Guiera senegalensis (GS) is a traditional plant distributed widely in
western Africa. In Burkina Faso traditional medicine, this plant is used to
treat some diseases like cough, dysentery, malaria, diabetes and hypertension.
The galls of G. senegalensis are used for the treatment of fowlpox and
have antiseptic, antifungal activities. In addition, recent studies demonstrate
that an aqueous acetone extract from galls of Guiera senegalensis inhibits
in vitro FowlPox Virus growth in secondary CES cells (Lamien
et al., 2005). Further pharmacological activities (antimicrobial
properties for example) of leaves of Guiera senegalensis have also been
shown (Kudi et al., 1999; Fiot
et al., 2006). The antiplasmodial activity of infusion, decoction
and chloroformic extract of stems and leaves of Guiera senegalensis has
been demonstrated on two strains of Plasmodium falciparum (Benoit
et al., 1996; Fiot et al., 2006).
The chloroformic extract of roots and the isolated alkaloids harman and tetrahydroharman
exhibited a significant antimalarial activity associated with a low cytotoxicity
(Ancolio et al., 2002). Aniagu
et al. (2005) demonstrate that the aqueous extract of Guiera senegalensis
protect the gastric mucosa of rats against ethanol-induced acute mucosal
damage with a reduction of the ulcer index; so indicating that Guiera senegalensis
could be an effective gastroprotective agent. The central sedative properties
of Guiera senegalensis have already been demonstrated (Amos
et al., 2001). Various chemical constituents are found in the extracts
from galls of Guiera senegalensis such as tannins, flavonoids, anthocyanidins,
alkaloids and steroids (Lamien et al., 2005).
Recently, a great interest has been given to naturally occurring antioxidants,
which may play important roles in inhibiting both free radicals and oxidative
chain-reactions within tissues and membranes (Nsimba et
al., 2008). Edible plants contain a wide variety of chemicals such as
flavonoids and other phenolic compounds that have potential antioxidant activity
through a number of different mechanisms. The proposed mechanisms for their
action include (1) direct radical scavenging, (2) inhibition of enzymes, such
as NO-synthase, xanthine oxidase, cyclooxygenase and lipoxygenase, (3) iron
chelation and (4) direct inhibition of lipid peroxidation (Xanthopoulou
et al., 2009). Several methods are available to evaluate antioxidant
activities of natural compounds in foods or biological systems. Methods commonly
used in antioxidant activity assays are the DPPH (2, 2-diphenyl-1-picrylhydrazyl),
FRAP (ferric reducing antioxidant power), β-carotene bleaching (BCB) procedures.
The DPPH method is based on the reduction of alcoholic DPPH solutions at 517
nm in the presence of an antioxidant that donate hydrogen or electron (Kulisic
et al., 2006). DPPH takes normally several hours for the reaction
to be completed and colour interference of the DPPH assay with samples that
contain anthocyanins leads to under-estimation of antioxidant activity (Teow
et al., 2007). DPPH method is independent of the substrate polarity.
The BCB method is usually used to evaluate the antioxidant activity of compounds
in emulsions, accompanied with the coupled oxidation of b-carotene and linoleic
acid. Ascorbic acid, is a well known antioxidant polar compound, its antioxidant
activity was not proved by this method. This can be explained by a phenomenon
formulated as the »polar paradox«: non-polar antioxidants exhibit
stronger antioxidative activities in emulsions because they concentrate at the
lipid/ air surface, thus ensuring high protection of the emulsion itself (Kulisic
et al., 2006). FRAP is used in assay to assess the metal (iron exclusively)
ions binding ability (Nsimba et al., 2008).
The reducing power is generally associated with the presence of reductones,
which exerts antioxidant action by breaking the free radical chain by donating
a hydrogen atom (Prasad et al., 2010). Xanthine
Oxidase (XO) catalyzes oxypurines (hypoxanthine and xanthine) to uric acid in
the purine catabolic pathway. Inhibition of XO activities decreases the uric
acid levels and results in an anti-hyperuricemic effect (Zhu
et al., 2004). The XO plays an important role in various ischemic
tissues, vascular injuries, inflammatory diseases, chronic heart failure, is
a major source of free radicals (superoxide), its activity contributes significantly
to the degree of oxidative stress, brain edema, thermal stress, respiratory
syndrome, viral infection and hemorrhagic shock in vivo and it is also
associated with gout (Naoghare et al., 2010).
