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Simultaneous Quantitative Determination of Six Caffeoylquinic Acids in Matricaria chamomilla L. with High-Performance Liquid Chromatography.

1. Introduction

Matricaria chamomilla L. (M. chamomilla) is a kind of famous herbaceous plant indigenous to Europe. It has been naturalized to many countries and regions of the world for thousands of years as one of the most popular medicinal plants in folk and traditional medicine [1, 2]. The capitula of M. chamomilla (CMC), named as "German Chamomile" in Europe, is included in the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), and British Pharmacopoeia (BP) to treat a series of diseases, such as digestive ailments, restlessness, mild insomnia due to nervous disorders, inflammation, and irritations of the skin and mucosa [3]. The dried whole herb of M. chamomilla (WHMC) named as "Yangganju" is recorded in Medicine and Pharmacy of Traditional Uyghur Medicine in Xinjiang China to treat stomach upset, dysuria, skin itching, blurred vision, cystitis, and stomatitis [4]. Moreover, essential oil of M. chamomilla is used extensively in cosmetics and aromatherapy in Europe [5]. In sight of its significant therapeutic applications, a large group of active constituents have been identified from M. chamomilla by researchers [6-9]. Our group has been devoted to the study on potential bioactive natural products in traditional medicinal plants for several decades [10-13]. In our previous chemical constituent study of M. chamomilla, except chlorogenic acid, five other caffeoylquinic acids (neochlorogenic acid, cryptochlorogenic acid, and isochlorogenic acid A, B, and C) were identified from M. chamomilla for the first time (Figure 1). The CAs represent a class of interesting natural products with wide pharmacological activities including antioxidant [14], anti-inflammatory [15,16], antimicrobial [17], enzyme inhibition [18], hepatocyte protection [19], platelet aggregation inhibition [20], antihepatic fibrosis [21], and anti-SARS [22]. According to these previous reports, the pharmacological activities of CAs were consistent with the efficacy of CMC and WHMC, and thus, the CAs should be the active ingredients. However, the method for the determination of CAs in M. chamomilla has not been reported. Herein, to develop improved solution for the quality evaluation of CMC and WHMC, a simple and effective HPLC method was developed for simultaneous quantitative analyses of the six CAs.

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2. Experimental

2.1. Chemicals, Reagents, and Materials. Acetonitrile (HPLC grade) was purchased from Fisher (Canada). Phosphoric acid (HPLC grade) was purchased from CNW Technology (Germany). Purified water was purchased from Wahaha Company (China). Syringe filter (0.45 [micro]m) was purchased from ANPEL (China). Standard compounds of neochlorogenic acid (NCA), cryptochlorogenic acid (CCA), and isochlorogenic acid A, B, and C (ICA, ICB, and ICC) were purchased from Chengdu Herbpurify CO., LTD. Standard compound of chlorogenic acid (CA) was purchased from National Institutes for Food and Drug Control. The purity of the six reference compounds was determined to be more than 98% by normalization of the peak areas detected by HPLC-DAD.

A total of 34 samples were collected from different geographical areas and identified as Matricaria chamomilla (L.) by Professor Chunsheng Liu and his group at Beijing University of Chinese Medicine, as shown in Table 1. The samples were airdried (indoor) at the origin of collection. Among them, 3 representative geographical batches (D1, D10, and D12) were categorized into different parts of M. chamomilla (roots, stems, and leaves) in the laboratory. These medicinal materials were deposited in Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

2.2. Apparatus. All medicinal materials were ground by high-speed disintegrator (Tianjin Taisite FW100). All sample extraction processes were carried out in a water bath (Tianjin Taisite DK-98-1). All medicinal materials and standard compounds were weighed by electronic analytical balances (Mettler Toledo AL204-IC and XS105 Dual Range). The HPLC system was SHIMADZU Prominence LC-20A (Shimadzu Corporation, Tokyo, Japan) equipped with CBM-20Alite system controller, LC-20AT pump, CTO-20A column oven, SPD-M20A UV-Vis detector, SIL-20A autoinjector, DGU-M20A5 degasser, and Shimadzu LC-solution work station.

2.3. Chromatographic Conditions. The separation of CAs was carried out on a Waters XBridge Shield RP [C.sub.18] column (4.6 mm x 250 mm, 5 [micro]m) at 35[degrees]C with a flow rate of 1.0 mL/ min. The detection wavelength was 327 nm with an injection volume of 10 [micro]L. The optimized mobile phase system was A (acetonitrile/phosphoric acid, 99.5/0.5, v/v) and B (water/ phosphoric acid, 99.5/0.5, v/v) with a gradient elution program: A/B = 12/82 (0-13 min), A/B = 12/82-25/75 (13-15 min), and A/B = 25/75 (15-30 min). All data were acquired and processed by Shimadzu LC solution software.

2.4. Preparation of Standard Solution. Standards were weighed accurately and dissolved in 10 mL of 70% aqueous methanol to prepare the standard mix stock solution of NCA (270 [micro]g/mL), CCA (212 [micro]g/mL), CA (240 [micro]g/mL), ICA (260 [micro]g/mL), ICB (218 [micro]g/mL), and ICC (246 [micro]g/mL). The standard mix stock solution was stored at 4[degrees]C and filtered through a 0.45 [micro]m syringe filter before HPLC analysis.

2.5. Preparation of Sample Solution. A conical flask was charged with 0.3 g of sample and 15 mL of 70% aqueous methanol. Then, the sample was refluxed for 45 min. After cooling to room temperature, replenish the loss of the solvent with 70% aqueous methanol. Finally, the sample solution was filtered through a 0.45 [micro]m syringe filter prior to HPLC analysis.

2.6. Method Validation. The proposed method was validated according to CFDA guidelines for the validation of analytical methods for pharmaceutical quality standard, with respect to linearity, lower limit of detection (LLOD) and quantification (LLOQ), precision, repeatability, stability, and accuracy.

The standard mix stock solutions at 12 different concentrations were injected for two replicates. The calibration curve was constructed by least square fit of the data with the peak area (y-axis) versus the injection amounts (x-axis) for each compound. The standard mix stock solution was further diluted to explore the LLOD and LLOQ. The LLOD and LLOQ were determined at a signal-to-noise (S/N) ratio of 3 and 10, respectively.

The precision was evaluated with standard solution under the selected optimal conditions in six replicates continuously. To further evaluate the repeatability of the developed assay, the sample was analyzed in six replicates. Stability was tested with the sample at room temperature (25[degrees]C) and analyzed at 0, 4, 12, 24, 48, and 72 h, respectively. Recovery tests were performed to evaluate the accuracy of the developed method. The accurate amounts of six CAs were weighed and spiked to certain amounts of the sample powder and were then extracted and analyzed in accordance with the method described above. The spiked amount of each standard was adjusted to provide a similar concentration present in the sample. The recovery rate (%) was measured for six replicates.

3. Results and Discussion

3.1. Optimization of the Extraction Procedure. During sample preparation, the extraction parameters, e.g., extraction method, solvent, extraction time, and solvent volume, were optimized. The efficiency of the extraction procedure was evaluated using different extraction methods, i.e., reflux and ultrasonic-assisted method. The results demonstrated that the reflux method provided the higher value in the content of the target compounds than the ultrasonic-assisted method. Then, the other factors were investigated using monofactor analysis, i.e., extraction solvent (30%, 50%, and 70% aqueous methanol (v/v) and pure methanol), extraction time (30 min, 45 min, and 60 min), and solvent volume (5mL, 10 mL, 15 mL, and 20 mL). As a result, the optimized extraction procedure was confirmed to be refluxed with 15 mL of 70% aqueous methanol solution (v/v) for 45 min.

3.2. Optimization of the Chromatographic Conditions. In order to separate the six CAs, a gradient method was developed to determine all the constituents in one analysis. Various mixtures of mobile phases were tested, such as methanol and water, methanol (0.1% formic acid) and water (0.1% formic acid), and methanol (0.5% phosphoric acid) and water (0.5% phosphoric acid), but the separation was unsatisfactory. However, by replacing methanol with acetonitrile, the special mobile phase system (acetonitrile (0.5% phosphoric acid) and water (0.5% phosphoric acid)) significantly improved the separation. We also tried to simplify the mobile phase system as acetonitrile and water (0.5% phosphoric acid), but the separation was unsatisfactory. Due to the similar structure, the UV absorption spectrograms of the six CAs were almost identical. The detection wavelength was selected at the maximum absorption of 327 nm.

3.3. Method Validation. The analytical method was validated with respect to the linearity, LLOD, LLOQ, precision, repeatability, stability, and accuracy. The linear ranges, regression equations, and correlation coefficients obtained from typical calibration curves and LLOD (S/N

= 3) and LLOQ (S/N = 10) are shown in Table 2. All calibration curves showed excellent linearity, and the correlation coefficients were higher than 0.999.

As shown in Table 3, the precision of the method was evaluated with peak areas obtained for each analyte and expressed as relative standard deviation (RSD). The RSD of intraday and interday was 0.49% and 0.09% for NCA, 0.65% and 0.03% for CCA, 0.06% and 0.03% for CA, 0.10% and 0.02% for ICA, 0.03% and 0.04% for ICB, and 0.12% and 0.05% for ICC, respectively. The method is repeatable, with the RSD was in the range of 1.0%~2.3%. The CAs were proved to be stable in sample solution within 72 h at room temperature with the RSD below 1.1%. As shown in Table 4, the extraction recoveries were performed to evaluate the accuracy of the developed method. The mean recoveries were in the range of 100.7%~101.5% with the RSD less than 3.0% for all the six CAs. In general, the developed method is precise, repeatable, and accurate for the simultaneous quantitative determination of the six CAs in M. chamomilla.

3.4. Sample Analysis. The established method has been successfully applied for the simultaneous determination of M. chamomilla samples collected from different geographical areas, as shown in Table 5 and Figure 2. The results showed that the contents of six CAs in CMC and WHMC collected from different geographical areas were different, and the contents of six CAs in different parts of specific M. chamomilla were also different.

Although the position of the substituent caffeoyl group in NCA, CCA, and CA is different, their mother nucleus structures are the same. In order to simplify the results, NCA, CCA, and CA were defined as total chlorogenic acids (TCAs). Similarly, ICA, ICB, and ICC were defined as total isochlorogenic acids (TICAs). The contents of six CAs, TCAs, and TICAs in CMC were generally higher than WHMC. The TCA contents in WHMC series and CMC series were in the range of 0.17~1.98 mg/g and 0.84~3.98 mg/ g, respectively. The TICA contents in these series were in the range of 0.47~5.84mg/g and 1.38~9.78 mg/g, respectively.

Compared with the literature report, the results showed that the contents of CAs in CMC were comparable to that of apigenin-7-O-glucoside (about 0.2~6.2 mg/g), higher than that of most flavonoids (such as luteolin, apigenin, and 7methoxycoumarin; far less than 1.0 mg/g) [8, 9, 23]. Because the pharmacological activities of CAs were consistent with the efficacy of CMC and WHMC, the CAs together with coumarins and flavonoids could all be considered as the main bioactive ingredients.

4. Conclusion

In this work, an HPLC method was established for the simultaneous determination of six CAs with pharmacological activities in M. chamomilla for the first time. The established method was validated by linearity, reproducibility, recovery, and precision; all parameters found satisfactory. This newly established HPLC method will be helpful in the quality assessment of CMC, WHMC, and related herbal formulas in future.

https://doi.org/10.1155/2019/4352832

Data Availability

The chromatographic data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was financially supported by the Key Project at Central Government Level: The Ability Establishment of Sustainable Use for Valuable Chinese Medicine Resources (2060302). The authors are grateful to Dr. Rizwan Elahi for providing language assistance and grammar check.

References

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Yifan Zhao [ID], Peng Sun [ID], Yue Ma [ID], Kun Wang [ID], Xiaoqiang Chang [ID], Yue Bai [ID], Dong Zhang [ID], and Lan Yang [ID]

Artemisinin Research Center and Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

Correspondence should be addressed to Dong Zhang; dzhang@icmm.ac.cn and Lan Yang; lyang@icmm.ac.cn

Received 12 April 2019; Revised 13 August 2019; Accepted 9 September 2019; Published 3 November 2019

Academic Editor: Artur M. S. Silva

Caption: Figure 2: HPLC chromatograms of the (a) standard mix stock solution and (b) sample (D10 roots). 1, NCA; 2, CCA; 3, CA; 4, ICB; 5, ICA; 6, ICC.
Table 1: Sample information of M. chamomilla collected from
different geographical areas.

Samples       Parts

D1             WHMC
D3             WHMC
D4             WHMC
D5             WHMC

D6             WHMC
D7             WHMC
D8             WHMC

D9             WHMC

D10            WHMC

D11            WHMC

D12            WHMC
H1             CMC
H2             CMC
H3             CMC
H4             CMC
H5             CMC
H6             CMC

H7             CMC
H8             CMC

H9             CMC

H10            CMC

H11            CMC

H12            CMC

H13            CMC
H14            CMC
D1 roots      Roots
D1 stems      Stems
D1 leaves     Leaves

D10 roots     Roots

D10 stems     Stems

D10 leaves    Leaves

D12 roots     Roots
D12 stems     Stems
D12 leaves    Leaves

Samples                            Sources

D1                Tacheng County, Xinjiang Autonomous Region
D3               Hotan Prefecture, Xinjiang Autonomous Region
D4                Jimsar County, Xinjiang Autonomous Region
D5               Yili Kazakh Autonomous Prefecture, Xinjiang
                              Autonomous Region
D6               Huo Cheng County, Xinjiang Autonomous Region
D7                Tacheng County, Xinjiang Autonomous Region
D8                Qapqal County, Yili Autonomous Prefecture,
                          Xinjiang Autonomous Region
D9            Hospital of Xinjiang Traditional Uyghur Medicine,
                          Xinjiang Autonomous Region
              Institute of Medicinal Plant Development, Sanming
D10             City Academy of Agricultural Sciences, Fujian
                                   Province
D11                Xuancheng Yueping Ecological Technology
                    Development Co., Ltd., Anhui Province
D12            Beijing University of Chinese Medicine, Beijing
H1                Tacheng County, Xinjiang Autonomous Region
H2                        Xinjiang Autonomous Region
H3                      Guangzhou, Guangdong Province
H4                        Qingdao, Shandong Province
H5                          Fuyang, Anhui Province
H6                Qapqal County, Yili Autonomous Prefecture,
                          Xinjiang Autonomous Region
H7                        Tai'an, Shandong Province
H8                   South of Xinjiang Autonomous Region
                   Germany import (grade 1), Woyang County
H9              Hongyang County Chinese Herbal Medicine Sales
                             Co., Ltd., Chongqing
                   Germany import (grade 2), Woyang County
H10             Hongyang County Chinese Herbal Medicine Sales
                             Co., Ltd., Chongqing
H11                       Xuancheng, Anhui Province
              Institute of Medicinal Plant Development, Sanming
H12             City Academy of Agricultural Sciences, Fujian
                                   Province
H13            Beijing University of Chinese Medicine, Beijing
H14            Beijing University of Chinese Medicine, Beijing
D1 roots          Tacheng County, Xinjiang Autonomous Region
D1 stems          Tacheng County, Xinjiang Autonomous Region
D1 leaves         Tacheng County, Xinjiang Autonomous Region
              Institute of Medicinal Plant Development, Sanming
D10 roots       City Academy of Agricultural Sciences, Fujian
                                   Province
              Institute of Medicinal Plant Development, Sanming
D10 stems       City Academy of Agricultural Sciences, Fujian
                                   Province
              Institute of Medicinal Plant Development, Sanming
D10 leaves      City Academy of Agricultural Sciences, Fujian
                                   province
D12 roots      Beijing University of Chinese Medicine, Beijing
D12 stems      Beijing University of Chinese Medicine, Beijing
D12 leaves     Beijing University of Chinese Medicine, Beijing

Samples        Collection time

D1               March, 2016
D3            September 19, 2016
D4            September 19, 2016
D5            February 17, 2017

D6               March, 2017
D7              March 23, 2017
D8              March 23, 2017

D9              April 07, 2017

D10             June 14, 2017

D11             June 14, 2017

D12             June 24, 2017
H1               March, 2016
H2             September, 2017
H3             September, 2017
H4             September, 2017
H5             September, 2017
H6             September, 2017

H7             September, 2017
H8             September, 2017

H9             September, 2017

H10            September, 2017

H11            September, 2017

H12             June 14, 2017

H13              May 13, 2017
H14             July 20, 2017
D1 roots         March, 2016
D1 stems         March, 2016
D1 leaves        March, 2016

D10 roots       June 14, 2017

D10 stems       June 14, 2017

D10 leaves      June 14, 2017

D12 roots       June 24, 2017
D12 stems       June 24, 2017
D12 leaves      June 24, 2017

Note. H1, D1, D1 roots, D1 stems, and D1 leaves were derived from
D1; H10, D10, D10 roots, D10 stems, and D10 leaves were derived
from D10; H13, H14, D12, D12 roots, D12 stems, and D12 leaves were
derived from D12.

Table 2: Linear ranges, LLOD, LLOQ, and characteristic
parameters of calibration curves.

Name   Calibration equation     r     Linear range (ng)

NCA    y = 2689399x + 8525    0.999    0.86-5.40 x [10.sup.3]
CCA     y = 2557852x + 46     0.999    0.68-4.24 x [10.sup.3]
CA      y = 3178855x - 953    0.999    0.77-4.80 x [10.sup.3]
ICA    y = 3567273x + 9390    0.999    0.83-5.20 x [10.sup.3]
ICB    y = 2821879x + 1015    0.999    0.70-4.36 x [10.sup.3]
ICC     y = 3200246x + 678    0.999    0.79-4.92 x [10.sup.3]

Name   LLOQ (ng)   LLOD (ng)

NCA      0.86        0.17
CCA      0.68        0.14
CA       0.77        0.15
ICA      0.83        0.17
ICB      0.70        0.14
ICC      0.79        0.16

Note. y: peak area at 327 nm; x: injection amount (ng); r:
correlation coefficient for 12 data points in the calibration
curves (n = 2); LLOQ: lower limit of quantification (S/N = 10);
LLOD: lower limit of detection (S/N = 3).

Table 3: Method validation for determination of six CAs.

             Precision (n = 6)    Repeatability    Stability (RSD %)
                  (RSD %)        (n = 6) (RSD %)

Analytes   Intraday   Interday

NCA          0.49       0.09          1.68               1.09
CCA          0.65       0.03          2.24               1.09
CA           0.06       0.03          1.28               0.16
ICA          0.10       0.02          1.11               0.63
ICB          0.03       0.04          1.65               0.35
ICC          0.12       0.05          2.05               0.62

Table 4: Recoveries of six CAs as determined by the standard
addition method (n = 6).

