Quantitative and Qualitative Analysis of Flavonoids and Phenolic Acids in Snow Chrysanthemum (Coreopsis Tinctoria Nutt.) by HPLC-DAD and UPLC-ESI-QTOF-MS

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Article Quantitative and Qualitative Analysis of Flavonoids and Phenolic Acids in Snow Chrysanthemum (Coreopsis tinctoria Nutt.) by HPLC-DAD and UPLC-ESI-QTOF-MS

Yinjun Yang 1,2, Xinguang Sun 2, Jinjun Liu 1, Liping Kang 2, Sibao Chen 1, Baiping Ma 2 and Baolin Guo 1,*

1 Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China; [email protected] (Y.Y.); [email protected] (J.L.); [email protected] (S.C.) 2 Department of Biology, Beijing Institute of Radiation Medicine, Beijing 100850, China; [email protected] (X.S.); [email protected] (L.K.); [email protected] (B.M.) * Correspondence: [email protected]; Tel.: +86-10-5783-3288

Academic Editor: Derek J. McPhee Received: 26 August 2016; Accepted: 25 September 2016; Published: 30 September 2016

Abstract: A simple, accurate and reliable high performance liquid chromatography coupled with photodiode array detection (HPLC-DAD) method was developed and then successfully applied for simultaneous quantitative analysis of eight compounds, including chlorogenic acid (1), 0 0 (R/S)-flavanomarein (2), butin-7-O-β-D-glucopyranoside (3), isookanin (4), taxifolin (5), 5,7,3 ,5 - tetrahydroxyflavanone-7-O-β-D-glucopyranoside (6), marein (7) and okanin (8), in 23 batches of snow chrysanthemum of different seed provenance and from various habitats. The results showed total contents of the eight compounds in the samples with seed provenance from Keliyang (Xinjiang, China), are higher than in samples from the other five provenances by 52.47%, 15.53%, 19.78%, 21.17% and 5.06%, respectively, which demonstrated that provenance has a great influence on the constituents in snow chrysanthemum. Meanwhile, an ultra performance liquid chromatography coupled with electrospray ionization and quadrupole time-of-flight-mass spectrometry (UPLC-ESI-QTOF-MS) was also employed to rapidly separate and identify flavonoids and phenolic acids in snow chrysanthemum from Keliyang. As a result, a total of 30 constituents, including 26 flavonoids and four phenolic acids, were identified or tentatively identified based on the exact mass information, the fragmentation characteristics, and retention times of eight reference standards. This work may provide an efficient approach to comprehensively evaluate the quality of snow chrysanthemum.

Keywords: snow chrysanthemum; HPLC-DAD; UPLC-ESI-QTOF-MS; ﬂavonoids and phenolic acids; provenance and habitat; quality evaluation

1. Introduction Coreopsis tinctoria Nutt. (Asteraceae), native to North America, is widely cultivated around the world [1]. The capitulum of Coreopsis tinctoria is called “snow chrysanthemum” (Xue Ju) or “Kunlun snow chrysanthemum” (Kun Lun Xue Ju) in China [2]. The whole plant has been employed in China for hundreds of years as a folk herb for heat-clearing and detoxifying , while the capitulum of C. tinctoria was taken as a drink to deter diabetes in Portugal [3]. Since the beginning of this century, it has been known that snow chrysanthemum was used by the Uyghur nationality in Xinjiang (China) to prevent cardiovascular and cerebrovascular diseases, such as hypoglycemia, hypolipidemia, and

Molecules 2016, 21, 1307; doi:10.3390/molecules21101307 www.mdpi.com/journal/molecules Molecules 2016, 21, 1307 2 of 19 hypotension. Modern pharmacological studies have not only confirmed these above effects, but also shown that it has good antioxidant activities [4–6]. Phytochemical studies have shown that snow chrysanthemum mainly contains flavonoids, phenolic acids, and terpenoids [7]. Flavanomarein and marein, the predominant flavonoids, were proved to have good hypoglycemic activities [3]. Meanwhile, satisfactory antioxidant activites were also reported for some of the other flavonoids, such as flavanokanin, okanin, coreopsin and flavanocorepsin [8–10]. The effectsMolecules on 2016 cardiovascular, 21, 1307 and cerebrovascular diseases was first discovered in C.2 tinctoriaof 18 from Keliyang, Xinjiang [11]. Since then, a large crop area has been dedicated to the cultivation of snow good antioxidant activities [4–6]. Phytochemical studies have shown that snow chrysanthemum mainly chrysanthemumcontains inflavonoids, Keliyang phenolic (Xinjiang) acids, and and terpenoids it is considered [7]. Flavanomarein a Xinjiang-specific and marein, the predominant medicinal material. The good hypoglycemicflavonoids, were proved and hypolipidemic to have good hypoglycemic effects of activitiesC. tinctoria [3]. Meanwhile,have attracted satisfactory wide antioxidant attention in recent years [12–14activites], but were the also excessive reported plantingfor some of has the other brought flavonoids, a series such of as problems.flavanokanin, The okanin, influx coreopsin of seeds from different sources,and flavanocorepsin including [8–10]. inter-mating of seeds from Keliyang, as well as imported seeds, and the The effects on cardiovascular and cerebrovascular diseases was first discovered in C. tinctoria from expansionKeliyang, to different Xinjiang areas [11]. of Since Xinjiang then, havea large led crop to area unevenness has been dedicated of the quality to the cultivation of snow of chrysanthemum snow on the market.chrysanthemum In general, in Keliyang the quality (Xinjiang) of C. and tinctoria it is conswasidered traditionally a Xinjiang-specifi evaluatedc medicinal by material. the color The and size of the flowers,good and hypoglycemic seeds from and Keliyang hypolipidemic were effects considered of C. tinctoria assuperior have attracted quality, wide attention and the in quality recent of snow chrysanthemumyears [12–14], was betterbut the withexcessive the planting higher has altitude. brought a series of problems. The influx of seeds from different sources, including inter-mating of seeds from Keliyang, as well as imported seeds, and the In recentexpansion years to theredifferent was areas some of Xinjiang research have toled verifyto unevenness this notion. of the qualit Therey of snow were chrysanthemum reports showing that the chemicalon the composition market. In general, of snow the chrysanthemumquality of C. tinctoria waswas traditionally related to evaluated the ecological by the color environment and size [15,16], however dueof the to flowers, the limited and seeds samples from Keliyang studied were and cons theidered lack asof superior major qua compoundslity, and the thisquality idea of was not convincing.snow A recentlychrysanthemum reported was better study with about the higher the antioxidantaltitude. activities and chemical characteristics In recent years there was some research to verify this notion. There were reports showing that of differentthe parts chemical from composition snow chrysanthemum of snow chrysanthemu madem thewas pointrelated that to the the ecological contents environment of flavonoids and phenolic acids[15,16], of however snow due chrysanthemum to the limited samples collected studied from and the Mt. lack Kunlun of major compounds were higher this idea than was those from other regionsnot convincing. [8,17]. To A our recently knowledge, reported study there about is no the reportantioxidant of relationshipactivities and chemical between characteristics seed provenance of of C. tinctoriadifferentand its parts quality. from snow chrysanthemum made the point that the contents of flavonoids and phenolic acids of snow chrysanthemum collected from Mt. Kunlun were higher than those from other regions In order[8,17]. to To further our knowledge, research there the effectsis no report of provenance of relationship or between habitat, seed six provenance batches ofof C. seeds tinctoria from clearly known sourcesand its werequality. planted at different altitudes in four regions. A consistent cultivation technique guaranteed thatIn theorder differences to further research in the the quality effects of provenan the snowce or chrysanthemums habitat, six batches of were seeds affected from clearly only by seed provenanceknown or habitat. sources Awere HPLC-DAD planted at different method altitudes was established in four regions. to theA consistent quantification cultivation of technique eight compounds, guaranteed that the differences in the quality of the snow chrysanthemums were affected only by seed namely chlorogenicprovenance or acidhabitat. (1 A), HPLC-DAD (R/S)-flavanomarein method was established (2)[17 to ],the butin-7- quantificationO-β of-D eight-gluco-pyranoside compounds, (3), 0 0 isookaninnamely (4), taxifolin chlorogenic (5), acid 5,7,3 (1), ,5(R/-tetrahydroxyflavanone-7-S)-flavanomarein (2) [17], butin-7-OO-β--βD--gluco-pyranosideD-gluco-pyranoside (3), isookanin (6), marein (7) and okanin(4), ( 8taxifolin) (Figure (5), 5,7,31) in′,5′ 23-tetrahydroxyflavanone-7- batches of snow chrysanthemumO-β-D-gluco-pyranoside grown (6), marein from (7) seeds and okanin of six (8) different provenances(Figure in 1) four in 23 differentbatches of snow habitats. chrysanthemum Meanwhile, grown from an seeds UPLC-ESI-QTOF-MS of six different provenances method in four was also different habitats. Meanwhile, an UPLC-ESI-QTOF-MS method was also developed to systematically developedidentify to systematically the flavonoidsidentify and phenolic the acids flavonoids in snow chrysanthemum. and phenolic acids in snow chrysanthemum.

Peak No.R1R2R3R4R5R6

2 OH H H H OGlu OH

3OHH H H OGluH

4OHH H H OHOH Peak No. R 5 OH H OH OH OH H 7OGlu 6HOHHOHOGluH 8OH

β Figure 1. StructuresFigure 1. Structures of chlorogenic of chlorogenic acid (acid1), ((R1),/ (SR)-flavanomarein/S)-flavanomarein ( (22),), butin-7- butin-7-O-βO-D--glucopyranoside-D-glucopyranoside (3), 0 0 isookanin( 34),), isookanin taxifolin (4 (),5 taxifolin), 5,7,3 ,5(5),-tetrahydroxyﬂavanone-7- 5,7,3′,5′-tetrahydroxyflavanone-7-OO--ββ-D-glucopyranoside-glucopyranoside (6), marein (6), marein (7), and okanin(7 (),8 and). okanin (8).

Molecules 2016, 21, 1307 3 of 19

2. Result and Discussion

2.1. Quantitative Analysis by HPLC-DAD

2.1.1. Optimization of the Extraction Method To optimize the extraction procedure, samples (0.5 g) were extracted for 30 min using six different extraction solvents (30%, 70%, and 100% methanol and 30%, 70%, and 95% ethanol). Comparing the peak areas of the HPLC chromatograms, 30% ethanol gave poor extraction efficiency for the eight target compounds, while 70% methanol showed the best extraction effiency for all of them. Besides, ultrasonic extraction and heat reflux were also compared and the results indicated that these two methods had similar performance. Additionally, different extraction times (30, 45, and 60 min) and extraction cycles (1–3 times) were tested, too. As a result, optimal extraction condition for the active ingredients of snow chrysanthemum was determined to be one cycle of 30 mins of ultrasonic extraction with 70% methanol [18,19].

