Effect of collection time on the chemical composition and levels of thiobarbituric acid reactive substance of Godulbaegi (Youngia sonchifolia M.)

YSM (leaf and root) is godulbaegi (Youngia sonchifolia M.) cultivated in open field after spraying seeds at early June in the Suncheon, South Jeolla Province, Korea. Samples were collected in the same area in early October and early December (Suncheon area weather (Data of Korea Meteorological Administration). : mean temperature/days; October→16.13℃ /days, December→0℃/days, precipitation/month; October→ 13.8 mm/month, December→10.6 mm/month). The samples used for the experiments were 25-30 cm length samples. The samples were washed and the roots and leaves were separated and stored at -54℃ deep freezer (FDA5508; Ilsinbiobase, Dongducheon, Korea).

Methanol, acetonitrile (HPLC grade), glacial acetic acid (analytical grade), monopotassium phosphate, and phosphoric acid were purchased from Merck (Germany). Sodium 1-hexanesulfonate (98% purity), anhydrous sodium carbonate (99% purity), and 2,4,6-trichloroanisole and 2,4,6-tribromoanisole were from Sigma-Aldrich (St. Louis, MO, USA). Standards of phenolic acids (arbutin, catechin, chlorogenic acid, p-coumaric acid), and standards of vitamins, organic acids, amino acids and free sugars were purchased from Sigma-Aldrich. To determine mineral and trace element contents, calibration standards were prepared by appropriate dilution from 1,000 ug/mL pure single element standard solutions (PerkinElmer, Shelton, CT, USA) while nitric acid (70%, Fluka 84380), hydrochloric acid (37%, Riedel-de- Haen30721), and hydrogen peroxide (30%, Fluka 95302) were of analytical purity.

A water extract of YSM was homogenized to a fine powder, followed by maceration of 100 g with 3 L of water at room temperature for 24 h. The extracts were filtered and stored at 4℃.

After the pH level of 0.1 M maleic acid buffer was adjusted to pH 6.5, 8 mL of the solution was put into a vessel. In addition, 50 Ul of Tween-20 and 0.5 mL of fish oil were added and the mixture was stirred for 15 minutes. Two or three pieces of KOH were added, and while the mixture was being stirred, an oil emulsion at pH 6.5 was prepared with 0.1 N HCl (15).

For oxygen species samples, 4 mM KO₂ was prepared for KO₂, 4 mM H₂O₂, for H₂O₂, 4 mM FeCl₂, for Fe²⁺, 4 mM H₂O₂+ 4 mM FeCl₂, for OH, and 4 mM CuSO₄, for Cu²⁺ (16). 0.1 mL of the oxygen species sample and 0.1 mL of YSM water extract were added to 0.5 mL of oil emulsion, and the mixture was adjusted by adding distilled water until the total amount of the mixture was 1 mL before it was used for experiments.

The moisture, crude protein, crude fat, crude ash, and crude fiber contents were determined by the AOAC official method (17). Moisture content was obtained by calculating the weight loss after powdered samples were oven-dried at 105℃ to a constant weight. Crude protein and crude fat were analyzed using the Kjeldahl and the Soxhlet extraction methods. Ash content was determined gravimetrically after the samples were ignited in a furnace at 600℃. Estimation of crude fiber was based on the loss upon ignition of the dried residue after samples were digested with 1.25% each of sulfuric acid and sodium hydroxide solutions. The following equation was used for calculation of carbohydrate contents: % carbohydrates= 100%-(% crude protein+% crude fat+% crude ash).

In the mineral analysis, all glassware was soaked overnight in a solution of 10% HCl in distilled water (v/v) prior to use. Ashed YSM samples (550℃, 6 h) were dissolved in 2 mL of 70% nitric acid. The acidified samples were neutralized in 5 mL of ddH₂O, filtered through Whatman No. 1 paper and diluted to volume with ddH₂O in a 50 mL volumetric flask. Major minerals: magnesium (Mg), potassium (K), calcium (Ca), iron (Fe), sodium (Na), and cooper (Cu) were determined using inductively coupled plasma optical emission spectrometry (ELEMENT 2*ICPMS, Thermo Fisher Scientific Inc., MA, USA).

