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PHCOG MAG.: Research Article
Antioxidant, Antihyperlipidaemic and Antidiabetic Activity of
Dendrophthoe falcata Leaves- A Preliminary study Tenpe C. R*., Upaganlawar A. B., Khairnar A. U. and Yeole P. G.
Department of Pharmacology, Institute of Pharmaceutical Education & Research, Wardha - 442 001, Maharastra,
India.
Author for Correspondence : [email protected]
ABSTRACT
Present study was carried out to evaluate antioxidant, antihyperlipidemic and antidiabetic activity of Petroleum
ether extract (PEE), Ethyl acetate extract (EAE) and ethanol (70%) extracts (EE) of Dendrophthoe falcata (DF)
leaves. Preliminary phytochemical analysis reveals the presence of sterols, flavonoids, carotenoids and phenols in
different extract. Ethanol extract showed better in vivo antioxidant activity in both DPPH radical scavenging
(IC50=16.78µg/ml) and nitric oxide radical scavenging activity (IC50=54.5µg/ml) in sodium nitroprusside/Griss
reagent system. Lipid lowering activity of Dendrophthoe falcata (DF) extract (300mg/kg/day, p.o) was tested in
high fat diet model for 42 days (six weeks). Treatment of Dendrophthoe falcata 70% ethanol extract for forty two
days along with high fat diet showed significant (P<0.01) reduction in serum total cholesterol (TC), triglyceride
(TG), low density lipoprotein (LDL), very low density lipoprotein (VLDL) and significant increased in the level of
high density lipoprotein (HDL) when compared with hyperlipedemic control. The lowered atherogenic index of
extract groups suggests antihyperlipedemic and cardioprotective potential. 70% ethanol extract (300mg/kg/day,
p.o) exhibited significant antihyperglycemic activity in alloxan induced diabetic rats with significant improvement
in body weight and reduction in blood glucose, serum creatinine and urea level. On the basis of above result we
can conclude that 70% ethanol extract of Dendrophthoe falcata leaves possesses good antioxidant,
antihyperlipidemic and antdiabetic activity than petroleum ether extract and ethyl acetate extract.
KEY WORDS: Dendrophthoe falcata, Antioxidant, Hyperlipedemia, Antidiabetic
INTRODUCTION
Oxygen involved in the respiratory process can be
transformed under some condition into superoxide
anion, hydroxyl radical, singlet oxygen and hydrogen
peroxide (1).These reactive oxygen species are
implicated in some diseases such as inflammation,
cancer, ageing, anemia, degenerative diseases and
atherosclerosis (2). Hyperlipidemia is one of the most
important risk factors for susceptibility to coronary
heart diseases and it is characterized by abnormally
elevated blood levels of lipids (triglyceride and
cholesterol) and lipoproteins (LDL, VLDL) (3).
Atherosclerosis is the underlying form in CHD that
affects the intima of the coronary arteries. Several risk
factors influence the pathogenesis viz. diabetes,
hypertension, obesity, high fat diet, lack of exercise
and stress. Oxidative stress has been shown to have a
role in the elevation of diabetes and related problems
(4). In diabetes, protein glycation and glucose
autooxidation may generate free radical in the body,
which in turn catalyze lipid peroxidation (5).
Dendrophthoe falcata (L. f.) Etting
(Family: Loranthaceae) is evergreen, large, bushy
parasitic plant, usually glabrous, branched parasite
with smooth gray bark, found almost throughout India,
Burma, Ceylon and Australia. The plant has the
properties as astringent, narcotic, bitter, diuretic and
used in the treatment of tuberculosis, asthma and
mania. It is used in menstrual disorder, wounds, in
prevention of stone in kidney & bladder, hemorrhage,
miscarriage and abortion during pregnancy. It is used
as an anti-ulcer, anthelmintic and blood purifier. It is
also used in fetus development and blood pressure
management. Plant grown on Ficus fistula host is used
for foetus development in Ayurveda. It is used in
Vatta, Kapha and Pitta. This plant is used for avoiding
abortion which occurs during 3rd month of pregnancy
(6-8).
