A simple and efficient fingerprinting method for chrysanthemum buds was developed with the aim of establishing a quality control protocol based on biochemical makeup. Chrysanthemum bud samples were successively extracted by water and alcohol. The fingerprints of the chrysanthemum buds samples were obtained using capillary electrophoresis and electrochemical detection (CE-ED) employing copper and carbon working electrodes to capture all of the chemical information. 10 batches of chrysanthemum buds were collected from different regions and various factories to establish the baseline fingerprint. The experimental data of 10 batches electropherogram buds by CE were analyzed by correlation coefficient and the included angle cosine methods. A standard chrysanthemum bud fingerprint including 24 common peaks was established, 12 from each electrode, which was successfully applied to identify and distinguish between chrysanthemum buds from 2 other chrysanthemum species. These results demonstrate that fingerprint analysis can be used as an important criterion for chrysanthemum buds quality control.
Chrysanthemums, colloquially known as mums, are herbaceous perennial flowering plants and have been cultivated for over 3 millennia. Chrysanthemums include more than 3000 varieties [
Significant amounts of biologically active compounds have been found in chrysanthemum buds that play important roles in human body, mainly including flavonoids, carbohydrate, and essential oils [
Fingerprinting is a method to capture total chemical information of herbs by chemical analytical techniques and is displayed as spectrograms, electropherograms, and other graphs. Fingerprint analyses produce a representative “fingerprint” that contains the greatest amount of information possible to accurately represent a sample and distinguish it from others. Fingerprint analysis of medicinal herbs has been the optimal measurement for identifying and assessing the variety and quality of the plants. Fingerprint analysis has been accepted as a strategy for the assessment of herbal medicines for the evaluation of medicinal products for herbal preparations by the U.S. Food and Drug Administration (FDA) [
Recently, several techniques have been developed which can characterize the nature and chemical composition of substances. HPLC [
CE technology has been widely applied to the characterization of diverse samples due to its low cost, minimal sample volume requirement, short analysis time, and high separation efficiency [
The purpose of this study is to establish chromatographic fingerprints of chrysanthemum buds by CE-ED analysis. In this analysis, water and alcohol extraction methods will be successively employed to enhance extraction efficiency. Copper and carbon electrodes will be both used to guarantee that the fingerprints produced can encompass the main bioactive compounds. Two distinct chrysanthemum samples will be identified by the utility of the proposed fingerprint.
Glucose and fructose were purchased from Sigma (St. Louis, MO, USA). Chlorogenic acid and luteolin were obtained from Shanghai Yuanye (Shanghai, China). Disodium tetraborate decahydrate (Na2B4O7·10H2O), H3BO3, phosphate salts, and sodium hydroxide (NaOH) were obtained from Shanghai Yuanye (Shanghai, China). All reagents were of analytical grade.
Glucose and fructose stock solutions were prepared in deionized water (Yancheng Chunyu Reagent Factory, Jiangsu, China). Chlorogenic acid and luteolin stock solutions were prepared with ethyl alcohol. The concentration of all stock solutions was 0.01 g mL−1. All analytes were diluted to the desired concentration in running buffer for CE analysis.
Twelve batches of chrysanthemum bud samples were purchased from supermarkets in five main cultivation areas located in China (Table
Resource of ten chrysanthemum buds’ sample.
Sample number | Name of sample | Source |
---|---|---|
1 | King of chrysanthemum buds | Hangzhou, Zhejiang |
2 | Chrysanthemum buds | Huangshan, Anhui |
3 | Chrysanthemum buds | Sheyang, Jiangsu |
4 | Chrysanthemum buds | Tongxiang, Zhejiang |
5 | Chrysanthemum buds | Jiaozuo, Henan |
6 | Chrysanthemum buds | Linyi, Shandong |
7 | Chrysanthemum buds | Lin’an, Zhejiang |
8 | Chrysanthemum buds | Kunming, Yunnan |
9 | Chrysanthemum buds | Yulin, Guangxi |
10 | Chrysanthemum buds | Bozhou, Anhui |
11 |
|
Hangzhou, Zhejiang |
12 |
|
Bozhou, Anhui |
First, the milled chrysanthemum buds (1 g) were suspended in 40 mL of deionized water and then ultrasonicated for 30 min to lyse the cells. Next, the mixture was heated at 90°C for 30 min to extract the water-soluble compounds. The suspension was cleared by centrifugation at 14800 rpm for 2 min using an Anke TGL-16C centrifuge (Shanghai Anting Instrument Factory, Shanghai, China), and the supernatant was filtered through a 0.22-
In this study, all employed electrodes were made in our laboratory.
A scrap copper wire (25 cm long, 0.3 mm diameter) was sealed into a soft glass capillary (10 cm long) with glue water. The capillary was cut perpendicular to its length to expose the wire at both ends. A copper electrode was used as soon as the glue solidified.
A lead inside a graphite pencil (4 cm long, 0.3 mm diameter) was first burnished and carefully wound with a polished copper wire. Then, the lead was sealed into a soft glass capillary with glue water. Finally, the capillary was cut perpendicular to its length to expose the lead and wire at each end of the capillary. The carbon electrode was used as soon as the glue solidified.
At the start of each experiment, both ends of the copper or carbon electrode were polished with extra fine carborundum paper followed by the sonication in deionized water using KQ-100KDE ultrasonic generator purchased from Kunshan Ultrasonic Instruments Co., Ltd. (Kunshan, China) before being placed in the cell.
CE analysis was performed on a laboratory-built CE-ED system [
The design of the CE-ED system was based on the end-column approach. The working electrode (either copper or carbon) was placed at the outlet of the separation capillary, and detection was carried out in the reservoir containing the grounding electrode for the CE instrument. Before use, the surface of the working electrode was positioned carefully opposite to the capillary outlet using a micropositioner (Shanghai Lianyi Instrument Factory, China). A three-electrode cell system composed of the working electrode, a platinum auxiliary electrode, and a saturated calomel electrode (SCE) was employed along with a BAS LC-3D amperometric detector (Biochemical System, West Lafayette, IN, USA). The electropherograms were processed with the HW-2000 software (Shanghai Qianpu Microsoftware, China).
As in previous CE-ED analyses [
The method was validated by identifying some key known compounds in chrysanthemum buds, such as chlorogenic acid, luteolin, glucose, and fructose. The relative standard deviations (RSDs), linearity, and detection limits of these compounds were calculated to determine the feasibility of this method.
Due to the novelty of fingerprinting analysis, only a few papers have been published on chemometrics [
The included angle cosine method considers the fingerprint spectrum data as a multidimensional space vector to convert the fingerprint spectrum similarity problem into the similarity between multidimensional vectors. The included angle cosine (
Under the optimum analysis conditions,
In order to achieve good separation of main components and quantify all of the bioactive chemical compounds in chrysanthemum buds, copper and carbon electrodes were utilized as the working electrode to analyze polysaccharides and flavonoids, respectively.
The carbon electrode was used as the working electrode mainly to analyze flavonoid compounds in chrysanthemum bud samples. Running buffer selection was considered first because of its significant effect on separation. Na2B4O7-NaOH was chosen as the running buffer for its greater elution effect after comparing with the separation efficiency of Na2B4O7-H3BO3, phosphate salts, and Na2B4O7-NaOH.
The acidity and concentration of the running buffer also plays a key role in CE due to its effects on the zeta-potential (
The potential applied to the working electrode directly affected the sensitivity and detection limit of this method. Separation voltage affects the velocity of the electroosmotic flow and the migration time of the analytes. In the following analyses, the potential applied to the carbon electrode was maintained at 0.95 V, where the background current was not too high, while the signal-to-noise (S/N) ratio was the highest. Moreover, the working electrode demonstrated good stability and high reproducibility at this optimum potential.
The effect of the separation voltage on the migration time of the analytes was also studied. The results show that a higher separation voltage resulted in shorter migration times for all analytes but also resulted in increased baseline noise. In the following analyses, the separation voltage was maintained at 14 V.
The copper electrode was used to analyze polysaccharide compounds in chrysanthemum buds. The optimal condition was selected with the same selection standards as for the carbon electrode. In order to obtain good separation and detection simultaneously [
Because the similarity analysis determined that the 10 batches of chrysanthemum buds were highly similar, they were used to produce mean electropherograms for chrysanthemum buds using the copper working electrode (Figure
Standard fingerprint for chrysanthemum buds obtained from CE with the carbon working electrode. Peak 5: chlorogenic acid; Peak 6: luteolin. Working potential is 0.95 V (versus SCE); running buffer: Na2B4O7-NaOH (pH 11.25, 3.1 × 10−3 g mL−1 boric acid ions); separation voltage: 14 kV, inject time: 8 s.
