Abstract: In this study the total phenolic and flavonoid content as well as the antioxidant activity of six Acanthaceae namely Blepharis lineariifolia PERS, Dicliptera verticillata (FORRSK.) C. CHRISTENS, Dyschoriste perrottetii (NEES) O. KTZE, Hygrophila auriculata (SCHUMACH.) HEINE, Lepidagathis anobrya NEES, Nelsonia canescens (LAM) SPRENG were evaluated. The total phenolic and flavonoid of their aqueous acetone extract were assessed by Folin-ciocalteu and AlCl3 method, respectively, whereas the antioxidant activities were determined by the DPPH method. Lepidagathis anobrya, Hygrophila auriculata and Nelsonia canescens which had the highest phenolic content, were found to possess the best antioxidant activities. The results suggest that these plants are good sources of antioxidants and support their use in cardiovascular and antiinflammatory diseases.
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.