The Effect of Strobilanthes crispus Extract on Serum Lipid

Profile and the Antioxidant Status of Hypercholesterolemic

Rabbits

Nurhafzan Anis Binti IsmailDepartment of Nutrition & Dietetics, Faculty of Medicine & Health Science,

Universiti Putra Malaysia

OUTLINEA.A. INTRODUCTIONINTRODUCTIONB.B. GENERAL & SPECIFIC OBJECTIVESGENERAL & SPECIFIC OBJECTIVESC.C. METHODOLOGYMETHODOLOGYD.D. RESULTS & DISCUSSIONSRESULTS & DISCUSSIONSE.E. CONCLUSIONS CONCLUSIONS F.F. RECOMMENDATIONSRECOMMENDATIONSG.G. REFERENCESREFERENCES

• CVD – most deadly disease to the populations in the developed countries (WHO Monica Project, 1988), involving pathological changes in blood vessels associated mainly with the heart and brain.

• The common, chronic condition underlying these diseases is the presence of atherosclerosis in the blood vessels.

• Atherosclerosis is characterized by a thickening of the internal lining of the arterial walls, causing a constriction of the vessel lumen and impeding blood flow and reducing vessel elasticity.

• This support the occlusive blood clots formation and can give effect in injury to heart, brain and lung tissue, which may cause death (Linder, 1991).

• Atherosclerosis is causally related to the elevated levels of plasma/serum LDL cholesterol and free radical reaction

A. INTRODUCTION

• These oxygen-derived free radicals are dangerous because it can interact readily with other biomolecules including nucleic acids, lipids, carbohydrates, protein and free amino acids.

• Those interactions can cause oxidative damage to protein, enzymes, unsaturated fatty acids, DNA, RNA and cellular membrane function.

• Antioxidants are materials which protect against free radical reaction and retard the progress of many chronic diseases.

• These antioxidants include the natural antioxidants and the synthetic antioxidants

• However, currently used synthetic antioxidants have been suspected to promote negative side effects (Branen, 1975; Barlow, 1990).

• Therefore, people are currently interested in products contains of natural antioxidants.

• Nowadays, many other plants species are being studied for their preventive action possibility against all kinds of diseases as well as in the search for novel antioxidants (Kim et al., 1994).

Also known as “jin batu” in Malaysia, “enyoh kelo” or “kejibeling” in Java and “daun picah beling” in Jakarta (Sunarto, 1977).

Has been used as a herbal remedies locally and in Indonesia as anti-diabetic, diuretic, hepatitis and cholesterol lowering agents.

Water extract of Strobilanthes crispus was found to have high content of mineral, such as potassium, calcium, sodium and iron (Maznah et al., 2000).

Earlier work have been conducted in rabbits by using lower concentration (2.5%) of Strobilanthes crispus water extract.

The study suggested that Strobilanthes crispus has an anti lipid peroxidation effect but no hypocholesterolemic effect at the experimental dose (Azlina D., 2000).

Strobilanthes crispus PLANT

General Objective

• To determine the hypocholesterolemic and toxicity effect of Strobilanthes crispus extract on animal model induced with atherosclerosis and to determine its antioxidant status.

Specific Objectives1. To determine the Total Dietary Fiber (TDF), antioxidant activity and

flavonoids content of Strobilanthes crispus.

2. To determine the effect of Strobilanthes crispus extract (0.2% w/w) on lipid profile of the experimental animal.

B. GENERAL & SPECIFIC OBJECTIVES

3. 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.

4. 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.

5. To measure the effect of Strobilanthes crispus extract (0.2% w/w) on atherosclerotic lesion in the aorta of the experimental animal.

6. 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.

Total Dietary Fiber (TDF)

• Samples - homogenized & dried overnight in an air oven (105 0C) • Ground finely. • Enzymatic-gravimetric method of AOAC (1997) to analysis:

1. Total Dietary Fiber (TDF),2. Insoluble Dietary Fiber (IDF) 3. Soluble Dietary Fiber (SDF)

C. METHODOLOGY

cont.

Sample preparation – Put 1 g of sample into tall beaker + 50 ml pH 6.0 phosphate buffer

Add 0.1 ml -amilase, incubate at 95 0C for 15 mins.

Cool solution to room temperature.

Protease incubation - pH 7.5 at 60 0C for 30 mins.

Cool solution to room temperature.

cont.

Amyloglucosidase incubation - pH 4.3 at 60 0C for 30 mins.

Ethanol prec ipitation

Filtration (alcohol & acetone wash)

Overnight drying in 105 0C air oven.

Cool to room temperature

Weigh

Protein determination Ash determination - macro K jeldahl

TDF calculation

cont.

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 0C 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

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 (Chang et al., 2002)

cont.

