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Research Article
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Anti-inflammatory and Antioxidant Activities of a Fraction I1 of Male Inflorescences of Borassus aethiopum Mart (Arecaceae) |
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J. Sakande,
E. Kabre,
M. Lompo,
E. Pale,
J.B. Nikiema,
O.G. Nacoulma,
M. Sawadogo
and
I.P. Guissou
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ABSTRACT
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In previous studies, using a bioassay-guided fractionation procedure; five
fractions (E1F1, E2F2, E3F3, E4F4 and E5F5) from powdered Borassus aethiopum
male inflorescences were extracted. Among these, the dichloromethane methanol
extract E2F2 was found to exert significant anti-inflammatory and antipyretic
activities and pro apoptotic effect. It seemed important to pursue the investigations
to understand the mechanism of the anti-inflammatory activity. The anti-inflammatory
activity was studied, C Reactive Protein (CRP) level in mice blood was immunoturbidimetry
after inflammation induction and antioxidant activity was studied using 1,1
diphenyl picrylhydrazyl (DPPH). Phytochemical screening was carried out according
to the methodology for chemical analysis for vegetable drugs. Among 3 fractions
(I1; I2, I3) of E2F2, I1 was the most active with a percentage of inhibition
(PI) of 80%. This anti-inflammatory activity was twice high than indometacin
(PI = 40%). The I1 fraction cause significant decline of concentration of CRP
compared with indometacin. The radical scavenging activities of I1 were approximately
4 times lower than ascorbic acid. Phytochemical analyses of Borassus aethiopum
extracts revealed the presence of terpenoids, steroids and saponins which all
have been shown to be potent anti-inflammatory and antioxidants. The present
study confirmed the anti-inflammatory and antioxidant potential of Borassus
aethiopum extracts with results comparable with those of standard compounds
such as indometacin. Further studies are needed to isolate, purify and identify
the chemical structure of the compounds responsible for anti-inflammatory and
antioxidant activity.
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How
to cite this article:
J. Sakande, E. Kabre, M. Lompo, E. Pale, J.B. Nikiema, O.G. Nacoulma, M. Sawadogo and I.P. Guissou, 2013. Anti-inflammatory and Antioxidant Activities of a Fraction I1 of Male Inflorescences of Borassus aethiopum Mart (Arecaceae). American Journal of Biochemistry and Molecular Biology, 3: 101-109.
DOI: 10.3923/ajbmb.2013.101.109
URL: https://scialert.net/abstract/?doi=ajbmb.2013.101.109
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Received: November 24, 2011;
Accepted: March 27, 2012;
Published: October 04, 2012
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INTRODUCTION
Borassus aethiopum Mart (Arecaceae) is a tropical plant widely spread
in Africa. This plant is used in Burkina Faso traditional medicine for the treatment
of inflammatory diseases (Cassou et al., 1997).
In previous studies, using a bioassay-guided fractionation procedure; five fractions
(E1F1, E2F2, E3F3, E4F4 and E5F5) from powdered Borassus aethiopum
male inflorescences were extracted. Among these, the dichloromethane methanol
extract E2F2 was found to exert significant anti-inflammatory and antipyretic
activities (Sakande et al., 2004a-b)
and pro apoptotic effect (Sakande et al., 2011).
It seemed important to pursue the investigations because other studies on Borassus
flabellifer flowers a plant belonging to the same family reported the isolation
of molecules with therapeutic interest (Revesz et al.,
1999; Yoshikawa et al., 2007). Furthermore,
the potential herbal sources of future drugs effective in oxidant-related diseases
are reported (Hasani-Ranjbar et al., 2009; Rahimi
et al., 2010; Kayode and Kayode, 2011; Malekirad
et al., 2011). The present study aimed at progressing towards the
isolation of the active substance by bioassay-guided fractionation of E2F2 and
to understand the mechanism of the anti-inflammatory activity.
MATERIALS AND METHODS
Vegetable drug: Borassus aethiopum male inflorescences.
Animals: Male NMRI mice weighing between 30 and 40 g were used. These
animals receiving food and water ad libitum were stabilized in an atmosphere
in 22°C with 70% of humidity. The photoperiod was 12-24 h.
