The fruits of
The fruit of
Phytochemicals in mume fruits have been reported as various kinds of benzaldehyde, benzyl alcohols, flavonoids [
Some of these components’ contents with various bioactivities in maesil were developed with simultaneous analytical methods using gas chromatography-mass spectroscopy (GC-MS) for volatile organic compounds such as benzaldehyde, 2-hexanal, isolongifololyl acetate, palmitic acid, linalool, butyl acetate, linoleic acid, and squalene [
In contrast, the simultaneous analytical method for some kinds of compounds such as mumefural using high-performance 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-
Therefore, 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.
Compounds 1 and 5 as reference compounds for this study were obtained from Adooq Bioscience (Irvine, CA, USA) and Toronto Research Chemicals (Toronto, ON, Canada), respectively. And other seven reference standards (compounds 2, 3, 4, 6, 7, 8, and 9) were received from Prof. Young-Ho Kim (College of Pharmacy, Chungnam National University, Daejeon, Korea) in July 2014. The purity of all standards was over 95.6%. The water used was ultrapure deionized water of 18 MΩ produced by the ultrapure water manufacturing device (Optimos SHRO-UP, Shinhan Science Tech, Daejeon, Korea). All other solvents used were of HPLC or highest grade available. The chemical structures of these reference compounds are shown in Figure
Structures of compounds 1–9 used as reference standards for
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
Sample lists of
Harvest regions | No. of samples | Sample weight (average g/each fresh fruit) | |
---|---|---|---|
Unripe fruitsa | Ripe fruitsb | ||
Daejeon | 1 | 12.96 ± 5.69 | 11.46 ± 2.54 |
Gwangyang | 2 | 13.58 ± 1.90 | 12.17 ± 0.40 |
Gyeryong | 4 | 17.12 ± 7.82 | 20.57 ± 9.61 |
Gurye | 4 | 18.08 ± 2.55 | 17.36 ± 2.52 |
Jangheung | 2 | 17.44 ± 4.41 | 18.33 ± 4.32 |
Jinju | 4 | 20.33 ± 2.66 | 22.65 ± 2.41 |
Nonsan | 2 | 20.75 ± 3.28 | 25.85 ± 4.87 |
Sacheon | 1 | 10.84 ± 2.97 | 13.03 ± 1.36 |
Suncheon | 1 | 17.70 ± 1.84 | 18.80 ± 2.36 |
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
The 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
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. The identification of each compound was carried out on the Kinetex F5, 2.6
The 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
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. The calculation of recovery ratio (%) was as follows:
All experiments were estimated in triplicates. All data were expressed by mean ± SD. The results were determined by one-way analysis of variance (ANOVA) using IBM SPSS statistic software (Ver. 22, IBM Co., Armonk, New York, USA). The posteriori tests were evaluated by Turkey’s method. The statistical significance level (
The 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
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. The 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
For compounds 5, 7, and 8 among nine compounds, the subfragmentation patterns of MS/MS for these compounds are newly showed as follows. The fragmentation pathway for compound 5 in positive mode showed four fragment intense peaks, that is, [M-161]+ at
Compound 5 showed the fragmentation pathway with two fragment intense peaks as [M-H]- at
For compounds 7 and 8, two compounds had same precursor ions such as [M + NH4]+ at m/
While compound 8 showed different fragment ion patterns with [M-107]+ at
Summary of UV, MS, and MS/MS spectra for nine compounds obtained from
No. | Retention time (min) | UV absorbance ( | Molecular formula | Molecular weight | Precursor ion (m/ | Product ion (m/ | Identified name |
---|---|---|---|---|---|---|---|
1 | 27.3 | 217, 233, 324 | C16H18O9 | 354 | [M+H]+ (355) | 89 (21.7), 117 (13.0), 135 (15.2), 145 (21.7), 163 (100.0) | NCA |
[M-H]− (353) | 85 (2.4), 135 (53.9), 173 (2.6), 179 (62.0), 191 (100.0) | ||||||
2 | 33.3 | 258 | C13H18O6 | 270 | [M + HCOO]− (315) | 269 (21.2) | BGP |
3 | 34.0 | 235 | C11H16O7 | 284 | [M+Na]+ (307) | 24 (2.6), 177 (2.3), 194 (52.3), 266 (100.0) | GPB |
4 | 36.6 | 203, 270 | C20H27NO11 | 457 | [M + NH4]+ (475) | 69 (70.6), 85 (100.0), 145 (41.0), 163 (47.7), 325 (56.8) | Amygdalin |
[M-H]− (356) | 323 (100.0) | ||||||
5 | 38.4 | 196, 233 | C15H22O9 | 346 | [M+H]+ (347) | 43 (1.1), 125 (15.9), 153 (31.7), 185 (100.0) | TMPGP |
[M + HCOO]−(391) | 153 (23.0), 345 (38.0) | ||||||
6 | 40.7 | 190, 260 | C14H17NO6 | 295 | [M + NH4]+ (313) | 231 (55.0), 272 (100.0) | Prunasin |
[M + HCOO]− (340) | 161 (100.0), 188 (57.4), 294 (55.3) | ||||||
7 | 41.1 | 202, 277 | C16H18O9 | 402 | [M + NH4]+ (420) | 73 (83.5), 91 (100.0), 97 (52.2), 163 (69.6), 295 (78.3) | BAPGP |
[M + HCOO]−(447) | 101 (8.1), 131 (4.3), 269 (13.5), 401 (100.0) | ||||||
8 | 52.1 | 210, 244 | C16H18O9 | 402 | [M + NH4]+ (420) | 57 (30.0), 73 (45.0), 91 (100.0), 97 (50.0), 295 (70.0) | BXPGP |
[M + HCOO]−(447) | 59 (4.8), 101 (14.3), 161 (19.0), 269 (59.5), 401 (100.0) | ||||||
9 | 52.4 | 210, 261 | C16H18O9 | 290 | [M+H]+ (291) | 95 (0.4), 123 (46.3), 133 (1.9), 139 (100.0) | (−)-Epicatechin |
The predicted MS/MS fragmentation pathways of compounds 1–9 in positive and negative modes.
