Effect of Aqueous Whole Plant Extract of Selaginella ...article.aascit.org/file/pdf/9230793.pdf73...
Transcript of 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.
GROUP 3 (600mg/kg b.w extract): A single daily dose of
600mg/kg b.w of aqueous whole plant extract of
Selaginellamyosuruswas orally administered to rats in this
groupfor 7 days.
GROUP 4 (800mg/kg b.w extract): A single daily dose of
800mg/kg b.w of aqueous whole plant extract of Selaginella
myosurus was orally administered to rats in this group for 7
days.
GROUP 5 (1000mg/kg b.w extract): A single daily dose of
1000mg/kg b.w of aqueous whole plant extract of Selaginella
myosurus was orally administered to rats in this group for 7
days.
GROUP 6 (400mg/kg b.w extract): A single daily dose of
400mg/kg b.w of aqueous whole plant extract of Selaginella
myosurus was orally administered to rats in this group for 14
days.
GROUP 7 (600mg/kg b.w extract): A single daily dose of
600mg/kg b.w of aqueous whole plant extract of Selaginella
myosurus was orally administered to rats in this groupfor 7
days.
GROUP 8 (800mg/kg b.w extract): A single daily dose of
800mg/kg b.w of aqueous whole plant extract of Selaginella
myosuruswas orally administered to rats in this group for 7
days.
GROUP 9 (1000mg/kg b.w extract): A single daily dose of
1000mg/kg b.w of aqueous whole plant extract of Selaginella
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.
75 Omeodu S. I. et al.: Effect of Aqueous Whole Plant Extract of Selaginella myosurus on Lipid Profile of Wistar Rats
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).
American Journal of Pharmacy and Pharmacology 2015; 2(5): 72-78 76
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,
77 Omeodu S. I. et al.: Effect of Aqueous Whole Plant Extract of Selaginella myosurus on Lipid Profile of Wistar Rats
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.
American Journal of Pharmacy and Pharmacology 2015; 2(5): 72-78 78
[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.