Chemicals:
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Extraction solvents: dichloromethane and methanol from Prolabo
(France) |
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Phytochemical screening reagents from Prolabo (France) |
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DPPH (1,1 diphenyl picrylhydrazyl) from Sigma-Aldrich (France) |
• |
Carrageenan from Sigma-Aldrich (France) |
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Ascorbic acid, Sigma-Aldrich (France) |
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Indometacin, Sigma-Aldrich (France) |
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C Reactive Protein (CRP) from Roche Diagnostics (Germany) |
Equipment:
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Column of chromatography (Kieselgel 63-160 μ, Prolabo,
France) |
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Lyophilisator alpha chris 1-2 |
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Preparative Thin layer chromatography (TLC) plate (silica gel G6F254),
Prolabo, France |
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Spectrophotometer Cobas c111 (Roche diagnostic) |
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Rotary evaporator (Rotavapor) |
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Plethysmometer (Ugo Basile) |
Plant material and extraction: Borassus aethiopum male inflorescences
were collected and identified by Professor Sita Guinko, Institute of natural
products research of Ouagadougou (Vegetal Biology and Ecology Laboratory of
UFR SVT). A voucher specimen AA1522 was deposited at the Herbarium.
The inflorescences were air-dried in the shade and powdered. The powder was
exhaustively extracted by percolation with 3 L of dichloromethane-methanol (50:50).
The fraction obtained (E2F2 extract) was evaporated under reduced pressure to
obtain 10 g of residue.
The extract E2F2 was split by preparative TLC in 3 fractions I1, I2 and I3.
The pharmacological screening of the anti-inflammatory activity by the method
of Winter et al. (1962) allowed to retain the
most active which was I1. Phytochemical screening was carried out according
to the methodology for chemical analysis for vegetable drugs (Ciulei,
1982; Bankole et al., 2011; Oseni
and Akindahunsi, 2011).
Carrageenan-induced mouse paw oedema: The method of Winter
et al. (1962) was used with slight modification to induce inflammation
(Hajarolasvadi et al., 2006; Bala
et al., 2011). Inflammation of mouse paw was induced by injecting
0.025 mL of carrageenan prepared in distilled water (1%, w/v) into the subplantar
surface of the right hind paw. Male mice were divided into six groups of six
animals each. The mice were deprived of food and water during the experiment.
Mice paw were measured before oedema induction. The control group was given
0.025 mL of Isotonic Saline Solution (ISS) by intraperitoneal (i.p.) injection.
The reference group received indometacin (1 mg kg-1, i.p.) and 4
groups receiving i.p. injection of Borassus aethiopum extracts (E2F2:
100 mg kg-1 , I1: 100 mg kg-1, I2: 100 mg kg-1,
I3: 100 mg kg-1). These drugs were injected into mice immediately
before the injection of carrageenan. The doses of plant extracts and indometacin
were chosen according to Choi et al. (2004).
The degree of oedema was measured at 0 and 3 h after injection. Volumes of right
hind paw of mice were measured with a plethysmometer (Ugo Basile). The percentage
of inhibition of the inflammatory was determined for each animal by comparing
with control and calculated by the following formula used by Karaca
et al. (2009):
where, dt is difference in paw volume in the drug treated group and dc the
difference in paw volume in the control group.
The median effective dose (ED50) of the most active fraction I1
was determined using the same method of Winter et al.
(1962). Male mice were divided into seven groups of six animals each. A
study on series of five concentrations of I1 (mg kg -1): 100; 50;
25; 12.5; 6.25 allowed to determine the ED50.
Effect of I1 on the kinetics of C reactive protein (CRP): Inflammation
of mice paw was induced by the method of Winter et al.
(1962). Male mice were divided into 4 groups of twenty five animals each.
The control group was given 0.025 mL of ISS by intraperitoneal (i.p.) injection.
The reference group received indometacin (1 mg kg-1, i.p.) and 2
groups receiving i.p. injection of Borassus aethiopum extracts (E2F2:
100 mg kg-1, I1: 100 mg kg-1). Before oedema induction
blood were collected from 5 mice of each group (T0). Drugs were injected into
mice immediately before the injection of carrageenan. After carrageenan injection
blood were collected at 3 h (T3), 9 h (T9), 24 h (T24) sand 48 h (T48) from
5 mice from each group. Mice were anesthetized by dichloromethane and the blood
was collected by cardiac draining in dry tubes. The serum was separated by centrifugation
at 2000 g during 5 min and kept at -20°C until the dosage.
CRP was measured by immunoturbidimetry using a spectrophotometer Cobas C111
(Roche Diagnostics).
