DAFTAR PUSTAKA Alfarabi M. 2008. Aktivitas antioksidasi ekstrak daun sirih merah [skripsi]. Bogor: Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Pertanian Bogor. Aqil F, Ahmad I, Mehmood Z. 2006. Antioxidant and free radical scavenging properties of twelve traditionally used Indian medicinal plants. Turk J Biol 30:177-183 Betteridge DJ. 2000. What is oxidative stress. Metabolism 49 (Suppl 1): 3-8 Bhattacharya S et al. 2007. Inhibitory property of Piper betel extract against photosensitization-induced damages to lipids and proteins. Food Chemistry 100: 1474-1480 Ceriello A. 2000. Oxidative stress and glycemic regulation. Metabolism 49 (Suppl 1): 27-29 Ceriello A. 2003. New insights on oxidative stress and diabetic complications may lead to a causal antioxidant therapy. Diabetes Care 26: 1589-1596 Cetto AA, Jimenez JB, Vazquez RC . 2008. Alfa-glucosidase inhibiting activity of some Mexican plants used in the treatment of type 2 diabetes. Journal of Ethnopharmacology 117: 27-32 Chang HJ, Ho MS, In WC, Hong YC. 2005. Antioxidant activities of cultivated and wild korean ginseng leaves. Food Chemistry 92: 535-540 Chen HM, Muramoto K, Yamauchi F, Nokihara K. 1996. Antioxidant activity of designed peptides base on the antioxidative peptide isolated from digest of soybean protein. J Agric Food Chem 44: 2619 Dobretsov M, Romanovsky D, Stimers JR. 2007. Early diabetic neuropathy: triggers and mechanism. World J Gastroenterol 13: 175-191 Duryatmo S. 2005. Dulu hiasan kini obat. Trubus 427:37 Evans CAR, Miller NJ, Paganga G. 1996. Sturcture antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine 20: 933-956 Guohua R, Mouming Z, Weifeng L, Haiyan L. 2007. Antioxidative activity of tobacco leaf protein hydrolysates. Food Technol Biotechnol 45: 80-84 Hans, Heldt W. 2005. Plant Biochemistry Third Edition. San Diego: Elsevier Academic Press
Harborne JB. 1987. Metode Fitokimia: Penuntun Cara Modern Menganalisis Tumbuhan. K Padmawinata, I Sudiro, penerjemah. Bandung: ITB. Terjemahan dari: Phytochemical Method Harborne JB, Williams CA. 2000. Advances in flavonoid research since 1992. Phytochemistry 55: 481-504 Hart H, Craine LE, Hart DJ. 2003. Kimia Organik: Suatu Kuliah Singkat. Achmadi S, penerjemah; Safitri A, editor. Jakarta: Erlangga. Terjemahan dari: Organic Chemistry: A Short Course Illanes A. 2008. Enzyme Biocatalysis: Principles and Applications. Chile: Springer Jankyova S et al. 2009. Pycnogenol efficiency on glycaemia, motor nerve conduction velocity, and markers of oxidative stress in mild type diabetes in rats. Phytotherapy Research 23: 1169-1174 Kalaivanam KN, Dharmalingam M, Marcus SR. 2006. Lipid peroxydation in type 2 diabetes mellitus. Int J Diab Dev Ctries 26: 30-32 Kikuzaki H, Nakatani N. 1993. Antioxidant effect of some ginger constituents. Journal of Food Science 58(6):1407-1410 Lan FW, Hong YZ. 2003. A theoretical investigation on DPPH radical scavenging mechanism of edaravone. Bioorganic and Medicinal Chemistry Letters 13: 3789-3792 Lau et al. 2009. Radix Astragali and Radix Rehmanniae, the principal components of two antidiabetic foot ulcer herbal formulae, elicit viability-promoting effects on primary fibroblast cultured from diabetic foot ulcer tissues. Phytotherapy Research 23: 809-815 Linder MC. 2006. Biokimia Nutrisi dan Metabolisme dengan Pemakaian Secara Klinis. Aminuddin Parakkasi, penerjemah. Jakarta: UI Press. Terjemahan dari: Nutritional Biochemistry and Metabolism Maritim AC, Sanders RA, Watkins JB. 2003. Diabetes, oxidative stress, and antioxidant: a review. J Biochem Molecular Toxicology 17: 24-38 Marlina PW. 2008. Konsentrasi flavonoid dan lethal concentration 50 (LC50) ekstrak daun sirih merah (Piper crocatum) [skripsi]. Bogor: Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Pertanian Bogor Memisogullari R, Taysi S, Bakan E, Capoglu I. 2003. Antioxidant status and lipid peroxidation in type II diabetes mellitus. Cell Biochem Funct 21: 291-296
Murray RK, Granner DK, Mayes PA, Rodwell VW. 2003. Biokimia Harper Edisi 25. Hartono, penerjemah. Jakarta: EGC. Terjemahan dari: Harper’s Biochemistry Myo JK et al. 1999. Comparative study of the inhibition of α-glucosidase, αamylase, and cyclomaltodextrin glucanosyltransferase by acarbose, isoacarbose, and acarviosine-glucose. Archives of Biochemistry and Biophysics 371: 277-283 Nakayama S, Katoh EM, Tsuzuki T, Kobayashi S. 2003. Protective effect αtocopherol-6-O-phosphate against ultraviolet B-induced damage in cultured mouse skin. The Journal Investigative Dermatology 121: 406-411 Panda S, Kar A. 2007. Antidiabetic and antioxidative effects of Annona squamosa leaves possibly mediated through quercetin-3-O-glucoside. BioFactors 31: 201-210 Parmar HS, Kar A. 2007. Antidiabetic potential of Citrus sinensis and Punica granatum peel extracts in alloxan treated meal mice. BioFactors 31: 17-24 Permata DA. 2006. Potensi rebusan daun sirih merah (Piper crocatum) terhadap perbaikan pankreas tikus putih hiperglikemia [skripsi]. Bogor: Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Pertanian Bogor Pokorny J, Yanishlieva N, Gordon M. 2001. Antioxidants In Food: Practical Applications. New York: CRC Press Pratama NR. 2009. Aktivitas antihiperglikemik ekstrak daging dan kulit buah salak (Salacca edulis Reinw) varietas bongkok [skripsi] Bogor: Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Pertanian Bogor Purwakusumah ED. 2003. Tumbuhan sebagai sumber biofarmaka. Di dalam: Pelatihan Tanaman Obat Tradisional (Swamedikasi). Prosiding Pertemuan Pengobatan Penyakit Diabetes Mellitus; Bogor, 3-4 Mei 2003. Bogor: Pusat Studi Biofarmaka Lembaga Penelitian IPB Rohman A, Riyanto S. 2005. Daya antioksidan ekstrak etanol daun kemuning (Murraya paniculata (L) Jack) secara in vitro. Majalah Farmasi Indonesia 16: 136-140 Saija A et al. 1995. Flavonoids as antioxidant agents: important of their interaction with biomembranes. Free Radical Biology and Medicine 19: 481486 Safithri M, Fahma F. 2005. Potensi rebusan daun sirih merah (Piper crocatum) Sebagai senyawa antihiperglikemia pada tikus putih galur Sprague Dawley. Laporan penelitian. Bogor: LPPM IPB
Salim A. 2006. Potensi rebusan daun sirih merah (Piper crocatum) sebagai senyawa antihiperglikemia pada tikus putih galur Sprague-Dawley [skripsi]. Bogor: Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Pertanian Bogor Sen CK, Packer L, Hanninen O. 2000. Handbook of Oxidants and Antioxidants in Exercise. Amsterdam: Elsevier Stryer L. 2000. Biokimia Edisi ke 4. Sadikin M et al., penerjemah; Soebianto S, Setiadi E, editor. Jakarta: EGC. Terjemahan dari: Biochemistry Stuart AR, Gulve EA, Wang M. 2004. Chemistry and biochemistry of type 2 diabetes. Chemical Reviews 104: 1255-1282 Sudewo B. 2005. Basmi Penyakit dengan Sirih Merah. Jakarta: Agromedia Pustaka Suteja L. 2003. Bioprospecting tumbuhan obat Indonesia sebagai sediaan fitofarmaka antidiabetes. Laporan kemajuan tahap II riset unggulan terpadu. Pusat Penelitian Kimia LIPI The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. 