33 A dvances in Environmental Biology, 3(1): 33-38, 2009 ISSN 1995-0756

33
A dvances in Environmental Biology, 3(1): 33-38, 2009
ISSN 1995-0756
© 2009, A merican-Euras ian Network for Scientific Information
T his is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Comparative Studies on the Hypoglycaemic, Hypoproteinaemic, Hypocholesterolaemic
and Hypolipidaemic Properties of Ethanolic and Normal Saline Extracts of the Root of
Vernonia Amygdalina in Diabetic Rats.
1
Igbak in, A.P. and Oloyede 2, O.B.
1
Department of Biochemistry, Adek unle Ajasin University, Ak ungba-Ak ok o, Ondo State, Nigeria.
Department of Biochemistry, University of Ilorin, Ilorin, Nigeria.
2
Igbakin, A .P. and Oloyede, O.B.; Comparative Studies on the Hypoglycaemic, H y p o p roteinaemic,
Hypocholes terolaemic and Hypolipidaemic Properties of Ethanolic and Normal Saline Extracts of the
Root of Vernonia Amygdalina in Diabetic Rats ., Am.-Eurasian J. Sustain. Agric., 3(1): 33-38, 2009
ABS TRACT
A comparative s t u d y of the hypoglycaemic, hypoproteinaemic, hypolipidaemic and hypocholes terolaemic
properties of the ethanolic and normal s aline extracts of the root of vernonia amy g d a l i n a w as carried out on
alloxanized diabetic rats treated for s even weeks . Res ults s howed that the ethanolic extract of the root of
vernonia amygdalina (EEVA ) is mo re potent as it lowered the blood glucos e by 68% while the Normal Saline
extract of the root of V. amygdalina (NEVA ) only reduce s ame by 24%. T h e lo w e ring effects of EEVA on
the s erum protein, choles terol a nd total lipid were als o s ignificant (p<0.05) when compared with rats on NEVA
and diabetic untreated groups . The res ults clearly indicate that the h y p o g lycaemic, hypocholes terolaemic,
hypolipidaemic and hypoproteinaemic active principles is contained in the ethan o lic extract of the root of
V e r n onia amygdalina and not the normal s aline extract. The various concepts as s ociated with metabolis m o f
s ugar, proteins , and lipids in diabetic s tate are als o dis cus s ed.
Key words: A llo xa n n ize d
d ia b e t ic rats , hypocholes terolaemic,
hypoproteinaemic and Vernonia amygdalina.
Introduction
Vernonia amygdalina (Compos itae) is one of the
edible vegetables in Nig e ria and other parts of
A frican s ub-regions . It is the name of an A frican
s hrub or s mall trees of the as ter family, called bitter
leaf [6]. A s a vegetable it contain s antinutrient
s ubs tances like alkaloids , s aponnins and flavones that
have medicinal properties [7,17] Studies have s hown
that extracts of the flowers of Vernonia amyg d a l i na
exhibited a pronounced antiviral effect agains t s everal
of the tes t virus es and it has been employed in the
treatment of A s thma and kidney problems in Brazil
a n d s o me part of A s ia [22]. Several other
antinutrients is olated from t h e p lant included
hypolip id a e mic , h y p o g ly c a e mic ,
vernoleptin which has s pamolytic activity in vitro on
s mooth mus cle of guinea pigs ; eramathine which
inhibits the penetration of cercariae of nematode
s chis tos oma mans onii [3]. Other phys iologically
active compounds is olated from this plant include
s es quiterpene lactones -vernodaline and ve rnomyelin,
a s t e ro l-7,24 (28)-s tigmas tadiene-3â -o l; t o xic
cardenolides and s aponin s . A ll thes e components
h a v e been proved to have phys iological and
metabolical effects on the body s ys tem.
In N igeria where the plant is found in
abundance, it performs both medic in al and nutritive
functions , it is us ed in the preparation of s oup,
prevention of malaria fever, elimination o f worms ,
treatment of s tomach ups et, ind u c t ion of fertility in
Corres ponding Author
Igbakin, A.P., Department of Biochemistry, Adekunle Ajasin University, Akungba-Akoko, Ondo
State, Nigeria.
Email: [email protected]
Adv. Environ. Biol., 3(1): 33-38, 2009
barren women and treatme nt of diabetic mellitus [11].
The leaves and the bark/s tem have been acclaimed to
have an t i-diabetic properties in different s olvents .
