Quantitation of Phenolic Compounds Related to Antioxidant and Antiosteoporosis Activities in Ripe and Unripe Maesil (Prunus mume)

College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon, Republic of Korea School of Chemistry & Pharmacy, Northwest A & F University, Xianyang, Shaanxi, China Division of Bioengineering, Dong-Eui University, Busan, Republic of Korea Clinical Trial and Bioequivalent Testing Centre, Vietnam Military Medical University, Hanoi, Vietnam

In contrast, the simultaneous analytical method for some kinds of compounds such as mumefural using highperformance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) was reported. In particular, the separation and isolation methods of phenolic compounds such as 4-O-caffeoylquinic acid methyl ester, prunasin, 5-O-caffeoylquinic acid methyl ester, benzyl-O-β-D-glucopyranoside, and liquiritigenin-7-O-β-D-glucopyranoside were studied by many researchers [32,33]. But, the simultaneous analytical methods for these compounds using HPLC and LC-MS in maesil were nominal. In particular, the analytical method to analyze the active compounds with antiosteoporosis such as neochlorogenic acid, benzylβ-D-glucopyranoside, and 3, 4, 5-trimethoxyphenyl-β-Dglucopyranoside was not reported until now. erefore, the aim of this study is to develop the simultaneous analytical methods for phenolic compounds with various bioactivities such as antioxidant and antiosteoporosis activities and evaluate the contents of these compounds in maesil for good quality control. e chemical structures of these reference compounds are shown in Figure 1.

Samples.
Unripe maesil (fresh fruits, 5∼10 kg per sample) for this study was directly purchased from the agrofishery market places in Korea during June-July, 2015 (Table 1). e ripe fruit of each sample was produced by natural ripening of 2∼5 kg of fresh fruits for 3∼4 days at 27 ± 3°C or some of them were directly collected by getting ripe fruits naturally matured after 80-90 days of flowering from the market. 100∼150 g per each sample was freeze-dried by a lyophilizer (Eyela FDU-540, Tokyo Rikakikai, Tokyo, Japan).

Preparation of Samples.
First, the freeze-dried samples were grinded and powdered by a grinder (Blender 7011G, Waring Commercial, Torrington, CT, USA), and then, these were filtered with the molecular sieve (No. 20). 0.5 g of each sample was weighed, and 10 mL of 50% methanol was added. Second, all samples were extracted by reflux for 30 min at 80°C and then centrifuged by a centrifuge (MF550, Hanil Science, Inchon, Korea) for 10 min at 3000 rpm. All supernatants were filtered by a syringe membrane filter (PVDF, 0.45 μm, SmartPor-II, Woonki Science, Seoul, Korea). Each sample was injected with 5 μL of volume of samples, using an HPLC autosampler.

HPLC Analytical Condition.
e HPLC analytical system for the quantization of compounds 1-9 in maesil was a ShimazduLC-20A system equipped with two LC-20AD pumps, a SPD-20A UV/Vis detector, a CTO-10ASvp column oven, and a Sil-20A autosampler linked to a Shimadzu LabSolutions software program (Ver. 1.25, Shimadzu, Kyoto, Japan). In order to optimize the simultaneous analytical method for quality control of compounds 19 in samples, the peak selectivity was tested by parameters as follows: the used columns were XDB-C18 (Zorbax Eclipse XDB-C18, 5 μm, pH parameters were checked at pH 2, 4, 6, and 8. And the concentration of phosphate buffer solution for the mobile phase was tested at 2, 4, and 8 mM within having no damage of column by high buffer concentration. Column temperatures were estimated at 25, 30, 40, 45, 50, and 60°C. Each peak of compounds 19 on HPLC chromatograms was scanned in the range of 190∼400 nm of wavelength (split 1.2 nm). e best analytical condition for quality control of compounds 19 in samples was selected through the estimation of above parameters.