2003. Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 26 (Suppl 1): 5-20 Udin LZ, Sutedja L, Soemardji AA, Dewi P, Sundawati U. 2005. Daun sukun (Artocarpus communis) sebagai fitofarmaka antidiabetes. Proyek Industri Bahan Baku Kimia Jadi Wenli et al. 2009. Triterpene acid isolated from Lagerstroemia speciosa leaves as α-glucosidase inhibitors. Phytotherapy Research 23: 614-618 Wild S, Roglic G, Green A, Sicree R, King H. 2004. Global prevalence of diabetes: estimates for the year 2000 and projections 2030. Diabetes Care 27: 1047-1053 Windyagiri A. 2006. Potensi hepatoprotektor air rebusan daun sirih merah (Piper crocatum) pada tikus putih hiperglikemia [skripsi]. Bogor: Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Pertanian Bogor
LAMPIRAN
Lampiran 1 Tahapan umum penelitian Daun sirih merah
Ekstraksi daun sirih merah dengan etanol 70%
Analisis aktivitas antioksidasi metode DPPH
Analisis aktivitas antioksidasi metode TBA
Analisis inhibitor α-glukosidase
Analisis komponen ekstrak dengan GC-MS
Lampiran 2 Ekstraksi daun sirih merah 25 gram daun sirih merah ditambah etanol 70% 100 mL, dimaserasi 24 jam suhu ruang
Rotavapor 50 °C
Ekstrak dikeringkan dengan freeze dryer -50 °C, 8 mbar
Lampiran 3 Analisis ekstrak etanol 70% daun sirih merah dengan GC-MS Ekstrak etanol 70% 842,1 mg dilarutkan dengan 1 mL etanol 99% dan diinjeksikan sebanyak 1 µL Kondisi GC-MS: kolom HP-5 (Agilent 19091J-433: 0,25 mm x 30 m x 0,25 µm mengandung 5% difenil 95% dimetilpolisilosan), laju alir 1,0 mL/menit, suhu injeksi 300 °C mode split, tekanan 10,47 psi, gas pembawa adalah He
Parameter MS: deteksi senyawa dengan massa 50800. Kondisi MS adalah suhu MS quad 150-200 °C dan suhu MS source 250-300 °C, hasil dianalisis dengan database
Lampiran 4 Penentuan waktu inkubasi maksimum asam linoleat 6 ml asam linoleat dalam etanol 99,8 % 6 mL buffer fosfat 0.1 M pH 7 3 mL akuades
Campuran dipipet ke dalam botol gelap (terdapat 11 botol dan 1 mL campuran untuk setiap botol), diinkubasi 40 °C
1 mL diambil setiap 24 jam, ditambahkan 2 mL TCA 20 % dan 2 mL TBA 1 % dalam asam asetat 50 % Larutan diinkubasi 100 °C, dinginkan Sentrifus 15,5 g 15 menit diukur serapannya pada λ 532 nm
Lampiran 5 Analisis aktivitas antioksidasi metode TBA 1 mL ekstrak etanol 70% daun sirih merah (25, 50, 75, 100, 200 ppm), vitamin E 200 ppm, dan akuades 2 mL buffer fosfat 0,1 M pH 7 2 mL asam linoleat 50 mM Inkubasi 40 °C. lama inkubasi optimum hasil pengukuran Masing-masing diambil 1 mL 2 mL TCA 20% 2 mL TBA 1 % dalam asam asetat 50%
Larutan Inkubasi 100 °C selama 10 menit, dinginkan, sentrifus 15,5 g selama 15 menit diukur serapannya pada λ 532 nm
Lampiran 6 Analisis aktivitas antioksidasi metode DPPH 2 mL ekstrak etanol 70 % daun sirih merah (25, 50, 75, 100, 200 ppm) dan vitamin E dicampur dengan 2 mL DPPH 0.1 mM
diinkubasi 30 menit dengan suhu ruang
diukur reduksi DPPH dengan λ = 517 nm
Lampiran 7 Analisis inhibisi α-glukosidase 0,5 g α-glukosidase + buffer fosfat 0,1 M pH 7 (mengandung 100 mg BSA)
diencerkan 25 kali
500 µL 20 mM p-nitrofenil-α-Dglukopiranosida + 980 µL buffer fosfat pH 7 + 20 µL ekstrak (0,1%, 0,25%, 0,5%, 0,75%, 1% b/v dalam DMSO) diinkubasi suhu 37 °C selama 5 menit. Pembanding acarbose 1%.
