Antioxidant Activity of Cinnamomum iners Leaves Standardized Extract

MATERIALS AND METHODS

Plant material: Cinnamomum iners leaf were collected at USM (University Sains Malaysia). The authentication work was carried out by a botanist from School of Biological Sciences, USM where, the plant material was deposited. The voucher specimen number is 11014.

Preparation of herb extracts: The plant leaf were washed with water to remove dirt prior to the drying process. The leaf were then crushed into fine powder. Powdered dried leaf (500 g) of the plant were serially macerated in petroleum ether (60-80, 2500 mL), chloroform (2500 mL) and methanol (2500 mL) for 3 days each. The residue after methanolic extraction was macerated in water for 24 h to obtain water extract. Then, the leaf extract was filtered and concentrated under reduced pressure at 55°C in a rotary evaporator. The concentrated extract obtained was placed in the oven at 60°C for 3 days to remove the remaining solvent. The aqueous extract was placed in freeze drier instead of oven.

Fractionation of the active extract (methanol extract): Methanol extract (2 g) was suspended in distilled water (500 mL). Then, the suspension obtained was placed into a 1 L separator funnel. Firstly, the solution was extracted with chloroform (3×250 mL). Next, the aqueous layer was extracted with ethyl acetate and n-butanol (3×250 mL) to obtain three respective fractions. All fractions obtained were concentrated using the rotary evaporator. Chloroform fraction, ethyl acetate fraction and n-butanol fraction were kept in oven at 60°C to remove the remaining solvents. The aqueous fraction was placed in freeze drier instead of oven.

Subfractionation of the active fraction (chloroform fraction): Chloroform fraction was further extracted in hexane-chloroform mixture (1:3). The supernatant formed was collected, filtered and concentrated using rotary evaporator and dried in oven at 60°C to obtain subfraction 1 (SF 1). The residue was dried and then similarly washed with chloroform until no color was formed. Again, this supernatant was filtered, concentrated using rotary evaporator and freeze dried to obtain subfraction 2 (SF 2).

Standardization of active extract, active fraction and active subfraction using cinnamic aldehyde: Sample solution of methanol extract, chloroform fraction and subfraction 1 (1 mg mL^–1) was prepared by dissolving in ethanol. Sample (1 μL) was injected and the profile was acquired using the GC-MS. The peak identification and quantification was carried out using the retention time and mass spectrum provided by the MS library.

Limit of quantification and detection: The ICH Harmonised Tripartite Guideline procedure was employed for the determination of Limit of Quantification (LOQ) and Limit of Detection (LOD). Signal-to-Noise (S/N) method was used for the determination of LOD and LOQ of cinnamic aldehyde. The LOD is the minimum concentration of the analyte that can be differentiated from assay background. The LOD is calculated in S/N ratio of 3:1. The LOQ is the minimum concentration of the analyze that can be quantified and determined by peak-to-peak noise, in ratio of 10:1 (ICH Q2B, 1996)

Precision: The ICH Harmonised Tripartite Guideline procedure was used for the inter-day and intra-day precision determination. Inter-day and intra-day precision were determined by calculating the percentage of relative standard deviation (RSD %) (ICH Q2B, 1996). Percentage of RSD of calculated using the following formula:

where, M is mean of experimentally determined concentration and SD is standard deviation of M.

Determination of total phenolic content: The Total Phenolic Content (TPC) was determined by spectrophotometric method (Wolfe et al., 2003). Firstly, 1 mL of sample (1 mg mL^–1) was mixed with 1 mL of Folin-Ciocalteu’s phenol Reagent followed by addition of 10 mL of a 7% Na₂CO₃ solution. Next, 13 mL of deionized distilled water was added and the mixture was kept in the dark for 90 min at 23°C, Finally, the absorbance was determined at 750 nm using spectrophotometer. The Mean±SD results of triplicate analyses were expressed as milligram gallic acid equivalents per gram plant material.

Estimation of total flavonoid content: Total flavonoid content was determined according to a method by Sakanaka et al. (2005). Firstly, 0.3 mL of extracts (1 mg mL^–1), 0.15 mL of NaNO₂ (0.5 M) and 0.15 mL of AlCl₃.6H₂O (0.3 M) were mixed. Thereafter, 1 mL of NaOH (1 M) was added. Lastly, the solution was mixed well and the absorbance was measured against the reagent blank at 506 nm. The standard curve for total flavonoids was made using catechin standard solution and expressed as milligram catechin equivalents per gram of plant material. The data represent as the mean of triplicate analyses.

