Research ArticleExploring Chemical Basis of Toxicity Reduction forProcessed Roots of Stellera chamaejasme L UsingUltraperformance Liquid ChromatographyndashTriple QuadrupoleTandem Mass Spectrometry

Wei Yang1 Xiaoli Ma2 LudiWang2 MengmengWei1 ShuyaoWang1 SiyangWu1

Chen Kang1 and Yingfei Li 1

1Center for DMPK Research of Herbal Medicines Institute of Chinese Materia Medica China Academy of Chinese Medical SciencesBeijing 100700 China2College of Traditional Chinese Medicine Hebei University Hebei 071000 China

Correspondence should be addressed to Yingfei Li dmpk20121201163com

Received 23 November 2018 Revised 2 February 2019 Accepted 18 February 2019 Published 6 March 2019

Academic Editor Anastasios S Economou

Copyright copy 2019 Wei Yang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Some herbal medicines are treated with various processing methods to ensure their safe and effective use However chemical basisof toxicity reducing for most herbal medicines remains unclear Stellera chamaejasme L particularly the root (ruixianglangdu)is toxic Thus ruixianglangdu is commonly processed with vinegar or milk to reduce toxicity Here with help of multiple-ion monitoring (MIM)-based metabolomics we comprehensively capture chemical information of ruixianglangdu Then 33differential components between crude drugs and processed products were identified or tentatively characterized by multiple-ionmonitoringndashinformation dependent acquiringndashenhanced product ion (MIM-IDA-EPI) whose level changed after being processedby vinegar or milk It was found that flavonoids especially biflavonones could be the important chemical basis of toxicity reductionfor processed ruixianglangdu In addition some coumarins and lignanoids could also play a role in reducing toxicity It is believedthat MIM-based metabolomics method was valuable for exploring chemical basis of toxicity reduction for processing The data iscritical to further study the mechanism of toxicity reducing for processed ruixianglangdu

1 Introduction

Processing of herbal medicinal materials is a pharmaceuticaltechnique based on traditional Chinese medicine (TCM)theory which aimed to enhance the efficacy andor reducethe toxicity of crude drugs [1] For herbal medicines manyprocessing approaches could be used to reduce the toxicityof crude drugs based on different clinical experience How-ever chemical basis of toxicity reduction for most herbalmedicines remains unclear Because of multiple componentsin herbal medicines exploring chemical basis is extensivelydampened by comprehensively and accurately capture infor-mation in complex multicomponent samples

Stellera chamaejasme L is a perennial herb belongingto the Thymelaeaceae family of flowering plants native toRussia China and Mongolia [2] Ruixianglangdu roots ofStellera chamaejasme L (ruixianglangdu) is one of the TCMmost commonly used as a remedy for furuncle carbuncle andtuberculosis of the lymph nodes with varied biological activ-ity including antiviral antitumor antibacterial and anti-inflammatory activities [3ndash5] Ruixianglangdu could poisonor even kill the cattle if ingested bymistake [6] By now someprocessing methods have been used to attenuate toxicityof ruixianglangdu Processing with milk is the special pro-cessing method of Mongolian medicine [7] Ruixianglangdutreated with an optimum amount of vinegar is the processing

HindawiInternational Journal of Analytical ChemistryVolume 2019 Article ID 4854728 9 pageshttpsdoiorg10115520194854728

2 International Journal of Analytical Chemistry

method described in Pharmacopoeia [8] They are all therepresentative processing methods of ruixianglangdu

Current methods including thin layer chromatogra-phy (TLC) and high-performance liquid chromatography-ultraviolet spectrophotometry (HPLC-UV) used for thedetermination of multiple components in ruixianglangduand its processed products are not enough selective andaccurate [9 10] They only concentrated on total componentor several components which could not comprehensivelyexplore the changes of multiple components For HPLCthe analytical time was long and the peak resolution wasnot well to determine multiple components Meanwhilethe HPLC-UV analysis of multiple components is affectedby low sensitivity and low signal-to-noise ratio Hence theultraperformance liquid chromatographyndashtriple quadrupoletandem mass spectrometry (UPLC-MSMS) method usingdifferent multiple reaction monitor (MRM) channels at thesame time would be theoretically suitable for simultaneousquantification of multiple components in complex matrixMultiple-ion monitoring (MIM) can be used in the cases inwhich the MSMS diagnostic fragment is the same in termsof intensity and selectivity of the parent ion [11 12] By nowthe metabolomics approach usingMIMhas been successfullyapplied to discover biomarkers in plasma rice and so on[13 14] In addition theMIMhas also been used to determinesome compounds in biological samples such as bicyclol andpeptides with acceptable accuracy sensitivity and selectivity[15 16]

In this study a MIM-based metabolomics method wasapplied to explore chemical basis of toxicity reducingfor processed ruixianglangdu using LC-MSMS equippedwith an ESI source Here we used raw ruixianglangduand two different procedures for processing ruixianglangdu(processed with vinegar or milk) as examples Threesteps are included to explore the chemical basis of toxi-city reduction (1) MIM-based metabolomics was appliedfor comprehensively capturing chemical information ofsamples (2) the analysis of the differential componentbetween the crude drugs and the products was per-formed by multiple-ion monitoringndashinformation dependentacquiringndashenhanced product ion (MIM-IDA-EPI)

2 Experimental

21 Reagents and Chemicals Neochamaejasmin A was pur-chased from Shanghai Jianglai Biological Technology CoLtd (Shanghai China) Neochamaejasmin B isochamae-jasmin and daphnoretin were purchased from ChengduChroma-Biotechnology Co Ltd (Sichuan China) Thepurity of the four standards was gt 95 Glutamic acidtyrosine isoleucine leucine phenylalanine and tryptophanwere purchased from the National Institutes for Food andDrug Control (Beijing China) HPLC-grade acetonitrilefrom Honeywell (Morris America) and formic acid (HPLCgrade) from Sigma-Aldrich (Steinheim Germany) were pur-chased Rice vinegar and fresh milk were obtained fromFoshan Haitian Flavoring amp Food Co Ltd (China) and Yili(Inner Mongolia Yili Industrial Group Co Ltd China)

respectively Ultrapure water was prepared using aMilli-Q SPsystem (Millipore Bedford MA USA)

22 PlantMaterials and Sample Preparation Ruixianglangduwas purchased in Anguo (Hebei China) and identified usingmorphological and histological methods by Dr Guihua Cuiwhich was deposited at Institute of Chinese Materia MedicaRaw ruixianglangdu was processed by vinegar or milk toobtain 3 processed products 350 g of raw ruixianglangdu wasmixed with rice vinegar or freshmilk left to soak for 6 h thenboiled for 30 min and eventually dried Ultimately 6 batchesof samples were obtained for subsequent analysis

According to the usage of TCM the raw or processedproducts (50 g)were boiled for 30minwith 500mLwater andmost solvent was removed through boiling Then the wholesupernatant was collected diluted to 100 mL with water andpassed through a 022 mm filterThe filtrate was stored at 4∘Cin a refrigerator before the LC-MS analysis

23 LC-MS Analysis AWaters ACQUITY UPLCndashQTRAP5500 mass spectrometer was used to perform multiple-ionmonitoringndashinformation dependent acquiringndashenhancedproduct ion (MIM-IDA-EPI) and MIM A WatersACQUITY UPLC (Waters USA) equipped with a binarysolvent delivery system an autosampler and a columncompartment was used Detection was performed usinga QTRAP 5500 system from Applied BiosystemsMDSSciex (Applied Biosystems Foster City CA USA) a hybridtriple quadrupole linear ion trap mass spectrometer Theinstrument was operated using an electrospray ionizationsource (ESI) The ion spray voltage was set to 55 kV andthe turbo spray temperature was maintained at 550∘CNebulizer gas (gas 1) and heater gas (gas 2) were set at50 and 50 psi respectively The curtain gas was kept at 35psi and the interface heater was on Nitrogen was usedas nebulizer and auxiliary gas Stepwise MIM in positiveand negative scan was adopted with 3 scans ranging frommz 51 to mz 350 from mz 351 to mz 650 and frommz 651 to mz 950 respectively with mass step 10 Daand dwell time 5 msec Three hundred MIM transitionsin a single run were obtained The declustering potential(DP) and collision energy (CE) of each MIM were set at60 V and 5 eV respectively For obtaining fragmentationions all the ions exceeding 5000 cps were used to triggerthe acquisition of EPI spectra The MS scan function wascontrolled with the Analyst software (versions 162) fromApplied BiosystemsMDS Sciex

The chromatographic separation was performed on aWaters HSS C

18column (21 times 100 mm 18 120583m) with the

column temperature set at 40∘C The mobile phase consistedof water containing 01 formic acid (A) and only acetonitrile(B) with a linear gradient elution at a flow rate of 03mLminThe gradient program was as follows 99-70 A (0-10 min)70-55A (10-25min) 1A (25-30min) 99A (30-33min)The volume of the sample injection was 1 120583L

24 Statistical Data Analysis The scores plot of PCA wasdrawn using SIMCA-P version 120 software (Umetrics

International Journal of Analytical Chemistry 3

0 13 260

0 13 260

0 13 260

0 13 260

0 13 260

0 13 260

mz 510 minus mz 3500 mz 510 minus mz 3500

mz 3510 minus mz 6500 mz 3510 minus mz 6500

mz 6510 minus mz 9500 mz 6510 minus mz 9500

ESI + ESI -

Time (min) Time (min)

Inte

nsity

(cps

)

90 times 106

45 times 106

8 times 106

40 times 106

65 times 106

35 times 106

1 times 107

5 times 106

96 times 106

48 times 106

1 times 107

5 times 106

Figure 1 Stepwise multiple-ion monitoring (MIM) chromatogram of ruixianglangdu

AB Umea Sweden) Furthermore an independent T-test(plt001) (Microsoft Office Excel 2010) was used to determineif the change in processing was statistically different at theunivariate analysis level Furthermore an independent T-test (plt001) (Microsoft Office Excel 2010) was used toanalyze the differential component between raw and milk-processed ruixianglangdu Meanwhile the differential com-ponent between raw and vinegar-processed ruixianglangduwas also analyzed with T-test Components with statisticaldifference were marked with asterisk

3 Results and Discussion

31 Metabolic Profiling of Ruixianglangdu Global profilingof stepwise scan MIM in positive and negative mode wasadopted to analyze 6 batches of raw vinegar-processedand milk-processed ruixianglangdu respectively The chro-matogram of ruixianglangdu is shown in Figure 1 On thechromatogram the ion peaks were easy to detect and theirpeak areas could be calculated by Analyst software The ionpeaks in the scan range frommz 651 tomz 950was relativelyless than those in the scan range frommz 51 tomz 650 andmost components were eluted before 13 min It was suggestedthat most of the compounds are hydrophilic with higherpolarity A total of 1175 ions with signal-to-noise ratio (SN) gt10 were obtained including 563 ions in positive mode and 612ions in negative mode The resultant 2D matrices includingpaired mzndashretention time sample names and peak areasof 1175 ions were analyzed by PCA to visualize general

clustering and trends As shown in Figure 2 an obviousseparation trend can be observed between ruixianglangduand its processed products And the 2 kinds of processedproducts were also obviously separated in the PCA

32 Identification of Components That Changed in ProcessingAfter filtering the fragment ions and isotope ions 37 compo-nents found in positive and 26 components found in negativemode were screened out which differ after processing withmilk or vinegar The fragmentation profile of changed con-stituents was rapidly analyzed using MIM-IDA-EPI Even-tually 33 compounds were identified or tentatively charac-terized in ruixianglangdu whose level changed after beingprocessed by vinegar or milk These components involved14 flavonoids 5 coumarins 8 lignanoids 5 amino acidsand 1 diterpenoid Their detailed information is presentedin Table 1 such as adduct ion fragmentation ions of MSnand retention time By now 22 compounds had been isolatedand identified from ruixianglangdu [2 17] Compounds 910 12 19 and 28-32 could be unambiguously identified asneochamaejasmin A neochamaejasmin B isochamaejasmindaphnoretin glutamic acid tyrosine isoleucine leucine andphenylalanine respectively by comparing their retentiontimes adduct ions and product ions with authentic stan-dards A detailed description of structural characterizationprocess was as follows

Flavonoids compound 1 was identified as epiafzelechin-7-O-glucoside with [M+H]+ mz 4371 a series of frag-ment ions [M+H-Glc]+ mz 2752 [M+H-H

2O-Glc-H

2O]+

4 International Journal of Analytical Chemistry

Table 1 Identification of compounds in ruixianglangdu

Noa tR(min)

Parent ion[M+H]+ or [M-H]-

(mz)Molecular formula

Fragmentation profile(mz)

(Relative abundance )Identification

Flavonoids (parent ions [M+H]+)

1lowast 481 4371 C21H24O10

2752 2572 1490 1390(100) 1070

Epiafzelechin-7-O-gluconoside

2lowast 585 7052 C36H32O15

5431 (100)

IsochamaejasmineChamaejasmine

Neochamaejasmine ANeochamaejasmineB-7-O- gluconoside

3lowast 641 2751 C15H14O5

2572 (100) 1390 Epiafzelechin4lowast 663 7052 C

36H32O15

5432 (100) 5271 3112 Same as no 2

5lowast 984 5451 C30H24O10

5272 4091 (100) 28321530

Naringenin-epiafzelechinor Naringenin-afzelechin

6lowast 1048 5451 C30H24O10

5272 4091 (100) 28321530 Same as no 5

7 1213 3411 C20H20O5

3232 (100) 2911 27121872 1370 8-Prenylnaringenin

8lowast 1483 5431 C30H22O10

3451 1992 (100) Chamaechromone

9lowast 1538 5431 C30H22O10

4490 3911 3112 19911530 (100) Neochamaejasmine A

10lowast 1719 5431 C30H22O10

4490 4172 3911 31122311 1992 1530 (100) Neochamaejasmine B

11lowast 1947 5431 C30H22O10

4490 4172 3911 31121530 (100) Chamaejasmine

12lowast 2004 5431 C30H22O10

4490 4172 3911 31121530 (100) Isochamaejasmine

13lowast 2135 5571 C31H24O10

4490 1530 (100)7-

MethoxylneochaejasminA

14lowast 2763 5712 C32H26O10

4631 4451 4192 (100)3551 3372 2452 2273 Chamaejasmenin A

Coumarins (parent ions of compounds 15 16 and 19 [M+H]+ parent ions of compounds 17 and 18 [M-H]-)

15lowast 481 1931 C10H8O4

1610 (100) 1330 11501050 765 Scopoletin

16lowast 489 3251 C15H16O8

2802 1630 (100) 11901070 Umbelliferone glucoside

17lowast 585 1770 C9H6O4

1490 (100) 1330 12111051 930 770 Daphnetin

18lowast 788 1610 C9H6O3

1331 (100) 1170 1051890 770 Umbelliferone

19lowast 1444 3531 C19H12O7

3382 2671 2512 1792(100) Daphnoretin

Lignanoids (parent ions of compounds 25-27 [M+H]+ parent ions of compounds 20-24 [M-H]-)

20 621 5212 C26H34O11

3592 (100) 1591 Isolariciresinol-4rsquo-O-glucoside

21 746 5232 C26H36O11

3613 (100) 1652 1362 Secoisolariciresinol-4-O-glucoside

22 798 5212 C26H34O11

3592 3293 (100)1781 Isolariciresinol-9-O-glucoside

23 841 5232 C26H36O11

3613 (100) 3132 Secoisolariciresinol-9-O-glucoside

24lowast 969 5192 C26H32O11

3572 (100) Pinoresinol-4-O-glucoside

25 1164 4012 C22H24O7

3832 2172 2072 16721350(100) Aschantin

International Journal of Analytical Chemistry 5

Table 1 Continued

Noa tR(min)

Parent ion[M+H]+ or [M-H]-

(mz)Molecular formula

Fragmentation profile(mz)

(Relative abundance )Identification

26lowast 1176 4192 C22H26O8

4011 3871 2361 (100)1821 Syringaresinol

27lowast 1342 3591 C20H22O6

3412 3232 3112 1370(100) Pinoresinol

Amino acids (parent ions [M+H]+)28 092 1481 C

5H9NO4

1300 (100) 1020 Glutamic acid

29 199 1821 C9H11NO3

1650 (100) 1470 13601230 Tyrosine

30 205 1321 C6H13NO2

860 (100) Isoleucine31 221 1321 C

6H13NO2

1140 860 (100) Leucine32 305 1661 C

9H11NO2

1490 1200 (100) 1030 PhenylalanineDiterpenoids (parent ions [M-H]-)