Quantification of Total Flavonoids Content

a) AlCl3 Colorimetric Method

0.5 ml Samples/Std (25, 50 & 100 µg/ml Quercetin)

Add 1.5 ml 95% ethanol

Add 0.1 ml 10% AlCl3

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 0C, 24 hrs

ground into fine powder

Soak in CHCl3 : 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 0C

Stored in dark ember bottle (-30 0C)

In vivo study

- 24 female New Zealand white rabbits (1.8-2.5 kg)

- Acclimatized for two weeks before assigned randomly into four groups (n=6/group).

- These four groups of rabbits were fed with:

1. An unmodified non-purified diet (control diet)

2. Control diet + 0.25% cholesterol

3. Control diet + 0.25% cholesterol + Strobilanthes crispus extract (0.2%/100g diet)

4. Control diet + 0.25% cholesterol + Simvastatin (22 mg/kg b.w.)

- 130 g rabbit pellet were given daily and tap water were given ad libitum for 12 wks.

Female New Zealand White rabbits (n=24) 

NC PC 0.25% (w/w) Cholesterol 0.25% (w/w)

(n=6) 0.25% (w/w) cholesterol + SC (0.2% extract Cholesterol (n=6) /100g diet) + SV (20 mg/kg)

(n=6) (n=6) 

food weigh blood samples kill (after 12 weeks) intake (weekly) (biweekly)(daily) - TC -brain weigh - HDL -liver MDA: b,l,k,h,

- TG -kidney histology:l,k,h - LDL -heart

- ALT -aorta atheroma plaque - GGT evaluation - Urea histology analysis

- Creatinine - MDA - TAS - CD

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*105M-1cm-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 0C 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*105M-1cm-1)

Conjugated Diene Assay (Recknagel and Glende, 1984)

250 µl serum + 1 ml MeOH

+ 3 ml CHCl3 (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 N2

Reconstitute with 2 ml hexane

Read absorbance at 234 nm (CD concentration was calculated by using an extinction coefficient of

2.95*104M-1cm-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

Histology analysis Tissue processing.

- Dehydrated with alcohol, cleared with xylene and impregnated with wax.

Tissue embedding.

Tissue sectioning.

Tissue staining.

- Haematoxylin and Eosin (H&E) staining.

Slides interpretation by using Image Analyser machine.

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

D. RESULTS & DISCUSSIONTDF in Strobilanthes crispus leaves

Sample Flavonoid Content (%)a AlCl3

b 2,4-Dc Total Fresh leaves 0.35 0.01 0.64 0.03 0.99 0.04 Dried leaves 0.10 0.01 0.36 0.05 0.46 0.06__a: Result were presented as mean SD (n=3)b: Flavonoid content (%) = quercetin equivalent (g/ml) total volume of ethanol extract (ml) sample weight (g) dilution factor 10-6 (g/g) 100c: Flavonoid content (%) = naringenin equivalent (g/ml) total volume of ethanol extract (ml) sample weight (g) dilution factor 10-6 (g/g) 100

Total Flavonoids Content

The method utilizing aluminium chloride reaction was specific only for flavones and flavonols, while another method utilizing 2,4-dinitrophenylhydrazine reaction was specific for flavonones.

Therefore, the sum of flavonoids content quantified by these two methods may represent the real content of total flavonoids.

Larger amount of flavonoids were found in the fresh leaves than in the ground, dried leaves.

A naturally occuring polyphenolic compounds, widely distributed in the leaves, seeds, bark and flowers of plants.

Derived from parent compounds, which is known as flavans (Singh, A. P., 2002).

Fig.2: Structure of flavonoidFig.1: Structure of flavan

The beneficial health effects of flavonoids are attributed mostly to their antioxidant and chelating abilities.

FLAVONOIDS

y = 370.12x + 324.81

R2 = 0.9999

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

0.00 5.00 10.00 15.00 20.00

Amount of Rutin (ug)

Peak

Are

a (m

Au*s

)

y = 62.649x + 50.673

R2 = 0.9993

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

0.00 5.00 10.00 15.00 20.00

Amount of Kaempferol (ug)

Pea

k A

rea

(mA

u*s)

y = 106.44x + 51.622

R2 = 0.9977

0.00

500.00

1000.00

1500.00

2000.00

2500.00

0.00 5.00 10.00 15.00 20.00 25.00

Amount of Luteolin (ug)

Pea

k A

rea

(mA

u*s)

y = 488.67x - 510.02

R2 = 0.9994

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

10000.00

0.00 5.00 10.00 15.00 20.00

Amount of Quercetin (ug)

Pea

k A

rea

(mA

u*s)

RP-HPLC Result

Sample Quercetin Kaempferol Luteolin Rutin

Leaves (mg/g) 104.55 0.82 101.80 68.36 5.68 2.39 1.79 1.61

Crude Extract (ug/g) 7.23 0.68 5.82 3.74 2.79 2.06 0.79 0.72

The highest amount of flavonoids in both Strobilanthes crispus ground, dried leaves and Strobilanthes crispus crude extracts were quercetin, followed by kaempferol, luteolin and rutin.

