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165
|Zoarilala Rinah Razafindrakoto
1*
|Nantenaina Tombozara
1,2
|David Ramanitrahasimbola
1,3
|
Dina Fitia
Raoelihajaina
3
| and | Dina Andriamahavola Rakotondramanana
3
|
1.
Institut Malgache de Recherches Appliquées | Laboratoire de Pharmacognosie Appliquée | Itaosy | Madagascar |
2.
University of Antananarivo| Faculty of Sciences, Organic Chemistry Department | Laboratory of Applied Chemistry to Natural Substances |
Antananarivo | Madagascar
3.
University of Antananarivo | Faculty of Medicine, Pharmacy Department | Antananarivo | Madagascar |
| Received March 27, 2020 | | Accepted April 13, 2020 |
| Published April 24, 2020 |
| ID Article | Zoarilala-Ref.1-ajira310320 |
ABSTRACT
Background:
Ageratum conyzoides
(ASTERACEAE) is traditionally used against asthma, articular rheumatism and arterial high
blood pressure in Malagasy folk medicine. Few studies are conducted on its antihypertensive activity
.
Objectives: This study aims
to validate the use of this plant as antihypertensive through the vaso-relaxing property of the aerial part methanol extract (ME) and
the action mechanism of ethyl acetate fraction (EF). Methods: Dried powder of
A. conyzoides
aerial part was extracted by
maceration in methanol. Methanolic solution was depigmented by activated charcoal then filtered on Whatman’s filter paper and
evaporated to dryness. This methanol extract was dissolved in distilled water and then successively partitioned with hexane,
dichloromethane, ethyl acetate and butanol. The vaso-relaxing activity of extract and fractions was assessed on the phenylephrine
pre-contracted isolated rat aorta. Action mechanism of ethyl acetate fraction (EF) was determined using three active reagents
including propranolol, indomethacin and L-NAME. Phytochemical screening was assessed in ME with common methods as well as
acute toxicity in mice. Results: Phytochemical screening shows the presence of phenolic compounds, flavonoids, tannins,
saponins, steroids, quinones and anthraquiniones. Tested on isolated rat aorta, ME exhibits a moderate activity (EC
50
=
383.44±17.34 μg/ml). After bio-guided fractionation, EF, with EC
50
of 204.07±8.50 μg/ml, was the most active. This activity wasn’t
modified by propranolol and indomethacin but with L-NAME, the EC
50
was increased to 584.04±30.98 μg/ml and the tested
maximal concentration does not allow achieving its maximum effect. The acute toxicity tests showed that ME is devoid of toxicity.
Conclusion: The anti-hypertensive activity of
A. conyzoides
is partly the result of the vaso-relaxing effect of bioactive molecules
dissolved in ethyl acetate. These results contribute to explain the antihypertensive virtue of
Ageratum conyzoides
.
Key words: Ageratum conyzoides, antihypertensive activity, vaso-relaxing activity
1. INTRODUCTION
Ageratum conyzoides
L. (ASTERACEAE) is a tropical plant growing in the western and eastern regions of the African
continent, as well as in some regions of Asia and South America
[1, 2]
. It is known as Ananjazavavy,
Hanitrinimpatsaka, Alonimpatsaka or Bemaimbo in different regions of Madagascar and known with other vernacular
name from other countries including Ageratum, Billygoat-weed, Goat weed, Chick weed and White weed
[3]
. In
northern of Madagascar, decoction of leaves of
A. conyzoides
is used during difficult childbirth to intimate feminine
dressing associated with Romba (
Ocimum gratissimum
) and Fagnivagna (
Aeschynomene sp.
), to wash the woman
and child after delivery. This decoction is also used to relieve painful periods and to attenuate vomiting and diarrhea,
to clean infected wounds and ulcers of the skin. The decoction of flowers is used to wash eyes and treat conjunctivitis
[4]
. Decoction of
A. conyzoides
is used to treat asthma and hypertension. The plant is crushed and rubbed on joints
to treat rheumatism. In India, flowers are used to treat cough and cold, headache and wormer
[5]
, and roots are
antilitic and antidiarrheal
[2]
. In Central Africa, Brazil and Congo,
A. conyzoides
is used to cure pneumonia, wounds
and burns
[6]
, its decoction is given to treat headache, fever and rheumatism
[2]
. In Cameroon, aqueous extracts of
the whole plant are known for their anti-diabetic properties
[7]
. In Ivory Coast, decoction of leaves is used to treat
malaria
[8]
. In Nigeria, seeds of
A. conyzoides
are anti-hyperglycemia
[9]
. Secondary metabolites of this plant were
widely studied including monoterpenes and sesquiterpenes, triterpenes, steroids, flavonoids, coumarins, tannins and
alkaloids
[10-16]
. Moreira et
al.