Reaksi enzimatis dimulai diinkubasi 37 °C selama 15 menit
Penghentian reaksi dengan 2 mL 200 mM Na2CO3
Diukur dengan λ= 400 nm
Lampiran 8 Analisis kinetika inhibisi enzim α-glukosidase Enzim + substrat (5, 10, 15, 20, 25 mM)
Enzim + substrat (5, 10, 15, 20, 25 mM) + ekstrak daun sirih merah 1%
Reaksi enzimatis dimulai diinkubasi 37 °C selama 15 Penghentian reaksi dengan 2 mL 200 mM Na2CO3 Diukur dengan λ= 400 nm
Lampiran 9 Rendemen hasil ekstraksi daun sirih merah dengan etanol 70% Ekstrak
Bobot daun
Bobot botol
Bobot botol + Bobot ekstrak Rendemen
(g)
kosong (g)
ekstrak (g)
(g)
(%)
Daun 1
25,0078
37,3183
38,2874
0,9691
3,87
Daun 2
25,8432
37,6933
38,6341
0,9408
3,64
Daun 3
25,1343
35,9828
37,4315
1,4487
5,76
Rerata
25,3284
1,1195
4,42
Contoh perhitungan: Rendemen (%) = bobot ekstrak x 100% bobot daun = 1,1195 x 100% 25,3284 = 4,42%
Lampiran 10 Absorban inkubasi kadar MDA (µM) terhadap waktu (λ = 532 nm) Hari
A1
A2
A3
[MDA]1
[MDA]2
[MDA]3
Rerata [MDA] ± SD
0
0,156
0,154
0,170
1,2746
1,2564
1,4024
1,3111 ± 0,0795
1
0,193
0,201
0,209
1,6122
1,6852
1,7582
1,6852 ± 0,0730
2
0,262
0,283
0,275
2,2417
2,4333
2,3604
2,3451 ± 0,0967
3
0,387
0,350
0,397
3,3823
3,0447
3,4735
3,3002 ± 0,2258
4
0,456
0,487
0,444
4,0118
4,2947
3,9024
4,0663 ± 0,2024
5
0,500
0,509
0,498
4,4133
4,4954
4,3951
4,4346 ± 0,0534
6
0,893
0,901
0,910
7,9991
8,0721
8,1542
8,0751 ± 0,0775
7
0,670
0,660
0,653
5,9644
5,8732
5,8093
5,8823 ± 0,0779
8
0,621
0,621
0,631
5,5173
5,5173
5,6086
5,5477 ± 0,0527
9
0,550
0,575
0,583
4,8695
5,0976
5,1706
5,0459 ± 0,1569
Contoh perhitungan : Kadar MDA hari ke-0 (A1)
y = 0,0163 + 0,1096 x 0,156 = 0,0163 + 0,1096 x x = 1,2746
Lampiran 11 Aktivitas antioksidasi ekstrak etanol 70% daun sirih merah metode TBA (532 nm) Larutan
A1
A2
A3
[MDA]1
[MDA]2
[MDA]3
Rerata [MDA] ±
Daya
(µM)
(µM)
(µM)
SD
hambat (%)
K (-)
0,650
0,600
0,575
5,7819
5,3257
5,0976
5,4017 ± 0,3484
0
K (+)
0,128
0,130
0,115
1,0192
1,0374
0,9005
0,9857 ± 0,0743
81,75
E1
0,345
0,328
0,365
2,9991
2,8439
3,1815
3,0082 ± 0,1689
44,31
E2
0,287
0,265
0,273
2,4698
2,2692
2,3422
2,3604 ± 0,1015
56,30
E3
0,228
0,248
0,230
1,9315
2,1141
1,9498
1,9984 ± 0,1005
63,00
E4
0,178
0,178
0,178
1,4753
1,4753
1,4753
1,4753 ± 0
72,68
E5
0,128
0,144
0,125
1,0192
1,1651
0,9917
1,0586 ± 0,0932
80,40
Keterangan: K = kontrol negatif (asam linoleat tanpa ekstrak dan vitamin E) dan positif (vitamin E 200 ppm), E1 = ekstrak 25 ppm, E2 = ekstrak 50 ppm, E3 = ekstrak 75 ppm, E4 = ekstrak 100 ppm, E5 = ekstrak 200 ppm Contoh perhitungan: Kadar MDA K (+) A1:
