 1,078
= 20,306
%SA rata-rata = %SA batch 1 + %SA batch 2 2
= 19,092 + 20,306 2
= 19, 700
Cara perhitungan yang sama digunakan pada perhitungan aktivitas antioksidan rimpang
C.longa dan C.zedoaria, C.longa, C.zedoaria, dan C.mangga, serta curcumin, bis-demethoxycurcumin, dan senyawa 19..
51 Lampiran 6. Perhitungan energi ikat
Energi ikat C – C masing-masing = 347,27 kJ/mol Energi ikat C = C masing-masing = 610,86 kJ/mol Energi ikat C – H masing-masing = 414,22 kJ/mol Energi ikat C – O masing-masing = 357,73 kJ/mol Energi ikat C = O masing-masing = 744,75 kJ/mol Energi ikat O – H masing-masing = 464,42 kJ/mol
Contoh perhitungan energi ikat:
Senyawa 1 = curcumin C – C 12 C = C 8 C – H 18 C – O 6 C = O 2 O – H 2 Senyawa 2 = demethoxycurcumin C – C 12 C = C 8 C – H 16 C – O 4 C = O 2 O – H 2
Energi ikat senyawa 1
= ( C–C x energi ikat C–C) + ( C=C x energi ikat C=C) + ( C–H x energi ikat C–H) + ( C–O x energi ikat C–O) + ( C=O x energi ikat C=O) + ( O–H x energi ikat O–H)
= (12 x 347,27 kJ/mol) + (8 x 610,86 kJ/mol) + (18 x 414,22 kJ/mol) + (6 x 357,73 kJ/mol) + (2 x 744,75 kJ/mol) + (2 x 464,42 kJ/mol) = (4167,24 + 4886,88 + 7455,96 + 2146,38 + 1489,50 + 928,84) kJ/mol = 21074,84 kJ/mol
Energi ikat senyawa 2
= ( C–C x energi ikat C–C) + ( C=C x energi ikat C=C) + ( C–H x energi ikat C–H) + ( C–O x energi ikat C–O) + ( C=O x energi ikat C=O) + ( O–H x energi ikat O–H)
= (12 x 347,27 kJ/mol) + (8 x 610,86 kJ/mol) + (16 x 414,22 kJ/mol) + (4 x 357,73 kJ/mol) + (2 x 744,75 kJ/mol) + (2 x 464,42 kJ/mol)
= 19530,94 kJ/mol
Cara perhitungan yang sama digunakan untuk menghitung aktivitas antioksidan senyawa 3 hingga 19.