LC-MS/MS Analytical Conditions.
Identification of compounds 1-9 in maesil were performed on a Shimazdu LCMS-8040 triple quadruple tandem mass spectrometry (Shimadzu, Kyoto, Japan) in negative and positive ESI interface modes. e identification of each compound was carried out on the Kinetex F5, 2.6 μm (PFP, 150 mm × 4.6 mm (I.D.), Phenomenex, Torrance, CA, USA) column by the mobile phase consisted of (A) 0.1% formic acid in water and (B) methanol. e elution condition of this mobile phase was hold to 5% B for 5 min, increased to 40% B for 70 min, increased to 60% B for 25 min, increased to 100% B for 1 min, hold to 100% B for 5 min, decreased to 5% B for 1 min, and then hold to maintain an enough equilibrium at 5% B for 13 min, successively. Operation conditions of a mass detector were applied as follows: that is, capillary voltage, 3.5 kV/−3.5 kV; desolation line (DL) temperature, 250°C; heat block temperature, 350°C; nebulizing gas (N 2 ) flow, 3 L/min; drying gas (N 2 ) flow, 15 L/min; and collision energy (CE) values for each reference compound were optimized by the multiple reaction monitoring (MRM) mode.

Method Validation.
e validation of the developed method was estimated by following parameters based on guidelines of the MFDS (Ministry of Food and Drugs Safety of Korea): linearity, limits of detection (LODs), limits of quantitation (LOQs), precision, accuracy, and recovery. Compounds 19 were dissolved in 50% methanol to a final concentration of 500 μg/mL as stock solutions. And then, the prepared stock solutions were serially diluted with seven different concentrations with the range of 16∼250 μg/mL (cf., compound 1, 16∼500 μg/mL) to generate calibration curves of each compound. Linearity was evaluated by plotting the correlation coefficient (r 2 ) with the integrated peak area of different standard concentrations. e evaluation method of LODs and LOQs was as follows: LODs were calculated based on the lowest detectable peak on the HPLC chromatogram containing signal-to-noise (S/N) ratio of 3. LOQs were evaluated based on the lowest quantitative level on the HPLC chromatogram having signal-to-noise (S/N) ratio of 10. Precision and accuracy of intraday/interday were evaluated by analyzing five injections within a day for intraday and repeating five injections in a day during five consecutive days for interday at three different concentrations (32, 63, and 93 μg/mL). Precision was expressed as the  17.70 ± 1.84 18.80 ± 2.36 * All unripe and ripe fruits were collected from agrofishery markets. a An unripe fruit is a fruit grown after 60-80 days of flowering. b Ripe fruits were artificially produced by after-ripening of fresh fruits for 3∼4 days at 27 ± 3°C or got ripe fruits naturally matured after 80-90 days of flowering from the market. relative standard deviations (%RSD) of intraday/interday, and accuracy of intraday/interday was calculated as follows: where conc (measured) is the measured concentration values of standards; and conc (nominal) is the nominal concentration values of standards. Finally, the recovery test of marker compounds in samples was measured and calculated at three different concentration levels (80%, 100%, and 120%) of the contents for marker compounds in samples. e calculation of recovery ratio (%) was as follows: where conc (found) is the founded concentration of marker compounds in samples; conc (original) is the original concentration of marker compounds in samples; and conc (spiked) is the spiked concentration of marker compounds in samples.

Statistical Analysis.
All experiments were estimated in triplicates. All data were expressed by mean ± SD. e results were determined by one-way analysis of variance (ANOVA) using IBM SPSS statistic software (Ver. 22, IBM Co., Armonk, New York, USA). e posteriori tests were evaluated by Turkey's method. e statistical significance level (p value) was set up at p < 0.05 (95%)

Optimization of HPLC Conditions.
e HPLC analytical condition was optimized for the effective quantification of compounds 1-9 with antiosteoporosis and antioxidant activities in maesil. In order to select the best HPLC analytical condition of compounds 1-9, it was considered to various parameters of mobile phase, such as various columns, different pHs, and different buffer concentrations. In results, pH and buffer concentration of the best mobile phase were pH 4.0 and 2 mM sodium phosphate aqueous solution in combination with methanol-acetonitrile (1 : 1) mixture solution (Supplementary Figures S1, S3). And the highest UV absorption for these compounds was observed at UV 210 nm. Also, the minimal peak tailing and high selectivity at a column temperature of 45°C were observed (Supplementary Figure S4). All standards in samples were clearly separated within 75 min. Each peak of these compounds was identified with comparison of retention time (t R ), and UV and MS spectra (Supplementary Table S1 and S2).

