2.1. HPLC Analysis of Flavonoids
The results obtained from the preliminary analysis of flavonoids are shown in
Table 1. Increasing the CO
2 concentration from 400 to 800 µmol mol
-1 resulted in enhanced quercetin, catechin, kaempferol and fisetin levels in the leaves and rhizomes of both varieties, and increase in naringenin content in just the leaves. On the other hand, the contents of rutin, epicatechin and morin decreased in ginger parts with rising of CO
2 concentration from ambient to 800 µmol mol
-1. It can be seen from the data in this table that quercetin content in ginger, when compared with other plants, for example red chilli (0.799 mg g
-1 DW), bird chilli (0.392 mg g
-1 DW), bell pepper (0.448 mg g
-1 DW), black tea (1.107 mg g
-1 DW), onion (1.49 mg g
-1 DW) and semambu (1.18 mg g
-1 DW) [
34] registered substantially high levels in both the leaves (1.33 mg g
-1 DW) and rhizomes (1.27 mg g
-1 DW) of Halia Bara exposed to elevated CO
2 concentration at 800 µmol mol
-1.
Table 1.
The concentrations of some flavonoids compounds in two varieties of Zingiber officinale, Halia Bentong (a) and Halia Bara (b) grown under different CO2 concentrations.
Table 1.
The concentrations of some flavonoids compounds in two varieties of Zingiber officinale, Halia Bentong (a) and Halia Bara (b) grown under different CO2 concentrations.
Flavonoid compounds | (a) Halia Bentong |
---|
400 | 800 |
---|
Leaves | Rhizomes | Leaves | Rhizomes |
---|
Quercetin | 0.972 ± 0.013c | 0.895 ± 0.039c | 1.22 ± 0.07b | 1.138 ± 0.023b |
Rutin | 0.171 ± 0.0028de | 0.452 ± 0.004a | 0.141 ± 0.031e | 0.388 ± 0.026b |
Epicatechin | 0.122 ± 0.018a | 0.083 ± 0.007bc | 0.073 ± 0.008c | 0.048 ± 0.018d |
Catechin | 0.409 ± 0.027d | 0.491 ± 0.019cd | 0.673 ± 0.044ab | 0.637 ± 0.034b |
Kaempferol | 0.042 ± 0.002e | 0.053 ± 0.003de | 0.118 ± 0.014c | 0.148 ± 0.023b |
Naringenin | 0.089 ± 0.0052c | 0.047 ± 0.003d | 0.127 ± 0.022b | 0.083 ± 0.004c |
Fisetin | 0.986 ± 0.012e | 0.633 ± 0.033f | 2.05 ± 0.27c | 2.82 ± 0.19a |
Morin | 0.514 ± 0.027e | 0.463 ± 0.014e | 0.49 ± 0.052e | 0.875 ± 0.036a |
Flavonoid compounds | (b) Halia Bara |
400 | 800 |
Leaves | Rhizomes | Leaves | Rhizomes |
Quercetin | 1.19 ± 0.122ab | 0.986 ± 0.032c | 1.33 ± 0.134a | 1.27 ± 0.01a |
Rutin | 0.174 ± 0.007d | 0.334 ± 0.009c | 0.151 ± 0.025de | 0.404 ± 0.016b |
Epicatechin | 0.12 ± 0.004a | 0.103 ± 0.0035ab | 0.096 ± 0.022bc | 0.037 ± 0.009d |
Catechin | 0.668 ± 0.079ab | 0.533 ± 0.034c | 0.733 ± 0.014a | 0.682 ± 0.05ab |
Kaempferol | 0.051 ± 0.002de | 0.068 ± 0.005d | 0.163 ± 0.011ab | 0.181 ± 0.009a |
Naringenin | 0.061 ± 0.004d | 0.028 ± 0.003e | 0.155 ± 0.027a | 0.121 ± 0.011b |
Fisetin | 1.53 ± 0.121d | 1.32 ± 0.12d | 2.38 ± 0.395b | 3.11 ± 0.185a |
Morin | 0.765 ± 0.024b | 0.606 ± 0.006d | 0.661 ± 0.029c | 0.515 ± 0.025e |
Kaempferol is a rare flavonoid component in plants, but it was detected in the leaves (0.042–0.163 mg g
-1 DW) and rhizomes (0.053–0.181 mg g
-1 DW) of both Halia Bara and Halia Bentong. These contents were slightly higher than those recorded in green chilli (0.039 mg g
-1 DW), sengkuang (0.037 mg g
-1 DW), white radish (0.0383 mg g