DPPH (2,2-diphenyl-2-picryl-hydrazyl) free radical scavenging method: The DPPH scavenging activity of C. iners standardized leaves extract was determined by the modified method of Brand-Williams et al. (1995) and Bursal and Gulcin (2011). Cinnamomum iners active portion (77 μL, 0.1-2.5 mg mL^–1) was added to DPPH (6×10-5 mol^–1) and vortex-mixed. Then, the mixture was placed in the dark for 15 min at room temperature. Thereafter, the absorbance was measured at 515 nm using spectrophotometer and DPPH scavenging activity was calculated using the following formula:

where, Ac is absorption of control (t = 0 min) and At is absorbance of tested extract or standard solution (t = 15 min). The DPPH solution without plant extract or standard served as control. Vitamin E was used as standard antioxidant.

Hydrogen peroxide (H₂O₂) scavenging activity: The H₂O₂ scavenging activity of C. iners leaves active portion was evaluated using the method of Ruch et al. (1989), Firstly, H₂O₂ solution (40 mM) was prepared in phosphate buffer (pH 7.4). Next, active portion of C. iners leaves or standard (0.1-4.8 mg mL^–1, 2.0 mL) was transferred into the test tubes containing H₂O₂ (1.2 mL). Finally, the absorbance of samples in the test tubes were determined at 230 nm using spectrophotometer. The abilities of samples to scavenge the hydrogen peroxide were calculated using the following equation:

where, Ac is absorption of control and At is absorbance of tested extract or standard solution. The H₂O₂ solution without plant extract or standard served as control. Vitamin C was used as standard antioxidant.

Reducing power assay: The reducing power was based on Fe (III) to Fe (II) transformation in the presence of the test samples were determined according to Oyaizu (1986). The formation of Fe (II) was monitored by measuring the formation of Perl’s Prussian blue at 700 nm using spectrophotometer. Briefly, different concentrations of the sample (0.05-2.5 mg mL^–1, 2 mL) were mixed with 2 mL of phosphate buffer (0.2 M, pH 6.6) and 2 mL of potassium ferricyanide (10 mg mL^–1). Next, the mixture was incubatedat 50°C for 20 min followed by addition of 2 mL of trichloroacetic acid (100 mg L^–1). Thereafter, the mixture was centrifuged at 3000 rpm for 10 min and the supernatant was collected. Then the supernatant was diluted with 2 mL of distilled water and 0.4 mL of 0.1% (w/v) fresh ferric chloride. Finally, the absorbance was measured after 10 min at 700 nm. Higher absorbance of the reaction mixture indicates a higher reducing power.

Statistical analysis: Data was analyzed by SPSS version 12.0.1 software program. Statistically significant difference between groups was calculated by the application of analysis of variance (ANOVA) followed by Dunnett’s test. A difference was considered significant at p<0.05. The IC₅₀ values were calculated from linear regression line.

RESULTS

Standardization of active extract, fraction and subfraction: Methanol extract, chloroform fraction and subfraction 1 of C. iners leaf was standardized using cinnamic aldehyde as the marker compound. The GC-MS profiles of methanol extract, chloroform fraction and subfraction 1 of C. iners leaf were compared with standard cinnamic aldehyde. The analysis show the presence of cinnamic aldehyde in all samples, but in varying proportions. Mass spectroscopy of cinnamic aldehyde identified in the samples showed major characteristic fragmentation pattern (m/z = 45, m/z = 59, m/z = 69, m/z = 77, m/z = 89, m/z = 103, m/z = 111, m/z = 119, m/z = 131, m/z = 152, m/z = 161 and m/z = 179 M+ = 204) exactly identical to the standard cinnamic aldehyde mass fragmentation pattern. The retention time of pure cinnamic aldehyde was 10.12 min whereas, the retention times of the samples were 10.2±0.2 min. The regression curve of cinnamic aldehyde is linear with y-intercept of 814 051 and r² value of 0.9921. The amount of cinnamic aldehyde in methanol extract, chloroform fraction and subfraction 1 is presented in Table 1.

LOD and LOQ: The LOD and LOQ of cinnamic aldehyde were 15.6 and 70.5 ppm, respectively.

Precision: The intra-day and inter-day precision for determination of cinnamic aldehyde was presented in Table 2. The RSD (%) value for intra-day were 0.0008, 0.0028, 0.0018 and 0.00097, respectively. The RSD (%) for inter-day precision were 0.006, 0.003, 0.002 and 0.00078, respectively.

Total phenolic and flavonoid content: Total phenolic and flavonoid content of C. iners active portion and cinnamic aldehyde was presented in Table 3. Results showed that increasing pattern of total phenolic and flavonoid content from methanol extract, chloroform fraction to subfraction 1.

DPPH and H₂O₂ scavenging activity: Cinnamomum iners active portion and cinnamic aldehyde was capable of scavenging hydrogen peroxide and DPPH which is shown in Table 4. Vitamin-C which is a natural antioxidants was used as positive control.