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
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
Finally, in the comparison of the extraction effect by two extraction methods using the reflux and sonication as shown in Supplementary Figure
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.
The validation of the developed method was performed using the following parameters in accordance with guidelines of the MFDS [
Method validation results of the developed method for analysis of compounds 1–9 in samples.
Compounds | Range | Linearity | Calibration | LODs | LOQs | Concentration | Intraday ( | Interday ( | Original concentration | Spiked concentration | Recovery | RSD | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Precision (%RSD) | Accuracy (%) | Precision (%RSD) | Accuracy (%) | |||||||||||
1 | 16–500 | 0.9994 | 2.12 | 7.06 | 16 | 2.1 | 98.8 ± 2.1 | 1.6 | 102.9 ± 1.6 | 214 | 171 | 98.1 | 2.9 | |
63 | 0.9 | 100.0 ± 0.9 | 1.6 | 102.6 ± 1.6 | 214 | 103.1 | 2.9 | |||||||
250 | 0.5 | 100.3 ± 0.5 | 0.1 | 100.1 ± 0.1 | 257 | 100.1 | 0.3 | |||||||
2 | 16–250 | 0.9996 | 0.72 | 2.41 | 16 | 0.0 | 100.0 ± 0.0 | 2.0 | 101.5 ± 2.0 | 34 | 27 | 100.0 | 1.9 | |
63 | 1.1 | 100.6 ± 1.1 | 1.8 | 98.6 ± 1.8 | 34 | 101.0 | 1.9 | |||||||
250 | 0.2 | 99.8 ± 0.2 | 0.4 | 99.7 ± 0.4 | 41 | 101.3 | 2.1 | |||||||
3 | 16–250 | 0.9997 | 3.80 | 11.53 | 16 | 2.9 | 101.9 ± 3.0 | 2.4 | 101.4 ± 2.5 | 120 | 96 | 102.1 | 3.7 | |
63 | 0.0 | 100.0 ± 0.0 | 2.7 | 102.7 ± 2.8 | 120 | 99.8 | 3.3 | |||||||
250 | 0.8 | 101.7 ± 0.8 | 2.3 | 101.4 ± 2.3 | 144 | 101.8 | 1.0 | |||||||
4 | 16–250 | 0.9992 | 0.37 | 1.24 | 16 | 0.0 | 100.0 ± 0.0 | 3.2 | 109.2 ± 3.4 | 45 | 36 | 100.0 | 0.9 | |
63 | 1.1 | 100.7 ± 1.1 | 2.9 | 99.6 ± 2.9 | 45 | 100.0 | 1.7 | |||||||
250 | 0.3 | 99.8 ± 0.3 | 1.2 | 100.3 ± 1.2 | 54 | 99.5 | 1.2 | |||||||
5 | 8–250 | 0.9995 | 0.64 | 1.95 | 16 | 1.4 | 99.2 ± 1.3 | 1.9 | 100.5 ± 2.0 | 11 | 9 | 101.0 | 3.9 | |
63 | 1.2 | 100.5 ± 1.2 | 1.5 | 98.9 ± 1.5 | 11 | 103.3 | 0.9 | |||||||
250 | 0.2 | 99.8 ± 0.2 | 1.3 | 99.5 ± 1.3 | 13 | 102.7 | 0.7 | |||||||
6 | 16–250 | 0.9996 | 1.96 | 6.55 | 16 | 0.0 | 100.0 ± 0.0 | 1.9 | 103.5 ± 1.9 | 49 | 39 | 102.6 | 3.6 | |
63 | 0.6 | 100.4 ± 0.6 | 1.7 | 99.1 ± 1.7 | 49 | 103.0 | 0.9 | |||||||
250 | 0.3 | 99.7 ± 0.3 | 0.3 | 99.8 ± 0.3 | 59 | 102.6 | 1.7 | |||||||
7 | 16–250 | 0.9996 | 0.61 | 2.04 | 16 | 0.0 | 100.0 ± 0.0 | 2.7 | 103.0 ± 2.7 | 21 | 17 | 101.6 | 3.2 | |
63 | 0.7 | 100.4 ± 0.7 | 2.8 | 99.8 ± 2.7 | 21 | 101.1 | 1.6 | |||||||
250 | 0.2 | 99.9 ± 1.2 | 1.7 | 100.6 ± 1.8 | 25 | 100.4 | 1.3 | |||||||
8 | 16–250 | 0.9995 | 1.60 | 5.33 | 16 | 0.0 | 100.0 ± 0.0 | 3.0 | 102.2 ± 3.0 | 106 | 85 | 104.3 | 5.2 | |
63 | 0.8 | 100.5 ± 0.8 | 2.2 | 99.4 ± 2.2 | 106 | 100.2 | 2.9 | |||||||