y = 0,0163 + 0,1096 x 0,128 = 0,0163 + 0,1096 x x = 1,0192
% daya hambat K (+) = [MDA] kontrol negatif – [MDA] ekstrak x 100% [MDA] kontrol negatif = 5,4017 – 0,9857 x 100% 5,4017 = 81,75%
Lampiran 12 Absorban kurva standar 1,1,3,3-tetrametoksipropana (TMP) [TMP] (µM)
A1
A2
A3
Rerata ± SD
0
0
0
0
0
1
0,111 0,115 0,114
0,113 ± 0,0021
2,5
0,350 0,343 0,360
0,351 ± 0,0085
5
0,523 0,530 0,519
0,524 ± 0,0055
7,5
0,838 0,850 0,860
0,849 ± 0,0110
10
1,108 1,112 1,114
1,111 ± 0,0031
Lampiran 13 Aktivitas antioksidasi ekstrak etanol 70% daun sirih merah metode DPPH (λ = 517 nm) Larutan
A1
A2
A3
Kontrol 0,502 0,556 0,500
Rerata ± SD
Daya hambat (%)
0,519 ± 0,0317
0
E1
0,469 0,431 0,454
0,451 ± 0,0191
13,10
E2
0,415 0,363 0,330
0,369 ± 0,0428
28,90
E3
0,299 0,206 0,314
0,273 ± 0,0552
47,47
E4
0,245 0,176 0,211
0,211 ± 0,0345
59,34
E5
0,112 0,110 0,194
0,138 ± 0,0479
73,41
Keterangan: K = kontrol (larutan DPPH tanpa ekstrak), E1 = ekstrak 25 ppm, E2 = ekstrak 50 ppm, E3 = ekstrak 75 ppm, E4 = ekstrak 100 ppm, E5 = ekstrak 200 ppm Contoh perhitungan: % daya hambat E1 = absorban kontrol – absorban ekstrak x 100% absorban kontrol = 0,519 – 0,451 x 100% 0,519 = 13,10%
IC50: y = -85,9440 + 30,5332 ln(x) 50 = -85,9440 + 30,5332 ln(x) ln(x) = 4,4523 x = 85,82 ppm
Lampiran 14 Aktivitas antioksidasi α-tokoferol (vitamin E) metode DPPH (λ = 517 nm) Larutan
A1
A2
A3
Kontrol 0,500 0,501 0,498
Rerata ± SD
Daya hambat (%)
0,499 ± 0,0015
0
E1
0,306 0,315 0,309
0,310 ± 0,0045
37,87
E2
0,247 0,208 0,250
0,235 ± 0,0223
52,91
E3
0,198 0,200 0,204
0,201 ± 0,0030
59,72
E4
0,119 0,130 0,128
0,126 ± 0,0058
74,75
E5
0,090 0,100 0,105
0,098 ± 0,0076
80,36
Keterangan: K = kontrol (larutan DPPH tanpa α-tokoferol), E1 = α-tokoferol 1 ppm, E2 = α-tokoferol 2.5 ppm, E3 = α-tokoferol 5 ppm, E4 = α-tokoferol 7.5 ppm, E5 = α-tokoferol 10 ppm Contoh perhitungan: % daya hambat E1 = absorban kontrol – absorban ekstrak x 100% absorban kontrol = 0,499 – 0,310 x 100% 0,499 = 37,87%
IC50: y = 36,3614 + 18,0913 ln(x) 50 = 36,3614 + 18,0913 ln(x) ln(x) = 0,7538 x = 2,12 ppm
Lampiran 15 Inhibisi ekstrak etanol 70% daun sirih merah terhadap α-glukosidase (λ = 400 nm) Larutan
A1
A2
A3
[pNP]1
[pNP]2
[pNP]3
Rerata [pNP] ±
Daya
(µM)
(µM)
(µM)
SD
hambat (%)
K (-)
0,437
0,440
0,437
10,0863
10,1535
10,0863
10,1087 ± 0,0387
0
K (+)
0,086
0,080
0,084
2,2185
2,0839
2,1736
2,586 ± 0,0685
78,64
E1
0,437
0,425
0,435
10,0863
9,8173
10,0414
9,9816 ± 0,1441
1,26
E2
0,323
0,334
