53
DAFTAR PUSTAKA Abuja PM, Albertini, R. 2001. Methods for monitoring oxidative stress, lipid peroxidation and oxidation resistance of lipoproteins. Clin Chim Acta 306:1-17. Amic D, Davidovic-Amic D, Beslo D, Trinajstic N. 2003. Structure-Radical Scavenging Activity Relationships of Flavonoids.Croat. Chem. Acta. 76 (1): 55-61 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. Behbahani M, Ali AM, Muse R, Mohd NB. 2007. Anti-oxidant and antiinflamatory activities of leaves of Barringtonia racemosa. J Med Plant Res 96-102. Best B, 2007. Mechanisms of aging. http://www.benbest.com/lifeext/LESurvey.php. [ 15 April 2007] Blokhina O, Virolainen E, Fagrstedt KV. 2003. Antioxidants, oxidative damage and oxygen deprivation stress : a review. Annals of Botany 91:179-194 Buhler DR, Miranda C. 2000. Antioxidant activities of flavonoids. Department of Environmental and molecule toxicology. Oregon University. Http://oregonstate.edu/infocancer/phytochemicals/flavonoid/index.html [ 15 April 2007] Carballo et al. 2002. A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products. BMC Biotechnology (2):1472-6750 Chatterjee ML, Katiyar SK, Mohan RR, Agarwal R. 1999. A flavonoid antioxidant, silymarin, affords exceptionally high protection against tumor promotion in the SENCAR mouse skin tumorigenesis model. Cancer Research 59:622-632. Choo CY, Chan KT, Takeya K, Itokawa H. 2000. Cytotoxic activity of Typhonium flagelliforme (Araceae) [Abstract] http://www3.interscience.wiley.com/cgibin/abstract/80503177/abstract?cretry=1&sretry=0 Contran RS, Kumar, Robbins SL. 1994. Pathologic basis of desease. WB. Sunders Company, Philadelfia USA.
54
Colic M, Pavelic K. 2004. Molecular mechanisms of anticancer activity of natural Dietetic products.[Abstract]. http://www.springerlink.com/content/yn817tbul8fp.html. Cooper GM. 1993. The Cancer Book. JB Publishers, Boston USA. Cragg GM, Suffness M. 1993. Cancer in Human Medicinal Agents from Plants. Di dalam: Kinghorn AD, Balandrin MF, editor. ACS Symposium Series 534, pp. 81- 95 Dean JD. 2003. Flavone: The Molecular and Mechanistic Study of How a Simple Flavonoid Protects DNA from Oxidative Damage. [Thesis]. Department of Biochemistry and Molecular Biology, East Tennessee State University. [Depkes] Departemen Kesehatan. 1987. Analisis Obat Tradisional. Jilid I. Jakarta Eridani SN. 2006. Potensi antioksidasi beberapa ekstrak senyawa bahan alam yang berkhasiat sebagai antikanker. [Skripsi]. Institut Pertanian Bogor. Faraouq 2003. Ekstrak sebagai salah satu pengembangan bentuk obat tradisional. Prosiding Seminar Nasional Tumbuhan Obat Indonesia XXIII, Jakarta. 45-52 Farnsworth NR. 1996. Biological and Phytochemical Screening of Plant. Journal of Pharmaceutical Sciences, 5:225-236. Farkya S, Bisaria V, Srivastava AK. Biotechnological aspects of the production of the anticancer drug podophyllotoxin. [Abstract] http://www.springerlink.com [ 12 april 2007]. Franzen H. 2001. How tumor-suppressor gene p53 keeps cancer at bay. Scientifc American.com. http://www.sciam.com/ [19 april 2007]. Frigo DE, et al. 2002. Flavonoid phytochemichals regulate activator protein-1 signal transduction pathways in endemetrial and kidney stable cell lines. J. Nutr. 132:1848-1853. Gill SMK, Balasioner N, Parte P. 2001. Intermitent Treatment With Taxmoxiven on Reproduction in Male Rat. Asian. J. Andri 3(2)-P155-158. Gondhowiarjo S. 2004. Proliferasi Sel dan Keganasan. Maj Kedokt Ind 54(7): 289-299. Go VW, Butrum RR, Wong DA. 2003. Diet, Nutrition, and Cancer Prevention: The Postgenomic Era. J Nutr 133 (11S-I):3830S-3836S. Greenwald. 1996. Chemopevention of Cancer. J Scientific American 9:96-99.
55
Hafid AF. 2003. Aktivitas Anti-Radikal Bebas DPPH Fraksi Metanol Fagraea auriculata dan Fagraea ceilanica. Majalah Farmasi Airlangga, III (1): 34-39. Hanani E, Mun’im A, Sekarini R. 2005. Identifikasi senyawa antioksidan dalam spons Callyspongia sp dari kepulauan seribu. Majalah Ilmu Kefarmasian II (3):127-133. Hakim EH, Rudiyansyah, Musthapa I, Takeya K. 2003. Bergenin suatu Dihidroksikumarin dari kayu dan kulit batang Shorea stenopthera Burk. Proc ITB Sains and Tek 35A:87-96. Harborne JB. 1996. Metode Fitokimia, penuntun dan cara modern menganalisis Tumbuhan. Penerjemah : Padmawinata K dan Soediro I. Penerbit ITB Bandung. Harliansyah. 2001. Mengunyah halia menyah penyakit. Paksi Jurnal pp 92-97. Hidayat MA. 2002. Uji Aktivitas Antikanker Ekstrak Heksana Daun Eupatorium triplinerve Vahl. terhadap Kultur Sel Mieloma. J Ilmu Dasar 3 (2):92-97 p. 92-97. Horinaka M et al. 2006. The dietary flavonoid apigenin sensitizes malignant tumor cells to tumor necrosis factor-related apoptosis-inducing ligand. Mol Cancer Ther 5(4): 945-951). Hosseinian F. 2006. Antioxidant properties of flaxseed lignans using in vitro model systems. [Thesis]. College of Pharmacy and Nutrition. University of Saskatchewan Saskatoon, Canada. Hussain A, Zia M, Mirza B. 2006. Cytotoxic and antitumor potential of Fagonia cretica L. Turk J Biol 31:19-24. Jujena P, et al. 2001. Anti Fertility Effect Estradiol in Adult Female Rat. J Endokrinol Invest 249(8): 598-607. Katzung BG. 1995. Basic & Clinical Pharmacology, 7th edition. Prentice Hall International, pp. 881. Keku TO, Burris TR, Millikan R. 2003. Gene Testing: What the Health Professional Needs to Know. J Nutr 133 (11S-I):3754S-3757S. Khantamat O, Chaiwangyen W, Limtrakul P. 2004. Screening of flavonoids for Their potential inhibitory effect on P-glycoprotein activity in human Cervical carcinoma KB-cells. Chiang Mai Med Bull 43(2):45-56 Kikuzaki H, Nakatani N. 1993. Antioxidant effects of some ginger constituents. J Food Sci 58:1407-10.
56
Kubo S, Mimaki Y, Terao M, Sashida Y, Nikaido T, Ohmoto T. 1992. Acylated cholestane glycosides from the bulbs of Ornithogalum saundersiae. Phytochemistry 31:3969–3973. Lee KW, Lee HJ, Lee CY. 2004. Vitamins, Phytochemicals, Diets and Their Implementation in Cancer Chemoprevention. Crit Rev Food Sci Nutr 44: 437-447. Lenny S. 2006. Isolasi dan uji bioaktifitas kandungan kimia utama puding merah dengan metoda uji brine shrimp. USU Repository Online. Mates JM, Gomez CP, De Castro IN. 1999.Antioxidant enzymes and human diseases. Clin Biochem 32(8) : 595-603. Maxwell SRJ, Lip GYH. 1997. Free radicals and antioxidants in cardiovascular desease. Br J Clin Pharmacol 44:307-317. Mc Cord JM. 2000. The evolution of free radicals and oxidative stress. The American J of Medicine 108 (8):652-659. Mc Laughlin JL. 1991. Crown Gall Tumours on Potato Disc and Brine Shrimp Lethality: Two Simple Bioassay for Higher Plant Screening and Fractination, Di dalam: Hostettman K, editor, Methods in Plants Biochemistry, Academic Press, 6, p. 1-32. Mc Laughlin JL et al, 1998. The use of biological assays to evaluate botanicals. Drug information journal, vol 32, pp 513-524. McKelvery KD, Evans JP. 2003. Cancer Genetics in Primary Care. J Nutr 133 (11S-I): 3767S-3772S. Milner JA. 2004. Molecular Targets for Bioactive Food Components. J Nutr 134(9): 2492S-2498S. Mimaki Y et al. 1997. Cholestane glycosides with potent cytostatic activities on various tumor cells from Ornithogalum saundersiae bulbs. Bioorg Med Chem Lett 7:633–636. Mosaddik MA. 2004. In vitro cytotoxicity of tanshinones isolated from Salvia miltiorrhiza Bunge against P388 lymphoctic leukimia cells. [abstract] http://www.sciencedirect.com/science/journal/09447113. Mosquera OM, Correa YM, Buitrago DC, Nino J. 2007. Antioxidant activity of twenty five plants from Colombian biodiversity. Mem Inst Oswaldo Cruz 102(5):631-634.