250 | 0.2 | 99.9 ± 0.2 | 1.4 | 99.6 ± 1.4 | 127 | 102.3 | 1.3 | |||||||
9 | 16–250 | 0.9997 | 3.85 | 12.82 | 16 | 3.9 | 96.7 ± 3.8 | 0.3 | 99.6 ± 0.3 | 40 | 32 | 101.6 | 2.6 | |
63 | 1.1 | 100.2 ± 1.1 | 0.3 | 99.7 ± 0.3 | 40 | 100.6 | 2.9 | |||||||
250 | 0.1 | 99.9 ± 0.1 | 0.2 | 99.8 ± 0.2 | 48 | 103.2 | 1.7 |
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
High-performance chromatograms of standard mixtures of compounds 1–9 in unripe and ripe
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
The contents of compounds 1–9 in each part of the unripe and ripe
Compounds | Ripeness | Content of marker compounds in each part of fruits (mg/g) | ||
---|---|---|---|---|
Whole | Seed | Flesh | ||
1 | Unripe fruits | 0.79 ± 0.23 | 0.19 ± 0.19 | 0.47 ± 0.26 |
Ripe fruits | 0.75 ± 0.44 | 0.17 ± 0.19 | 0.61 ± 0.38 | |
2 | Unripe fruits | 1.18 ± 0.35 | 1.18 ± 0.12∗ | 0.57 ± 0.24 |
Ripe fruits | 0.56 ± 0.27 | 0.27 ± 0.12 | 0.43 ± 0.20 | |
3 | Unripe fruits | 0.62 ± 0.27 | 0.28 ± 0.20 | 0.36 ± 0.17 |
Ripe fruits | 0.59 ± 0.35 | 0.29 ± 0.17 | 0.38 ± 0.25 | |
4 | Unripe fruits | 1.81 ± 1.06 | 1.09 ± 0.64 | 0.18 ± 0.15 |
Ripe fruits | 1.66 ± 1.29 | 1.32 ± 0.98 | 0.11 ± 0.09 | |
5 | Unripe fruits | 0.16 ± 0.09 | 0.10 ± 0.03 | 0.06 ± 0.05 |
Ripe fruits | 0.09 ± 0.04 | 0.09 ± 0.03 | 0.03 ± 0.01 | |
6 | Unripe fruits | 0.29 ± 0.19 | 0.32 ± 0.26 | 0.17 ± 0.11 |
Ripe fruits | 0.22 ± 0.08 | 0.44 ± 0.37 | 0.17 ± 0.07 | |
7 | Unripe fruits | 1.15 ± 0.53 | 0.42 ± 0.46 | 0.93 ± 0.48 |
Ripe fruits | 1.38 ± 0.57 | 0.26 ± 0.14 | 0.84 ± 0.20 | |
8 | Unripe fruits | 0.90 ± 0.57 | 0.66 ± 0.69 | 0.47 ± 0.24 |
Ripe fruits | 0.66 ± 0.34 | 0.50 ± 0.36 | 0.54 ± 0.22 | |
9 | Unripe fruits | 0.90 ± 0.46 | 0.94 ± 0, 18 | 0.22 ± 0.27 |
Ripe fruits | 0.68 ± 0.37 | 0.88 ± 0.08 | 0.18 ± 0.10 |
All data were repeated with the triplet, nonpair
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. The 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. That 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.
Antioxidants and antiosteoporosis activites of main compounds used in this study were already reported with the articles by the co-author. The results of bioactivities in articles are available at doi 10.1007/s12272-014-0389-2, 10.1016/j.foodchem.2014.01.078, and 10.1016/j.bmcl.2014.01.028.
Young Sik Park and Chong Woon Cho are the co-first authors.
The authors declare that there are no conflicts of interest.
Young Sik Park and Chong Woon Cho contributed equally.
The authors are very grateful to Professor Young Ho Kim at Chungnam National University for providing reference standards in the experiment. This work was supported by the research fund of Chungnam National University.
Data of both Tables S1 and S2 explain the contents of MS/MS and UV spectra to support the identification of compounds 1–9 in