chrysanthemums , colloquially known as mums , are herbaceous perennial flowering plants and have been cultivated for over 3 millennia .
chrysanthemums include more than 3000 varieties , including ammobium alatum , perennial chamomile , aster novi - belgii , and calendula officinalis , which come from different regions , flower in different seasons , and may contain different active compounds .
chrysanthemum buds are an important component in many traditional chinese medicine ( tcm ) formulas   for its therapeutic effects , which include antioxidant , anti - inflammatory , antiviral ( including hiv ) , antimutagenic , anticarcinogenic , antihepatotoxic , and antiaging activities .
chrysanthemum buds are also a common health food / supplement used by many consumers   for  scattering cold ,   cleaning heat and toxin ,  and  brightening eyes ,  which are considered beneficial to human health .
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 . among these compounds , chlorogenic acid , luteolin , and glucoside
traditionally , these active components were used to evaluate the quality of raw plant material . however , owing to the fact that there are hundreds of complex active components in chrysanthemum buds , it has been extremely difficult to identify all these substances and carry out quantitative analyses on them individually .
what is more , the chemical composition of chrysanthemum buds can differ between different varieties . as a result
, it became necessary to develop a new technology to capture the total chemical composition of chrysanthemum buds and to identify chrysanthemum varieties and verify their authenticity .
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 )   and the european medicines agency . in china ,
the former state drug administration ( sda ) also began to develop fingerprints of raw materials as a standard of quality control in 2000 . recently , several techniques have been developed which can characterize the nature and chemical composition of substances .
hplc   and gc , prime techniques used for fingerprint analysis , have high precision , sensitivity , and reproducibility .
however , sample preparations , including preconcentration and derivatization , are often time - consuming , complicated , and troublesome .
thin layer chromatography ( tlc )   is a commonly used technique for screening of herbal liquid extracts .
the ultra - performance liquid chromatography ( uplc )   approach has some advantages over hplc , gc , and tlc , including a large decrease in analysis time and solvent consumption , the possibility of obtaining high efficiencies , and the ability to resolve coeluting compounds .
however , its drawbacks include increased back - pressure and the availability of only few stable stationary phases .
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 [ 1416 ] .
electrochemical detection ( ed ) is a commonly used chemical detection method because of the small size of both the detector and control instrumentation and low power demands .
ce coupled to electrochemical detection ( ce - ed ) is a useful technology offering high sensitivity and good selectivity for electroactive analytes .
based on the two main kinds of active compounds in chrysanthemum buds , flavonoids and polysaccharides including the hydroxyl ( oh ) groups are electroactive at carbon and copper electrodes , respectively , which suggests that ce - ed is an appropriate method to investigate the chemical fingerprint of chrysanthemum buds .
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
copper and carbon electrodes will be both used to guarantee that the fingerprints produced can encompass the main bioactive compounds .
disodium tetraborate decahydrate ( na2b4o710h2o ) , h3bo3 , phosphate salts , and sodium hydroxide ( naoh ) were obtained from shanghai yuanye ( shanghai , china ) .
glucose and fructose stock solutions were prepared in deionized water ( yancheng chunyu reagent factory , jiangsu , china ) .
the concentration of all stock solutions was 0.01  g  ml . 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 1 ) .
the chrysanthemum buds were dried at room temperature and finely ground using a blender ( joyoung limited by share ltd . , shandong , china ) .
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 90c 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-m membrane to produce the polysaccharide fraction . to obtain the flavonoid fraction ,
the filtered residue was extracted with 50  ml 95% ethanol solution and ultrasonicated for 30  min .
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 lead inside a graphite pencil ( 4  cm long , 0.3  mm diameter ) was first burnished and carefully wound with a polished copper wire .
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 .
a 30  kv high voltage power supply ( shanghai institute of nuclear research , china ) supplied the voltage between the ends of the capillary .
the inlet end of the capillary was held at cathodic potential and the outlet end was maintained at ground .
the inlet cell was filled with the separation running buffer , and the outlet end was placed in the detection cell filled with detection running buffer .
a fused - silica capillary of 25  m ( inner diameter ) obtained from hebei yongnian factory ( handan , china ) was used for the separation .