0,313
7,5309
7,7775
7,3068
7,5384 ± 0,2354
25,43
E3
0,279
0,283
0,288
6,5446
6,6343
6,7464
6,6417 ± 0,1011
34,30
E4
0,270
0,268
0,263
6,3429
6,2981
6,1860
6,2756 ±0,0808
37,92
E5
0,262
0,268
0,248
6,1636
6,2981
5,8497
6,1038 ± 0,2301
39,62
Keterangan: K = kontrol negatif (enzim dan substrat) dan positif (acarbose 1%), E1 = ekstrak 0,1%, E2 = ekstrak 0,25%, E3 = ekstrak 0,5%, E4 = ekstrak 0,75%, E5 = ekstrak 1% Contoh perhitungan: Kadar pNP K (+) A1:
y = -0,012971 + 0,044612 x 0.086 = -0,012971 + 0,044612 x x = 2,2185
% daya hambat K (+) = [pNP] kontrol negatif – [pNP] ekstrak x 100% [pNP] kontrol negatif = 10,1087 – 2,1586 x 100% 10,1087 = 78,64%
Lampiran 16 Hasil kurva standar p-nitrofenol (pNP) [pNP] (µM)
A1
A2
A3
Rerata ± SD
0
0
0
0
0
1
0,034
0,034
0,034
0,034 ± 0
2,5
0,101
0,106
0,100
0,102 ± 0,0032
5
0,206
0,206
0,210
0,207 ± 0,0023
7,5
0,312
0,300
0,308
0,306 ± 0,0061
10
0,400
0,400
0,400
0,400 ± 0
12,5
0,560
0,575
0,550
0,562 ± 0,0125
15
0,691
0,660
0,665
0,672 ± 0,0166
Lampiran 17 Hasil analisis kinetika inhibisi ekstrak etanol 70% daun sirih merah terhadap α-glukosidase (λ = 400 nm) Reaksi enzim substrat tanpa inhibitor (ekstrak 1%) [S]
A1
A2
(mM)
[pNP]1
[pNP]2
Aktivitas1
Aktivitas2
Rerata aktivitas ±
(µM)
(µM)
(U/ml.menit)
(U/ml.menit)
SD
5
0,177
0,179
4,2583
4,3031
0,5677
0,5737
0,5707 ± 0,0042
10
0,221
0,220
5,2445
5,2222
0,6993
0,6963
0,6978 ± 0,0021
15
0,270
0,270
6,3429
6,3429
0,8457
0,8457
0,8457 ± 0
20
0,298
0,300
6,9705
7,0154
0,9294
0,9354
0,9324 ± 0,0042
25
0,320
0,325
7,4637
7,5757
0,9952
1,0101
1,0026 ± 0,0105
Reaksi enzim substrat dengan inhibitor (ekstrak 1%) [S]
A1
A2
(mM)
[pNP]1
[pNP]2
Aktivitas1
Aktivitas2
Rerata aktivitas ±
(µM)
(µM)
(U/ml.menit)
(U/ml.menit)
SD
5
0,032
0,028
1,0080
0,8883
0,1344
0,1184
0,1264 ± 0,0113
10
0,057
0,058
1,5684
1,5908
0,2091
0,2121
0,2106 ± 0,0021
15
0,098
0,099
2,4874
2,5098
0,3316
0,3346
0,3331 ± 0,0021
20
0,110
0,109
2,7564
2,7340
0,3675
0,3645
0,3660 ± 0,0021
25
0,136
0,135
3,3393
3,3168
0,4452
0,4422
0,4437 ± 0,0021
Nilai 1/[S], 1/V tanpa dan dengan inhibitor 1/[S]
1/V
1/V*
0,2
1,7522
7,9114
0,1
1,4331
4,7483
0,067
1,1825
3,0021
0,05
1,0725
2,7322
0,04
0,9974
2,2537
Keterangan: (*) dengan inhibitor
Persamaan garis: tanpa inhibitor:
y = 4,643 x + 0,863; r = 99,00
dengan inhibitor: y = 35,55 x + 0,879; r = 99,50
Contoh perhitungan: Aktivitas enzim: [pNP] (µM) V.t
V = volume enzim dalam sistem reaksi t = waktu inkubasi
Aktivitas enzim dengan [S] 5 mM =
1,0080 µM 0,5 mL.15 menit
