Abstract: Aqueous acetone extracts prepared from five Indigofera species of Burkina Faso, namely Indigofera colutea (Burm.) Murril., I. macrocalyx Guilld et Perr., I. nigritana Hook f., I. pulchra willd. and I. tinctoria L., were investigated for their phytochemical composition and their antioxidant activities. Standard methods and TLC were used to screen the phytochemical composition. The total phenolic and flavonoid content of extracts were assessed by Folin-Ciocalteu and AlCl3 methods, respectively. These extracts were also evaluated for their antioxidant potentials using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-l-picrylhydrazyl (DPPH) and 2,2`-azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) assays. Flavonoids, saponins, quinones, sterols/triterpenes and tannins were present in all these species except for I. pulchra where quinones were not found. Gallic acid, caffeic acid, rutin and myricetin in I. colutea; gallic acid, quercitrin, myricetin in I. tinctoria; galangin and myricetin in I. macrocalyx were identified by thin layer chromatography. Among these, I. colutea, I. tinctoria, I. nigritana and I. macrocalyx, which had the highest phenolic content, were also found to possess the best antioxidant activities. The results indicated a good correlation between antioxidant activities and total phenolic content (p<0.05 for FRAP/DPPH and DPPH/ABTS and p<0.01 for FRAP/ABTS). These plants represent promising sources of natural antioxidants and these findings give scientific bases to their ethnopharmacological uses.
INTRODUCTION
The genus Indigofera comprises around 700 species that are distributed geographically in tropical regions. In Burkina Faso, Nigeria and India, Indigofera colutea (Burm.) Murril., I. macrocalyx Guilld et Perr., I. nigritana Hook f., I. pulchra willd. and I. tinctoria L. have intensive popular use in the treatment of malaria, dysentery, constipation, stomach ache, fatigue, skin disease and wounds (Table 1).
Rotenoids isolated from I. tintoria were found to be toxic to larvae of Anopheles stephensi and adults of Callosobruchus chinensis (Kamal and Mangla, 1993).
| Table 1: | Medicinal uses of Indigofera species |
Recently, antidyslipidemic activity (Narender et al., 2006) and hepatoprotective effects (Singh et al., 2006) of I. tinctoria have been reported. Except these data, little is known on the biological properties of Indigofera species despite their intensive uses by local inhabitants in West Africa and India. Therefore, investigation on the biological properties of Indigofera species is needed for their safe and efficient use.
Among the biological potential of medicinal plants, the antioxidant activity has gained an increase interest these last years because of the role that they play in the prevention of chronic ailments such as heart disease, cancer, diabetes, hypertension, stroke and Alzheimer`s disease by combating oxidative stress (Cole et al., 2005; Liu, 2003; Riboli and Norat, 2003).
According to some researchers, vitamins, phenolic and carotenoids are the major natural antioxidant groups (Rice-Evans et al., 1997; Gülçin, 2006; Thaipong et al., 2006). Phenolic compounds are secondary metabolites widespread in the plant kingdom and which differ widely in terms of structure and biological properties.
The aim of the present study was to examine the total phenolic and flavonoid contents as well as the antioxidant activity of five Indigofera aqueous acetone extracts. The antioxidant potential has been determined using ferric reducing antioxidant power assay (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical and the 2,2-azinobis-(3- ethylbenzthiazoline-6-sulphonic acid) (ABTS) free radical scavenging assays.
MATERIALS AND METHODS
Chemicals: The Folin-Ciocalteu reagent, NaH2PO4, Na2HPO4, sodium carbonate, aluminium trichloride (AlCl3), diphenylboric acid 2-aminoethyl Ester (diphenyl- boryloxyethylamine), gallic acid and quercetin were purchased from Sigma aldrich chemie, Steinheim, Germany. 2,2-diphenyl-l-picrylhydrazyl (DPPH); 2,2`-azinobis(3-ethylbenzothiazoline-6-sulphonate) ABTS, polyethylene glycol 4000, trichloroacetic acid, potassium persulfate and solvents used were from Fluka Chemie, Buchs, Switzerland. Potassium hexacyanoferrate [K3Fe(CN)6] was from Prolabo, Paris, France and ascorbic acid was from Labosi, Paris, France. All chemicals used were of analytical grade.