Nothing has been documented reas onably on the antidiabetic properties of the root. This paper attempts to
as s es s a n d compare the hypocholes terolaemic,
hypoglycaemic, hypolipidaemic and hypoproteinaemic
effect of different s olvent extracts of t h e root of
Vernonia amygdalina.
34
Materials and methods
hours to a llow extraction to take place. The res ulting
mixtures were vigorous ly s haken for 6 hours by wris t
action s haker for complete extraction to be achiev e d .
The extracts were s eparately filtered and evaporated
under reduc ed pres s ure The res ulting res idues were
s eparately re-extracted with 2% (v/v) e t h a nol and
0.9%(w/v) NaCl s olu t ion and filtered again. The two
filtrate s were pull together and the weight of the
remaining res idues computed. The filtrate was
recons tituted to a final concentration of 500 mg/ml in
each cas e and kept frozen until ready for us e.
Equipments and chemicals
Experimental design
Bovine s erum albumin (BSA ), copper(II)ulphate ,
potas s ium iodide, choles t e ro l c o lo u r reagent,
c h o le s t e rol s tanda rd , v a n illin , a llo xa n a n d
bromocres ol green are produ c t s of s igma chemical
company M o, USA while chloroform, ethanol,
phos phoric acid, s u lp h u ric acid, glacial acetic acid
and ferric chloride were purchas ed from BDH
chemicals P oole, England, olive oil is a product of
M ay and Baker. A ll reagents are of analytical grade,
s pectro 21 Baus ch a n d Lomb made in USA ,
laboratory table ce ntrifuge M odel SM 800B unis cope
SM 8001A laboratory water bath and wris t action
s haker made in USA were us ed for the analys is .
The rats were div id e d into s ix groups with each
group cons is ting o f nine rats . Five of thes e groups
were made diabetic b y a s ingle intraperitoneal
injection (100mg/kg body weight) o f alloxan in
no rmal s aline. Blood glucos e of rats more than
180mg/dl was chos en as criteria for diabetes [13].
The grouping of the animals are as follows :
Animals
Pos t weanlin g healthy albino rats of the wis ter
s train (Rattus norvegicus) weighing between 145 and
158g were us ed fo r t he s tudy. They were fed
commercially produced diet (Bendel feed Nig. Ltd.
Benin city, Nigeria) ad l i b i t u m . T hey were
maintained at 25-27o C and ke p t under 12 hours of
light and 12 hours of darknes s . They were als o
allowed free acces s to drinkin g w a ter in a well
ventilated animal cage.
Plant samples
The roots of Vernonia amygdalina was obtained
from various locations in Ilorin s ub-towns during the
month of many 2002; it was air-dried in the
laboratory for 20 days at 26-29o C. the plants root
was identified and au t henticated by Profes s or M .A .
F a lu y i of the plant s cience Department of univers it y
of A do-Ekiti, Ekiti State, Nigeria.
Preparation of root extract of Vernonia amygdalina
The dried root was finely grounded into powder
after w h ich 5g portions were s eparately macerated
with 250cm 3 of 2% ethanol (v/v) and 0.9% NaCl
(w/v) s olutions and kept at room temperature for 24
NDDW : Non diabetic rats o n dis tilled water (control)
DEVA : Diabetic rats on 500mg/kg bod y weight of
ethanolic extract of Vernonia amygdalina
DNVA : Diabetic rats on 500mg/kg body weight of
n o rma l s a line e xt ra c t o f V e r n o n i a
amygdalina
DETH: Diabetic rats on 2% ethanol
DNOS: Diabetic rats on normal s aline
DDSW :Diabetic rats on dis tilled water.
The extracts were adminis tered orally t o the
animals for s even weeks . A t the end of the s e v e n th
w e e k the rats were s acrificed and their s era collected
for biochemical analys is .
Biochemical analysis
A fter 24hours of alloxan adminis tration to induce
diabetes , plas ma glucos e w a s determined from the
whole blood drawn from the tail vein us in g T rinder
methods o f 1969; plas ma and tis s ue proteins were
determined by the method of Gornall et al [9].
Plas ma choles terol was determined by enzymatic
method of Trinder [25] and plas ma lipids was
extracted and es timated by the method report e d b y
Strova and M arkarova [24].
Statistical analysis
The data obtained were expres s ed as mean ±
SEM and analys e d s tatis tically. The differences in
va lu es between pairs of diabetic (untreated) rats and
diabetic rats treated with ethanolic extract of the root
o f Vernonia amygdalina (EEVA ), between pairs of
control rats and diabetic treated with normal s aline
Adv. Environ. Biol., 3(1): 33-38, 2009
extract of the root of Vernonia amy g d a lina (NEVA ),
between rats on EEVA and NEVA w e re compared.
