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
To determine the effect of Strobilanthes crispus extract (0.2%) on lipid peroxidation index, the malondialdehyde (MDA) and conjugated diene (CD), in animal model induced with atherosclerosis.
To determine the effect of Strobilanthes crispus extract (0.2% w/w) on toxicity parameters; alanine aminotransferase (ALT), gamma glutamyl transpeptidae (GGT), urea and creatinine in animal model induced with atherosclerosis.
To measure the effect of Strobilanthes crispus extract (0.2% w/w) on atherosclerotic lesion in the aorta of the experimental animal.
To measure the effect of Strobilanthes crispus extract (0.2%) on the morphology changes in the aorta, heart, kidney and liver of the animal model induced with atherosclerosis.
The same method have been used for IDF determination, but instead of ethanol ,water was used for filtration and washing.
For SDF determination, filtrate from the IDF was precipitated using ethanol and then filtrated using the same method as TDF.
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.
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
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
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.
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
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)
TC, HDL, LDL, TG, ALT, GGT, BUN, Creatinine level was measured by a Chemistry Analyser (Hitachi 902, Roche Diagnostic).
TAS was measured by Automated Clinical Chemistry Analyser (Selectra E).
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 )
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.
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 )
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))
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
The differences of the flavonoids amount might be due to the processing methods applied in order to gain the ground, dried leaves and the crude extracts.
These results support the previous study that Strobilanthes crispus plant have potential as natural antioxidant, mainly by quercetin and kaempferol, as well as by catechin as shown from our previous study (Maznah et al. , 2000).
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
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
Liver consists of hepatocytes; epithelial cells with a unique configuration.
Organized into lobules; typically hexagonal in cross section and is centered on a branch of the hepatic vein (the central vein).
Within each lobule, hepatocytes are arranged into hepatic cords separated by vascular sinusoids.
Sinusoids – vascular spaces lined by a fenestrated endothelium.
Central veins – conspicuous spaces, with no associated connective tissue, located roughly midway between portal areas. Mark the centers of lobules.
Lipofuscin – in the form of yellow or brown pigment granules, sometimes evident in hepatocytes, especially toward the centers of lobules. Represents the presence of lysosomes that have accumulated a noticeable amount of indigestible residue.
The high TDF obtained in this study, 58.05 1.08% indicated that the SC plant has a potential as a rich source of dietary fiber and thus may be developed into TDF powder and potentially be used as a dietary fiber supplement.
SC extract showed 3.76 7.45% to 56.72 2.49% scavenging effect of DPPH radical with the EC 50 = 2.21 mg/ml. Thus, SC is also a free radical inhibitor, particularly of the peroxy radical, which is the major propagator of the autoxidation chain of fat, thus terminating the chain reaction. The antioxidative properties of SC extract is mainly due to quercetin, kaempferol, luteolin and rutin with the total flavonoids content of 0.99 0.04% in the fresh leaves and 0.46 0.06% in the dried leaves.
Feeding diet enriched with 0.25% cholesterol has significantly increased (p<0.05) TC, LDL and HDL levels, HTR, LDL/HDL ratio, atherogenic index, serum, heart, liver and kidney MDA concentrations, serum CD concentration and relative liver weight in the PC group as compared to the NC group. Besides, the PC group has also exhibited slightly elevated level of TG, ALT, GGT, creatinine, urea concentrations and relative kidney, heart and brain weight as well. However, the supplementation of 0.25% hypercholesterolemic diet have slightly decreased the body weight, average food consumption and TAS in the PC group compared to the NC group. Nevertheless, by feeding on 0.25% cholesterol enriched diet has
successfully produced atherosclerotic lesion in the PC group and thus increased the intima to media ratio compared to the NC group. The cholesterol exposure has resulted in remarkable changes of the liver histology in PC group such as a moderate to fairly intense lymphocyte infiltration, slight macrovesicular lipid droplets, ballooning degeneration of hepatocytes, an irregularly bi-nucleated cells and vacuolated cytoplasm. Furthermore, no remarkable changes of the heart and kidney tissues have been observed in all the experimental rabbits.
Feeding rabbits with 0.25% cholesterol enriched diet supplemented with 0.2 g SC extract/100g diet has resulted in a significantly elevated (p<0.05) HDL levels plus a significant reduction of the kidney MDA levels, although the rabbits showed only slight increment of total antioxidant status, body weight, relative brain weight, HTR, TC, LDL, ALT, GGT as well as serum and brain MDA levels. On the other hand, following SC treatment have slightly decreased the atherogenic index, LDL/HDL ratio, TG level, heart and liver MDA levels, serum CD concentration, average food consumption and relative liver, kidney and heart weight, GGT, creatinine and urea concentrations as compared to the PC group. In addition, compared to the PC group, lesser intimal surface lesion which leads to the lesser thickening of aortas and the inhibition of hepatic macrovesicular lipid droplets, ballooning degeneration of hepatocytes and lymphocyte infiltration have also been observed in the SC group.
These results support the previous study that SC plant may possess the hypocholes-terolemic properties that can slower down the development of atherosclerosis due to the antioxidative properties of flavonoids, particularly quercetin, kaempferol, luteolin, rutin and catechin as previously reported exist in the SC plant. Also, the hypocholesterolemic effect of SC may possibly due to the mechanism of dietary fiber content. Efficient amount of fiber content might have not been extracted in the extraction method for the in vivo study and thus the hypocholesterolemic effect of SC does not occur effectively as shown in the lipid profile of the experimental rabbits in this study.
The treatment with 20 mg SV/kg body weight have successfully reduced the TC, LDL, heart, liver and serum MDA concentrations significantly (p<0.05). Besides, following SV treatment have slightly reduced the kidney serum CD, MDA level, atherogenic index, HTR and LDL/HDL ratio, ALT, GGT, creatinine, urea concentrations, average food consumption, body weight and relative liver weight as well. Even so, significantly elevated (p<0.05) brain MDA concentrations as well as slightly increased TAS, relative brain, kidney and heart weight and TG levels have been observed subsequent to SV treatment. Moreover, the treatment with SV have shown similar histological changes as the SC group, in which lesser thickening of aortas due to lesser intimal surface lesion and the inhibition of hepatic macro-vesicular lipid droplets, ballooning degeneration of hepatocytes and lymphocyte infiltration have also been detected in the SC group compared to the SV group.
The following recommendations can be withdrawn for future work:
The effect of processing parameters (e.g.: solvent-to-solid ratio, operating temperature, raw material particle size, pre-processing treatment) to the yield of SC extract and flavonoids content.
The stimulatory effects of different concentrations of SC extract on LDL receptor activity in vitro , i.e. on HepG 2 or Chang cells and in vivo . The effective concentration from the in vitro shall be used in the in vivo study in order to get clear view of the hypocholesterolemic mechanism exhibited by the extract.
The screening of other SC bio-active constituents and the correlation with hypocholesterolemic effect and other therapeutic actions such as anti-inflammatory and diuretic action.
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