                         

        Quercetin

                            

     Kaempferol

                             

    Rutin

                         

        

Luteolin

According to Singh, A. P. (2002), quercetin, kaempferol and quercitrin are common flavonoids present in almost 70 % of the plants.

From this analysis, flavonoids level were found higher in the ground, dried leaves compared to the crude extract.

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).

In vivo Results

Food Consumption

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 0.27Chol + 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

Body Weight

Relative Organ Weight (ROW)

Total Cholesterol

Triglycerides

High Density Lipoprotein

Low Density Lipoprotein

LDL/HDL Ratio

HDL/TC

Atherogenic Index

Alanine Amino Transferase

Gamma Glutamyl Transpeptidase

Creatinine

Urea

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 0.01Chol + 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 0.02Values are means sd (mmol/l), for n=3

Total Antioxidant Status in rabbits serum

TAS level in each group

1.001.051.101.151.201.251.301.351.401.451.501.551.601.651.70

Week 0 Week 6 Week 12

TA

S l

evel

(m

mo

l/L

)

-ve Control +ve Control Chol + S. crispus Chol + Simvastatin

Malondialdehyde in Serum

Malondialdehyde in Heart, Liver, Brain and Kidney

Conjugated Diene in Serum

NC

PC

SC

SV

Histology analysis of the rabbits aorta

A B

C DA: 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

E

HG

F

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

LIVER

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.

Hepatocytes are commonly binucleate.  

Portal areas (also called portal triads or portal canals) – located at the corners of liver lobules. Normally surrounded by much larger areas packed with hepatic cords and sinusoids.

Each portal area contains three more-or-less conspicuous tubular structures all wrapped together in connective tissue.

i) a branch of the bile duct.

ii) a branch of the portal vein.

iii) a branch of the hepatic artery.

Bile duct – most easily recognized by the conspicuous round nuclei in its cuboidal epithelium.

Portal vein – brings much more blood to the liver. – each branch of the portal vein is typically much

larger than the associated branch of the hepatic artery.

HEART & KIDNEY

Despite the notable changes in the liver tissues, no histological changes have been observed in the heart and kidney tissues in all the experimental rabbits.

NC, (4x)

NC, (40x)

NC, (10x)

LIVER

PC, (4x)

PC, (10x)

PC, (40x)

PC, (10x)PC, (40x)

PC, (4x)PC, (40x)

SC, (4x)

SC, (10x)

SC, (40x)

SC, (10x)

SC, (4x)

SC, (10x)

SC, (40x)

SV, (4x)

SV, (10x)

SV, (40x)

SV, (4x)

SV, (10x)

SV, (40x) SV, (40x)

SV, (10x)

E. CONCLUSIONS1) 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.

2) SC extract showed 3.76 7.45% to 56.72 2.49% scavenging effect of DPPH radical with the EC50 = 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.

3) 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.

4) 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.

5) 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.

6) 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.

F. RECOMMENDATIONSThe following recommendations can be withdrawn for future work:

1) 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.

2) The stimulatory effects of different concentrations of SC extract on LDL receptor activity in vitro, i.e. on HepG2 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.

3) 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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Kim SY, Kim SK, Oh MJ and Jung MY. 1994. Antioxidant activity of selected oriental herb extracts. J. Am. Oil. Chem. Soc. 71: 633-640.

Linder, M.C. 1991. Nutritional biochemistry and metabolism with clinical applications.2nd ed.; Elsevier: New York.

Maznah, I., Manickam, E., Azlina, M. D., Asmah, R. and Asmah, Y. 2000. Chemical composition and antioxidant activity of Strobilanthes crispus leaf extract. J. Nutr. Biochem 11:536-542.

Recknagel, R. O. and Glende Jr., E. A. 1984. Spectrophotometric detection of lipid conjugated dienes. Methods Enzymology. 105: 331-337.

Singh, A. P.2002. Flavones, flavonoids and xanthones. In A treatise on phytochemistry. United Kingdom: Emedia Science Ltd.

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The WHO Monica Project. Geographical variation in the major risk factors of coronary heart disease in men and women aged 35-64 years. World Health Stat., 1988, 41, 115-40.

Yamaguchi, T., Takamura, H., Matoba, T., Terao, J. HPLC method for evaluation of the free radical-scavenging activity of foods by using 1,1,-diphenyl-2-picrylhydrazyl. Biosci., Biotechnol., Biochem. 1998. 62. 1201-1204.

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