Plant material: Indigofera colutea (Burm.) Murril., I. macrocalyx Guilld et Perr., I. nigritana Hook f., I. pulchra willd. and I. tinctoria L. were collected in the Ouagadougou region of Burkina Faso in August 2005 and identified by Pr. J. Millogo, a botanist from the University of Ouagadougou. Voucher specimen numbers 01, 02, 03, 04 and 05, (respectively for I. colutea, I. marcrocalyx, I. nigritana, I. pulchra and I. tinctoria) were deposited in the Herbarium of Laboratoire de Biologie et d`Ecologie Végétales UFR/SVT University of Ouagadougou.
Preparation of plant extracts: For each plant, the freshly cut stems with leaves were dried at room temperature and ground to fine powder using a grinder. The extraction was processed using 50 g of powder in 500 mL of acetone:water (80:20) during 48 h under mechanical agitation (SM 25, Edmund BÜHLER, Germany, shaker), at room temperature. After filtration, acetone was removed under reduced pressure in a rotary evaporator (BÜCHI Rotavapor R-200, Switzerland) and the remaining aqueous solutions were lyophilised using a freeze drying system (Cryodos 50, TELSTAR, Spain).
Phytochemical screening and thin-layer chromatography (TLC): The aqueous acetone extract obtained from each plant was used to screen alkaloids, tannins, anthraquinones, flavonoids, saponins, triterpenoids, steroids and coumarins using the method described by Ciulei (1982). The thin layer chromatography analysis of phenolic acids and flavonoids were performed according to Medié-Sarié et al. (2004) and Wagner and Bladts (1996) methods.
The thin layer chromatography plates (Silica gel 60F254, Kiesel gel, 10x10 cm) were spotted with standards or plant extracts and developed in solvent system S1 (Ethyl acetate:formic acid:glacial acetic acid:water, 100:11:11:26) or S2 (n-hexane:ethyl acetate:acetic acid, 62:24:10). Phenolic acids and flavonoids were revealed by spraying the plate with NEU-reagent (Natural products-polyethylene glycol reagent).
Determination of total phenolic and total flavonoid contents: Total phenolic content of each plant extract was determined by Folin-Ciocalteu method (Singleton et al., 1999). The diluted aqueous solution of each extract (0.5 mL) at a concentration of 100 μg mL-1 was mixed with 2.5 mL of Folin Ciocalteu reagent (0.2 N). This mixture was allowed to stand at room temperature for 5 min and then, 2 mL of sodium carbonate solution (75 g L-1 in water) was added. After 2 h of incubation, the absorbencies were measured at 760 nm against a water blank using a spectrophotometer (CECIL CE 2041, CECIL Instruments, England). The standard calibration curve was plotted using gallic acid (0-200 mg L-1). The determination was performed in triplicate and the results were expressed as mg of Gallic Acid Equivalents (GAE)/100 mg of extract
The total flavonoid content of the plant extract was estimated according to Dowd method, as adapted by Arvouet-Grand et al. (1994). For each extract, 2 mL of methanolic solution (100 μg mL-1) was mixed with 2 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 2 mL of methanol and 2 mL of plant extract without AlCl3.
Quercetin was used as reference compound to produce the standard curve and the average of three readings was used and expressed as mg of Quercetin Equivalents (QE)/100 mg of plant extract.