-1 DW), and pegaga (0.0205 mg g
-1 DW). Nevertheless, both ginger varieties had lower kaempferol contents when compared to cekur manis (0.323 mg g
-1 DW), pumpkin (0.371 mg g
-1 DW), and carrot (0.140 mg g
-1 DW) [
35]. Tolonen
et al. [
36] reported very low kaempferol contents (9 mg/g DW) in white cabbages, and it was the only flavonoid found. Meanwhile, Kim [
37] detected about 0.1–0.8 mg/g fm of quercetin and kaempferol aglycone contents in green cabbages. Exposing ginger plants to 800 µmol mol
-1 of CO
2 concentration saw the synthesis of kaempferol enhanced to 0.163 and 0.181 mg g
-1 DW in Halia Bara leaves and rhizomes, respectively.
Fisetin is another rare yet well known flavonoid component in plants. Previous studies showed that fisetin had anti-inflammatory [
35,
38], anti-carcinogenic [
39] and strong antioxidant [
39] effects. Ginger leaves and rhizomes exhibited good potential levels of this flavonoid. It seemed that fisetin content could also be improved by increasing CO
2 concentration in both of varieties, especially in Halia Bara (leaves: 1.53 increased to 2.38 mg g
-1 DW; rhizome: 1.32 increased to 3.11 mg g
-1 DW). Morin is another flavonoid belonging to the flavonols group. Morin acts as a chemopreventive agent
in vitro and
in vivo against oral carcinogenesis [
40,
41]. The importance of morin and related compounds as anti-tumour drugs has also been widely recognized [
42]. In comparison with old fustic (
Chlorophora tinctoria), osage orange (
Maclura pomifera) [
43], almonds (
P. guajava L.) [
44], mill (
Prunus dulcis), fig (
Chlorophora tinctoria) [
43], onion and apple [
44], both the leaves and rhizomes local ginger varieties showed good levels of morin when grown under both 400 and 800 µmol mol
-1 CO
2 conditions, indicating that the plant is naturally a good source of morin, although the content of the latter was variable. For example, the content of morin in the leaves decreased in both varieties with increasing of CO
2 concentration, while a high content of morin (0.875
vs. 0.463 mg g
-1 DW) was obtained from extract of Halia Bentong rhizomes grown under elevated CO
2.
Similar trends of increasing concentration of flavonoid components with increasing CO
2 concentration was observed in
Betula pendula and strawberry [
2,
9]. Wang
et al. [
9], reported growing strawberry plants under CO
2 enrichment conditions (950 µmol mol
-1) significantly enhanced fruit
p-coumaroylglucose, dihydroflavonol, quercetin 3-glucoside, quercetin 3-glucuronide, and kaempferol 3-glucoside contents, as well as cyanidin 3-glucoside, pelargonidin 3-glucoside, and pelargonidin 3-glucoside succinate content. This finding is in agreement with Sttute
et al. [
32] who showed the ability of elevated CO
2 concentrations to enhance flavonoid components (apygenin, baicalin, scutellarein) in
Scutellaria species. The percentages of increase or decrease in flavonoid contents of ginger when exposed to 800 µmol mol
-1 concentrations of CO
2 are tabulated in
Table 2.
Table 2.
Percent of increase or decrease of flavonoid compounds in two varieties of Zingiber officinale grown under elevated CO2 concentration (800 µmol mol-1).