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 running buffer was selected from na2b4o7-h3bo3 , phosphate salts , na2b4o7-naoh , and naoh ; ph and the concentration of the running buffer varied from 9 to 13 and from 10 to 50  mm , respectively ; separation voltage ranged from 10 to 25  kv ; the potential applied to copper working electrode ranged from 0.5 to 0.8  v , and the potential applied to carbon working electrode ranged from 0.8 to 1.1  v. 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 . in this study , data were analyzed with the professional software computer - aided similarity evaluation , which was developed based on chemometrics by the research center for the modernization of traditional chinese medicines ( central south university , changsha , china ) .
ten batches of chrysanthemum buds were analyzed to establish the mean chromatograph as a representative standard fingerprint electropherogram .
data was analyzed using included angle cosine   and correlation coefficient   methods in order to compare their suitability for discriminating between chrysanthemum fingerprints .
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 ( cos ) is calculated by the following equation:(1)cos=i=1nxiyii=1nxi2i=1nyi2,while the correlation coefficient ( 1 ) , which measures the relationship between the two properties , is calculated by the following equation:(2)1=i=1nxixiyiyii=1nxixi2i=1nyiyi2,where xi and yi are the ith elements in the two different electropherograms ( namely , x and y , resp . ) and n is the number of the elements in the electropherograms . x and y are the mean values of the n elements in electropherograms x and y , respectively . under the optimum analysis conditions , chrysanthemum morifolium and chrysanthemum indicum obtained from local supermarkets
the electropherograms were compared with the standard fingerprint of chrysanthemum buds to distinguish between various chrysanthemums .
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 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 electroosmotic flow ( eof ) , and the overall charge of the analytes , all of which impact the separation and migration time of the analytes . when the ph of the same running buffer in separation cell and detection cell was lower than 9.89 , two standard compounds ( chlorogenic acid and luteolin ) could not be separated and there were few peaks , demonstrating poor separation efficiency . on the other hand ,
when the ph of the running buffer was higher than 12 , the migration time was over 1  h. so ph 11.25 na2b4o7-naoh ( including 3.1  10  g  ml boric acid ions ) was selected as the optimum running buffer , balancing good separation with reasonable separation times .
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 ,
naoh ( ph 13.0 ) was selected as the optimal detection buffer because of the good response of the copper electrode in strong basic solution , and na2b4o7 ( ph 9.24 , 7.63  10  g  ml ) was selected as the best separation buffer because it resulted in a good separation efficiency .
the optimal potential to the copper working electrode was determined to be 0.67  v , and the optimal separation voltage was determined to be 20  kv .
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 1 ) and the carbon working electrode ( figure 2 ) .
together , these electropherograms comprise a comprehensive fingerprint of the bioactive compounds in chrysanthemum buds .
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 1 represent glucose and fructose , respectively . peaks 5 and 6 in figure 2 represent chlorogenic acid and luteolin , respectively .
the reproducibility of the peak current was evaluated by repeatedly injecting a standard solution under the optimum conditions .
the rsds of the migration time were 1.83% , 0.57% , 2.13% , and 1.41% , respectively .
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 .
as predicted , the observed reproducibility and detection limits of the four analytes were satisfactory .
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  > 0.90 and cos > 0.95 , the samples are assigned to the same origin . from the data analysis in tables 3 and 4 , ten chrysanthemum bud samples ( numbers 110 ) had high similarity even though the concentration of active compounds among samples was not at the same level . from the results
it can be concluded that the ten samples belong to the same species even though they are obtained from different place and different years .
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
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 ( chrysanthemum morifolium and chrysanthemum indicum )
were analyzed by this ce - ed method . by comparing their electropherograms with the standardized fingerprint of chrysanthemum buds ( tables 3 and 4 ) ,
some of the fingerprint common peaks are not found ( carbon peaks 1 , 2 , 9 , and 11 ; copper peaks 2 , 5 , 7 , and 9 ) from both species .
what is more , similarity analysis demonstrated that these species are significantly different from  < 0.90 and cos < 0.92 , which are considered to be very different by the chinese pharmacopoeia commission and in accordance with the actual varieties we bought from the supermarket .
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 .
 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 . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion