Biochemical characterization of radical scavenging polyphenols from Nyctanthes arbortristis.

CONTEXT: Antioxidants are quenchers of free radical that are responsible for inducing oxidative stress generated via reactive oxygen species-induced degenerative diseases such as cancer, diabetes, and cardiovascular diseases etc. Plant and plant products are recognized as safe and potential health promoting and nutritive sources. AIMS: To investigate the antioxidant potency of polyphenol extract (PE) of Nyctanthes arbortristis leaves and identification of the active constituent by HPLC.
MATERIALS AND METHODS: PE of N. arbortristis leaves was investigated for antioxidant activity employing various established in vitro systems, such as lipid peroxidation in liposome, DPPH and hydroxyl radical scavenging, reducing power assay, and iron ion chelation. Identification of active constituent in PE of N. arbortristis responsible for antioxidant activity by HPLC. STATISTICAL ANALYSIS USED: All experiments were carried out in triplicates. Data were shown as mean ± standard deviation (SD). SPSS 10.0.5 version for windows (SPSS software Inc., USA) computer program was used for statistical analysis.
RESULTS: Identification of active constituent in PE revealed gallic acid 75.8 ± 0.21, protocatechuic acid 14.6 ± 0.5, chlorogenic acid 6.79 ± 0.43, and caffeic acid 5.34 ± 0.2 μg/ml. PE showed strong inhibitory activity of 73% at 200 μg/ml toward lipid peroxidation in egg lecithin, concentration-dependent inhibition of deoxyribose oxidation at 200 μg/ml was 85% inhibition, and considerable antioxidant activity in DPPH radical assay system at 200 μg/ml was 79% inhibition. BHA and gallic acid showed significant observations.
CONCLUSION: The antioxidant potency significantly correlated with the phenolic content of PE. Considering that medicinal herbs contain potent phytochemicals, which is effectively utilized for various degenerative disease, these in vitro results showed that N. arbortristis leaves could be effectively employed in functional food, to alleviate oxidative stress.

Free radicals have noxious effects on cells and is believed that the damage caused by the free radicals is reported in the etiology of several diseases.[1] Reactive oxygen species (ROS) in the form of hydroxyl radical (OH) and hydrogen peroxide (H2O2) are generated by normal metabolic processes or from exogenous factors and agents that can easily initiate the peroxidation of membrane lipids, which leads to the accumulation of lipid peroxides, these ROS are capable of damaging a wide range of essential biomolecules.[2] In recent years, there has been an increasing interest in finding natural antioxidants, especially of plant origin, used in the treatment of several human diseases, and their pharmacological and therapeutic properties have been attributed to different chemical constituents isolated from their crude extracts.[3] Of particular importance, chemical constituents with antioxidant activity can be found at high concentrations in plants and are responsible for their preventive effects including cancer, neurological, and cardiovascular diseases.[4] Thus, the antioxidant properties of plants have a full range of perspective applications in human healthcare. Interestingly, literature data have indicated that the pharmacological properties of crude extracts of plants can be lost after the isolation of specific compounds, indicating that part of their pharmacological properties can be related to a combination of different classes of compounds.[56]

Nyctanthes arbortristis L (Oleaceae) is widely used in ayurvedic medicine to treat sciatica, arthritis, fever, various painful swelling and as laxative.[7] Of particular importance, some studies have demonstrated immunostimulant effects of N. arbortristis.[8] Particularly, the dried leaves is used in traditional and herbal medicine because of its anti-inflammatory and anti-nociceptive,[8] anti-leishmanial,[9] anti-microbial, and anti-viral properties.[10] Nonetheless, the mechanism involved in the therapeutic properties of this plant is not still elucidated. Hence, the present study investigates the anti-oxidative effects of polyphenol extract (PE), employing various in vitro systems such as lipid peroxidation in egg lecithin, 1,1 diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, hydroxyl radical scavenging, reducing power, and iron ion chelation and identification of active constituents by HPLC.