33lowast 718 6133 C35H50O9

5672 4052 3092 1790(100) 1192 1130 Stelleramacrin B

a The compounds isolated from ruixianglangdu are marked with lowast

minus20

minus10

0

10

20

minus30 minus20 minus10 0 10 20 30t[1] (412)

Raw ruixianglangdu

Milk-processed ruixianglangdu

Vinegar-processed ruixianglangdu

t[2] (

332

)

Figure 2 Principle component analysis scores plot for ruixianglangdu and its processed products

mz 2572 and characteristic fragments of RDA cleav-age mz 1390 In the MS2 spectrum of flavonoids (nos2 and 4) the ion at mz 70518 fragmented to ion[M+H-Glc]+ mz 5431 Considering that isochamaejasmin-7-O-glucoside has been isolated from roots of Stellerachamaejasme compounds 2 and 4 were tentatively identi-fied as isochamaejasminchamaejasmineneochamaejasminAneochamaejasmin B-7-O-glucoside Based on [M+H]+mz 2751 a series of fragment ions [M+H-H

2O]+ mz 2572

and characteristic fragments of RDAcleavagemz 1390 com-pound 3 was identified as epiafzelechin Compounds 5 and6 were tentatively identified as naringenin-epiafzelechin ornaringenin-afzelechin in agreement with [M+H]+ mz 5451Their MS2 spectrum gave the fragment ions mz 4091 withfragments of RDA cleavage mz 1530 Compound 7 exhib-ited the deprotonated molecule [M+H]+ mz 3411 Major

fragment ions [M+H-H2O]+ mz 3232 [M+H-C

5H9]+ mz

2712 a fragment of RDA cleavage mz 1370 were shown inthe MSMS spectrumThus it was tentatively identified as 8-prenylnaringenin In the MS2 spectrum of compound 8 theion at mz 5431 fragmented to the ion mz 3451 losing onemolecule of bis(4-hydroxyphenyl)methane and mz 1992 Itwas identified as chamaechromone Four biflavonones (nos9 10 11 and 12) had a series of the same fragment ionsmz 4490 3911 3112 and 1530 The peak at mz 3911and 1530 corresponded to the RDA cleavage Compoundsnos 9 10 and 12 were confirmed as neochamaejasmin Aneochamaejasmin B and isochamaejasmin by comparisonwith standards Compound 11 was identified as chamaejas-mine due to the similar fragment ions with compounds9 10 and 12 Compound 13 exhibited [M+H]+ mz 5571major fragment ions mz 4490 and characteristic fragments

6 International Journal of Analytical Chemistry

mz 1530 which was similar to compounds 9-12 whichwas identified as 7-methoxylneochaejasmin A Compound14 had a series fragment ions mz 4451 4192 3551 and3372 The peak at mz 4192 corresponded to the RDAcleavage Compound 14 was tentatively identified as chamae-jasmenin A All the 14 flavonoids except for compound 7had been isolated from roots of Stellera chamaejasme L[17ndash23]

Coumarins compound 15 exhibited [M+H]+ mz 1931Based on major fragment ions [M+H-CH

3OH]+ mz 1610

[M+H-CH3OH-CO]+mz 1330 and [M+H-CH

3OH-2CO]+

mz 1050 this compound was tentatively assigned to scopo-letin Compound 16 was tentatively identified as umbellifer-one glucoside with a series of fragment ions [M+H-Glc]+mz 1630 and [M+H-Glc-CO

2]+ mz 1191 Compound 17

was identified as daphnetin with [M+H]+ mz 1770 anda series of fragment ions [M-H-CO]minus mz 1490 [M-H-CO2]minus mz 1330 and [M-H-CO-CO

2]minus mz 1051 The MS2

spectrum of compound 18 with [M-H]minus mz 1610 gave thecharacteristic fragment ions of mz 1331 1170 and 1051which corresponded to [M-H-CO]minus [M-H-CO

2]minus and [M-

H-2CO]minus It was tentatively identified as umbelliferoneThe MSMS mass spectrum of compound 19 [M+H]+ mz3531 presented the specific fragments of [M+H-CH

3]+ mz

3382 It was confirmed as daphnoretin by comparison withstandard All the 5 coumarins had been isolated from roots ofStellera chamaejasme L [21 24 25]

Lignanoids base peak of compounds 20 and 22 was mz3592 Compounds 20 and 22 were tentatively identifiedas isolariciresinol-4rsquo-O-glucoside and isolariciresinol-9-O-glucoside based on their polarity Two compounds (no 21and 23) had the same [M-H]minus mz 5232 and characteristicfragment [M-H-Glc]+ mz 3613 Based on their differentpolarity compounds 21 and 23 were tentatively identified assecoisolariciresinol-4-O-glucoside and secoisolariciresinol-9-O-glucoside respectively based on their polarity Basedon the characteristic fragment [M-H-Glc]minus mz 3572 com-pound 24 with [M-H]minus mz 5192 was tentatively identifiedas pinoresinol-4-O-glucoside Compound 25 was tentativelyidentified as aschantin with a series of fragment ions [M+H-H2O]+ mz 3832 and characteristic fragment ion mz 1672

Compound 26 with [M+H]+mz 4192 was tentatively identi-fied as syringaresinol which fragmented to ion [M+H-H

2O]+

mz 4011 and [M+H-HOCH3]+mz 3871 Compound 27was

tentatively identified as pinoresinol with [M+H]+ mz 3591and a series of fragment ions [M+H-H

2O]+mz 3412 [M+H-

2H2O]+ mz 3232 and characteristic fragment ion mz 1370

Only 3 lignanoids (no 24 26 and 27) had been isolated fromroots of Stellera chamaejasme L [21 26]

The 5 amino acids compounds (28-32) were confirmedby comparison with standard Compound 33 was tentativelyidentified as stelleramacrin B with [M+H]+ mz 6133 and acharacteristic fragment ion [M+H-C

14H24-H2O]+mz 4052

which had been isolated from roots of Stellera chamaejasmeL [20]

33 Exploring Chemical Basis of Toxicity reducing for Pro-cessing The extracted ion chromatogram (XIC) of the 33

components was found to be significantly altered after beingprocessed by vinegar or milk as shown in Figure 3 T-test ofthe 33 components between raw and milkvinegar-processedruixianglangdu was performed As shown in Figure 4 whenthe raw ruixianglangdu was processed by vinegar 25 compo-nents exhibited significant changes including 11 flavonoids(no 1-4 7-9 and 11-14) 4 coumarins (no 15 and 17-19)7 lignanoids (no 20-21 and 23-27) 2 amino acids (no 28and 29) and 1 diterpenoid Eleven of the 25 componentsunderwent a concentration decrease after vinegar processingincluding 8 flavonoids (no 2-4 8 11-14) 1 coumarin (no18) and 2 lignanoids (no 21 and 24) The change ratioof components in raw to vinegar-processed ruixianglangduwas in the range 03sim29 When raw ruixianglangdu wasprocessed by milk 26 components exhibited significantchanges including 13 flavonoids (no 1-8 10-14) 2 coumarins(no 16 and 19) 5 lignanoids (no 21 22 24 25 and 27) 5amino acids (no 28-30) and 1 diterpenoid Sixteen of the26 components exhibited decreasing tendency including 11flavonoids (no 2-6 7 10-14) 2 coumarins (no 16 and 19)and 3 lignanoids (no 21 22 and 24) It is worth notingthat 10 components underwent a concentration decrease afterprocessing with bothmilk and vinegar including 8 flavonoids(chamaejasmine-7-O-glucoside epiafzelechin neochamae-jasmin A-7-O-glucopyranoside chamaechromone chamae-jasmine isochamaejasmin 7-methoxylneochaejasminA andchamaejasmenin A) and 2 lignanoids Decreased flavonoidcould be due to the thermic treatment performed during theraw ruixianglangdu extraction For example total flavonoidcontent of sea buckthorn extract reduction of 9004 wasobserved as a result of heating at 100∘C after 25 min [27]The total and five flavonoids were also significantly decreasedafter fresh Cara Cara juice heating at 90∘C for 120 min [28]Quercetinwas also unstable in aqueous solution and aqueousstability of flavonol was associated with B-ring substitution[29]

Total flavonoids obtained from Stellera chamaejasmehave showed strong toxicity to mice and rabbit [30]Chamaechromone (no 8) and isochamaejasmin (no 12)were major components in ruixianglangdu with cytotox-icity to canine kidney MDCK cells [31 32] Coumarindaphnoretin (no 19) was found to be a cytotoxic con-stituent of Dirca occidentalis (Thymelaeaceae) [33] Basedon these changing components and literature for the pro-cessed ruixianglangdu flavonoids especially biflavononescould be the important chemical basis of toxicity reduc-tion In addition some coumarins and lignanoids thatdecreased after processing could also play a role in reducingtoxicity

Compared with milk-processed ruixianglangdu groupsthe 10 components involving 8 flavonoids had a lowerlevel in the vinegar-processed ruixianglangdu group It wasinferred that processing method withmilk could be better fortoxicity reduction of ruixianglangdu According to literaturethe mortality of zebrafish embryo was higher after treat-ing with vinegar-processed ruixianglangdu than milk-proc-essed ruixianglangdu [34] which was consistent with ourinference

International Journal of Analytical Chemistry 7

0 4 8 12 16 20 24 28 32Time (min)

Rela

tive i

nten

sity

()

0

1

2

3

4

7

5

68

9

10

11

12 13

1415

16

17

18 1920

21

2223 24

25

26

2728

293031

3233

50

100

4371437170517051

54515451

54315431

27512751

34113411

5551555157115711

1931193132513251

17711771 16111611

35313531 5211521152315231

5191519140114011

3591359114811481

1821182113211321 16611661

61316131

41914191

Figure 3 The extracted ion chromatogram (XIC) of the 33 components found to be significantly altered after being processed by vinegaror milk All the ion pairs are monitored in positive mode except for 17711771 16111611 52115211 52315231 51915191 and 61316131Compound number is consistent with the compound number in Table 1

0

50000000

100000000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0

50000000

100000000

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Peak

Are

a

Compound ID

Coumarins Lignanoids Amino acids Diterpenoids

Flavonoids Coumarins

Raw ruixianglangdu Vinegar-processed ruixianglangdu Milk-processed ruixianglangdu

lowastlowast lowast lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Figure 4 Peak area of 33 compounds changed in processed ruixianglangdu Compound ID is consistent with the number in Table 1Compounds with statistical difference between raw and milkvinegar-processed ruixianglangdu were marked with asterisk (lowast)

8 International Journal of Analytical Chemistry

4 Conclusions

With the help of MIM-based metabolomics this studyillustrated the chemical basis of toxicity reducing for pro-cessed ruixianglangdu Flavonoids especially biflavononescould be the important chemical basis of toxicity reducingIn addition some coumarins and lignanoids that decreasedafter processing could also play a role in reducing toxicityCompared with vinegar processing processing method withmilk could be better for toxicity reduction of ruixianglangduIt is believed that MIM-based metabolomics method wasvaluable for exploring chemical basis of toxicity reductionfor processed TCM The data is critical to further study thetoxicity reduction mechanism of processed ruixianglangdu

Data Availability

The data supporting the findings of this study are availablewithin the article Rawdata and additional information of thisstudy are available from the corresponding author on request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Wei Yang and Xiaoli Ma contributed equally to this work

Acknowledgments

The study has been financially supported by Natural Sci-ence Foundation of Beijing (7174325) National Science andTechnology Major Project (2015ZX09501004-003-005) andNational Natural Science Foundation Youth Science Founda-tion (81803722)

References

[1] Z Z Zhao Z T Liang K Chan et al ldquoA unique issue in thestandardization of Chinese materia medica processingrdquo PlantaMedica vol 76 no 17 pp 1975ndash1986 2010

[2] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y Chaildquoharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

[3] G Yang andD Chen ldquoBiflavanones flavonoids and coumarinsfrom the roots of Stellera chamaejasme and their antiviral effecton hepatitis B virusrdquo Chemistry amp Biodiversity vol 5 no 7 pp1419ndash1424 2008

[4] M Kim H J Lee A Randy J H Yun S Oh and CW Nho ldquoStellera chamaejasme and its constituents inducecutaneous wound healing and anti-inflammatory activitiesrdquoScientific Reports vol 7 Article ID 42490 2017

[5] X Liu Q Yang G Zhang et al ldquoAnti-tumor pharmacologicalevaluation of extracts from stellera chamaejasme L basedon hollow fiber assayrdquo BMC Complementary and AlternativeMedicine vol 14 p 116 2014

[6] Y Wang J Li M Han W Wang and Y Li ldquoPrevention andtreatment effect of total flavonoids in Stellera chamaejasme Lon nonalcoholic fatty liver in ratsrdquo Lipids in Health and Diseasevol 14 p 85 2015

[7] T Bai and D J L H Wang ldquoProcessing difference betweenmongolian medicine and traditional chinese medicinerdquo WorldLatest Medicine Information vol 18 p 182 2018

[8] P E C o M O Health Chinese Pharmacopoeia PeoplersquosMedical PublishingHouse Beijing China Edition 1977 edition1979

[9] Z F Jiang X L Ma B Han S Y Cao and Y Y Gen ldquoInitialstudy on effection of different processing methods on theStellera chamaejasme chemical constituentsrdquoMedical Researchand Education vol 32 pp 1ndash6 2015

[10] X W Ji M L Hao X Yang and Y H Wang ldquoRP-HPLCfingerprint of milk Stellera chamaejasmerdquo Zhong Yao Cai vol35 pp 1754ndash1757 2012

[11] W Yang M Ye M Liu et al ldquoA practical strategy for thecharacterization of coumarins in Radix Glehniae by liquidchromatography coupled with triple quadrupole-linear ion trapmass spectrometryrdquo Journal of Chromatography A vol 1217 no27 pp 4587ndash4600 2010

[12] M Yao LMaWGHumphreys andM Zhu ldquoRapid screeningand characterization of drug metabolites using a multiple ionmonitoring-dependentMSMS acquisitionmethod on a hybridtriple quadrupole-linear ion trapmass spectrometerrdquo Journal ofMass Spectrometry vol 43 no 10 pp 1364ndash1375 2008

[13] P Luo W Dai P Yin et al ldquoMultiple reaction monitoring-ion pair finder a systematic approach to transform nontar-geted mode to pseudotargeted mode for metabolomics studybased on liquid chromatographyndashmass spectrometryrdquo Analyt-ical Chemistry vol 87 no 10 pp 5050ndash5055 2015

[14] W Chen L Gong Z Guo et al ldquoA novel integrated methodfor large-scale detection identification and quantification ofwidely targeted metabolites Application in the study of ricemetabolomicsrdquo Molecular Plant vol 6 no 6 pp 1769ndash17802013

[15] L Sheng J Hu B Wang H Chen and Y Li ldquoDeterminationof bicyclol in dog plasma using liquid chromatographyndashtandemmass spectrometryrdquo Journal of Chromatography B vol 878 no23 pp 2106ndash2110 2010

[16] DV Rossetti CMartelli R Longhi F IavaroneMCastagnolaand C Desiderio ldquoQuantitative analysis of thymosin beta4 inwhole saliva by capillary electrophoresis-mass spectrometryusing multiple ions monitoring (CE-MIM-MS)rdquo Electrophore-sis vol 34 no 18 pp 2674ndash2682 2013

[17] C Jin R G Michetich and M Daneshtalab ldquoFlavonoids fromStellera chamaejasmerdquo Phytochemistry vol 50 no 3 pp 505ndash508 1998

[18] W Chen X Luo Z Wang Y Zhang L Liu and H WangldquoA new biflavone glucoside from the roots of Stellera chamae-jasmerdquo Chinese Journal of Natural Medicines vol 13 no 7 pp550ndash553 2015

[19] Z Yan H Guo J Yang et al ldquoPhytotoxic flavonoids from rootsof Stellera chamaejasme L (Thymelaeaceae)rdquo Phytochemistryvol 106 pp 61ndash68 2014

[20] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y ChaildquoCharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