2.1.2. Validation of the Method

Calibration Curves, Limits of Detection and Limits of Quantification The calibration curves were generated from six different concentrations of the standard working solutions. Each concentration was analyzed in triplicate. The limits of detection (LOD) and limits of quantification (LOQ) were measured on the basis of the corresponding concentrations at signal-to-noise ratios of 3 and 10, respectively. All analytes showed good linearity of the correlation curves with correlation coefficients better that 0.9990. The LOD and LOQ values of the eight analytes varied from 3.55 to 9.25 ng and 12.46 to 35.88 ng, respectively, indicating a good sensitivity (Table1).

Table 1. Linearity of calibration curve for the eight target compounds.

Target Compounds Regression Equation R2 Linear Range (µg) LOD (ng) LOQ (ng) 1 Y = 1.308 × 105X + 17129 0.9998 0.15–6.00 8.33 31.22 2 Y = 1.299 × 105X + 45064 0.9990 0.60–24.00 6.76 16.38 3 Y = 1.641 × 105X + 53463 0.9993 0.02–1.00 7.96 30.52 4 Y = 1.800 × 105X − 58985 0.9995 0.20–8.00 6.66 25.86 5 Y = 4.328 × 105X − 70904 0.9990 0.05–2.00 3.55 12.46 6 Y = 2.004 × 105X + 13368 0.9990 0.18–7.00 5.28 21.08 7 Y = 8.303 × 105X + 41070 0.9996 0.80–32.00 9.25 35.88 8 Y = 7.594 × 105X + 15260 0.9993 0.35–14.00 6.88 23.58

Precision, Stability, Repeatability and Accuracy The intra-day and inter-day precision were determined by analyzing the six replicates of the eight target standard solutions on the same day and three consecutive days, measured by calculating the relative standard deviation (RSDs) of the contents of all analytes. The RSDs of the precision of intra-day and inter-day analyses ranged from 1.76% to 4.18% and 1.63% to 4.26%, respectively (Table2).

Table 2. Intra-day and inter-day precisions, repeatability, stability, recovery tests of 8 target compounds.

Target Precision (RSD, %) Repeatability Stability Recovery (%) (n = 6) Compounds (RSD, %) Intra-Day (n = 6) Inter-Day (n = 6) (RSD, %) Average RSD 1 2.92 2.78 2.51 2.03 95.75 4.58 2 3.53 4.06 3.38 3.15 99.67 4.88 3 2.58 1.92 1.77 3.52 99.50 2.21 4 2.97 1.63 2.62 1.73 96.35 2.87 5 4.16 3.02 3.74 3.03 98.52 2.79 6 3.21 2.87 2.89 2.64 98.48 2.34 7 1.76 3.69 1.58 1.24 102.51 3.32 8 4.18 4.26 1.37 2.19 103.50 1.33 Molecules 2016, 21, 1307 4 of 19

To test the repeatability of the method, six independently prepared samples (USA, Minfeng, Xinjiang,Molecules batch 2016, No. 21, 1307 S1) were analyzed in parallel. Variations were expressed as RSDs of4 the of 18 eight target compounds, and the results were 2.51%, 3.38%, 1.77%, 2.62%, 3.74%, 2.89%, 1.58% and 1.37%, To test the repeatability of the method, six independently prepared samples (USA, Minfeng, respectively.Xinjiang, The batch results No. S1) showed were analyz gooded repeatability in parallel. (TableVariations2). were expressed as RSDs of the eight Fortarget the compounds, stability test, and the the same results sample were 2.51%, was analyzed 3.38%, 1.77%, at 0, 2,2.62%, 4, 8, 3.74%, 12, and 2.89%, 24 h at1.58% room and temperature. 1.37%, The RSDsrespectively. of the peak The results areas ofshowed the eight good constituents repeatability in(Table the same2). sample ranged from 1.24% to 3.22%, indicatingFor that the the stability sample test, was the stablesame sample under was the analyzed experimental at 0, 2, conditions4, 8, 12, and (Table24 h at2 room). temperature. InThe order RSDs to of examine the peak theareas recovery of the eight of theconstituents extraction in the method, same sample high amounts ranged from of the 1.24% standards to 3.22%, were addedindicating into a sample that the that sample wasanalyzed was stable as unde describedr the experimental in Sections conditions3.2.1 and 3.3.1(Table. Recovery 2). was calculated as follows:In order to examine the recovery of the extraction method, high amounts of the standards were added into a sample that was analyzed as described in Sections 3.2.1 and 3.3.1. Recovery was calculatedRecovery as follows: (%) = (amount found − original amount)/spiked amount × 100% Recovery (%) = (amount found – original amount)/spiked amount × 100% The results showed that the mean recovery ranged from 95.75% to 103.50% with RSDs less than The results showed that the mean recovery ranged from 95.75% to 103.50% with RSDs less 4.88%than for the4.88% eight for the compounds eight compounds (Table2 (Table). 2).

2.1.3.2.1.3. Quantitative Quantitative Analysis Analysis of Flavonoidsof Flavonoids and and Phenolic Phenolic Acid in in Snow Snow Chrysanthemum Chrysanthemum by HPLC-DADby HPLC-DAD The establishedThe established HPLC-DAD HPLC-DAD method method was was successfully successfully applied applied to to the the determination determination of chlorogenic of chlorogenic acid (acid1), (R(1/),S ()-ﬂavanomareinR/S)-flavanomarein (2 ),(2), butin-7- butin-7-OO--ββ--D-glucopyranoside (3), (3 isookanin), isookanin (4), (taxifolin4), taxifolin (5), (5), 0 0 5,7,3 ,55,7,3-tetrahydroxyﬂavanone-7-′,5′-tetrahydroxyflavanone-7-O-Oβ--βD--glucopyranosideD-glucopyranoside ( (66),), marein marein (7 ()7 and) and okanin okanin (8) in (8 )23 in batches 23 batches of snowof snow chrysanthemum. chrysanthemum. Figure Figure2 shows 2 shows typivcal typivcal chromatrograms. chromatrograms.

Figure 2. Typical HPLC chromatograms of mixed standards (A) and crude extract of snow Figurechrysanthemum 2. Typical HPLC(B), detected chromatograms at 285 nm (left) of and mixed 378 nm standards (right). (A) and crude extract of snow chrysanthemum (B), detected at 285 nm (left) and 378 nm (right). Due to the different seed provenance and habitat, there were remarkable differences between the Amongsamples thein terms four flavoneof the total glycosides, contents of flavanomarein the investigated and compounds marein, whichwhich bothranged exhibited from 73.99 various mg/g– good biological136.38 activities mg/g, asin shown vitro ,in accounted Table 3. In for addition, a large percentagethe contents ofof thefour flavonoids flavone glycosides, (Table3). including As shown in (R/S)-flavanomarein (2), butin-7-O-β-D-glucopyranoside (3), 5,7,3′,5′-tetrahydroxy-flavanone-7-O-β-D- Table3, through the 23 batches of samples, the content of flavanomarein or marein was mostly highest glucopyranoside (6), and marein (7), accounted 74.46%–83.69% of the eight investigated compounds in allin samples all tested with samples. a seed source from Keliyang, and the the contents of both compounds represented more thanAmong 52% of the the four investigated flavone glycosid compounds.es, flavanomarein Thus, flavanomarein and marein, which and mareinboth exhibited could playsvarious a key role ingood the qualitybiological assessment activities in of vitr snowo, accounted chrysanthemum. for a large percentage of the flavonoids (Table 3). DueAs shown to the in different Table 3, seedthrough provenance the 23 batches and habitat,of samples, there the werecontent remarkable of flavanomarein differences or marein between the sampleswas mostly in termshighest ofin all the samples total contents with a seed of sour thece investigated from Keliyang, compounds and the the which contents ranged of both from 73.99compounds mg/g–136.38 represented mg/g, as more shown than in 52% Table of 3the. In investigated addition, thecompounds. contents Thus, of four flavanomarein flavone glycosides, and 0 0 includingmarein ( Rcould/S)-flavanomarein plays a key role in (the2), quality butin-7- assessmentO-β-D-glucopyranoside of snow chrysanthemum. (3), 5,7,3 ,5 -tetrahydroxyflavanone-7-O-β-D-glucopyranoside (6), and marein (7), accounted 74.46%–83.69% of the eight investigated compounds in all tested samples.

Molecules 2016, 21, 1307 5 of 19

Table 3. Content determination of eight compounds in 23 batches of snow chrysanthemum (mg·g−1).

Flavonoid Flavonoid Total Sum NO. 1 2 3 4 5 6 7 8 Glycosides Aglycones (1 + 2 + ... + Molecules 2016, 21, 1307 2 + 7 5 of 18 (2 + 3 + 6 + 7) (4 + 5 + 8) 7 + 8)

S1 1.72Table 17.29 3. Content 0.41 determination 5.34 2.85 of eight 4.35 compounds 35.25 in 6.7823 batches 52.54 of snow 57.30chrysanthemum 14.96 (mg·g−1). 73.99 S2 10.09 28.59 1.09 5.07 1.83 5.84 50.02 5.66 78.61 85.54 12.56 108.19 Flavonoid Flavonoid Total S3 4.02 29.95 1.11 5.66 2.34 4.53 51.22 6.85Sum 81.17 86.81 14.86 105.69 NO. 1 2 3 4 5 6 7 8 Glycosides Aglycones (1 + 2 + … S5 5.27 26.50 1.34 6.55 2.42 12.51 70.45 7.362 96.95 + 7 110.80 16.32 132.39 S6 14.73 30.71 1.27 5.46 2.99 9.33 54.06 6.21 84.77(2 + 3 +95.36 6 + 7) (4 + 5 + 8) 14.66 + 7 + 8) 124.75 S7 2.08S1 18.611.72 17.29 0.40 0.41 6.11 5.34 3.19 2.85 3.334.35 36.8635.25 8.926.78 52.54 55.47 57.30 59.20 14.96 18.22 73.99 79.51 S2 10.09 28.59 1.09 5.07 1.83 5.84 50.02 5.66 78.61 85.54 12.56 108.19 S8 5.86 25.96 1.63 6.81 3.69 7.74 54.30 9.02 80.26 89.63 19.52 115.01 S3 4.02 29.95 1.11 5.66 2.34 4.53 51.22 6.85 81.17 86.81 14.86 105.69 S9 5.36S5 24.045.27 26.50 0.96 1.34 4.61 6.55 1.96 2.42 4.1412.51 49.6670.45 7.437.36 96.95 73.7 110.80 78.80 16.32 14.00 132.39 98.17 S10 10.19S6 19.3614.73 30.71 0.80 1.27 4.43 5.46 1.88 2.99 4.659.33 48.4954.06 5.266.21 84.77 67.85 95.36 73.30 14.66 11.56 124.75 95.06 S11 13.93S7 19.762.08 18.61 0.66 0.40 4.44 6.11 1.35 3.19 11.013.33 57.2436.86 5.438.92 55.47 77.00 59.20 88.67 18.22 11.22 79.51 113.83 S12 12.79S8 23.075.86 25.96 1.07 1.63 4.50 6.81 1.52 3.69 8.927.74 55.1054.30 4.999.02 80.26 78.17 89.63 88.17 19.52 11.01 115.01 111.96 S13 2.35S9 18.75 5.36 24.04 0.44 0.96 3.89 4.61 2.30 1.96 2.994.14 45.6749.66 7.167.43 73.7 64.42 78.80 67.85 14.00 13.34 98.17 83.55 S14 9.26S10 22.8510.19 19.36 1.02 0.80 4.14 4.43 2.37 1.88 4.99 4.65 53.1448.49 7.545.26 67.85 75.99 73.30 82.00 11.56 14.05 95.06 105.31 S15 7.58S11 23.0513.93 19.76 1.27 0.66 3.53 4.44 1.87 1.35 3.7011.01 57.3657.24 8.725.43 77.00 80.41 88.67 85.37 11.22 14.12 113.83 107.07 S16 8.04S12 26.3712.79 23.07 1.49 1.07 3.93 4.50 2.12 1.52 4.55 8.92 57.7355.10 5.864.99 78.17 84.1 88.17 90.14 11.01 11.92 111.96 110.10 S13 2.35 18.75 0.44 3.89 2.30 2.99 45.67 7.16 64.42 67.85 13.34 83.55 S17 12.99 29.05 1.54 3.46 1.83 15.50 65.42 6.59 94.47 111.51 11.88 136.38 S14 9.26 22.85 1.02 4.14 2.37 4.99 53.14 7.54 75.99 82.00 14.05 105.31 S18 12.01S15 21.597.58 23.05 1.24 1.27 3.56 3.53 1.81 1.87 6.473.70 52.7757.36 4.468.72 80.41 74.36 85.37 82.07 14.12 9.84 107.07 103.92 S19 3.75S16 17.458.04 26.37 0.58 1.49 4.28 3.93 2.60 2.12 3.964.55 45.2657.73 4.895.86 84.1 62.71 90.14 67.25 11.92 11.77 110.10 82.78 S20 10.03S17 14.6812.99 29.05 0.94 1.54 5.69 3.46 1.55 1.83 4.5815.50 51.1465.42 4.996.59 94.47 65.82 111.51 71.33 11.88 12.22 136.38 93.59 S21 8.33S18 16.9412.01 21.59 1.64 1.24 4.83 3.56 1.37 1.81 6.62 6.47 51.1352.77 5.324.46 74.36 68.07 82.07 76.33 9.84 11.51 103.92 96.17 S22 9.79S19 16.083.75 17.45 1.08 0.58 4.56 4.28 1.55 2.60 5.373.96 53.3445.26 4.914.89 62.71 69.42 67.25 75.87 11.77 11.02 82.78 96.68 S23 11.11S20 17.8910.03 14.68 0.75 0.94 4.55 5.69 1.22 1.55 9.90 4.58 55.3451.14 4.274.99 65.82 73.23 71.33 83.88 12.22 10.04 93.59 105.04 S24 16.56S21 27.728.33 16.94 1.07 1.64 5.16 4.83 1.52 1.37 11.286.62 55.4651.13 4.725.32 68.07 83.18 76.33 95.53 11.51 11.41 96.17 123.51 S22 9.79 16.08 1.08 4.56 1.55 5.37 53.34 4.91 69.42 75.87 11.02 96.68 S23 11.11 17.89 0.75 4.55 1.22 9.90 55.34 4.27 73.23 83.88 10.04 105.04 S24 16.56 27.72 1.07 5.16 1.52 11.28 55.46 4.72 83.18 95.53 11.41 123.51 As shown in Figure3A, there was signiﬁcant difference in the total contents of the eight compoundsAs betweenshown in theFigure six 3A, snow there chrysanthemum was significant difference seed sources. in the Accordingtotal contents to of the the folk eight tradition, snow chrysanthemumcompounds between with the seedsix snow provenance chrysanthemum from Keliyangseed sources. is consideredAccording to to the be folk the tradition, genuine herbal medicine,snow whichchrysanthemum is supported with seed by theprovenance fact that from the Keliyang total contents is considered of the to eightbe the compoundsgenuine herbal in snow chrysanthemummedicine, which with is Keliyang supported provenance by the fact that seed the were total muchcontents higher of the thaneight thatcompounds of the fourin snow other seed chrysanthemum with Keliyang provenance seed were much higher than that of the four other seed source samples (p < 0.05). There was no difference in the total contents of the eight compounds between source samples (p < 0.05). There was no difference in the total contents of the eight compounds between Keliyang-1Keliyang-1 and Keliyang-2and Keliyang-2 provenance. provenance. Meanwhile,Meanwhile, the the contents contents of eight of eight compounds compounds in three in other three other seed sourcesseed sources of snow of snow chrysanthemum chrysanthemum (seeds(seeds from Ni Ningxia,ngxia, Jiangsu, Jiangsu, and andXinjiang) Xinjiang) also showed also showed no no difference.difference. As the As majorthe major contributors contributors of of thethe components,components, the the contents contents of flavone of ﬂavone glycosides glycosides followed followed the samethe same rule asrule the as totalthe total components components (Figure (Figure3 3B).B).

AC 140 140 130 130 120 ＊＊ 120 110 ＊ 110 100 100 90 ＊＊

80 90

70 80 Keliyang-1 Keliyang-2 Ningxia Jiangsu Xinjiang USA

Total contents of 8 compounds(mg/g) 70 Total contents of 8 compounds(mg/g) Minfeng.Xinjiang Sihai.Beijing Shenjiaying.Beijing Implad.Beijing B 110

105 D 17 100

95 16 ＊＊ 90 ＊ 15 ＃ 85 14 80 13 75 ＃＃ 70 ＊＊ 12 contents of flavonoid of contents glycosides (mg/g) contents of flavonoid of contents (mg/g) aglycones 65 11 60 10 Keliyang-1 Keliyang-2 Ningxia Jiangsu Xinjiang USA Minfeng.Xinjiang Sihai.Beijing Shenjiaying.BeijingImplad.Beijing Provenance Habitat FigureFigure 3. Content 3. Content determination determination ofof eighteight compounds compounds in insnow snow chrysanthemum chrysanthemum of six ofbatches six batchesof of differentdifferent provenance provenance (A); ( ﬂavonoidA); flavonoid glycosides glycosides ( 2(2+ + 33 + 66 ++ 77) )in in snow snow chrysanthemum chrysanthemum of six of batches six batches of of different provenance (B); eight compounds in snow chrysanthemums from four different habitats different provenance (B); eight compounds in snow chrysanthemums from four different habitats (C); (C); and flavonoid aglycones (4 + 5 + 8) (D) in snow chrysanthemums from four different habitats. and ﬂavonoidValues are aglycones mean ± S.D. ( 4**+ p 5< +0.01,8)( *D p )< in0.05 snow vs. Keliyang-1; chrysanthemums ## p < 0.01, # fromp < 0.05 four vs. Minfeng different Xinjiang. habitats. Values are mean ± S.D. ** p < 0.01, * p < 0.05 vs. Keliyang-1; ## p < 0.01, # p < 0.05 vs. Minfeng Xinjiang. Molecules 2016, 21, 1307 6 of 19 Molecules 2016, 21, 1307 6 of 18

On the otherother hand,hand, thethe total total contents contents of of the the eight eight compounds compounds in in snow snow chrysanthemum chrysanthemum showed showed no significantno significant regularity regularity in planting in planting in the fourin the different four different habitats. Thehabitats. total contentsThe total of contents eight compounds, of eight flavonoidcompounds, glycosides flavonoid and glycosides two major and flavonoid two major glycosides flavonoid in glycosides samples fromin samples the habitat from ofthe Minfeng habitat (Xinjiang)of Minfeng with (Xinjiang) higher with altitude higher were altitude not higher were than not thosehigher from than other those three from habitats. other three Also habitats. the total contentsAlso the oftotal the contents eight compounds of the eight in samples compounds from thein samples habitat of from IMPLAD the habitat (Beijing) of withIMPLAD lower (Beijing) altitude werewith lower also not altitude lower were than thosealso not from lower three than other those higher from altitude three other habitats higher (Figure altitude3C). habitats Therefore, (Figure the idea3C). thatTherefore, the higher the theidea attitude, that the the higher better the the attitude, quality remainsthe better to the be confirmed.quality remains What to is interesting,be confirmed. however, What is thatinteresting, in Figure however,3D, the total is that contents in Figure of flavones 3D, th aglycones,e total contents including of isookaninflavones aglycones, ( 4), taxifolin including (5), and okaninisookanin (8) ( in4), snowtaxifolin chrysanthemum (5), and okanin planted (8) in insnow Minfeng chrysanthemum (Xinjiang) were planted typically in Minfeng higher (Xinjiang) than that inwere samples typically from higher IMPLAD than (Beijing,that in samples China) (fromp < 0.01). IMPLAD That may(Beijing, be causedChina) by(p the< 0.01). different That habitats,may be butcaused there by were the lessdifferent differences habitats, between but there the sampleswere less planted differences in Minfeng, between Xinjiang the samples and Sihai, planted Beijing in (Minfeng,p > 0.05). Xinjiang and Sihai, Beijing (p > 0.05). Besides, there were two batches of seedsseeds fromfrom inter-matinginter-mating fromfrom seedsseeds ofof Keliyang.Keliyang. One was amphichrome, andand the the other other had had red red flowers. flowers. In mostIn most snow snow chrysanthemums, chrysanthemums, the outside the outside of the ligulate of the flowerligulate is flower yellow, is and yellow, the center and isthe red, center so the is flowers red, so are the basically flowers amphichrome. are basically Theamphichrome. phenomomenon The thatphenomomenon the whole of that flower the is whole red is of also flower seen is in red a few is also individuals. seen in a few individuals. ForFor consumptionconsumption as a tea,tea, thethe completelycompletely red flowersflowers are consideredconsidered the most desirable, not the amphichrome ones. In the study, the red flowerflower samplessamples were selected from the autologous breeding of the samesame batchbatch ofof amphichromeamphichrome plants.plants. The similar genetic background made them comparable. According to report, the red parts of flowerflower are due to the presence of anthocyanins which have the same upstream metabolic pathways as flavonoidsflavonoids [20].20]. As shown in Figure 44,, thethe contentscontents ofof sevenseven flavonoidsflavonoids in amphichrome samples (S5, S11, and S17)S17) werewere comparativelycomparatively higher than those in red flowerflower ones (S6, S12, and S18) in this study. This may be explained by the fact that more anthocyanins were synthesized in redred flowersflowers andand lessless flavonoidsflavonoids werewere synthesizedsynthesized inin thethe biosyntheticbiosynthetic pathway.pathway.

130

125

120

115

110

105

100

90 S5 S6 S11 S12 S17 S18

Figure 4. Content determination of seven flavonoids in samples which seeds were from Figure 4. Content determination of seven flavonoids in samples which seeds were from Keliyang (Xinjiang). Keliyang (Xinjiang). 2.2. Identification of Flavonoids and Phenolic Acid in Snow Chrysanthemum by UPLC-MS 2.2. Identification of Flavonoids and Phenolic Acid in Snow Chrysanthemum by UPLC-MS Optimized chromatographic conditions, including mobile phase, elution program and spectrometerOptimized conditions chromatographic were determned conditions, through including trials in this mobile study. phase, As a result, elution a linear program gradient and spectrometerelution with acetonitrile conditions wereand water determned containing through formic trials acid in thisas the study. mobile As phase a result, gave a linear the best gradient peak elutionresolution. with The acetonitrile parameters and of water flow containingrate of gas, formicgas pressure, acid as spray the mobile voltage, phase capillary gave the temperature best peak resolution.and voltage The of entrance parameters were of optimized flow rate of to gas, obtain gas appropriate pressure, spray ionization. voltage, Figure capillary 5 presents temperature a typical and voltagechromatogram of entrance of a weresample optimized with mass to obtainspectrometric appropriate detection ionization. in negative Figure 5ion presents mode using a typical the chromatogramoptimum condition. of a A sample total of with 30 compounds mass spectrometric (Figures detection5 and 7), including in negative four ion phenolic mode using acids, the11 optimumflavanones, condition. six chalcones, A total two of flavones, 30 compounds four flav (Figuresonols and5 and three7), including aurones were four phenolicidentified acids, in the snow chrysanthemum samples. Among them, eight compounds were unambiguously identified by comparing their retention time, the accurate masses and fragment ions with those of reference

Molecules 2016, 21, 1307 7 of 19

11 flavanones, six chalcones, two flavones, four flavonols and three aurones were identified in the snow chrysanthemumMolecules 2016, 21, 1307 samples. Among them, eight compounds were unambiguously7 of identified 18 by comparing their retention time, the accurate masses and fragment ions with those of reference compoundscompounds and 22 compoundsand 22 compounds were tentativelywere tentatively assigned assigned by matching by matching the th empiricale empirical molecular molecular formula with thatformula of the knownwith that compoundsof the known compounds previously prev reportediously reported in the literature in the literature (Table (Table4). 4). Molecules 2016, 21, 1307 7 of 18 XJ-3-YP-CS 20160329-YLC-11 1: TOF MS ES- 100 BPI compounds and 22 compounds were tentatively assigned by matching the empirical4.32e4 molecular 14 formula with that of the known compounds previously reported in the literature (Table 4).

XJ-3-YP-CS 20160329-YLC-11 1: TOF MS ES- 100 BPI 5 4.32e4 14

% 4 28 21

8 19 % 28 20 21 29 6 2 24 22 23 30 12 18 26 13 19 1 8 10 27 7 15 16 25 3 11 17 20 9 29 0 6 Time 2.00 4.00 6.002 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 22 23 30 12 26 Figure 5. The base peak chromatograms of snow 13chrysanthemum18 by UPLC-ESI-QTOF-MS in 1 Figure 5. The base peak chromatograms of snow chrysanthemum10 by UPLC-ESI-QTOF-MS27 in negative negative ion mode. 7 15 16 25 3 ion mode. 9 11 17 0 Time 2.2.1. Identification2.00 of 4.00 Phenolic 6.00 Acids 8.00 in 10.00 Snow 12.00 Chrysanthemum 14.00 16.00 18.00 by ESI-Q-TOF-MS 20.00 22.00 24.00 26.00 Figure 5. The base peak chromatograms of snow chrysanthemum by UPLC-ESI-QTOF-MS in 2.2.1. IdentiﬁcationChlorogenic of Phenolic acid (peak Acids 1, 6.34 in min) Snow showed Chrysanthemum a major molecular by ESI-Q-TOF-MSion at m/z 353.0855 [M − H]− negative ion mode. Chlorogenic(calculated acidfor C16 (peakH17O9, 1,353.0873). 6.34 min) At high showed CE, two a majorcharacteristic molecular fragment ion ions at m/zat m/z353.0855 191.0542 and [M − H]− 161.0266 were observed due to the cleavage of the caffeoyl bond, as shown in Figure 6. Likewise, peaks (calculated2.2.1. for Identification C H O , of 353.0873). Phenolic Acids At high in Snow CE, Chrysanthemum two characteristic by ESI-Q-TOF-MS fragment ions at m/z 191.0542 and 18, 19 and16 22 all17 gave9 [M − H]− ions at m/z 515.1188, corresponding to C25H24O12. These peak produced 161.0266two were successiveChlorogenic observed neutral acid due losses(peak to the of1, caffeic6.34 cleavage min) acid, showed yielding of the a caffeoyltwo major stable molecular bond,fragment asion ion shown at at m/z m/z 353.0855353.0845 in Figure [M6 −. HH] Likewise, −− (calculated− for C16H17O9, 353.0873). At−− high CE, two characteristic fragment ions at m/z 191.0542 and peaks 18,C9 19H6O and3] , 191.0536 22 all [M gave − H [M − 2C−9HH]6O3] . ionsAccording at m to/z their515.1188, empirical corresponding molecular formulae to and C25 Hliterature24O12. These peak produceddata161.0266 [21], were two peaks observed successive 18, 19 anddue to22 neutral thebelonged cleavage losses to of caffeoyl the of caffeoyl caffeic quinic bond, acid, acids as yielding andshown were in Figure twotentatively stable 6. Likewise, identified fragment peaks as ion at 18, 19 and 22 all gave [M − H]− ions at m/z 515.1188, corresponding to C25H24O12. These peak produced m/z 1,3-dicaffeoylquinic− − acid, 3,5-dicaffeoylquinic− acid,− 3,4-dicaffeoylquinic− − acid, respectively. 353.0845two successive [M H neutralC9 Hlosses6O3 of] caffeic, 191.0536 acid, yielding [M twoH stable2C9 fragmentH6O3] ion. According at m/z 353.0845 to their[M − H empirical − molecularC9 formulaeH6O3]−, 191.0536 and [M literature − H − 2C data9H6O3 []−21. According], peaks 18,to their 19 andempirical 22 belonged molecular toformulae caffeoyl and quinic literature acids and XChlorogenicJ-JMD acid were tentativelydata 20160329-YLC-10[21], identiﬁedpeaks 18,339 (6.367)19 as and 1,3-dicaffeoylquinic 22 belonged to caffeoyl acid, quinic 3,5-dicaffeoylquinic acids and were tentatively acid,2: TOF 3,4-dicaffeoylquinic MS identified ES- as 191.0570 100 51.5 一 acid, respectively.1,3-dicaffeoylquinic135.0454 acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic-H acid, respectively. 一 134.0377 -H ! XJ-JMD% Chlorogenic133.0250 acid ! 20160329-YLC-10 339 (6.367) ! 179.0282 2: TOF MS ES- 216.9796 ! 161.0266 191.0570 ! ! ! ! ! 51.5 100 !;105.0320 m/z=161.0266248.9910 258.9612276.9776 320.9704 一 310.9808 353.1162 135.0454 -H m/z=353.0855 0 m/z 一 100 120134.0377 140 160 180 200 220 240 260 280 300 320 340 360 -H 20160329-YLC-10 340 (6.376) Cm (324:348) 1: TOF MS ES- ! 一 d % 191.0542 ci 2.35e4 100 ! -H a 133.0250 ! 179.0282 ic 216.9796 ! ffe 161.0266 ! ! a ! ! ! !;105.0320 m/z=161.0266248.9910 258.9612276.9776- C 320.9704 310.9808m/z=353.0855353.1162 0 m/z

100% 120 140 160 180 200 220 240 260 280 300 320 340 360 353.0855 20160329-YLC-10 340 (6.376) Cm (324:348) 一 1: TOF MS ES- 191.0542192.0489 cid 2.35e4 100 -H a 219.8792m/z=260.9375191.0542 ic 127.0351 160.9248179.0018 193.9515 276.0203 ffe302.9942 331.0164 0 Ca m/z 100 120 140 160 180 200 220 240 260 280- 300 320 340 360 % 353.0855 192.0489 219.8792m/z=260.9375191.0542 127.0351 160.9248179.0018 193.9515 276.0203 302.9942 331.0164 0 m/z 100 120 140 160 180 200 220 240 260 280 300 320 340 360

Figure 6. Cont.

Molecules 2016, 21, 1307 8 of 19

Figure 6. MS spectra and the proposed fragmentation pathways of chlorogenic acid, ﬂavanomarein and marein.

Molecules 2016, 21, 1307 9 of 19

Table 4. Identiﬁcation of 30 compounds in snow chrysanthemum.

[M − H]− Peak tR (min) Formula Fragments Ion MS (m/z) in Negative Ion Mode Identification Experimental Mass (m/z) Theoreticalmass (m/z) Mass Error (ppm) 353.0855 [M − H]− 1 6.34 C16H18O9 353.0855 353.0873 −1.6 − Chlorogein acid 191.0542 [M − H − C9H6O3] 465.1045 [M − H]− 303.0477 [M − H − glucose]− 2 6.68 C21H22O12 465.1045 465.1033 2.2 − Taxifolin-7-O-β-D-glucoside 285.0390 [M − H − glucose − H2O] − 151.0021 [M − H − glucose − H2O − C8H6O2] 447.0912 [M − H]− 285.0385 [M − H − glucose]− − 3 8.14 C21H20O11 447.0912 447.0927 −3.4 267.0663 [M − H − glucose − H2O] Luteoloside − 151.0024 [M − H − glucose − H2O − C8H4O] − 135.0417 [M − H − glucose − H2O − C8H4O − O] 449.1079 [M − H]− 287.0534 [M − H − glucose]− − 4 9.95 C21H22O11 449.1079 449.1084 −0.7 269.0465 [M − H − glucose − H2O] R or S-flavanomarein − 151.0012 [M − H − glucose − H2O − C8H6O] − 135.0428 [M − H − glucose − H2O − C8H6O − O] 449.1079 [M − H]− 287.0534 [M − H − glucose]− − 5 10.03 C21H22O11 449.1079 449.1084 −0.7 269.0465 [M − H − glucose − H2O] R or S-flavanomarein − 151.0012 [M − H − glucose − H2O − C8H6O] − 135.0428 [M − H − glucose − H2O − C8H6O − O] 433.1136 [M − H]− 271.0587 [M − H − glucose]− 6 10.63 C21H22O10 433.1136 433.1135 0.2 − Butin-7-O-β-D-gluco-pyranoside 253.0482 [M − H − glucose − H2O] − 135.0231 [M − H − glucose − H2O − C8H6O] 611.1619 [M − H]− 449.1105 [M − H − glucose]− 287.0395 [M − H − 2glucose]− 7 11.13 C27H32O16 611.1619 611.1612 1.1 − Luteolin 7-O-β-D-sophoroside 269.0334 [M − H − 2glucose − H2O] − 151.0042 [M − H − 2glucose − H2O − C8H6O] − 135.0437 [M − H − 2glucose − H2O − C8H6O − O] 479.0843 [M − H]− 317.0262 [M − H − glucose]− 8 12.10 C21H20O13 479.0843 479.0826 3.5 − Quercetagitin-7-O-glucoside 151.0023 [M − H − glucose − C8H10O4] − 135.0428 [M − H − glucose − C8H10O4 − O] 287.0545 [M − H]− − 269.0441 [M − H − H2O] 9 12.15 C15H12O6 287.0545 287.0556 −1.1 − Isookain 151.0025 [M − H − H2O − C8H6O] − 135.0440 [M − H − H2O − C8H6O − O] Molecules 2016, 21, 1307 10 of 19

Table 4. Cont.

[M − H]− Peak tR (min) Formula Fragments Ion MS (m/z) in Negative Ion Mode Identification Experimental Mass (m/z) Theoreticalmass (m/z) Mass Error (ppm) 433.1135 [M − H]− − 7,3’,5’-Trihydroxy-flavanone-7-O- 10 12.35 C21H22O10 433.1135 433.1135 0 271.0605 [M − H − glucose] − β-D-glucopyranoside 135.0424 [M − H − glucose − C8H8O2] 303.0481 [M − H]− − 285.0375 [M − H − H2O] 11 12.74 C15H12O7 303.0492 303.0505 −4.3 − Taxifolin 151.0034 [M − H − H2O − C8H6O2] − 135.0475 [M − H − H2O − C8H6O2 − O] 449.1059 [M − H]− 287.0522 [M − H − glucose]− 5,7,3’,5’-Tetra-hydroxyflavanone- 12 14.15 C21H22O11 449.1059 449.1084 −3.2 − 151.0021 [M − H − glucose − C8H8O2] 7-O-β-D-gluco-pyranoside − 135.0443 [M − H − glucose − C8H8O2-O] 463.0878 [M − H]− − 301.0349 [M − H − glucose] Quercetin-7-O-β-D- 13 14.83 C21H20O12 463.0878 463.0877 0.1 − 151.0021 [M − H − glucose − H2O − C8H6O3] glucopyranoside − 135.0443 [M − H − glucose − H2O − C8H6O3 − O] 449.1082 [M − H]− 287.0561 [M − H − glucose]− − 14 15.40 C21H22O11 449.1082 449.1084 −0.2 269.0445 [M − H − glucose − H2O] Marein − 151.0013 [M − H − glucose − H2O − C8H6O] − 135.0426 [M − H − glucose − H2O − C8H6O − O] 447.0928 [M − H]− 285.0414 [M − H − glucose]− 15 15.85 C21H20O11 447.0928 447.0927 0.2 − Maritimein 151.0015 [M − H − glucose − C8H6O2] − 135.0443 [M − H − glucose − C8H6O2 − O] 463.0878 [M − H]− 301.0449 [M − H − glucose]− 16 16.23 C21H20O12 463.0878 463.0877 0.1 − Hyperoside 151.0021 [M − H − glucose − H2O − C8H6O3] − 135.0443 [M − H − glucose − H2O − C8H6O3 − O] 271.0605 [M − H]− 17 16.94 C15H12O5 271.0605 271.0606 −0.1 − 7,3’,5’-Trihydroxy-flavanone 135.0229 [M − H − C8H8O2] 515.1188 [M − H]− − 18 17.40 C25H24O12 515.1188 515.1190 −0.4 353.0845 [M − H − C9H6O3] 1,3-Dicaffeoylquinic acid − 191.0536 [M − H − 2C9H6O3] 515.1188 [M − H]− − 19 17.65 C25H24O12 515.1188 515.1190 −0.4 353.0845 [M − H − C9H6O3] 3,5-Dicaffeoylquinic acid − 191.0536 [M − H − 2C9H6O3] Molecules 2016, 21, 1307 11 of 19

Table 4. Cont.

[M − H]− Peak tR (min) Formula Fragments Ion MS (m/z) in Negative Ion Mode Identiﬁcation Experimental Mass (m/z) Theoreticalmass (m/z) Mass Error (ppm) 285.0396 [M − H]− − 267.0078 [M − H − H2O] 20 18.05 C15H10O6 285.0396 285.0399 −0.3 − Luteolin 151.0080 [M − H − H2O − C8H4O] − 135.0432 [M − H − H2O − C8H4O − O] 433.1135 [M − H]− 271.0605 [M − H − glucose]− 21 18.66 C21H22O10 433.1135 433.1135 0 − Coreopsin 253.0494 [M − H − glucose − H2O] − 135.0424 [M − H − glucose − H2O − C8H6O] 515.1188 [M − H]− − 22 19.20 C25H24O12 515.1188 515.1190 −0.4 353.0845 [M − H − C9H6O3] 3,4-Dicaffeoylquinic acid − 191.0536 [M − H − 2C9H6O3] 491.1191 [M − H]− 287.0522 [M − H − AcGlu]− − 23 19.32 C23H24O12 491.1191 491.1190 0.2 269.0430 [M − H − AcGlu − H2O] Acetylmarein − 151.0021 [M − H − AcGlu − H2O − C8H6O] − 135.0443 [M − H − AcGlu − H2O − C8H6O − O] 287.0558 [M − H]− − 269.0430 [M − H − H2O] 24 19.45 C15H12O6 287.0558 287.0556 0.7 − Okanin 151.0021 [M − H − H2O − C8H6O] − 135.0443 [M − H − H2O − C8H6O − O] 287.0534 [M − H]− − 25 20.79 C15H12O6 287.0534 287.0556 −2.2 151.0042 [M − H − C8H8O2] 5,7,3’,5’-Tetrahydroxy-ﬂavanone − 135.0426 [M − H − C8H8O2 − O] 475.1246 [M − H]− 271.0614 [M − H − AcGlu]− 26 21.40 C23H24O11 475.1246 475.1240 0.6 − Acetylcoreopsin 253.0498 [M − H − AcGlu − H2O] − 135.0424 [M − H − AcGlu –H2O − C8H6O] 301.0330 [M − H]− − 27 21.67 C15H10O7 301.0330 301.0348 −1.8 285.0385 [M − H − H2O] Quercetin − 151.0021 [M − H − H2O − C8H6O2] 285.0385 [M − H − ]− − 28 21.79 C15H10O6 285.0385 285.0399 −1.3 151.0025 [M − H − C8H6O2] 6,7,3’,4’-Tetrahydroxy-aurone − 135.0428 [M − H − C8H6O2 − O] 271.0597 [M − H]− − 29 22.76 C15H12O5 271.0597 271.0606 −0.9 253.0496 [M − H − H2O] Butein − 135.0428 [M − H − H2O − C8H6O] 269.0414 [M − H]− 30 23.03 C15H10O5 269.0414 269.0450 −3.6 − Sulfuretin 135.0357 [M − H − C8H6O2] Molecules 2016, 21, 1307 12 of 19

2.2.2. Identiﬁcation of Flavonoids in Snow Chrysanthemum by ESI-Q-TOF-MS

The Fragmentation Rules of Standards The ion-fragmentation pathways of flavonoids often show a retro-Diels-Alder rearrangement in ring C, and the loss of neutral molecules of H2O, sugar and carbonyl groups. Among the seven flavonoid standards, there were two major aglycones, flavanones (flavanomarein, butin-7-O-β-D-glucopyranoside, 0 0 isookanin, taxifolin, 5,7,3 ,5 -tetrahydroxyflavanone-7-O-β-D-glucopyranoside) and chalcones (marein, 0 0 0 okanin). The two aglycones have hydroxyls at C-3 , 4 , or 5 in the B ring and the loss of H2O caused by the 30,40-dihydroxyl disposition. In addition, these two aglycones have a hydroxyl and a sugar at C-5 and C-7 in the A ring, usually identified as being glucose. Flavanomarein (peak 5, 10.03 min) showed a predominant molecular ion at m/z 449.1079 [M − H]− in negative ion mode. Other fragment ions at m/z 287.0534 [M − H − glucose]−, 269.0465 [M − H − − − glucose − H2O] , 151.0012 [M − H − glucose − H2O − C8H6O] , 135.0428 [M − H − glucose − H2O − − C8H6O − O] were assigned to the loss of H2O or loss of sugar, as well as the retro-Diels-Alder rearrangement, as proposed in Figure6. The sugar at the 7 position was identified as glucose and the 0 0 loss of water arose from the 3 ,4 -dihydroxy substitution. The loss of C8H6O suggested that no hydroxyl − was at C-3 in ring C. Similarly, the [M − H] fragmentation pathway of butin-7-O-β-D-glucopyranoside (peak 6, 10.63 min) at m/z 433.1136 indicated no hydroxyls at the 5, 6 or 8 position in ring A. Isookanin (peak 9), as the aglycone of flavanomarein, exhibited the same fragment ions as flavanomarein. Likewise, taxifolin (peak 11) eluted at 12.74 min, exhibiting [M − H]− ions at m/z 303.0492. The loss of 0 0 − sugar, 3 ,4 -dihydroxyl and 3-hydroxyl caused the fragment ions at m/z 285.0375 [M − H − H2O] , − 151.0034 [M − H − H2O − C8H6O2] . The loss of C8H6O2 indicated the existence of a 3-hydroxyl 0 0 substitution pattern. In addition, 5,7,3 ,5 -tetrahydroxyflavanone-7-O-β-D-glucopyranoside (peak 12), another flavanone references, showed similar but different fragmentation patterns from other types of flavanones. It eluted at 14.15 min, showing four predominant fragment ions at m/z 449.1059 [M − H]−, − − 0 0 287.0522 [M − H − glucose] , 151.0021 [M − H − glucose − C8H8O2] . 3 ,5 -dihydroxyl and retro-Diels-Alder rearrangement resulted in the loss of C8H8O2, without the loss of H2O. Marein (peak 14, 15.40 min) produced a minor protonated ion [M − H]− at m/z 449.1082 and a dominant fragment ion [M − H − glucose]− at m/z 287.0561 in negative mode. The m/z 269.0445 [M − H − 0 0 − glucose − H2O] peak was due to the loss of water between ring the B 3 -OH and 4 -OH. In addition, − a fragment ion at m/z 151.0013 [M − H − glucose − H2O − C8H6O] , 135.0426 [M − H − glucose − − H2O − C8H6O − O] was produced by a retro-Diels-Alder rearrangement. The detailed fragmentation pathway is shown in Figure6. Okanin (peak 24) gave an [M − H]− ion at m/z 287.0558, which was 162 Da less than marein, proving the loss of a sugar from the C-7 position compared with marein. In addition, okanin showed the similar fragmentation rules as marein. In summary, these fragmentation behaviors of standards could be used to obtain the fragmentation rules of different types of flavonoids and the 3-OH, 5-OH or 30,40-dihydroxyl, 30,50-dihydroxyl substitution patterns could be identified by the anundance of characteristic fragments.

Identification of Flavanones in Snow Chrysanthemum by ESI-Q-TOF-MS Eleven flavanones have been identified from snow chrysanthemum, six of which (peaks 4, 5, 6, 9, 11 and 12) were unambiguously identified by comparison with the reference standards. Peak 2 − − produced a [M − H] ion at m/z 465.1045 [M − H] (calculated for C21H21O12, 465.1033). Moderately abundant product ions at m/z 303.0477 and 285.0390 were formed by the neutral losses of glucose and 0 0 H2O. The loss of H2O confirmed the existence of a 3 ,4 -dihydroxyl substitution pattern. The fragment ions at m/z 151.0021 yielded by retro-Diels-Alder rearrangement in ring C and the loss of C8H6O2 showed the existence of a 3-OH group. By comparing its fragment ions with those of the taxifolin − standard, it was identified as taxifolin-7-O-β-D-glucoside. Peak 7 gave an [M − H] ion at m/z 611.1619, corresponding to a molecular formula C27H32O16. At high CE, ions at m/z 449.1105 and 287.0395 were produced by two successive neutral losses of glucose from the deprotonated [M − H]− Molecules 2016, 21, 1307 13 of 19

molecule of peak 7. In addition, the fragment ion at m/z 269.0334 resulted from the loss of H2O. Likewise, the fragment ion at m/z 151.0042 was produced by the characteristic RDA cleavage and neutral loss of C8H6O from the fragment ion at m/z 269.0334. Therefore, peak 7 was tentatively − identified as luteolin 7-O-β-D-sophoroside [22]. Similarly, peak 10 exhibited an ion [M − H] at m/z 433.1135 (C21H22O10), which produced a major fragment ion at m/z 271.0605 by loss of glucose, indicating the presence of a glucose unit in its structure. A fragment ion at m/z 135.0424 resulted from the loss of C8H8O2 from the fragment ion at m/z 271.0605. That indicated the existence of a 30,50-dihydroxy disposition in peak 10 without the loss of water, so peak 10 was identified as 0 0 7,3 ,5 -trihydroxyflavanone-7-O-β-D-glucopyranoside by comparison with an authentic standard. Peak 17 gave an [M − H]− at m/z 271.0605, corresponding to the fragment ion of peak 10. According to the fragment ions, peak 17 was identified as 7,30,50-trihydroxyflavanone. Similarly, by comparison with the available data, peak 25 was also identified having a 30,50-dihydroxyl susbtituion pattern and the major fragment ions at m/z 287.0534 and 151.0042 displayed the existence of a 5-OH group, so peak 25 was tentatively assigned as 5,7,30,50-tetrahydroxyflavanone.

Identification of Chalcones in Snow Chrysanthemum by ESI-Q-TOF-MS Chalcones, one of the major type of flavonoids in the snow chrysanthemum, have similar fragment rules as flavanones, but there is a tremendous difference in chromatographic behavior between chalcones and flavanones in that chalcones have longer retention times than flavanones [18]. Peaks 14 and 24 were unambiguously identified as marien and okanin by comparison with reference standards. Peak 21 showed an obvious ion of [M − H]− at m/z 433.1135, which was 16 Da less than that of peak 14, indicating removal of an atom of oxygen from the C-8 position of marein. Otherwise it showed similar fragment ions as marein. Thus, the structure of peak 21 was tentatively identified as − coreopsin. Likewise, peak 29 displayed an ion [M − H] at m/z 271.0597 (C15H11O5). By comparison with peak 21, we found that its molecular weight was 162 Da less than that of compound 21, indicating the loss of a glucose unit from the C-7 position of peak 21. Peak 23 produced a dominant fragment − ion [M − H] at m/z 491.1191(C21H22O10) in negative ion mode, which was 42 Da more than that of marein. Besides, its spectrum at high CE showed a series of ions at m/z 287.0522 [M − H − AcGlu]−, − − 269.0430 [M − H − AcGlu − H2O] , 151.0021 [M − H − AcGlu − H2O − C8H6O] , 135.0443 [M − H − − AcGlu − H2O − C8H6O − O] , indicating the existence of acetylation-glucose substitution. On the basis of the available reference data [23], peak 23 was tentatively identified as acetylmarein. Similarly, peak 26 was identified as acetylcoreopsin due to an ion at m/z 475.1246 [M − H]− and a fragment ion at m/z 271.0614 produced by losses of acetylation/glucose unit (204 Da) from the deprotonated molecule ion of [M − H]−, which were in agreement with the data reported in the literature [24].

Identification of Flavones in Snow Chrysanthemum by ESI-Q-TOF-MS In addition, based on the similar fragmentation rules of flavanones and chalcones, two flavones (peaks 3 and 20) and four flavanols (peak 8, 13, 16, and 27) were also identified. Flavones with a C-2–C-3 double bond are another type of compounds found in snow chrysanthemum. Because of the C=C in flavones, the losses of C8H4O (116 Da) or C8H6O2 (132 Da) were the characteristic fragment ions. Peak 3 showed a major ion at 447.0912 [M − H]−, which was 2 Da less than peak 4 (flavanomarein), indicating the existence of a C-2/C-3 double bond. Based on other fragment ions at − − m/z 285.0385 [M − H − glucose] , 267.0663 [M − H − glucose − H2O] , 151.0024 [M − H − glucose − − − H2O − C8H4O] , 135.0417 [M − H − glucose − H2O − C8H4O − O] , peak 3 was identified as luteoloside. Peak 20 gave a major molecule ion at 285.0396 [M − H]−, which was 162 Da less than that of peak 3, showing a glucose unit was removed from the C-7 position of peak 3. According to other fragment ions, peak 20 was identified as luteolin. Peak 8 showed an ion of [M − H]− at m/z 479.0843, corresponding to the molecular formula − C21H20O13. At high CE, the ions at m/z 317.0262 [M − H − glucose] , 151.0023 [M − H − glucose − − − C8H10O4] , 135.0428 [M − H − glucose − C8H10O4 − O] were produced by the loss of glucose and Molecules 2016, 21, 1307 14 of 19

RDA rearrangement in ring A. Peak 8 was tentatively identified as quercetagitin-7-O-glucoside on the basis of the empirical molecular formula and literature data [25]. Peak 27 gave a major molecular ion at m/z 301.0330 [M − H]−, which was 162 Da less than that of peak 8, showing a glucose unit was removed from the C-7 position of peak 8. Accroding to other fragment ions, peak 27 was identified as − quercetin. Peak 13 produced an ion of [M − H] at m/z 463.0878 (C21H20O12) and its molecular weight was 16 Da less than that of peak 8, indicating removal of an atom of oxygen from the C-3 position of peak 8. Based on the other fragment ions at m/z 301.0349 [M − H − glucose]−, 151.0021 [M − H − − − glucose − H2O − C8H6O3] , 135.0443 [M − H − glucose − H2O − C8H6O3 − O] and a literature report [26], peak 13 was tentatively identified as quercetin-7-O-β-D-glucopyranoside. Peak 16 gave the same [M − H]− ion at m/z 463.0878 of peak 13, and other fragment ions at m/z 301.0349 [M − H − − − glucose] , 151.0021 [M − H − glucose − H2O − C8H6O3] , 135.0443 [M − H − glucose − H2O − − C8H6O3 − O] were also consistent with those of peak 13, therefore, peak 16 was tentatively identified as hyperoside based on literature data [27].

Identification of Aurones in Snow Chrysanthemum by ESI-Q-TOF-MS Peak 15 produced an [M − H]− ion at m/z 447.0928, which indicated a molecular formula of C21H22O11. At high CE, two characteristic fragment ions at m/z 285.0414 and 151.0015 were observed caused by the loss of glucose and a RDA rearrangement in ring C. Hence, peak 15 was identified as maritimein and this identification was confirmed by comparsion with the literature [28]. Likewise, peak 28 gave a major molecule ion at m/z 285.0385 [M − H]−, which was 162 Da less than that of peak 15, showing a glucose unit was removed from the C-7 position of peak 15. According to other fragment ions, peak 28 was identified as 6,7,3’,4’-tetrahydroxyaurone. Peak 30 showed an [M − H]− ion at m/z 269.0414 and its molecular weight was 16 Da less than that of peak 28, indicating an atom of oxygen was removed from the C-8 position of peak 28. Based on other fragment ions at m/z 135.0357 [M − H − − C8H6O2] and literature date [29], peak 30 was tentatively identified as sulfuretin. All the structure assigments are summarized in Figure7.

Figure 7. Chemical structures of the compounds identiﬁed in snow chrysanthemum.

2 Molecules 2016, 21, 1307 15 of 19

3. Experimental Section

3.1. Samples, Standards and Reagents Six batches of seeds were collected from the USA, Ningxia, Jiangsu, and Xinjiang. Then we planted them in four different regions: Minfeng (Xinjiang), Sihai (Beijing), Shenjiaying (Beijing), and Implad in Beijing. Detailed information about the seeds and regions is shown in Table5. Molecules 2016, 21, x 15 of 18 Table 5. Summary of the analyzed samples. Table 5. Summary of the analyzed samples.

SampleSample Codes Provenance Provenance Habitat Habitat S1 USA, USA, purchased purchased from Jiangsu from Jiangsu Pacific Seeds, Pacific Chenmei. Seeds,Chenmei. S2 Ningxia, Ningxia, purchased purchased from Jiangsu from JiangsuPacific Seeds, Pacific Chenmei. Seeds, Chenmei. S3 Jiangsu, Jiangsu, purchased purchased from Jiangsu from Jiangsu Pacific Seeds, Pacific Chenmei. Seeds, Chenmei. Minfeng S4S4 ※ Xinjiang,Xinjiang, purchased purchased from Xinjiang from Xinjiang CoreopsisCoreopsis tinctoria Company tinctoria Company (Xinjiang,Minfeng H = 2700 m) Xinjiang,Xinjiang, intermating intermating from seeds from of seeds Keliyang, of Keliyang, amphichrome, amphichrome, purchased from purchased (Xinjiang, H = 2700 m) S5 Xinjiangfrom Coreopsis Xinjiang tinctoriaCoreopsis Company. tinctoria Company. Xinjiang,Xinjiang, intermating intermating from seeds from of seeds Keliyang, of Keliyang, red flowers, red purchased flowers, from purchased S6 Xinjiangfrom Coreopsis Xinjiang tinctoriaCoreopsis Company. tinctoria Company. S7 USA, purchased from Jiangsu Pacific Seeds, Chenmei. S7S8 Ningxia, USA, purchased purchased from from Jiangsu Jiangsu Pacific Pacific Seeds, Seeds, Chenmei. Chenmei. S8S9 Jiangsu, Ningxia, purchased purchased from Jiangsu from JiangsuPacific Seeds, Pacific Chenmei. Seeds, Chenmei. S9 Jiangsu, purchased from Jiangsu Pacific Seeds, Chenmei. Sihai S10 Xinjiang, purchased from Xinjiang Coreopsis tinctoria Company. Sihai S10 Xinjiang, purchased from Xinjiang Coreopsis tinctoria Company. (Beijing, H = 725 m) Xinjiang, intermating from seeds of Keliyang, amphichrome, purchased from (Beijing, H = 725 m) S11 Xinjiang, intermating from seeds of Keliyang, amphichrome, purchased S11 Xinjiang Coreopsis tinctoria Company. from Xinjiang Coreopsis tinctoria Company. Xinjiang, intermating from seeds of Keliyang, red flower, purchased from S12 Xinjiang, intermating from seeds of Keliyang, red flower, purchased from S12 Xinjiang Coreopsis tinctoria Jiangsu Pacific Seeds, Chenmei. Xinjiang Coreopsis tinctoria Jiangsu Pacific Seeds, Chenmei. S13 USA, purchased from Jiangsu Pacific Seeds, Chenmei. S13S14 Ningxia, USA, purchased purchased from from Jiangsu Jiangsu Pacific Pacific Seeds, Seeds, Chenmei. Chenmei. S14S15 Jiangsu, Ningxia, purchased purchased from Jiangsu from JiangsuPacific Seeds, Pacific Chenmei. Seeds, Chenmei. S15S16 Xinjiang,purchased Jiangsu, purchased from Xinjiang from Jiangsu Coreopsis Pacific tinctoria Seeds, Company Chenmei. Shen ShenJiaying Jiaying S16Xinjiang, Xinjiang, intermating purchased from fromseeds Xinjiangof Keliyang,Coreopsis amphichrome, tinctoria purchasedCompany from (Beijing,(Beijing, H = 514 H m) = 514 m) S17 Xinjiang, intermating from seeds of Keliyang, amphichrome, purchased S17 Xinjiang Coreopsis tinctoriac Company. Xinjiang,from intermating Xinjiang Coreopsis from seeds tinctoria of Keliyang,c Company. red flower, purchased from S18 Xinjiang, intermating from seeds of Keliyang, red flower, purchased from S18 Xinjiang Coreopsis tinctoria Company. S19 USA,Xinjiang purchasedCoreopsis from Jiangsu tinctoria PacificCompany. Seeds, Chenmei. S19S20 Ningxia, USA, purchased purchased from from Jiangsu Jiangsu Pacific Pacific Seeds, Seeds, Chenmei. Chenmei. S20S21 Jiangsu, Ningxia, purchased purchased from Jiangsu from JiangsuPacific Seeds, Pacific Chenmei. Seeds, Chenmei. S21S22 Xinjiang, Jiangsu, purchased purchased from Xinjiang from Jiangsu Coreopsis Pacific tinctoria Seeds, company. Chenmei. IMPLADIMPLAD Xinjiang, intermating from seeds of Keliyang, amphichrome, purchased from (Beijing, H = 50 m) S22S23 Xinjiang, purchased from Xinjiang Coreopsis tinctoria company. (Beijing, H = 50 m) XinjiangXinjiang, Coreopsis intermating tinctoria Company. from seeds of Keliyang, amphichrome, purchased S23 Xinjiang, intermating from seeds of Keliyang, red flower, purchased from S24 from Xinjiang Coreopsis tinctoria Company. XinjiangXinjiang, Coreopsis intermating tinctoria Company. from seeds of Keliyang, red flower, purchased from S24 Xinjiang Coreopsis tinctoria※ SampleCompany. S4 was lost. Sample S4 was lost. Chlorogenic acid (≥98%) was purchased from Muenster (Chengdu, China). Flavanomarein, marein, isookanin, okanin, 5,7,3′,5′-tetrahydroxyflavanone-7-O-β-D-glucopyranoside, taxifolin, and butin-7-ChlorogenicO-β-D-glucopyranoside acid (≥98%) was (≥98%) purchased were separated from Muenster and purified (Chengdu, in our China). laboratory. Flavanomarein, Their 0 0 marein,structure isookanin, were also okanin, confirmed 5,7,3 based,5 -tetrahydroxyflavanone-7- on their MS and NMR dataO [30]-β-D (Figure-glucopyranoside, 1). Acetonitrile taxifolin, (HPLC and butin-7-grade)O-β was-D-glucopyranoside purchased from Fisher (≥98%) Scientific were separatedCo. (Loughborough, and purified UK). in Formic our laboratory. acid (HPLC Their grade) structure werewas also purchased confirmed from based Acros on Co. their Ltd. MS (Fair and Lawn, NMR NJ, data USA). [30] (FigureWater was1). Acetonitrilepurified with (HPLC a Mill Q-Plus grade) was purchasedsystem from(Millipore, Fisher Billerica, Scientific MA, Co. USA). (Loughborough, Other reagents UK). used Formic for extraction acid (HPLC and separation grade) was were purchased AR fromgrade, Acros and Co. purchased Ltd. (Fair from Lawn, Beijing NJ, Chemical USA). Water Plant was(Beijing, purified China). with a Mill Q-Plus system (Millipore, Billerica, MA, USA). Other reagents used for extraction and separation were AR grade, and purchased 3.2. Instruments and Conditions from Beijing Chemical Plant (Beijing, China). 3.2.1. HPLC-DAD Analysis Conditions 3.2. Instruments and Conditions HPLC-DAD analysis was performed on a Flexar system (Perkin Elmer, Waltham, MA, USA) 3.2.1.equipped HPLC-DAD with a Analysis pump, a diode Conditions array detector (DAD), and a Totalchrom chromatographic workstation, with an Ageal Innoval ODS-2 column (4.6 mm × 250 mm, 5 µm). Gradient elution of 0.2% formic acidHPLC-DAD solution (solvent analysis A) wasand acetonitrile performed (solvent on a Flexar B) at a system flow rate (Perkin of 1 mL/min Elmer, was Waltham, employed: MA, 0–5 USA) equippedmin, 13%–15% with a pump, B; 5–12 a diodemin, 15%–18% array detector B; 12–20 (DAD), min, 18%–20% and a Totalchrom B; 20–24 min, chromatographic 20%–22% B; 24–29 workstation, min, 22%–40% B. The injection volume was 20 µL, and the detection wave-length was set at 285 nm, 378 nm. Resolution factor (R) of the eight compounds with the closest peak from analysis of sample was ≥ 1.5. Number of theoretical plates (n) of the eight compounds was ≥11,000. Tailing factor (T) of the eight compounds was within 0.90 and 1.10. The chromatograms are shown in Figure 2.

Molecules 2016, 21, 1307 16 of 19 with an Ageal Innoval ODS-2 column (4.6 mm × 250 mm, 5 µm). Gradient elution of 0.2% formic acid solution (solvent A) and acetonitrile (solvent B) at a ﬂow rate of 1 mL/min was employed: 0–5 min, 13%–15% B; 5–12 min, 15%–18% B; 12–20 min, 18%–20% B; 20–24 min, 20%–22% B; 24–29 min, 22%–40% B. The injection volume was 20 µL, and the detection wave-length was set at 285 nm, 378 nm. Resolution factor (R) of the eight compounds with the closest peak from analysis of sample was ≥ 1.5. Number of theoretical plates (n) of the eight compounds was ≥11,000. Tailing factor (T) of the eight compounds was within 0.90 and 1.10. The chromatograms are shown in Figure2.

3.2.2. UPLC-ESI-QTOF-MS Analysis Conditions UPLC-ESI-QTOF-MS analysis was carried out on an Acquity UPLCTM system (Waters Corp., Milford, MA, USA) coupled with a Synapt G-1 MS system (Waters Corp., Manchester, UK). A Waters Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm) was used for the analysis with the column temperature set at 45 ◦C. The mobile phase was water with 0.1% formic acid (A) and acetonitrile (B). The gradient used as follows: 0–5 min, 3%–5% B; 5–8 min, 5%–10% B; 8–15 min, 10%–14% B; 15–20 min, 14%–20% B; 20–22 min, 20%–30% B; 22–28 min, 30%–35% B .The ﬂow of rate was 0.5 mL/min, and the injection volume was 2 µL. The TOF MS detection was performed on a Synapt MS system with the data acquisition modern MSE. The tandem mass experiment was performed in negative ESI ionization modes with the data acquisition ranging from 100 to 1500 Da. The source temperature was 100 ◦C, and the desolvation temperature was 450 ◦C with desolvation gas ﬂow of 900 L/h. Leucine Enkephalin (200 pg/µL) was used as the lock mass. The capillary voltage was 2.5 KV, and the cone voltage was 40 V. The collision energy was 6 eV (trap) and 4 eV (transfer) for low-energy scans, and 45–60 eV ramp (trap) and 12 eV (transfer) for high-energy scans. Finally, the instrument was controlled by Waters Masslynx 4.1 software.

3.3. Sample and Standard Solution Preparation for Analysis

3.3.1. Sample Preparation Sample (about 0.5 g) were accurately weighed into a covered 50 mL conical flask. Then, 70% methanol (20 mL) was accurately added into the flask. The mixture was ultrasonicated (500 W, 40 KHz) for 30 min, and filtered into a 25 mL volumetric flask. Afterward, the residue was washed with 70% methanol (3 mL). Finally, the sample solution was made up to 25 mL and filtered through a 0.22 µm filter for further analysis.

3.3.2. Standard Solution Preparation Each of standards were accurately weighed, dissolved in methanol and diluted with methanol to an appropriate concentration. A mixed solution of eight standards, containing 300 µg/mL of chlorogenic acid, 1000 µg/mL of ﬂavanomarein, 1600 µg/mL of marein, 400 µg/mL of isookanin, 0 0 700 µg/mL of okanin, 350 µg/mL of 5,7,3 ,5 -tetrahydroxyﬂavanone-7-O-β-D-glucopyranoside, 100 µg/mL of taxifolin, and 50 µg/mL of butin-7-O-β-D-glucopyranoside, was prepared in methanol and stored in the refrigerator at 4 °C until required for analysis.

3.4. Method Validation

3.4.1. Calculations Calibration curves were plotted by the peak area (Y) of analytes against concentration (X) of standards. The linear range was evaluated by linear regression analysis calculated by the least square regression method. Limits of detection (LOD) and quantiﬁcation (LOQ) under the present chromatographic conditions were determined on the basis of response and slope of each regression equation at a signal-to-noise ratio (S/N) of 3 and 10, respectively. Molecules 2016, 21, 1307 17 of 19

3.4.2. Precision, Repeatability, Stability and Accuracy Intra-day and inter-day variations were chosen to determine the precision of the method. They were determined by analyzing mixed standards solution six times within one day and on three consecutive days. Then the relative standard deviation (RSD) was taken as a measure of precision. To assess the repeatability of the method, a sample was extracted and analyzed six times parallelly. Then calculate the RSDs of the contents of eight compounds. The stability test was performed by analysis of the sample at 0, 2, 4, 6, 8, 12 and 24 h, and calculating the RSDs for peak area ratio of each analyte. The recovery test was used to evaluate the accuracy of this method. Accurate amounts of eight standards were added to the known amounts of eight compounds in samples. The above-prepared samples (n = 6) were extracted and analyzed as described as in Sections 3.2.1 and 3.3.1. The average recoveries were determined by the formula: Recovery (%) = (amount found − original amount)/spiked amount × 100%.

4. Conclusions In this study, a high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) method was established and validated in terms of linarity, sensitivity, precision, accuracy and stability, and then successfully applied for simultaneous quantitative analysis of eight characteristic compounds in snow chrysanthemum. The results of the 23 batches of samples demonstrate the influence of seed provenance and habitat. Flavanomarein and marein are found to be abundant in 23 batches of samples and could be used as suitable markers for quality control of snow chrysanthemum. In addition, a high resolution UPLC-ESI-QTOF-MS/MS method was developed for the systemic analysis of the flavonoids and phenolic acids in snow chrysanthemum. Based on their exact mass, fragmentation behaviors and retention timee, a total of 30 ingredients were identified in the crude extract of snow chrysanthemum, providing a deeper understanding of the chemical constituents of snow chrysanthemum.

Acknowledgments: This research work was supported by Program for Innovative Research Team in IMPLAD (PIRTI). Author Contributions: Yinjun Yang, Sibao Chen, and Baolin Guo conceived and designed the experiments; Yinjun Yang, Jinjun Liu and Baiping Ma performed the isolation and structure elucidation of the constituents; Yinjun Yang, Xinguang Sun and Liping Kang analyzed the data and wrote this paper. All authors read and approved the final manuscript. Conflicts of Interest: The authors declar no conflict of interest.

References

1. Guo, L.; Zhang, W.; Li, S.; Ho, C.T. Chemical and nutraceutical properties of Coreopsis tinctoria. J. Funct. Foods 2015, 13, 11–20. [CrossRef] 2. Wang, T.; Xi, M.; Guo, Q.; Wang, L.; Shen, Z. Chemical components and antioxidant activity of volatile oil of a Compositae tea (Coreopsis tinctoria Nutt.) from Mt. Kunlun. Ind. Crop. Prod. 2015, 67, 318–323. [CrossRef] 3. Dias, T.; Bronze, M.R.; Houghton, P.J.; Mota-Filipe, H.; Paulo, A. The flavonoid-rich fraction of Coreopsis tinctoria promotes glucose tolerance regain through pancreatic function recovery in streptozotocin-induced glucose-intolerant rats. J. Ethnopharmacol. 2010, 132, 483–490. [CrossRef][PubMed] 4. Dias, T.; Liu, B.; Jones, P.; Houghton, P.J.; Mota-Filipe, H.; Paulo, A. Cytoprotective effect of Coreopsis tinctoria extracts and flavonoids on tBHP and cytokine-induced cell injury in pancreatic MIN6 cells. J. Ethnopharmacol. 2012, 139, 485–492. [CrossRef][PubMed] 5. Yang, Q.; Sun, Y.H.; Zhang, L.; Xu, L.; Hu, M.Y.; Liu, X.Y.; Shi, F.Y.; Gu, Z.Y. Antihypertensive effects of extract from flower buds of Coreopsis tinctoria on spontaneously hypertensive rats. Chin. Herb. Med. 2014, 6, 103–109. [CrossRef] 6. Li, N.; Meng, D.; Pan, Y.; Cui, Q.; Li, G.; Ni, H.; Sun, Y.; Qing, D.; Jia, X.; Pan, Y.; Hou, Y. Anti-neuroinflammatory and NQO1 inducing activity of natural phytochemicals from Coreopsis tinctoria. J. Funct. Foods 2015, 17, 837–846. [CrossRef] Molecules 2016, 21, 1307 18 of 19

7. Zhang, Y.; Shi, S.; Zhao, M.; Chai, X.; Tu, P. Coreosides A–D, C14-polyacetylene glycosides from the capitula of Coreopsis tinctoria and its anti-inflammatory activity against COX-2. Fitoterapia 2013, 87, 93–97. [CrossRef] [PubMed] 8. Chen, L.X.; Hu, D.J.; Lam, S.C.; Ge, L.; Wu, D.; Zhao, J.; Long, Z.R.; Yang, W.J.; Fan, B.; Li, S.P. Comparison of antioxidant activities of different parts from snow chrysanthemum (Coreopsis tinctoria Nutt.) and identification of their natural antioxidants using high performance liquid chromatography coupled with diode array detection and mass spectrometry and 2,20-azinobis(3-ethylbenzthiazoline-sulfonic acid) diammonium salt-based assay. J. Chromatogr. A 2016, 1428, 134–142. [PubMed] 9. Yao, L.; Zhang, Y.; Mao, X.; Li, L.; Zhang, R.; Wang, J.; Li, X.; Li, H. The anti-inflammatory and antifibrotic effects of Coreopsis tinctoria Nutt on high-glucose-fat diet and streptozotocin-induced diabetic renal damage in rats. BMC Complement. Altern. Med. 2015, 15, 314. [CrossRef][PubMed] 10. Ma, Z.; Zheng, S.; Han, H.; Meng, J.; Yang, X.; Zeng, S.; Zhou, H.; Jiang, H. The bioactive components of Coreopsis tinctoria (Asteraceae) capitula: Antioxidant activity in vitro and profile in rat plasma. J. Funct. Foods 2016, 20, 575–586. [CrossRef] 11. Wang, J.; Aierken, G.; Li, X.; Li, L.; Mao, X. Testing the absorption of the extracts of Coreopsis tinctoria Nutt. in the intestinal canal in rats using an Ussing chamber. J. Ethnopharmacol. 2016, 186, 73–83. [CrossRef][PubMed] 12. Li, Y.; Yan, S.; Li, X.; Xu, C.; Liu, S.; Sun, S. Discrimination of Kunlun chrysanthemum from different regions by infrared spectroscopy. Mod. Instrum. 2012, 18, 71–73. 13. Aimaiti, M.; Zhao, W.; Abulizi, M.; Li, X. Determination of chlorogenic acid in Kunlun chrysanthemum by reversed phase high performance liquid chromatography. J. Xinjiang Med. Univ. 2011, 34, 1370–1372. 14. Zhang, Y.; Wang, Y.; Li, X.; Zhi, L.; Eyitulun, S. Determination of chlorogenic acid and baicalin in Kunlun chrysanthemum by HPLC. Chin. J. Exp. Tradit. Med. Formul. 2012, 18, 107–109. 15. Sun, Z.Z.; Biken, A.; Zhang, Y.l.; Aytulun, S. Quantitative chemical composition and chemical pattern recognition in Coreopsis tinctoria from different areas. Chin. J. Exp. Tradit. Med. Formul. 2012, 18, 174–178. 16. Guzhalinuer, A.; Li, X.; Mao, X.; Li, L.; Wang, Y.; Tao, Y.; Ma, X.; Zhu, M.; Jiao, Y. Determination and data analysis of chlorogenic acid and flavonoids in Coreopsis tinctoria from different areas in Xinjiang. J. Northwest Pharm. 2013, 28, 248–251. 17. Lam, S.C.; Liu, X.; Chen, X.Q.; Hu, D.-J.; Zhao, J.; Long, Z.R.; Fan, B.; Li, S.P. Chemical characteristics of different parts of Coreopsis tinctoria in China using microwave-assisted extraction and high-performance liquid chromatography followed by chemometric analysis. J. Sep. Sci. 2016, 39, 2919–2927. [CrossRef] [PubMed] 18. Wu, Y.; Wang, X.F.; Yuan, S.L. Process optimization by response surface methodology for extraction of water-soluble flavonoids from Coreopsis tinctoria flowers. Food Sci. Technol. 2013, 34, 129–133. 19. Zhang, R.; Li, X.; Mao, X.; Li, L. Determination of flavanomarein and marein in extracts of Coreopsis tinctoria by HPLC. J. Anhui Agric. Sci. 2015, 43, 73–74. 20. Bai, X.; Hu, K.; Dai, S.; Wang, L. Components of flower pigments in the petals of different color Chrysanthemum morifolium Ramat. cultivars. J. Beijing For. Univ. 2006, 28, 84–89. 21. Ling, Y.; Liu, K.; Zhang, Q.; Liao, L.; Lu, Y. High performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight–mass spectrometry as a powerful analytical strategy for systematic analysis and improved characterization of the major bioactive constituents from Radix Dipsaci. J. Pharm. Biomed. Anal. 2014, 98, 120–129. [PubMed] 22. Es-Safi, N.E. Plant polyphenols: Extraction, structural characterization, hemisynthesis and antioxidant properties. In Phytochemicals as Nutraceuticals-Global Approaches to Their Role in Nutrition and Health; InTech: Rijeka, Croatia, 2012; pp. 181–206. 23. Zhu, N.; Li, X.W.; Liu, G.Y.; Shi, X.L.; Gui, M.Y.; Sun, C.Q.; Jin, Y.R. Constituents from aerial parts of Bidens ceruna L. and their DPPH radical scavenging activity. Chem. Res. Chin. Univ. 2009, 25, 328–331. 24. Li, S.; Kuang, H.X.; Okada, Y.; Okuyama, T. New flavanone and chalcone glucosides from Bidens bipinnata Linn. J. Asian Nat. Prod. Res. 2005, 7, 67–70. [CrossRef][PubMed] 25. Zhang, Y.; Shi, S.; Zhao, M.; Jiang, Y.; Tu, P. A novel chalcone from Coreopsis tinctoria Nutt. Biochem. Syst. Ecol. 2006, 34, 766–769. [CrossRef] 26. Cui, Q.L.; Ni, H.; Pan, Y.N.; Li, N.; Jia, X.G.; Liu, X.Q. Isolation and identification of chemical constituents from capitula of Coreopsis tinctoria. J. Shenyang Pharm. Univ. 2015, 32, 14–17. Molecules 2016, 21, 1307 19 of 19

27. Khallouki, F.; Haubner, R.; Ricarte, I.; Erben, G.; Klika, K.; Ulrich, C.M.; Owen, R.W. Identiﬁcation of polyphenolic compounds in the ﬂesh of Argan (Morocco) fruits. Food Chem. 2015, 179, 191–198. [CrossRef] [PubMed] 28. Shimokoriyama, M. Anthochlor Pigments of Coreopsis tinctoria. J. Am. Chem. Soc. 1957, 79, 214–220. [CrossRef] 29. Chao, J.; Yin, Z.; Ye, W.; Zhao, S. Chemical constituents from branch of Broussonetia papyrifera. China J. Chin. Mater. Med. 2006, 31, 1078–1080. 30. Liu, J.J.; Yang, Y.J.; Zhu, Y.D.; Li, G.Z.; Huang, W.H.; Guo, B.L. A new bisabolane-type sesquiterpenoid from Coreopsis tinctoria. China J. Chin. Mater. Med. 2015, 40, 2132–2137.

Sample Availability: Samples of the compounds 1–8 in this paper and 23 batches of snow chrysanthemum are available from the authors.

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