Materials and Methods

Chemicals

Butylated hydroxy anisole (BHA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), gallic acid, and deoxyribose were obtained from Sigma Chemicals, St. Louis, MO, USA. Thiobarbituric acid (TBA), Ethylenediaminetetra acetic acid (EDTA), Trichloroacetic acid (TCA), ascorbic acid, ferrozine, ferric chloride, and potassium ferricyanide were obtained from M/s Sisco Research laboratories, Mumbai, India. All the reagents used were analytical grade.

Extraction of polyphenols

Fresh and healthy leaves of N. arbortristis were collected in and around Mysore, Karnataka, India during November 2008. The harvested plant material was dried at room temperature, then samples were powdered to 60 meshes. Polyphenol extraction was done according to Yu and Dahlgren.[11]

Determinations performed for polyphenol extract

Polyphenols were quantified spectrophotometrically at 750 nm using Folin-Ciocalteau reagent[12] and gallic acid as standard.

Identification of polyphenols in the extract was carried out by RP-HPLC (LC-10A liquid chromatography LC: Shimadzu, Japan) in C18 column (4.6 × 250 mm) using diode array UV detector at 280 and 296 nm. A binary gradient of 4.5% formic acid in deionized water (solvent A), and acetonitrile (solvent B) was as follows: From 10% to 20% solvent B over 20 min, from 20% to 25% solvent B over 10 min, from 25% to 35% solvent B over 10 min and then isocratic condition for a further 10 min. The flow rate was 1 ml/min. Quantification was done by comparing with known standards (gallic acid, protocatechuic, caffeic and chlorogenic acids were from Sigma Chemicals, St. Louis, MO, USA).

Inhibition of lipid peroxidation

Lipid peroxidation (LPO) inhibitory activity was measured according to Kulkarni et al.,[13] egg lecithin (3 mg/ml in phosphate buffer, pH 7.4) was sonicated (Hielscher GmbH UP 50H ultra-challprozessor sonicator) for 30 min to obtain small membrane liposome vesicles. Different concentrations of PE were added to 0.5 ml of liposome mixture. Lipid peroxidation was induced by adding 400 mM FeCl 3 and 200 mM L- ascorbic acid and incubated for 60 min at 37°C. The reaction was stopped by the addition of 0.25N HCl containing 15% TCA and 0.375% TBA and incubated in boiling water bath for 15 min, and the supernatant absorbance was measured at 532 nm. The synthetic antioxidant BHA was used as a positive control in all the assays. The scavenging effect was measured using the following equation:

Scavenging effect (%) = [(Acontrol – Asample)/Acontrol] × 100

where A = Absorbance.

Scavenging of DPPH radical

DPPH radical scavenging activity was done according to Meghashri et al.,[14] Briefly, 1 ml of DPPH solution (0.1 mM in 95% ethanol (v/v) was incubated with different concentrations of PE. The reaction mixture was incubated for 20 min at room temperature (RT), and the absorbance was read at 517 nm. The scavenging effect was calculated using the equation as described in LPO.

Hydroxyl radical scavenging assay

The reaction mixture containing different concentrations of PE was incubated with deoxyribose (10 mM), H2O2 (10 mM), FeCl3 (5 mM), EDTA (1 mM), and ascorbic acid (5 mM) in potassium phosphate buffer (50 mM, pH 7.4) for 60 min at 37°C and read at 535 nm.[15]

Metal ion chelating assay

The Fe2+ chelating ability of PE was measured by the ferrous iron-ferrozine complex.[16] The reaction mixture containing FeCl2 (2 mM) and ferrozine (5 mM) along with PE was adjusted to a total volume of 0.8 ml with methanol, mixed and incubated for 10 min at RT, and absorbance was read at 562 nm.