Dendrophthoe falcata, is reported to contain
biologically active substances such as flavonoid,
quercetin, kempferol, rutin (9) tannins, β-sitosterol, β-
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amyrin, oleanolic acid (10) chlorophyll (11) and
Phenolic contents (12). Indranin et al found (+)
catechin and leucocyanidin from bark of D. falcata
(13). Based on the presence of different active
constituents mainly flavonoids, tannins and phenolic a
study was designed to screen the different extracts of
Dendrophthoe falcata for its in vitro antioxidant
activity, antihyperlipidemic and antidiabetic activity.
MATERIALS AND METHODS
Collection and extraction of the Plant
Dendrophthoe falcata leaves were collected from
Nashik District, Maharashtra, India and a voucher
specimen (No. BSI/WC/Tech/2006/197) was deposited
with the, Botanical survey of India, Pune .The shade
dried and powdered leaves of Dendrophthoe falcate,
were subjected to successive extraction in a soxhlet
apparatus with petroleum ether, ethyl acetate and
ethanol (70%) extracts. All extracts were individually
filtered, through Whatmann filter paper # 42 and
evaporated to dryness at 50º C in oven. The extracts
were then stored in desiccators till further use.
Percentage yield of the extracts was found to be 5.8%,
14.7%, and 16.3% w/w respectively.
Chemicals
Diagnostic kit used for estimation of Triglyceride,
Cholesterol, HDL-C and glucose were obtained from
MERCK LTD, Mumbai. Alloxan hyderate was procured
from Loba chemicals. All the other chemicals used
were of AR grade.
In Vitro antioxidant activity
DPPH scavenging activity (14,15)
The free radical scavenging activity of petroleum
ether, ethyl acetate and ethanol 70 % extracts of
Dendrophthoe falcata leaves and ascorbic acid (ASC)
was measured in terms of hydrogen donating ability
using the stable radical DPPH.
0.1 mM solution of DPPH in ethanol was prepared and
absorbance was measured at 517 nm in
spectrophotometer, said as absorbance of control
reaction, further 1.0 ml of this solution was added to
3.0 ml of extracts solution in ethanol and water for
ethanol (70%), petroleum ether and ethyl acetate
extract respectively, at different concentration [5-80
µg/ml], 30 minutes later, the absorbance was
measured at 517 nm in spectrophotometer (Make and
model used). Lower absorbance of the reaction
mixture indicates higher free radical scavenging
activity.
Nitric oxide radical scavenging activity (16-17)
The reaction mixture (3 ml) containing sodium
nitroprusside (10mM, 2 ml), phosphate buffer saline
(0.5 ml) and 0.5 ml of different concentration (5-80
µg/ml) of extracts were dissolved in respective solvent
system and incubated at 250C for 150 minute. After
incubation, 0.5 ml of the reaction mixture containing
nitrite was pipette and mixed with 1 ml of sulfanilic
acid reagent (0.33% in 20% glacial acetic acid) and
allowed to stand for 5 minute for completing
diazotization. Then, 1 ml of napthylethylendiamine
dihydrochloride (0.1%) was added, mixed and allowed
to stand for 30 minutes at 250C. A pink colored
chromophore was formed in diffused light. The
absorbance of this solution was measured at 547 nm
against the corresponding blank solution using
spectrophotometer. Ascorbic acid was used as positive
control. IC 50 value is the concentration of sample
required to inhibit 50 % of nitric oxide radical. All
determinations were performed in triplicates.
Acute oral toxicity test:
Acute oral toxicity test was carried out as per the
protocol given in the OECD guidelines (18) and a dose
of 300 mg/kg was selected for the animal activity.
Experimental animals:
Adult albino rats (Wistar strain), of either sex
weighing 120-200 gm were used in the entire study.
The animals were housed in the polypropylene cages
with 12 hours dark-light cycle, at appropriate
temperature and humidity conditons. Animals were
fed with a balanced diet and water ad libitum. All
animal experiments were approved by the
Institutional Animal Ethical Committee (Registration
No.535 / 02 / a / CPCSEA / Jan 2002) of Institute of
Pharmaceutical Education and Research, Wardha.