(2007) found that methoxyflavone isolated from hexane extract of leaves of
A.
conyzoides
had insecticidal activity
[12]
. Adetutu et
al.
(2012) and Odeleye et
al. (2014)
reported the plant extracts
antibacterial activities then Morais et
al.
(2014) highlighted antifungal and Teixeira et
al.
(2014) the anti-parasitic
activity
[17,
18,
19, 20]
. Others properties such as anti-inflammatory
[21]
, antalgic
[22]
, wound healing
[23]
and
cytotoxicity properties
[17]
have been demonstrated. The essential oil of the leaves and aerial parts of the plant has
been widely investigated for its components and biological activities. The major constituents generally found are the
chromenes, precocene I and precocene II, and the sesquiterpenes caryophyllene and germacrene-D
[1,2]
. The main
activity described in the literature for the essential oil is the insecticide
[15]
, but
A. conyzoydes
can also exert
allelopathic
[24]
and antifungal activities
[25, 26]
. Among these biological activities, few studies have been conducted
ORIGINAL ARTICLE
VASO-RELAXING ACTIVITY OF
Ageratum Conyzoides
Linn.
(ASTERACEAE) AERIAL PARTS ON ISOLATED RAT AORTA
*
Corresponding Author: |Dina Andriamahavola Rakotondramanana | Author Copyright © 2020:
|Zoarilala Rinah Razafindrakoto |
.
All Rights Reserved. All articles published in American
Journal of Innovative Research and Applied Sciences are the property of Atlantic Center Research Sciences, and is protected by copyright laws CC-BY. See:
http://creativecommons.org/licenses/by-nc/4.0/
.
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regarding its antihypertensive activity. Thus, this work aims to demonstrate the antihypertensive activity of
A.
conyzoides
through its vaso-relaxing activity and its mechanism of action.
2. MATERIALS AND METHODS
2.1 Plant material
Aerial parts of
A. conyzoides
were harvested at Ambohimanambola - Antananarivo in December 2018. They were
dried in cool and aerated place away of sunlight before to be grinded. The taxonomic botany of the plant was
identified and authenticated by
Dr.
Benja Rakotonirina, the botanist of the “
Institut Malgache de Recherches
Appliquées
” (IMRA) and the voucher specimen was deposited at the IMRA Botany Department under the identification
code NT-009/LPA.
2.2 Animals
Adults males or females wistar rats, weighing between 150-200 g and aged between 5-6 months, are used for
in vitro
pharmacological tests. Male OF
1
mice (25±2 g, aged between 3-4 months) are used for
in vivo
acute toxicity test.
Animals are provided from IMRA animal house and allowed free access to standard pellets (1420, Livestock Feed Ltd.)
and tap water. They were exposed to day-night light cycle (12h) and room temperature. All experiments were carried
out in accordance with the European Parliament and the Council of 22 September 2010 on the protection of animals
used for scientific purposes (DIRECTIVE 2010/63/EU).
2.3
Extraction and fractionation
Three hundred grams of vegetable material powder of
A. conyzoides
were macerated in methanol for 24 hours with
intermittent shaking. The protocol was repeated three times to maximize extraction efficiency. The filtrates were
gathered and depigmented through a layer of activated charcoal then depigmented methanolic solution were
evaporated to dryness under reduced pressure at a temperature of 40° C, with a rotavapor (Buchi-R114) in order to
obtain the methanol extract (ME). Then, 15 g of ME were partitioned by the liquid-liquid fractionation method using
distillated water - hexane, dichloromethane and ethyl acetate successively to allow to hexane (HF), dichloromethane
(DF), ethyl acetate (EF) and aquoeus (AF) fractions.
2.4 Phytochemical screening
The major classes of secondary metabolites were detected in ME and EF of
A. conyzoides
using specific reagents as
described in our previous work
[27]
.
