Antioxidant and antibacterial activities of hibiscus (Hibiscus rosa-sinensis L.) and Cassia (Senna bicapsularis L.) flower extracts

2.2.1 Extract preparation

Distilled water and ethanol (99.7%) were used as solvents for extraction of antioxidant compounds. A known weight of the powdered sample was mixed individually with the solvent (500 mg extracted in 50 mL, as 3 individual replicates) and was shaken in an orbital shaker (Lab Companion, Model SI 600R Bench top shaker) at 160 rpm for 24 h at room temperature. Extracts were filtered through Whatman filter paper (Whatman No. 41, UK). All the filtrates were collected and transferred to screw-top glass bottles (with Teflon caps) and were covered with aluminum foil to avoid exposure to light.

2.2.2 DPPH radical scavenging activity and ferric reducing antioxidant power assay (FRAP)

The capacity of the flower extracts to scavenge DPPH radicals (2,2-diphenyl-1-picrylhydrazyl) was measured based on the method described by Sanchez-Moreno et al. (1998). The results obtained were expressed as the percentage inhibition of DPPH based on the following formula: $$\text{Percent inhibition of DPPH}=[({\text{A}}_{\text{control}}-{\text{A}}_{\text{sample}})/{\text{A}}_{\text{control}}]\times 100$$ where A_control is the absorbance of the DPPH solution without sample extract and A_sample is the absorbance of the sample with DPPH solution.

For FRAP assay, a modified method described by Benzie and Strain (1996) was adapted to measure the ability of extracts to reduce ferric ions. Ferrous sulfate solution (concentration ranging from 0.1 to 1 μM) was used for preparing the standard calibration curve. FRAP activity was expressed as micromoles of Fe (II)/100 g of dry weight of samples.

2.2.3 Determination of total phenolic content, tannins, flavonoids and flavonols

The total phenolic content of the flower extracts were determined based on the Folin–Ciocalteu (FC) method (Singleton and Rossi, 1965). In brief, 400 μL of the sample extract was mixed with 2.0 mL of FC reagent (10 times pre-diluted). Further, after incubation for 5 min at room temperature, 1.6 mL of (7.5%, w/v) sodium carbonate solution was added and the solution was mixed thoroughly and incubated for 60 min at room temperature. Followed by this, absorbance was measured using a UV–visible spectrophotometer (Shimadzu UV-160A, Kyoto, Japan) at 765 nm. A suitable calibration curve was prepared using standard gallic acid solution. All the results were expressed as mg Gallic acid equivalents (GAE) per gram of sample.

For total tannins, the vanillin–HCl method was employed (Bhat et al., 2007). Briefly, 1 mL of the sample extracts was treated with 5 mL of reagent mixture (4% vanillin in methanol and 8% concentrated HCl in methanol, 1:1 ratio). The color developed was read after 20 min. at 500 nm using a UV–visible spectrophotometer (Shimadzu UV-160A). Suitable standard calibration curve was prepared using catechin (20–400 μg/mL) and results were expressed as mg Catechin equivalent (CE) per 100 g dry weight of the samples, respectively.

Total flavonoids in the sample extracts were determined using the aluminum chloride method as described in the report of Liu et al. (2008). In brief, for 500 mL of the sample extract solution, 2.5 mL of distilled water and sodium nitrite solution (5%, w/v, 150 mL) were added to the mixture. This mixture was maintained for 5 min., followed by addition of 300 mL of aluminum chloride (10%, w/v) and again incubated for 6 min. Followed by this, 1 mL of sodium hydroxide (1 M) was added and the mixture was diluted with 550 mL of distilled water. This solution was mixed vigorously and the absorbance of the mixture was measured immediately at 510 nm using a UV–visible spectrophotometer (Shimadzu UV-160A, Shimadzu Corporation, Kyoto, Japan). Results of the total flavonoid content were expressed as mg Catechin equivalents (CE) per 100 g of dry weight of the sample.

Total flavonols in the sample extracts were evaluated based on the method described by Miliauskas et al. (2004) with slight modifications. Briefly, 1 mL of 0.15–0.05 mg/mL quercetin methanol solution with 1 mL of 2% aluminum trichloride and 3 mL of 5% sodium acetate were mixed to obtain a quercetin calibration curve. After 150 min and incubation at 20 °C, the absorption was read at 440 nm using a UV–visible spectrophotometer (Shimadzu UV-160A). This procedure was repeated using 1 mL of the sample extract (1 mg/mL) instead of quercetin solution. Results obtained were expressed as mg Quercetin equivalent (QE) per 100 g dry weight of samples.

To determine total anthocyanins, the spectrophotometric method detailed by Abdel-Aal and Hucl (1999) was employed. Briefly, anthocyanins were extracted using acidified methanol (methanol and 1 M HCl, 85:15, v/v) with a solvent to sample extract ratio of 10:1. This was centrifuged and the absorbance was measured at 525 nm using a UV–visible spectrophotometer (UV-160A, Shimadzu, Japan) against a reagent blank. Cyanidin-3-glucoside (5, 10, 15, 20, and 25 mg/L, r² = 0.9982) was used to prepare for the standard calibration curve. Total anthocyanin contents in the flower extracts were expressed as mg cyanidin-3-glucoside equivalents (c-3-gE) per 100 g dry weight of samples.

2.3.1 Microorganisms and growth conditions

A total of eight food-borne pathogenic bacteria (obtained from the culture collection of the Food Microbiology Laboratory, School of Industrial Technology, Universiti Sains Malaysia) was used for evaluating the antibacterial activities of the flower extracts. Four Gram-positive (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes) and four Gram-negative (Escherichia coli, Salmonella typhimurium, Salmonella enteritidis, Klebsiella pneumoniae) bacteria were used to determine antibacterial properties of flower extracts. Pathogens obtained from respective glycerol stock cultures were inoculated (1%, v/v) into Brain Heart Infusion (BHI) media followed by incubation at 37 °C (for 18 h) to activate cultures. All the tested pathogens were standardized to a concentration of 10⁸ CFU/mL for antibacterial susceptibility test.

2.3.2 Extract preparation for microbial analysis

Extraction using either distilled water or ethanol (99.7%) was performed following the modified method described by Alade and Irobi (1993). Briefly, 1.0 g of each powdered sample was soaked individually in 100 mL of distilled water or ethanol and mixed thoroughly using a magnetic stirrer at room temperature for 3 h. After filtration, the residues were again soaked in 100 mL of the solvent for re-extraction for 12 h. The filtrates were pooled together followed by filtering using Whatman filter paper (Whatman No. 41). The alcoholic filtrates obtained were concentrated using a rotary vacuum evaporator (Rotary evaporator IKA, Model RV06-M1-1-B, IKA-WERKE, Germany) at 50 °C under room atmospheric pressure (approximately 100 kPa), while aqueous filtrates were evaporated under pressure of 43 kPa at 40 °C. A stock solution of 100 mg/mL was prepared by dissolving the semi-solid materials of each crude extract in the solvent used in extraction and stored at −20 °C until further use in the disk diffusion assay.

2.3.3 Culture preparation

Suspension of tested pathogens (10⁸ CFU/mL) was spread on BHI agar (using sterile-glass hockey stick) to ensure even distribution of the suspension on the agar plate. The agar plates were left to be fully diffused after each application. Based on the modified agar disk diffusion method (Washington, 1981), inhibitory potential of microbial growth was determined. Sterile blank paper disks (Oxoid, England, 6 mm diameter) were impregnated with 50 μl of the extracts (100 mg/mL and 50 mg/mL), left to be dried and gently pressed on the inoculated agar plates. Distilled water and ethanol served as the negative control while chloramphenicol (Oxoid, England, 10 μg/disk) served as positive control. Agar plates were incubated at 37 °C for 24 h. Antibacterial activity was determined by measuring the diameter of the clear zones of inhibition.

3.1.1 Percent inhibition of DPPH radical and FRAP assay

Determining antioxidant activity of a sample extract based on the overall scavenging effects of DPPH radical is one of the routinely employed antioxidant assays. This method is considered to be rapid, simple and most convenient. The method is widely accepted as it is independent of sample extracts’ polarity and is effective while screening large volume of samples (Magalhaes et al., 2008; Alam et al., 2012). In the present study, both ethanolic and aqueous extracts of hibiscus and Cassia flowers exhibited rich scavenging effects on DPPH. Overall comparison showed ethanol extracts of Cassia to exhibit stronger scavenging effects on DPPH radicals, while ethanolic extracts of hibiscus had the lowest. Higher radical scavenging activity might be attributed to the presence of high phenolics, tannins or flavonols in the sample extracts.

FRAP assay like the DPPH assay, is considered to be rapid and sensitive and is more of a semi-quantitative assay. In FRAP assay, antioxidant capacity is evaluated based on the capability of the sample extracts to reduce ferric tripyridyltriazine (Fe (III)-TPTZ) complexes to ferrous tripyridyltriazine (Fe (II)-TPTZ). This assay is performed using freshly prepared FRAP reagent consisting of 2,4,6-tris (1-pyridyl)-5-triazine (pH 3.6). A blue product (ferrous–TPTZ complex) is formed due to the reduction of ferric iron in FRAP reagent (Benzie and Strain, 1996). The higher the FRAP value, the greater will be the antioxidant activity of sample extracts. Results obtained revealed hibiscus to exhibit higher reducing power in aqueous extracts compared to ethanolic extract, while in Cassia it was vice versa. For ethanol and aqueous extracts of hibiscus, the reducing powers were 2349.06 and 2883.23 μmoles Fe (II) per 100 g of samples, respectively. In Cassia, reducing power of ethanolic and aqueous extracts were 2403.15 and 1966.30 μmoles Fe (II)/100 g, respectively.

These results are on par with earlier reports on various flower extracts, which have been shown to exhibit high antiradical activities (Özkan et al., 2004; Wijekoon et al., 2011).

3.1.2 Total phenolic content, tannins, flavonoids, favonols and anthocyanins

Plant based phenol compounds exhibit rich antioxidant activity by scavenging the free radicals generated during the normal metabolism process. This group encompasses a wide diversity of compounds, which mainly includes: flavonoids and pro-anthocyanidins (condensed tannins) (Shahidi and Naczk, 2004). In the present study, the amount of total phenolics significantly varied between the two flower extracts and was dependent on the solvents used for extraction. Total phenols ranged between 4598.16 to 26223.78 mg GAE/100 g (see Table 1). Both ethanolic and aqueous extracts of Cassia and hibiscus had high phenolic content with ethanolic extracts of Cassia exhibiting the highest.

In both the flower extracts, amount of tannins differed significantly and ranged between 103.28 to 4420.87 mg CE/100 g (see Table 1). The aqueous extract of hibiscus showed higher tannins, while aqueous extract of Cassia had the lowest. Tannins in hibiscus flowers extracted in ethanol and water were higher compared to Cassia. Presence of tannins (high molecular weight phenols) in adequate amounts can be advantageous as they are able to quench free radicals very effectively, which in turn depended on the number of aromatic rings, molecular weight, and nature of the hydroxyl group substitution (Cai et al., 2006).

With regard to total flavonoid or bio-flavonoid content, hibiscus showed high content in aqueous extracts (2768.06 mg CE/100 g) compared to ethanolic extracts (2155.39 mg CE/100 g). In Cassia, total flavonoids were higher in ethanolic extract (3839.91 mg CE/100 g) compared to aqueous extracts (1133.60 mg CE/100 g). Flavonoids possess rich antioxidant properties and are produced as natural secondary metabolites in plants that encompass 6 sub-classes such as: isoflavones, flavonols, flavones and anthocyanins which vary in their structural characteristics. These flavonoids are capable of effectively interact and scavenge free radicals, which damage cell membranes and biological molecules (Rice-Evans and Miller, 1997).

Total flavonols, which are the most widespread sub-class of flavonoids in plant-based food-stuffs significantly varied between the flowers and their extracts. High flavonol content was recorded in ethanolic extracts of Cassia (1293.58 mg QE/100 g), while its aqueous extract had the lowest (22.19 mg QE/100 g). Similar trend was observed in hibiscus, indicating ethanol to be more suitable for extracting flavonols compared to distilled water.

With regard to total anthocyanin content, between the two flower extracts, hibiscus exhibited a higher value in both extracted solvents compared with Cassia. In hibiscus, high total anthocyanin were recorded in aqueous extract (205.76 mg c-3-gE/100 g) compared to ethanolic extracts (155.28 mg c-3-gE/100 g). Whereas, in Cassia, ethanolic extracts had higher total anthocyanin than aqueous extracts (77.11 and 49.83 mg c-3-gE/100 g, respectively). Of late, anthocyanins are becoming increasingly important not only due to their antioxidant properties, but also because of their antibacterial properties and use as a natural food colorant (Naz et al., 2007).

Presence of high level of total phenols, flavonols, flavonoids, and anthocyanins has been reported in different flowers and their extracts (Cai et al., 2004; Gouveia et al., 2013; Yang et al., 2012; Wijekoon et al., 2011), thus supporting the results and observations done in this study. Additionally, studies have reported a positive correlation to occur between antioxidant compounds and antioxidant activities in plant parts (e.g. flowers, fruits, leaves, seeds) and their extracts (Shyu et al., 2009; Wijekoon et al., 2011; Voon et al., 2012). Earlier, it has been reported that different solvent extraction systems can contribute significantly to differences in the antioxidant activities of the extracts (Tian et al., 2009; Wijekoon et al., 2011), which holds true in the present study also. Overall, our results clearly illustrated that phenols including flavonoids, tannins, flavonol and anthocyanins are most probably the major contributor to the observed antioxidant properties in both hibiscus and Cassia flower extracts.