INTRODUCTION
It is now well known that free radicals play a fundamental
role in several diseases. The biochemical damage that they cause to cells
and tissues, lead to the development of diseases such as arteriosclerosis,
hypertension, aging, cancer, diabetes mellitus, inflammation, renal failure,
liver disease, AIDS etc. (Allard et al., 1998; Cheng et al.,
2003; Ellnain-Wojtaszek, 2003; Govindarajan et al., 2005; Tiwari,
2004). Several epidemiological studies have shown that compounds that
can scavenge free radicals are effective in ameliorating the progress
of these related diseases. Of these, phenolic substances present in foods
and plants possess strong antioxidant properties, and thus, are being
increasingly investigated.
Blepharis lineariifolia, Dicliptera verticillata,
Dyschoriste perrottetii, Hygrophila auriculata, Lepidagathis anobrya,
Nelsonia canescens are plants that belong to the Acanthaceae family
used in traditional medicine in Burkina Faso for the treatment of several
diseases (Table 1) (Nacoulma, 1996)
Pharmacological investigations have shown that the aqueous
extracts of Hygrophila auriculata possess significant hepatoprotective
and antioxidant activities (Shanmugasundaram and Venkataraman, 2005).
The ethanolic extracts of the leaves of Nelsonia canescens have
been shown to possess analgesic and anti-inflammatory activities (Oweyele
et al., 2005). The efficiency of Dicliptera verticillata
in combination with Aloe buettneri, Justicia insularis,
and Hibiscus macranthus to induce in vitro the production
of oestradiol have been shown (Telefo et al., 2004).
However, little is known on the pharmacology of Blepharis
lineariifolia, Dyschoriste perrottetii, Lepidagathis anobrya, and
scientific information on the phenolic content and the antioxidant potential
of Acanthaceae of Burkina Faso are still rather scarce.
The objective of the present study was to evaluate the
antioxidant activity and quantify the total phenolic and flavonoid contents
of plants of the Acanthaceae family.
MATERIALS AND METHODS
This study was carried out during the year 2005
at Laboratoire de Biochimie et Chimie Appliquées, UFR/SVT, University
of Ouagadougou, Burkina Faso.
Chemicals: The Folin Ciocalteu reagent, gallic acid and quercetin
were purchased from Sigma-Aldrich Chemie, Steinheim, Germany; sodium carbonate,
ascorbic acid and
| Table 1: |
Medicinal uses of the six investigated plants |
 |
aluminium trichloride (AlCl3) were from Labosi,
Paris, France; 2.2-diphenyl-picrylhydrazyl (DPPH) and solvents used were
from Fluka Chemie, Switzerland. All chemicals used were of analytical
grade.
Plant material: Stems with leaves of Dicliptera verticillata
were collected in August 2004 in the botanical garden of the Research
institute in Health Science of Ouagadougou (Burkina Faso) and voucher
specimen was deposited in herbarium of University of Ouagadougou. Stems
with leaves of Blepharis lineariifolia, Dyschoriste perrottetii, Hygrophila
auriculata, Lepidagathis anobrya, Nelsonia canescens were bought from
practitioner of traditional medicine in “Nabi Yaar” market
of Ouagadougou in January 2005. All these plants were identified by Prof.
Millogo, a botanist from university of Ouagadougou.
Preparation of plant extracts: The dried plants were pulverised
into fine powder using a grinder. For each plant, 25 g of powder were
extracted with 80% aqueous acetone (250 mL) for 48 h under agitation using
a mechanical shaker (SM 25, Edmund BÜHLER, Germany). After filtration,
acetone was removed under reduced pressure in a rotary evaporator (BÜCHI
Rotavapor R-200, Switzerland) and the remained aqueous solution lyophilised
using a freeze drying system (Cryodos 50, TELSTAR, Spain).
Determination of total phenolic content: The Singleton et al.
(1999) method, using Folin-Ciocalteu reagent, was used to determine the
total phenolic content. Each plant extract was prepared at a concentration
of 1 mg mL-1. The absorbances of all samples were measured
at 760 nm against a methanol blank using a spectrophotometer (CECIL CE
2041, CECIL Instruments, England). The standard calibration curve was
plotted using gallic acid. The mean of three readings was used and the
results expressed as g of Gallic Acid Equivalents (GAE) per 100 g of lyophilised
extract.
Determination of total flavonoid content: The total flavonoid
content was determined using the Dowd method as adapted by Arvouet-Grand
et al. (1994). Briefly, 2 mL of plant extract (2 mg mL-1)
were mixed with 2 mL of aluminium trichloride (AlCl3) in methanol
(2%). The absorbance readings at 415 nm were taken after 10 min against
a blank consisted in 2 mL of plant extract and 2 mL of methanol without
AlCl3. Quercetin was used as a reference compound to produce
the standard curve. The mean of three readings was used and expressed
as g of quercetin equivalents per 100 g of lyophilised extract.
Determination of antioxidant activity: The free radical scavenging
activity of plants extract for DPPH (2,2-diphenyl-picrylhydrazyl), was
determined as described by Velazquez et al. (2003) with some modifications.
