Antioxidant Activity of Syzygium oleana

MATERIALS AND METHODS

Raw material: Young leaves and fruits of S. oleana.

Chemicals: Methanol, diphenylpicrylhydrazyl (DPPH), distilled water, gallic acid, Na₂CO_3, Folin-Ciocalteu reagent, HCl, CH₃CO₂Na·3H₂O.

DPPH radical scavenging activity: The DPPH radical scavenging activity was monitored using the method originally developed by Blois⁷. A portion (0.1 mL) of the methanol extract solution (1.0 mg mL^–1) in a test tube was mixed well with 3.9 mL of methanol and 1.0 mL of a DPPH solution (1.0 mM in methanol). The mixture was kept at ambient temperature for 30 min prior to measurement of the absorbance at 517 nm (A517 nm). All measurements were done in triplicate.

Total polyphenol: The total phenolic content of the extract was measured by the Folin-Ciocalteu method⁸, using gallic acid as a standard. A 0.1 mL sample of the extract solution was added to 2 mL of 2% Na₂CO₃ and mixed for 3 min. After adding 0.1 mL of 50% Folin-Ciocalteu reagent, the final mixture was left for 30 min before measuring the absorbance at 750 nm. All measurements were conducted in triplicate and the data were expressed as Gallic Acid Equivalent (GAE) per kg extract based on the calibration curve of gallic acid.

Anthocyanin content⁹: About 1.86 g of KCl was added to a beaker and approximately 980 mL of distilled water was added to it. Next, the pH was measured and adjusted it to (1.0±0.05) with HCl (approximately 6.3 mL). The mixture was transferred to 1 L volumetric flask and diluted to volume with distilled water. Furthermore, 54.43 g of CH₃CO₂Na·3H₂O was weighed in a beaker and added distilled water to approximately 960 mL. Next, the pH was measured and adjusted it to (4.5±0.05) with HCl (approximately 20 mL). Then, the mixture was transferred to a 1 L volumetric flask and diluted to volume with distilled water.

Next, anthocyanin pigment concentration was calculated, expressed as cyanidin-3-glucoside equivalents, as follows:

where, A = (A520-A700 nm) pH 1.0-(A520-A700 nm) pH 4.5.

where, Molecular Weight (MW) = 449.2 g mol^–1 for cyanidin-3-glucoside (cyd-3-glu), DF is dilution factor established in D, l is path length in cm, ε is 26,900 molar extinction coefficient, in L and mol^–1 and cm^–1, for cyd-3-glu and 103 is factor for conversion from g to mg.

Statistical analysis: Standard Deviation (SD) was calculated using Excel 2003 for the antioxidant, total polyphenols and anthocyanin results.

RESULTS AND DISCUSSION

Antioxidant activity: The DPPH was used to measure the antioxidant activities of S. oleana fruit and leaf extracts, which have a deep purple color. The DPPH disappears upon reduction by an antioxidant compound, yielding a yellow colored solution. This could be taken as an indication of the hydrogen-donating ability of the samples¹⁰. The use of DPPH can estimate the antioxidant activity in a short time and the incubation time for DPPH solution and sample is 30 min. Thirty minutes of incubation is enough time for polyphenols and other antioxidants to react optimally with DPPH. Some compounds react very fast (2 min), but some antioxidants need more time (30-60 min). The change in the purple color shows the antioxidant property in plant, the purple color becomes less intense or even disappears because of the ability of antioxidants to scavenge the radical solvent. The antioxidant activities of the water, methanol and ethanol extracts in the S. oleana fruit are shown in Figs. 1 and 2. Antioxidant activities of the water, methanol and ethanol extracts in the S. oleana leaves are shown in Fig. 3.