The vitamin B group and vitamin C in YSM were assayed by the methods from Wimalasiri and Wills (18), and Hefferan et al. (19) with a slight modification. Concentrations of vitamin B group (Niacin, Vit. B₆, Vit. B₂, Vit. B₁) and vitamin C were analyzed using a high performance liquid chromatography system (Waters M510, Waters Co., Milford, MA, USA) with a Radial Pak C18 column (ID 0.8×300 mm, Waters Co., Milford, MA, USA) operated at 25℃ and UV 486 detector (Waters Co.) at 280 nm. The mobile phase was composed of 75% methanol with 5 mM sodium 1-hexanesulfonate (Pic B₆, Fluka), and the flow rate was set to 1.0 mL/min.

One hundred grams of YSM was homogenized in 3 L of water. The solution was then incubated for 20 min in a 35℃ water bath and centrifuged at 10,000 ×g for 10 min.. The supernatants were collected and filtered through membrane filters (Millipore Co., Billerica, MA, USA) with a pore size of 0.45 μm for the organic acid tests. Concentrations of the six main organic acids (lactic, citric, maleic, succinic, acetic and tartaric acids) were analyzed using a high performance liquid chromatography system (Waters M510, Waters Co.) with a Rspak KC-811 column (ID 0.8×300 mm, Waters Co.,) operated at 25℃ and UV 486 detector (Waters Co.,) at 220 nm. The mobile phase was composed of 95% (v/v) 3.3 mM KH₂PO₄ and 5% methanol with the pH adjusted to 2.5 with phosphoric acid, and the flow rate was set to 1.0 mL/min.

In the amino acid analysis, the YSM was hydrolyzed in 6 N HCl for 24 h. Amino acids were quantified using the amino acid auto analyzer (S433, Sykam Co., Eresing, Germany) employing sodium citrate buffers as step gradients with the cation exchange post column ninhydrin derivatization method. The data were described as grams of amino acid per 100 g.

Free sugar contents in YSM were identified by methods from Damon and Pettitt (20) with a slight modification. Concentrations of the four main free sugars (fructose, glucose, sucrose and maltose) were analyzed using a high performance liquid chromatography system (Waters M510, Waters Co.,) with a carbohydrate analysis column (ID 4.6×250 mm, Grace Co., Deerfield, IL, USA) operated at 30℃ and ELSD 2000ES detector (ELSD 2000ES detector, Alltech Co., Vienna, VA, USA). The mobile phase was composed of 75% acetonitrile, and the flow rate was set to 1.0 mL/min.

The polyphenol compounds in YSM were identified by the methods from Liu et al. (21) with a slight modification. The high performance liquid chromatography (HPLC) system is composed of a Waters M510 pump system (Waters M510, Waters Co.,) pump system and a 486 detector (Waters Co.). A u-Bondpak C18 column (ID 3.9×250 mm, Waters Co.) and a gradient solvent system consisting of methanol (solvent A) and water with 2% glacial acetic acid (solvent B) (conditions: 5-17% A from 0 to 5 min and kept at 17% A from 5 to 25 min; 17-31% A from 25 to 40 min and kept at 31% A from 40 to 76 min; 31-40 % A from 76 to 80 min and kept at 40% A from 80 to 120 min; flow rate=1 mL/min) were used for separation of components whose UV spectra were recorded from 280 nm. Polyphenol compounds included arbutin, catechin, chlorogenic acid, p-coumaric acid.

The TBARS method was conducted by allowing the test tube filled with 1 mL of the reaction mixture to react in 37℃ water bath for one hour following the method of Buege and Aust (22). Immediately after the reaction was terminated, 50 Ul of 7.2% BHT was added to the sample to stop the oxidation reaction, and 2 mL of the TCA/TBA reagent was added. The mixture was heated in boiling water for 15 minutes, cooled in cold water, and centrifuged at a rate of 2,000 ×g for 15 min. The supernatant was measured at 531 nm, and the blank sample was measured in the same manner after distilled water was added to them in place of the sample. The TBARS values were represented as the malondialdehyde (MDA) values for the reaction mixture.