International Journal of Analytical Chemistry 9

[21] Z Jiang T Tanaka T Sakamoto I Kouno J Duan and RZhou ldquoBiflavanones diterpenes and coumarins from the rootsof stellera chamaejasme lrdquoChemical amp Pharmaceutical Bulletinvol 50 no 1 pp 137ndash139

[22] Y Li C Li L Lv et al ldquoA UPLC-MSMSmethod for simultane-ous determination of five flavonoids from Stellera chamaejasmeL in rat plasma and its application to a pharmacokinetic studyrdquoBiomedical Chromatography vol 32 no 6 Article ID e41892018

[23] G Liu H Tatematsu M Kurokawa M Niwa and Y HirataldquoNovel C-3C-31015840rsquo-biflavanones from Stellera chamaejasme LrdquoChemical amp Pharmaceutical Bulletin vol 32 pp 362ndash365 1984

[24] X Li K Rahman J Zhu andH Zhang ldquoChemical Constituentsand Pharmacological Activities of Stellera chamaejasmerdquo Cur-rent Pharmaceutical Design vol 24 no 24 pp 2825ndash2838 2018

[25] X Su R Lin S Wong S Tsui and S Kwan ldquoIdentificationand characterisationof the Chinese herb Langdu by LC-MSMSanalysisrdquo Phytochemical Analysis vol 14 no 1 pp 40ndash47 2003

[26] L Qiao L Yang D Zhang J Zou and J Dai ldquoStudies on chem-ical constitutes from callus cultures of Stellera chamaejasmerdquoZhongguo Zhongyao Zazhi vol 36 no 24 pp 3457ndash3462 2011httpswwwncbinlmnihgovpubmed22368856

[27] F M Ursache I O Ghinea M Turturica I Aprodu GRapeanu andN Stanciuc ldquoPhytochemicals content and antiox-idant properties of sea buckthorn (Hippophae rhamnoides L)as affected by heat treatment - Quantitative spectroscopic andkinetic approachesrdquo FoodChemistry vol 233 pp 442ndash449 2017

[28] Q Lu Y Peng C Zhu and S Pan ldquoEffect of thermal treatmenton carotenoids flavonoids and ascorbic acid in juice of orangecv Cara Carardquo Food Chemistry vol 265 pp 39ndash48 2018

[29] S Maini H L Hodgson and E S Krol ldquoTheUVA and aqueousstability of flavonoids is dependent on b-ring substitutionrdquoJournal of Agricultural and Food Chemistry vol 60 no 28 pp6966ndash6976 2012

[30] Z Yan L Zeng H Jin and B Qin ldquoPotential ecological rolesof flavonoids from Stellera chamaejasmerdquo Plant Signaling andBehavior vol 10 Article ID e1001225 2015

[31] Y Lou J Zheng B Wang X Zhang X Zhang and SZeng ldquoMetabolites characterization of chamaechromone invivo and in vitro by using ultra-performance liquid chro-matographyXevo G2 quadrupole time-of-flight tandem massspectrometryrdquo Journal of Ethnopharmacology vol 151 no 1 pp242ndash252 2014

[32] Y H Wang X Yang L J Sun and L M Yang ldquoStudies onthe cytotoxicity of Stellera chamaejasme L in vitrordquo ChineseMedicinal Biotechnology vol 7 pp 9ndash13 2012

[33] M M Badawi S S Handa A D Kinghorn G A Cordelland N R Farnsworth ldquoPlant anticancer agents XXVIIAntileukemic and cytotoxic constituents of Dirca occiden-talis (thymelaeaceae)rdquo Journal of Pharmaceutical Sciences vol72 no 11 pp 1285ndash1287 1983 httpswwwncbinlmnihgovpubmed6644588

[34] J F Yang W J Dong J C Zong et al ldquoToxicity detectionof Stella Chamaejasme in zebrafish embryo with differentextraction methodsrdquo Journal of Inner Mongolia University forNationalities vol 31 pp 236ndash239 2016

International Journal of Analytical Chemistry 3

0 13 260

0 13 260

0 13 260

0 13 260

0 13 260

0 13 260

mz 510 minus mz 3500 mz 510 minus mz 3500

mz 3510 minus mz 6500 mz 3510 minus mz 6500

mz 6510 minus mz 9500 mz 6510 minus mz 9500

ESI + ESI -

Time (min) Time (min)

Inte

nsity

(cps

)

90 times 106

45 times 106

8 times 106

40 times 106

65 times 106

35 times 106

1 times 107

5 times 106

96 times 106

48 times 106

1 times 107

5 times 106

Figure 1 Stepwise multiple-ion monitoring (MIM) chromatogram of ruixianglangdu

AB Umea Sweden) Furthermore an independent T-test(plt001) (Microsoft Office Excel 2010) was used to determineif the change in processing was statistically different at theunivariate analysis level Furthermore an independent T-test (plt001) (Microsoft Office Excel 2010) was used toanalyze the differential component between raw and milk-processed ruixianglangdu Meanwhile the differential com-ponent between raw and vinegar-processed ruixianglangduwas also analyzed with T-test Components with statisticaldifference were marked with asterisk

3 Results and Discussion

31 Metabolic Profiling of Ruixianglangdu Global profilingof stepwise scan MIM in positive and negative mode wasadopted to analyze 6 batches of raw vinegar-processedand milk-processed ruixianglangdu respectively The chro-matogram of ruixianglangdu is shown in Figure 1 On thechromatogram the ion peaks were easy to detect and theirpeak areas could be calculated by Analyst software The ionpeaks in the scan range frommz 651 tomz 950was relativelyless than those in the scan range frommz 51 tomz 650 andmost components were eluted before 13 min It was suggestedthat most of the compounds are hydrophilic with higherpolarity A total of 1175 ions with signal-to-noise ratio (SN) gt10 were obtained including 563 ions in positive mode and 612ions in negative mode The resultant 2D matrices includingpaired mzndashretention time sample names and peak areasof 1175 ions were analyzed by PCA to visualize general

clustering and trends As shown in Figure 2 an obviousseparation trend can be observed between ruixianglangduand its processed products And the 2 kinds of processedproducts were also obviously separated in the PCA

32 Identification of Components That Changed in ProcessingAfter filtering the fragment ions and isotope ions 37 compo-nents found in positive and 26 components found in negativemode were screened out which differ after processing withmilk or vinegar The fragmentation profile of changed con-stituents was rapidly analyzed using MIM-IDA-EPI Even-tually 33 compounds were identified or tentatively charac-terized in ruixianglangdu whose level changed after beingprocessed by vinegar or milk These components involved14 flavonoids 5 coumarins 8 lignanoids 5 amino acidsand 1 diterpenoid Their detailed information is presentedin Table 1 such as adduct ion fragmentation ions of MSnand retention time By now 22 compounds had been isolatedand identified from ruixianglangdu [2 17] Compounds 910 12 19 and 28-32 could be unambiguously identified asneochamaejasmin A neochamaejasmin B isochamaejasmindaphnoretin glutamic acid tyrosine isoleucine leucine andphenylalanine respectively by comparing their retentiontimes adduct ions and product ions with authentic stan-dards A detailed description of structural characterizationprocess was as follows

Flavonoids compound 1 was identified as epiafzelechin-7-O-glucoside with [M+H]+ mz 4371 a series of frag-ment ions [M+H-Glc]+ mz 2752 [M+H-H

2O-Glc-H

2O]+

4 International Journal of Analytical Chemistry

Table 1 Identification of compounds in ruixianglangdu

Noa tR(min)

Parent ion[M+H]+ or [M-H]-

(mz)Molecular formula

Fragmentation profile(mz)

(Relative abundance )Identification

Flavonoids (parent ions [M+H]+)

1lowast 481 4371 C21H24O10

2752 2572 1490 1390(100) 1070

Epiafzelechin-7-O-gluconoside

2lowast 585 7052 C36H32O15

5431 (100)

IsochamaejasmineChamaejasmine

Neochamaejasmine ANeochamaejasmineB-7-O- gluconoside

3lowast 641 2751 C15H14O5

2572 (100) 1390 Epiafzelechin4lowast 663 7052 C

36H32O15

5432 (100) 5271 3112 Same as no 2

5lowast 984 5451 C30H24O10

5272 4091 (100) 28321530

Naringenin-epiafzelechinor Naringenin-afzelechin

6lowast 1048 5451 C30H24O10

5272 4091 (100) 28321530 Same as no 5

7 1213 3411 C20H20O5

3232 (100) 2911 27121872 1370 8-Prenylnaringenin

8lowast 1483 5431 C30H22O10

3451 1992 (100) Chamaechromone

9lowast 1538 5431 C30H22O10

4490 3911 3112 19911530 (100) Neochamaejasmine A

10lowast 1719 5431 C30H22O10

4490 4172 3911 31122311 1992 1530 (100) Neochamaejasmine B

11lowast 1947 5431 C30H22O10

4490 4172 3911 31121530 (100) Chamaejasmine

12lowast 2004 5431 C30H22O10

4490 4172 3911 31121530 (100) Isochamaejasmine

13lowast 2135 5571 C31H24O10

4490 1530 (100)7-

MethoxylneochaejasminA

14lowast 2763 5712 C32H26O10

4631 4451 4192 (100)3551 3372 2452 2273 Chamaejasmenin A

Coumarins (parent ions of compounds 15 16 and 19 [M+H]+ parent ions of compounds 17 and 18 [M-H]-)

15lowast 481 1931 C10H8O4

1610 (100) 1330 11501050 765 Scopoletin

16lowast 489 3251 C15H16O8

2802 1630 (100) 11901070 Umbelliferone glucoside

17lowast 585 1770 C9H6O4

1490 (100) 1330 12111051 930 770 Daphnetin

18lowast 788 1610 C9H6O3

1331 (100) 1170 1051890 770 Umbelliferone

19lowast 1444 3531 C19H12O7

3382 2671 2512 1792(100) Daphnoretin

Lignanoids (parent ions of compounds 25-27 [M+H]+ parent ions of compounds 20-24 [M-H]-)

20 621 5212 C26H34O11

3592 (100) 1591 Isolariciresinol-4rsquo-O-glucoside

21 746 5232 C26H36O11

3613 (100) 1652 1362 Secoisolariciresinol-4-O-glucoside

22 798 5212 C26H34O11

3592 3293 (100)1781 Isolariciresinol-9-O-glucoside

23 841 5232 C26H36O11

3613 (100) 3132 Secoisolariciresinol-9-O-glucoside

24lowast 969 5192 C26H32O11

3572 (100) Pinoresinol-4-O-glucoside

25 1164 4012 C22H24O7

3832 2172 2072 16721350(100) Aschantin

International Journal of Analytical Chemistry 5

Table 1 Continued

Noa tR(min)

Parent ion[M+H]+ or [M-H]-

(mz)Molecular formula

Fragmentation profile(mz)

(Relative abundance )Identification

26lowast 1176 4192 C22H26O8

4011 3871 2361 (100)1821 Syringaresinol

27lowast 1342 3591 C20H22O6

3412 3232 3112 1370(100) Pinoresinol

Amino acids (parent ions [M+H]+)28 092 1481 C

5H9NO4

1300 (100) 1020 Glutamic acid

29 199 1821 C9H11NO3

1650 (100) 1470 13601230 Tyrosine

30 205 1321 C6H13NO2

860 (100) Isoleucine31 221 1321 C

6H13NO2

1140 860 (100) Leucine32 305 1661 C

9H11NO2

1490 1200 (100) 1030 PhenylalanineDiterpenoids (parent ions [M-H]-)

33lowast 718 6133 C35H50O9

5672 4052 3092 1790(100) 1192 1130 Stelleramacrin B

a The compounds isolated from ruixianglangdu are marked with lowast

minus20

minus10

0

10

20

minus30 minus20 minus10 0 10 20 30t[1] (412)

Raw ruixianglangdu

Milk-processed ruixianglangdu

Vinegar-processed ruixianglangdu

t[2] (

332

)

Figure 2 Principle component analysis scores plot for ruixianglangdu and its processed products

mz 2572 and characteristic fragments of RDA cleav-age mz 1390 In the MS2 spectrum of flavonoids (nos2 and 4) the ion at mz 70518 fragmented to ion[M+H-Glc]+ mz 5431 Considering that isochamaejasmin-7-O-glucoside has been isolated from roots of Stellerachamaejasme compounds 2 and 4 were tentatively identi-fied as isochamaejasminchamaejasmineneochamaejasminAneochamaejasmin B-7-O-glucoside Based on [M+H]+mz 2751 a series of fragment ions [M+H-H

2O]+ mz 2572

and characteristic fragments of RDAcleavagemz 1390 com-pound 3 was identified as epiafzelechin Compounds 5 and6 were tentatively identified as naringenin-epiafzelechin ornaringenin-afzelechin in agreement with [M+H]+ mz 5451Their MS2 spectrum gave the fragment ions mz 4091 withfragments of RDA cleavage mz 1530 Compound 7 exhib-ited the deprotonated molecule [M+H]+ mz 3411 Major

fragment ions [M+H-H2O]+ mz 3232 [M+H-C

5H9]+ mz

2712 a fragment of RDA cleavage mz 1370 were shown inthe MSMS spectrumThus it was tentatively identified as 8-prenylnaringenin In the MS2 spectrum of compound 8 theion at mz 5431 fragmented to the ion mz 3451 losing onemolecule of bis(4-hydroxyphenyl)methane and mz 1992 Itwas identified as chamaechromone Four biflavonones (nos9 10 11 and 12) had a series of the same fragment ionsmz 4490 3911 3112 and 1530 The peak at mz 3911and 1530 corresponded to the RDA cleavage Compoundsnos 9 10 and 12 were confirmed as neochamaejasmin Aneochamaejasmin B and isochamaejasmin by comparisonwith standards Compound 11 was identified as chamaejas-mine due to the similar fragment ions with compounds9 10 and 12 Compound 13 exhibited [M+H]+ mz 5571major fragment ions mz 4490 and characteristic fragments

6 International Journal of Analytical Chemistry

mz 1530 which was similar to compounds 9-12 whichwas identified as 7-methoxylneochaejasmin A Compound14 had a series fragment ions mz 4451 4192 3551 and3372 The peak at mz 4192 corresponded to the RDAcleavage Compound 14 was tentatively identified as chamae-jasmenin A All the 14 flavonoids except for compound 7had been isolated from roots of Stellera chamaejasme L[17ndash23]

Coumarins compound 15 exhibited [M+H]+ mz 1931Based on major fragment ions [M+H-CH

3OH]+ mz 1610

[M+H-CH3OH-CO]+mz 1330 and [M+H-CH

3OH-2CO]+

mz 1050 this compound was tentatively assigned to scopo-letin Compound 16 was tentatively identified as umbellifer-one glucoside with a series of fragment ions [M+H-Glc]+mz 1630 and [M+H-Glc-CO

2]+ mz 1191 Compound 17

was identified as daphnetin with [M+H]+ mz 1770 anda series of fragment ions [M-H-CO]minus mz 1490 [M-H-CO2]minus mz 1330 and [M-H-CO-CO

2]minus mz 1051 The MS2

spectrum of compound 18 with [M-H]minus mz 1610 gave thecharacteristic fragment ions of mz 1331 1170 and 1051which corresponded to [M-H-CO]minus [M-H-CO

2]minus and [M-

H-2CO]minus It was tentatively identified as umbelliferoneThe MSMS mass spectrum of compound 19 [M+H]+ mz3531 presented the specific fragments of [M+H-CH

3]+ mz

3382 It was confirmed as daphnoretin by comparison withstandard All the 5 coumarins had been isolated from roots ofStellera chamaejasme L [21 24 25]

Lignanoids base peak of compounds 20 and 22 was mz3592 Compounds 20 and 22 were tentatively identifiedas isolariciresinol-4rsquo-O-glucoside and isolariciresinol-9-O-glucoside based on their polarity Two compounds (no 21and 23) had the same [M-H]minus mz 5232 and characteristicfragment [M-H-Glc]+ mz 3613 Based on their differentpolarity compounds 21 and 23 were tentatively identified assecoisolariciresinol-4-O-glucoside and secoisolariciresinol-9-O-glucoside respectively based on their polarity Basedon the characteristic fragment [M-H-Glc]minus mz 3572 com-pound 24 with [M-H]minus mz 5192 was tentatively identifiedas pinoresinol-4-O-glucoside Compound 25 was tentativelyidentified as aschantin with a series of fragment ions [M+H-H2O]+ mz 3832 and characteristic fragment ion mz 1672

