2.1. Antioxidant Activities of Crude Extract and Fractions
The DPPH radical assay is a suitable model for estimating total antioxidant potential of antioxidants [
12].
Figure 1A shows the dose-response curves of the DPPH radical scavenging activities of crude extract and fractions from
R. apiculata. All samples have antioxidant activity against DPPH and the reducing power increased as the sample concentration increased from 2.09 to 33.34 µg/mL. The butanol fraction (BF) exhibits the highest scavenging activity of 89.3% at the concentration of 33.34 µg/mL, whereas the ethanol extract (EE), ethyl ester fraction (EF) and water fraction (WF) show 77.9%, 79.9% and 67.21% at the same concentration, respectively. IC
50 values, defined as the concentration with 50% activity, were calculated for comparison. The IC
50 values of scavenging activities for EE, EF, BF, WF and positive control BHT were 13.56 ± 1.79, 19.31 ± 1.56, 9.68 ± 1.86, 23.72 ± 1.94 and 52.20 ± 1.57 μg/mL, respectively. According to these IC
50 values, the DPPH radical scavenging ability was found in the order of BF > EF > EE > WF > BHT (
P < 0.05).
ABTS is another widely used synthetic radical for both the polar and non-polar samples [
13]. The ABTS
•+ scavenging abilities of the crude extract and fractions of
R. apiculata were plotted in
Figure 1B. EE, EF and BF exhibit a maximum scavenging activity of above 90% at the concentration of 13.33 μg/mL while WF shows 87.22% inhibition ability at the same concentration. The IC
50 values of the scavenging activities of EE, EF, BF, WF and BHT were 1.71 ± 0.39, 3.01 ± 0.75, 1.26 ± 0.05, 4.32 ± 0.96 and 9.63 ± 0.15 μg/mL, respectively. The order of ABTS radical scavenging ability was BF > EF > EE > WF> BHT (
P < 0.05).
Hydroxyl radicals are highly reactive and short-lived species causing damage to virtually all adjacent biomolecules [
14]. Its radical scavenging abilities of extract/fractions of
R. apiculata were investigated and are shown in
Figure 1C. Like in the DPPH and ABTS assays, the hydroxyl radical scavenging abilities increase with the increased concentration of the test samples. The IC
50 values of EE, EF, BF, WF and BHT were 13.57 ± 1.59, 17.93 ± 1.51, 9.07 ± 0.99, 33.59 ± 1.66 and 45.58 ± 2.14 µg/mL, respectively. According to these IC
50 values, the order of hydroxyl radical scavenging abilities is BF > EE >EF > WF > BHT (
P < 0.05).
Figure 1.
Antioxidant activities of extract and fractions of R. apiculata.(A) DPPH; (B) ABTS•+; (C) Hydroxyl radicals.
Figure 1.
Antioxidant activities of extract and fractions of R. apiculata.(A) DPPH; (B) ABTS•+; (C) Hydroxyl radicals.
2.2. Total Phenolic Content
Phenolic compounds are a large group of phytochemical components widespread in the plant kingdom and characterized by having at least one aromatic ring with one or more hydroxyl groups attached which directly contribute to the antioxidant properties [
15]. Therefore, it is important to evaluate the total phenolic in the crude extract and fractions of the
R apiculata. The total phenolic content was expressed in milligrams equivalents of gallic acid per gram of each fraction. The amount of phenolic compounds among different fractions were in the order of BF (181.84 ± 2.56 mg/g) > EE (127.81 ± 4.5 mg/g) > EF (121.16 ± 2.57 mg/g) > WF (106.88 ± 9.87 mg/g), results in our study show that the extent of antioxidant activity of fraction is in accordance with the total phenolic content.
2.3. Separation of Compounds
According to the above results, the BF contains the largest amount of phenolic compounds and exhibits the strongest radical scavenging activities. Therefore the BF was further fractionated to identify the compounds responsible for the antioxidant activity. Compounds
1–
3 were obtained from BF and identified as lyoniresinol-3α-
O-β-arabinopyranoside, lyoniresinol-3α-
O-β-rhamnoside and afzelechin-3-
O-
L-rhamnopyranoside by comparing their NMR data with literature data [
16,
17,
18]. To the best of our knowledge, it is first report of the separation of lignan and flavonol compounds from
R. apiculata.
2.4. Activity and Quantity Analysis of Isolated Compounds
Lignans and flavonols are a group of compounds which show several biological activities. In our experiment, all separated compounds showed remarkable antioxidant activity and the IC
50 values were listed in
Table 1. The results indicated that compounds
1–
3 were active ingredients in
R. apiculata.
Table 1.
Antioxidant activity of compounds isolated from R. apiculata.
Table 1.
Antioxidant activity of compounds isolated from R. apiculata.
Compound | IC50 (μg/mL) |
---|
DPPH | ABTS•+ | OH |
---|
Lyoniresinol-3α-O-β-arabinopyranoside | 2.06 | 1.64 | 5.83 |
Lyoniresinol-3α-O-β-rhamnoside | 2.64 | 2.09 | 9.07 |
Afzelechin-3-rahmnoside | 2.26 | 1.96 | 7.05 |
BHT | 55.20 | 9.63 | 45.58 |
R. apiculata is a protected marine plant in China. To reasonably utilize the mangrove plant resource, a HPLC method was developed to study the distribution of the three compounds in different parts of
R. apiculata. Excellent separation was achieved under the optimized chromatographic conditions and the HPLC chromatogram of the crude extract is shown in
Figure 2. Results (
Table 2) showed the highest content was located in the bark and lowest content was in leaves, suggesting that bark of
R. apiculata was a better resource of
1–
3 than the other two parts of the plant.
Figure 2.
Chromatogram of crude extract of R. apiculata.
Figure 2.
Chromatogram of crude extract of R. apiculata.
Table 2.
Content of compounds in different part of R. apiculata.
Table 2.
Content of compounds in different part of R. apiculata.
Compound | Content (%) |
---|
twig | leaf | bark |
---|
Lyoniresinol-3α-O-β-arabinopyranoside | 0.047 | 0.012 | 0.068 |
Lyoniresinol-3α-O-β-rhamnoside | 0.059 | 0.016 | 0.066 |
Afzelechin-3-rahmnoside | 0.010 | 0.006 | 0.011 |