Data were expressed as mean±SD and statistical analysis for single comparisons was performed using Duncan’s multiple range test. Each experiment was repeated at least three times to yield comparable results. Null hypotheses of no difference were rejected if p-values were less than 0.05.

The results of dividing the roots and leaves of YSM and analysis of the changes in the contents of general components by collection periods are shown in Table 1. The moisture contents of leaves and roots were 86.12% and 84.12%, respectively, in the October test plot (hereafter, October) and decreased slightly to 85.32% and 82.44%, respectively, in the December test plot (hereafter, December). The crude ash content increased from 0.70% in October to 0.61% in December in the leaves and increased from 0.64% to 1.12% in the roots. Like the crude ash content, the crude protein content increased from 1.83% in October to 2.10% in December in the leaves and increased from 1.83% to 2.74% in the roots. The crude fat content decreased in both the leaves and roots in December compared to that of October, but the crude fiber increased in the leaves and roots from 4.34% and 4.63% in October to 4.93% and 4.81% in December. The carbohydrate contents of both the leaves and roots increased slightly from 6.21% and 8.42% in October and to 7.03% and 8.70% in December. In the content analysis of the general components of Lactuca indica from June to August, Kim et al. (14) reported that the moisture content decreased and the crude protein content increased, while no significant difference was observed in the contents of the crude fat, crude ash, and crude fiber. The results of this research were similar in regard to the changes in the moisture and crude protein contents, but different from those of Kim et al. (14) in the contents of crude fat, crude ash, and crude fiber. These results may be because this research differed from Kim et al. (14) in the varieties used and harvesting periods, from June to August versus from October to December.

The presence of minerals in plants is largely dependent on growing conditions including cultivation techniques, abiotic or biotic stress and nutrient status. The changes in the mineral content of YSM (leaf and root) by collection time are presented in Table 1. The main minerals of the leaves and roots were K (potassium), Na (sodium), Mg (magnesium), and Ca (calcium), in descending order of contents. Fe (iron) and Cu (copper) were minimal. The Na content decreased in the leaves from 44.43 mg% in October to 34.82 mg% in December, while it increased in the roots from 23.84 mg% in October to 30.23 mg% in December. Thus, Na changed the most. Nour V et al. (23) analyzed the mineral contents of the leaves of blackcurrants (Ribes nigrum L.) by harvest time and reported delayed harvest decreased Na. The results of this study are consistent with their research. In addition, Kim et al. (14) reported that potassium was high irrespective of the growth conditions, as a result of analyzing minerals of wild and cultivated Lactuca indica.

The changes in vitamin contents of YSM (leaf and root) by collection time are in Table 2. Vitamin C in the leaves and roots was 87.12 mg% and 15.87 mg% in October, and 78.68 mg% and 13.56 mg% in December, respectively, so decreasing slightly in December compared to October. The vitamin C content was approximately five times higher in the leaves than in the roots, and the amount of reduction was higher in the leaves. The content of vitamin C varies greatly depending on the growth age and climate conditions of the plant. Choi and Han (24) reported that vitamin C content of perilla leaves by leaf age was high in young leaves. In this study, samples collected at early October were higher. The niacin contents of the leaves and roots was 4.63 mg% and 3.78 mg% in October, and 3.29 mg% and 3.33 mg% in December, respectively, so decreased slightly in December compared to October and the niacin content was higher in the leaves. The vitamin B group was slightly higher in December than in October in the leaves, but B₂ and B₁ tended to decrease slightly in the roots. Content differences by the parts of YSM were generally not seen, except that the B₆ content was nearly double in the leaves than in the roots.

The changes in the organic acid contents of YSM (leaf and root) by collection time are presented in Table 2. The main organic acids of the leaves and roots of YSM were citric, maleic and tartaric acid. For the leaves, the maleic acid content was the highest with 1.86 mg% in October and 1.97 mg% in December, and for the roots, the citric acid content was highest with 1.20 mg% in October and 1.12 mg% in December. Tartaric acid was detected in the leaves, but a very small amount was detected in the roots. For the leaves, an overall increase in the contents was observed in December, the later harvest time, compared to the measurements of October. However, root content tended to decrease overall. The total content of organic acids of YSM was higher in the leaves than in the roots. Kim et al. (14) measured the organic acid contents of Lactuca indica and reported that they were higher in the leaves than in the roots.