The products of 5-lipoxygenase including leukotrienes (LTs) constitute an important
class of inflammatory mediators of asthma and various inflammatory diseases,
contribute to vascular changes in inflammation (Li et
The ethnomedicinal uses activities from the galls of GS suggest that the galls might possess antioxidant activities. The aim of the present study was to screen the phytochemical analysis and the antioxidant activities from galls of GS. This was performed by assessing the lipoxygenase (LOX) and the Xanthine Oxidase (XO) activities.
MATERIALS AND METHODS
Plant material: The galls of Guiera senegalensis were collected in kadiogo (province of Burkina Faso) in August 2009 and authenticated by professor Millogo-Rasolodimby, botanist at Ouagadougou University.
Chemical and reagents: Folin-Ciocalteu reagent, aluminium trichloride (AlCl3), 2,2-diphenyl-1-picrylhydrazyl (DPPH), xanthine, gallic acid, quercetin, allopurinol, trichloroacetic acid, tween 40, 2-thiobarbituric acid, Xanthine oxidase (E.C. 22.214.171.124), Urethane were purchased from Sigma-Aldrich Chemie, Steinheim (Germany ). Sodium carbonate, potassium hexacyanoferrate [K3Fe (CN)6 ], ascorbic acid, ferric chloride ( FeCl3), ferrous chloride (FeCl2 ) were from Prolabo, Paris (France). Solvents used were supplied by Fluka Chemie, Buchs (Switzerland). β-Carotene type I, Linoleic acid, (+)-α-tocopherol from vegetable oil type V were from sigma (USA). 15-Lipoxygenase (EC 126.96.36.199) type I-B (Soybean) was purchased from Sigma (St. Louis, MO). All other reagents were of analytical and HPLC grades.
Animals: A group of 5 adult wistar rats (155-201 g) of either sex were used. The animals were obtained from the Laboratory of institute of research in health science (IRSS, Burkina Faso). The animals had free access to food and water and kept in a regulated environment at 25±1°C under 12 h light/12 h dark conditions during 2 days for acclimatization. Rats were subjected to urethane anesthesia (1.5 g kg-1 of b.wt.). Liver was removed, washed with cold saline, quickly blotted and weighed. Liver was stored at -70°C for estimation of extracts/fractions inhibition of lipid peroxidation. Throughout the experiments, animals were processed according to the suggested international ethical guidelines for the care of laboratory animals.
Extraction and fractionation: The galls were dried and ground to powder. The obtained powder was extracted with selected solvents: acetone 80% and distilled water.
The first extraction was processed, using 50 g of powder in 500 mL of acetone/water (80/20) during 48 h under mechanical agitation at room temperature.
Fifty gram of powder were also extracted with 500 mL of distilled water by heating (100°C) for 30 min. The filtrate obtained using whatman filter paper was concentrated under reduced pressure in a rotary evaporator and lyophilized using a freeze drying system to give the hydroacetonic extract (HAE) and aqueous decoction extract (ADE).
The hydroacetonic and the aqueous decoction extracts were respectively dissolved in distilled water and successively extracted with ethyl acetate, butanol. Each extract was dried to give: Ethyl Acetate Fraction (EAF), Butanol Fraction (BF) and final Water Fraction (WF). The obtained extracts were stored in a refrigerator at +4°C until use.
Determination of totals phenol, totals flavonoid content in the extracts:
The total phenol content of extracts and fractions was determined as described
by Singleton et al. (1999) using gallic acid
as standard. The diluted aqueous solution of each extract or fraction (0.25
mL) was mixed with 1.25 mL of Folin Ciocalteu reagent (0.2 N). This mixed solution
was allowed to stand at room temperature for 5 min and then 1 mL of sodium carbonate
solution (75 g L-1) was added. After 2 h incubation, the absorbance
was measured at 760 nm against blank. A standard curve was plotted using gallic
acid (0-200 mg L-1). The results were expressed as mg of Gallic Acid
Equivalents (GAE) per 100 mg of extract or fraction (mg GAE/100 mg extract or
Totals flavonoid content was determined using the Dowd method as adapted by
Arvouet-Grand et al. (1994). For each extract,
1 mL of methanolic solution (100 μg mL-1) was mixed with 1 mL
of aluminium trichloride (AlCl3) in methanol (2%). The absorbance
was read at 415 nm after 10 min against a blank sample consisting of a 1 mL
of methanol and 1 mL of plant extract without AlCl3. The totals flavonoid
content was determined on a standard curve using quercetin as a standard. The
mean of three readings was used and expressed as mg of quercetin equivalents
(QE) per 100 mg of extract or fraction (mg QE/100 mg extract or fraction).