Standard fingerprint for chrysanthemum buds obtained from CE with the copper working electrode. Peak 8: glucose; Peak 11: fructose. Working potential: 0.67 V (versus SCE); separation buffer: Na2B4O7 (pH 9.24, 7.63 × 10−3 g mL−1); detection buffer: NaOH (pH 13.0); separation voltage: 20 kV, inject time: 8 s.
Some compounds found in chrysanthemum buds, such as chlorogenic acid, luteolin, glucose, and fructose, were selected as marker compounds to validate this technology. Peaks 8 and 11 in Figure
Additionally, a dilution series of standard solutions was also tested to measure the linearity of the current response for each of the four standard analytes. The linearity and detection limits are summarized in Table
Results of the regression analysis on the calibration curves and the detection limits.
Compound | Regression equation1 | Correlation coefficient ( |
Linear range (×10−4 g mL−1) | Detection2 limits (10−7 g mL−1) |
---|---|---|---|---|
Glucose |
|
0.997 | 0.02–2.0 | 3.5 |
Fructose |
|
0.998 | 0.02–2.0 | 1.8 |
Chlorogenic acid |
|
0.995 | 0.02–2.0 | 6.2 |
Luteolin |
|
0.998 | 0.02–2.0 | 5.1 |
2The detection limits correspond to the concentrations of the signal-to-noise ratio of 3.
Peaks found in all samples were assigned as “common peaks,” standing for the main characteristic compounds and representing the chemical profile of sample. According to the selection criterion of common peaks in Chinese herbs, common peaks were selected as those with a migration time RSD lower than 5% and a minimum relative peak area of less than 0.5%. Ten chrysanthemum bud samples were analyzed by CE with both the copper and carbon electrodes. 12 common peaks were chosen from each of the two CE electropherograms as the common peaks for chrysanthemum buds for a total of 24.
The similarity of 10 batches of chrysanthemum bud samples from various locations was evaluated by correlation coefficient and the included angle cosine. The similarity between samples was calculated by the correlation coefficient method with the average of all samples as a standard, and the similarities between samples were calculated by the included angle cosine method with the average of all samples taken as a standard. If the value of angle cosine and correlation coefficient from samples are similar with
From the data analysis in Tables
Peak area of various samples analyzed by CE with carbon electrode.
Peak | Sample1 | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
1# | 2# | 3# | 4# | 5# | 6# | 7# | 8# | 9# | 10# | 11# | 12# | |
1 | 1412023 | 1645374 | 1663717 | 1458483 | 1546122 | 1354982 | 1841631 | 1567651 | 1103240 | 1534023 | No found | No found |
2 | 907835 | 851391 | 813486 | 921323 | 923131 | 921354 | 835468 | 947313 | 994135 | 903712 | No found | No found |
3 | 2149706 | 2216378 | 2484610 | 2462123 | 2237155 | 2148461 | 1975232 | 2567855 | 2657165 | 1956742 | 1944541 | 2383874 |
4 | 1171446 | 1942103 | 1431231 | 1321330 | 903493 | 1579132 | 913485 | 1683256 | 1134902 | 991324 | 1076725 | 959534 |
5 | 16923651 | 18792134 | 19715642 | 13544132 | 18713543 | 15463449 | 19738394 | 18974667 | 13598120 | 14792134 | 6937854 | 7194685 |
6 | 679752 | 721564 | 643485 | 624836 | 681324 | 647122 | 594321 | 646794 | 679123 | 708616 | 1441354 | 2273234 |
7 | 14524829 | 15461365 | 11763465 | 18741561 | 13675645 | 10764475 | 18467132 | 19467486 | 13268785 | 15679815 | 10645381 | 8269314 |
8 | 5137047 | 5464132 | 5164725 | 5844154 | 5132112 | 5487612 | 5616912 | 581361 | 512641 | 541341 | 4251672 | 4586133 |
9 | 2166917 | 1774315 | 1843242 | 2354987 | 2465135 | 1949314 | 1891654 | 1676312 | 2546138 | 1687105 | No found | 2156515 |
10 | 3685649 | 4013651 | 4164623 | 3348971 | 3254854 | 3845624 | 3945215 | 3521265 | 3842946 | 4015689 | 3498665 | 3372452 |
11 | 4633980 | 5163435 | 4831656 | 4137568 | 4513981 | 4861635 | 5013826 | 4254682 | 4821591 | 4879612 | 4157674 | No found |
12 | 5547956 | 5312486 | 5461358 | 5782923 | 5978613 | 5428913 | 5342381 | 5138198 | 6021567 | 6223365 | 4978784 | 5445845 |
|
0.997 | 0.995 | 0.969 | 0.957 | 0.990 | 0.978 | 0.996 | 0.981 | 0.973 | 0.976 | 0.890 | 0.841 |
|
0.998 | 0.998 | 0.984 | 0.977 | 0.994 | 0.987 | 0.997 | 0.985 | 0.986 | 0.986 | 0.910 | 0.903 |
Peak area of various samples analyzed by CE-copper electrode.