Antihyperlipidaemic activity
Hyperlipidaemia was induced by administration of
High fat diet (HFD) having composition wheat flour 30
g, sucrose 25 g, milk powder 22 g, hydrogenated
ground nut oil 22 g, cholesterol 1% of 100 g of diet.
Petroleum ether, ethyl acetate and ethanol (70%)
extracts of Dendrophthoe falcata leaves were
suspended in Tween 80 (2.5 %) in water and
administered in a dose of 300 mg/kg, p.o. to the
animals for six weeks (42 days). The rats were divided
in seven groups.
Group A: Animals served as control and received 0.3
ml 2.5 % tween 80 /kg/day, p.o.
Group B: Animals received HFD.
Group C: Animals received HFD and Lovastatin
(10mg/kg, p.o).
Group D: Animals received HFD and Petroleum ether
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extracts (300 mg/ kg, p.o).
Group E: Animals received HFD and Ethyl acetate
extracts (300 mg/kg, p.o).
Group F: Animals received HFD and Ethanol (70%)
extracts (300 mg /kg, p.o).
Antidiabetic activity
Rats were made diabetic by a single intraperitoneal
injection of Alloxan monohydrate (Loba Chemicals,
Bombay) 70 mg/kg. Two days after Alloxan injection
rats with plasma glucose level of > 200 mg/dl were
included in the study.
All extracts of Dendrophthoe falcata Linn, leaves were
suspended in Tween 80 and was orally (p.o)
administered in dose 300 mg/kg to the animals by
gastric intubation for the period of 21 days. In the
experiment Wistar albino rats were used (36 Nos). The
rats were divided in six groups of six animals in each
group.
Group I: Animals served as normal and received 0.3
ml tween 80% /kg/day, p.o. .
Group II: Animals were treated with single dose of
Alloxan (70mg/kg, i.p).
Group III: Standard reference drug Glibenclamide
(10mg/kg, p.o) + Alloxan (70mg/kg, i.p) .
Group IV: Petroleum extracts (300 mg/kg, p.o) +
Alloxan (70mg/kg, i.p)).
Group V: + Ethyl acetate extracts (300 mg/kg, p.o)
+ Alloxan (70mg/kg, i.p).
Group VI: Ethanol (70%) extracts (300 mg/kg) p.o)+
Alloxan (70mg/kg, i.p).
Biochemical analysis
Blood samples were collected at the end of treatment
period from individual rat by orbital sinus under ether
anesthesia and were centrifuged by using table top
centrifuged at 2000 rpm for 30 minute so as to
separate the serum. Quantitative determination of
serum total cholesterol (TC), triglyceride (TG) and HDL
was done by using Diagnostic Kits. Level of low density
lipoprotein (LDL) and very low density lipoproteins
(VLDL) were calculated by the method of Friedwald’s
formula (19) and Atherogenic index was determined
using following equation.
LDL= Total Cholesterol-(VLDL+ HDL)
VLDL=Triglyceride/5
A.I= VLDL+LDL / HDL
In case of antidiabetic study the protocol was of 21
days. Blood glucose level and body weights were
analyzed on 1, 7, 14, 21st day of study. Analysis of
serum creatinine and urea was done at the end of
study on 21th days using diagnostic kit.
Statistical analysis
The results was analyzed by one-way analysis of
variance (ANOVA) followed by Dunnett’s t-test. P<0.05
was considered as significant. All data are expressed as
mean ± SEM.
RESULTS AND DISCUSSION
Preliminary phytochemical investigation was done by
chemical tests. Pet. ether extract showed presence of
sterol and carotenoids. Ethyl acetate extract showed
presence of flavonoids and phenolic compounds.
Ethanol (70%) extract showed presence of sterol,
flavonoids, tannins and phenolic compounds.
Free radical scavenging activity:
Diphenyl picryl hydrazyl (DPPH) is a nitrogen-centered
free radical. It reacts similar to the peroxyl radical and
the reaction rates correlate directly with the
antioxidant activity.
