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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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