Effect of Aqueous Whole Plant Extract of Selaginella ...article.aascit.org/file/pdf/9230793.pdf73...

American Journal of Pharmacy and Pharmacology 2015; 2(5): 72-78

Published online February 26, 2016 (http://www.aascit.org/journal/ajpp)

ISSN: 2375-3900

Keywords Total Cholesterol (T-CHOL),

Triglyceride (TG),

Low Density Lipoprotein

Cholesterol (LDL-CHOL) and

High Density Lipoprotein

Cholesterol (HDL-CHOL)

Received: December 29, 2015

Revised: January 14, 2016

Accepted: January 16, 2016

Effect of Aqueous Whole Plant Extract of Selaginella myosurus on Lipid Profile of Wistar Rats

Omeodu S. I., Peters D. E.*, Oki J.

Department of Biochemistry, Faculty of Science, University of Port Harcourt, Rivers State, Nigeria

Email address [email protected] (Peters D. E.), [email protected] (Peters D. E.)

Citation Omeodu S. I., Peters D. E., Oki J. Effect of Aqueous Whole Plant Extract of Selaginellamyosurus

on Lipid Profile of Wistar Rats. American Journal of Pharmacy and Pharmacology.

Vol. 2, No. 5, 2015, pp. 72-78.

Abstract Cardiovascular disease continues to be the leading cause of death across the globe today.

Treatment with synthetic drugs is associated with several adverse effects. This study was

designed to investigate the effect of aqueous whole plant extract of selaginella myosurus

on lipid profile of wistar rats. A total of thirty six (36) wistar rats of both sexes weighing

between 100.5g-149.5g were divided into nine groups of four rats each. Group 1

received distilled water, while groups 2-5 and 6-9 received 400, 600, 800, and

1000mg/kg BW of extract for 7 and 14 days respectively. Rats were sacrificed 24hours

after the last treatment and blood samples collected for determination of total

cholesterol(T-CHOL), triglyceride(TG), high density lipoprotein-cholesterol(HDL-

CHOL) and low density lipoprotein-cholesterol(LDL-CHOL), histological investigation

on the heart tissue and phytochemical screening of the plant. Significant reductions

(p<0.05) were observed in T-CHOL and TG in all the extract-treated groups when

compared to control value and non significant reduction (p>0.05) HDL-CHOL and LDL-

CHOL in all the groups except in groups 3 and 5 respectively. Phytochemical screening

of plant revealed the presence of flavonoids, triterpenoids, saponins, tannin, steroid,

cardiac glycoside and phenol in decreasing order (32.19±0.23, 26.24±0.12, 23.74±0.20,

18.74±0.17, 16.53±0.12, 15.28±0.23 and 13.10±0.11 mg/100g respectively).Histological

result revealed that all heart muscles were in good histological conditions in control and

all extract treated groups. Hence, aqueous whole plant extract of selaginella myosurus

has hypolipidemic effect, could protect the heart against cardiovascular disease (CVD)

and hence a promising potential herbal pharmaceutical agent.

1. Introduction

Cardiovascular disease continues to be the leading causeof death across the globe

today the major ones being coronary heart diseases, stroke and hypertension [1].

Elevated plasma lipids are riskfactors in cardiovascular problems. Hyperlipidemia and

other abnormal blood lipid profile are largely of genetic origin or due to unwholesome

nutritional habits. Lipids and other substances accumulates on arterial wall, forming

plague, which occlude the vascular lumen and obstruct the blood flow to vital organs

such as the heart, brain, liver or kidney. Obstruction of blood supply to the heart, brain,

liver or kidney cause coronary heart diseases, stroke or kidney failure.

Hyperlipidemia refers to elevated levels of lipids and cholesterol in the blood, and is

also identified as dyslipidemia, to describe the manifestations of different disorders of

lipoprotein metabolism. Although elevated low density lipoprotein cholesterol (LDL) is

73 Omeodu S. I. et al.: Effect of Aqueous Whole Plant Extract of Selaginella myosurus on Lipid Profile of Wistar Rats

thought to be the best indicator of atherosclerosis risk,

dyslipidemia can also be described as elevated total

cholesterol (TC) or triglycerides (TG), or low levels of high

density lipoprotein cholesterol (HDL).