2.5 Pharmacological experiments
2.5.1 Chemicals:
All reference products used for pharmacological tests such as phenylephrine, acetylcholine,
indomethacin, L-Nitro-Arginine Methyl Ester (L-NAME) and propranolol are purchased from Sigma-Aldrich and all salts
including KCl, NaCl, NaHCO
3
, MgSO
4
, KH
2
PO
4
and CaCl
2
and Glucose used to prepare survival solution of Krebs-
Heinseleit are purchased from Prolabo.
2.5.2 Organ preparation:
Animal was anesthesied with petrolium ether and then exsanguinated by carotid artery
transection. The thoracic aorta was removed and carefully cleaned of adhering fat and connective tissue, and cut into
rings (2-3 mm length). The rings were then mounted in standard organ baths filled with a physiological salt solution
called Krebs-Henseleit solution composed (in mM) by KCl: 4.8; NaCl: 118; NaHCO
3
: 25; MgSO
4
: 1.2; KH
2
PO
4
: 1.2;
CaCl
2
: 1.25 and Glucose: 11, maintained at 37°C and continuously bubbled with carbogen (95% O
2
- 5% CO
2
).
Resting tension was adjusted to 2 g. Tension developped by the organe trip was measured with an isometric force
transducer. After an equilibration period of 90 min, with a renewal of survival medium every 20 min, the vessels were
maximally contracted with phenylephrine (10
-5
M) in order to test their contractile capacity and the integrity of each
aorta ring was verified with acetylcholine (10
-6
M).
2.5.3 Effect of extract and fractions on the pre-contracted wistar rat aorta:
Vessels were pre-contracted
with 10
-6
M of Phenylephrine. At the contraction plateau, sample was cumulatively and increasingly tested at different
concentrations including 125, 250, 500, 750, and 1000 μg/ml. The relaxing effect of each concentration was
calculated and expressed as a percentage by considering the contraction plateau as 100%. The EC
50
which is the
concentration giving 50% of the maximum relaxing effect (E
max
), was calculated by linear regression.
2.5.4 Effect of propranolol on the vaso-relaxing activity of EF:
Isolated organ vessels were divided into two
groups. Group I (n = 6) were submerged for 30 min in 10
-5
M propranolol while group II (n = 6) was submerged in
the survival solution. Then, all rings were pre-contracted with 10
-6
M of phenylephrine. At the plateau contraction, EF
was cumulatively and increasingly tested at different concentrations ranging from 125 to 1000 μg/ml. Its EC
50
was
calculated in each condition then compared.
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2.5.5 Effect of indomethacin on the vaso-relaxing activity of EF:
As previously, the aorta rings were divided
into 2 groups so that the first group (n = 6) were in contact for 30 min with 10
-5
M of indomethacin and the second
group (n = 6) without indomethacin. After pre-contraction with 10
-6
M of phenylephrine, EF was tested at different
concentrations ranging from 125 to 1000 μg/ml at the plateau contraction in a cumulative and increasing manner.
EC
50
was calculated for both conditions and compared.
2.5.6 Effect of L-NAME on the vaso-relaxing activity of EF:
As previously, the isolated aorta rings were divided
into two groups. The first group (n = 6) were in contact for 30 min with 10
-4
M of L-NAME and the second group were
left without L-NAME. After pre-contraction with 10
-6
M phenylephrine, EF was tested at different concentrations as
previously and its EC
50
was calculated in these two conditions.
2.5.7 Acute toxicity:
OF
1
male mice were fasted 12 hours before the beginning of the experiment.
Oral use
:
Animals were divided into 5 groups of 5 mice. Group A: received only 0.25 ml of distilled water, while
group B, C, D and E received respectively 0.5, 1, 1.5, and 2 g/kg of ME administered by gavage at the rate of 0.25
ml/animal. After treatment, animals had free access to water and food. During the 72 hours of observation, all
abnormal behavior of the animals relative to the controls and mortalities were noted.
Intra-peritoneal method
:
Animals were divided into 6 groups of 5 mice. ME was administered by intra-peritoneal
injection at a rate of 0.2 ml/animal. Animals of the first group received distilled water, whereas those of the other
groups received respectively 0.2, 0.4, 0.6, 0.8, and 1 g/kg of ME. General behaviours of animals as well as the
possible mortality were recorded.