Measurement of reducing power

The reducing power of PE was measured by incubating the reaction mixture (1 ml) containing the PE in phosphate buffer at pH 6.6 with potassium ferricyanide (1%) at 50°C for 20 min. The reaction was terminated by adding TCA (10%), centrifuged at 3,000 rpm for 10 min at RT and the supernatant was mixed with ferric chloride (0.1%), the absorbance was measured at 700 nm.[14]

Statistical analysis

All experiments were carried out in triplicates. Data were shown as mean ± standard deviation (SD). SPSS 10.0.5 version for windows (SPSS software Inc., USA) computer program was used for statistical analysis. The level of significance was P < 0.05.

Results and Discussion

The antioxidant activity of polyphenolic compounds in N. arbortristis leaves to scavenge free radicals has not been comprehensively investigated. Quantitative analysis of phenolic content in PE yielded 1240 ± 0.10 μg gallic acid/equivalents/g. However, antioxidant activity of plant extract is often associated with the phenolic compounds present in them.[6] Gallic acid was the most abundant phenolic acid [Table 1 and Figure 1] found in PE. Caffeic acid was also detectable in small quantities, whereas appreciable quantities of chlorogenic and protocatechuic acids (6.79 ± 0.43 μg/ml and 14.6 ± 0.5 μg/ml, respectively) were found. In addition, series of other compounds that could not be identified were also observed to be present, based on their absorption spectra, these may be hyroxybenzoic acid derivatives and various flavonoids (primarily flavone derivatives). According to literature mining, not much work has been carried out in N. arbortristis, only few components are identified, such as arbortristoside-A[17] and iridoids.[9]

Table 1

Phenolics detectable in the polyphenol extract (5 g/100 ml)

Figure 1

RP-HPLC chromatograms for the polyphenol extract. Peak 1: Gallic acid, Peak 2: Protocatechuic acid, Peak 4: Chlorogenic acid, Peak 5: Caffeic acid, Peaks 3, 6-15: Unidentified substances.

In biological systems, lipid peroxidation generates a number of degradation products, such as malondialdehyde (MDA) and is found to be an important cause of cell membrane destruction and cell damage, also extensively studied and measured as an index of LPO and as a marker of oxidative stress.[15] In this study, the inhibition of LPO by PE and BHA at 200 μg/ml was found to be 73% and 79%, respectively [Figure 2a]. The antioxidant activity of PE in DPPH radical assay, which primarily evaluates proton radical-scavenging ability. The DPPH free radical scavenging potential of PE and BHA at 200 μg/ml was found to be 79% and 82%, respectively [Figure 2b]. It is well accepted that the DPPH radical scavenging by antioxidants is due to their hydrogen donating ability.[18] Further, the hydroxyl radical scavenging activity of PE was obtained from deoxyribose system [Figure 2c]. The PE and BHA at 200 μg/ ml exhibited concentration-dependent inhibition of 85% and 90%, respectively. Earlier, worker[19] has employed this system to assess the biological activity of various natural plant-derived biomolecules. It is likely that the chelating effect of PE on metal ions may be responsible for the inhibition of deoxyribose oxidation.[19] The iron chelating ability of PE and EDTA at 200 μg/ml was found to be 72% and 79%, respectively [Figure 2d]. Earlier, authors have observed the direct correlation between antioxidant activity and reducing power of certain plant extracts.[14] The reducing properties are generally associated with the presence of reductones, which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom.[20] Our data on reducing power of PE suggest that it is likely to contribute significantly towards the observed antioxidant effect [Figure 2e].

Figure 2

Antioxidant Activity of PE of N. arbortristis leaves and BHA at different concentrations

Conclusion

In conclusion, the results obtained in this study clearly revealed that the PE of N. arbortristis leaves contains a number of antioxidant molecules, which can effectively scavenge free radicals. Although we have not isolated a single compound responsible for antioxidant activity, these effects may be due to the presence of gallic acid, caffeic acid, chlorogenic acid, and protocatechuic acid, which was revealed by qualitative and quantitative analysis.[6] The multiple antioxidant activity of PE clearly indicates its potential application in combating oxidative stress. However, further studies should focus on isolation of antioxidant molecules and their health promoting potential and mammalian safety.