Free radical scavenging activity, kinetic behaviour and phytochemical constituents of Aristolochia clematitis L. roots

2.1 Plant material

A. clematitis roots were purchased from a local market of Bechar department (southern Algeria). A voucher specimen of the plant was identified and authenticated at the laboratory of botany at the biology Institute. The roots were ground by an electrical mill mesh and powdered part was stored in a nylon bag in a deep freeze until the time of use.

2.2 Preparation of the extracts

2.3 Phytochemical screening

The diethyl ether, methanol and water extracts were screened for phytochemical constituents (flavonoids, free alkaloids, alkaloid salts, saponins, steroids, terpenoids, tannins, anthraquinones, anthracenosides, anthocyanosides, coumarins, reducing sugars, fatty acids, volatile oils, emodols) using standard procedures to identify the constituents as described by Sofowara (1993) and Harborne (1978).

2.4 Determination of free radical scavenging activity by the DPPH method

The antioxidant potential of the tannins and methanolic crude extracts were determined on the basis of their scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. Briefly, 100 μL of various concentrations of the each extract in methanol was added to 1.9 mL of a methanol solution of DPPH (0.004%). The mixture was vigorously shaken and then allowed to stand at room temperature for 30 min in the dark. The absorbance of the mixture was measured at 517 nm by using a double-beam UV–vis Camspec M550 spectrophotometer. A mixture of 100 μL of methanol and 1.9 ml of DPPH solution was used as the control. The scavenging activity on the DPPH radical was expressed as inhibition percentage using the following equation: $$\%\text{Inhibition}=[({\text{A}}_{\text{B}}-{\text{A}}_{\text{S}})/{\text{A}}_{\text{B}}]\times 100$$ (Blois, 1958). where A_B is the absorbance of the control reaction (containing all reagents except the test compound), and A_S is the absorbance of the test compound. The commercial known antioxidant, ascorbic acid was used for comparison or as a positive control.

The tests were carried out in triplicate. The extract concentration providing 50% inhibition (IC₅₀) was calculated from the graph of inhibition percentage plotted against extract concentration (0.5; 0.25; 0.125; 0.0625; 0.0312; 0.0156; 0.0078 mg/ml).

2.5 High performance thin layer chromatography (HPTLC) study of extracts and DPPH

Tannins and methanolic crude extracts of A. clematitis were subjected to High performance thin-layer chromatography (HPTLC) on a silica gel plate (20 × 20 cm, Silica gel F254, Merck). The solvent system optimized for crude extracts of A. clematitis was methanol and Chloroform (10: 90 v/v). The tannins and methanolic crude extracts were loaded on a TLC silica gel plate and the plate was developed in a sandwich TLC chamber to a distance of 70 mm. After 15 min air-drying, the plate was sprayed with 0.004% (w/v) DPPH reagent prepared in methanol and 10% (v/v) sulphuric acid respectively. The spots on the plates were observed after the plate had been heated at 60 °C for 30 min exactly after spraying (Subramanion et al., 2011).

2.6 Kinetic analysis

Fitting of the experimental data was carried out by using the Levenberg–Marquardt method (Marquardt, 1963) implemented in Origin v. 6.0 programme for Windows. Recording of spectrophotometric data was taken until the disappearance of DPPH^• in the presence of the tannins or methanolic crude extracts occurred.

DPPH radical: DPPH was obtained from Fluka (Buchs, Switzerland). 1 mg DPPH was solved in 25 mL methanol (mother solution (1) 0.04%). This stock solution was used for the measurements, and kept in the dark at ambient temperature when not used.

On the other hand, 5 mg of each extract was solved in 4 mL methanol (mother solution (2) 1,25 mg/mL). From this solution, different concentrations were prepared (0.6250; 0.3125; 0. 1562; 0.0780 mg/mL) and used for kinetic behaviour.

To fellow the kinetic behaviour of DPPH^• radical scavenging activity of extracts under study, 1,5 mL of solution (2) was mixed with 1,5 mL of solution (1), the absorbance was measured after each 30 s until it becomes constant.

3.1 Phytochemical screening

Phytochemical screening of the crude water, diethyl ether and methanolic extract revealed the presence of free alkaloids, alkaloid salts, steroids, terpenoids, tannins, anthracenosides, anthocyanosides, reducing sugars, fatty acids and volatile oils. Whereas, flavonoids, saponins, anthraquinones, coumarins and emodols were not detected; the results of phytochemical tests have been summarized in Table 1.

3.2 Extraction of tannin extracts

Tannins are the natural polyphenolic compounds which can influence the nutritive value of different food stuffs utilized by human and other animals. Tannins also have large influence on the phytochemical and phytotherapeutical value of medicinal plants. Various methods have been used to increase the extraction efficiency of tannins from different medicinal plants for their use in pharmaceutical field (Cobzac et al., 2005).

The mixture acetone/water has been found to be the appropriate used solvent for the extraction of tannins. The results of extraction revealed that the roots of A. clematitis L. were found to contain a moderate content of tannins (yield: 0.34% (v/w)) based on the dry weight of plant.

3.3 Determination of free radical scavenging activity by HPTLC and DPPH methods

On the basis of the fact that the phenolic compounds and terpens are known for their properties to trap the free radicals (Jukié and Milos, 2005), it appeared important to us to make a study of the antioxidant activity of tannins and crude methanolic extracts.

Using the HPTLC bioautography technique, we observe on the TLC plate, the appearance of zones of antiradicalaire activity of pale yellow colour on purple bottom (Molyneux, 2004) for the tannins and methanol extracts as for the vitamin C.

Three purple colour spots were visualized after spraying the plates with sulphuric acid demonstrating the presence of several bioactive compounds in the methanolic crude extract. Furthermore, three yellow spots were observed also after spraying the plates with DPPH solution indicating, the presence of antioxidant compounds in the extract of tannins.

In conclusion, the bioautography HPTLC method revealed that tannins possessed antioxidant effect. Besides, the antioxidant activity observed in the crude methanolic extract probably contributes by phenolic substances it contained.

The evaluation of the oxidizing activity with the DPPH by spectrophotometric measurement gave the following results illustrated in Figs. 1–3.

The model of scavenging the stable DPPH^• radical is a widely used method to evaluate the free radical scavenging ability of various samples (Lee et al., 2003). DPPH^• is a stable nitrogen-centred free radical, the colour of which changes from violet to yellow upon reduction by either the process of hydrogen- or electron-donation. Substances which are able to perform this reaction can be considered as antioxidants and therefore radical scavengers (Brand-Williams et al., 1995).

The reading of Fig. 1 showed a very fast reaction between ascorbic acid (vitamin C) and free radical DPPH^•. The scavenging reaction was carried out in short time and attaints the steady state (100% of inhibition). These results are in agreement with the findings of Raquibul Hasan (Raquibul Hasan et al., 2009). We concluded that acid ascorbic has a potent antioxidant effect with IC₅₀ value of 0.0331 mg/mL.

Close

Figure 1

Antioxidant activity of vitamin C.

Export to PPT

Regarding antioxidant activity of A. clematitis crude methanolic extract (Fig. 2), it seems interesting that it showed good antioxidant activity with IC₅₀ value of 0.142 mg/mL. The antioxidant effectiveness of this extract is mainly attributed to the active compounds present in it such as phenolic compounds, alkaloids and others. Moreover, this effect can be due to the high percentage of main constituents, but also to the presence of other constituents in small quantities or to synergy among them. As shown in Fig. 2, the scavenging potency of extract increased with the concentration which correlated well with the findings of Popovici et al. (2009), Prabu et al. (2011) and Xiaoli et al. (2008).

Close

Figure 2

Antioxidant activity of methanol extract.

