Antimicrobial, Antioxidant, cytotoxic activity and Free Radical Scavenging Effect of Acanthus ilicifolius

Acanthus ilicifolius is a mangrove plant and it used coastal village peoples for traditional folk medicine method for varies disease. In general salt tolerance plants have a more antioxidant constitute. India is a rich source of medicinal plants and a number of plant extracts are used against diseases in various systems of medicine such as Ayurveda, Unani and Sidha. Only few of them were scientifically explored. Acanthus ilicifolius Linn. (Acanthaceae) is a folklore medicinal plant used against rheumatism, paralysis, asthma and snake bite (Kathiresan and Ramanathan, 1997; Ramanathan, 2000). The whole plant

extract has been reported to possess analgesic and anti-inflammatory activities (Agshikar et al., 1979) while no such report has been published in reference to the leaves of Acanthus ilicifolius. Methanolic extract of the leaves has been reported to exhibit hepatoprotective (Babu et al., 2001) and tumor reducing activities (Babu et al., 2002). Leishmanicidal activity of 2-benzoxazolinone, isolated from the leaves of this plant has also been documented (Kapil et al., 1994). Phytochemical studies with the plant revealed the presence of lignans (Kanchapoom et al., 2001) and megastigmane glycosides (Wu et al., 2003). According to various medical literatures, several adverse.Anti-oxidants are vital substances which possess the ability to protect the body from damage caused by free radical induced oxidative stress (Ozsoy et al., 2008). There is an increasing interest in natural anti-oxidants e.g., polyphenols, present in medicinal and dietary plants which might help prevent oxidative damage (Silva et al., 2005). Polyphenols possess ideal structural chemistry for free radicalscavenging activity and they have been shown to be more effective anti-oxidants in vitro than tocopherols and ascorbate. In the present study to carried out antioxidant and free radical scavenging potential of A. ilicifolius.

Antimicrobial activity of Acanthus ilicifolius

To investigate the antimicrobial activity of Acanthus ilicifolius against the skin infecting bacterial and fungal pathogens. Through the literature survey, the mangrove plant Acanthus ilicifolius was used in skin infection diseases and have potential anti-inflammatory activity.

Methods

Antimicrobial activity of the leaf extracts was tested using agar well diffusion method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were carried out.

Results

Among the different extracts, chloroform extract showed maximum activity against the bacterial pathogens methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Candida albicans and Trichophyton rubrum. Methanol and acetone extracts showed maximum activity against Staphylococcus epidermis and Lactobacillus plantarum respectively. Chloroform extracts showed the lowest MIC (0.5 mg/mL) and MBC (2 mg/mL) values against the skin pathogens compared with other extracts. Phytochemical screening revealed the presence of resins, steroids, tannins, glycosides, sugars, carbohydrates, saponins, sterols, terpenoids, phenol, alkaloids, cardiac glycosides and catechol.

Antioxidant and cytotoxic activity of Acanthus ilicifolius flower

To investigate the antioxidant and cytotoxic activity of the flower of Acanthus ilicifolius (A. ilicifolius).

Methods

Antioxidant activity was determined as antiradical efficiency with diphenyl picrylhydrazil (DPPH) method and cytotoxic assay was undertaken using brine shrimp lethal toxicity test.

Results

A. ilicifolius flower contained terpenoid, phenolic compounds, and alkaloid. The methanol extract of A. ilicifolius flower showed the highest antiradical efficiency (AE=1.41×10⁻³) against DPPH radicals and the highest cytotoxicity (LC₅₀=22 μg/mL) against brine shrimp nauplii.

Conclusions

It is suggested that active compounds of A. ilicifolius flower solved in methanol play a role to inhibit free radical activity and kill Artemia salina nauplii. The substances can be considered as potential antioxidant and cytotoxic agents as well as imminent candidate for cancer therapy.

Free Radical Scavenging Effect of Acanthus ilicifolius

Determination of total phenolic content:

Total soluble phenolics in the extracts were determined with Folin-Ciocalteau reagent according to the method using gallic acid as a standard phenolic compound (Slinkard and Singleton, 1977), 1.0 mL of extract solution containing 1.0 g extract in a volumetric flask was diluted with 45 mL of methanol. The 1.0 mL of Folin-ciocalteau reagent was added and mixed thoroughly. About 3 min later 3.0 mL of 2% sodium carbonate was added and the mixture was allowed to stand for 3 h with intermittent shaking. The absorbance of the blue color that developed was read at 760 nm. The concentration of total phenols was expressed as mg g^-1 of dry extract (Kim et al., 2003). All determinations were performed in triplicate.

Determination of DPPH (1-1-diphenyl 2-picryl hydroxyl) radical-scavenging activity:

 The free radical-scavenging activity of the A. ilicifolius chloroform extract was measured in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH (Blois, 1958). The 0.1 mM solution of DPPH in ethanol was prepared and 1.0 mL of this solution was added to 3.0 mL of extract solution in methanol at different concentrations (0.1-5 mg mL^-1). The 30 min later, the absorbance was measured at 517 nm. Ascorbic acid was used as the reference compound. Lower absorbance of the reaction mixture indicated higher free radical-scavenging activity.

Determination of hydroxyl radical-scavenging activity:

The hydroxyl radical scavenging capacity was measured using modified method as described previously (Halliwell et al., 1987). Stock solutions of EDTA (1 mM), FeCl₃ (10 mM), ascorbic acid (1 mM), H₂O₂ (10 mM) and deoxyribose (10 mM) were prepared in distilled de-ionized water. The assay was performed by adding 0.1 mL EDTA, 0.01 mL of FeCl₃, 0.1 mL of H₂O₂, 0.36 mL of deoxyribose, 1.0 mL of extract (0.1-5 mg mL^-1) each dissolved in methanol, 0.33 mL of phosphate buffer (50 mM, pH 7.4) and 0.1 mL of ascorbic acid in sequence. The mixture was then incubated at 37°C for 1 h. The 1.0 mL portion of the incubated mixture was mixed with 1.0 mL of (10%) trichloroacetic acid and 1.0 mL of (0.5%) thiobarbituric acid (in 0.025 M NaOH containing 0.025 MNaOH butyl hydroxyl anisol) to develop the pink chromogen which was measured at 532 nm. The hydroxyl radical-scavenging activity of the extract was reported as the percentage of inhibition of deoxyribose degradation. Ascorbic acid was used as a positive control. 2.7. Determination of nitric oxide radical-scavenging activity nitric oxide was generated from sodium nitroprusside and measured by the greiss reaction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide (Marcocci et al., 1994) which interacts with oxygen to produce nitric ions that can be estimated by using greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduce production of nitric oxide. Sodium nitroprusside (5 mM) in Phosphate Buffer Saline (PBS) was mixed with 3.0 mL of different concentrations (0.1-5 mg mL^-1) of the A. ilicifolius extract and incubated at 25°C for 150 min. The samples were added to Greiss reagent (1% sulphanilamide, 2% H₃PO₄ and 0.1% napthylethylenediamine dihydrochloride). The absorbance of the chromaphore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamme was read at 546 nm and referred to the absorbance of standard solutions of ascorbic acid treated in the same way with Griess reagent as a positive control.

ABTS radical scavenging assay:

The ABTS assay was employed to measure the anti-oxidant activity of the leaf extract. ABTS was dissolved in de-ionized water to 7 mM concentration and potassium persulphate added to a concentration of 2.45 mM. The reaction mixture was left to stand at room temperature overnight (12-16 h) in the dark before usage. The 0.5 mL of leaf extract was diluted with 0.3 mL ABTS solution and made up to the volume with methanol. Absorbance was measured spectro- photometrically at 745 nm. The assay was performed at least in triplicates. Fresh stocks of ABTS solution were prepared every 5 days due to self-degradation of the radical. The assay was first carried out on ascorbic acid which served as a standard.

CONCLUSION

In this study, using various in vitro assay systems, the antioxidant potential of A. ilicifolius extract was evaluated based on DPPH, superoxide and hydroxyl radical scavenging activities and total phenolic activity, inhibition of lipid peroxidation in linoleic acid emulsion. In addition, researchers further evaluated the inhibition of protein oxidation as well as reducing power of the extract. The results clearly confirmed confirmed the antioxidative and free radical scavenging activity of the extract. Identification of the antioxidative constituents of the plant and evaluation of their probable anti-diabetic, anti cancer and cardiac vascular disease properties is in progress.