High level of blood cholesterol is a contributory factor of

atherosclerosis and many lipid associated ailments like

obesity, heart attacks and stroke and kidney failure.[2]have

shown that lipid associated disorders are not only attributed

to the total serum cholesterol, but also to its distribution

among different lipoproteins.. The low density lipoproteins

(LDLs) are the major carriers of cholesterol towards tissues

having atherogenic potential, while the high density

lipoproteins (HDLs) carry cholesterol from peripheral tissues

to the liver [3]. HDLs thus give protection against many

cardiac problems and obesity [4]. Although genetic factors

recline behind these lipid disorders, in most of the cases it is

allied with diets high in saturated fats or trans fats.

The clinical consequences of these disease conditions are

serious and meaningful research efforts to improve the

knowledge and understanding of the pathogenesis is

essential, in order to provide a more rational approach to

their prophylaxis and treatment. [5-6].

Selaginellamyosurus Alston with an alternative name of

Stachygynandrum, wide spread in all continents

predominantly terrestrial plant of lowland to mid-montane

primarily rainforest but preferring more open glades and river

banks and therefore a frequent component of secondary

forest in these areas.Selaginella myosurus (Sw.) Alston

(Lycopodiummyosurus Sw.; Selaginella scandens P. Beauv.;

Stachygynandrumscandens P. Beauv.) are creeping or

ascendant plants with simple, scale-like leaves (microphylls)

on branching stems from which roots also arise. It appears

that the traditional use of Selaginella myosurus in Nigeria

and other part of West Africa is still relatively rare, compared

to the number of species that are grown in this region. It is

commonly referred to by indigenes of Rivers State as Akoro,

ukor. It is a powerful plant that is used in many regions due

to different beliefs, it is found in swamp forests, disturbed

areas, along roadsides, edge of forests.

Selaginella is traditionally used to treat several diseases

such as injury, treatment of post childbirth, cancer, skin

disease, headaches, fever, respiratory infections, urinary tract

infections, menstrual disorders, liver disorders, fractures and

arthritis. All parts of the plant can be used, although

sometimes they are called only a leave (herb) [7].

Its use can be solely or in combination, fresh or dried,

eaten immediately or cooked. The plant sweet and have

warm effects [8].

Selaginella contains a variety of secondary metabolites

such as alkaloids, phenol (flavonoids, tannins, and saponins)

and terpenoids [9]. The main secondary metabolite of this

plant is bioflavonoid, whose type is varied depending on the

species. Bioflavonoid, once known as “Vitamin P” is useful

in treatment and prevention of many health conditions. It is

referred to as “nature’s biological response modifiers”

modify body’s reaction to compounds such as aoikjllergens,

viruses and carcinogens. These compounds act as

antioxidants, anti-inflammatory, antimicrobial, antifungal,

antiviral etc. [10]. It is well known that plants generally

contain secondary metabolites and some of these secondary

metabolites have been shown to be highly biologically active

[11] and as well as exhibiting physiological activity [12].

Saponins cause hypocholesterolemia by binding cholesterol,

making it unavailable for absorption [13].

Flavonoids are a group of phytochemicals found in varying

amounts in foods and medicinal plants which have been

shown to exert potent anti-oxidant activity against the

superoxide radical [14]. Its consumption has been

documented not to be associated with mortality due to

coronary heart disease. This may be as a result of its

antioxidant activity and subsequent inhibitions of Low

Density Lipoproteins (LDL) oxidation known to have been

attributed to the dietary and supplemental intake of

flavonoids and other micronutrients. Tannins hasten the

healing of wounds and inflamed mucous membrane [15]. The

lowering level of serum cholesterol using diet or drugs

decreases the incidence of coronary heart disease [16, 17].

Steroids are a group of cholesterol derived lipophilic, low-

molecular weight compounds and can be grouped on the

basis of type of organism in which they are present.Steroids

are classified into three broad categories; insect steroids,

vertebrate steroids and plant steroids. Plant steroids are of

two broad categories: phytosterols and brassinosteroids.