Identification of Compounds 1-9 by LC-MS/MS.
All analysts were identified by the full scanning (scan range was 100∼1000 a.m.u.) in both positive and negative modes using LC-ESI-MS. e different collision energy voltages (−30∼18 V) were applied for characterizing these compounds 19 using full-scan mass spectra and multiple reaction monitoring (MRM) data, respectively (Supplementary Table S1). Compounds 1, 4, 5, 6, 7, and 8 were observed in precursor ions on MS spectra and their product ions of MS/MS spectra in both positive and negative modes. In particular, for compounds 19, the MS/MS fragment intense ions were further fragmented by multiple reaction monitoring (MRM), which showed major fragment intense ions. Among them, compounds 1, 7, and 8 were further fragmented with two kinds of fragmentation pathways (pathway I and II) in both of ionization mode.

Optimization of Sample Preparation for Active Compounds in Maesil.
To optimize the extraction condition of nine active compounds from maesil, the parameters such as species of extraction solvents, extraction time, and extraction method were estimated. In extraction effect comparison of various solvents as shown in Supplementary Figure S5, the content of active compounds in each solvent extract was different.
at is, each content of compounds 1-4 in methanol extracts was 0.84 ± 0.02, 0.25 ± 0.02, 0.44 ± 0.11, and 0.06 ± 0.01%, which were the highest than those of three compounds in other solvent extracts. Also, in statistical analysis using a one-way ANOVA test, p value for methanol extracts was shown as p < 0.05, and it indicated that the content of other solvents was different. And, the content of compounds 3 and 8 in methanol extract was 0.25 ± 0.02 and 0.05 ± 0.01%, respectively. It was similar to those of two compounds in ethanol and water extracts. Also, the content of compounds 5 and 7 in methanol extracts was 0.01 ± 0.00 and 0.12 ± 0.00%, and it was similar with those of ethanol and water extracts from the analysis results of the one-way ANOVA test in the range of 95% confidence interval (p < 0.05). While the content of compounds 6 and 9 in methanol extracts was 0.00 ± 0.00 and 0.04 ± 0.01%, respectively.
ey were similar to the contents of these compounds in other solvents except for n-butanol and acetone extracts, and their results were not distinguished in statistical analysis using a one-way ANOVA as p < 0.05. However, the total content of all active compounds in methanol extract was 1.82 ± 0.02%, and it was the highest than those of all active compounds in other solvent extracts. Also, it was distinguished to other solvent extracts in statistical analysis using a one-way ANOVA test. In the comparison of the extraction effectiveness by extraction solvents, the best extraction solvent for active compounds from mume fruit was verified as methanol.
On the other hand, in the comparison of extraction effectiveness by methanol-water ratio (water and 30%, 50%, 70%, and 100% methanol extracts) as shown in Supplementary Figure S6, the content of compounds 2, 4, and 6 in water solvent was 0.02 ± 0.00%, 0.05 ± 0.00%, and 0.03 ± 0.00%, respectively, and it was different as p < 0.05 in   Journal of Food Quality comparison of the content of compounds 1-9 among methanol-water solvent extracts by statistical analysis using a one-way ANOVA test. While the content of other compounds except for above three compounds in methanolwater solvent extracts was similar and was verified to be no significant as p> 0.05 in a one-way ANOVA test. And the total content of compounds 1-9 in 50% methanol and 70% methanol extracts among methanol-water solvent extracts was higher than other solvent extracts, but it was not significant as p > 0.05 in a one-way ANOVA test. However, compound 1 peak among compounds 1-9 in 70% and 100% methanol solvents was shown as fronting or a part of broken peak.
Finally, in the comparison of the extraction effect by two extraction methods using the reflux and sonication as shown in Supplementary Figure S7, the total content of compounds 1-9 in the sonication extraction method was increased from 1.30 ± 0.10% for 0 min to 1.61 ± 0.17% for 30 min, but it was quickly decreased to 0.77 ± 0.06% for 60 min, and that of each compound except for compound 2 was shown as a similar pattern for 30 min and 60 min. However, the total content of these compounds in the reflux method was 1.87 ± 0.0% higher than the sonication method, and it was similar from 15 min to 90 min except for 0 min. Also, it was not significant as p > 0.05 in the one-way ANOVA test.
When inclusively considering above results, the best condition to extract compounds 1-9 in maesil was 50% methanol using the reflux method for 30 min.

