Abortifacient and Antioxidant Activities Of: Avicennia Marina
Abortifacient and Antioxidant Activities Of: Avicennia Marina
Abortifacient and Antioxidant Activities Of: Avicennia Marina
Online: 2015-01-27
ABSTRACT
The current study was intended to evaluate abortifacient and antioxidant activity of Avicennia
marina leaves extracts. Abortifacient activity was evaluated in rats, compared with standard drug
(Mifepristone) and antioxidant activity was evaluated by using three free radicals (Superoxide,
Hydroxyl and DPPH) compared with Ascorbic acid. The extracts were showed pre implantation loss,
post implantation loss of implantations and decreased the survival ration of foetuses. Among all
extracts hydroalcoholic extract showed better activity. The selected plant extracts showed
concentration dependent percentage inhibition of free radicals. Among three extracts hydroalcoholic
extract showed better activity with IC50 values on superoxide, hydroxyl and DPPH radicals were
203g, 237g and 143g. From the results obtained during the study it the hydroalcoholic extract was
used for the isolation of pure compounds, and isolated the different compounds (-sitosterol, Lupeol
and Betulinic acid).
Keywords: Avicennia marina; Abortifacient activity; Free radicals; Antioxidant Activity
1. INTRODUCTION
Medicinal plants will continue to provide a source for generating novel drug
compounds. Plants may become the base for the development of a new medicine or they may
be used as phyto-medicine for the treatment of disease [1].
Majority of the present day diseases are due to the shift in the balance of the pro-oxidant
and the antioxidant homeostatic phenomenon in the body. The antioxidant defense systems
can only protect the body when the amount of free radicals are within the normal
physiological level; but when this balance is shifted towards more of free radicals, it leads to
oxidative stress which may result in tissue injury and subsequent diseases [2].
Abortifacient agents obtained from indigenous medicinal plants would be of immense
benefit especially to inhabitants of developing countries, since the cost of these drugs would
be within their reach. The antifertility plants with estrogenic property can directly influence
pituitary action through peripheral modulation of luteinizing (LH) and follicle-stimulating
hormones (FSH) by decreasing the secretion of these hormones and blocking ovulation. In
addition, the plants may also intercept the synchronized development of the ovum and
endometrium while others may have abortifacient or antiprogestational effects. In different
parts of the world, herbal substances have been used in abortion for unwanted pregnancy
conditions and to restrict human population by exploring their abortifacient properties because
of the use of synthetic drugs leads to vigorous systemic illness or even death [3-6]. In recent
days many researchers reported the folklore claims of abortifacient plant used by the people
scientifically [7-12].
On ethnobotanical survey on different medicinal plants which are used by the tribal
people in araku valley region of Visakhapatnam District, Andhra Pradesh, India claimed to be
used to abortion was Avicennia marina [13]. There is no information in the open scientific
literature that has substantiated or refuted the abortifacient claim of this. Therefore, the aim of
this study was to evaluate the abortifacient activity of different extracts of A. marina with a
view to validating their acclaimed use by the tribal people of Araku.
2. MATERIALS AND METHODS
2.1. Collection of plant material and preparation of extracts
The plant material was collected at Araku valley, Visakhapatnam district, Andhra
Pradesh, India and the plant was authenticated by taxonomist Prof. M. Venkaiah, Department
of Botany, Andhra University. The collected plant material was shade dried and pulverized
into powder. The powdered material was used for extraction with different solvents (Hexane,
Ethyl acetate, and Hydro alcoholic) using maceration process. Then the extracts were used for
screening antioxidant and abortifacient activities.
2.2. Selection of Animals
Wistar albino rats of weighing between 150-200 g were obtained from National Institute
of Nutrition, Hyderabad, Andhra Pradesh, India. The animals were housed under standard
environmental conditions (temperature of 25 20c with an alternating 12h light-dark cycle
and relative humidity of 50 15%), one week before the start and also during the experiment
as per the rules and regulations of the Institutional Animal Ethics Committee and of the
regulatory body of the government (Regd no. 516/01/A/CPCSEA). They were fed with
standard laboratory diet during the experiment.