Phytosterols covers both plant sterols and stanols [18]. They

are plant components with a structure similar to cholesterol

[19], although they are more poorly absorbed by the

intestine. They are classified into different groups depending

on their structure and biosynthesis [20]. Their exact

mechanism of action and cholesterol lowering properties are

not known, but, because their structure is similar to that of

cholesterol, they compete for solubilization in the micelles

and therefore inhibit intestinal absorption of both dietary and

endogenous cholesterol [21] lowering postprandial

cholesterol level in the blood.

2. Materials and Methods

2.1. Apparatus/Equipment

Spectrophotometer (BSA 3000), SFRI France,Rotary

evaporator, Centrifuge (Universal laboratory century),

Hettich Zentrifugen, Metlar weighing balance,SIEMENS

Advia 2120 Automated Analyzer.

2.2. Reagents/Chemicals

All reagents and chemicals are of analytical grade.

2.3. Collection/Identification of Plant

Selaginella myosurus was collected in the surrounding

bush of the University of Port Harcourt in Choba community

of Obio/Akpor Local Government Area of Rivers state. A

voucher specimen (UPH-NO.C-129) was authenticated by a

American Journal of Pharmacy and Pharmacology 2015; 2(5): 72-78 74

botanist, Dr. N. L. Edwin-Nwosu and deposited at the

herbarium unit of the Department of Plant Science and

Biotechnology (PSB), University of Port Harcourt.

2.4. Extract Preparation

The whole plant of Selaginella myosurus was washed with

running tap water and air dried for 2 weeks before grinding

into powdered form. The coarsely powdered plant material

was macerated in a maceration jar for 24hours, with distilled

water. Filtration was done usingWhatman filter paper in a

glass funnel placed in a retort stand.The filtrate was allowed

for about 1-2 hours to observe any residue or sediment. After

having a clear filtrate, it was put in a rotary evaporator which

separated the water from the extract, leaving the extract in a

paste form. The extract was then poured into a crucible plate

for drying on a steam bath at 40°C to 50°C. The crude extract

was stored in a refrigerator pending usage.

2.5. Phytochemical Screening

Phytochemical screening of the whole plant of Selaginella

myosurus was done using standard procedure as described by

[22] in the Department of Pharmacognosy, Faculty of

Pharmacy, University of Port Harcourt.

2.6. Source of Animals

A total of thirty six (36) wistar rats of both sexes weighing

between 100.5g-149.5g were purchased from an animal

breeding facility in Choba community, and were kept in the

Department of Biochemistry, University of Port Harcourt

Animal House, Choba park for one week acclimatization.

The rats were fed with normal rat feed and water ad libitum.

2.7. Lethal Dose (LD50) Determination

LD50determination was done using an “up-and-down”

procedure described by[23]. Three doses of 1000mg/kg,

3000mg/kg, and 5000mg/kg were orally administered to 3

groups of rats (n=2 rats per group). The rats were observed

for 24hours and for a period of 1 week. No death was

recorded; therefore, safe doses of 400,600, 800 and

1000mg/kgBW were selected.

2.8. Experimental Design

The rats were divided into nine (9) groups (n=4rats).

GROUP 1 (Control): 0.5ml of distilled water was orally

given to the animals in this group dailyfor 14 days.

GROUP 2 (400mg/kg b.w extract): A single daily dose of

400mg/kg b.w of aqueous whole plant extract of

Selaginellamyosuruswas orally administered torats in this

group for 7 days.


600mg/kg b.w of aqueous whole plant extract of

Selaginellamyosuruswas orally administered to rats in this

groupfor 7 days.


800mg/kg b.w of aqueous whole plant extract of Selaginella

myosurus was orally administered to rats in this group for 7

days.




days.




days.



myosurus was orally administered to rats in this groupfor 7

days.



myosuruswas orally administered to rats in this group for 7

days.



myosurus was orally administered to rats in this groupfor 7

days.

Sacrifice, Collection and Preparation of Plasma

At the end of 7 and 14 days, all the animals were

anaesthetized with chloroform before decapitated for

collection of blood. The blood was stored in heparinised

sample bottle, spun at 5000rpm using MSE centrifuge to

obtain plasma for biochemical investigations.

