Antioxidant properties of crude extract, partition extract, and fermented medium from
Normal cell metabolism continuously produces dangerous by-product substances known as free radicals. Free radicals are groups of atoms with unpaired electrons and can be formed when oxygen interacts with certain molecules. These organic molecules are highly reactive radicals and can start a chain reaction once they formed in the body just like the dominoes effect. Due to the unstable electron, the radicals tend to bond with other molecules to capture the electron in order to become stable, thus destroying healthy tissue and further continuing the damaging process. The antioxidant assays chosen for this study are 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric ion reducing antioxidant power (FRAP) assays which were used to measure the relative antioxidant ability to scavenge the free radicals produced in the reagents. These three assays are based on electron transfer (ET) reaction principle, where the color change will act as an indicator to the capacity of antioxidant in reducing the radicals [
Emotional and physical stress, poor diets, and even pollution can all contribute to free radical formation in our system. Natural antioxidants produced within the cells are acting energetically to neutralize the harmful effect of free radicals. However, the additional antioxidants substances are still required by the body to fight the worst consequence of free radicals [
Fermentation is a conventional method to preserve something using microbial cultures. It has been found that fermented soybean paste in Korea has better total phenolic contents (TPC) and comprises stronger antioxidant capacity as compared to unfermented soybean because the fermentation is believed to aid in producing more aglycone isoflavone and malonylglycoside isoflavone [
The data were analyzed using SPSS 19.0 and Tukey’s multiple comparison test was used. Significant differences among the treatments and dosages were determined by
The extraction yield and total phenolic content of DSF methanolic crude extract (DSF MCE), DSF ethanolic crude extract (DSF ECE), and DSF water crude extract (DSF WCE) are presented in Table
The extraction yield, TPC, and antioxidant capacity of crude extract.
Crude extract | Water |
Methanol |
Ethanol |
---|---|---|---|
Yield extract (%) | 45.8 | 18.7 | 6.9 |
TPC (mg GAE/g extract) | 31.64 ± 0.17 |
|
|
The TPC results are presented as mean ± SD. Post hoc (Tukey) test shows significant difference in TPC between DSF WCE and DSF ECE at
The antioxidant assays (DPPH, ABTS, and FRAP) measure the relative antioxidant ability enclosed in DSF to scavenge the free radicals produced in the reagents. Figure
DPPH, ABTS, and FRAP scavenging activity of
Since DSF MCE showed the best performance in antioxidant capacity, it was further tested for partition extraction. 100% methanol was compared to acidified methanol in which both were used for extracting 40 g of grounded DSF, respectively. Table
The extraction yield, TPC, and antioxidant capacities of partitioned extract.
Crude extract using methanol | 100% methanol (C 100) | Acidified methanol (C A) | ||||
---|---|---|---|---|---|---|
Yield extract (%) | 18.9 | 22.5 | ||||
TPC (mg GAE/g extract) | 26.82 ± 5.02 | 27.43 ± 0.38 | ||||
|
||||||
Further partition method | EA 100 | W 100 | H 100 | EA A | W A | H A |
|
||||||
Yield extract (%) | 1.2 | 13.4 | 1.2 | 1.5 | 15.3 | 1.3 |
TPC (mg GAE/g extract) |
|
21.30 ± 1.80 | 17.46 ± 0.62 |
|
18.66 ± 1.10 | 22.74 ± 1.30 |
The TPC results are presented as mean ± SD. Post hoc (Tukey) test shows no significant difference between TPC of 100% methanol (C 100) and acidified methanol (C A) crude extracts but shows significant difference in TPC between ethyl acetate extract (EA 100; EA A) and water layer extract (W 100; W A) and hexane extract (H 100; H A) at
The antioxidant properties of partition extract are presented in Figure
Antioxidant capacity assays (DPPH, ABTS, and FRAP) between partition extracts. The DPPH, ABTS, and FRAP results are presented as mean ± SD. Post hoc (Tukey’s) test shows significant difference in all DPPH, ABTS, and FRAP assays between ethyl acetate extract (EA 100; EA A) with water layer extract (W 100; W A) and hexane extract (H 100; H A) at
There was some bacterial growth in the medium based on the pH changes of the collected medium and also the optical density result as presented in Figure
pH changes and optical density (OD) of the fermented medium over the 84-hour period.
Figures
TPC of fermented medium from
DPPH scavenging activity of fermented medium from
The extraction yield showed differences which suggest that different solvent used for extraction would extract different compounds depending on the polarity of the solvent, the chemical nature of extracted phenolic compound, the extraction method, plant matrix, and the presence of interfering substances [
The TPC of crude extract result shows that the DSF MCE has the significantly highest total phenolic content followed by DSF ECE and DSF WCE. The TPC result suggested that the polarity of the solvent has an effect on extracting different phenolic compounds from the DS flower and therefore would have different total phenolic content. Methanol extracted extra phenolic compound as compared to ethanol and water. Methanol and ethanol have similar polarity but gave distinctive result, where ethanol was less efficient than methanol. This was because ethanol has low solvation of antioxidant compound which was caused by the presence of longer ethyl radical than the methyl radical in methanol [
The TPC of partitioned crude extract showed that EA 100 and EA A both had very significantly high total phenolic contents as compared to water (W 100 and W A) and hexane (H 100 and H A). The solvent-solvent partition was done on methanolic crude extract using different polarity solvent from polar (water), semipolar (ethyl acetate), and nonpolar (hexane) solvents in order to acquire the faction of difference classes of phenolic compound. The water layer could extract polar compound (anthocyanins) with some flavonoids, while the ethyl acetate could extract phenolic compounds but not anthocyanin. A partition study done on
The antioxidant assays (DPPH, ABTS, and FRAP) measured the relative antioxidant ability contained in DS flower to scavenge the free radicals produced in the reagents. All crude extracts were reconstituted in their corresponding solvent prior to the experiment but all the blank, reagents, and controls (Trolox and gallic acid) were dissolved in methanol. TPC and all antioxidant assays (DPPH, ABTS, and FRAP) showed a strong correlation between total phenolic content and the antioxidant capacity as shown in Figure
Correlation between TPC and antioxidant capacity (DPPH, FRAP, and ABTS).
It can be concluded that there is some bacteria growth in the medium based on the optical density result which shows an increasing trend. The highest value of OD at
The TPC got increased which indicated that the fermentation of the DS flower medium has positive effect in multiplying the phenolic content, where the TPC has a significant increase starting at
The DPPH result showed sudden increase at
In summary,
The authors declare that they have no conflicts of interest.
The authors would like to thank all staff of Laboratory of Anatomy and Histology for all advices, help, and guidance throughout this study. This work was supported by Research University Grant Scheme (RUGS) Initiative 4, Universiti Putra Malaysia (UPM), with Project Code of 04-01-11-1162RU.