57
Mun’im A, Andrajati R, Susilowati H. 2006. Uji hambatan tumorigenesis sari Buah merah (Pandanus conoideus Lam.) terhadap tikus putih betina yang diinduksi 7,12 Dimetilbenz(a)antrasen (DMBA). Majalah Ilmu Kefarmasian III (3):153-161. Murray RK, Granner DK, Mayes PA, Rodwell VW. 2003. Harper’s illustrated Biochemistry 21st edition. Lange Medical Books/McGraw-Hill. Nambiar Pr, Jackson JA. Chelack BJ, Kidney BA, Jaines, DM. 2001. Immunohistochemical Detection of Tumor Suppressor Gene p53 Protein in Feline Injection Site-associated Sarcomas. Vet Pathol 38:236–238. Nowell SA, Ahn J, Ambrosone CB. 2004. Gene-Nutrient Interaction in Cancer Etiology. Nutr Rev 62 (11): 427-434. Okawa M, Kinjo J, Nohara T, Ono M. 2001.DPPH (1,1-Diphenyl-2Picrylhydrazyl) Radical Scavenging Activity of Flavonoids Obtained from Some Medicinal Plants. Biol Pharm Bull 24(10):1202-1205. Oteiza PI, Erlejman AG, Verstraeten SV, Keen CL, Franga CG. 2005. Flavonoidmembrane interactions : a protective role of flavonoid at the membrane surface? Clinical & Development Immunology 12(1):19-25. Peng IW dan Kuo SM. 2003. Flavonoid structure affects the inhibition of lipid peroxidation in Caco-2 intestinal cells at physiological concenterations. American Society for Nutrional Sciences. 2184-2187. Pitot H, Dragan Y. 1991. Facts and theories concerning the mechanism of carcinogenesis. J. Faseb. 4(10):112-118. Pourmorad F, Hosseinimehr SJ, Shahabimajd N. 2006. Antioxidant activity, phenol and flavonoid contents of some selected Iranian medicinal plants. African Journal of Biotech 5(11):1142-1145. Puspitasari HP, Sukardiman, Widyawayuranti. 2003. Uji Aktivitas Sitotoksik Ekstrak Metanol Herba Ageratum conyzoides L. Pada Kultur Sel Mieloma Mencit. Majalah Farmasi Airlangga 3:93-95. Ren et al. 2003. Medicinal Research. Reviews Medical. 23 (4): 519-534. Ricardo MAG et al. 2004. Bioactive pyrones and flavonoids from Cryptocarya ashersoniana seedlings. Issue in honor of Prof. Otto Gottlieb.ISSN 14246376 Arkat USA. Rusmarilin H. 2003. Aktivitas Anti-Kanker Ekstrak Rimpang Lengkuas Lokal (Alpinia galanga (L) Sw ) Pada Galur Sel Kanker Manusia Serta Mencit Yang Ditransplantasi Dengan Sel Tumor Primer. [Disertasi]. Bogor : Sekolah Pascasarjana Institut Pertanian Bogor.
58
Sampels S. 2005. Fatty Acids and Antioxidants in Reindeer and Red Deer. [Dissertation]. Swedish University of Agricultural Sciences. Sarjono PAR. 2004. Potensi Isoflavon Asal Limbah Tahu Sebagai Antikanker Dalam Penghambatannya Terhadap Enzim Tirosin Kinase. [Tesis]. Bogor : Sekolah Pascasarjana Institut Pertanian Bogor. Silalahi J, Tambunan ML. 2003. Zat Bersifat Antikanker di Dalam Makanan. Medika; 39 (7): 440-446. Silalahi J. 2006. Antioksidan dalam Diet dan Karsinogenesis. Cermin Dunia Kedokteran 153:39-42. [SIBS] Salk Institute For Biological Studies. 2005. Cancer related gene p53 not regulated as indicated by previous tissue culture research; results may be relevant to drug development. http://www.salk.edu/index.php [ 19 April 2007]. Simbala EI. Rondonuwu SJ. Achmad AS. De Queljoe E. 2004. Pemberdayaan Keragaman Hayati Tumbuhan Obat di Sulawesi Utara : Kajian Botani, Etnobotani, Fitokimia dan Konservasi. Laporan Penelitian RISTEK, Kementerian Riset dan Teknologi. 77 -158. Simpson JA. 2006. Antioxidant properties of peanut plant leaves and roots and contribution of specific phenolic compounds to antioxidant capacity. [Thesis]. Food Science. North Carolina State University. Silva et al. 2002. Structure antioxidant activity relationship of flavonoid : a reexamination. Free Radical Research, 36 (11):1219-1227. Su X, Duan J, Jiang Y, Duan X, Chen F. 2007. Polyphenolic profile and antioxidant activities of Oolong tea infusion under various steeping conditions. Int J Mol Sci 8:1196-1205. Suffness M. 1987. Biologically Active Natural Products. Di dalam : Hostettmann K, Lea PJ, editor. Oxford University Press, Oxford, pp.85–104. Sukardiman, Santa IGP, Rahmaday. 1995. Efek antikanker isolat flavonoid dari herba benalu mangga (Dendeophytoe petandra). Cermin Dunia Kedokteran 122:5-8 Sukardiman, Rahman A, Pratiwi NF. 2004. Uji praskrining aktivitas antikanker ekstrak eter dan ekstrak metanol Marchantia cf. planiloba Steph.Dengan Metode Uji Kematian Larva Udang dan Profil Densitometri Ekstrak Aktif. Majalah Farmasi Airlangga 4(3): 97-100.
59
Sukardiman, Ekasari W, Hapsari PP. 2006. Aktivitas antikanker dan indukasi apoptosis fraksi kloroform daun pepaya (Carica papaya L.) terhadap kultur sel kanker mieloma. Media Kedokteran Hewan 22:104-111. Trusheva B, Trunkova D, Bankova V. 2007. Different extraction methods of biologically active components from propolis : a preliminary study. Chemistry Central Journal. 1 (13):1186/1752. Tukozkan N, Erdamar H, Seven I. 2006. Measurement of total malondialdehyde in plasma and tissues by High-Performance Liquid Chromatography and Thiobarbituric acid assay. Fırat Tıp Dergisi. 11(2): 88-92 Usman H, Hakim EH, Achmad SA, Harlim T. 2005. 2’,4’-Dihidroksi-3’,5’,6’Trimetoksi Calkon suatu Senyawa Antitumor dari Kulit Batang Tumbuhan Cryptocarya costata (Lauraceae). J Matematika dan Sains. 10(3):97-100. Valko M et al. 2006. Free radical, metals and antioxidants in oxidative stressinduced cancer. Chem Biol Interact. 160(1):1-40. Yang D, Wang Q, Ke L, Jiang J, Ying T. 2007. Antioxidant activities of various extracts of lotus (Nelumbo nucifera Gaertn) rhizome. Asia Pac J Clin Nutr. 16(Suppl 1):158-163 Yokota , Sugimura T. 1993. Multiple steps in carcinogenesis involving alterations of multiple tumor suppressor genes. The FASEB Journal 7:920-925. Young HY, Chiang, CT, Huang, YL, Pan FP, Chen GL. 2002. Analytical and stability studies of ginger preparations. J Food and Drug Anal 10(3):145-153f. Walker WA, Blackburn G. 2004. Symposium Introduction: Nutrition and Gene Regulaton. J. Nutr. 134(9): 2434S-2436S. Webb MR, Ebeler SE. 2004. Comparative analysis of topoisomerase IB inhibition and DNA intercalation by flavonoids and similar compounds : structural determinates of activity. Biochem J 384: 527–541. Williams RB. 2005. Searching For Anticancer Natural Products From The Rainforest Plants Of Suriname And Madagascar. [Dissertation] Virginia Polytechnic Institute and State University. Zhu J, Xiong L, Yu B, Wu J. 2005. Apoptosis induced by a new member of saponin family mediated through caspase-8-dependent cleavage of Bcl-2. Mol Pharmacol (68):1831-1836.
Lampiran 1 Diagram Alir Penelitian Ekstraksi
Filtrat
Freeze dryer
Ekstrak Kasar
KLBBetoh
Analisa Fitokimia (Harborne 1996)
KLBBka
KLBKetoh
Uji antioksidan dengan Metode DPPH dan Metode TBA
KLBBka
-
Uji toksisitas Metode BSLT Uji in vitro antikanker pada sel Murine Leukimia P-388
Keterangan : KLBBetoh (kulit batang basah ekstraksi dengan etanol 70%), KLBBka (kulit batang basah ekstraksi dengan kloroform:air), KLBKetoh (kulit batang kering ekstraksi dengan etanol 70 %), dan KLBKka (kulit batang kering ekstraksi dengan kloroform:air)
60
Lampiran 2 Ekstraksi Kulit Batang Lansium domesticum L. (Harborne, 1996) Kulit Batang L. domesticum L
Kulit Langsat Batang Basah
Kulit Langsat Batang Kering
Dihaluskan
Dikeringanginkan & Haluskan
1:5 ( 40 gr : 200 ml etanol )
Di meserasi dengan etanol 70 %, selama 24 jam pada suhu 25 0 C
Filtrat
Residu
Di meserasi dengan etanol 70 %, selama 24 jam pada suhu 25 0 C
Filtrat
Residu
Kloroform : air (1 : 1)
Filtrat
Filtrat
Filtrat
Filtrat
Freeze dryer/Rotapavor
Freeze dryer/Rotapavor
Ekstrak Kasar : KLBB EtOH dan KLBB kloroform:air
Ekstrak Kasar : KLBK EtOH dan KLBK kloroform:air
61
Lampiran 3 Analisis antioksidasi metode TBA Buffer fosfat, asam linoleat dan air bebas ion
Buffer fosfat, asam linoleat dan ekstrak kasar 200 ppm terdiri :
KLBK etoh
Buffer fosfat, asam linoleat dengan vit. E 200 ppm, air bebas ion
KLBB etoh
Oksidasi dengan udara selama 6-7 hari pada suhu 400C
Pengukuran Kadar MDA dengan uji TBA
62
Analisis Konsenterasi MDA menggunakan Metode TBA Vit. E
KLBKetoh
Botol gelap berulir
Tiap larutan diambil 1 mL
Inkubasi 400C selama 8 hari
Tabung reaksi KLBBetoh
Asam Linoleat
+ TCA 20 % + TBA 1 % dalam asam asetat 50 %
Inkubasi pada 1000 C selama 10 menit, dinginkan
Sentrifugasi pada 3000 rpm selama 15 menit
Ukur absorbansi pada λ 522 nm
63
64
Pembuatan Kurva Standar TMP
TMP 6 M
0.15 µM
0.15 µM
0.15 µM
0.15 µM
Tiap larutan dipipet 0.5 mL
Tabung reaksi
Inkubasi 100 0 C, 10 menit, dinginkan
Sentrifugasi 3000 rpm 15 menit
Diukur absorbansi pada λ 532 nm
0.15 µM
65
Analisis hidroperoksida dari asam linoleat menggunakan metode FTC yang dimodifikasi (Aqil et. al. 2006)
2 mL buffer fosfat 0.1 M pH 7.2 asam linoleat 50 mM dalam etanol 99.98 % 1 mL air bebas
Botol gelap berulir
Diambil 50 µL setiap hari
Tabung Reaksi + 6 mL etanol 75 % + 50 µL amonium tiosianat 30% + 50 µL FeCl2 20 mM dalam HCL 3,5 %
Diamkan 3 menit tepat
Diukur absorbansi pada λ 532 nm
66
Lampiran 4 Pengambilan sampel dan ekstraksi
Pegambilan sampel kulit batang di perkebunan rakyat Kabupaten Minahasa Utara, Propinsi Sulawesi Utara
Simplisia kulit batang langsat basah (KBLB) dan kulit langsat batang kering (KBLK)
Penyaringan hasil maserasi dan penguapan residu pelarut dengan Rotapavor
Ekstrak kasar KBLK dan KBLB
67
Lampiran 5 Hasil ekstraksi dan analisis fitokimia Hasil ekstraksi Simplisia Pelarut KBLK Etanol 70% Kloroform:air KBLB Etanol 70% Kloroform:air n-heksan
Bobot sampel (g) 40,03 40,33 40,17 40,24 50,12
Bobot ekstrak (g) 2,3722 1,7210 1,4665 0,8628 1,7602
% Rendemen = Berat ekstrak / berat sampel x 100 %
Foto hasil analisis fitokimia Flavonoid : KBLKEtOH (+), KBLK klor:air (-), KBLBEtOH (+),KBLB klor:air (++) (Indikasi : warna kuning, merah dan jingga pada lapisan amil alkohol)
Saponin : KBLKEtOH (+),KBLK klor:air (-), KBLBEtOH (++),KBLB klor:air (+++) (Indikasi : adanya busa yang stabil selama ± 10 menit)
Tanin : KBLKEtOH (-),KBLK klor:air (-), KBLBEtOH (-),KBLB klor:air (-) (Indikasi : Warna biru tua atau hitam kehijauan menunjukkan adanya tanin)
% Rendemen 5,9205 4,2625 3,6603 2,1410 3,5104
68
Alkaloid : Dari kiri ke kanan : Pareaksi Meyer, Pareaksi Wagner dan Pareaksi Dragendorff Dari atas ke bawah KBLKEtOH (-/+), KBLK klor:air (+), KBLBEtOH (+) KBLB klor:air (-)
(Indikasi : Pareaksi Meyer (enndapan putih); pareaksi Wagner (endapan cokelat); pareaksi Dragendorff (endapan merah jingga) Berturut-turut kiri atas ke kanan : Triterpenoid : KBLKEtOH (+),KBLK klor:air (-), KBLBEtOH (-),KBLB klor:air (-) (Indikasi : warna merah atau ungu menunjukkan adanya triterpenoid)
Berturut-turut kiri atas ke kanan : Steroid : KBLKEtOH (+),KBLK klor:air (-), KBLBEtOH (-),KBLB klor:air (-) (Indikasi : warna hijau atau biru menunjukkan adanya Steroid)
69
Lampiran 6 Analisis hasil uji toksisitas metode BSLT Jenis Ekstrak
Konsenterasi
Kontrol Tween 80 KBLK etoh
1000 500 100 10 1000 500 100 10
KBLB etoh
Jumlah Larva yang mati Ulangan Ulangan Ulangan 1 2 3 0 0 0 0 0 0 10 9 10 9 8 9 5 4 4 3 2 3 10 10 9 8 7 8 7 3 7 2 3 2
Persen mortalitas