2.3. Chemicals
Test drugs
: Different extracts of A. marina leaves (at 125 and 250 mg/kg b.w
doses)
Standard drug
: Mifepristone (2.85 mg/Kg b.w)
Drug vehicle
: 1% Gum acacia
1,1- diphenyl-2-picrylhydrazyl was purchased from Sigma chemicals, USA. Nitroblue
tetrozolium was purchased from Sisco Research Laboratories Pvt Ltd., Mumbai. Riboflavin
was purchased from Loba Chemie Pvt Ltd., Bombay.
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in the evening. Female rats exhibiting thick clumps of spermatozoa in the vaginal smear were
chosen for the study and that day was considered as day one of pregnancy. The pregnant rats
were divided into twenty groups of six each. Group I served as a control, which received 2mL
of 1% gum acacia (p.o. daily). Groups II received standard drug (Mifepristone) and groups III
to VIII received different extracts of A. marina leaves (at 250 and 500 mg/kg b.w doses) (p.o.
daily) respectively, six females with vaginal plug/sperm-positive rats will be sacrificed on
gestational day 6 for determination of number of corpora lutea and implantations. On 18th day
of pregnancy six rats were laparotomised under light ether anesthesia. The number of
implantation sites and live fetuses were noted in both horns of the uterus. The observations of
the drug-treated groups were compared with control group. The following parameters will be
observed.
Number of corpora lutea
Number of implantations
Implantation index = Total number of implantation sites x 100
Number of corpora lutea
Number of live foetuses
Number of dead fetuses
Survival ratio =
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Avicennia marina
Ethyl acetate extract
Hydro alcoholic extract
+
++
++
+
+
++
+
+
+
+
+
++
+
++
+
++
Hexane
Ethyl acetate
24.681.22
22.381.66
36.421.46
30.022.12
Assignment
methyls
3-H
H-5
O-H
17
18
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Position
13
position
13
38.7
16
35.6
27.4
17
43.0
3
4
5
79.0
38.7
55.3
18
19
20
48.3
48.0
150.9
18.3
21
29.8
34.3
22
40.0
40.8
23
28.0
50.4
24
15.3
10
37.1
25
16.1
11
20.9
26
16.0
12
25.1
27
14.5
13
38
28
18.0
14
42.8
29
109.3
15
27.4
30
19.3
19
m.p 294-296C, [] D + 8.6 (CHCl3, C=0.5) and analyzed for the formula C30H48O3 by mass
spectral data and elemental analysis. It gave pink colour in L.B. reaction. The IR spectrum
showed absorptions at 3460 (-OH), 1690 (carbonyl of COOH), 1640 (double bond), 1380 and
30
-1
C=0.5) with AC2O/PY, a monoester with diazomethane, m.p 220-221C, [] D +8.5 (CHCl3,
30
C=0.5), a methyl ester acetate with AC2O/Pyridine, m.p 195-198C [] D +14.2 (CHCl3,
C=0.5). With the 1H NMR data of the compound was identified as betulinic acid (Fig 4).
H
COOH
HO
BETULINIC ACID
20
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(survival ratio), while no live fetus was observed with standard drug. The hydroalcoholic
extract at a dose of 500mg/kg b.w has shown significantly decreased the survival of foetuses
i.e. 13.79%, the preimplantation and postimplantation loss were 54.69% and 86.21%
respectively.
The implantation index of all extracts was not more different from each other in all the
treated groups but in hydroalcoholic extract at a dose of 500mg/kg b.w show significant i.e,
45.31%, but the percentage of survival ratio and postimplantation loss was varies. The
postimplantation loss was more significant than preimplantation loss at both doses of three
extracts (Hydroalcoholic, Ethyl Acetate and Hexane). All the extracts of A. marina more
significantly decreased the number of live foetuses in all treated groups. The results were
showed in Table 5.
The order of survival ratio of all extracts was
Mifepristone (0%) < A m Hyd. alc Ext 500mg/kg b.w (13.79%) < A m Hyd. alc Ext
250mg/kg b.w (24.39%) < A m E. A Ext 500mg/kg b.w (25.64%) < A m Hex. Ext 500 mg/kg
b.w (31.71%) < A m E. A Ext 250mg/kg b.w (36.59%) < A m Hex. Ext 250 mg/kg b.w
(40.42%) < Control (100%).
The order of preimplantation loss of all extracts was
Mifepristone (90.91%) > A m Hyd. alc Ext 500mg/kg b.w (54.69%) > A m E. A Ext
500mg/kg b.w (35.00%) > A m Hyd. alc Ext 250 mg/kg b.w (31.67%) > A m E. A Ext 250
mg/kg b.w (29.31%) > A m Hex. Ext 500mg/kg b.w (28.07%) > A m Hex. Ext 250 mg/kg b.w
(24.19%).> Control (15.38%).
The order of postimplantation loss was
Mifepristone (100%) > A m Hyd. alc Ext 500 mg/kg b.w (86.21%) > A m Hyd. alc Ext
250 mg/kg b.w (75.61%) > A m E. A Ext 500 mg/kg b.w (74.36%) > A m Hex. Ext 500 mg/kg
b.w (68.29%) > A m E. A Ext 250mg/kg b.w (63.41%) > A m Hex. Ext 250 mg/kg b.w
(55.57%).> Control (0%).
The order of abortifacient activity of different extracts of on the basis of
postimplantation loss and survival ratio
A.m Hyd. alc Ext 500 mg/kg b.w > A.m E. A Ext 500 mg/kg b.w > A.m Hex. Ext 500
mg/kg b.w.>Lupeol at 100mg/K.g b.w.
Table 5. Abortifacient activity of different extracts of A. marina at 250 mg/kg b.w and 500 mg/kg b.w
and isolated compound AMS-2 (Lupeol) at 100mg/Kg b.w.
S.no
Parameter
Control
Standard
(Mifepristone)
A m Hyd. alc
Ext
250
500
mg/
mg/
kg
kg
b.w
b.w
A m E. A
A m Hex.
AMS-2
Ext
Ext
250 500 250
500
100
mg/ mg/ mg/
mg/
mg/
kg
kg
kg
kg
kg
b.w b.w b.w
b.w
b.w
9.8
10.33
10
9.50 10.7
0.35
0.31
1.21
0.20
0.25
1.
Number of
corpora lutea
8.66
0.36
9.17
0.29
10 10.67
0.24
0.20
2.
Number of
implantations
7.33
0.23
0.83
0.16
9.3
0.52
3.
Implantation
index (%)
84.62
9.09
68.33
45.31
70.7
65.0
87.44
4.
Number of live
foetuses
7.33
0.23
0.00
1.67
0.09
0.67
0.09
2.5
0.09
1.67
3.17 2.17 8.33
0.16
0.13
0.82
0.20
75.8
71.9
5.
6.
7.
8.
Number of dead
foetuses
Preimplantation
loss (%)
Postimplantation
loss (%)
Survival ratio
(%)
21
4.17 4.33
0.07
0.14
4.83
4.67 4.67
0.09
0.17
0.16
1.0
0.63
31.67
54.69
29.3
35.0
24.2
28.1
12.56
100
75.6
86.2
63.4
74.4
55.5
68.3
10.72
0.00
24.39
13.79
36.6
25.5
40.3
31.7
89.28
0.00
0.83
0.16
5.17
0.20
15.38
90.9
0.0
100
Superoxide radical
Hydroxyl radical
DPPH radical
Hydroalcoholic extract
203
237
143
279
274
231
Hexane extract
349
440
436
Ascorbic acid
59
70
53.1
22
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Hydroalcoholic extract
Ethyl acetate extract
Hexane extract
Ascorbic acid
500
IC50(g)
400
300
200
100
0
e
id
ox
r
pe
Su
d
ra
al
ic
o
dr
Hy
l
ca
di
a
lr
xy
PH
DP
l
ca
di
a
r
Fig. 5. 50% Inhibition concentrations (IC50) of different extracts of A. marina against Superoxide,
Hydroxyl and DPPH radicals.
4. DISCUSSION
The rural and tribal women are well acquainted with the wild resources around them.
They seem to depend on plants for curing various diseases including abortion, sterility,
conception disorders, menstrual troubles, leucorrhoea etc prevailing among them. However
many of the uses of plants as abortifacient and antifertility herbals documented from the area,
have also been reported from different countries by various researchers and also people use
the plants for the diseases they suffer (abortion, sterility, conception disorders, menstrual
troubles, leucorrhoea), it is very difficult to judge the effectiveness of plants [13, 22, 25, 26].