3. Biochemical Investigation

Total Cholesterol (TC) and Triacylglycerol (TG), were

estimated by enzymatic methods described by[24],using

assay kits (Randox Laboratories Ltd, UK). High-Density

Lipoprotein Cholesterol (HDL-C) was determined by

enzymatic methodsdescribed by [25]using assay kits (Randox

Laboratories Ltd, UK). Low-Density Lipoprotein Cholesterol

(LDL-C) was calculated using the formular by [26].

3.1. Histopathogical Studies

The rats were dissected using a set of dissection kit and

heartsfrom control and extract treated groups were collected

and fixed in 10% freshlyprepared formalin for 48 hours and

subsequentlydehydrated in alcohol, cleared with xylem

andembedded in paraffin wax. Sections of lobe at about5µm

were mounted on glass slides and stained withhaematoxylin

and eosin [27].

3.2. Statistical Analysis

All the values were reported as mean ± standard error of

mean (M ± SEM). Statistical analysis was performed using

SPSS version 20.0 (IBM, U.S.A). The data were analyzed

using one-way analysis of variance (ANOVA) and significant

difference were determined using post Hoc Turkey’s test for

multiple comparisons at p< 0.05.


4. Result

Table 1. Effect of Aqueous Whole Plant Extract of Selaginella myosurus on

Lipid Profile in Wistar Rats.

TREATMENT

GROUPS

(Mg/kgBW)

PARAMETERS

TG

umol/L

CHOL

umol/L

HDL-CHOL

umol/L

LDL

umol/L

WATER

CONTROL 1.65±0.07a 3.40±0.08a 1.08±0.05a 1.53±0.10a

400mg/kgBW

EXT FOR 7

DAYS

1.38±0.05a 2.73±0.14a 0.93±0.05 1.15±0.16

600mg/kgBW

EXT FOR 7

DAYS

1.33±0.05a 2.73±0.15a 0.83±0.05a 1.25±0.10

800mg/kgBW

EXT FOR 7

DAYS

1.40±0.04a 2.73±0.08a 0.85±0.06 1.20±0.07

1000mg/kgBW

EXT FOR 7

DAYS

1.28±0.05a 2.45±0.06a 0.90±0.04 0.95±0.5a

400mg/kgBW

EXT FOR 14

DAYS

1.48±0.48 3.05±0.06 1.03±0.05 1.30±0.09

600mg/kgBW

EXT FOR 14

DAYS

1.35±0.03a 2.85±0.06a 0.90±0.04 1.30±0.08

800mg/kgBW

EXT FOR 14

DAYS

1.38±0.05a 2.78±0.09a 0.88±0.05 1.18±0.05

1000mg/kgBW

EXT FOR 14

DAYS

1.33±0.05a 2.68±0.09a 0.88±0.05 1.13±0.09

Data are represented in Mean±Standard Error of Mean (M±SEM)

Similar superscripts represent significant different (p<0.05) in the same row

Table 2. Qualitative Phytochemical Screening of Whole Plant Extract of

Selaginella myosurus.

SECONDARY

METABOLITES TEST RESULT

Alkaloids

Drangedorff -ve

Mayer -ve

Hager -ve

Flavonoids

Shinoda -ve

Lead acetate -ve

Alkali +ve

Tannins

FeCl3 +ve

Phlobatannins +ve

Gelatin ND

Albumin ND

Anthraquinone Free Anthraquinone -ve

Combined Anthraquinone -ve

Triterpenoid/steroids Liebermann-Buchard +ve

Salwoski +ve

Fixed oil +ve

Carbohydrates Molisch +ve

Fehlings +ve

Cardenolide Keller Killani +ve

Kedde +ve

Cyanogenic

glycosides Frothing -ve

Saponins

Frothing +ve

Haemolysis -ve

Emulsion +ve

Note:+Ve means present, -ve means absent, while ND is Not Determined

Table 3. Quantitative Photochemical Screening of Whole Plantof Selaginella

myosorus.

mean± standard error of mean (M + SEM)

Flavonoid 32.19± 0.23

Saponins 23.74± 0.20

Cardiac glycoside 15.28± 0.23

Steroid 16.53± 0.12

Terpenoid 26.24± 0.12

Tannin 18.74± 0.17

Phenol 13.10± 0.11

HISTOLOGICAL EXAMINATION OF HEART

SECTIONS OF RATS TREATED WITH DISTILLED

WATER AND VARYING CONCENTRATIONS OF

AQUEOUS WHOLE PLANT EXTRACT OF

SELAGENELLA MYOSURUS FOR 7 AND 14 DAYS

Fig. 1. Heart section of control rat showing good histological

conditions(H&E, x 400).