Peak | Sample | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
1# | 2# | 3# | 4# | 5# | 6# | 7# | 8# | 9# | 10# | 11# | 12# | |
1 | 3747425 | 3624777 | 4358598 | 6876961 | 5709162 | 4521216 | 4452454 | 4021416 | 5601451 | 4335254 | 7511364 | 4679831 |
2 | 153880 | 85758 | 141249 | 105365 | 95080 | 98623 | 131639 | 108698 | 150123 | 106415 | No found | 65264 |
3 | 1080579 | 978309 | 655783 | 629236 | 896236 | 91636 | 970861 | 800012 | 609357 | 879412 | 1064891 | 501310 |
4 | 104520 | 75693 | 67969 | 95421 | 90413 | 102576 | 89563 | 101233 | 91975 | 84489 | 98699 | 58956 |
5 | 238491 | 147583 | 219547 | 187573 | 219576 | 167089 | 197456 | 129046 | 148643 | 177533 | 287789 | No found |
6 | 149385 | 237952 | 288587 | 189234 | 190643 | 289456 | 190121 | 188965 | 159758 | 189765 | 159137 | 251987 |
7 | 58153 | 73624 | 55581 | 74285 | 69210 | 57459 | 61361 | 57868 | 71698 | 69124 | No found | No found |
8 | 1478427 | 1881504 | 1675532 | 1086598 | 789865 | 979853 | 1376068 | 1678754 | 1586985 | 975097 | 2457040 | 1178621 |
9 | 2746590 | 2088749 | 1286953 | 2089796 | 2083654 | 1875916 | 2987542 | 2858155 | 2074764 | 3585875 | No found | No found |
10 | 268517 | 361587 | 291786 | 301765 | 369866 | 308716 | 345425 | 222752 | 357146 | 291578 | 368271 | 407981 |
11 | 406312 | 295709 | 454106 | 305136 | 416918 | 309572 | 451986 | 312476 | 281461 | 331653 | 334586 | 321786 |
12 | 454742 | 562069 | 356123 | 501685 | 542352 | 441875 | 501153 | 608754 | 409852 | 588743 | 541657 | 487819 |
|
0.976 | 0.975 | 0.971 | 0.978 | 0.983 | 0.986 | 0.989 | 0.980 | 0.992 | 0.960 | 0.877 | 0.876 |
|
0.983 | 0.981 | 0.980 | 0.980 | 0.987 | 0.989 | 0.992 | 0.986 | 0.993 | 0.973 | 0.902 | 0.902 |
Fingerprinting analysis can be used to assess the quality of chrysanthemum buds that come from different sources. By examining the relative retention time and the relative peak area of the common peaks in a fingerprint, we can determine whether a raw herb is genuine. But the most important application of fingerprints is that they can be used to separate different chrysanthemum varieties from each other.
Under the optimal analysis conditions, two other chrysanthemum species (
The above-mentioned results indicate that this method is accurate, sensitive, and reproducible foridentification and quality assessment of chrysanthemum buds. Furthermore, these methods may be used in further research in other natural agricultural products.
In this study, an efficient fingerprinting of chrysanthemum buds was developed by CE coupled with double detection electrodes, which established a quality control protocol based on biochemical makeup for chrysanthemum buds. We hope that this study has provided an appropriate method not only to generate fingerprints of herbs, but also to identify and asses the quality of chrysanthemum buds.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by the Natural Scientific Foundation of the Higher Education Institutions of Jiangsu Province, China. The authors are grateful for the financial and instrumental support by the Yancheng Institute of Technology.