Compound 26 with [M+H]+mz 4192 was tentatively identi-fied as syringaresinol which fragmented to ion [M+H-H

2O]+

mz 4011 and [M+H-HOCH3]+mz 3871 Compound 27was

tentatively identified as pinoresinol with [M+H]+ mz 3591and a series of fragment ions [M+H-H

2O]+mz 3412 [M+H-

2H2O]+ mz 3232 and characteristic fragment ion mz 1370

Only 3 lignanoids (no 24 26 and 27) had been isolated fromroots of Stellera chamaejasme L [21 26]

The 5 amino acids compounds (28-32) were confirmedby comparison with standard Compound 33 was tentativelyidentified as stelleramacrin B with [M+H]+ mz 6133 and acharacteristic fragment ion [M+H-C

14H24-H2O]+mz 4052

which had been isolated from roots of Stellera chamaejasmeL [20]

33 Exploring Chemical Basis of Toxicity reducing for Pro-cessing The extracted ion chromatogram (XIC) of the 33

components was found to be significantly altered after beingprocessed by vinegar or milk as shown in Figure 3 T-test ofthe 33 components between raw and milkvinegar-processedruixianglangdu was performed As shown in Figure 4 whenthe raw ruixianglangdu was processed by vinegar 25 compo-nents exhibited significant changes including 11 flavonoids(no 1-4 7-9 and 11-14) 4 coumarins (no 15 and 17-19)7 lignanoids (no 20-21 and 23-27) 2 amino acids (no 28and 29) and 1 diterpenoid Eleven of the 25 componentsunderwent a concentration decrease after vinegar processingincluding 8 flavonoids (no 2-4 8 11-14) 1 coumarin (no18) and 2 lignanoids (no 21 and 24) The change ratioof components in raw to vinegar-processed ruixianglangduwas in the range 03sim29 When raw ruixianglangdu wasprocessed by milk 26 components exhibited significantchanges including 13 flavonoids (no 1-8 10-14) 2 coumarins(no 16 and 19) 5 lignanoids (no 21 22 24 25 and 27) 5amino acids (no 28-30) and 1 diterpenoid Sixteen of the26 components exhibited decreasing tendency including 11flavonoids (no 2-6 7 10-14) 2 coumarins (no 16 and 19)and 3 lignanoids (no 21 22 and 24) It is worth notingthat 10 components underwent a concentration decrease afterprocessing with bothmilk and vinegar including 8 flavonoids(chamaejasmine-7-O-glucoside epiafzelechin neochamae-jasmin A-7-O-glucopyranoside chamaechromone chamae-jasmine isochamaejasmin 7-methoxylneochaejasminA andchamaejasmenin A) and 2 lignanoids Decreased flavonoidcould be due to the thermic treatment performed during theraw ruixianglangdu extraction For example total flavonoidcontent of sea buckthorn extract reduction of 9004 wasobserved as a result of heating at 100∘C after 25 min [27]The total and five flavonoids were also significantly decreasedafter fresh Cara Cara juice heating at 90∘C for 120 min [28]Quercetinwas also unstable in aqueous solution and aqueousstability of flavonol was associated with B-ring substitution[29]

Total flavonoids obtained from Stellera chamaejasmehave showed strong toxicity to mice and rabbit [30]Chamaechromone (no 8) and isochamaejasmin (no 12)were major components in ruixianglangdu with cytotox-icity to canine kidney MDCK cells [31 32] Coumarindaphnoretin (no 19) was found to be a cytotoxic con-stituent of Dirca occidentalis (Thymelaeaceae) [33] Basedon these changing components and literature for the pro-cessed ruixianglangdu flavonoids especially biflavononescould be the important chemical basis of toxicity reduc-tion In addition some coumarins and lignanoids thatdecreased after processing could also play a role in reducingtoxicity

Compared with milk-processed ruixianglangdu groupsthe 10 components involving 8 flavonoids had a lowerlevel in the vinegar-processed ruixianglangdu group It wasinferred that processing method withmilk could be better fortoxicity reduction of ruixianglangdu According to literaturethe mortality of zebrafish embryo was higher after treat-ing with vinegar-processed ruixianglangdu than milk-proc-essed ruixianglangdu [34] which was consistent with ourinference

International Journal of Analytical Chemistry 7

0 4 8 12 16 20 24 28 32Time (min)

Rela

tive i

nten

sity

()

0

1

2

3

4

7

5

68

9

10

11

12 13

1415

16

17

18 1920

21

2223 24

25

26

2728

293031

3233

50

100

4371437170517051

54515451

54315431

27512751

34113411

5551555157115711

1931193132513251

17711771 16111611

35313531 5211521152315231

5191519140114011

3591359114811481

1821182113211321 16611661

61316131

41914191

Figure 3 The extracted ion chromatogram (XIC) of the 33 components found to be significantly altered after being processed by vinegaror milk All the ion pairs are monitored in positive mode except for 17711771 16111611 52115211 52315231 51915191 and 61316131Compound number is consistent with the compound number in Table 1

0

50000000

100000000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0

50000000

100000000

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Peak

Are

a

Compound ID

Coumarins Lignanoids Amino acids Diterpenoids

Flavonoids Coumarins

Raw ruixianglangdu Vinegar-processed ruixianglangdu Milk-processed ruixianglangdu

lowastlowast lowast lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Figure 4 Peak area of 33 compounds changed in processed ruixianglangdu Compound ID is consistent with the number in Table 1Compounds with statistical difference between raw and milkvinegar-processed ruixianglangdu were marked with asterisk (lowast)

8 International Journal of Analytical Chemistry

4 Conclusions

With the help of MIM-based metabolomics this studyillustrated the chemical basis of toxicity reducing for pro-cessed ruixianglangdu Flavonoids especially biflavononescould be the important chemical basis of toxicity reducingIn addition some coumarins and lignanoids that decreasedafter processing could also play a role in reducing toxicityCompared with vinegar processing processing method withmilk could be better for toxicity reduction of ruixianglangduIt is believed that MIM-based metabolomics method wasvaluable for exploring chemical basis of toxicity reductionfor processed TCM The data is critical to further study thetoxicity reduction mechanism of processed ruixianglangdu

Data Availability

The data supporting the findings of this study are availablewithin the article Rawdata and additional information of thisstudy are available from the corresponding author on request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Wei Yang and Xiaoli Ma contributed equally to this work

Acknowledgments

The study has been financially supported by Natural Sci-ence Foundation of Beijing (7174325) National Science andTechnology Major Project (2015ZX09501004-003-005) andNational Natural Science Foundation Youth Science Founda-tion (81803722)

References

[1] Z Z Zhao Z T Liang K Chan et al ldquoA unique issue in thestandardization of Chinese materia medica processingrdquo PlantaMedica vol 76 no 17 pp 1975ndash1986 2010

[2] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y Chaildquoharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

[3] G Yang andD Chen ldquoBiflavanones flavonoids and coumarinsfrom the roots of Stellera chamaejasme and their antiviral effecton hepatitis B virusrdquo Chemistry amp Biodiversity vol 5 no 7 pp1419ndash1424 2008

[4] M Kim H J Lee A Randy J H Yun S Oh and CW Nho ldquoStellera chamaejasme and its constituents inducecutaneous wound healing and anti-inflammatory activitiesrdquoScientific Reports vol 7 Article ID 42490 2017

[5] X Liu Q Yang G Zhang et al ldquoAnti-tumor pharmacologicalevaluation of extracts from stellera chamaejasme L basedon hollow fiber assayrdquo BMC Complementary and AlternativeMedicine vol 14 p 116 2014

[6] Y Wang J Li M Han W Wang and Y Li ldquoPrevention andtreatment effect of total flavonoids in Stellera chamaejasme Lon nonalcoholic fatty liver in ratsrdquo Lipids in Health and Diseasevol 14 p 85 2015

[7] T Bai and D J L H Wang ldquoProcessing difference betweenmongolian medicine and traditional chinese medicinerdquo WorldLatest Medicine Information vol 18 p 182 2018

[8] P E C o M O Health Chinese Pharmacopoeia PeoplersquosMedical PublishingHouse Beijing China Edition 1977 edition1979

[9] Z F Jiang X L Ma B Han S Y Cao and Y Y Gen ldquoInitialstudy on effection of different processing methods on theStellera chamaejasme chemical constituentsrdquoMedical Researchand Education vol 32 pp 1ndash6 2015

[10] X W Ji M L Hao X Yang and Y H Wang ldquoRP-HPLCfingerprint of milk Stellera chamaejasmerdquo Zhong Yao Cai vol35 pp 1754ndash1757 2012

[11] W Yang M Ye M Liu et al ldquoA practical strategy for thecharacterization of coumarins in Radix Glehniae by liquidchromatography coupled with triple quadrupole-linear ion trapmass spectrometryrdquo Journal of Chromatography A vol 1217 no27 pp 4587ndash4600 2010

[12] M Yao LMaWGHumphreys andM Zhu ldquoRapid screeningand characterization of drug metabolites using a multiple ionmonitoring-dependentMSMS acquisitionmethod on a hybridtriple quadrupole-linear ion trapmass spectrometerrdquo Journal ofMass Spectrometry vol 43 no 10 pp 1364ndash1375 2008

[13] P Luo W Dai P Yin et al ldquoMultiple reaction monitoring-ion pair finder a systematic approach to transform nontar-geted mode to pseudotargeted mode for metabolomics studybased on liquid chromatographyndashmass spectrometryrdquo Analyt-ical Chemistry vol 87 no 10 pp 5050ndash5055 2015

[14] W Chen L Gong Z Guo et al ldquoA novel integrated methodfor large-scale detection identification and quantification ofwidely targeted metabolites Application in the study of ricemetabolomicsrdquo Molecular Plant vol 6 no 6 pp 1769ndash17802013

[15] L Sheng J Hu B Wang H Chen and Y Li ldquoDeterminationof bicyclol in dog plasma using liquid chromatographyndashtandemmass spectrometryrdquo Journal of Chromatography B vol 878 no23 pp 2106ndash2110 2010

[16] DV Rossetti CMartelli R Longhi F IavaroneMCastagnolaand C Desiderio ldquoQuantitative analysis of thymosin beta4 inwhole saliva by capillary electrophoresis-mass spectrometryusing multiple ions monitoring (CE-MIM-MS)rdquo Electrophore-sis vol 34 no 18 pp 2674ndash2682 2013

[17] C Jin R G Michetich and M Daneshtalab ldquoFlavonoids fromStellera chamaejasmerdquo Phytochemistry vol 50 no 3 pp 505ndash508 1998

[18] W Chen X Luo Z Wang Y Zhang L Liu and H WangldquoA new biflavone glucoside from the roots of Stellera chamae-jasmerdquo Chinese Journal of Natural Medicines vol 13 no 7 pp550ndash553 2015

[19] Z Yan H Guo J Yang et al ldquoPhytotoxic flavonoids from rootsof Stellera chamaejasme L (Thymelaeaceae)rdquo Phytochemistryvol 106 pp 61ndash68 2014

[20] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y ChaildquoCharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

International Journal of Analytical Chemistry 9

[21] Z Jiang T Tanaka T Sakamoto I Kouno J Duan and RZhou ldquoBiflavanones diterpenes and coumarins from the rootsof stellera chamaejasme lrdquoChemical amp Pharmaceutical Bulletinvol 50 no 1 pp 137ndash139

[22] Y Li C Li L Lv et al ldquoA UPLC-MSMSmethod for simultane-ous determination of five flavonoids from Stellera chamaejasmeL in rat plasma and its application to a pharmacokinetic studyrdquoBiomedical Chromatography vol 32 no 6 Article ID e41892018

[23] G Liu H Tatematsu M Kurokawa M Niwa and Y HirataldquoNovel C-3C-31015840rsquo-biflavanones from Stellera chamaejasme LrdquoChemical amp Pharmaceutical Bulletin vol 32 pp 362ndash365 1984

[24] X Li K Rahman J Zhu andH Zhang ldquoChemical Constituentsand Pharmacological Activities of Stellera chamaejasmerdquo Cur-rent Pharmaceutical Design vol 24 no 24 pp 2825ndash2838 2018

[25] X Su R Lin S Wong S Tsui and S Kwan ldquoIdentificationand characterisationof the Chinese herb Langdu by LC-MSMSanalysisrdquo Phytochemical Analysis vol 14 no 1 pp 40ndash47 2003

[26] L Qiao L Yang D Zhang J Zou and J Dai ldquoStudies on chem-ical constitutes from callus cultures of Stellera chamaejasmerdquoZhongguo Zhongyao Zazhi vol 36 no 24 pp 3457ndash3462 2011httpswwwncbinlmnihgovpubmed22368856

[27] F M Ursache I O Ghinea M Turturica I Aprodu GRapeanu andN Stanciuc ldquoPhytochemicals content and antiox-idant properties of sea buckthorn (Hippophae rhamnoides L)as affected by heat treatment - Quantitative spectroscopic andkinetic approachesrdquo FoodChemistry vol 233 pp 442ndash449 2017

[28] Q Lu Y Peng C Zhu and S Pan ldquoEffect of thermal treatmenton carotenoids flavonoids and ascorbic acid in juice of orangecv Cara Carardquo Food Chemistry vol 265 pp 39ndash48 2018

[29] S Maini H L Hodgson and E S Krol ldquoTheUVA and aqueousstability of flavonoids is dependent on b-ring substitutionrdquoJournal of Agricultural and Food Chemistry vol 60 no 28 pp6966ndash6976 2012

[30] Z Yan L Zeng H Jin and B Qin ldquoPotential ecological rolesof flavonoids from Stellera chamaejasmerdquo Plant Signaling andBehavior vol 10 Article ID e1001225 2015

[31] Y Lou J Zheng B Wang X Zhang X Zhang and SZeng ldquoMetabolites characterization of chamaechromone invivo and in vitro by using ultra-performance liquid chro-matographyXevo G2 quadrupole time-of-flight tandem massspectrometryrdquo Journal of Ethnopharmacology vol 151 no 1 pp242ndash252 2014

[32] Y H Wang X Yang L J Sun and L M Yang ldquoStudies onthe cytotoxicity of Stellera chamaejasme L in vitrordquo ChineseMedicinal Biotechnology vol 7 pp 9ndash13 2012

[33] M M Badawi S S Handa A D Kinghorn G A Cordelland N R Farnsworth ldquoPlant anticancer agents XXVIIAntileukemic and cytotoxic constituents of Dirca occiden-talis (thymelaeaceae)rdquo Journal of Pharmaceutical Sciences vol72 no 11 pp 1285ndash1287 1983 httpswwwncbinlmnihgovpubmed6644588

[34] J F Yang W J Dong J C Zong et al ldquoToxicity detectionof Stella Chamaejasme in zebrafish embryo with differentextraction methodsrdquo Journal of Inner Mongolia University forNationalities vol 31 pp 236ndash239 2016

4 International Journal of Analytical Chemistry

Table 1 Identification of compounds in ruixianglangdu

Noa tR(min)

Parent ion[M+H]+ or [M-H]-

(mz)Molecular formula

Fragmentation profile(mz)

(Relative abundance )Identification

Flavonoids (parent ions [M+H]+)

1lowast 481 4371 C21H24O10

2752 2572 1490 1390(100) 1070

Epiafzelechin-7-O-gluconoside

2lowast 585 7052 C36H32O15

5431 (100)