Name   Sample weight (g)   Original (mg)   Spiked (mg)   Found (mg)

             0.251             0.022          0.021        0.043
             0.250             0.021          0.021        0.043
NCA          0.251             0.022          0.021        0.044
             0.252             0.022          0.021        0.043
             0.251             0.022          0.021        0.043
             0.250             0.021          0.021        0.043
             0.251             0.012          0.011        0.023
             0.250             0.012          0.011        0.023
CCA          0.251             0.012          0.011        0.024
             0.252             0.012          0.011        0.024
             0.251             0.012          0.011        0.024
             0.250             0.012          0.011        0.024
             0.251             0.204          0.204        0.410
             0.250             0.204          0.204        0.408
CA           0.251             0.205          0.204        0.415
             0.252             0.205          0.204        0.409
             0.251             0.205          0.204        0.409
             0.250             0.204          0.204        0.407
             0.251             0.410          0.401        0.815
             0.250             0.409          0.401        0.813
ICA          0.251             0.411          0.401        0.826
             0.252             0.411          0.401        0.815
             0.251             0.411          0.401        0.820
             0.251             0.128          0.119        0.248
             0.250             0.128          0.119        0.248
ICB          0.251             0.128          0.119        0.251
             0.252             0.128          0.119        0.249
             0.251             0.128          0.119        0.249
             0.250             0.127          0.119        0.246
             0.251             0.188          0.175        0.366
             0.250             0.188          0.175        0.365
ICC          0.251             0.189          0.175        0.369
             0.252             0.189          0.175        0.366
             0.251             0.188          0.175        0.364
             0.250             0.187          0.175        0.362

Name   Recovery (a) (%)   Average recovery (%)   RSD (%)

            100.70
            99.58
NCA         102.95               100.74           1.18
            100.48
            100.87
            99.85
            98.26
            96.40
CCA         102.25               100.92           2.83
            102.49
            103.05
            103.08
            100.84
            100.10
CA          102.94               100.77           1.09
            100.28
            100.44
            100.05
            100.99
            100.76
ICA         103.57               101.52           1.11
            100.63
            102.08
            101.07
            101.34
ICB         103.17               101.53           0.94
            101.37
            101.92
            100.32
            101.89
            101.52
ICC         103.37               101.55           1.06
            101.59
            100.68
            100.26

Note. (a) Recovery (%) = [(found-original)/spiked] x 100; RSD:
relative standard deviation.

Table 5: Heatmap of six CA contents in M. chamomilla samples (n = 2).

Sample lists                  Content (mg/g)

                  NCA        CCA        CA         ICA

D1              0.059#     0.045#     0.289##     0.826
D3              0.117##    0.076##    1.306*      3.360
D4              0.118#     0.028#    0.667###     0.940
D5              0.040#     0.031#     0.216#      0.584
D6              0.037#     0.030#    0.446###     1.107
D7              0.060##    0.061##   0.437###     0.971
D8              0.031#     0.021#     0.119#      0.395
D9              0.259##    0.164##    1.206*      0.807
D10            0.409###    0.049#     1.526*      1.364
D11             0.084##    0.017#     0.218##     0.469
D12             0.124##    0.009#     0.181##     0.228
H1              0.211##    0.173##    2.706**     4.445
H2             0.386###    0.180##    1.837*      2.411
H3             0.406###    0.273##    1.652*      1.786
H4             0.401###    0.157##    1.951*      1.923
H5             0.415###    0.206##    1.374*      1.615
H6              0.325##    0.170##    0.994*      0.847
H7              0.314##    0.240##    2.398**     1.523
H8             0.403###    0.183##    1.180*      1.196
H9              0.350##    0.226##    1.686*      1.525
H10            0.529###    0.251##    1.501*      2.095
H11            0.557###    0.339##    3.081**     2.529
H12             0.726*     0.227##    1.703*      4.615
H13             0.145#     0.043#    0.662###     0.284
H14            0.389###    0.047#    0.400###     0.398
D1 roots        0.082#     0.050#    0.418###     0.860
D1 stems        0.038#     0.024#     0.167#      0.493
D1 leaves       0.045#     0.040#     0.135#      0.229
D10 roots       0.086#     0.045#     0.815*      1.603
D10 stems       0.201##    0.024#     0.876*      0.699
D10 leaves      1.683**    0.088##   4.086***     5.152
D12 roots       0.022#     0.005#     0.038#      0.105
D12 stems       0.081##    0.007#     0.092#      0.149
D12 leaves      0.180##    0.009#     0.212##     0.247