Table 2.
Percent of increase or decrease of flavonoid compounds in two varieties of Zingiber officinale grown under elevated CO2 concentration (800 µmol mol-1).
Flavonoid compounds | Halia Bentong | Halia Bara |
---|
Leaves | Rhizomes | Leaves | Rhizomes |
---|
Quercetin | +25.5 | +27.2 | +9.2 | +28.8 |
Rutin | -17.5 | -14.2 | -13.2 | +21.0 |
Epicatechin | -40.2 | -42.2 | -20.0 | -64.1 |
Catechin | +64.5 | +29.7 | +9.7 | +28.0 |
Kaempferol | +181.0 | +179.2 | +219.6 | +166.2 |
Naringenin | +42.7 | +76.6 | +154.1 | +332.1 |
Fisetin | +107.9 | +345.5 | +55.6 | +135.6 |
Morin | -4.7 | +89.0 | -13.6 | -15.0 |
According to data from this table, kaempferol levels were more enhanced in varieties grown under elevated carbon dioxide conditions and after that, fisetin and naringenin were more enhanced. On average flavonoid compounds increased 44.9% in leaves and 86.3% in rhizomes of Halia Bentong and 50.1 % in leaves and 79% in rhizomes of Halia Bara when exposed to elevated carbon dioxide conditions. To the best of our knowledge the current study is the first report of fisetin, morin and naringenin in young ginger varieties.
Figure 1 shows the HPLC chromatogram of flavonoids analysis in Halia Bentong extract (leaves).
Figure 1.
HPLC chromatogram of Halia Bentong ginger (Zingiber officinale) leaves extracts at wavelengths of 360 nm (a), and 280 nm (b). Identification of compounds: quercetin (1), rutin (2), kaempferol (3), fisetin (4), morin (5), catechin (6), epicatechin (7), naringenin (8).
Figure 1.
HPLC chromatogram of Halia Bentong ginger (Zingiber officinale) leaves extracts at wavelengths of 360 nm (a), and 280 nm (b). Identification of compounds: quercetin (1), rutin (2), kaempferol (3), fisetin (4), morin (5), catechin (6), epicatechin (7), naringenin (8).
2.2. HPLC Analysis of Phenolic Compounds
Like the alteration of flavonoid accumulation in both varieties of ginger when they were exposed to 400 to 800 µmol mol
-1 CO
2 the phenolic contents also increased in the leaves more than in the rhizomes. Phenolic contents are influenced by the interaction between varieties and parts of the plants. Partitioning and accumulation of phenolics in different parts of ginger grown under ambient CO
2 followed the trend of leaves > rhizomes. Rhizomes in both of varieties had more phenolic content when exposed to elevated CO
2. Among the phenolic acid compounds, gallic acid had a higher content in both ginger varieties (
Table 3). Elevated CO
2 had significant effects (p ≤ 0.001) on the synthesis of phenolics. What is interesting in this data is that vanillic acid, cinnamic acid and salicylic acid were not detected in ginger grown under ambient (400 µmol mol
-1) conditions. Conversely, tannic acid was not detected in gingers grown under elevated CO
2 (800 µmol mol
-1). Results imply that different CO
2 concentrations have variable effects on each of the phenolic components. Among the studied phenolic compounds vanillic acid, cinnamic acid and salicylic acid were not detected in Halia Bentong grown under ambient CO
2. Also cinnamic acid and salicylic acid were not detected in Halia Bara grown under ambient CO
2. Tannic acid was not detected in those varieties grown under elevated (800 µmol mol
-1) CO
2.
Table 3.
The concentrations of some phenolics compounds in two varieties of Zingiber officinale, Halia Bentong (a) and Halia Bara (b) grown under different CO2 concentrations.
Table 3.
The concentrations of some phenolics compounds in two varieties of Zingiber officinale, Halia Bentong (a) and Halia Bara (b) grown under different CO2 concentrations.