Measurement of DPPH (1,1 diphenyl picrylhydrazyl) radical scavenging activity:
The method of DPPH (Everette and Islam, 2012; Hemalatha
et al., 2012) was used for the determination of free radical scavenging
activity of the extracts. Five hundred microliter of extracts, were introduced
into 3 mL of solution of DPPH (1 mM). The mixture was shaken and incubated in
dark for 10 min. The decrease in absorbance at 517 nm was then measured using
spectrophotometer. Ascorbic acid was used as reference. All analysis were performed
in duplicate and the ability to scavenge the DPPH radical was calculated using
the following formula:
Scavenging effect (%) = [(A0-A1)/A0]x100 |
where, A0 is the absorbance of the control, A1 is the absorbance in the presence
of the sample of extract or reference.
A curve of calibration allowed determining the concentration of extracts corresponding
to a decrease of 50% of absorbance (IC50).
Statistical analysis: All data were represented as Mean±SD or
as percentage. Data were analyzed by ANOVA test using SPSS software. The differences
were considered as significant for p<0.05. Curves were drawn by MS Excel
2000 software.
RESULTS
Phytochemical screening: The results of phytochemical screening (Table
1) showed the presence of sterols, triterpenes and saponins in E2F2 extract
and its fraction I1. A Chromatogram of E2F2 and its fractions after revelation
with Liebermann-Buchard reagent showed 3 spots of E2F2 fractions: I1 (Rf
= 68), I2 (Rf = 57), I3 (Rf = 43). The I1 fraction with
a yield of 8% reported to 750 g of dry powder of Borassus aethiopum male
inflorescences was the main compound.
Carrageenan-induced mouse paw oedema: The results of anti-inflammatory
activity of Borassus aethiopum extracts are presented in Table
2. Bioassay-guided fractionation procedure using Winter
et al. (1962) method allowed to show that the fraction I1 was the
most active with 80% of oedema inhibition. This anti-inflammatory activity was
twice high than indometacin (PI = 40%) used as reference substance. The anti-inflammatory
activity of I1 fraction was less important than E2F2 extract (PI: 82%). The
median effective dose (ED50) value of I1 fraction was 33 mg kg-1.
Table 1: |
Chemical screening of Borassus aethiopum extracts |
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+++: Moderate quantity, +++: High quantity, -: Absent |
Table 2: |
Anti-inflammatory activity of Borassus aethiopum fractions |
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Values are Mean±SEM, n = 6 animals in each group, ED50
of I1 was 33 mg kg-1, *p<0.05 when compared to Indometacin
group |
Table 3: |
Effect of extracts on the kinetic of CRP (mg L-1) |
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*p<0.05 when compared to isotonic saline solution (ISS)
group |
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Fig. 1(a-c): |
Antioxidant activity of (a) E2F2 extracts, (b) I1 fraction
and (c) Ascorbic acid using DPPH method. The linear regressions showed that
radical scavenging activities of extracts were dose dependant |
Effect of I1 on the kinetics of CRP: The Table 3 presents
the effect of Borassus aethiopum extracts on the kinetics of protein
C reactive (CRP). These extracts cause significant decline of concentration
of this protein of acute inflammation phase compared with indometacin. From
24 h the decline of CRP was more important with I1 fraction than indometacin.
Measurement of DPPH (1,1 diphenyl picrylhydrazyl) radical scavenging activity:
Figure 1 presents the antioxidant activity of E2F2 extracts,
I1 fraction and ascorbic acid used as reference. E2F2 linear regression equation
was y = -0.005x+1.0787 with a correlation coefficient: R2 0.8866,
I1 linear regression equation was y = -0.006x+1.0967 with correlation coefficient
R2 = 0.9115. The anti-oxidizing activity of the ascorbic acid linear
regression equation was y = -0.0163x+1.0367 with correlation coefficient R2
= 0.9654. These linear regressions showed that radical scavenging activities
of extracts were dose dependant. Decreases of 50% of absorbance (IC50) values
were: E2F2 (87.6 μg), I1 (72.2 μg) and ascorbic acid (24.3). The radical
scavenging activities of extracts were approximately 4 times lower than ascorbic
acid.
