LAMPIRAN. Lampiran 1. Metode analisa antioksidan

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1 LAMPIRAN Lampiran 1. Metode analisa antioksidan No Metode analisa In vitro/ in vivo 1. DPPH scavenging activity In vitro 2. Hydrogen peroxide scavenging (H 2 O 2 ) 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 In vitro decolorization assay 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

2 43 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 Total KAdb Test of Homogeneity of Variances Levene Statistic df1 df2 Sig. KAdb ANOVA Sum of Squares df Mean Square F Sig. Between Groups Within Groups Total Duncan KAdb Subset for alpha =.05 uji N Sig Means for groups in homogeneous subsets are displayed. a Uses Harmonic Mean Sample Size = 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

3 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 Total KAdb Test of Homogeneity of Variances Levene Statistic df1 df2 Sig. Duncan KAdb Subset for alpha =.05 uji N Sig Means for groups in homogeneous subsets are displayed. a Uses Harmonic Mean Sample Size = 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

4 45 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 y = x R² = 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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6 46 Lampiran 4. Perhitungan curcuminoid absorbansi curcuminoid basis KA Curcuminoid & dlm 100 mg batch 1 batch 2 (y) pengenceran sampel rimpang b1.1 b1.2 b1.3 rata2b1 b2.1 b2.2 b2.3 rata2b.2 rata2 y = x CL CZ CM absorbansi curcuminoid basis KA Curcuminoid & dlm 100 mg batch 1 batch 2 (x) pengenceran sampel ekstrak b1.1 b1.2 b1.3 rata2b1 b2.1 b2.2 b2.3 rata2b.2 rata2 y = x CL CZ CM absorbansi curcuminoid Curcuminoid prosentase dlm 100 mg batch 1 batch 2 (x) sampel fraksi b1.1 b1.2 b1.3 rata2b1 b2.1 b2.2 b2.3 rata2b.2 rata2 y = x Curcumin BDMC Seny Jml

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8 47 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, = 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.

9 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 x bis-demethoxycurcumin x senyawa 19 Total = 1,450 mg = 0,868 mg = 1,116 mg = 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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11 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 CZ CM 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 CZ CM 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 BDMC seny

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14 50 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, ,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..

15 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, , , , , ,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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