Phenolic compounds | (a) Halia Bentong |
---|
400 | 800 |
---|
Leaves | Rhizomes | Leaves | Rhizomes |
---|
Gallic acid | 0.173 ± 0.0091d | 0.141 ± 0.031d | 0.576 ± 0.049b | 0.489 ± 0.043c |
Vanillic acid | nd | nd | 0.229 ± 0.058b | 0.335 ± 0.028a |
Ferulic acid | 0.081 ± 0.022f | 0.116 ± 0.016ef | 0.117 ± 0.026de | 0.21 ± 0.022b |
Tannic acid | 0.388 ± 0.072a | nd | nd | nd |
Cinnamic acid | nd | nd | 0.134 ± 0.027a | 0.0336 ± 0.255b |
Salicylic acid | nd | nd | 0.22 ±0.021b | 0.037 ± 0.0125c |
Phenolic compounds | (b) Halia Bara |
400 | 800 |
Leaves | Rhizomes | Leaves | Rhizomes |
Gallic acid | 0.191±0.008d | 0.152+0.0081d | 0.645±0.066a | 0.537±0.034bc |
Vanillic acid | 0.082±0.016c | nd | 0.24±0.052b | 0.357±0.038a |
Ferulic acid | 0.071±0.017f | 0.148+0.017cd | 0.162±0.014c | 0.285±0.038a |
Tannic acid | 0.224±0.041b | nd | nd | nd |
Cinnamic acid | nd | nd | 0.125±0.027a | 0.0457±0.01b |
Salicylic acid | nd | nd | 0.269±0.027a | 0.0417±0.044c |
Figure 2.
HPLC chromatogram of Halia Bentong ginger (Zingiber officinale) leaves extracts. Identification of compounds: gallic acid (1), vanillic acid (2), ferulic acid (3), cinnamic acid (4), salicylic acid (5).
Figure 2.
HPLC chromatogram of Halia Bentong ginger (Zingiber officinale) leaves extracts. Identification of compounds: gallic acid (1), vanillic acid (2), ferulic acid (3), cinnamic acid (4), salicylic acid (5).
Salicylic acid belongs to plant phenolics group and is found in some plant species, with the highest levels being observed in the inflorescences of thermogenic plants and in spice herbs [
45]. A high content of salicylic acid (0.269 mg g
-1 DW) was detected in extract of Halia Bara leaves grown under 800 µmol mol
-1 CO
2. The results of previous studies showed that salicylic acid could enhance plant growth and yield. Jeyakumar
et al. [
46] reported that salicylic acid was able to enhance the dry matter production in black gram, while Nagasubramaniam
et al. [
45] stated that salicylic acid increased plant height, leaf area, crop growth rate, and total dry matter production in baby corn. Salicylic acid was able to enhance plant growth by improving nutrition uptake. According to previous studies we could say that increasing of cinnamic acid in ginger might be one of the reasons for increased ginger growth under elevated carbon dioxide.
Table 4.
Percent of increase or decrease of phenolic compounds in two varieties of Zingiber officinale grown under elevated CO2 concentration (800 µmol mol-1).
Table 4.
Percent of increase or decrease of phenolic compounds in two varieties of Zingiber officinale grown under elevated CO2 concentration (800 µmol mol-1).
Phenolic compounds | Halia Bentong | Halia Bara |
---|
Leaves | Rhizomes | Leaves | Rhizomes |
---|
Gallic acid | +232.4 | +246.8 | +252.4 | +262.8 |
Vanillic acid | +100 | +100 | +192.6 | +100 |
Ferulic acid | +44.4 | +81 | +128.2 | +92.5 |
Tannic acid | -100 | 0 | -100 | 0 |
Cinnamic acid | +100 | +100 | +100 | +100 |
Salicylic acid | +100 | +100 | +100 | +100 |
According to data from
Table 4, gallic acid more enhanced in those varieties grown under elevated carbon dioxide conditions and after that vanillic acid and ferulic acid were more enhanced. On average phenolic compounds increased 79.4% in leaves and 107.6% in rhizomes of Halia Bentong and 112.2% in leaves and 109.2% in rhizomes of Halia Bara when exposed to elevated carbon dioxide conditions.
