1. The Effect of Strobilanthes crispus Extract on Serum Lipid Profile and the Antioxidant Status of Hypercholesterolemic Rabbits Nurhafzan Anis Binti Ismail Department of Nutrition & Dietetics, Faculty of Medicine & Health Science, Universiti Putra Malaysia

9. cont.

10. cont.

12. Free Radical Scavenging Method (Yamaguchi et al .,1998) mix aliquot of Strobilanthes crispus extracts (0.32, 0.62, 1.24, 2.48, and 4.96 mg/ml),  -tocopherol (0.04, 0.08, 0.16, 0.32, 0.64 and 1.28 mg/ml) or BHT (0.04, 0.08, 0.16, 0.32, 0.64 and 1.28 mg/ml) with 800  l of 100 mM Tris – HCl buffer (pH 7.4). add mixture to 1 ml of 500  m DPPH in ethanol (final concentration of 250  m). shake mixture vigorously and left in the dark at room temperature for 20 mins. measured absorbance of the resulting solution at 517 nm spectrophotometrically.

13. Flavonoids Analysis by RP-HPLC Method ( Hertog et al ., 1992) Put 0.5 g of dried sample into conical flask quick fit 24/29 Add 20 ml of 62.5% aqueous methanol containing 2 g/L TBHQ and 5 ml of 6M HCl Refluxed at 90 0 C for 2 hrs and let it cool at room temperature Filter using Buchner filter (no. 4) Make up to 25 ml with methanol before filter again with 0.45  m Whatman membrane filter Inject using HPLC

14. Quantification of Total Flavonoids Content ( Chang et al ., 2002 ) cont. Strobilanthes crispus leaves washed with distilled water Strobilanthes crispus Wiped with clean cloth ground, dried leaves Add 25 ml 95% ethanol Extracted under 200 rpm shaking , 24 hrs Filtered Adjusted to 25 ml with 80% ethanol Stored in dark amber bottle

15. Quantification of Total Flavonoids Content a) AlCl 3 Colorimetric Method 0.5 ml Samples/Std (25, 50 & 100 µg/ml Quercetin) Add 1.5 ml 95% ethanol Add 0.1 ml 10% AlCl 3 Add 0.1 ml potassium acetate Add 2.8 ml distilled water Left 30 mins at room temperature Read absorbance at 415 nm cont.

16. b) 2,4-Dinitrophenylhydrazine Colorimetric Method 1 ml Samples / Std (0.5, 1.0 & 2.0 mg/ml (  )-Naringenin) Add 2 ml 1% 2,4-Dinitrophenylhydrazine Add 2 ml MeOH Incubate at 500C for 50 mins Cooled at room temperature Add 5 ml 1% potassium hydroxide in 70% MeOH Incubate at room temperature for 2 mins After 2 mins, take 1 ml and add with 5 ml MeOH Centrifudge at 1000 g, 10 mins Collect supernatant and adjust to 25 ml Read absorbance at 495 nm

17. Strobilanthes crispus leaves extraction Strobilanthes crispus leaves washed thoroughly with tap water rinsed with distilled water wiped with clean cloth & air dried overnight dried in air oven at 40 0 C, 24 hrs ground into fine powder Soak in CHCl 3 : MeOH (2:1), 24 hrs, room temperature Filtered with Whatman filter paper no. 1 Reextracted twice (every 24 hrs) Pool filtrate Evaporated by using rotary evaporator at 40 0 C Stored in dark ember bottle (-30 0 C)

21. Malondialdehyde (MDA) level in rabbits serum (Buege and Aust, 1978) 0.5 ml serum + 1.0 ml TBA-TCA-HCl Mixed thoroughly Heat for 15 mins in a boiling waterbath Cooled Centrifudge (1000 g , 10 mins) Read absorbance against a blank at 535 nm (MDA concentration was calculated by using an extinction coefficient of 1.56*10 5 M -1 cm -1 )

22. Malondialdehyde level in rabbits liver, brain, heart and kidney Tissue homogenates (Ohkawa et al ., 1979) Organs Wash with 0.9% NaCl Homogenised with Ultra Turrax homogeniser (1 g wet tissue in 9 ml 1.55M KCl in 0.05M PBS, pH 7.4) cont.