The mean diffe re nces were analys ed for s ignificance
by us ing Duncan multiple range te s t (DM RT) for the
s amples [20]. Only p values les s than 0.05 were
cons idered s tatis tically s ignificant.
Res ults and dis cus s ions
Figure 1 s ummarize s t h e plas ma glucos e of the
an imals employed in this s tudy. Hyperglycaemia is
the major s ymptom in diabetics an d when glucos e
e limination occurs in the urine it becomes glucos uria
[14]. A ll the diabetic rats in this pres ent s tudy were
hyperglycaemic except the control. DNVA , DN O S,
DETH and DDSW had their plas ma glucos e levels
above 200mg/dl throughout the s even weeks of the
experiment. Thes e values are high and s tatis t ic a lly
s ignifican t (p<0.05) when compared with the control
(NDDW ) and DEVA . M any res earch e rs had reported
this trend of increas ed plas ma gluc o s e level in
diabetic animals [4,19,15]. P la s ma hyperglycaemia
t h a t o ccurred in diabetics has to do with th e
c o mplete abs ence or relative deficiency of ins ulin
which s uppos e to activate uptake of glucos e from the
plas ma, utilization of glucos e by peripheral tis s ues ,
inhibition of gluconeogenes is a n d many other
fu n c t ions that enable quick removal of glucos e from
the plas ma [18].
The mechanis m by which the ethanolic extract of
the ro o t of vernonia amygdaliona (EEVA ) reduced
plas ma glucos e level as ma n ifes ted in this pres ent
s tudy is y e t to be elucidated. A lthough there are
many pos s ibilities , s ome of which may include the
followings : enhancemen t of glucos e utilization,
pos s es s ion of ins ulin-like properties and therefore
induce trans port of glucos e into the peripheral tis s ues ,
ability to repair the pancreas to be res pons ive to
gluco s e concentration for ins ulin production, may be
acted upon and trans ferred by enzymes to an
intermediate which pos s es s es a pharmacolo g ical
property s uch as hypoglycaemic property , it may
contain biomolecules that can modify or s timulate
ins ulin receptors , may modify the s tructure of glucos e
trans port protein (GLUT 4) and it may inhibit ins ulin
antagonis t within the body
Plasma protein concentration
Increas ed plas ma prot ein was obs erved in this
pres ent s tudy (Figures 2). A nimals in various groups
s uch as DNVA , DETH, DNOS and DDSW had their
plas ma protein increas ed s ignificantly (p<0.05) when
compared with DEVA , and NDDW (contro l). There
was no s ignificant different (p<0.05) between the
control g ro u p (NDDW ) and DEVA . The various
functions of ins ulin whic h is deficient in diabetics
35
s u ch as promoter of protein s ynthes is , enhanceme n t
of amino acids uptake by peripheral t is s ues , and
ribos omes , decreas e protein catabolis m and decreas ed
releas e of gluconeoge n ic amino acids etc res ulted in
the main tenance of cons tancy in the plas ma protein
c o ncentration [27]. W hen ins ulin is not available o r
wh en there is availability of non-functional ins ulin,
the revers e of all the proces s es above occurs . W ith
this , the plas ma protein level is s uppos ed to decreas e
too. W hat actually increas ed plas ma protein level in
diabetics is not unconnected with altera tion in
phys iological p roces s es and complicative damage of
the s y n d ro me o n t h e b o d y t is s u e s [16].
Hyperpro teinaemia had been implicated to occur
under the following conditions : c hronic nephritis at
the s tage of polyuria w h ic h as s ociate tightly with
diabetes me llitus , renal tubular damage at the s tage
of recovery with gros s diures is as found in diabetic
p a t ie nts , uncomplicated liver dis eas e s uch as acute
hepatitis , hepatic necros is , cirrh o s is , biliary or portal
together with vas cular dis eas es and s evere infections
which characterized diabetic c o n dition and s evere
diabetic ketos is res ulted in elevated plas ma protein
[21,5,2].
Ethan o lic extract of the root of vernonia
amygdalina (EEVA ) was able to re d uce the plas ma
protein level to appreciable level of the control
probably becaus e of their abilities to prevent diabetic
c omplications s uch as kidney and liver damage or t o
prevent hyperglycaemia which is the origin of all the
diabetic abnormalities .