Antioxidant activity
Iron (III) to iron (II) reduction activity (FRAP): The total antioxidant capacity of plant extract was determined using
iron (III) reduction method (Hinneburg et al., 2006). The diluted
aqueous solution of each extract (1 mL) at a concentration of 100 μg
mL-1 was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6)
and 2.5 mL of a 1% aqueous potassium hexacyanoferrate [K3Fe(CN)6]
solution. After 30 min incubation at 50°C, 2.5 mL of trichloroacetic
acid (10 %) was added and the mixture was centrifuged at 3000 rpm for
10 min. Then, 2.5 mL of the upper layer solution was mixed with 2.5 mL
of water and 0.5 mL of an aqueous FeCl3 (0.1%) solution. Absorbencies
were read at 700 nm and ascorbic acid was used to produce the calibration
curve. The iron (III) reducing activity determination was performed in
triplicate and expressed in mmol Ascorbic Acid Equivalents per gram of
extract.
DPPH radical method: The ability of the extract to scavenge DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical was determined according to the method of Velazquez et al. (2003) with some modifications. Briefly, 1.5 mL of a freshly prepared methanolic solution of DPPH (20 mg L-1) was mixed with 0.75 mL of extract solution (0.003-1 mg mL-1). After 15 min of incubation in the dark, at room temperature, absorbencies were read at 517 nm against a blank sample consisting of a 1.5 mL of methanol and 0.75 mL of extract solution.
Quercetin, ascorbic acid and gallic acid were used as positive controls. All determinations were performed in triplicate. DPPH radical inhibition percentage was calculated according to the formula of Miliauskas et al. (2004):
Inhibition (%) = [(AB-AA)/AB]x100 |
where, AB is the blank absorbance and AA the sample absorbance (tested extract solution), IC50 value was obtained by graphically determination. A lower IC50 value indicates greater antioxidant activity.
ABTS radical cation decolorization assay: The radical scavenging capacity of antioxidants for ABTS radical cation was carried out as described by Re et al. (1999). The ABTS+ was generated by reacting 7 mM aqueous solution of ABTS with 2.5 mM potassium persulfate (final concentration) followed by storage in the dark, at room temperature, for 12 h before use. The mixture was diluted with ethanol to give an absorbance of 0.70±0.02 unit at 734 using spectrophotometer.
For each extract, a 20 mg mL-1 solution in methanol was prepared and further 100 fold diluted in ethanol. Ten microliter of this diluted sample was allowed to react with 990 μL of fresh ABTS+ solution and then absorbance was taken 6 min exactly after initial mixing. Ascorbic acid was used as standard and the capacity of free radical scavenging was expressed as mmol ascorbic acid equivalents g-1 of extract.
Statistical analysis: For statistical analysis, MS Excel software (CORREL Statistical function) was used to calculate quercetin, ascorbic acid and gallic acid equivalents, to determine inhibition percentage and to establish linear regression equations. Pearson Product Moment function and One way ANOVA (Tukey test) of SigmaStat 2.0 (Jandel Scientific software) were used to determine correlation coefficients (R) and the level of statistical significance, respectively.
RESULTS AND DISCUSSION
Different Indigofera species from Burkina Faso folk medicine have been studies, in order to support and confirm their uses (Table 1).
Firstly, aqueous acetone extracts prepared from studied plants were screened for their phytochemical composition. The dry residues obtained were 5.57 g for I. colutea, 6.02 g for I. macrocalyx, 5.96 g for I. nigritana, 5.28 g for I. pulchra and 12.63 g for I. tinctoria.
Flavonoids, saponins, quinones, sterols/triterpenes and tannins were detected in all these species with an exception being the absence of quinones in I. pulchra (Table 2). The presence of saponin, flavonoid and tannin in some Indigofera species such as I. arrecta, I. aspalathoides and I. dendroides has already been reported (Christina et al., 2003; Esimone et al., 1999; Nacoulma, 1996).
Contrasting with the findings of a previous study on a close related species (Leite et al., 2006), namely I. suffruticosa, alkaloids and coumarins were not detected in the plants used in this study.