96.667 86.667 43.333 26.667 96.667 76.667 56.667 23.333
Penentuan Nilai IC50 dengan Analisis Probit menggunakan Minitab 14 ————— 6/12/2007 18:01:28 ———————————————————— Welcome to Minitab, press F1 for help. Retrieving project from file: 'C:\Documents and Settings\MOKOSULI TUEGEH\My Documents\BSLT\BSLT_1.MPJ'
Results for: Worksheet 3 Probit Analysis: MATI, LARVA versus KONSENTERASI, EKSTRAK Distribution: Weibull Response Information Variable Value Count MATI Success 361 Failure 239 LARVA Total 600 Factor Information Factor Levels Values EKSTRAK 5 KBK etoh, KBK ka, KLB etoh, KLB ka, KLB nh Estimation Method: Maximum Likelihood
Regression Table Variable Constant KONSENTERASI EKSTRAK KBLK ka KBLB etoh KBLB ka KBLB nh Natural Response
Coef -3.36150 0.660010 -0.204194 -0.0487059 0.0093225 -0.258712
Test for equal slopes:
Standard Error 0.305680 0.0488364 0.200459 0.195681 0.196857 0.200974
Z -11.00 13.51 -1.02 -0.25 0.05 -1.29
P 0.000 0.000 0.308 0.803 0.962 0.198
0 Chi-Square = 15.4067
DF = 4
P-Value = 0.004
70
Log-Likelihood = -260.267 Multiple degree of freedom test Term Chi-Square DF P EKSTRAK 3.00590 4 0.557 Goodness-of-Fit Tests Method Chi-Square DF P Pearson 27.8779 14 0.015 Deviance 32.3364 14 0.004
EKSTRAK = KBLK etoh Tolerance Distribution Parameter Estimates Standard Parameter Estimate Error Shape 0.660010 0.0488364 Scale 162.894 34.9713
95.0% Normal CI Lower Upper 0.570910 0.763017 106.945 248.112
Table of Percentiles Percent 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 91 92 93 94 95 96 97 98 99
Percentile 0.153092 0.440944 0.821380 1.28006 1.80918 2.40381 3.06064 3.77736 4.55232 5.38439 16.7852 34.1618 58.8708 93.4834 142.686 215.799 334.998 576.378 616.805 663.089 716.921 780.819 858.752 957.506 1090.11 1286.67 1647.43
Standard Error 0.0903340 0.228306 0.391091 0.572157 0.768189 0.977190 1.19785 1.42926 1.67077 1.92192 4.92651 8.87831 14.0597 21.0404 30.8918 45.8117 71.2224 126.653 136.393 147.686 161.004 177.054 196.965 222.688 258.023 311.914 414.858
95.0% Fiducial CI Lower Upper 0.0401004 0.424487 0.136586 1.08195 0.280309 1.87892 0.467604 2.78818 0.696392 3.79578 0.965374 4.89327 1.27371 6.07515 1.62086 7.33768 2.00652 8.67828 2.43054 10.0952 8.82085 28.3859 19.5058 54.8200 35.4863 91.6046 58.5157 142.891 91.7037 216.307 141.129 327.264 221.022 513.184 379.225 906.291 405.277 974.001 434.960 1052.11 469.300 1143.71 509.817 1253.43 558.897 1388.65 620.592 1562.06 702.629 1798.26 822.725 2154.85 1039.11 2826.84
Table of Survival Probabilities 95.0% Fiducial CI Stress Probability Lower Upper 1000 0.0364263 0.0101878 0.0833207
EKSTRAK = KBLB etoh Tolerance Distribution Parameter Estimates Standard Parameter Estimate Error Shape 0.660010 0.0488364 Scale 175.369 36.9535
95.0% Normal CI Lower Upper 0.570910 0.763017 116.035 265.043
71
Table of Percentiles
Percent 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 91 92 93 94 95 96 97 98 99
Percentile 0.164817 0.474714 0.884287 1.37810 1.94774 2.58791 3.29504 4.06665 4.90097 5.79676 18.0707 36.7781 63.3796 100.643 153.613 232.326 360.655 620.521 664.044 713.873 771.828 840.619 924.521 1030.84 1173.59 1385.22 1773.60
Standard Error 0.0974290 0.246166 0.421557 0.616540 0.827524 1.05234 1.28957 1.53822 1.79758 2.06713 5.28144 9.48535 14.9687 22.3245 32.6733 48.3208 74.9662 133.175 143.415 155.293 169.306 186.201 207.169 234.273 271.529 328.392 437.127