IsochamaejasmineChamaejasmine

Neochamaejasmine ANeochamaejasmineB-7-O- gluconoside

3lowast 641 2751 C15H14O5

2572 (100) 1390 Epiafzelechin4lowast 663 7052 C

36H32O15

5432 (100) 5271 3112 Same as no 2

5lowast 984 5451 C30H24O10

5272 4091 (100) 28321530

Naringenin-epiafzelechinor Naringenin-afzelechin

6lowast 1048 5451 C30H24O10

5272 4091 (100) 28321530 Same as no 5

7 1213 3411 C20H20O5

3232 (100) 2911 27121872 1370 8-Prenylnaringenin

8lowast 1483 5431 C30H22O10

3451 1992 (100) Chamaechromone

9lowast 1538 5431 C30H22O10

4490 3911 3112 19911530 (100) Neochamaejasmine A

10lowast 1719 5431 C30H22O10

4490 4172 3911 31122311 1992 1530 (100) Neochamaejasmine B

11lowast 1947 5431 C30H22O10

4490 4172 3911 31121530 (100) Chamaejasmine

12lowast 2004 5431 C30H22O10

4490 4172 3911 31121530 (100) Isochamaejasmine

13lowast 2135 5571 C31H24O10

4490 1530 (100)7-

MethoxylneochaejasminA

14lowast 2763 5712 C32H26O10

4631 4451 4192 (100)3551 3372 2452 2273 Chamaejasmenin A

Coumarins (parent ions of compounds 15 16 and 19 [M+H]+ parent ions of compounds 17 and 18 [M-H]-)

15lowast 481 1931 C10H8O4

1610 (100) 1330 11501050 765 Scopoletin

16lowast 489 3251 C15H16O8

2802 1630 (100) 11901070 Umbelliferone glucoside

17lowast 585 1770 C9H6O4

1490 (100) 1330 12111051 930 770 Daphnetin

18lowast 788 1610 C9H6O3

1331 (100) 1170 1051890 770 Umbelliferone

19lowast 1444 3531 C19H12O7

3382 2671 2512 1792(100) Daphnoretin

Lignanoids (parent ions of compounds 25-27 [M+H]+ parent ions of compounds 20-24 [M-H]-)

20 621 5212 C26H34O11

3592 (100) 1591 Isolariciresinol-4rsquo-O-glucoside

21 746 5232 C26H36O11

3613 (100) 1652 1362 Secoisolariciresinol-4-O-glucoside

22 798 5212 C26H34O11

3592 3293 (100)1781 Isolariciresinol-9-O-glucoside

23 841 5232 C26H36O11

3613 (100) 3132 Secoisolariciresinol-9-O-glucoside

24lowast 969 5192 C26H32O11

3572 (100) Pinoresinol-4-O-glucoside

25 1164 4012 C22H24O7

3832 2172 2072 16721350(100) Aschantin

International Journal of Analytical Chemistry 5

Table 1 Continued

Noa tR(min)

Parent ion[M+H]+ or [M-H]-

(mz)Molecular formula

Fragmentation profile(mz)

(Relative abundance )Identification

26lowast 1176 4192 C22H26O8

4011 3871 2361 (100)1821 Syringaresinol

27lowast 1342 3591 C20H22O6

3412 3232 3112 1370(100) Pinoresinol

Amino acids (parent ions [M+H]+)28 092 1481 C

5H9NO4

1300 (100) 1020 Glutamic acid

29 199 1821 C9H11NO3

1650 (100) 1470 13601230 Tyrosine

30 205 1321 C6H13NO2

860 (100) Isoleucine31 221 1321 C

6H13NO2

1140 860 (100) Leucine32 305 1661 C

9H11NO2

1490 1200 (100) 1030 PhenylalanineDiterpenoids (parent ions [M-H]-)

33lowast 718 6133 C35H50O9

5672 4052 3092 1790(100) 1192 1130 Stelleramacrin B

a The compounds isolated from ruixianglangdu are marked with lowast

minus20

minus10

0

10

20

minus30 minus20 minus10 0 10 20 30t[1] (412)

Raw ruixianglangdu

Milk-processed ruixianglangdu

Vinegar-processed ruixianglangdu

t[2] (

332

)

Figure 2 Principle component analysis scores plot for ruixianglangdu and its processed products

mz 2572 and characteristic fragments of RDA cleav-age mz 1390 In the MS2 spectrum of flavonoids (nos2 and 4) the ion at mz 70518 fragmented to ion[M+H-Glc]+ mz 5431 Considering that isochamaejasmin-7-O-glucoside has been isolated from roots of Stellerachamaejasme compounds 2 and 4 were tentatively identi-fied as isochamaejasminchamaejasmineneochamaejasminAneochamaejasmin B-7-O-glucoside Based on [M+H]+mz 2751 a series of fragment ions [M+H-H

2O]+ mz 2572

and characteristic fragments of RDAcleavagemz 1390 com-pound 3 was identified as epiafzelechin Compounds 5 and6 were tentatively identified as naringenin-epiafzelechin ornaringenin-afzelechin in agreement with [M+H]+ mz 5451Their MS2 spectrum gave the fragment ions mz 4091 withfragments of RDA cleavage mz 1530 Compound 7 exhib-ited the deprotonated molecule [M+H]+ mz 3411 Major

fragment ions [M+H-H2O]+ mz 3232 [M+H-C

5H9]+ mz

2712 a fragment of RDA cleavage mz 1370 were shown inthe MSMS spectrumThus it was tentatively identified as 8-prenylnaringenin In the MS2 spectrum of compound 8 theion at mz 5431 fragmented to the ion mz 3451 losing onemolecule of bis(4-hydroxyphenyl)methane and mz 1992 Itwas identified as chamaechromone Four biflavonones (nos9 10 11 and 12) had a series of the same fragment ionsmz 4490 3911 3112 and 1530 The peak at mz 3911and 1530 corresponded to the RDA cleavage Compoundsnos 9 10 and 12 were confirmed as neochamaejasmin Aneochamaejasmin B and isochamaejasmin by comparisonwith standards Compound 11 was identified as chamaejas-mine due to the similar fragment ions with compounds9 10 and 12 Compound 13 exhibited [M+H]+ mz 5571major fragment ions mz 4490 and characteristic fragments

6 International Journal of Analytical Chemistry

mz 1530 which was similar to compounds 9-12 whichwas identified as 7-methoxylneochaejasmin A Compound14 had a series fragment ions mz 4451 4192 3551 and3372 The peak at mz 4192 corresponded to the RDAcleavage Compound 14 was tentatively identified as chamae-jasmenin A All the 14 flavonoids except for compound 7had been isolated from roots of Stellera chamaejasme L[17ndash23]

Coumarins compound 15 exhibited [M+H]+ mz 1931Based on major fragment ions [M+H-CH

3OH]+ mz 1610

[M+H-CH3OH-CO]+mz 1330 and [M+H-CH

3OH-2CO]+

mz 1050 this compound was tentatively assigned to scopo-letin Compound 16 was tentatively identified as umbellifer-one glucoside with a series of fragment ions [M+H-Glc]+mz 1630 and [M+H-Glc-CO

2]+ mz 1191 Compound 17

was identified as daphnetin with [M+H]+ mz 1770 anda series of fragment ions [M-H-CO]minus mz 1490 [M-H-CO2]minus mz 1330 and [M-H-CO-CO

2]minus mz 1051 The MS2

spectrum of compound 18 with [M-H]minus mz 1610 gave thecharacteristic fragment ions of mz 1331 1170 and 1051which corresponded to [M-H-CO]minus [M-H-CO

2]minus and [M-

H-2CO]minus It was tentatively identified as umbelliferoneThe MSMS mass spectrum of compound 19 [M+H]+ mz3531 presented the specific fragments of [M+H-CH

3]+ mz

3382 It was confirmed as daphnoretin by comparison withstandard All the 5 coumarins had been isolated from roots ofStellera chamaejasme L [21 24 25]

Lignanoids base peak of compounds 20 and 22 was mz3592 Compounds 20 and 22 were tentatively identifiedas isolariciresinol-4rsquo-O-glucoside and isolariciresinol-9-O-glucoside based on their polarity Two compounds (no 21and 23) had the same [M-H]minus mz 5232 and characteristicfragment [M-H-Glc]+ mz 3613 Based on their differentpolarity compounds 21 and 23 were tentatively identified assecoisolariciresinol-4-O-glucoside and secoisolariciresinol-9-O-glucoside respectively based on their polarity Basedon the characteristic fragment [M-H-Glc]minus mz 3572 com-pound 24 with [M-H]minus mz 5192 was tentatively identifiedas pinoresinol-4-O-glucoside Compound 25 was tentativelyidentified as aschantin with a series of fragment ions [M+H-H2O]+ mz 3832 and characteristic fragment ion mz 1672

Compound 26 with [M+H]+mz 4192 was tentatively identi-fied as syringaresinol which fragmented to ion [M+H-H

2O]+

mz 4011 and [M+H-HOCH3]+mz 3871 Compound 27was

tentatively identified as pinoresinol with [M+H]+ mz 3591and a series of fragment ions [M+H-H

2O]+mz 3412 [M+H-

2H2O]+ mz 3232 and characteristic fragment ion mz 1370

Only 3 lignanoids (no 24 26 and 27) had been isolated fromroots of Stellera chamaejasme L [21 26]

The 5 amino acids compounds (28-32) were confirmedby comparison with standard Compound 33 was tentativelyidentified as stelleramacrin B with [M+H]+ mz 6133 and acharacteristic fragment ion [M+H-C

14H24-H2O]+mz 4052

which had been isolated from roots of Stellera chamaejasmeL [20]

33 Exploring Chemical Basis of Toxicity reducing for Pro-cessing The extracted ion chromatogram (XIC) of the 33

components was found to be significantly altered after beingprocessed by vinegar or milk as shown in Figure 3 T-test ofthe 33 components between raw and milkvinegar-processedruixianglangdu was performed As shown in Figure 4 whenthe raw ruixianglangdu was processed by vinegar 25 compo-nents exhibited significant changes including 11 flavonoids(no 1-4 7-9 and 11-14) 4 coumarins (no 15 and 17-19)7 lignanoids (no 20-21 and 23-27) 2 amino acids (no 28and 29) and 1 diterpenoid Eleven of the 25 componentsunderwent a concentration decrease after vinegar processingincluding 8 flavonoids (no 2-4 8 11-14) 1 coumarin (no18) and 2 lignanoids (no 21 and 24) The change ratioof components in raw to vinegar-processed ruixianglangduwas in the range 03sim29 When raw ruixianglangdu wasprocessed by milk 26 components exhibited significantchanges including 13 flavonoids (no 1-8 10-14) 2 coumarins(no 16 and 19) 5 lignanoids (no 21 22 24 25 and 27) 5amino acids (no 28-30) and 1 diterpenoid Sixteen of the26 components exhibited decreasing tendency including 11flavonoids (no 2-6 7 10-14) 2 coumarins (no 16 and 19)and 3 lignanoids (no 21 22 and 24) It is worth notingthat 10 components underwent a concentration decrease afterprocessing with bothmilk and vinegar including 8 flavonoids(chamaejasmine-7-O-glucoside epiafzelechin neochamae-jasmin A-7-O-glucopyranoside chamaechromone chamae-jasmine isochamaejasmin 7-methoxylneochaejasminA andchamaejasmenin A) and 2 lignanoids Decreased flavonoidcould be due to the thermic treatment performed during theraw ruixianglangdu extraction For example total flavonoidcontent of sea buckthorn extract reduction of 9004 wasobserved as a result of heating at 100∘C after 25 min [27]The total and five flavonoids were also significantly decreasedafter fresh Cara Cara juice heating at 90∘C for 120 min [28]Quercetinwas also unstable in aqueous solution and aqueousstability of flavonol was associated with B-ring substitution[29]

Total flavonoids obtained from Stellera chamaejasmehave showed strong toxicity to mice and rabbit [30]Chamaechromone (no 8) and isochamaejasmin (no 12)were major components in ruixianglangdu with cytotox-icity to canine kidney MDCK cells [31 32] Coumarindaphnoretin (no 19) was found to be a cytotoxic con-stituent of Dirca occidentalis (Thymelaeaceae) [33] Basedon these changing components and literature for the pro-cessed ruixianglangdu flavonoids especially biflavononescould be the important chemical basis of toxicity reduc-tion In addition some coumarins and lignanoids thatdecreased after processing could also play a role in reducingtoxicity

Compared with milk-processed ruixianglangdu groupsthe 10 components involving 8 flavonoids had a lowerlevel in the vinegar-processed ruixianglangdu group It wasinferred that processing method withmilk could be better fortoxicity reduction of ruixianglangdu According to literaturethe mortality of zebrafish embryo was higher after treat-ing with vinegar-processed ruixianglangdu than milk-proc-essed ruixianglangdu [34] which was consistent with ourinference

International Journal of Analytical Chemistry 7

0 4 8 12 16 20 24 28 32Time (min)

Rela

tive i

nten

sity

()

0

1

2

3

4

7

5

68

9

10

11

12 13

1415

16

17

18 1920

21

2223 24

25

26

2728

293031

3233

50

100

4371437170517051

54515451

54315431

27512751

34113411

5551555157115711

1931193132513251

17711771 16111611

35313531 5211521152315231

5191519140114011

3591359114811481

1821182113211321 16611661

61316131

41914191

Figure 3 The extracted ion chromatogram (XIC) of the 33 components found to be significantly altered after being processed by vinegaror milk All the ion pairs are monitored in positive mode except for 17711771 16111611 52115211 52315231 51915191 and 61316131Compound number is consistent with the compound number in Table 1

0

50000000

100000000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0

50000000

100000000

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Peak

Are

a

Compound ID

Coumarins Lignanoids Amino acids Diterpenoids

Flavonoids Coumarins

Raw ruixianglangdu Vinegar-processed ruixianglangdu Milk-processed ruixianglangdu

lowastlowast lowast lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Figure 4 Peak area of 33 compounds changed in processed ruixianglangdu Compound ID is consistent with the number in Table 1Compounds with statistical difference between raw and milkvinegar-processed ruixianglangdu were marked with asterisk (lowast)

8 International Journal of Analytical Chemistry

4 Conclusions

With the help of MIM-based metabolomics this studyillustrated the chemical basis of toxicity reducing for pro-cessed ruixianglangdu Flavonoids especially biflavononescould be the important chemical basis of toxicity reducingIn addition some coumarins and lignanoids that decreasedafter processing could also play a role in reducing toxicityCompared with vinegar processing processing method withmilk could be better for toxicity reduction of ruixianglangduIt is believed that MIM-based metabolomics method wasvaluable for exploring chemical basis of toxicity reductionfor processed TCM The data is critical to further study thetoxicity reduction mechanism of processed ruixianglangdu

Data Availability

The data supporting the findings of this study are availablewithin the article Rawdata and additional information of thisstudy are available from the corresponding author on request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Wei Yang and Xiaoli Ma contributed equally to this work

Acknowledgments

The study has been financially supported by Natural Sci-ence Foundation of Beijing (7174325) National Science andTechnology Major Project (2015ZX09501004-003-005) andNational Natural Science Foundation Youth Science Founda-tion (81803722)

References

[1] Z Z Zhao Z T Liang K Chan et al ldquoA unique issue in thestandardization of Chinese materia medica processingrdquo PlantaMedica vol 76 no 17 pp 1975ndash1986 2010

[2] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y Chaildquoharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

[3] G Yang andD Chen ldquoBiflavanones flavonoids and coumarinsfrom the roots of Stellera chamaejasme and their antiviral effecton hepatitis B virusrdquo Chemistry amp Biodiversity vol 5 no 7 pp1419ndash1424 2008

[4] M Kim H J Lee A Randy J H Yun S Oh and CW Nho ldquoStellera chamaejasme and its constituents inducecutaneous wound healing and anti-inflammatory activitiesrdquoScientific Reports vol 7 Article ID 42490 2017

[5] X Liu Q Yang G Zhang et al ldquoAnti-tumor pharmacologicalevaluation of extracts from stellera chamaejasme L basedon hollow fiber assayrdquo BMC Complementary and AlternativeMedicine vol 14 p 116 2014

[6] Y Wang J Li M Han W Wang and Y Li ldquoPrevention andtreatment effect of total flavonoids in Stellera chamaejasme Lon nonalcoholic fatty liver in ratsrdquo Lipids in Health and Diseasevol 14 p 85 2015

[7] T Bai and D J L H Wang ldquoProcessing difference betweenmongolian medicine and traditional chinese medicinerdquo WorldLatest Medicine Information vol 18 p 182 2018

[8] P E C o M O Health Chinese Pharmacopoeia PeoplersquosMedical PublishingHouse Beijing China Edition 1977 edition1979

[9] Z F Jiang X L Ma B Han S Y Cao and Y Y Gen ldquoInitialstudy on effection of different processing methods on theStellera chamaejasme chemical constituentsrdquoMedical Researchand Education vol 32 pp 1ndash6 2015