Further confirmation for the presence of phenolic compounds was made by thin layer chromatography.
| Table 2: | Phytochemical screening of acetone extract of Indigofera species |
| Table 3: | Polyphenol contents and antioxidant activities of Indigofera acetone extracts |
| Results are mean±SD (n = 3), Values with the same letter(s) are not significantly different (p>0.05) | |
Galangin in I. macrocalyx extract, gallic and caffeic acids in I. tinctoria and gallic acid in I. colutea were identified using the solvent system 2. With the system 1, rutin in I. pulchra, rutin and myricetin in I. tinctoria, quercitrin and myricetin in I. pulchra and myricetin in I. macrocalyx were identified (Table 2).
The result shows a qualitative difference in phenolic acids and flavonoids composition between these species. As the detected phenolics have different known biological activities, their presence can partially justify some of the medicinal uses of Indigofera species in Burkina Faso. Examples are the anti-inflammatory and antimicrobial activities of galangin, rutin and gallic acid (Bruneton, 1993; Carnat et al., 2004; Elliott et al., 2000; Rajkapoor et al., 2004; Raj Narayana et al., 2001; Ueda et al., 2002) which can justify the uses of these Indigofera species to cure inflammation and skin diseases.
Because the structure of phenolic compounds, mainly the position of the hydroxyl radical, plays a major role in their antioxidant properties (Miliauskas et al., 2004), these five Indigofera species were studied, keeping in mind the difference in their qualitative composition. For this purpose, the total phenolic and flavonoid content of the different extracts was determined.
Total phenolic and flavonoid content estimated from the calibration curves (Y = 104.83 X, R2 = 0.9969 for total phenolic and Y= 40.23 X, R2 = 0.9999 for total flavonoid content) are shown in Table 3. The highest total phenolic content was recorded in I. colutea extract (54.27±4.87 mg GAE/100 mg) while the highest amount of flavonoids (9.63±0.45 mg QE/100 mg of dried extract) was found in the extract of I. nigritana.
High levels of phenolic were also found in I. nigritana and I. tinctoria and the lowest flavonoid content which found in I. colutea and I. tinctoria. These results showed that I. macrocalyx and I. nigritana have comparable amounts of total phenolic and total flavonoid. No significant correlation was found between the total phenolic and the total flavonoid content.
The FRAP assay determined the reducing power of plant extracts resulting from the ability of their components to donate electrons and, therefore, participate in redox reactions. Using the standard curve of ascorbic acid (Y = 126.9X, R2 = 0.9999), the best activities were found with I. colutea (2.54± 0.14 mmol g-1) and I. tinctoria (2.04±0.09 mmol g-1) extract followed by I. nigritana (1.57±0.06 mmol g-1) and I. macrocalyx (1.41±0.02 mmol g-1) (Table 3). For these four plants, the amounts of ascorbic acid equivalents were significantly higher than that of I. pulchra (0.81±0.01 mmol g-1).
The DPPH assay is based on the measurement of the relative inhibition of the extract tested at various concentrations. Chemicals which are able to change the colour of the DPPH free radical from violet to yellow can be considered as antioxidants and therefore, radical scavengers (Hinneburg et al., 2006). Table 3 shows that values of the 50% inhibition concentration (IC50) varied from 2.45 0.15 to 12.37 0.32 μg mL-1.
The best antioxidant activity was obtained from I. colutea (2.45 ± 0.15 μg mL-1) followed by I. nigritana (3.68±0.87 μg mL-1) and I. tinctoria (3.79±0.08 μg mL-1), which with the first one, were also found to possess the highest phenolic content. I. pulchra with low phenolic content exhibited a relatively weak antioxidant activity. The IC50 values for the references were 1.80±0.43 μg mL-1 for ascorbic acid, 0.88±0.11 μg mL-1 for quercetin and 0.61±0.14 μg mL-1 for gallic acid.
