LAMPIRAN
Lampiran 1. Metode analisa antioksidan
No Metode analisa 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
DPPH scavenging activity Hydrogen peroxide scavenging (H2O2) assay Nitric oxide scavenging activity Peroxynitrite radical scavenging activity Trolox equivalent antioxidant capacity (TEAC) method / ABTS radical cation decolorization assay Total radical-trapping antioxidant parameter (TRAP) method Ferric reducing-antioxidant power (FRAP) assay Superoxide radical scavenging activity (SOD) Hydroxyl radical scavenging activity Hydroxyl radical averting capacity (HORAC) method Oxygen radical absorbance capacity (ORAC) Method Reducing power method (RP) Phosphomolybdenum method Ferric thiocyanate (FTC) method Thiobarbituric acid (TBA) method DMPD (N,N-dimethyl-p-phenylene diamine dihydrochloride) method b-carotene linoleic acid method/conjugated diene assay Xanthine oxidase method Cupric ion reducing antioxidant capacity (CUPRAC) method Metal chelating activity Ferric reducing ability of plasma Reduced glutathione (GSH) estimation Glutathione peroxidase (GSHPx) estimation Glutathione-S-transferase (GSt) Superoxide dismutase (SOD) method Catalase (CAT) c-Glutamyl transpeptidase activity (GGT) assay Glutathione reductase (GR) assay Lipid peroxidation (LPO) assay LDL assay
Sumber: Alam et al, 2013, Prior et al 2005
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In vitro/ in vivo In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vitro In vivo In vivo In vivo In vivo In vivo In vivo In vivo In vivo In vivo In vivo
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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
1.00
6
18.9857
.75990
.31023
Lower Bound 18.1882
Upper Bound 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 1.509
df1
df2 2
Sig. .253
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ANOVA KAdb Sum of Squares
df
Mean Square
Between Groups
38.421
2
19.211
Within Groups
56.146
15
3.743
Total
94.568
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KAdb Duncan Subset for alpha = .05 uji 2.00
N 6
1 15.4145
3.00
6
16.9992
1.00
6
2 16.9992 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
F 5.132
Sig. .020
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Kadar air berat kering (dry basis) Descriptives KAdb N
Mean
Std. Deviation
Std. Error
.31023
17.87
19.81
.85505
13.2165
17.6125
12.44
17.89
2.50303
1.02186
14.3724
19.6259
15.41
22.03
2.35856
.55592
15.9602
18.3060
12.44
22.03
6
18.9857
.75990
6
15.4145
2.09444
3.00
6
16.9992
Total
18
17.1331
Test of Homogeneity of Variances KAdb df1
df2 2
Sig. 15
.253
KAdb Duncan Subset for alpha = .05 uji 2.00
N
Maximum
Upper Bound 19.7831
2.00
1.509
Minimum
Lower Bound 18.1882
1.00
Levene Statistic
95% Confidence Interval for Mean
1 6
15.4145
3.00
6
16.9992
1.00
6
2 16.9992 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
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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. absorbansi 1.6 y = 0.3776x + 0.0033 R² = 0.9928
1.4 1.2 1 0.8 0.6 0.4 0.2 0 0
1
2
3
4
5
Konsentrasi μg/ml
Nilai kandungan curcuminoid dalam sampel didapatkan dengan memasukkan nilai absorbansi sampel ke dalam persamaan sebagai y sehingga didapatkan besarnya konsentrasi sampel (x).