2.6 statistical analysis
All the results are expressed as mean ± s.e.m. calculated from the values obtained on n experiments or n animals.
The means were compared statistically using the Student's
t
-test. A value of
p
< 0.05 among the degree of freedom
used was considered a statistically significant different.
3. RESULTS
3.1 Extraction and bio-guided fractionation
The extraction from drug powder yields 5.82% (17.8 g) of ME which exhibited a moderate activity on pre-contracted
isolated rat aorta by phenilephrine with a EC
50
of 383.44 ± 17.34 μg/ml. After liquid-liquid partition, EF (1.79 g) is the
most active with an EC
50
of 204.07 ± 8.50 μg/ml (table 1, figure 1).
Table 1: The table presents the result of the Yield of extraction and bio-guided fractionation of
A.
conyzoides
Tested fraction
Mass (g)
Yield
a
(%)
EC
50
values
b
(μg/ml)
E
max
b
(%)
ME
17.8
5.82
383.44 ± 17.34
98.73 ± 0.84
HF
7.89
1.58
492.91 ± 29.24
c,e
85.01 ± 2.29
d,e
DF
1.42
0.28
898.72 ± 100.63
c,e
55.78 ± 5.13
d,e
EF
1.79
0.36
204.07 ± 8.50
d
98.51 ± 1.49
AF
5.85
1.17
< 1000
45.80 ± 2.76
d,e
a: yield are relative to plant powder; b: values are expressed as mean ± s.e.m. of 6 independent experiments (n = 6); c:
p < 0.01 vs ME ; d: p < 0.001 vs ME ; e: p < 0.001 vs EF.
Figure 1: The figure shows the vaso-relaxing effect of the extract and fractions of
A. conyzoides
on the phenylephrine pre-contracted isolated rat aorta (n = 6).
3.2 Phytochemical screening
0
25
50
75
100
0
250
500
750
1000
%
o
f
re
la
x
a
tio
n
[Sample] (µg/mL)
ME
HF
DF
EF
AF
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Phytochemical investigation on ME and EF are reported in table 2. Flavonoids, phenolic compounds and steroids are
found in both ME and EF. On the other hand, saponins, tannins, quinones, anthraquinones and cardiac glycosides are
only present in ME but not found in EF.
Table 2: The table presents the secondary metabolites in ME and EF.
Secondary metabolite
ME
EF
Flavonoids
+
+
Unsaturated sterols
-
-
Phenolic compounds
+
+
Steroids
+
+
Lactonic steroids
-
-
Cardio-tonic glycosides
+
-
Alkaloids
-
-
Terpenoids
-
-
Anthraquinones
+
-
Quinones
+
-
Tannins
+
-
Saponins
+
-
(+): presence of phytochemical compounds; (-): absence of phytochemical compounds.
ME: methanol extract; EF: ethyl acetate fraction.
3.3 Action mechanism study of EF
The EC
50
and E
max
of EF of
A. conyzoides
in presence or absence of antagonists are reported in table 3. One the first
hand, the presence of propranolol or indomethacin didn’t affect significantly the vaso-relaxing activity of EF (figure 2
and 3). On the other hand, the presence of L-NAME increases significantly (
p
< 0.001) the EC
50
value of EF from
231.15 ± 13.99 μg/ml to 584.04 ± 30.98 μg/ml and decrease significantly (
p
< 0.001) the maximal effect (Emax) of
EF of 17.10 % (figure 4) showing its vascular effect in the presence of the e-NOS enzyme inhibitor.
Table 3: The table presents the EC
50
of EF of
A.conyzoides
in presence or in
absence of antagonists
Sample
EC
50
(µg/mL)
Emax (%)
EF alone
231.15 ± 13.99
97.46 ± 1.15
EF in presence of propranolol
223.94 ± 48.07
98.16 ± 0.88
EF in presence of indomethacin
236.22 ± 10.55
96.37 ± 0.61
EF in presence of L-NAME
584.04 ± 30.98*
80.36 ± 1.15*
Values express the mean ± s.e.m.; *: p < 0.0001
vs EF alone. EF: Ethyl acetate fraction; EC
50
:
median effective concentration; Emax: maximal effect
Figure 2: The figure shows the vaso-relaxing effect of EF on the isolated rat aorta pre-
contracted to phenylephrine in the absence (♦ ) and presence (■ ) of 10
-4
M L-NAME (n = 6).