Export to PPT

For the tannin extract, Fig. 3 showed the curve of DPPH^• radical scavenging activity of these phytoconstituents, compared with ascorbic acid. At a concentration of 0.5 mg/ml, the scavenging activity of tannins reached 60%, while at the same concentration that of the ascorbic acid 98%. A lower value of IC₅₀ indicates greater antioxidant activity. IC₅₀ value of tannins (0.196 mg/mL) was superior to that of the reference ascorbic acid (0.0331 mg/mL). The effect of antioxidants on DPPH is thought to be due to their hydrogen donating ability (Baumann et al., 1979). The study showed that the tannins have proton donating ability and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants (Muir, 1996; Brand-Williams et al., 1995 and Nenandis et al., 2003).

Close

Figure 3

Antioxidant activity of tannin extract.

Export to PPT

In conclusion, and from the results, it is shown clearly that the tannin extract had a good antioxidant activity as ascorbic acid. Besides, our results are in analogy with several reports describing the antioxidant activity of tannins (Ryszard and Agnieszka, 2003; Radosław et al., 2006; Shang-Ju et al., 2010; Andréia Cristina et al., 2005).

3.4 Kinetic analysis

Kinetic studies of DPPH-extract reaction were carried out to estimate the scavenging activity as a function of time. Besides, our experience aimed to study the effect of the concentration of the methanol and tannin extracts on scavenging reaction of the free radical DPPH^•. In fact, different concentrations were tested (0.625; 0.312; 0.156 and 0.078 mg/mL). The free DPPH^• radical is quite stable for more than 110 min at 25 °C in the reaction medium. So it allows the evaluation of the radical scavenging activity of the extracts within that time.

The trap of the free radical was evaluated via the calculation of the remaining DPPH^• concentration at the end of the reaction taking into account its initial one. The remaining (DPPH^•)_R percent was calculated from the following equation: $$\%{({\text{DPPH}}^{•})}_R=({[{\text{DPPH}}^{•}]}_{t=T}/{[{\text{DPPH}}^{•}]}_{t=0})\times 100$$ where [DPPH^•]_t=T was the concentration of DPPH at steady state and [DPPH^•]_t=0 was the initial concentration of DPPH^•.

Table 2 summarized the time reaction at steady state, half life (t^1/2) and the remaining DPPH^• percent.

The results given in Table 2 showed clearly that the tannin extract exhibited a significant antioxidant effect than the methanolic extract where the times of reaction at steady state were 8; 25; 49 and 58 min for the tannin extract, whereas, 13, 60; 89 and 112 min were unregistered for the methanolic extract at concentration of 0.625; 0.312; 0.156 and 0.078 mg/mL respectively. Furthermore, we observed well that the remaining DPPH^• in case of tannin extract (5.90; 6.85; 7.64 and 8.41%) were less than those of methanolic one (8.10; 10.11; 15.04 and 17.20%). The effectiveness of tannins was expressed also via the values of half life t^1/2 which were shorter in comparison with those of methanolic extract.

For more details, Figs 4 and 5 illustrated the kinetic profile of DPPH^• annihilation by the studied extracts.

Close

Figure 4

Reaction kinetics of methanolic extract with DPPH^• radical.

Export to PPT

Close

Figure 5

Reaction kinetics of tannin extract with DPPH^• radical.

Export to PPT

Immediately after the addition of the methanolic or tannin extracts to the reaction medium, the absorbance of DPPH^• at 517 nm dropped (data not shown), due to the decrease of DPPH^• concentration in the medium (Figs. 4 and 5).

In case of rapid kinetic behaviour, practically all samples at high concentrations reacted within a very short time, and a steady state was reached almost immediately. On the other hand, slow kinetic behaviour (lowest concentration for each sample) implied longer periods before the steady state was reached (Table 2). These results are in harmony with those obtained by Popovici et al. (2009), Sladjana et al. (2012), Qian and Nihorimbere (2004), Dongmei et al. (2007), Nikolaos and Maria (2002).