This stimulated to the present study i.e the isolation of bioactive compounds and evaluation of
antioxidant and abortifacient activity of A. marina leaves extracts with using different
solvents which has been used by the tribal people for abortion [27-29].
The effect of different extract of A. marina on the duration of different phases of estrus
cycle were studied in rats. During estrus cycle many physiological, biochemical,
morphological, and histological changes occur in the ovaries. Follicular growth and ovulation
are regulated by endocrine (follicle stimulating hormone, luteinizing hormone, and prolactin)
and ovarian hormones (such as progestin, estrogen, and androgen). Ovarian hormones are
produced by different cell types of the ovary like granulosa cells of the mature follicles and
the corpus luteum [30]. Imbalance in these hormones leads to irregularity in the ovarian
functions and duration of the estrus cycle [31].
In our study, the number of days the rats of groups II and III spent in the estrus cycle
showed a significant decrease in the median duration of estrus and metestrus phases (as
compared to the control group). Reduction in the estrus and metestrus phases indicates
nonavailability of the matured graafian follicles or nonmaturation of secondary follicles.
Therefore, ovulation was inhibited. There was an increase in the mean number of days the rats
spent in proestrus phase in groups II and III, which was significant as compared with the
control group. The prolongation of the proestrus phase indicates that maturation of the
follicles in the preovulatory phase was delayed leading to nonmaturation of graafian follicles.
This was due to nonavailability of estrogen produced by the granulosa cells which is essential
for the maturation and differentiation of the ovarian follicles, or imbalance in the endogenous
steroid, protein, and hormones.
In the present study abortifacient and anti oxidant activities were screened for different
extracts of A. marina. The different extracts of A. marina showed concentration dependent
percentage inhibition on tested free radicals (Superoxide, Hydroxyl and DPPH). The free
radicals are produced in different metabolic process of the body and they can damage a wide
range of biomolecules such as proteins, DNA and amino acids in the body [30, 32-35].
Recently, many researchers have been reported that many medicinal plants possess more
potential antioxidant activity and their phytochemical constituents (Phenolic acids, flavonoids
and tannins, etc) have potential biological activities. The present studies have shown that the
extracts of A. marina have free radicals scavenging ability. Among all the extract of plants the
hydroalcoholic extract showed the better percentage inhibition on free radicals.
The abortifacient activity of A. marina plant extracts (at 250 and 500 mg/kg b.w) was
screened using rats by measuring the preimplantation loss, postimplantation loss and
percentage of survival ratio of live foetuses. The extracts were found more active in
preimplantation loss, postimplantation loss at dose of 500mg/kg b.w when compared with
control group. Abortifacient plants, block, alter, or interfere in the production of hormones
(estrogen and progesterone). The lining of the uterus does not grow enough to be supportive
or nourishing to a fertilized egg, thus preventing implantation [22, 25, 26, 31]. The
abortifacient activity (based on pre and post implantation loss) of tested different extracts of
A. marina was may be due to the above said reasons. From the results obtained in the present
study, it could be concluded that the different extracts of selected plant extracts possesses
abortifacient activity and justifies the folkloric claim of as an abortifacient. Different bioactive
compounds have been conformed abortifacient activity in animal models [7]. Therefore, it is
assuming that based on phytochemical analysis of selected plant different phytochemicals
(Alkaloids, Phenolics, Steroids and Glycosides etc.,) present in this plant may act either alone
or on combination responsible for the observed abortifacient activity in the present study.
Furthermore, the mechanism of abortifacient activity of selected plant extracts is wrathful
study to identify the bioactive principle of abortifacient activity.
5. CONCLUSION
The current study was intended to evaluate abortifacient and antioxidant activity of
Avicennia marina leaves extracts. Abortifacient activity was evaluated in rats, compared with
standard drug (Mifepristone) and antioxidant activity was evaluated by using three free
radicals (Superoxide, Hydroxyl and DPPH) compared with Ascorbic acid. The extracts were
showed pre implantation loss, post implantation loss of implantations and decreased the
survival ration of foetuses. Among all extracts hydroalcoholic extract showed better activity.
The selected plant extracts showed concentration dependent percentage inhibition of free
radicals.
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