95.0% Fiducial CI Lower Upper 0.0430401 0.457630 0.146681 1.16578 0.301150 2.02366 0.502540 3.00195 0.748645 4.08558 1.03809 5.26545 1.36999 6.53560 1.74379 7.89197 2.15918 9.33177 2.61601 10.8531 9.51090 30.4627 21.0630 58.7433 38.3703 98.0297 63.3485 152.727 99.3881 230.940 153.110 349.051 239.999 546.859 412.100 965.025 440.439 1037.05 472.728 1120.14 510.083 1217.59 554.155 1334.32 607.539 1478.18 674.640 1662.68 763.859 1914.00 894.456 2293.45 1129.72 3008.60
Table of Survival Probabilities 95.0% Fiducial CI Stress Probability Lower Upper 1000 0.0426387 0.0133225 0.0919216
Table of Relative Potency Factor: EKSTRAK Relative Comparison Potency KBK etoh VS KBK ka 1.36258 KBK etoh VS KLB etoh 1.07659 KBK etoh VS KLB ka 0.98597 KBK etoh VS KLB nh 1.47991 KBK ka VS KLB etoh 0.79011 KBK ka VS KLB ka 0.72361 KBK ka VS KLB nh 1.08611 KLB etoh VS KLB ka 0.91583 KLB etoh VS KLB nh 1.37463 KLB ka VS KLB nh 1.50096
95.0% Fiducial CI Lower Upper 0.749852 2.50365 0.598018 1.93491 0.545875 1.77946 0.813779 2.72911 0.432155 1.42622 0.394573 1.31131 0.590225 2.00429 0.510689 1.64380 0.760840 2.52266 0.827563 2.76276
Nilai IC50 Uji Toksisitas BSLT Ekstrak
LC50 (ppm)
KBK Etoh
93.4834
KBB EtoH
100.643
72
Lampiran 7 Analisis hasi uji antioksidasi metode DPPH inhibisi abs. blanko – abs pada konsenterasi x % Inhibisi = ------------------------------------------------------------ x 100 % abs. blanko 1.081 – 0.959 Misalnya : % inhibisi KBLK EtOH 10 ppm = ------------------- x 100% = 11,28585 1.081
1. KBLKetoh No 1 2
Sampel Blanko KBLK etoh
Kons 0 10 50 100 200 250
Abs 1,081 0,959 0,73 0,598 0,567 0,457
% Inhibisi 11,28585 32,46994 44,68085 47,54857 57,72433
2. KBLBetoh No 3
Sampel KLBB etoh
Kons 10 50 100 200 250
Abs 0,957 0,729 0,678 0,558 0,478
% Inhibisi 11,47086 32,56244 37,2803 48,38113 55,78168
73
3. Kontrol BHT No 4
Sampel BHT
Kons 10 50 100 200 250
Abs 0.998 0.892 0.663 0.62 0.612
% Inhibisi 7.678076 17.48381 38.6679 42.6457 43.38575
Pengitungan Nilai IC50 No
Sampel KBLK etoh KLBB etoh BHT
y 50 50 50
a 13,55 12,96 12,067
b 19,92 19,01 22,252
lnx 5,160147601 5,324845679 5,987569404
x 174,19 205,38 398,44
74
Nilai IC50 diperoleh dari persamaan regresi 1. KBLK EtOH : y = 13,55ln(x) - 19,22 2. KBLB EtOH : y = 12,96ln(x) - 19,01 3. BHT : y = 12,06ln(x) - 22,25
; R² = 0,974 ; R² = 0,882 ; R² = 0,978
Nilai x adalah nilai IC50 apabila y=50. Misalnya : Hasil persamaan regresi KBLK adalah nilai x dari ln (x). lnx ln x x
y = 15,36ln(x) - 26,99. Maka nilai IC50 KBLK
50 + 19,22 = ------------------13,55 = 5,160 = 174,19 = IC50 KBLK
Nilai IC50 KODE SAMPEL KBLK etoh KBLK etoh BHT
IC50 174,19 205,38 398.44
75
Lampiran 8 Analisis hasil uji antioksidan metode TBA Tabel Analisis Hidroperoksida Hari ke 1 2 3 4 5 6
1 0.742 0.908 1.325 1.068 1.358 1.002
Absorbansi 2 0.678 0.939 1.208 1.136 1.289 0.988
3 0.789 0.953 1.336 1.234 1.431 1.042
Aborbansi rata-rata 0.736 0.933 1.290 1.146 1.359 1.011
76
Absorbansi Standar TMP Konsenterasi 0.00 0.50 1.00 1.50 2.00 2.50 3.00
Absorbansi Absorbansi rata-rata 1 2 0.000 0.000 0.000 0.019 0.007 0.013 0.018 0.010 0.014 0.028 0.026 0.027 0.042 0.038 0.040 0.073 0.064 0.069 0.124 0.111 0.118
Tabel Konsenterasi MDA yang terbentuk dan % daya hambat oksidasi