3.4 Acute toxicity
During the three consecutive days of behavioral observation of animals treated with different doses of ME, no
significant changes were observed concerning the gross behavior of the treated animals compared with the control
animals. All tested doses did not cause any mortality in both administration methods. Thus,
A. conyzoides
is not toxic.
5. DISCUSSION
Phytochemical screening methods showed that the aerial part of
A. conyzoides
consists on the following major
chemical families: polyphenols, flavonoids, tannins, saponins, steroids and anthraquinone. Several works carried out
by Amadi et
al.
(2012) and Odeleye et
al.
(2014) on
A. conyzoides
from Nigeria reported the presence of high
concentration of alkaloids and low concentrations of leucoanthocyanins and steroids in the aerial parts extract
[28,
0
25
50
75
100
0
250
500
750
1000
%
o
f
re
la
x
a
tio
n
[EF] (µg/mL)
In absence of L-NAME
In presence of L-NAME
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18]
. Those carried out by Kamboj and Saluja (2008),
A. conyzoides
from India have shown the presence of alkaloids,
coumarins, flavonoids, triterpenoids and sterols
[29]
. In addition of these chemical families, steroids, tannins and
phenolic compounds are reported by Dash and Murthy (2011)
[30]
. The absence of alkaloids in
A. conyzoides
from
Madagascar may be due to ecological factors which influences the metabolism of organic molecules of the plant.
Presence of phenolic compounds is a promising results for isolation of active compounds from this plant because they
constitute a family of organic molecules widely present in the plant kingdom which contribute to the decrease in the
incidence of cardiovascular diseases including kaempferol, quercetin
[31]
, gallic acid
[27,32]
and more others as a
vaso-relaxing compounds.
Concerning the vaso-relaxing activity of the ME and fractions, the significant difference between the EC
50
of ME and
EF can be due to the influence of the other secondary metabolites absent in EF however, the maximal effect of has
not been influenced. Propranolol is a non-selective β-adrenergic receptor antagonist with no intrinsic
sympathomimetic activity
[33]
. It was used to study the involvement or not of β2-adrenergic receptors in the vascular
effect of EF. The vaso-relaxing effect of EF wasn’t affected by propranolol indicating that this effect didn’t implicate
this adrenergic receptor. Indomethacin is an inhibitor of cyclooxygenases (COXs), enzymes responsible for the
biosynthesis of prostaglandins including prostacyclin (PGI2), which is one of the endothelial relaxation factors
[34]
.
Thus, it was used to study the involvement of prostacyclin in the vascular effect of EF. The difference of the EF EC
50
and its maximal effect levels is not statistically significant. It could indicate that PGI2 isn’t implicated in its vaso-
relaxing activity. L-NAME is an inhibitor of the enzyme e-NOS or endothelial NO synthase
[35]
. The Nitric oxide (NO)
is one of the endothelial relaxation factors
[36]
. Therefore, L-NAME was used to study the involvement of endothelial
NO in the vascular effect of EF. In the presence of this eNOS inhibitor, the pharmacological parameters of the EF
vaso-relaxing activity were significantly modified (Fig.2 and Tab 3).
EF of
A. conyzoides
produces a concentration-dependent vaso-relaxing effect whose mechanism of action would
involve nitrogen monoxide (NO) which is a vasodilating substance produced by endothelium from L-Arginine under
the action of the enzyme nitrogen synthase (NOS). The NO after its intercellular diffusion will stimulate the guanylate
cyclase which synthesizes the cyclic guanosine monophosphate (cGMP) in the vascular smooth muscle cells (VSMC).