In the present study IC 50 values of different extracts
were calculated and compared with IC 50 value of
ascorbic acid as a standard (IC 50=12.42 µg/ml).
Ethanol (70%) extract showed significant activity with
IC 50 value 16.78 µg/ml. The results indicate efficacy of
extract in following order ASC>ET>EAE>PET. (Fig.1)
Nitric oxide radical generated from sodium
nitroprusside at physiological pH was found to be
inhibited by Ethanol (70%), Ethyl acetate and Pet.
ether extracts. Determination of IC 50 values of
different extracts and ascorbic acid as a standard were
done. Ethanol (70%) extract had shown significant
activity with IC 50 value 54.5 µg/ml as compared to IC
50 of ascorbic acid (Fig. 2).
Antihyperlipidaemic activity:
HFD treatment for 42 days shows marked elevated
levels of serum TC, TG, LDL VLDL, and reduction in
level of HDL as compared to control group fed with
normal diet (Table 1). Administration of Dendrophthoe
falcata leaves extracts with daily dose of 300 mg/kg,
p.o significantly altered the levels of serum TC, TG,
LDL, VLDL and serum HDL level at different degrees.
Elevated level of blood cholesterol especially LDL is a
known major risk factor for coronary heart disease
where as HDL is cardioprotective (20). Among the
different extracts, ethanol (70%) extract at 300mg/kg
showed significant activity (p<0.01). While the Ethyl
acetate extract had shown considerable reduction in
the TG &VLDL level (p<0.05). Lovastatin 10 mg/kg was
used as a standard drug and showed significant results
(p<0.01).
Rats treated with HFD and ethanol (70%), Ethyl acetate
and petroleum extract showed reducing effects on
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Fig 1- DPPH Scavenging activity.
Std – Ascorbic acid, pet-Petrolium ether extract,
eta-Ethyl acetate extract and eth-Ethanolic extract
Fig 2: Nitric oxide radical scavenging activity
Std – Ascorbic acid, pet-Petrolium ether extract,
eta-Ethyl acetate extract and eth-Ethanolic extract
Table 1: Effects of D. falcata leaves extracts on lipid profile in rats treated with HFD
Groups TC (mg/dl) TG
(mg/dl)
HDL-C
(mg/dl)
LDL
(mg/dl)
VLDL
(mg/dl)
A I
(mg/dl)
Group A
Normal
86.15
±1.98
78.49
±1.43
49.28
±2.85
52.85
±1.51
15.00
±0.16
2.76
±0.54
Group B
HFD control 227.32
±1.92
173.65
±2.02
33.65
±1.69
225.51
±2.58
37.02
±2.14
13.86
±1.23
Group C
HFD + standard
(10 mg/kg)
155.99
±2.33**
112.12
±2.78**
45.17
±1.32
134.68
±1.56**
24.59
±0.70**
7.65
±0.97**
Group D
HFD + Pet. ether
extract
(300mg/kg)
208.22
±2.867
145.47
±3.07
34.56
±1.33
192.14
±3.56
33.95
±1.33 ns
12.89
±1.51
Group E
HFD+Ethylacetate
extract (300
mg/kg)
177.67
±1.82
129.46
±3.53 **
34.43
±0.77
172.13
±2.02
27.19
±1.50
**
11.75
±1.47
Group F
HFD + Ethanol
(70 %) extract
(300 mg/kg)
162.22
±1.59**
122.42
±3.99**
39.16
±2.14**
151.61
±2.81**
24.72
±0.79**
9.46
±1.98**
Values are expressed as Mean ± S.D. (n=6). The results was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s
t-test. p<0.05* , p<0.01** Considered as significant
Table 2: Effect of D. falcata leaves extracts on glucose level and body weight
Groups Days Glucose (mg/dl) Weight (g)
Group I
Normal group
1
7
14
21
84.14±2.269
84.95± 2.65
80.91±2.529
77.5±2.671
201.4±3.586
203.5±4.661
203.6±5.632
204.3±3.146
Group II 1 312.5±2.27 197.5±4.68
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Diabetic control 7
14
21
303.8±3.69
295.8±2.98
293.2±3.584
180.4±5.12