Fig. 2. Heartsection of rat treated with 600mg/kgbw extract for 7days

showinggood histological conditions (H&E, x 400).


Fig. 3. Heart section of rat treated with 800mg/kgbw extract for7days

showing good histological conditions (H&E, x 400).

Fig. 4. Heart section of rat treated with 1000mg/kgbw extractfor 7days

showinggood histological conditions (H&E, x 400).

Fig. 5. Heartsection of rat treated with 600mg/kgbw extractfor

14daysShowing good histological conditions (H&E, x 400).

Fig. 6. Heartsection of rat treated with 800mg/kgbw extractfor

14daysShowing good histological conditions (H&E, x 400).

Fig. 7. Heart section of rat treated with 1000mg/kgbw extract for14days

showing good histological conditions (H&E, x 400).

5. Discussion and Conclusion

It is widely accepted that a plant based diet with high

intake of fruits and vegetables may reduce the risk of

oxidative stress-related diseases such ascancer and

cardiovascular diseases. Most bioactive food constituents are

derived from plants and are collectively called

phytochemicals. The majority of these phytochemicals are

antioxidants.Antioxidants are defined as compounds that can

delay, inhibit, or prevent the oxidation ofoxidizable materials

by scavenging free radicals and diminishing oxidative stress.

Oxidative stress isan imbalanced state where excess

quantities of reactive oxygen and/or nitrogen species

(ROS/RNS,e.g., superoxide anion, hydrogen peroxide,


hydroxyl radical, peroxynitrite) overcome endogenous

antioxidantcapacity, leading to oxidation of a varieties of

biomacromolecules, such as enzymes, proteins, DNA and

lipids. Oxidative stress is important in the development of

chronic degenerative diseasesincluding coronary heart

disease, cancer and aging[28].

Significant reductions (p<0.05) were observed in T-CHOL

and TG concentrations in all the extract treated groups when

compared to control value and non significant reduction

(p>0.05) inHDL-CHOL and LDL-CHOL concentrations in

all the groups except in groups 3 and 5 respectively in Table

1.Atherogenicitydevelops when LDL cholesterol,

triacylglycerols and total cholesterol are elevated relative to

plasma HDL-C[29]. Although elevated low density

lipoprotein cholesterol (LDL) is thought to be the best

indicator of atherosclerosis risk, [30] dyslipidemia can also

be describe aselevated total cholesterol (TC) or

triglycerides(TG), or low levels of high density lipoprotein

cholesterol (HDL). Hence the plasma lipid profile lowering

effect of the extract indicates protection against

atherosclerotic cardiovascular disease (ASCVD) which

includes stroke, peripheral arterial disease and coronary heart

disease.