Konsentrasi Asam linoleat 1 Asam linoleat 2 Asam linoleat 3 Vitamin E 200 ppm 1 Vitamin E 200 ppm 2 Vitamin E 200 ppm 3 KLBK etoh 50 ppm 1 KLBK etoh 50 ppm 2 KLBK etoh 50 ppm 3 KLBK etoh 100 ppm 1 KLBK etoh 100 ppm 2 KLBK etoh 100 ppm 3 KLBK etoh 200 ppm 1 KLBK etoh 200 ppm 2 KLBK etoh 200 ppm 3 KLBK etoh 500 ppm 1 KLBK etoh 500 ppm 2 KLBK etoh 500 ppm 3 KLBK etoh 1000 ppm 1 KLBK etoh 1000 ppm 2 KLBK etoh 1000 ppm 3 KLBB etoh 50 ppm 1 KLBB etoh 50 ppm 2 KLBB etoh 50 ppm 3
A hari ke-0
A hari ke-7
[MDA] hari ke0 (M)
[MDA] hari ke-7 (M)
[MDA] (M)
0.053 0.045 0.043 0.054 0.056 0.073 0.047 0.053 0.047 0.057 0.056 0.047 0.053 0.063 0.067 0.053 0.059 0.062 0.058 0.062 0.053 0.058 0.063 0.058
1.073 0.983 0.934 0.248 0.241 0.326 0.611 0.711 0.543 0.381 0.383 0.325 0.215 0.232 0.225 0.245 0.267 0.347 0.348 0.348 0.246 0.310 0.311 0.312
1.238 1.031 0.979 1.264 1.316 1.756 1.083 1.238 1.083 1.342 1.316 1.083 1.238 1.497 1.601 1.238 1.394 1.472 1.368 1.472 1.238 1.368 1.497 1.368
27.663 25.332 24.062 6.290 6.109 8.311 15.694 18.285 13.933 9.736 9.788 8.285 5.435 5.876 5.694 6.212 6.782 8.855 8.881 8.881 6.238 7.896 7.922 7.948
26.425 24.301 23.083 5.026 4.793 6.554 14.611 17.047 12.850 8.394 8.472 7.202 4.197 4.378 4.093 4.974 5.389 7.383 7.513 7.409 5.000 6.528 6.425 6.580
Ratarata [MDA] (M) 24.603
Daya hambat oksidasi (%) 0.000
5.458
77.817
14.836
39.698
8.022
67.392
4.223
82.836
5.915
75.956
6.641
73.008
6.511
73.535
77
KLBB etoh 100 ppm 1 KLBB etoh 100 ppm 2 KLBB etoh 100 ppm 3 KLBB etoh 200 ppm 1 KLBB etoh 200 ppm 2 KLBB etoh 200 ppm 3 KLBB etoh 500 ppm 1 KLBB etoh 500 ppm 2 KLBB etoh 500 ppm 3 KLBB etoh 1000 ppm 1 KLBB etoh 1000 ppm 2 KLBB etoh 1000 ppm 3
0.083 0.083 0.087 0.067 0.065 0.063 0.067 0.072 0.063 0.057 0.053 0.063
0.280 0.280 0.283 0.210 0.198 0.208 0.278 0.276 0.263 0.267 0.256 0.246
2.016 2.016 2.119 1.601 1.549 1.497 1.601 1.731 1.497 1.342 1.238 1.497
7.119 7.119 7.197 5.306 4.995 5.254 7.067 7.016 6.679 6.782 6.497 6.238
5.104 5.104 5.078 3.705 3.446 3.756 5.466 5.285 5.181 5.440 5.259 4.741
5.095
79.291
3.636
85.223
5.311
78.413
5.147
79.080
Rata-rata MDA linoleat yang terbentuk Daya hambat oksidasi = ------------------------------------------------------------------ x 100 % Rata-rata MDA tiap perlakuan yang terbentuk
78
Gambar 16 Pembentukan warna pada uji TBA KLBK dari kanan ke kiri : Asam linoleat, KLBK50 ppm, KLBK 100 ppm, KLBK 200 ppm, KLBK 500 ppm, KLBK 1000 ppm dan α-tokoferol.
Gambar 17 Pembentukan warna pada uji TBA KLBB dari kiri kekanan : Asam linoleat, KLBB50 ppm, KLBB 100 ppm, KLBB 200 ppm, KLBB 500 ppm, KLBB 1000 ppm dan α-tokoferol.
79
80
Lampiran 9 Hasil uji sitotoksisitas pada sel murine leukimia P388
Hasil Bioassay Sitotoksik terhadap Sel Murine Leukemia P388 [ ex HSRRB Lot Number : 113098 seed (JCRB0017) ]
Hasil Optical Dencity (OD) perlakuan ekstrak pada berbagai konsenterasi Konsenterasi (ppm) 100 30 10 3 1 0,3 0,1
OD (optical dencity) KLBK EtOH - 0,008 0,098 0,112 0,110 0,161 0,190 0,224
Positive Control = 0,219
Nilai IC50 Ekstrak Sampel KLBB EtOH KLBK EtOH
Nilai IC50 15,8 ug/mL 12,0 ug/mL
Catatan : Menurut National Cancer Institute *Aktif untuk senyawa murni =< 4,00 ug/mL *Aktif untuk ekstrak =< 20,0 ug/mL
KLBB EtOH 0.001 0.011 0.185 0,190 0,259 0,190 0,187
81
Lampiran 10 Formulasi Medium RPMI