DISCUSSION
The results showed that dichloromethane-methanol extract of Borassus aethiopum
E2F2 exercises an anti-inflammatory activity. The percentage of inhibition (PI)
on mice paw oedema treated with E2F2 was 82% against 40% of inhibition obtained
with indometacin. The interesting activity of E2F2 extract led to its fractionation
in order to progress towards the isolation of the active principle. I1 fraction
was the most active with a percentage of inhibition of 80%. The decline of activity
of I1 (80%) compared to E2F2 (82%) from which it arises shows that there would
be a synergy of action between the constituents of E2F2 (I1, I2 and I3). The
anti-inflammatory activity of Borassus aethiopum extracts are comparable
to other plants reported by authors (Okokon et al.,
2008; Usman et al., 2008; Karaca
et al., 2009; Gill et al., 2011).
The phytochemical screening allowed attributing the anti-inflammatory activity
to the group of sterols, triterpenes and saponins. The anti-inflammatory activity
of these compounds was reported by many authors (Sawadogo
et al., 2006; Kawabata et al., 2011;
Wu et al., 2011; Zha et
al., 2011; Zeng et al., 2011).
To understand better the mechanism of the anti-inflammatory activity of extracts,
we studied their effect on CRP, an acute phase protein of inflammation. Indeed
CRP rises from the 6th hour of the inflammation, is openly pathological 24 h
after the beginning of the inflammation and quickly normalizes after its disappearance
(7-14 days). So, carrageenan induced mice paw oedema in group receiving
ISS, increased CRP level from 2.1 to 22.5 mg L-1 after 3 h and this
level was 24.1 mg L-1 after 48 h. In group treated with extracts
a decrease of CRP was observed after 24 h of treatment with I1 (CRP = 2.75 mg
L-1). Decreasing effects of hydro alcoholic extract of plant (Urtica
dioica) on IL-6 and hs-CRP levels in patients with type 2 diabetes is also
reported (Namazi et al., 2011). The administration
of indometacin (Anti-inflammatory drug of reference) exhibit a decrease of CRP
(6.42 mg L-1) after 24 h.
The role played by non-steroidal anti-inflammatory drugs (NSAIDs) on CRP kinetics
is known. After 2 weeks of treatment with flurbiprofen (200 mg day-1)
and with ibuprofen to subjects presenting a rheumatoid arthritis, a significant
reduction of CRP was observed (Cush et al.,1990).
The decline of CRP led by the extracts of Borassus aethiopum opens a
way for the clarification of the mechanism of action. Indeed the replication
of the CRP mRNA by hepatocytes is stimulated by interleukin-6 which is stimulated
in his turn by tumor necrosis factor (TNF-α) and interleukin 1 Mostafa
et al. (2005) and Salari and Abdollahi (2011).
Furthermore, it was observed that serum C-reactive Protein (CRP) positively
correlates with serum concentrations of IL-6 and TNF-α in healthy (Yudkin
et al., 2000). Present study is the first that demonstrated effects
of Borassus extract on inflammatory indicators as CRP, so exact mechanism
on inflammatory cytokine is unknown. More studies are essential to discover
mechanism of I1 fraction. The dosage of these cytokines is necessary to understand
the mechanism. The role of extracts on CRP could present an interest in the
treatment of cardiovascular diseases. Indeed an influencing role of atorvastatin
on C-Reactive protein profile is reported (Anand et al.,
2009).
The study of the anti-oxidizing activity of Borassus aethiopum extracts
had for objective to understand the anti-inflammatory mechanism. Indeed several
works on the extracts of plants showed that the anti-inflammatory mechanism
was bound to their antioxidant power (Karaca et al.,
2009; Alam et al., 2011; Karou
et al., 2011; Sombie et al., 2011).
The study showed a weak dose dependant radical scavenging activities of extracts.
Phytochemical screening of Borassus aethiopum extracts revealed the presence
of terpenoids, steroids and saponins. All of these compounds have been shown
to be potent antioxidants (Chanwitheesuk et al.,
2005; Hafidh et al., 2009; Arora
et al., 2011; Roy et al., 2011). This
weak anti-oxidizing activity obtained allows saying that the anti-inflammatory
activity of Borassus aethiopum extracts fear to be bound to an anti-free
radical mechanism but would not be the only explanation.
CONCLUSION
The present study confirmed the anti-inflammatory and antioxidant potential
of Borassus aethiopum extracts with results comparable with those of standard
compounds such as indometacin. Further studies are needed to isolate, purify
and identify the chemical structure of the compounds responsible for anti-inflammatory
and antioxidant activity.
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