The antioxidant activities of S. oleana fruit extracts ranged from 3-63% with water, methanol or ethanol. For concentrations of 1000 and 10,000 ppm, the water extract of S. oleana had almost no antioxidant property. The extract of S. oleana showed higher antioxidant activity for the ethanol extract than the methanol extract. Ethanol can better dissolve the antioxidants in S. oleana fruit than methanol and water. Ethanol is the best solvent to extract the antioxidants in the red leaves and fruit of S. oleana and ethanol is less toxic than methanol. The use of methanol is also dangerous for the environment and ethanol is more safe for the environment and for health. Water is a very safe solvent, but it cannot dissolve antioxidant or polyphenols in this plant well. Anthocyanin is water soluble but best dissolves in organic solvents such as ethanol and methanol. When using ethanol, before using the food colorant, the solvent should be evaporated in rotary evaporator. The deep purple color of the fruit of S. oleana could be due to the presence of anthocyanin. However, Fig. 5 shows that the anthocyanin content in S. oleana fruit was not influenced by the solvents used for extraction, meaning that the anthocyanin content in S. oleana fruit was not the only polyphenol responsible for the antioxidant activity. There might exist was another compound in S. oleana fruit that is a potent antioxidant besides anthocyanin.

Fig. 1:	Antioxidant Activities of water, methanol and ethanol extract of the S. oleana fruit

Fig. 2:	Antioxidant Activities of S. oleana leaf water, methanol and ethanol extracts

Fig. 3:	Total Polyphenol contents of S. oleana fruit water, methanol and ethanol extracts

The antioxidant activities of S. oleana leaf water, methanol and ethanol extracts showed higher antioxidant activity than the fruit extract. The S. oleana leaf water extract showed the lowest antioxidant activity compared with the methanol and ethanol extracts, as was seen with antioxidant activity in the fruit extracts. In contrast to the S. oleana fruit, the leaves were potent as an antioxidant, as seen from the extract. The color of the leaves is red and the color of fruit extract is deep purple. The different colors mean there is a different anthocyanin content in the leaves and fruit. There is a need for investigation about the kinds of anthocyanin in this plant, because they have different capacities as antioxidants. From the antioxidant activity presented in Fig. 2, it is seen that S. oleana leaves have more antioxidant properties than the fruit extract. This study used the young, red colored leaves of S. oleana. The red color could be due to the presence of anthocyanin or another compound that gives a red color.

In candies made of fruit extract enriched with grape skin powder, the anthocyanin, flavonol and procyanidin contents increased and the candies remained stable during processing¹¹. Grape skin powder is reported to be an important source of phenolic compounds, which act as natural antioxidant sources. Phenolic compounds are responsible for scavenging free radicals. Their correlation with antioxidant agents has been studied in fruits or vegetables¹².

Based on Fig. 3, it is evident that ethanol is the best solvent to dissolve the polyphenols in the fruit of S. oleana compared to methanol and water. The total polyphenol content in S. oleana leaves was not influenced by the solvents used for extraction, where the total polyphenols obtained with the use of water, methanol and ethanol did not vary much. This could be due to the presence of a larger portion of major polyphenol compounds in the leaves than in fruit. Fruits have more varied polyphenols that are dissolved well in ethanol. It is easier to separate them using ethanol and the rotary vacuum evaporator. This way the anthocyanin pigments in plants could be used for food coloring. Polymeric color is a widely used index for measuring anthocyanin degradation in aqueous extracts and juice¹³.

Figure 4 shows that the highest total polyphenol content was in the ethanol extract. A previous study showed that the ethanol extract exhibited the highest antioxidant and phenolic content in the Kenitu fruit¹⁴. Other results regarding total polyphenols in leaves and fruit showed that, the polyphenol in fruit is easy to dissolve with all three kinds of solvent. However, the polyphenol in fruit has slight activity as an antioxidant. Antioxidant activity is very important to human health and is important for food product if we use them as colorants. For food products with high fat, this will help to make the shelf life longer because of the function of polyphenol as antioxidant.

Fig. 4:	Total polyphenol contents of S. oleana leaf water, methanol and ethanol extracts

Fig. 5:	Anthocyanin contents of S. oleana fruit water, methanol and ethanol extracts

The use of S. oleana red leaves will increase the value for food products because of the unique color produced by it. The color will make the food eye catching and will function as an antioxidant that will reduce oxidative stress in humans.