[10] X W Ji M L Hao X Yang and Y H Wang ldquoRP-HPLCfingerprint of milk Stellera chamaejasmerdquo Zhong Yao Cai vol35 pp 1754ndash1757 2012

[11] W Yang M Ye M Liu et al ldquoA practical strategy for thecharacterization of coumarins in Radix Glehniae by liquidchromatography coupled with triple quadrupole-linear ion trapmass spectrometryrdquo Journal of Chromatography A vol 1217 no27 pp 4587ndash4600 2010

[12] M Yao LMaWGHumphreys andM Zhu ldquoRapid screeningand characterization of drug metabolites using a multiple ionmonitoring-dependentMSMS acquisitionmethod on a hybridtriple quadrupole-linear ion trapmass spectrometerrdquo Journal ofMass Spectrometry vol 43 no 10 pp 1364ndash1375 2008

[13] P Luo W Dai P Yin et al ldquoMultiple reaction monitoring-ion pair finder a systematic approach to transform nontar-geted mode to pseudotargeted mode for metabolomics studybased on liquid chromatographyndashmass spectrometryrdquo Analyt-ical Chemistry vol 87 no 10 pp 5050ndash5055 2015

[14] W Chen L Gong Z Guo et al ldquoA novel integrated methodfor large-scale detection identification and quantification ofwidely targeted metabolites Application in the study of ricemetabolomicsrdquo Molecular Plant vol 6 no 6 pp 1769ndash17802013

[15] L Sheng J Hu B Wang H Chen and Y Li ldquoDeterminationof bicyclol in dog plasma using liquid chromatographyndashtandemmass spectrometryrdquo Journal of Chromatography B vol 878 no23 pp 2106ndash2110 2010

[16] DV Rossetti CMartelli R Longhi F IavaroneMCastagnolaand C Desiderio ldquoQuantitative analysis of thymosin beta4 inwhole saliva by capillary electrophoresis-mass spectrometryusing multiple ions monitoring (CE-MIM-MS)rdquo Electrophore-sis vol 34 no 18 pp 2674ndash2682 2013

[17] C Jin R G Michetich and M Daneshtalab ldquoFlavonoids fromStellera chamaejasmerdquo Phytochemistry vol 50 no 3 pp 505ndash508 1998

[18] W Chen X Luo Z Wang Y Zhang L Liu and H WangldquoA new biflavone glucoside from the roots of Stellera chamae-jasmerdquo Chinese Journal of Natural Medicines vol 13 no 7 pp550ndash553 2015

[19] Z Yan H Guo J Yang et al ldquoPhytotoxic flavonoids from rootsof Stellera chamaejasme L (Thymelaeaceae)rdquo Phytochemistryvol 106 pp 61ndash68 2014

[20] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y ChaildquoCharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

International Journal of Analytical Chemistry 9

[21] Z Jiang T Tanaka T Sakamoto I Kouno J Duan and RZhou ldquoBiflavanones diterpenes and coumarins from the rootsof stellera chamaejasme lrdquoChemical amp Pharmaceutical Bulletinvol 50 no 1 pp 137ndash139

[22] Y Li C Li L Lv et al ldquoA UPLC-MSMSmethod for simultane-ous determination of five flavonoids from Stellera chamaejasmeL in rat plasma and its application to a pharmacokinetic studyrdquoBiomedical Chromatography vol 32 no 6 Article ID e41892018

[23] G Liu H Tatematsu M Kurokawa M Niwa and Y HirataldquoNovel C-3C-31015840rsquo-biflavanones from Stellera chamaejasme LrdquoChemical amp Pharmaceutical Bulletin vol 32 pp 362ndash365 1984

[24] X Li K Rahman J Zhu andH Zhang ldquoChemical Constituentsand Pharmacological Activities of Stellera chamaejasmerdquo Cur-rent Pharmaceutical Design vol 24 no 24 pp 2825ndash2838 2018

[25] X Su R Lin S Wong S Tsui and S Kwan ldquoIdentificationand characterisationof the Chinese herb Langdu by LC-MSMSanalysisrdquo Phytochemical Analysis vol 14 no 1 pp 40ndash47 2003

[26] L Qiao L Yang D Zhang J Zou and J Dai ldquoStudies on chem-ical constitutes from callus cultures of Stellera chamaejasmerdquoZhongguo Zhongyao Zazhi vol 36 no 24 pp 3457ndash3462 2011httpswwwncbinlmnihgovpubmed22368856

[27] F M Ursache I O Ghinea M Turturica I Aprodu GRapeanu andN Stanciuc ldquoPhytochemicals content and antiox-idant properties of sea buckthorn (Hippophae rhamnoides L)as affected by heat treatment - Quantitative spectroscopic andkinetic approachesrdquo FoodChemistry vol 233 pp 442ndash449 2017

[28] Q Lu Y Peng C Zhu and S Pan ldquoEffect of thermal treatmenton carotenoids flavonoids and ascorbic acid in juice of orangecv Cara Carardquo Food Chemistry vol 265 pp 39ndash48 2018

[29] S Maini H L Hodgson and E S Krol ldquoTheUVA and aqueousstability of flavonoids is dependent on b-ring substitutionrdquoJournal of Agricultural and Food Chemistry vol 60 no 28 pp6966ndash6976 2012

[30] Z Yan L Zeng H Jin and B Qin ldquoPotential ecological rolesof flavonoids from Stellera chamaejasmerdquo Plant Signaling andBehavior vol 10 Article ID e1001225 2015

[31] Y Lou J Zheng B Wang X Zhang X Zhang and SZeng ldquoMetabolites characterization of chamaechromone invivo and in vitro by using ultra-performance liquid chro-matographyXevo G2 quadrupole time-of-flight tandem massspectrometryrdquo Journal of Ethnopharmacology vol 151 no 1 pp242ndash252 2014

[32] Y H Wang X Yang L J Sun and L M Yang ldquoStudies onthe cytotoxicity of Stellera chamaejasme L in vitrordquo ChineseMedicinal Biotechnology vol 7 pp 9ndash13 2012

[33] M M Badawi S S Handa A D Kinghorn G A Cordelland N R Farnsworth ldquoPlant anticancer agents XXVIIAntileukemic and cytotoxic constituents of Dirca occiden-talis (thymelaeaceae)rdquo Journal of Pharmaceutical Sciences vol72 no 11 pp 1285ndash1287 1983 httpswwwncbinlmnihgovpubmed6644588

[34] J F Yang W J Dong J C Zong et al ldquoToxicity detectionof Stella Chamaejasme in zebrafish embryo with differentextraction methodsrdquo Journal of Inner Mongolia University forNationalities vol 31 pp 236ndash239 2016

International Journal of Analytical Chemistry 5

Table 1 Continued

Noa tR(min)

Parent ion[M+H]+ or [M-H]-

(mz)Molecular formula

Fragmentation profile(mz)

(Relative abundance )Identification

26lowast 1176 4192 C22H26O8

4011 3871 2361 (100)1821 Syringaresinol

27lowast 1342 3591 C20H22O6

3412 3232 3112 1370(100) Pinoresinol

Amino acids (parent ions [M+H]+)28 092 1481 C

5H9NO4

1300 (100) 1020 Glutamic acid

29 199 1821 C9H11NO3

1650 (100) 1470 13601230 Tyrosine

30 205 1321 C6H13NO2

860 (100) Isoleucine31 221 1321 C

6H13NO2

1140 860 (100) Leucine32 305 1661 C

9H11NO2

1490 1200 (100) 1030 PhenylalanineDiterpenoids (parent ions [M-H]-)

33lowast 718 6133 C35H50O9

5672 4052 3092 1790(100) 1192 1130 Stelleramacrin B

a The compounds isolated from ruixianglangdu are marked with lowast

minus20

minus10

0

10

20

minus30 minus20 minus10 0 10 20 30t[1] (412)

Raw ruixianglangdu

Milk-processed ruixianglangdu

Vinegar-processed ruixianglangdu

t[2] (

332

)

Figure 2 Principle component analysis scores plot for ruixianglangdu and its processed products

mz 2572 and characteristic fragments of RDA cleav-age mz 1390 In the MS2 spectrum of flavonoids (nos2 and 4) the ion at mz 70518 fragmented to ion[M+H-Glc]+ mz 5431 Considering that isochamaejasmin-7-O-glucoside has been isolated from roots of Stellerachamaejasme compounds 2 and 4 were tentatively identi-fied as isochamaejasminchamaejasmineneochamaejasminAneochamaejasmin B-7-O-glucoside Based on [M+H]+mz 2751 a series of fragment ions [M+H-H

2O]+ mz 2572

and characteristic fragments of RDAcleavagemz 1390 com-pound 3 was identified as epiafzelechin Compounds 5 and6 were tentatively identified as naringenin-epiafzelechin ornaringenin-afzelechin in agreement with [M+H]+ mz 5451Their MS2 spectrum gave the fragment ions mz 4091 withfragments of RDA cleavage mz 1530 Compound 7 exhib-ited the deprotonated molecule [M+H]+ mz 3411 Major

fragment ions [M+H-H2O]+ mz 3232 [M+H-C

5H9]+ mz

2712 a fragment of RDA cleavage mz 1370 were shown inthe MSMS spectrumThus it was tentatively identified as 8-prenylnaringenin In the MS2 spectrum of compound 8 theion at mz 5431 fragmented to the ion mz 3451 losing onemolecule of bis(4-hydroxyphenyl)methane and mz 1992 Itwas identified as chamaechromone Four biflavonones (nos9 10 11 and 12) had a series of the same fragment ionsmz 4490 3911 3112 and 1530 The peak at mz 3911and 1530 corresponded to the RDA cleavage Compoundsnos 9 10 and 12 were confirmed as neochamaejasmin Aneochamaejasmin B and isochamaejasmin by comparisonwith standards Compound 11 was identified as chamaejas-mine due to the similar fragment ions with compounds9 10 and 12 Compound 13 exhibited [M+H]+ mz 5571major fragment ions mz 4490 and characteristic fragments

6 International Journal of Analytical Chemistry

mz 1530 which was similar to compounds 9-12 whichwas identified as 7-methoxylneochaejasmin A Compound14 had a series fragment ions mz 4451 4192 3551 and3372 The peak at mz 4192 corresponded to the RDAcleavage Compound 14 was tentatively identified as chamae-jasmenin A All the 14 flavonoids except for compound 7had been isolated from roots of Stellera chamaejasme L[17ndash23]

Coumarins compound 15 exhibited [M+H]+ mz 1931Based on major fragment ions [M+H-CH

3OH]+ mz 1610

[M+H-CH3OH-CO]+mz 1330 and [M+H-CH

3OH-2CO]+

mz 1050 this compound was tentatively assigned to scopo-letin Compound 16 was tentatively identified as umbellifer-one glucoside with a series of fragment ions [M+H-Glc]+mz 1630 and [M+H-Glc-CO

2]+ mz 1191 Compound 17

was identified as daphnetin with [M+H]+ mz 1770 anda series of fragment ions [M-H-CO]minus mz 1490 [M-H-CO2]minus mz 1330 and [M-H-CO-CO

2]minus mz 1051 The MS2

spectrum of compound 18 with [M-H]minus mz 1610 gave thecharacteristic fragment ions of mz 1331 1170 and 1051which corresponded to [M-H-CO]minus [M-H-CO

2]minus and [M-

H-2CO]minus It was tentatively identified as umbelliferoneThe MSMS mass spectrum of compound 19 [M+H]+ mz3531 presented the specific fragments of [M+H-CH

3]+ mz

3382 It was confirmed as daphnoretin by comparison withstandard All the 5 coumarins had been isolated from roots ofStellera chamaejasme L [21 24 25]

Lignanoids base peak of compounds 20 and 22 was mz3592 Compounds 20 and 22 were tentatively identifiedas isolariciresinol-4rsquo-O-glucoside and isolariciresinol-9-O-glucoside based on their polarity Two compounds (no 21and 23) had the same [M-H]minus mz 5232 and characteristicfragment [M-H-Glc]+ mz 3613 Based on their differentpolarity compounds 21 and 23 were tentatively identified assecoisolariciresinol-4-O-glucoside and secoisolariciresinol-9-O-glucoside respectively based on their polarity Basedon the characteristic fragment [M-H-Glc]minus mz 3572 com-pound 24 with [M-H]minus mz 5192 was tentatively identifiedas pinoresinol-4-O-glucoside Compound 25 was tentativelyidentified as aschantin with a series of fragment ions [M+H-H2O]+ mz 3832 and characteristic fragment ion mz 1672

Compound 26 with [M+H]+mz 4192 was tentatively identi-fied as syringaresinol which fragmented to ion [M+H-H

2O]+

mz 4011 and [M+H-HOCH3]+mz 3871 Compound 27was

tentatively identified as pinoresinol with [M+H]+ mz 3591and a series of fragment ions [M+H-H

2O]+mz 3412 [M+H-

2H2O]+ mz 3232 and characteristic fragment ion mz 1370

Only 3 lignanoids (no 24 26 and 27) had been isolated fromroots of Stellera chamaejasme L [21 26]

The 5 amino acids compounds (28-32) were confirmedby comparison with standard Compound 33 was tentativelyidentified as stelleramacrin B with [M+H]+ mz 6133 and acharacteristic fragment ion [M+H-C

14H24-H2O]+mz 4052

which had been isolated from roots of Stellera chamaejasmeL [20]

33 Exploring Chemical Basis of Toxicity reducing for Pro-cessing The extracted ion chromatogram (XIC) of the 33

components was found to be significantly altered after beingprocessed by vinegar or milk as shown in Figure 3 T-test ofthe 33 components between raw and milkvinegar-processedruixianglangdu was performed As shown in Figure 4 whenthe raw ruixianglangdu was processed by vinegar 25 compo-nents exhibited significant changes including 11 flavonoids(no 1-4 7-9 and 11-14) 4 coumarins (no 15 and 17-19)7 lignanoids (no 20-21 and 23-27) 2 amino acids (no 28and 29) and 1 diterpenoid Eleven of the 25 componentsunderwent a concentration decrease after vinegar processingincluding 8 flavonoids (no 2-4 8 11-14) 1 coumarin (no18) and 2 lignanoids (no 21 and 24) The change ratioof components in raw to vinegar-processed ruixianglangduwas in the range 03sim29 When raw ruixianglangdu wasprocessed by milk 26 components exhibited significantchanges including 13 flavonoids (no 1-8 10-14) 2 coumarins(no 16 and 19) 5 lignanoids (no 21 22 24 25 and 27) 5amino acids (no 28-30) and 1 diterpenoid Sixteen of the26 components exhibited decreasing tendency including 11flavonoids (no 2-6 7 10-14) 2 coumarins (no 16 and 19)and 3 lignanoids (no 21 22 and 24) It is worth notingthat 10 components underwent a concentration decrease afterprocessing with bothmilk and vinegar including 8 flavonoids(chamaejasmine-7-O-glucoside epiafzelechin neochamae-jasmin A-7-O-glucopyranoside chamaechromone chamae-jasmine isochamaejasmin 7-methoxylneochaejasminA andchamaejasmenin A) and 2 lignanoids Decreased flavonoidcould be due to the thermic treatment performed during theraw ruixianglangdu extraction For example total flavonoidcontent of sea buckthorn extract reduction of 9004 wasobserved as a result of heating at 100∘C after 25 min [27]The total and five flavonoids were also significantly decreasedafter fresh Cara Cara juice heating at 90∘C for 120 min [28]Quercetinwas also unstable in aqueous solution and aqueousstability of flavonol was associated with B-ring substitution[29]

Total flavonoids obtained from Stellera chamaejasmehave showed strong toxicity to mice and rabbit [30]Chamaechromone (no 8) and isochamaejasmin (no 12)were major components in ruixianglangdu with cytotox-icity to canine kidney MDCK cells [31 32] Coumarindaphnoretin (no 19) was found to be a cytotoxic con-stituent of Dirca occidentalis (Thymelaeaceae) [33] Basedon these changing components and literature for the pro-cessed ruixianglangdu flavonoids especially biflavononescould be the important chemical basis of toxicity reduc-tion In addition some coumarins and lignanoids thatdecreased after processing could also play a role in reducingtoxicity