= 0,1344 U/mL.menit
Lampiran 18 Hasil analisis GC-MS Abundance TIC: SIRIH MERAH.D 1.4e+07
1.2e+07
1e+07
8000000
6000000
4000000
2000000
0 10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
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Pk# RT Area% Library/ID Qual _____________________________________________________________________________ 1 9.87 1.80 C:\Database\wiley7n.l n-Hexadecanoic acid 98 Myristic acid 98 Palmitic acid 95
2 11.68 1.78
3 12.07 6.13
4 12.28 1.93
9 21.15 1.81
C:\Database\wiley7n.l trans-Phytol Phytol Phytol
91 90 90
C:\Database\wiley7n.l Linolenic acid ethyl linoleolat 9,12,15-Octadecatrien-1-ol
91 91 90
C:\Database\wiley7n.l Stearic acid Octadecanoic acid Octadecanoic acid
99 99 98
C:\Database\Willey and Nist.L myricetin $ 4'-hydroxy-3',5'-dimethoxyphenyl
43
4-amino-2-isopropyl-5-methylphenol [2,2'-Binaphthalene]-1,4,5',8'-tetrone 10 22.05 2.06 C:\Database\Willey and Nist.L Pyrazol N-[2-(2-Isopropyl-5-methyl-phenoxy)-ethyl]2-methylsulfanyl-benzamid 2-Propenoic Acid
35 30
25 25 25
12 23.56 4.96 C:\Database\Willey and Nist.L 2,4,6(1H,3H,5H)-Pyrimidenetrione Benzenemethanol Benzofuran
59 59 58
13 23.87 2.67 C:\Database\Willey and Nist.L Naphthalene Naphthalene Naphthalene
46 46 46
14 24.03 4.05 C:\Database\Willey and Nist.L 2,4,6(1H,3H,5H)-Pyrimidinetrione Benzo[B]Thiophene Benzene
59 53 50
15 24.89 12.19 C:\Database\Willey and Nist.L t-4-Methylstilbene oxide 3,5-Dimethoxybenzamide 3,4-Dimethoxybenzaldehyde oxime
30 25 25
16 26.12 4.52 C:\Database\wiley7n.l methyl (25R)-5-oxo-A-nor-3,5-secos pirostan-3-oate Endosulfan 4-(Ethoxycarbonyl)dibenzo[f,h]quin oline-2,3-dicarboxylate Acid N-Phenylimide
90 55 47
17 27.20 44.69 C:\Database\Willey and Nist.L 4,4'-Stilbenediamine Phenol C-1-(4-methoxyphenyl)-2-phenylethene
60 38 38
18 28.42 1.53 C:\Database\Willey and Nist.L Pyrimidine, 4-amino-6-methoxy-2-(t rifluoromethyl) Pyrimidine, 4-amino-6-methoxy-2-(t rifluoromethyl) Pyrimidine, 4-amino-6-methoxy-2-(t rifluoromethyl)
44 44 44
19 28.85 1.83
C:\Database\wiley7n.l 4-Allyloxy-6-methoxy-N,N-dimethyl-1,3,5-triazin-2-amine (E)-2-hydroxy-4'-methylstilbene tricyclo[4.3.3.0(1,6)]dodeca-7,10- diene-7,11-dinitrile
23 36.46 1.65 C:\Database\wiley7n.l Vitamin E dl-.alpha.-Tocopherol Vitamin E
91 64 55
99 98 96
Lampiran 19 Analisa statistika data aktivitas antioksidasi dan inhibisi enzim Kadar MDA terhadap waktu ANOVA
Between Groups Within Groups Total
Sum of Squares 118.085
df 9
Mean Square 13.121
.311
20
1.554E-02
118.396
29
F
Sig.