These data show that all these extracts displayed significant antioxidant activity (IC50 <15 μg mL-1), but the reference substances were more radical scavengers than the Indigofera extracts.
The free radical scavenging ability of Indigofera species was also determined using ABTS radical cation. Ascorbic acid was used to produce the dose response curve.
Table 3 shows ascorbic acid equivalents antioxidant capacity of the different extracts in ABTS assay which were estimated from the standard curve of ascorbic acid (Y = 20.06-33.48X, R2 = 0.9982).
The strongest antioxidant activities were obtained from I. colutea (3.74±0.14 mmol g-1) and I. tinctoria (3.00±0.37 mmol g-1) which possess the highest phenolic content. I. pulchra with a low phenolic content has shown the weakest antioxidant capacity (1.36±0.25 mmol g-1).
In this study, ascorbic acid has been used as standard instead of trolox, because these two chemicals have almost the same inhibition percentage at 734 nm for ABTS radical cation (Katalinic et al., 2006). Several study confirm the usefulness of ascorbic acid (Lee et al., 2003; Soong and Barlow, 2004).
In the present study, the highest phenolic content and the best antioxidant activities, using 3 different methods, were recorded in I. colutea. These results suggest that it can be a good source of antioxidants.
The antioxidant activities of these different Indigofera species can be attributed, in part, to their gallic acid, rutin and myricetin content, due to the strong antioxidant capacity of these chemicals. The high total phenolic content of these plants can also explain, in part, these good antioxidant activities. This last assertion is strongly supported by the correlation studies (Fig. 1) which show good correlations between the total phenolic content and iron reduction (R = 0.95, p<0.05), DPPH inhibition (R = 0.99, p<0.001) and ABTS reduction (R = 0.92, p<0.05). Otherwise, more than 90% of the antioxidant capacity of these plant extracts derives from the contribution of phenolic compounds and the antioxidant activity is not limited to phenolics. The activity may also come from the presence of other antioxidant compounds which, in this case, are contributing for 9% of the antioxidant capacity (Javanmardi et al., 2003).
These correlations also suggest that phenolic compounds contribute well to the antioxidant capacity of the Indigofera species. Similar correlation were also found
| Fig. 1: | Correlations between total phenolic content and antioxidants capacities of Indigofera extracts |
with plant extracts, honey and fruits in earlier study confirming the interest of polyphenols as natural antioxidant from plants and foods (Cai et al., 2006; Ivanova et al., 2005; Lee et al., 2003; Meda et al., 2005; Sawadogo et al., 2006; Zheng and Wang, 2001). This antioxidant capacity is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers (Javanmardi et al., 2003).
In contrast to the total phenolic content, no significant correlation was found between total flavonoid content and the antioxidant capacities.
A strong correlation have been found between the different antioxidant assays (R = 0.95, p<0.05 for FRAP/ DPPH and DPPH/ABTS and R = 0.98, p<0.01 for FRAP/ ABTS).
The good antioxidant activities of the studied plants can also justify their uses for the treatment of inflammation, as the anti-inflammatory properties of some polyphenols derive mainly from their free radical scavenging activities (Fenglin et al., 2004). Nevertheless, further investigations are required to confirm this hypothesis.
This study has shown that Indigofera species, used in folk medicine in Burkina Faso, have some phytochemicals with known pharmacological activities. The aqueous acetone extracts of I. colutea, I. tinctoria and I. nigritana which had the highest total phenolic content were found to possess the strongest radical scavengers in both DPPH and ABTS assays and also, the best reduction power in FRAP assay. Good correlations were obtained between DPPH, FRAP and ABTS antioxidant assays and also between each of these assays and the total phenolic content. According to the total phenolic content and antioxidant activities of their extracts, the plants used in this study represent a good source of antioxidant. These results give further support to the therapeutical uses of these Indigofera species.
ACKNOWLEDGMENT
We are grateful to the International Atomic Energy Agency for providing the facilities through the technical cooperation project BKF 5002.