Figure 5 shows that the anthocyanin content in S. oleana fruit was not influenced by the solvent used for extraction, but the total polyphenol content in Fig. 3 shows that ethanol is the best solvent to extract the S. oleana fruit. It can be concluded that there were other polyphenol compounds besides anthocyanin presence in the S. oleana fruit that dissolved well in ethanol. Based on above figure, anthocyanin dissolves well in water, methanol and ethanol, but compared with fruit, the red leaves have small amounts of anthocyanin.

Figure 6 shows that the anthocyanin content in S. oleana leaves dissolved in ethanol>methanol>water: 30.57, 17.27 and 13.49 mg mL^–1 respectively. Based on Fig. 4, the polyphenols present in S. oleana leaves is the same in the various extractions but Fig. 6 shows that the anthocyanin content in S. oleana leaves varies depending on the solvent.

Fig. 6:	Anthocyanin contents of S. oleana leaf water, methanol and ethanol extracts

Fig. 7:	Basic structure of anthocyanin

It showed that anthocyanin is not the major compound in S. oleana leaves. It is suggested to investigate the polyphenols present in S. oleana leaves. Further investigation about the antioxidant properties both in leaves and fruit of S. oleana is necessary because Figs. 6 and 5 show that fruit with high anthocyanin content do not have high levels of antioxidants. There are seven kinds of anthocyanins: delphinidin (C₁₅H₁₁O₇), cyanidin (C₁₅H₁₁O₆), pelargonidin (C₁₅H₁₁O₅), malvidin (C₁₇H₁₅O₇), petudinin (C₁₆H₁₃O₇), petunidin (C₁₆H₁₃O₆) and fragarin (C₂₁H₂₁O₁₀).

Anthocyanin in colored rice (red and black rice) in West Sumatra and its application in a food product called ‘Kembang Loyang’ a traditional food that is made from rice, sugar and coconut milk and is deep fried, still has antioxidant activity and polyphenols^15,16. Thus, the application of the S. oleana fruit will be a very good resource of food colorant and antioxidant in foods, such as bakery products, ice cream, bread, or fried food, because anthocyanin is quite stable at high temperatures, although the color of anthocyanin will change based on the pH. The basic structure of anthocyanin can be seen in Fig. 7.

By using the Brine Shrimp Test, it was observed that the extract of young leaves and fruit of S. oleana is not toxic (data not shown).

Fig. 8:	Plant of Syzygium oleana

Fig. 9:	Fruit of Syzygium oleana

Thus, the fruit of S. oleana which has not been consumed before, could be consumed freshly like common fruits including blueberries or blackberries. Additionally, they can be extracted optimally by using ethanol as a solvent, which is also environmentally safe. Natural, safe and functional foods provide a preventive, protective function against disease, in addition to adequate nutritional benefits¹⁷. The plant and fruit of S. oleana can be seen in Fig. 8 and Fig. 9.

SIGNIFICANCE STATEMENTS

This study discovered the antioxidant activity of S. oleana young leaves and fruit. The results showed that this plant could be a promising source of natural colorant that is safe and whose contents provide functional benefits for human health. The use of the pigment can be applied to some food products or the beverage industry. This study will help the researcher to find new information and a new source about the benefits of natural pigments from S. oleana young leaves and fruits.

CONCLUSION

Syzygium oleana fruit and leaves contain antioxidants, total polyphenols and anthocyanins and can be applied to food products (nontoxic). The best solvent extraction process used is ethanol. The results showed that the antioxidant content is activated up to 3.87, 26.4 and 65.65% with water, methanol and ethanol as a solvent, respectively. The total polyphenol content in water, methanol and ethanol are 55.37, 78.87 and 122.10 mg mL^–1, respectively. Anthocyanin content measured in water, methanol and ethanol are 16.42, 19.20 and 15.16%, respectively. Other polyphenol compounds in S. oleana should be investigated further.