The changes in the contents of component amino acids of YSM (leaf and root) by collection time are in Table 3. Sixteen kinds of component amino acids of YSM were identified, including aspartic acid, and they contained all the eight essential amino acids. In the leaves, total component amino acids increased significantly from 94.87 mg% in October to 145.97 mg% in December, and increased in the roots from 127.91 mg% in October to 171.79 mg% in December. Thus, amino acid levels were higher in the roots than in the leaves. The main amino acids of the YSM leaves in December detected were Ile at 64.01 mg%, Leu at 52.03 mg%, Glu at 3.62 mg%, Ser at 3.52 mg%, and Pro at 3.36 mg%, and the isoleucine content was the highest. For the YSM roots in October, the main amino acids were Ile, Leu, Arg, Thr, Phe, and Glu from the highest to the lowest, and following those, other amino acids detected were Ser, Ala, Asp, Val, and Lys.

The changes in the free sugar contents of the YSM (leaf and root) by collection time are in Table 4. Four kinds of free sugars contained in the leaves and roots of YSM were identified: fructose, glucose, sucrose, and maltose. The fructose content of the YSM leaves was 0.23% in October and 0.33% in December, while the fructose content of the roots was 0.43% in October and 0.73% in December, so the fructose content was higher in December. The glucose content was higher in the leaves than in the roots, and while there were no differences in the content by collection time in the roots, the glucose content increased five times in the leaves from 0.20% in October to 1.04% in December, a significant difference. Liu S et al. (25) measured the free sugar content of Arundo donax L. by harvest time and reported that the glucose content increased in all samples as the harvest time was delayed. Thus, their research is consistent with this study. Sucrose comprises most of the free sugars contained in the YSM, and its content was higher in the samples collected in December in both the leaves and roots. Maltose was higher in the roots.

Among the polyphenolic compounds, flavonoid reacts with 1O² or O^2-, forms a stable complex, and prevents lipid peroxidation by scavenging them. Consequently, the phenolic content and antioxidant activity are associated (26). The changes in the content of polyphenol compounds of YSM (leaf and root) by collection time are in Table 5. For the YSM samples collected in October, polyphenol was 1.02 mg% in the leaves and 2.04 mg% in the roots. For the samples collected in December, it was 1.81 mg% in the leaves and 3.80 mg% in the roots, so the contents were higher in the samples collected in December. In addition, the root contents were more than twice as high as the leaves. The main components of both roots and leaves were arbutin and p-coumaric acid. The arbutin content was more than 2.5 times higher in the samples collected in December than those collected in October, a significant difference. The catechin content tended to decrease in the samples collected in December in comparison to those collected in October in the leaves, but increased by more than 35 times in the roots of samples collected in December. Chlorogenic acid was detected only in the roots collected in December, and the p-coumaric acid content was higher in the samples collected in October, tending to decrease as the collection time is delayed.

The effect of the addition of 0.05% YSM (leaf and root) water extracts on reactive oxygen species that reacted in the oil emulsion was measured by thiobarbituric acid reactive substances (TBARS) (Table 6). Adding KO₂ resulted in a measurement of 0.15 mg MDA/L in the YSM root extract, excellent antioxidative activity. Other samples did not differ significantly. For the effect on H₂O₂, the TBARS values of the YSM root extract were lower than those of the YSM leaf extract, strongly antioxidative. The TBARS values did not differ by collection time. The YSM root extract possessed higher capture capacity for hydroxyl radical (·OH, which causes fat oxidation and accelerates the aging process) compared with the YSM leaf extract and exhibited antioxidative activity. As to Fe²⁺ and Cu²⁺, the YSM root extract showed better antioxidative activity than the YSM leaf extract. The December samples showed stronger antioxidative activity than those of October. Lee and Han (27) measured TBARS values of the butanol extract of Ulmus devidiana and reported that the measured values were 0.14 -0.44 mg MDA/L, indicating the highest antioxidative activity. In this study, TBARS measurements ranged from 0.13 to 0.25 mg MDA/L, so YSM extracts showed similar antioxidant activity to Ulmus devidiana butanol extracts.