Iron (III) to iron (II) reduction activity: The ability of the extracts
or fractions to reduce iron from the form (III) to the form (II) was assessed
with the method of Hinneburg et al. (2006). A
0.5 mL aliquot of each extract dissolved in water was mixed with 1.25 mL of
phosphate buffer (0.2 M, pH 6.6) and 1.25 mL of a 1% aqueous potassium hexacyanoferrate
[K3Fe (CN)6 ] solution. After 30 min incubation at 50°C,
1.25 mL of 10% trichloroacetic acid was added and the mixture was centrifuged
at 2000 g for 10 min.
A 1.25 mL aliquot of the upper layer was mixed with 1.25 mL of water and 0.25 mL of aqueous FeCl3 (0.1%) and the absorbance was recorded at 700 nm. Iron (III) reducing activity was determined as mmol ascorbic acid equivalents per gram of extract or fraction (mmol AEAC g-1 extract or fraction). The values are presented as the means of triplicate analyse.
Free radical-scavenging activity in vitro: The ability of the
extracts or fractions to scavenge the DPPH (2, 2-diphenyl-1-picrylhydrazyl)
radical was evaluated as described by Lamien-Meda et
Extracts or fractions were dissolved in methanol and 0.75 mL of each was mixed with 1.5 mL of a 0.02 mg mL-1 solution of DPPH in methanol. The mixtures were left for 15 min at room temperature and the absorbance was measured at 517 nm. The blank sample consisted of 0.75 mL of extract or fraction solution with 1.5 mL of methanol. The antioxidant content was determined using standard curves for ascorbic acid (0-10 mg L-1). The means of three values were obtained, expressed as mmol of ascorbic acid equivalent per g of extract or fraction antioxidant content (mmol AEAC g-1 extract or fraction).
Antioxidant assay using the β-carotene linoleate model system:
The antioxidant activity of extracts and fractions was evaluated by the method
of Shon et al. (2003) with some modifications.
A solution of β-carotene was prepared by dissolving 8 mg of β-carotene
in 10 mL of chloroform. The 0.5 mL of this solution was pipetted into a 100
mL round-bottom flask. After the chloroform was removed at 40°C under vacuum.
Forty seven microliter of linoleic acid, 362 μL of Tween 40 emulsifier
and 100 mL of distilled water were added to the flask with vigorous shaking.
Aliquots (4.8 mL) of this emulsion were transferred into different test tubes containing 0.2 mL of different concentrations of the extracts. The tubes were shaken and incubated at 50°C in a water bath. As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm using a spectrophotometer. Absorbance readings were then recorded at 20 min intervals until the control sample had changed colour. A blank, devoid of β-carotene, was prepared for background subtraction. α-Tocopherol, quercetin and gallic acid were used as positive controls. Antioxidant activity was calculated using the following equation:
The assays were carried out in triplicate and the results expressed as mean
Inhibition of lipid peroxidation in rat liver homogenate: The inhibition
activity of extracts or fractions on lipid peroxidation (LPO) was determined
according to the thiobarbituric acid method. FeCl2H2O2
was used to induce the liver homogenate peroxidation to the method of Su
et al. (2009) with slightly modification. In this method, 0.2 mL
of extract or fraction at the concentration of 1.25 mg mL-1 was mixed
with 1.0 mL of 1% liver homogenate (each 100 mL homogenate solution contains
1.0 g rat liver), then 50 μL of FeCl2 (0.5 mM) and 50 μL
of H2O2 (0.5 mM) was added. The mixture was incubated
at 37°C for 60 min, then 1.0 mL of trichloroacetic acid (15%) and 1.0 mL
of 2-thiobarbituric acid (0.67%) was added and the mixture was heated up in
boiled water for 15 min. The absorbance was recorded at 532 nm. Quercetin and
gallic acid were used as the positives controls. The percentage of inhibition
effect was calculated according to following equation:
where, A0 is the absorbance of the control (without extract), A1
is the absorbance of the extract addition and A2 is the absorbance
without liver homogenate.
Xanthine oxidase inhibitory: The XO activities with xanthine as the
substrate were measured spectrophotometrically using the procedure reported
by Filha et al. (2006) with some modifications.
The assay mixture consisted of 50 μL of extract or fraction solution at
final concentration of 100 μg mL-1, 150 μL of 1:15 M phosphate
buffer (pH 7.5) and 50 μL of enzyme solution (0.28 U mL-1 in
buffer). After pre-incubation of the mixture at 25°C for 1 min, the reaction
was initiated by adding 250 μL of xanthine substrate solution (0.6 mM )
and the absorbance was measured for 120 sec. A negative control was prepared
without extract. Allopurinol, a known inhibitor of XO, quercetin and gallic
acid were used as positive controls, in a final concentration of 100 μg
mL-1 in the reaction mixture.
Lipoxygenase inhibitory activity: Lipoxygenase inhibitory activity was
measured by slightly modifying the spectrometric method as developed by Malterud
and Rydland (2000).
Four hundred microliter of lipoxygenase solution (167 U mL-1), 100 μL of the sample solution (50 μg mL-1 at final concentration) were mixed and incubated for 1 min at 25°C. The reaction was initiated by the addition of 500 μL of linoleic acid substrate solution (134 μM) and the absorption change at 234 nm with the formation of (9Z, 11E)-13S)-13-hydroperoxyoctadeca-9, 11-dienoate was followed for 3 min. All the reactions were performed in triplicate. Quercetin and gallic acid were used as positive control for lipoxygenase inhibition.
Statistical analysis: Chemical and enzymatic analyses of individual samples were performed in triplicates. The results presented are the mean and standard deviations of the obtained values. Data manipulation was performed by means of Microsoft Excel. Data were analyzed using ANOVA test (Fisher). The p<0.05 was considered significant.
RESULTS AND DISCUSSION
Extraction yields: All extracts were analyzed for polyphenols compounds content. The yield of aqueous decoction and hydroacetonic extracts was determined. The yield extraction is 17.6% for the aqueous decoction and 16.9% for the hydroacetonic (80:20) extraction. We obtained almost the same yield for the two types of extractions.
Determination of total phenol and flavonoid content in the extracts: Table 1 shows the totals phenol, totals flavonoid content in the extracts and fractions from galls of G. senegalensis.
The hydroacetonic extract (HAE) exhibited the highest total phenolic content
(73.9±2.34 mg GAE/100 mg of extract). This extract is significantly higher
than when compared to the others extract and fractions. The result of hydroacetonic
extract is higher than that obtained by Lamien et al.
(2005) on the same extract. This result could be explained by the fact that
the total phenol content of the plants varies as a function of the plant part
collected and the season (De Sousa Araújo et al.,
2008). The amounts of total phenol contents were increased by polarity of
the extraction solvent. Polyphenols are secondary plant metabolites that are
present in many plant and plant product. Many of the phenolics have been shown
to contain high levels of antioxidant activities.
||Totals phenolic and totals flavonoid content
Data are Mean±SEM (n = 3). HAE: Hydroacetonic
extract, EAF/HAE: Ethyl acetate fraction from HAE, BF/HAE: Butanol fraction
from HAE, WF/HAE: Water fraction from HAE, ADE: aqueous decoction extract,
EAF/ADE: Ethyl acetate fraction from ADE, BF/ADE: Butanol fraction from
ADE, WF/ADE: Water fraction from ADE. Values showing the same letter are
not significantly different (p>0.05) from one other in the same columns
The butanol fraction of aqueous decoction (BF/ADE) contains the significant
highest of flavonoid (8.44±0.05 mg QE/100 mg of fraction). Totals flavonoid
content did not change according to the polarity of the extraction solvents.
Flavonoids are good free radical scavengers that donate hydrogen and have lipid
peroxidation, anti-mutagenic and anti-inflammatory activities (Tunalier
et al., 2007). The totals flavonoid content of HAE and ADE are also
higher than these obtained by Lamien et al. (2005)
on the same extracts (respectively 1.61±0.01 mg QE/100 mg for HAE and
0.79±0.00 mg QE/100 mg for ADE).
The galls of Guiera senegalensis contain high polyphenols content than
that six species of acanthaceae from Burkina Faso (Sawadogo et
Antioxidant activity: The principle of the antioxidant activity is the availability of electrons to neutralize any so-called free radicals. Since, antioxidant mechanisms are diverse, a variety of in vitro techniques has been developed. It is better to use different assays based on different mechanisms to evaluate the antioxidant capacity. In this study, the antioxidant activity of the extracts and fractions was evaluated using DPPH scavenging, FRAP and β-carotene bleaching assays.
The results of antioxidant activity of extracts from Guiera senegalensis are summarized in Table 2.
The FRAP assay measured the ability of antioxidant components to reduce Fe (3+) to Fe (2+). Related FRAP values ranged from 04.73±0.10 to 10.88±0.86 mmol AEAC g-1 extract or fraction. Hydroacetonic extract show the strongest and significantly activity to reduce Fe (3+) to Fe (2+) than the others fractions except the aqueous decoction extract and water fraction of hydroacetonic extract.
The radical-scavenging activities of the extracts and fractions from galls
of G. senegalensis were estimated by comparing the inhibition of formation
of DPPH radicals by the extracts and those of ascorbic acid. The water fraction
of hydroacetonic extract (WF/HAE) from galls of G. senegalensis (6.012±0.13
mmol AEAC/g extract) which has the best free radical scavengers is significantly
higher than aqueous decoction extract and it fractions (WF/ADE, EAF/ADE and
BF/ADE). The ability to scavenge DPPH radicals of various solvent extracts from
GS was in the order of water fraction (HAE)>butanol fraction (HAE)>hydroacetonic
extract>ethyl acetate fraction (HAE)> Aqueous decoction extract>Water
fraction (ADE)>butanol fraction (ADE)>Ethyl acetate fraction (ADE).
and anti-lipid peroxidation activity
are Mean±SEM (n = 3). HAE: Hydroacetonic extract, EAF/HAE: Ethyl
acetate fraction from HAE, BF/HAE: Butanol fraction from HAE, W/HAE: Water
fraction from HAE, ADE: aqueous decoction extract, EAF/ADE: Ethyl acetate
fraction from ADE, BF/ADE: Butanol fraction from ADE, WF/ADE: Water fraction
from ADE. β-Carotene bleaching expressed as percentage of inhibition
at a concentration of 100 μg mL-1. Anti-Lipid peroxidation
(Anti-LPO) of rat liver expressed as percentage of inhibition at a concentration
of 1.25 mg mL-1 of extract or fraction. Values showing the
same letter are not significantly different (p>0.05) from one other
in the same columns
The antioxidant activities of GS extracts and fractions were also evaluated
by the β-carotene bleaching method, in which the oxidation of β-carotene
in the presence of linoleic acid takes place. The percentage inhibition of β-carotene
values varied from 31.15±1.76 to 59.00±3.27%. β-Carotene
method demonstrated the significant higher antioxidant activity for the butanol
fraction of hydroacetonic extract. This activity is significant higher than
α-tocopherol antioxidant activity which is a reference inhibition of β-carotene
compound. The antioxidant activity of carotenoids is based on the radical adducts
of carotenoids with free radicals from linoleic acid. The linoleic acid free
radical attacks the highly unsaturated β-carotene models. The presence
of different antioxidants can hinder the extent of β-carotene-bleaching
by neutralizing the linoleate-free radical and other free radicals formed in
the system (Suja et al., 2005).
Total phenols content versus the antioxidant activity: In this study, the correlation between the antioxidant activity and totals phenolic content of extracts and fractions was studied using a linear regression analysis. As demonstrated in Fig. 1, the good and significant correlation coefficient between totals phenolic and FRAP values (R2 =0.9702, y = 0.1127x+2.4818, p = 0.003), total phenolic and DPPH. radical scavenging (R2 = 0.9519, y = 0.712x-1. 6532, p = 0.006) were found. The correlation coefficient between totals phenol and β-carotene assay (y = 0.0583x+37.634; R2 = 0.019, p>0.05) was found to be very weak, less than 0.5 (Fig. 1).
An almost linear correlation between DPPH radical-scavenging activity and concentrations
of totals phenolic compounds in various vegetable and fruits have been reported
(Lamien-Meda et al., 2008; Bakasso
et al., 2008). This low correlation values between totals phenolic
content and the β-carotene assay suggest that the totals phenol from galls
of G. senegalensis do not take part in the inhibition of β-carotene
oxidation. We can also suggest that the strong free radical-scavenging and iron
(III) to iron (II) reduction capacities of these extracts and fractions were
due to their high phenolic contents. This last assertion is confirmed by the
correlation studies between the total phenolic and antioxidant activities (DPPH
and FRAP). Sofidiya et al. (2006) also reported
a strong relationship between total phenol content and reducing power.