Changes in plasma total lipids
Total lipids levels in the plas ma of diabetic rats
in t h is pres ent s tudy is high (Figure 3). There was
s ignificant (p<0.05) difference in the level of total
lipids of DNVA , DETH, DNOS and DDSW as
c o mpared with NDDW (control) and DEVA . Hig h
level of tot a l lipids in diabetics (hyperlipidaemia) had
earlier been reported by A doga [1] and Godwin and
M ohammed [8]. Total lipids in the s e ru m include
FFA , choles terol, triglycerides and fractionation of
lipoproteins . A ll thes e plas ma lipids component s a re
elevated in diabetic s tate as a res ult of relative abs ent
or deficiency of ins u lin [12]. Ins ulin activates
lipoproteins lipas e that clears triglycerides fro m t he
plas ma. It inhibits hormon e s ens itive lipoprotein
lipas e in the adipos e tis s ues that releas e fatty acids
into the p la s ma. W ith high concentration of lipids in
the plas ma lipoprotein s ynthes is equally occurs in the
liver. A ll thes e res ult ed into accumulation of lipids in
the plas ma. W ith the treat ment of diabetic rats with
ethanolic extracts of the root of vernonia amygdalina
(EEVA ) t he cells may revert to glucos e as a s ource
of energy or enhance res pons ivenes s to ins ulin action
thereby improving trig ly c e rid e c le a ra nce and
decreas ing lipolytic ac t ivities , res ulting in lowered
plas ma total lipids and choles terol as re ported in this
pres ent s tudy.
Adv. Environ. Biol., 3(1): 33-38, 2009
36
Fig. 1:
Plas ma glucos e concentration (mg/dl) of alloxan-induced diabetic rats adminis tered norma l s a line and
ethanolic extracts of the root of Vernonia amygdalina over a period of s even weeks .
Fig. 2:
Plas ma protein concentration (mg/dl) of alloxan-induced diabetic rats adminis tered normal s aline and
ethanolic extract of the root of Vernonia amygdalina over a period of s even weeks .
Cholesterols in the plasma
Choles terol in the plas ma of the animals in the
DNVA , DETH , DNOS and DDSW groups increas ed
s ignificantly (p < 0.05) as compared with thos e in the
NDDW (control) and DEVA (Figure 4). It ha s e a rlier
been reported that experimentally-induced diabetes is
accompanied by a concomitan t increas e in the s erum
choles terol and lipid i.e. hypercholes terola emia and
h y p e rlip id a e mia re s p e c t ively [26,10,23]. T h e
hypercholes terolaemia is a cons equence of the body
reverting to the us e of lipids for energy production in
t h e a bs ence of glucos e. This res ulted to accelerated
fatty acids â-oxidation that led to enormous
Adv. Environ. Biol., 3(1): 33-38, 2009
37
Fig. 3:
Plas ma total lipid concentration (mg/dl) of alloxan-induced diabetic rats adminis tered n o rmal s aline
and ethanolic extracts of the root of Vernonia amygdalina over a period of s even weeks .
Fig. 4:
Plas ma choles terol concentration (mg/dl) of alloxan-induced diabetic ra t s adminis tered normal s aline
and ethanolic extracts of the root of Vernonia amygdalina over a period of s even weeks .
pro d u ction of acetyl-coA , exces s of which is
channeled to choles tero l bios ynthes is . A bility of the
ethanolic extracts of the root of vernonia amygdalina
(EEVA ) to reduce the choles terol levels in d iabetes
may be probably due t o t h e fact that thes e plant
materials contain bioactive molecules that pos s es s e d
choles terol lowering action or ins ulin-like ac t ion or
ac t ion which can inhibit oxidation of fatty acids for
energy production.
Conclus ively this work has been able to s how
that the ethanolic extra c t o f the root of Vernonia
amygdalina has hypoglycaemic, hypolipida emic,
h y p oproteina e mic a n d h y p o c h o le s t e ro la e mic
properties more than the normal s aline extra c t o f the
Adv. Environ. Biol., 3(1): 33-38, 2009
s ame plant. It equally s howed that the bioactive
ingredient us eful in the treatme n t of diabetic mellitus
is pres ent in the e t h anolic extract of the root of
Vernonia amygdalina (EEVA ) and not in the n o rmal
s aline extract. This ethanolic ext ract can be further
s eparated and purified s o as to identify and determine
the s tructure of the bioactive compound.
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