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Lampiran 4. Perhitungan curcuminoid absorbansi curcuminoid batch 1 rimpang CL CZ CM
b1.1 0.616 0.007 0.015
b1.2 0.654 0.013 0.012
batch 2
(y)
y = 0.3776x b1.3 rata2b1 b2.1 b2.2 b2.3 rata2b.2 rata2 + 0.0033 0.671 0.647 0.575 0.604 0.645 0.608 0.628 1.653 0.012 0.011 0.008 0.014 0.009 0.010 0.011 0.019 0.019 0.015 0.017 0.017 0.015 0.016 0.016 0.033 absorbansi curcuminoid
batch 1 ekstrak CL CZ CM
b1.1 1.724 0.000 0.085
b1.2 1.732 0.073 0.074
batch 2
(x)
y = 0.3776x b1.3 rata2b1 b2.1 b2.2 b2.3 rata2b.2 rata2 + 0.0033 1.723 1.726 1.717 1.723 1.719 1.720 1.723 4.554 0.038 0.056 0.011 0.013 0.016 0.013 0.035 0.083 0.084 0.081 0.089 0.096 0.099 0.095 0.088 0.224 absorbansi curcuminoid
batch 1 fraksi Curcumin BDMC Seny.19
b1.1 0.213 0.135 0.147
b1.2 0.271 0.127 0.109
batch 2
(x)
y = 0.3776x b1.3 rata2b1 b2.1 b2.2 b2.3 rata2b.2 rata2 + 0.0033 0.263 0.249 0.153 0.244 0.124 0.174 0.551 1.450 0.135 0.133 0.138 0.107 0.128 0.124 0.331 0.868 0.151 0.136 0.197 0.193 0.185 0.192 0.425 1.116 Jml 3.435
basis KA Curcuminoid & dlm 100 mg pengenceran sampel
0.278 0.017 0.028
13.892 0.837 1.414
basis KA Curcuminoid & dlm 100 mg pengenceran sampel
0.765 0.072 0.191
38.270 3.582 9.570
Curcuminoid prosentase dlm 100 mg sampel
42.227 25.279 32.494 100.000
42.227 25.279 32.494 100.000
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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 C.longa, C.zedoaria, dan C.mangga.
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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 Cara perhitungan yang sama digunakan pada curcumin dan bis-demethoxycurcumin.
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Lampiran 5. Perhitungan aktivitas antioksidan batch 1 rimpang DPPH b1.1 b1.2 b1.3 rata2b1 %SA CL 1.063 0.776 0.776 0.776 0.776 27.011 CZ 1.063 0.863 0.876 0.901 0.880 17.238 CM 1.063 0.856 0.996 0.728 0.860 19.093
batch 2 %SA DPPH b2.1 b2.2 b2.3 rata2b.2 %SA rata2 1.079 0.760 0.746 0.776 0.761 29.478 28.244 1.079 0.863 0.876 0.901 0.880 18.432 17.835 1.079 0.858 0.863 0.858 0.860 20.307 19.700
ekstrak CL CZ CM
batch 1 DPPH b1.1 b1.2 b1.3 rata2b1 %SA DPPH 1.063 0.356 0.330 0.289 0.325 69.428 1.079 1.063 0.714 0.125 0.156 0.332 68.816 1.079 1.063 0.125 0.112 0.084 0.107 89.951 1.079
batch 2 %SA b2.1 b2.2 b2.3 rata2b.2 %SA rata2 0.298 0.337 0.363 0.333 56.271 62.849 0.060 0.068 0.077 0.069 92.200 80.508 0.116 0.121 0.115 0.117 86.386 88.169
fraksi C BDMC seny.19
batch 1 DPPH b1.1 b1.2 b1.3 rata2b1 %SA DPPH 1.063 0.213 0.271 0.263 0.249 67.921 1.079 1.063 0.135 0.127 0.135 0.133 84.938 1.079 1.063 0.147 0.109 0.151 0.136 84.228 1.079
batch 2 %SA b2.1 b2.2 b2.3 rata2b.2 %SA rata2 0.153 0.244 0.124 0.174 77.158 72.540 0.138 0.107 0.128 0.124 85.889 85.413 0.197 0.193 0.185 0.192 77.701 80.965
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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, bisdemethoxycurcumin, dan senyawa 19..
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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=C8 C – H 18 C–O6 C=O2 O–H2
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
Senyawa 2 = demethoxycurcumin C – C 12 C=C8 C – H 16 C–O4 C=O2 O–H2
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.
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