acclimatized under control conditio

acclimatized under control condition of humidity 70–80% with 12 h light/dark cycle and free access to food and water under room temperature in individual cages for one week before use. The animals used were in concordance with procedure accepted by Animal Care and Use Committee, Faculty of Medicine, University of Malaya (Animal Ethic No: ISB/11/03/2009/MFM(R)).PREPARATION OF SIMVASTATIN DRUG AND PLANTS EXTRACT FOR ANIMAL TREATMENTSCommercial Simvastatin drug was purchased from Pharmaniaga. The dosage given to the treatment of rabbit in group D and water extract of R. tomentosa was 5 and 50 mg/kg, respectively. Treatment was given to the subject orally through force-feeding needle.PREPARATION OF ANIMALS BLOOD SAMPLEBlood were taken from ear vein of non-anaesthetized rabbit at week 0, week 5 and week 10 of the experimental period. Rabbits were kept under fasting condition at least 12 h before blood sampling to allow the relevant estimation of lipid profile levels. Blood volume was collected minimally at 7 mL using 21 G syringe. The animals were kept in rabbit restrainers and the ears were disinfected by wiping the central vein area thoroughly with 10% alcohol swab. Then, the needle was inserted at ¼ the length of the needle distally into the central vein, with the tips of the needle pointing toward the base of the ear. When needle is in place, blood was collected into open plain tube, as it should begin to flow immediately through the needle. EDTA tube for serum collection and Gel Tube for plasma collection was used. In order to obtain the serum, blood in the tube were leave clot for 30 min. Then, the tubes were centrifuged at 3000 rpm for 10 min. Serum and plasma obtained from the centrifugation was separated into 3 appendorf tubes for duplicate and kept under -80ºC before further analysis.ANALYSIS OF LIPID PEROXIDATION WITH TBARS-MELONDIALDEHYDE (MDA)
The standard MDA was prepared by adding together 0.01 mL 1,1,3,3-tetraetoxipropane (malondialdehyde tetraetil asetal) 4.05 M or MDA reagent into 1 L distilled water to form 0.04 mM MDA. Five different MDA standard concentrations (0.25, 0.5, 1.0, 2.0 and 4.0 nmol/mL) were prepared.
0.5 mL of standard MDA was pippetted out into 5 test tubes. Then, 2.5 mL trichloroasetic acid 1.22 M (TCA 1.22 M in HCl 0.5 M) and 1.5 mL tiobarbituric acid (0.67% TBA in 0.05 M NaOH) was added into each test tubes. Test tubes were tightly closed and heated in water bath at 100ºC for 30 min to allow the formation of MDA-TBA complex. Then the test tubes were taken out and allowed cooling at room temperature.
The MDA-TBA complex was extracted with 4 mL n-butanol and vortexes vigorously for 3 min. The mixture was centrifuged for 10 min at 3000 rpm (to separate

diaklimatisasi di bawah kondisi kontrol kelembaban 70-80% dengan 12 h cahaya / siklus gelap dan akses gratis ke makanan dan air di bawah suhu kamar dalam kandang individu selama satu minggu sebelum digunakan. Hewan-hewan yang digunakan berada di konkordansi dengan prosedur diterima oleh Perawatan Hewan dan Komite Penggunaan, Fakultas Kedokteran, Universitas Malaya (Animal Etika No: ISB / 11/03/2009 / MFM (R)).
PERSIAPAN SIMVASTATIN OBAT DAN TANAMAN EKSTRAK UNTUK HEWAN PERAWATAN
obat Commercial Simvastatin dibeli dari Pharmaniaga. Dosis yang diberikan kepada pengobatan kelinci di grup D dan ekstrak air R. tomentosa adalah 5 dan 50 mg / kg, masing-masing. Pengobatan diberikan kepada subjek secara lisan melalui kekuatan-makan jarum.
PERSIAPAN HEWAN SAMPEL DARAH
darah diambil dari telinga vena kelinci non-dibius pada minggu 0, minggu 5 dan minggu 10 dari periode eksperimental. Kelinci disimpan di bawah kondisi puasa setidaknya 12 jam sebelum pengambilan sampel darah untuk memungkinkan estimasi yang relevan dari tingkat profil lipid. Volume darah dikumpulkan minimal di 7 mL menggunakan 21 G jarum suntik. Hewan-hewan itu disimpan di restrainers kelinci dan telinga yang didesinfeksi dengan menyeka area vena sentral secara menyeluruh dengan 10% kapas alkohol. Kemudian, jarum dimasukkan di ¼ panjang jarum distal ke dalam vena sentral, dengan ujung jarum menunjuk ke arah pangkal telinga. Ketika jarum di tempat, darah dikumpulkan ke dalam tabung dataran terbuka, seperti yang seharusnya mulai mengalir segera melalui jarum. Tabung EDTA untuk koleksi serum dan Gel Tabung untuk koleksi plasma digunakan. Dalam rangka untuk mendapatkan serum, darah di dalam tabung yang meninggalkan gumpalan selama 30 menit. Kemudian, tabung disentrifugasi pada 3000 rpm selama 10 menit. Serum dan plasma yang diperoleh dari sentrifugasi dipisahkan menjadi 3 tabung appendorf duplikat dan disimpan di bawah -80ºC sebelum analisis lebih lanjut.
ANALISIS peroksidasi lipid DENGAN TBARS-MELONDIALDEHYDE (MDA)
MDA standar dibuat dengan menambahkan bersama-sama 0,01 mL 1,1,3 , 3-tetraetoxipropane (malondialdehid tetraetil asetal) 4.05 M atau MDA reagen ke dalam 1 L air suling untuk membentuk 0,04 mM MDA. Lima konsentrasi standar MDA yang berbeda (0,25, 0,5, 1,0, 2,0 dan 4,0 nmol / mL) disusun.
0,5 ml standar MDA pippetted keluar ke 5 tabung reaksi. Kemudian, 2,5 mL asam trichloroasetic 1,22 M (TCA 1,22 M di HCl 0,5 M) dan 1,5 mL asam tiobarbituric (0,67% TBA di 0,05 M NaOH) ditambahkan ke dalam setiap tabung reaksi. Tabung uji tertutup rapat dan dipanaskan dalam air mandi di 100 º C selama 30 menit untuk memungkinkan pembentukan kompleks MDA-TBA. Kemudian tabung reaksi dibawa keluar dan diizinkan pendinginan pada suhu kamar.
MDA-TBA kompleks diekstraksi dengan 4 mL n-butanol dan pusaran penuh semangat selama 3 menit. Campuran disentrifugasi selama 10 menit pada 3000 rpm (untuk memisahkan