Phytochemical screening of plant from Tables 2 and 3

revealed the presence of flavonoids, triterpenoids, saponins,

tannin, steroid, cardiac glycoside and phenol in decreasing

order (32.19± 0.23, 26.24± 0.12, 23.74± 0.20, 18.74± 0.17,

16.53± 0.12, 15.28± 0.23 and 13.10± 0.11 mg/100g

respectively). It is widely accepted that a plant based diet

with high intake of fruits andvegetables may reduce the risk

of oxidative stress-related diseases such ascancer and

cardiovascular diseases. Most bioactive food constituents are

derived from plants. Result from the study demonstrated

significant reduction in plasma T-CHOL,and TG, and non

significantreduction of LDL-CHOL without a corresponding

increase in HDL-CHOL suggesting enhanced inhibition in

intestinal absorption of both dietary and endogenous

cholesterol by phytochemicals present in plant extract and

not necessarily increased transport of plasma lipids to the

liver via HDL-CHOL. Saponins cause hypocholesterolemia

by binding cholesterol, making it unavailable for absorption

[13]. Specifically, saponin is known to elicit serum

cholesterol lowering activity by causing resin-like action,

thereby reducing the enterohepatic circulation of bile acids

[31]. In the process, the conversion of cholesterol to bile acid

is enhanced in the liver resulting in concomitant

hypocholesterolemia [32,33].Also plant steroids such as

sterols and stanols exhibits cholesterol lowering

propertiesbecause of their structural similarity to that of

cholesterol, they compete for solubilization in the micelles

and therefore inhibit intestinal absorption of both dietary and

endogenous cholesterol [21]. Flavonoids are a group of

phytochemicals found in varying amounts in foods and

medicinal plants which have been shown to exert potent anti-

oxidant activity against the superoxide radical [14]. Its

consumption has been documented not to be associated with

mortality due to coronary heart disease. This may be as a

result of its antioxidant activity and subsequent inhibitions of

Low Density Lipoproteins (LDL) oxidation known to have

been attributed to the dietary and supplemental intake of

flavonoids and other micronutrients.

Figures 1-7 showing histological results revealed that all

the heart muscles were in good histological conditions both

in the control and all extract treated groups. Hence, aqueous

whole plant extract of selaginella myosurus has

hypolipidemic effect and could protect the heart against

atherosclerotic cardiovascular disease (ASCVD), hence

would benovel herbalpharmaceutical agent.

References

[1] Medrano, M.J., Cerrato, E., Boix, R., Delgado-Rodriguez, M(2005). Cardiovascular riskinSpanish population: metaanalysis of cross-sectional studies. Med.Clin. (Bar), 124:606-612.

[2] Pushpavalli, G., Veeramani, C. and Pugalendi, K.V. (2010). Influence of chrysin on hepatic marker enzymes and lipid profile against D-galactosamine-induced hepatotoxicity rats. Food. Chem. and Toxicol., 48: 1654-1659.

[3] Agellon, L.B., Walsh, A., Hayek, T., Moulin, P., Jiang, X.C., Shelanski, S.A. Breslow, J.L. and Tall, A.R. (1991). Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice. Journal. of Biological. Chem., 266: 10796- 10801.

[4] Brinton, E.A., Eisenberg, S. and Breslow, J.L. (1990). A low fat diet decreases high-density lipoprotein (HDL) cholesterol level by decreasing HDL apolipoprotein transport rates. J. Clin. Invest., 85: 144-151.

[5] Kritchersky, S., (1970). The role of cholesterol as vehicles experimental atherosclerosis. Am. J.Clin., Nutr., 23: 1105-1110.

[6] Kucera, M., V.O. Marqis and Kuceroahk, 1972. Contribution to the knowledge of Nigeria medical plant J.L.C separation and quantitative evaluation of alstoniaboonei alkaloid plants. Medica., 21: 343-346.

[7] Setyawan, A.D. &Darusman, L.K. (2008). Review: Biflavonoid compounds of Selaginella Pal. Beauv. and its benefit. Biodiversitas 9 (1): 64-81.

[8] Bensky, D., Clavey, S. andStöger, E. (2004).. Chinese herbal medicine; material medica. 3rd ed. Eastland Press. Seattle, WA.

[9] Chikmawati, T., Setyawan, A.D. &Miftahudin. (2008). KandunganfitokimiaekstraktumbuhanSelaginella di pulauJawa. Seminar danKonggres PTTI ke-VIII. Cibinong Science Center, Bogor-Indonesia, 21-23.

[10] Setyawan AD. (2009). Traditionally utilization of Selaginella; field research and literature review. Nusantara Bioscience 1: 146-158

[11] Zenk, H.M., (1991). Chasing the enzymes of secondary metabolism: Plant cell cultures as a pot of goal. Phytochemistry, 30(12): 3861-3863.

[12] Sofowora, A. (1982). Medicinal Plants and Traditional Medicine in Africa. Wiley, Ann Arbor, Mich., pp: 256, ISBN: 0471103675.


[13] Price, K.R., Johnson, L.I. and Feriwick, H. (1987). The chemical and biological significance of saponins in foods and feeding stuffs. CRC Critical Revigar. Food Sci. Nutr., 26: 127-135.