Compared with milk-processed ruixianglangdu groupsthe 10 components involving 8 flavonoids had a lowerlevel in the vinegar-processed ruixianglangdu group It wasinferred that processing method withmilk could be better fortoxicity reduction of ruixianglangdu According to literaturethe mortality of zebrafish embryo was higher after treat-ing with vinegar-processed ruixianglangdu than milk-proc-essed ruixianglangdu [34] which was consistent with ourinference

International Journal of Analytical Chemistry 7

0 4 8 12 16 20 24 28 32Time (min)

Rela

tive i

nten

sity

()

0

1

2

3

4

7

5

68

9

10

11

12 13

1415

16

17

18 1920

21

2223 24

25

26

2728

293031

3233

50

100

4371437170517051

54515451

54315431

27512751

34113411

5551555157115711

1931193132513251

17711771 16111611

35313531 5211521152315231

5191519140114011

3591359114811481

1821182113211321 16611661

61316131

41914191

Figure 3 The extracted ion chromatogram (XIC) of the 33 components found to be significantly altered after being processed by vinegaror milk All the ion pairs are monitored in positive mode except for 17711771 16111611 52115211 52315231 51915191 and 61316131Compound number is consistent with the compound number in Table 1

0

50000000

100000000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0

50000000

100000000

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Peak

Are

a

Compound ID

Coumarins Lignanoids Amino acids Diterpenoids

Flavonoids Coumarins

Raw ruixianglangdu Vinegar-processed ruixianglangdu Milk-processed ruixianglangdu

lowastlowast lowast lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Figure 4 Peak area of 33 compounds changed in processed ruixianglangdu Compound ID is consistent with the number in Table 1Compounds with statistical difference between raw and milkvinegar-processed ruixianglangdu were marked with asterisk (lowast)

8 International Journal of Analytical Chemistry

4 Conclusions

With the help of MIM-based metabolomics this studyillustrated the chemical basis of toxicity reducing for pro-cessed ruixianglangdu Flavonoids especially biflavononescould be the important chemical basis of toxicity reducingIn addition some coumarins and lignanoids that decreasedafter processing could also play a role in reducing toxicityCompared with vinegar processing processing method withmilk could be better for toxicity reduction of ruixianglangduIt is believed that MIM-based metabolomics method wasvaluable for exploring chemical basis of toxicity reductionfor processed TCM The data is critical to further study thetoxicity reduction mechanism of processed ruixianglangdu

Data Availability

The data supporting the findings of this study are availablewithin the article Rawdata and additional information of thisstudy are available from the corresponding author on request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Wei Yang and Xiaoli Ma contributed equally to this work

Acknowledgments

The study has been financially supported by Natural Sci-ence Foundation of Beijing (7174325) National Science andTechnology Major Project (2015ZX09501004-003-005) andNational Natural Science Foundation Youth Science Founda-tion (81803722)

References

[1] Z Z Zhao Z T Liang K Chan et al ldquoA unique issue in thestandardization of Chinese materia medica processingrdquo PlantaMedica vol 76 no 17 pp 1975ndash1986 2010

[2] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y Chaildquoharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

[3] G Yang andD Chen ldquoBiflavanones flavonoids and coumarinsfrom the roots of Stellera chamaejasme and their antiviral effecton hepatitis B virusrdquo Chemistry amp Biodiversity vol 5 no 7 pp1419ndash1424 2008

[4] M Kim H J Lee A Randy J H Yun S Oh and CW Nho ldquoStellera chamaejasme and its constituents inducecutaneous wound healing and anti-inflammatory activitiesrdquoScientific Reports vol 7 Article ID 42490 2017

[5] X Liu Q Yang G Zhang et al ldquoAnti-tumor pharmacologicalevaluation of extracts from stellera chamaejasme L basedon hollow fiber assayrdquo BMC Complementary and AlternativeMedicine vol 14 p 116 2014

[6] Y Wang J Li M Han W Wang and Y Li ldquoPrevention andtreatment effect of total flavonoids in Stellera chamaejasme Lon nonalcoholic fatty liver in ratsrdquo Lipids in Health and Diseasevol 14 p 85 2015

[7] T Bai and D J L H Wang ldquoProcessing difference betweenmongolian medicine and traditional chinese medicinerdquo WorldLatest Medicine Information vol 18 p 182 2018

[8] P E C o M O Health Chinese Pharmacopoeia PeoplersquosMedical PublishingHouse Beijing China Edition 1977 edition1979

[9] Z F Jiang X L Ma B Han S Y Cao and Y Y Gen ldquoInitialstudy on effection of different processing methods on theStellera chamaejasme chemical constituentsrdquoMedical Researchand Education vol 32 pp 1ndash6 2015

[10] X W Ji M L Hao X Yang and Y H Wang ldquoRP-HPLCfingerprint of milk Stellera chamaejasmerdquo Zhong Yao Cai vol35 pp 1754ndash1757 2012

[11] W Yang M Ye M Liu et al ldquoA practical strategy for thecharacterization of coumarins in Radix Glehniae by liquidchromatography coupled with triple quadrupole-linear ion trapmass spectrometryrdquo Journal of Chromatography A vol 1217 no27 pp 4587ndash4600 2010

[12] M Yao LMaWGHumphreys andM Zhu ldquoRapid screeningand characterization of drug metabolites using a multiple ionmonitoring-dependentMSMS acquisitionmethod on a hybridtriple quadrupole-linear ion trapmass spectrometerrdquo Journal ofMass Spectrometry vol 43 no 10 pp 1364ndash1375 2008

[13] P Luo W Dai P Yin et al ldquoMultiple reaction monitoring-ion pair finder a systematic approach to transform nontar-geted mode to pseudotargeted mode for metabolomics studybased on liquid chromatographyndashmass spectrometryrdquo Analyt-ical Chemistry vol 87 no 10 pp 5050ndash5055 2015

[14] W Chen L Gong Z Guo et al ldquoA novel integrated methodfor large-scale detection identification and quantification ofwidely targeted metabolites Application in the study of ricemetabolomicsrdquo Molecular Plant vol 6 no 6 pp 1769ndash17802013

[15] L Sheng J Hu B Wang H Chen and Y Li ldquoDeterminationof bicyclol in dog plasma using liquid chromatographyndashtandemmass spectrometryrdquo Journal of Chromatography B vol 878 no23 pp 2106ndash2110 2010

[16] DV Rossetti CMartelli R Longhi F IavaroneMCastagnolaand C Desiderio ldquoQuantitative analysis of thymosin beta4 inwhole saliva by capillary electrophoresis-mass spectrometryusing multiple ions monitoring (CE-MIM-MS)rdquo Electrophore-sis vol 34 no 18 pp 2674ndash2682 2013

[17] C Jin R G Michetich and M Daneshtalab ldquoFlavonoids fromStellera chamaejasmerdquo Phytochemistry vol 50 no 3 pp 505ndash508 1998

[18] W Chen X Luo Z Wang Y Zhang L Liu and H WangldquoA new biflavone glucoside from the roots of Stellera chamae-jasmerdquo Chinese Journal of Natural Medicines vol 13 no 7 pp550ndash553 2015

[19] Z Yan H Guo J Yang et al ldquoPhytotoxic flavonoids from rootsof Stellera chamaejasme L (Thymelaeaceae)rdquo Phytochemistryvol 106 pp 61ndash68 2014

[20] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y ChaildquoCharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

International Journal of Analytical Chemistry 9

[21] Z Jiang T Tanaka T Sakamoto I Kouno J Duan and RZhou ldquoBiflavanones diterpenes and coumarins from the rootsof stellera chamaejasme lrdquoChemical amp Pharmaceutical Bulletinvol 50 no 1 pp 137ndash139

[22] Y Li C Li L Lv et al ldquoA UPLC-MSMSmethod for simultane-ous determination of five flavonoids from Stellera chamaejasmeL in rat plasma and its application to a pharmacokinetic studyrdquoBiomedical Chromatography vol 32 no 6 Article ID e41892018

[23] G Liu H Tatematsu M Kurokawa M Niwa and Y HirataldquoNovel C-3C-31015840rsquo-biflavanones from Stellera chamaejasme LrdquoChemical amp Pharmaceutical Bulletin vol 32 pp 362ndash365 1984

[24] X Li K Rahman J Zhu andH Zhang ldquoChemical Constituentsand Pharmacological Activities of Stellera chamaejasmerdquo Cur-rent Pharmaceutical Design vol 24 no 24 pp 2825ndash2838 2018

[25] X Su R Lin S Wong S Tsui and S Kwan ldquoIdentificationand characterisationof the Chinese herb Langdu by LC-MSMSanalysisrdquo Phytochemical Analysis vol 14 no 1 pp 40ndash47 2003

[26] L Qiao L Yang D Zhang J Zou and J Dai ldquoStudies on chem-ical constitutes from callus cultures of Stellera chamaejasmerdquoZhongguo Zhongyao Zazhi vol 36 no 24 pp 3457ndash3462 2011httpswwwncbinlmnihgovpubmed22368856

[27] F M Ursache I O Ghinea M Turturica I Aprodu GRapeanu andN Stanciuc ldquoPhytochemicals content and antiox-idant properties of sea buckthorn (Hippophae rhamnoides L)as affected by heat treatment - Quantitative spectroscopic andkinetic approachesrdquo FoodChemistry vol 233 pp 442ndash449 2017

[28] Q Lu Y Peng C Zhu and S Pan ldquoEffect of thermal treatmenton carotenoids flavonoids and ascorbic acid in juice of orangecv Cara Carardquo Food Chemistry vol 265 pp 39ndash48 2018

[29] S Maini H L Hodgson and E S Krol ldquoTheUVA and aqueousstability of flavonoids is dependent on b-ring substitutionrdquoJournal of Agricultural and Food Chemistry vol 60 no 28 pp6966ndash6976 2012

[30] Z Yan L Zeng H Jin and B Qin ldquoPotential ecological rolesof flavonoids from Stellera chamaejasmerdquo Plant Signaling andBehavior vol 10 Article ID e1001225 2015

[31] Y Lou J Zheng B Wang X Zhang X Zhang and SZeng ldquoMetabolites characterization of chamaechromone invivo and in vitro by using ultra-performance liquid chro-matographyXevo G2 quadrupole time-of-flight tandem massspectrometryrdquo Journal of Ethnopharmacology vol 151 no 1 pp242ndash252 2014

[32] Y H Wang X Yang L J Sun and L M Yang ldquoStudies onthe cytotoxicity of Stellera chamaejasme L in vitrordquo ChineseMedicinal Biotechnology vol 7 pp 9ndash13 2012

[33] M M Badawi S S Handa A D Kinghorn G A Cordelland N R Farnsworth ldquoPlant anticancer agents XXVIIAntileukemic and cytotoxic constituents of Dirca occiden-talis (thymelaeaceae)rdquo Journal of Pharmaceutical Sciences vol72 no 11 pp 1285ndash1287 1983 httpswwwncbinlmnihgovpubmed6644588

[34] J F Yang W J Dong J C Zong et al ldquoToxicity detectionof Stella Chamaejasme in zebrafish embryo with differentextraction methodsrdquo Journal of Inner Mongolia University forNationalities vol 31 pp 236ndash239 2016

6 International Journal of Analytical Chemistry

mz 1530 which was similar to compounds 9-12 whichwas identified as 7-methoxylneochaejasmin A Compound14 had a series fragment ions mz 4451 4192 3551 and3372 The peak at mz 4192 corresponded to the RDAcleavage Compound 14 was tentatively identified as chamae-jasmenin A All the 14 flavonoids except for compound 7had been isolated from roots of Stellera chamaejasme L[17ndash23]

Coumarins compound 15 exhibited [M+H]+ mz 1931Based on major fragment ions [M+H-CH

3OH]+ mz 1610

[M+H-CH3OH-CO]+mz 1330 and [M+H-CH

3OH-2CO]+

mz 1050 this compound was tentatively assigned to scopo-letin Compound 16 was tentatively identified as umbellifer-one glucoside with a series of fragment ions [M+H-Glc]+mz 1630 and [M+H-Glc-CO

2]+ mz 1191 Compound 17

was identified as daphnetin with [M+H]+ mz 1770 anda series of fragment ions [M-H-CO]minus mz 1490 [M-H-CO2]minus mz 1330 and [M-H-CO-CO

2]minus mz 1051 The MS2

spectrum of compound 18 with [M-H]minus mz 1610 gave thecharacteristic fragment ions of mz 1331 1170 and 1051which corresponded to [M-H-CO]minus [M-H-CO

2]minus and [M-

H-2CO]minus It was tentatively identified as umbelliferoneThe MSMS mass spectrum of compound 19 [M+H]+ mz3531 presented the specific fragments of [M+H-CH

3]+ mz

3382 It was confirmed as daphnoretin by comparison withstandard All the 5 coumarins had been isolated from roots ofStellera chamaejasme L [21 24 25]

Lignanoids base peak of compounds 20 and 22 was mz3592 Compounds 20 and 22 were tentatively identifiedas isolariciresinol-4rsquo-O-glucoside and isolariciresinol-9-O-glucoside based on their polarity Two compounds (no 21and 23) had the same [M-H]minus mz 5232 and characteristicfragment [M-H-Glc]+ mz 3613 Based on their differentpolarity compounds 21 and 23 were tentatively identified assecoisolariciresinol-4-O-glucoside and secoisolariciresinol-9-O-glucoside respectively based on their polarity Basedon the characteristic fragment [M-H-Glc]minus mz 3572 com-pound 24 with [M-H]minus mz 5192 was tentatively identifiedas pinoresinol-4-O-glucoside Compound 25 was tentativelyidentified as aschantin with a series of fragment ions [M+H-H2O]+ mz 3832 and characteristic fragment ion mz 1672

Compound 26 with [M+H]+mz 4192 was tentatively identi-fied as syringaresinol which fragmented to ion [M+H-H

2O]+

mz 4011 and [M+H-HOCH3]+mz 3871 Compound 27was

tentatively identified as pinoresinol with [M+H]+ mz 3591and a series of fragment ions [M+H-H

2O]+mz 3412 [M+H-

2H2O]+ mz 3232 and characteristic fragment ion mz 1370

Only 3 lignanoids (no 24 26 and 27) had been isolated fromroots of Stellera chamaejasme L [21 26]

The 5 amino acids compounds (28-32) were confirmedby comparison with standard Compound 33 was tentativelyidentified as stelleramacrin B with [M+H]+ mz 6133 and acharacteristic fragment ion [M+H-C

14H24-H2O]+mz 4052

which had been isolated from roots of Stellera chamaejasmeL [20]

33 Exploring Chemical Basis of Toxicity reducing for Pro-cessing The extracted ion chromatogram (XIC) of the 33

components was found to be significantly altered after beingprocessed by vinegar or milk as shown in Figure 3 T-test ofthe 33 components between raw and milkvinegar-processedruixianglangdu was performed As shown in Figure 4 whenthe raw ruixianglangdu was processed by vinegar 25 compo-nents exhibited significant changes including 11 flavonoids(no 1-4 7-9 and 11-14) 4 coumarins (no 15 and 17-19)7 lignanoids (no 20-21 and 23-27) 2 amino acids (no 28and 29) and 1 diterpenoid Eleven of the 25 componentsunderwent a concentration decrease after vinegar processingincluding 8 flavonoids (no 2-4 8 11-14) 1 coumarin (no18) and 2 lignanoids (no 21 and 24) The change ratioof components in raw to vinegar-processed ruixianglangduwas in the range 03sim29 When raw ruixianglangdu wasprocessed by milk 26 components exhibited significantchanges including 13 flavonoids (no 1-8 10-14) 2 coumarins(no 16 and 19) 5 lignanoids (no 21 22 24 25 and 27) 5amino acids (no 28-30) and 1 diterpenoid Sixteen of the26 components exhibited decreasing tendency including 11flavonoids (no 2-6 7 10-14) 2 coumarins (no 16 and 19)and 3 lignanoids (no 21 22 and 24) It is worth notingthat 10 components underwent a concentration decrease afterprocessing with bothmilk and vinegar including 8 flavonoids(chamaejasmine-7-O-glucoside epiafzelechin neochamae-jasmin A-7-O-glucopyranoside chamaechromone chamae-jasmine isochamaejasmin 7-methoxylneochaejasminA andchamaejasmenin A) and 2 lignanoids Decreased flavonoidcould be due to the thermic treatment performed during theraw ruixianglangdu extraction For example total flavonoidcontent of sea buckthorn extract reduction of 9004 wasobserved as a result of heating at 100∘C after 25 min [27]The total and five flavonoids were also significantly decreasedafter fresh Cara Cara juice heating at 90∘C for 120 min [28]Quercetinwas also unstable in aqueous solution and aqueousstability of flavonol was associated with B-ring substitution[29]