2.3. Radical Scavenging Activity (DPPH)
Under ambient conditions (400 µmol mol
-1 CO
2), ginger leaves exhibited higher radical scavenging activity than rhizomes (
Figure 3). At a concentration of 30 µg mL
-1 leaves of Halia Bara showed a 50.0% inhibition of free radicals, and at 45 µg mL
-1, the scavenging activity of the methanolic extract of Halia Bara leaves grown under ambient CO
2 concentration (400 µmol mol
-1) reached 62.1%, while at the same extract concentration, that of the rhizomes was 42.0% (
Figure 4), while 50% free radical scavenging activity was observed for Halia Bentong leaves at 45 µg mL
-1 extract concentration, implying that Halia Bara, under natural environmental conditions, had higher antioxidant properties than Halia Bentong, and that was found in the leaves.
Figure 3.
DPPH scavenging activities of the methanolic extracts in different parts of two varieties of Zingiber officinale (error bar represents standard deviation).
Figure 3.
DPPH scavenging activities of the methanolic extracts in different parts of two varieties of Zingiber officinale (error bar represents standard deviation).
The effect of antioxidants on DPPH scavenging is due to their hydrogen donating ability. The results of the current study showed that DPPH radical scavenging abilities of the extracts of ginger parts were less than those of butylated hydroxytoluene (BHT) (83.7%) and α-tocopherol (92.3%) at 45 µg mL
-1. Antioxidant activity in the leaves and rhizomes were enhanced by increasing the CO
2 concentration (
Figure 3). When the CO
2 concentration was increased from 400 to 800 µmol mol
-1, the free radical scavenging power increased about 30.0% in Halia Bentong and 21.4% in Halia Bara. When comparing the ginger parts, it was found that the free radical scavenging power was more enhanced in the rhizomes than in the leaves (44.9% in Halia Bentong; 46.2% in Halia Bara). The above results also suggested that Halia Bentong seemed to be more responsive to increased CO
2 concentration than Halia Bara, although the rhizomes of the latter were more receptive to elevated CO
2 in the improvement of the antioxidative power. At low concentrations (10 and 15 µg mL
-1) the DPPH activities of Halia Bara leaves was higher (35% and 38%, respectively) than BHT (20% and 32%, respectively). The DPPH scavenging activities of rhizomes in both varieties also increased after the concentration was increased to 35 µg mL
-1, and these activities were higher than those recorded from the leaves using the same concentration. Our finding is in agreement with that of Wang
et al. [
9], who reported the increase in free radical scavenging power of strawberry by elevated CO
2 concentrations (950 µmol mol
-1). This study demonstrated that ginger has good free radical scavenging ability and, hence, can be used as a radical inhibitor or scavenger, possibly acting as a primary antioxidant. At the same time, with the anticipated rise in CO
2 concentrations in the future with the current climate change scenario, it is anticipated that the antioxidant properties of ginger extracts could be enhanced, as the results indicate that an increased atmospheric carbon dioxide concentration could have a major effect on the antioxidant capacities of young ginger varieties.
Figure 4.
DPPH radical scavenging activity of the methanolic extracts in different parts of two varieties of Zingiber officinale compared with positive controls butylated hydroxytoluene (BHT) and α-tocopherol. L and R represent the leaves and rhizomes at 400 µmol mol-1 CO2 (a), and 800 µmol mol-1 CO2 (b).
Figure 4.
DPPH radical scavenging activity of the methanolic extracts in different parts of two varieties of Zingiber officinale compared with positive controls butylated hydroxytoluene (BHT) and α-tocopherol. L and R represent the leaves and rhizomes at 400 µmol mol-1 CO2 (a), and 800 µmol mol-1 CO2 (b).