High and significant correlation (R2 = 0.93, p<0.05, y = 0.712x-1.6532)
was found between data from the DPPH and FRAP antioxidant assays. However, found
no correlation between the FRAP and β-carotene assays or DPPH and β-carotene
assays on extracts from galls of G. senegalensis.
Present results indicate that 93% of the radical scavenging compounds from
galls of Guiera senegalensis also take part in iron (III) to iron (II)
reduction activity and dont contribute to β-carotene bleaching assays.
On the contrary Hinneburg et al. (2006) did not
find any correlation with the DPPH. and the iron chelation assay.
Anti-lipid peroxidation activity: The percent inhibition of lipid peroxidation
was quantified by measuring the reduction of TBARS production with respective
controls in the presence of extracts or fractions. Quercetin and gallic acid
were used as standard antioxidant. The role of peroxidative processes in disease
is a subject of intense research interest. Lipid peroxidation of cell membranes
is associated with various pathological events such as atherosclerosis, inflammation
and liver injury (Roome et al., 2008). FeCl2-
H2O2 system was used to induce lipid peroxidation in rat
Water fraction (WF/ADE) from the galls of GS demonstrated a strong anti-lipid
peroxidative effect (87.25±1.27%) at a concentration of 1.25 mg mL-1.
All the extracts or fractions except water fraction (WF/HAE) and ethyl acetate
fraction (EAF/ADE) produced greater inhibition as compared to the quercetin.
Hydroacetonic extract, butanol fraction (BF/HAE), aqueous decoction extract
and water fraction (WF/ADE) produced greater inhibition as compared to gallic
acid. The lipid peroxidation inhibition activity of extracts and fractions from
galls of G. senegalensis is stronger when compared to the findings of
study concerning Pinus koraiensis seed extract (Su
et al., 2009).
The involvement of iron in lipid peroxidation is well established. In fact,
ferrous ions can precipitate the formation of oxygen radicals and either initiate
or take part in peroxidative process (Dixit and Kar, 2009).
The low correlation values between extracts or fractions activities to reduce
Fe(3+) to Fe(2+) and anti-lipid peroxidation (R2
= 0.265; p>0.05, y = 0.0538x+5.5479), DPPH radical-scavenging activity and
anti-lipid peroxidation (R2 = 0.1684x; p = 0.805, y = 0.0317x+2.6705),
β-carotene bleaching assay and lipid peroxidation (R2 = 0.0063,
p>0.005, y = 0.0307x+39.055) were obtain (Fig. 2). The
anti-peroxidative effects of the extracts were not also correlated with their
totals phenolic and totals flavonoid contents.
The low correlation obtained between antioxidant and anti-lipid peroxidation
activities suggest that the anti-peroxidation activity from galls of Guiera
senegalensis is not related to their radical scavenging and ferric reduction
activities. Ozgova et al. (2003) did not fund
correlation between IC50 values with the quantitatively assayed chelation
potency of different polyphenols. Decrease in lipid peroxidation by extracts
from galls of Guiera senegalensis may be a result of it scavenging OH
produced by FeCl2-H2O2 and H2O2
in the reaction system (Wang et al., 2008).
oxidase (XO) and of soybean lipoxygenase (LOX) inhibitory activity
are Mean±SEM (n = 3). HAE: Hydroacetonic extract, EAF/HAE: Ethyl
acetate fraction from HAE, BF/HAE: Butanol fraction from HAE, WF/HAE:
Water fraction from HAE, ADE: aqueous decoction extract, EAF/ADE: Ethyl
acetate fraction from ADE, BF/ADE: Butanol fraction from ADE, WF/ADE:
Water fraction from ADE. For XO inhibitory activity extracts, fractions
and references were tested at a concentration of 100 μg mL-1.
For LOX inhibitory activity extracts and fractions were tested at a concentration
of 50 μg mL-1 references were tested at a concentration
of 25 μg mL-1
Our extracts reduced lipid peroxidation in liver, the primary target organ
of drug metabolism. These results suggest that the extracts of G. senegalensis
may not cause hepatotoxicity; but acts as protective agent by preventing oxidative
Xanthine oxidase and of soybean lipoxygenase inhibitory activity: Xanthine
oxidase inhibitors are known to be therapeutically useful for the treatment
of gout, hepatitis and brain tumor (Song et al., 2003).