Method Validation of Active Compounds.
e validation of the developed method was performed using the following parameters in accordance with guidelines of the MFDS [40][41][42]. at is, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy, and recovery. e results of each parameter are shown in Table 3. LOD and LOQ levels of each compound were 0.37∼3.85 μg/mL and 1.24∼12.82 μg/mL, respectively. Range value of each standard was 16∼250 μg/mL (compound 1, 16∼500 μg/mL). Linearity (r 2 value) was shown by passing the starting point as over 0.999 in all standards. Precision of intraday and interday of each compound was 0.0∼3.9% and 0.1∼3.0% at three different concentrations (16.0, 63.0, and 250.0 μg/mL), respectively. In addition, accuracy of intraday and interday was 98.8 ± 2.1∼101.9 ± 0.9% and 98.6 ± 1.8∼109.2 ± 3.4%, respectively. Finally, the results of recovery tests as shown in Table 3 was 98.1∼104.4% (RSD values were 0.3∼5.2%) at three different concentrations (31.3, 63.0, and 125.0 μg/mL), respectively. In results, all data of these parameters were satisfied with the criteria of the MFDS guidelines.

Quantitation Analysis for Active Compounds in Unripe and Ripe Fruits.
In order to compare the content change of compounds 1-9 by the ripeness between unripe and ripe fruits, the quantitation for compounds 1-9 was carried out using a HPLC. Figure 3 shows typical HPLC chromatograms of compounds 1-9 in unripe and ripe fruits of maesil. e contents of these compounds 19 in whole of unripe fruits were at a range of 016∼1.81 mg/g with species of marker compounds. However, in ripe fruits, contents of compounds 19 were at a range of 0.09∼1.66 mg/g. In comparison of the compounds 19 contents for each part of fruits, the contents of compounds 4 and 6 with cytotoxicity in seed part of unripe fruits were increased as 20.8 and 40.1% rather than in that of unripe fruits. Also, that of compound 2 in seed part of ripe fruits during ripeness of unripe fruits was relatively decreased higher than in fresh part of ripe fruits. On the one hand, the contents of compounds 2, 5, 8, and 9 in both seed part of fruits were relatively higher than in flesh part. In contrast, those of compounds 1, 3, and 7 in flesh part of fruits were relatively higher than that in seed part (Table 4).

Conclusion
A simultaneous analytical method to analyze nine compound contents using HPLC to estimate effective sources and processing products of maesil related to antioxidant and antiosteoporosis activities successfully developed. e developed analytical method was estimated by validation parameters such as linearity, LODs, LOQs, precision, and accuracy of intraday and interday, and the recovery test was based on guidelines of MFDS. To check the change of nine compounds contents by ripeness between unripe and ripe maesil, the quantitation of compounds 1-9 was carried out using HPLC. Compounds 1-9 contents in ripe fruits were generally reduced rather than that in unripe fruits. While those of compounds 4 and 6 with cytotoxicity in seed part of ripe fruits were increased as 20.8% and 40.1% rather than in that of unripe fruits. Also, that of compound 2 in seed part of ripe fruits during ripeness of unripe fruits was relatively decreased higher than that in fresh part of ripe fruits. at is, all of these compounds in ripe fruits were reduced more than in unripe fruits. On the one hand, the contents of compounds 2, 5, 8, and 9 in seed part of fruits were relatively higher than that in flesh part. In contrast, those of compounds 1, 3, and 7 in flesh part of fruits were relatively higher than that in seed part. Generally, the contents of compounds 19 in unripe fruits were higher than that in ripe fruits. However, the contents of compounds 1-9 in each part (seed and fresh) of fruits were different according to species of compounds. It indicates that the selection of harvesting time and process part of fruits as the source of foods and medicines are important.

Data Availability
Antioxidants and antiosteoporosis activites of main compounds used in this study were already reported with the articles by the co-author.

Disclosure
Young Sik Park and Chong Woon Cho are the co-first authors.

Conflicts of Interest
e authors declare that there are no conflicts of interest.