Reducing power assay: The reducing power of active portion of C. iners leaves and cinnamic aldehyde was monitored by its reduction capability of Fe³⁺ ferricyanide complex to a ferrous form and the result is shown in Fig. 1. Cinnamomum iners active portion and cinnamic aldehyde showed statistically significant difference compared to vitamin E at the highest concentration (2.5 mg mL^–1).

DISCUSSION

Medicinal plants have been used throughout human history to cure various diseases. However, owing to the fact that there is lack of both qualitative and quantitative standards of herbal medications, standardization serves as a technique for authentication of herbs to assure the quality control of these materials (WHO., 1991). Hence, C. iners leaf active portion had been standardized using cinnamic aldehyde, which acts as its bio active compound.

Table 1:	Amount of cinnamic aldehyde in methanol extract, chloroform fraction and subfraction 1 of Cinnamomum iners leaf

Table 2:	Precision and recovery for determination of cinnamic aldehyde

Fig. 1:	Reducing power of Cinnamomum iners active portion, cinnamic aldehyde and vitamin E at various concentrations, values are the Means±SD of three replicates, *Significant difference with vitamin E at p<0.05, data was analyzed using one way ANOVA followed by post hoc Dunnett’s test

Table 3:	Total phenolic and flavonoid content of Cinnamomum iners active portion and cinnamic aldehyde
Values are expressed as Mean±SD, GAE: Gallic acid equivalent, CE: Catechin equivalent

Table 4:	IC50 values of Cinnamomum iners active portion and cinnamic aldehyde
Values are expressed as Mean±SD, DPPH: 2,2-diphenyl-2-picryl-hydrazyl, H2O2: Hydrogen peroxide

Antioxidant screening of C. iners active portion was evaluated using DPPH scavenging method, H₂O₂ decomposition and reducing power assay. The DPPH assay is a rapid and sensitive method which has been widely used for the screening of scavenging activity of antioxidant. The ability to scavenge DPPH free radical indicate hydrogen donating capability of plant extracts (Baumann et al., 1979). The H₂O₂ scavenging activity is closely related to the ability of plant extract in accelerating the conversion of H₂O₂ to H₂O to prevent accumulation of H₂O₂ in body. Accumulation of H₂O₂ in body may cause formation of harmful hydroxyl radicals that lead to oxidative DNA damage (Miller et al., 1993). Table 4 represent the IC₅₀ value for DPPH and H₂O₂ scavenging activity of C. iners active portion along with its bio active compound, cinnamic aldehyde. The present results indicate that DPPH and H₂O₂ scavenging activity of C. iners leaves increase from methanol extract, chloroform fraction to subfraction 1 (Table 4). This might be due to increasing amount of cinnamic aldehyde which is well known for its antioxidant activity (Wondrak et al., 2010). The IC₅₀ value of methanol extract in scavenging DPPH is in agreement with previous studies by Mustaffa et al. (2010b). The DPPH scavenging activity of subfraction 1 (0.21 mg mL^–1) is as good as other plant with good antioxidant activity (Centella asiatica, 0.2 mg mL^–1, Pisonia alba, 0.18 mg mL^–1, Orthosiphon stamineus, 0.21 mg mL^–1, Mentha arvensis, 0.22 mg mL^–1; Ocimum basilicum, 0.19 mg mL^–1) (Subhasree et al., 2009; Zakaria et al., 2008). The IC₅₀ value of methanol extract to scavenge H₂O₂ is in agreement with previous study of antioxidant activity of C. iners leaves (Mustaffa et al., 2010b).

The reducing power a compound is related on its ability in donating electrons (Zou et al., 2004). The reducing power of C. iners active portion is shown in Fig. 1. As illustrated in Fig. 1, the reducing power of C. iners active portion and cinnamic aldehyde increase in concentration dependent manner. At the highest concentration, (2.5 mg mL^–1), chloroform fraction and subfraction 1 of C. iners leaves showed significantly higher reducing power than standard antioxidant (Vitamin E). Furthermore, the reducing power of chloroform fraction and subfraction 1 was found to be as good as Smilax excelsa leaves extract (5.0 mg mL^–1, Absorbance, 1.6) which is used as natural antioxidant source in Turkey (Ozsoy et al., 2008). This goes to say that C. iners leaves has a good electron donating capability to scavenge free radicals. Flavonoids and phenolic compounds might contribute to the remarkable reducing activity of C. iners leaves as phenolic hydroxyl groups present in them act as a reducing agent by donating electron (Shahidi et al., 1992).

CONCLUSION

The present study clearly showed that C. iners leaves showed promising antioxidant activity and served as potential natural antioxidant source to prevent formation of free radicals.

ACKNOWLEDGMENTS

Thanks are expressed to En. Rosli from School of Pharmaceutical Sciences for his expert technical assistance. We are grateful to Ministry of Education Malaysia and USM for providing fellowships and grant (RU Grant a/c 1001/PFARMASI/815080).