The increase in intracellular cGMP leads to activation of type 1 protein kinase G (PKG) which would reduce the
intracellular calcium concentration by opening the membrane potassium channels that would lead to hyperpolarization
of the plasmatic membrane and closure of the Ca
2+
channels type L
[37]
, the same mechanism of action as
acetylcholine
[38]
and bradykinin
[39]
or by stimulating a serine/threonine protein phosphatase 2A which, in turn,
dephosphorylates the Ca
2+
channel and thus inactivates it
[37]
. The activated PKG can also activate the calcium/ATP-
ases pumps (Ca
2+
/ATP-ases) for the expulsion of the calcium from the cell through the PMCA and its recovery in the
sarcoplasmic reticulum and therefore a decrease in the concentration in intracellular calcium in favor of relaxation
[40]
. Previous studies have shown that polyphenols such as quercetin, epicatechin increase NO production to improve
endothelium-dependent vascular relaxation
[41-44]
, while high-level gallic acid can induce vascular relaxation
[27,
32]
. In the longer term, polyphenols can increase the level of expression of endothelial NOS, leading to sustain NO
formation and therefore persistent vascular protection
[45]
. Indeed, quercetin induces a rapid phosphorylation of
endothelial NOS to serine 1179 (Ser1179) via an Akt-independent pathway and a cyclic adenosine
monophosphate/protein kinase A dependent pathway to increase NO production and to promote vasodilation
[46]
.
Epicatechin increases NO in endothelial cells via inhibition of nicotinamide adenine dinucleotide phosphate oxidase
(NADPH oxidase)
[47]
. Epicatechin and quercetin can also act as antioxidants by reducing nitrite and nitrate to NO
[42,48]
. This type of mechanism of action which increases the expression of endothelial NOS and decreases the
activity of NADPH oxidase was also observed for the case of the plant
Salva
miltorrhiza
. This plant is also used in
cases of arterial hypertension due to its vasodilatory properties
[49]
. Other factors, such as low-density lipoprotein
cholesterol (LDL-cholesterol), which are bad cholesterol, could influence the integrity of blood vessels. The
accumulation of these lipids in the vessels and arteries causes a vascular restriction, the main cause of hypertension.
The presence of phytosterols in particular sitosterol in
A. conyzoides
would be beneficial in hypertensive patients.
Because of their very similar structure to that of cholesterol, phytosterols, when present in sufficient amounts in the
intestine, compete with cholesterol in the formation of micelles necessary for the absorption of cholesterol
[50,51]
.
Therefore, a 50% reduction in intestinal absorption of cholesterol has been demonstrated with sitosterol
[52]
.
Although sitosterol significantly reduces LDL-cholesterol by 10-20%
[53]
, Becker et
al.,
(1964) reported that taking
sitosterol or sitostanol in children with familial hypercholesterolemia increases fecal excretion neutral sterols of 45%
and 88%
[54]
. Heinemann et
al.
(1991) directly measured the effects of sitosterol and sitostanol on endogenous
cholesterol absorption in healthy intestinal infusion volunteers and found a 50% reduction in bile cholesterol
absorption with sitosterol and 85% Sitostanol
[50]
. Thus, the anti-hypertensive activity of
A. conyzoides
is the result
of the vaso-relaxing effect of polyphenols and flavonoids and the hypocholesterolemic effect of phytosterols.
5. CONCLUSION
Ageratum conyzoides
is used by Malagasy traditional healers for the treatment of high blood pressure. The
phytochemical screening of the plant revealed the presence of phenolic compounds, flavonoids, steroids, terpenes,
saponins and anthraquinones. Results obtained on the ethyl acetate extract of the aerial parts of
Ageratum
conyzoides
made it possible to demonstrate its antihypertensive activity dependent-dose on the vascular level via a
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vaso-relaxation mechanism dependent on endothelial NO. This activity may be due to the phenolic compounds and
phytosterols present in the plant. Its traditional use as an antihypertensive agent is proven; however, this work did
not define extactly the action mechanism of EF of
A. conyzoides
. Therefore, further studies are necessary to elucidate
this mechanism and isolate the active compounds of this plant. In view of these tremendous activities of
Ageratum
conyzoides
, this plant is certainly an interesting medication.
Acknowledgment:
This work was funded by Albert and Suzanne Rakoto-Ratsimamanga Foundation.
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Cite this article: Razafindrakoto Z. R., Tombozara N., Ramanitrahasimbola D., Raoelihajaina D. F., and
Rakotondramanana A. D. VASO-RELAXING ACTIVITY OF AGERATUM CONYZOIDES LINN. (ASTERACEAE) AERIAL PARTS ON ISOLATED
RAT AORTA. Am. J. innov. res. appl. sci. 2020; 10(4): 165-171.
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