Sample lists                 Content (mg/g)

                 ICB        ICC       TCAs      TICAs

D1             0.390**    0.466###    0.393     1.681
D3             1.093***   1.384**     1.499     5.837
D4              0.164#    0.282##     0.813     1.386
D5             0.310##    0.334##     0.287     1.228
D6             0.454###   0.501###    0.513     2.061
D7             0.329##    0.546###    0.558     1.846
D8              0.157#     0.166#     0.171     0.718
D9              0.150#    0.346##     1.629     1.303
D10            0.466###    0.588*     1.984     2.418
D11             0.137#    0.263##     0.320     0.869
D12             0.052#    0.187##     0.313     0.467
H1             0.541###    2.102*     3.090     7.088
H2             1.912**     1.879*     2.403     6.202
H3             2.395**    2.481**     2.330     6.662
H4              1.649*     1.706*     2.509     5.278
H5             2.389**    2.239**     1.995     6.243
H6              1.107*     1.281*     1.488     3.236
H7             1.596**    1.527**     2.952     4.646
H8              1.023*     1.269*     1.766     3.488
H9              1.616*     1.900*     2.263     5.041
H10             1.851*    2.496**     2.281     6.441
H11             2.451*    2.802**     3.976     7.783
H12             1.996*    3.171**     2.656     9.782
H13            0.440###   0.660###    0.850     1.384
H14            0.538###    0.828*     0.837     1.764
D1 roots       0.383###    0.653*     0.550     1.896
D1 stems       0.220##    0.256##     0.229     0.970
D1 leaves      0.147##    0.188##     0.220     0.564
D10 roots       0.474*     0.698*     0.946     2.776
D10 stems       0.168#    0.301##     1.101     1.167
D10 leaves      0.706*    2.289**     5.857     8.147
D12 roots       0.032#     0.065#     0.064     0.202
D12 stems       0.054#    0.153##     0.180     0.356
D12 leaves      0.069#    0.282##     0.402     0.599

Note. The deeper the green, the lower the content; the
deeper the red, the higher the content.

Figure 1: Chemical structures of six CAs.

Substance                 Abbreviation   [R.sub.1]

Neochlorogenic acid           NCA           OH
Cryptochlorogenic acid        CCA           OH
Chlorogenic acid               CA        Caffeoyl
Isochlorogenic acid A         ICA        Caffeoyl
Isochlorogenic acid B         ICB        Caffeoyl
Isochlorogenic acid C         ICC           OH

Substance                 [R.sub.2]   [R.sub.3]

Neochlorogenic acid          OH       Caffeoyl
Cryptochlorogenic acid    Caffeoyl       OH
Chlorogenic acid             OH          OH
Isochlorogenic acid A        OH       Caffeoyl
Isochlorogenic acid B     Caffeoyl       OH
Isochlorogenic acid C     Caffeoyl    Caffeoyl
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Article Details
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Title Annotation:Research Article
Author:Zhao, Yifan; Sun, Peng; Ma, Yue; Wang, Kun; Chang, Xiaoqiang; Bai, Yue; Zhang, Dong; Yang, Lan
Publication:Journal of Chemistry
Geographic Code:9CHIN
Date:Nov 1, 2019
Words:4912
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