lampiran lampiran 1. metode analisa antioksidanrepository.unika.ac.id/482/9/08.95.0002 yovita dyah...
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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
Sumber: Alam et al, 2013, Prior et al 2005
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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
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
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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
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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.
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
40
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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
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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 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
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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, bis-
demethoxycurcumin, 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 = 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.
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