23. MDA Assay 4 ml tissue homogenates Incubate at 37 0 C for 1 hr Add 1 ml aliquote to 2 ml 7.5% TCA Centrifudge 1000g, 10 mins Add 2 ml of supernatant to 1 ml 0.7% TBA Boiling, 10 mins Read absorbance at 532 nm (MDA concentration was calculated by using an extinction coefficient of 1.56*10 5 M -1 cm -1 )

24. Conjugated Diene Assay (Recknagel and Glende, 1984) 250 µ l serum + 1 ml MeOH + 3 ml CHCl 3 (shake vigorously) + 3 ml KCl (0.05 M) and shake Centrifudge 3000 rpm, 5 mins Discard top layer, remove bottom layer into test tube by drying under N 2 Reconstitute with 2 ml hexane Read absorbance at 234 nm (CD concentration was calculated by using an extinction coefficient of 2.95*10 4 M -1 cm -1 , (Brizzi et al ., 2003))

25. Evaluation of Atherosclerosis Plaque (Blumel et al. , 2001) Disected free aortas Clean with normal saline Opened longitudinally and stretched onto a piece of paper Fixed by 10% formalin Left in 70% ethanol for 24 hrs Dyed in 70% ethanol with 2 g Sudan IV for 24 hrs Washed in 70% ethanol Washed in distilled water Take photograph Analyzed by SIS software

27. D. RESULTS & DISCUSSION TDF in Strobilanthes crispus leaves Weight % TDF 0.5805  0.0108 58.05  1.08 IDF 0.5461  0.0392 54.61  3.92 SDF 0.0601  0.0082 6.01  0.82

33. In vivo Results Food Consumption

34. Body Weight Group Initial body wt Final body wt Weight gain (kg) (kg) (kg) -ve Control 2.04  0.21 2.33  0.50 0.29  0.31 +ve Control 1.98  0.25 2.19  0.42 0.21  0.27 Chol + S. crispus 2.04  0.20 2.23  0.49 0.19  0.46 Chol + Simvastatin 2.16  0.26 1.98  0.32 -0.18  0.41

35. Relative Organ Weight (ROW)

36. Total Cholesterol Triglycerides

37. High Density Lipoprotein Low Density Lipoprotein

38. LDL/HDL Ratio

39. HDL/TC Atherogenic Index

40. Alanine Amino Transferase Gamma Glutamyl Transpeptidase

41. Creatinine Urea

42. Total Antioxidant Status in rabbits serum Group Week 0 Week 6 Week 12 -ve Control 1.52  0.02 1.62  0.01 1.56  0.01 +ve Control 1.51  0.02 1.62  0.04 1.55  0.01 Chol + S. crispus 1.57  0.03 1.63  0.01 1.60  0.05 Chol + Simvastatin 1.52  0.73 1.63  0.08 1.58  0.02 Values are means  sd (mmol/l), for n=3

43. Malondialdehyde in Serum Malondialdehyde in Heart, Liver, Brain and Kidney

44. Conjugated Diene in Serum

46. NC PC SC SV

47. Histology analysis of the rabbits aorta

48. A: A photomicrograph of a section in aorta of a –ve control rabbit, H&E, 4x. B: A section in Fig A, 10x. C: A photomicrograph of a section in aorta of a +ve control rabbit, H&E, 4x. D: A section in Fig B, 10x L : Lumen TI : Tunica Intima TM: Tunica Media TA: Tunica Adventitia A B C D

49. E: A photomicrograph of a section in aorta of a S. crispus treated rabbit , H&E, 4x. F: A section in Fig E, 10x. G: A photomicrograph of a section in aorta of a Simvastatin treated rabbit, H&E, 4x. H: A section in Fig G, 10x L : Lumen TI : Tunica Intima TM: Tunica Media TA: Tunica Adventitia E H G F

53. NC, (4x) NC, (40x) NC, (10x) LIVER

54. PC, (4x) PC, (10x) PC, (40x)

55. PC, (10x) PC, (40x) PC, (4x) PC, (40x)

56. SC, (4x) SC, (10x) SC, (40x) SC, (10x)

57. SC, (4x) SC, (10x) SC, (40x)

58. SV, (4x) SV, (10x) SV, (40x)

59. SV, (4x) SV, (10x) SV, (40x) SV, (40x) SV, (10x)

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66. THANK YOU FOR YOUR KIND ATTENTION QUESTION AND ANSWER SESSION