844.185
.000
Duncan N alpha = .05 Hari 1 2 3 4 5 6 7 8 9 10 0 3 1.3111 1 3 1.6852 2 3 2.3451 3 3 3.3002 4 3 4.0696 5 3 4.4346 9 3 5.0459 8 3 5.5477 7 3 5.8823 6 3 8.0751 Sig. 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed. a Uses Harmonic Mean Sample Size = 3.000.
Aktivitas antioksidasi ekstrak etanol 70% daun sirih merah ANOVA
Between Groups Within Groups Total
Sum of Squares 42.481
df 6
Mean Square 7.080
.369
14
2.637E-02
42.850
20
F
Sig.
268.490
.000
Duncan N alpha = .05 Konsentrasi 1 2 3 4 5 6 Vitamin E 3 .9857 200 ppm 3 1.0587 100 ppm 3 1.4753 75 ppm 3 1.9985 50 ppm 3 2.3604 25 ppm 3 3.0082 5.4017 Kontrol negatif 3 Sig. .591 1.000 1.000 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed. a Uses Harmonic Mean Sample Size = 3.000.
Aktivitas inhibisi enzim ekstrak etanol 70% daun sirih merah ANOVA
Between Groups Within Groups Total
Sum of Squares 131.201
df 6
Mean Square 21.867
.304
14
2.173E-02
131.505
20
F
Sig.
1006.349
.000
Duncan N alpha = .05 Konsentrasi 1 2 3 4 5 acarbose 3 2.1587 1% 3 6.1038 0.75% 3 6.2757 0.50% 3 6.6418 0.25% 3 7.5384 0.1% 3 9.9817 Kontrol negatif 3 10.1087 Sig. 1.000 .175 1.000 1.000 .309 Means for groups in homogeneous subsets are displayed. a Uses Harmonic Mean Sample Size = 3.000.
Kinetika Inhibisi enzim ANOVA Sum of Squares 1.695
Between Groups Within 2.975E-04 Groups Total 1.695
df 9
Mean Square .188
10
2.975E-05
F
Sig.
6331.022
.000
19
Duncan N alpha = .05 [S] 1 2 3 4 5 6 7 8 9 10 5 mM* 2 .1264 10 mM* 2 .2106 15 mM* 2 .3331 20 mM* 2 .3660 .4437 25 mM* 2 5 mM 2 .5707 10 mM 2 .6978 15 mM 2 .8457 20 mM 2 .9324 25 mM 2 1.0027 Sig. 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Means for groups in homogeneous subsets are displayed. a Uses Harmonic Mean Sample Size = 2.000 *) with inhibitor.
Lampiran 20 Dokumentasi hasil penelitian
larutan hasil maserasi
waktu inkubasi hari ke-6
Aktivitas antioksidan metode DPPH (konsentrasi ekstrak dari kiri ke kanan): kontrol negatif, 25, 50, 75, 100, dan 200 ppm
Aktivitas antioksidan metode TBA (konsentrasi ekstrak dari kiri ke kanan): 0, 25, 50, 75, 100, 200 ppm, dan vitamin E 200 ppm
Aktivitas antioksidan metode DPPH (konsentrasi vitamin E dari kiri ke kanan): kontrol negatif, 1, 2.5, 5, 7.5, dan 10 ppm
inhibisi enzim α-glukosidase (konsentrasi kiri ke kanan): acarbose 1% , 1%, 0.75%, 0.5%, 0.25%, 0.1% dan kontrol negatif