At a concentration of 100 μg mL-1 of hydroacetonic extract,
uric acid formation was partially suppressed (50.90±6.91%).
Hydroacetonic extract may contain bioactive substances useful in the treatment
of gout or other XO-induced diseases justifying the traditional use of this
specie as diuretic, depurative. Table 3 shows the inhibitory
effects of the extracts and fractions from galls of GS on Xanthine Oxidase (XO)
activities. Allopurinol (95.7±1.52%), the positive control is similar
to published data at a concentration of 100 μg mL-1 (Umamaheswari
et al., 2007). These clearly showed that hydroacetonic extract from
galls of G. senegalensis has the best inhibitory activities of the xanthine
oxidase enzyme. Ethyl acetate fraction (ADE) showed any inhibitory effects.
Umamaheswari et al. (2009) obtained a better
inhibitory activity (84.75±0.54%) of the chloroform fraction of Erythrina
stricta on the xanthine oxidase at a concentration of 100 μg mL-1.
The 5-LOX pathway generates an important class of inflammatory mediators, such
as leukotrienes (LTs), which plays a major part in the inflammatory process
(Li et al., 2006).
Table 3 also shows the inhibitory effects of the extracts
and fractions on lipoxygenase (LOX) activity at a concentration of 50 g mL-1.
All the extracts showed LOX inhibitory effects, enzyme involved in generating
free radicals. According to the inhibition percentage values, the most effective
LOX inhibitory extracts were, in order of efficacy, HAE>BF (HAE)>WF(HAE)>ADE
>EAF(HAE) WF (ADE)>BF (ADE) and EAF (ADE). Hydroacetonic extract from
galls of Guiera senegalensis(HAE) showed stronger inhibitory activity
towards lipoxygenase at a concentration of 50 μg mL-1 comparatively
of ethanol extracts from nine vine plants used in traditional Chinese medicine
at a concentration of 340 μg mL-1 (Li et
between LOX, XO inhibition and totals phenolic content
between LOX inhibition and XO inhibition activity
between LOX, XO inhibition and FRAP
Regression equations and coefficients of correlation between the totals phenolic
and the inhibition of XO and LOX activities by extracts and fractions are represented
on Fig. 3.
These results suggest that totals phenolic of extracts from galls of GS contribute for more than 90% to the inhibition of the XO and the LOX.
A good correlation (R2 = 0.8931, p = 0.008) between the ability of galls extracts to inhibit lipoxygenase action and their ability to inhibit xanthine oxidase was obtained (Fig. 4).
The anti-inflammatory effect could be also related to antioxidant activity.
All the extracts from galls of GS possess anti-inflammatory activity correlated
to its high antioxidant/free radical-scavenging capacities (Fig.
5, 6). Maiga et al. (2006)
also show a correlation between the antioxidant and anti-inflammatory activity
of the extracts.
between LOX, XO inhibition and DPPH
Lows correlations were obtained between the antioxidant activity by the β-carotene
bleaching method and the activities of inhibition of the xanthine oxidase and
The non-polar antioxidants which exhibit stronger antioxidative activities in emulsions do not take part in xanthine oxidase and lipoxygenase inhibition activities.
In this study HAE, BF (HAE), WF (HAE) and ADE extracts derived from galls of Guiera senegalensis demonstrated pronounced antioxidant potential. These extracts contain polyphenols compounds that can scavenge free radicals, chelate metal ions and inhibit the lipid peroxidation activity. The antioxidant activity is correlated with the total phenolic content. However, extracts from galls of Guiera senegalensis demonstrated effective anti-inflammatory activity which probably be related at least in part to its antioxidant activity that justifies its traditional use against inflammatory and xanthine oxidase-induced diseases. It also provides useful information for pharmacological activities associated with free radicals. However, there is no correlation between totals phenolic content and anti-lipid peroxidation activity. More detailed phytochemical studies are thus necessary to identify the extracts active principle(s) responsible for the β-carotene and the anti-lipid peroxidation activities.
The International Atomic Energy Agency (AIEA) is gratefully acknowledged for financial assistance by Co-operation Project BKF/5002.