Extract solutions were prepared by dissolving 100 mg of each extract in
10 mL of methanol. The solution of DPPH in methanol (20 mg L-1)
was prepared daily, before absorbances measurements. 1.5 mL of this solution
were mixed with 0.75 mL of various concentration of each extract (3.9-500
μg mL-1) except for Dicliptera verticillata where
higher concentrations were used. Methanol was used as blank sample. The
mixtures were kept in the dark for 15 min at room temperature and then
the decrease in absorbance was measured at 517 nm. Quercetin (0-50 μg
mL-1) and ascorbic acid (0-40 μg mL-1) were
used as positive controls. The radical scavenging activity was calculated
as follows as (Motalleb et al., 2005).
% Inhibition = [(AB – AA)/AB]
x 100, where: AB is the blank absorbance and AA
the sample absorbance. The mean of three IC50 (concentration
causing 50% inhibition) value of each extract was determined graphically.
Correlation coefficients between phenolic content and
antioxidant activity were calculated using the Sigma
| Table 2: |
Total phenolic, total flavonoid contents and radical scavenging
activity of the 6 plants studied |
 |
Stat 2.0 Jandel Scientific software (Person product moment
correlation function).
RESULTS
Phenolic and flavonoid content: The amount of phenolic compounds
was determined from regression equation of calibration curve (Y = 89.59X,
R2 = 0.99). Values were expressed in gallic acid equivalents
(GAE) and varied from 2.82 to 23.67g GAE/100 g of lyophilised extract(Table
2).
The highest phenolic content was found in the extract
of Lepidagathis anobrya (23.67±0.85 g GAE/100 g).
The flavonoid content expressed in quercetin equivalents
(QE)/100 g of lyophilised extract, were determined from regression equation
of calibration curve Y= 40.55X, R2 = 0.99. The values varied
from 0.03 to 2.4 g QE/100 g of lyophilised extract (Table
2).
The highest amounts of flavonoids were found in extracts
of Dyschoriste perrottetii (2.4±0.57 g QE/100 g)
and Dicliptera verticillata (2.33±0.33) g QE/100
g.
Antioxidant activity: This investigation was based on the measurement
of the relative inhibitory effect of extract tested at different concentration.
Table 2 shows capacity of each plant extract to scavenge
the DPPH radical. Values of the 50 % inhibition concentration (IC50)
varied between 16.33 and 785.67 μg mL-1. The IC50
of quercetin and ascorbic acid were 0.87±0.06 and 1.8±0.43
μg mL-1, respectively.
Lepidagathis anobrya extract have shown the best
scavenging activity with IC50 value of 16.33 μg mL-1.
Dicliptera verticillata is one of the six Acanthaceae that possess
a weak antioxidant activity with an IC50 value of 785.67±6.03
μg mL-1.
The correlation between total phenolic and 1/IC50
was 0.95 (p<0.005). No significant correlation was found between total
flavonoid and 1/IC50.
DISCUSSION
Among the six investigated plants, only five have
presented a remarkable radical scavenging activity with IC50
value ranging from 16.33 to 45.82 μg mL-1. The chemical
composition of these plants indicates the presence of phenolic compounds
including tannins and flavonoids (Nacoulma, 1996) which are known to possess
antioxidant activities (Aderogba et al., 2005; Badami et al.,
2003; Motalleb et al., 2005). Present study shows that the extract
with an amount of phenolic compounds higher than 10 g/100 g present good
antioxidant activity. Dicliptera verticillata which was found to
have a weak antioxidant activity possess the lowest phenolic content of
the six investigated Acanthaceae.
The scavenging activity of all samples on the DPPH radical
was found to be strongly dependent on the extract concentration. It has
been shown that the scavenging effects on the DPPH radical increase sharply
with increasing concentration of the samples and standards to a certain
extent (Motalleb et al., 2005). Some authors found a correlation
between the phenolic content and the antioxidant activity, while others
found no such relationship. Velioglu et al. (1998) have reported
a strong relationship between total phenolic content and antioxidant activity
in selected fruits, vegetables and grain products. Javanmardi et al.
(2003) have found a significant correlation (R2 = 0.71)
between the total antioxidant activity and total phenolic contents of
Iranian Ocimum accessions. In contrast, Kahkonen et al. (1999)
do not find such kind of correlation between antioxidant activity and
phenolic content in plant extracts. In this study, we have found a significant
correlation between antioxidant activity and total phenolic content. In
contrast, such a kind of relationship was not observed with the flavonoids
contents. Hygrophila auriculata, with a very low concentration
of flavonoids (0.033 g QE/100 g) presents an antioxidant activity higher
than Dyschoriste perrottetii which has the highest flavonoid content
(2.4 g QE/100 g). These are in agreement with the findings of Miliauskas
et al. (2004) and Garcia-Alonso et al. (2004) who found
a weak correlation between antioxidant activity and flavonoid content
in fruits.
These results indicate that the studied plants among
Acanthaceae family, especially Lepidagathis anobrya, Hygrophila
auriculata and Nelsonia canescens could possess therapeutical
effects arising from their antioxidant activity, in area such as inflammatory
diseases and cardiovascular protection. Such kind of anti-inflammatory
effects (Oweyele et al., 2005) and hepatoprotective effects (Shanmugasundaram
and Venkataraman, 2005) have already been shown for Nelsonia canescens
and Hygrophila auriculata, respectively.
These findings give a scientific basis to the traditional
uses of the investigated plants.
ACKNOWLEDGMENTS
We are grateful to the International Atomic Energy
Agency for providing the facilities through the technical cooperation
project BKF 5002.
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