163.4±4.21
142.7±3.47
Group III
Standard drug
1
7
14
21
296.5±1.325
225.3±2.54
153.8±3.511
125.4±2.82**
203.7±4.34
194.2±3.46
190.5±2.14
189.5±3.47**
Group IV
Pet. Ether extract
1
7
14
21
301.8±2.061
292.5±3.147
266.47±2.165
213.85±1.66 **
206.7±2.14
195.4±3.54
178.5±4.31
160.4±2.54 **
Group V
Ethyl acetate
extract
1
7
14
21
303.5±2.502
266.54±1.365
217.36±2.043
193.02±3.54**
201.4±3.69
190.4±1.36
183.8±3.85
180.6±2.44**
Group VI
Ethanol (70 %)
extract
1
7
14
21
306.86±1.458
252.87±2.547
173.7±1.525
147.35±2.69**
199.5±3.58
193.4±2.89
187.7±5.13
183.0±2.17**
Values are expressed as Mean ± S.D. (n=6). The results was analyzed by one-way analysis of variance (ANOVA) followed by
Dunnett’s t-test. p<0.05* , p<0.01** Considered as significant
Gro
up I
Gro
up II
Gro
up II
I
Gro
up IV
Gro
up V
Gro
up V
I0.0
0.5
1.0
1.5
GroupsGroupsGroupsGroups
CR
EA
TIN
INE
mg
/dl
CR
EA
TIN
INE
mg
/dl
CR
EA
TIN
INE
mg
/dl
CR
EA
TIN
INE
mg
/dl
***
*****
GROUP I
GROUP II
GROUP II
I
GROUP IV
GROUP V
GROUP V
I0
25
50
75
GroupsGroupsGroupsGroups
UR
EA
mg
/dl
UR
EA
mg
/dl
UR
EA
mg
/dl
UR
EA
mg
/dl
***
******
Fig 3: Effect of D. falcata leaves extracts on serum creatinine
level.
The results was analyzed by one-way analysis of variance
(ANOVA) followed by Dunnett’s t-test (n=6). *** p<0.01
Fig 4: Effect of D. falcata leaves extracts on serum urea level
The results was analyzed by one-way analysis of variance
(ANOVA) followed by Dunnett’s t-test (n=6). *** p<0.01
atherogenic index. Ethanol (70%) extract shows
significant (p<0.01) reduction in atherogenic index.
These results suggest that the D. falcata leaves
extracts had significant antihyperlipidaemic activity in
HFD induced hyperlipidaemia.
Antidiabetic Activity
Alloxan produces oxygen free radical in the body,
which cause pancreatic injury (21) and could be
responsible for increased blood glucose level (22). In
our study there is marked increase in blood glucose
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level in the diabetic control group as analysis done on
1, 7, 14, 21th day of study (312.5, 303.8, 295.8, 293.2
mg/dl) as compared with normal group. (84.1, 84.9,
80.9, 77.5 mg/dl). Treatment with ethanol extracts
(70%) 300mg/kg had significant (p<0.01) reduction in
elevated blood glucose level (306.8, 252.8, 173.7,
147.3 mg/dl) as compared with diabetic control group
(Table 2).
Glibenclamide 10 mg/kg p.o for 42 days also
significantly decreased blood glucose (296.5, 225.3,
153.8, 125.4 mg/dl) level as compared with diabetic
control.
Body weights of animals were recorded during the
study period and there was a marked reduction in body
weight of animals of diabetic control group. Treatment
with ethanol (70%) extract and glibenclamide
significantly (p<0.01) protects reduction in body
weight as compared with diabetic control group. The
level of creatinine and urea was significantly increase
in diabetic rats. At the end of study period estimation
of creatinine and urea were also carried out and
among the extracts. Ethanol (70%) extract had shown
significant (p<0.01) reduction in elevated levels of
urea and creatinin as compared with diabetic control
group (Fig.3 & 4).
In conclusion 70% ethanol extract of D. falcata leaves
showed good in-vitro antioxidant activity, in-vivo
antihyperlipidemic and antdiabetic activity which need
to be evaluated further.
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