[14] Hertog, M.G.L., Feskens, E.J.M., Hollman, D.C.H., Katan, M.B.and Kromhout, D. (1993). Dietary antioxidant flavonoids and risk of coronary heart disease. Zutphen Elderly Stud. Lanc., 342: 1077-1011.

[15] Okwu, D.E. and Okwu, M.E. (2004). Chemical composition of SpondiasmombinLinn plant parts. J. Sustain. Agric. Environ., 6: 140-147.

[16] Treasure, C.B., J.L. Klein and W.S. Weintraub, 1995. Beneficial effects of cholesterol lowering therapy on the coronary endothelium in patients with coronary heart diseases. N. Engl. J. Med., 332: 481-487.

[17] Steiner, M. and W. Li, 2001. Aged garlic extract, modulator of cardiovascular risk factor: A dose finding study on the effect of age on platelet function. J. Nutri., 131(3): 9805-9845.

[18] Silveira, M.B., Monereo, S. and Molina, B. (2003). Funtional nutrition and optimal nutricion. Near or far? Rev. Esp. Salud. Pública.,77(3):317-331.

[19] Plaza, I (2001). Los fitosteroles, el colesterol y la prevención de lasenfermedadescardiovasculares. Clin. Invest Arterioesclerosis, 5:209-18.

[20] Piironen, V., Lindsay, D.G., Miettinen, T.A., Toivo, J., Lampi, A.M.(2000) Plant sterols: biosynthesis: biological function and their importance to human nutrition. J. Sci. Food Agric., 80:939-966.

[21] Heinemann, T., Kullabak-Ublick, A., Pietruck, B. and von Bergmann, K.(1991). Mechanism of action of plant sterols on inhbition of cholesterol absorption. Eur. J. Clin. Pharmacol., 20:59-63.

[22] Sofowara A. (1993). Medicinal Plants and Traditional Medicine in Africa. p. 289, Spectrum Books Ltd., Ibadan, Nigeria.

[23] Bruce, R.D. (1985) An up-and-down procedure for acute toxicity testing. Fund.Appl.Toxicol. 5:151-157.

[24] Allain, C.C., Poon, L.S. Chan, C.S.G. Richmond, W. and Fu, P.C. (1974). Enzymatic determination of total serum cholesterol.Clin. Chem., 20: 470-475.

[25] Stein, E.A.(1987). Lipids, Lipoproteins and Apolipoproteins. In: Tietz, N.W. (Ed.), 3rd Edn., Fundamentals of Clinical Chemistry. WB Saunders, Philadelphia, pp: 470-479.

[26] Friedewald, W.T., Levy, R.I.and Frederickson,D.S. (1972). Estimation of the concentration of low-density lipoprotein cholesterol in plasma without the use of preparative ultra-centrifuge. Clin. Chem., 18: 499-502.

[27] Lillie, R.D. (1965). Nuclei, nucleic acid, general oversight stains. In: Histopathology Technique andPracticalHistochemistry, 3rd edition, McGraw Hill Book Company, pp.142-179.

[28] Ames, B.N.; Shigenaga, M.K. and Hagen, T.M. (1993). Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA,90, 7915-7922.

[29] James, D.B., Kadejo, O.A., Nwochiri, C. and Luca, C.D. (2013).Determination of Phytochemical Constituents of the Aqueous Extracts of the Leaves, Stem Bark and Root Bark of Vitexdonianaand its Effects on Lipid Profile of Albino Rats. British Journal of Pharmacology and Toxicology, 4(6): 210-214.

[30] Jacobson, M.S. (1998). Heart healthy diets for all children: no longer controversial. J. Pediatr., 133(1):1-2.

[31] Topping, D.L., Storer, G.B., Calvert, G.D., Illman, R.J., Oakenfull, D.G. and Weller, R.A. (1980). Effects of dietary saponins on fecal bile acids and neutral sterols, plasma lipids and lipoprotein turnover in the pig. The American Journal of Clinical Nutrition, 33: 783-786.

[32] Kritchevsky, D (1977). Dietary fiber and other dietary factors in hypocholesterolemia. The American Journal of Clinical Nutrition, 30: 979-984.

[33] Potter, D.P., Topping, D.L. and Oakenfull, D. (1979). Soya saponins and plasma cholesterol. Lancet, 1: 223.

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