Total flavonoids obtained from Stellera chamaejasmehave showed strong toxicity to mice and rabbit [30]Chamaechromone (no 8) and isochamaejasmin (no 12)were major components in ruixianglangdu with cytotox-icity to canine kidney MDCK cells [31 32] Coumarindaphnoretin (no 19) was found to be a cytotoxic con-stituent of Dirca occidentalis (Thymelaeaceae) [33] Basedon these changing components and literature for the pro-cessed ruixianglangdu flavonoids especially biflavononescould be the important chemical basis of toxicity reduc-tion In addition some coumarins and lignanoids thatdecreased after processing could also play a role in reducingtoxicity

Compared with milk-processed ruixianglangdu groupsthe 10 components involving 8 flavonoids had a lowerlevel in the vinegar-processed ruixianglangdu group It wasinferred that processing method withmilk could be better fortoxicity reduction of ruixianglangdu According to literaturethe mortality of zebrafish embryo was higher after treat-ing with vinegar-processed ruixianglangdu than milk-proc-essed ruixianglangdu [34] which was consistent with ourinference

International Journal of Analytical Chemistry 7

0 4 8 12 16 20 24 28 32Time (min)

Rela

tive i

nten

sity

()

0

1

2

3

4

7

5

68

9

10

11

12 13

1415

16

17

18 1920

21

2223 24

25

26

2728

293031

3233

50

100

4371437170517051

54515451

54315431

27512751

34113411

5551555157115711

1931193132513251

17711771 16111611

35313531 5211521152315231

5191519140114011

3591359114811481

1821182113211321 16611661

61316131

41914191

Figure 3 The extracted ion chromatogram (XIC) of the 33 components found to be significantly altered after being processed by vinegaror milk All the ion pairs are monitored in positive mode except for 17711771 16111611 52115211 52315231 51915191 and 61316131Compound number is consistent with the compound number in Table 1

0

50000000

100000000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0

50000000

100000000

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Peak

Are

a

Compound ID

Coumarins Lignanoids Amino acids Diterpenoids

Flavonoids Coumarins

Raw ruixianglangdu Vinegar-processed ruixianglangdu Milk-processed ruixianglangdu

lowastlowast lowast lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Figure 4 Peak area of 33 compounds changed in processed ruixianglangdu Compound ID is consistent with the number in Table 1Compounds with statistical difference between raw and milkvinegar-processed ruixianglangdu were marked with asterisk (lowast)

8 International Journal of Analytical Chemistry

4 Conclusions

With the help of MIM-based metabolomics this studyillustrated the chemical basis of toxicity reducing for pro-cessed ruixianglangdu Flavonoids especially biflavononescould be the important chemical basis of toxicity reducingIn addition some coumarins and lignanoids that decreasedafter processing could also play a role in reducing toxicityCompared with vinegar processing processing method withmilk could be better for toxicity reduction of ruixianglangduIt is believed that MIM-based metabolomics method wasvaluable for exploring chemical basis of toxicity reductionfor processed TCM The data is critical to further study thetoxicity reduction mechanism of processed ruixianglangdu

Data Availability

The data supporting the findings of this study are availablewithin the article Rawdata and additional information of thisstudy are available from the corresponding author on request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Wei Yang and Xiaoli Ma contributed equally to this work

Acknowledgments

The study has been financially supported by Natural Sci-ence Foundation of Beijing (7174325) National Science andTechnology Major Project (2015ZX09501004-003-005) andNational Natural Science Foundation Youth Science Founda-tion (81803722)

References

[1] Z Z Zhao Z T Liang K Chan et al ldquoA unique issue in thestandardization of Chinese materia medica processingrdquo PlantaMedica vol 76 no 17 pp 1975ndash1986 2010

[2] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y Chaildquoharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

[3] G Yang andD Chen ldquoBiflavanones flavonoids and coumarinsfrom the roots of Stellera chamaejasme and their antiviral effecton hepatitis B virusrdquo Chemistry amp Biodiversity vol 5 no 7 pp1419ndash1424 2008

[4] M Kim H J Lee A Randy J H Yun S Oh and CW Nho ldquoStellera chamaejasme and its constituents inducecutaneous wound healing and anti-inflammatory activitiesrdquoScientific Reports vol 7 Article ID 42490 2017

[5] X Liu Q Yang G Zhang et al ldquoAnti-tumor pharmacologicalevaluation of extracts from stellera chamaejasme L basedon hollow fiber assayrdquo BMC Complementary and AlternativeMedicine vol 14 p 116 2014

[6] Y Wang J Li M Han W Wang and Y Li ldquoPrevention andtreatment effect of total flavonoids in Stellera chamaejasme Lon nonalcoholic fatty liver in ratsrdquo Lipids in Health and Diseasevol 14 p 85 2015

[7] T Bai and D J L H Wang ldquoProcessing difference betweenmongolian medicine and traditional chinese medicinerdquo WorldLatest Medicine Information vol 18 p 182 2018

[8] P E C o M O Health Chinese Pharmacopoeia PeoplersquosMedical PublishingHouse Beijing China Edition 1977 edition1979

[9] Z F Jiang X L Ma B Han S Y Cao and Y Y Gen ldquoInitialstudy on effection of different processing methods on theStellera chamaejasme chemical constituentsrdquoMedical Researchand Education vol 32 pp 1ndash6 2015

[10] X W Ji M L Hao X Yang and Y H Wang ldquoRP-HPLCfingerprint of milk Stellera chamaejasmerdquo Zhong Yao Cai vol35 pp 1754ndash1757 2012

[11] W Yang M Ye M Liu et al ldquoA practical strategy for thecharacterization of coumarins in Radix Glehniae by liquidchromatography coupled with triple quadrupole-linear ion trapmass spectrometryrdquo Journal of Chromatography A vol 1217 no27 pp 4587ndash4600 2010

[12] M Yao LMaWGHumphreys andM Zhu ldquoRapid screeningand characterization of drug metabolites using a multiple ionmonitoring-dependentMSMS acquisitionmethod on a hybridtriple quadrupole-linear ion trapmass spectrometerrdquo Journal ofMass Spectrometry vol 43 no 10 pp 1364ndash1375 2008

[13] P Luo W Dai P Yin et al ldquoMultiple reaction monitoring-ion pair finder a systematic approach to transform nontar-geted mode to pseudotargeted mode for metabolomics studybased on liquid chromatographyndashmass spectrometryrdquo Analyt-ical Chemistry vol 87 no 10 pp 5050ndash5055 2015

[14] W Chen L Gong Z Guo et al ldquoA novel integrated methodfor large-scale detection identification and quantification ofwidely targeted metabolites Application in the study of ricemetabolomicsrdquo Molecular Plant vol 6 no 6 pp 1769ndash17802013

[15] L Sheng J Hu B Wang H Chen and Y Li ldquoDeterminationof bicyclol in dog plasma using liquid chromatographyndashtandemmass spectrometryrdquo Journal of Chromatography B vol 878 no23 pp 2106ndash2110 2010

[16] DV Rossetti CMartelli R Longhi F IavaroneMCastagnolaand C Desiderio ldquoQuantitative analysis of thymosin beta4 inwhole saliva by capillary electrophoresis-mass spectrometryusing multiple ions monitoring (CE-MIM-MS)rdquo Electrophore-sis vol 34 no 18 pp 2674ndash2682 2013

[17] C Jin R G Michetich and M Daneshtalab ldquoFlavonoids fromStellera chamaejasmerdquo Phytochemistry vol 50 no 3 pp 505ndash508 1998

[18] W Chen X Luo Z Wang Y Zhang L Liu and H WangldquoA new biflavone glucoside from the roots of Stellera chamae-jasmerdquo Chinese Journal of Natural Medicines vol 13 no 7 pp550ndash553 2015

[19] Z Yan H Guo J Yang et al ldquoPhytotoxic flavonoids from rootsof Stellera chamaejasme L (Thymelaeaceae)rdquo Phytochemistryvol 106 pp 61ndash68 2014

[20] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y ChaildquoCharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

International Journal of Analytical Chemistry 9

[21] Z Jiang T Tanaka T Sakamoto I Kouno J Duan and RZhou ldquoBiflavanones diterpenes and coumarins from the rootsof stellera chamaejasme lrdquoChemical amp Pharmaceutical Bulletinvol 50 no 1 pp 137ndash139

[22] Y Li C Li L Lv et al ldquoA UPLC-MSMSmethod for simultane-ous determination of five flavonoids from Stellera chamaejasmeL in rat plasma and its application to a pharmacokinetic studyrdquoBiomedical Chromatography vol 32 no 6 Article ID e41892018

[23] G Liu H Tatematsu M Kurokawa M Niwa and Y HirataldquoNovel C-3C-31015840rsquo-biflavanones from Stellera chamaejasme LrdquoChemical amp Pharmaceutical Bulletin vol 32 pp 362ndash365 1984

[24] X Li K Rahman J Zhu andH Zhang ldquoChemical Constituentsand Pharmacological Activities of Stellera chamaejasmerdquo Cur-rent Pharmaceutical Design vol 24 no 24 pp 2825ndash2838 2018

[25] X Su R Lin S Wong S Tsui and S Kwan ldquoIdentificationand characterisationof the Chinese herb Langdu by LC-MSMSanalysisrdquo Phytochemical Analysis vol 14 no 1 pp 40ndash47 2003

[26] L Qiao L Yang D Zhang J Zou and J Dai ldquoStudies on chem-ical constitutes from callus cultures of Stellera chamaejasmerdquoZhongguo Zhongyao Zazhi vol 36 no 24 pp 3457ndash3462 2011httpswwwncbinlmnihgovpubmed22368856

[27] F M Ursache I O Ghinea M Turturica I Aprodu GRapeanu andN Stanciuc ldquoPhytochemicals content and antiox-idant properties of sea buckthorn (Hippophae rhamnoides L)as affected by heat treatment - Quantitative spectroscopic andkinetic approachesrdquo FoodChemistry vol 233 pp 442ndash449 2017

[28] Q Lu Y Peng C Zhu and S Pan ldquoEffect of thermal treatmenton carotenoids flavonoids and ascorbic acid in juice of orangecv Cara Carardquo Food Chemistry vol 265 pp 39ndash48 2018

[29] S Maini H L Hodgson and E S Krol ldquoTheUVA and aqueousstability of flavonoids is dependent on b-ring substitutionrdquoJournal of Agricultural and Food Chemistry vol 60 no 28 pp6966ndash6976 2012

[30] Z Yan L Zeng H Jin and B Qin ldquoPotential ecological rolesof flavonoids from Stellera chamaejasmerdquo Plant Signaling andBehavior vol 10 Article ID e1001225 2015

[31] Y Lou J Zheng B Wang X Zhang X Zhang and SZeng ldquoMetabolites characterization of chamaechromone invivo and in vitro by using ultra-performance liquid chro-matographyXevo G2 quadrupole time-of-flight tandem massspectrometryrdquo Journal of Ethnopharmacology vol 151 no 1 pp242ndash252 2014

[32] Y H Wang X Yang L J Sun and L M Yang ldquoStudies onthe cytotoxicity of Stellera chamaejasme L in vitrordquo ChineseMedicinal Biotechnology vol 7 pp 9ndash13 2012

[33] M M Badawi S S Handa A D Kinghorn G A Cordelland N R Farnsworth ldquoPlant anticancer agents XXVIIAntileukemic and cytotoxic constituents of Dirca occiden-talis (thymelaeaceae)rdquo Journal of Pharmaceutical Sciences vol72 no 11 pp 1285ndash1287 1983 httpswwwncbinlmnihgovpubmed6644588

[34] J F Yang W J Dong J C Zong et al ldquoToxicity detectionof Stella Chamaejasme in zebrafish embryo with differentextraction methodsrdquo Journal of Inner Mongolia University forNationalities vol 31 pp 236ndash239 2016

International Journal of Analytical Chemistry 7

0 4 8 12 16 20 24 28 32Time (min)

Rela

tive i

nten

sity

()

0

1

2

3

4

7

5

68

9

10

11

12 13

1415

16

17

18 1920

21

2223 24

25

26

2728

293031

3233

50

100

4371437170517051

54515451

54315431

27512751

34113411

5551555157115711

1931193132513251

17711771 16111611

35313531 5211521152315231

5191519140114011

3591359114811481

1821182113211321 16611661

61316131

41914191

Figure 3 The extracted ion chromatogram (XIC) of the 33 components found to be significantly altered after being processed by vinegaror milk All the ion pairs are monitored in positive mode except for 17711771 16111611 52115211 52315231 51915191 and 61316131Compound number is consistent with the compound number in Table 1

0

50000000

100000000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0

50000000

100000000

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Peak

Are

a

Compound ID

Coumarins Lignanoids Amino acids Diterpenoids

Flavonoids Coumarins

Raw ruixianglangdu Vinegar-processed ruixianglangdu Milk-processed ruixianglangdu

lowastlowast lowast lowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

Figure 4 Peak area of 33 compounds changed in processed ruixianglangdu Compound ID is consistent with the number in Table 1Compounds with statistical difference between raw and milkvinegar-processed ruixianglangdu were marked with asterisk (lowast)

8 International Journal of Analytical Chemistry

4 Conclusions

With the help of MIM-based metabolomics this studyillustrated the chemical basis of toxicity reducing for pro-cessed ruixianglangdu Flavonoids especially biflavononescould be the important chemical basis of toxicity reducingIn addition some coumarins and lignanoids that decreasedafter processing could also play a role in reducing toxicityCompared with vinegar processing processing method withmilk could be better for toxicity reduction of ruixianglangduIt is believed that MIM-based metabolomics method wasvaluable for exploring chemical basis of toxicity reductionfor processed TCM The data is critical to further study thetoxicity reduction mechanism of processed ruixianglangdu

Data Availability

The data supporting the findings of this study are availablewithin the article Rawdata and additional information of thisstudy are available from the corresponding author on request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Wei Yang and Xiaoli Ma contributed equally to this work

Acknowledgments

The study has been financially supported by Natural Sci-ence Foundation of Beijing (7174325) National Science andTechnology Major Project (2015ZX09501004-003-005) andNational Natural Science Foundation Youth Science Founda-tion (81803722)

References

[1] Z Z Zhao Z T Liang K Chan et al ldquoA unique issue in thestandardization of Chinese materia medica processingrdquo PlantaMedica vol 76 no 17 pp 1975ndash1986 2010

[2] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y Chaildquoharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

[3] G Yang andD Chen ldquoBiflavanones flavonoids and coumarinsfrom the roots of Stellera chamaejasme and their antiviral effecton hepatitis B virusrdquo Chemistry amp Biodiversity vol 5 no 7 pp1419ndash1424 2008

[4] M Kim H J Lee A Randy J H Yun S Oh and CW Nho ldquoStellera chamaejasme and its constituents inducecutaneous wound healing and anti-inflammatory activitiesrdquoScientific Reports vol 7 Article ID 42490 2017

[5] X Liu Q Yang G Zhang et al ldquoAnti-tumor pharmacologicalevaluation of extracts from stellera chamaejasme L basedon hollow fiber assayrdquo BMC Complementary and AlternativeMedicine vol 14 p 116 2014

[6] Y Wang J Li M Han W Wang and Y Li ldquoPrevention andtreatment effect of total flavonoids in Stellera chamaejasme Lon nonalcoholic fatty liver in ratsrdquo Lipids in Health and Diseasevol 14 p 85 2015

[7] T Bai and D J L H Wang ldquoProcessing difference betweenmongolian medicine and traditional chinese medicinerdquo WorldLatest Medicine Information vol 18 p 182 2018

[8] P E C o M O Health Chinese Pharmacopoeia PeoplersquosMedical PublishingHouse Beijing China Edition 1977 edition1979

[9] Z F Jiang X L Ma B Han S Y Cao and Y Y Gen ldquoInitialstudy on effection of different processing methods on theStellera chamaejasme chemical constituentsrdquoMedical Researchand Education vol 32 pp 1ndash6 2015

[10] X W Ji M L Hao X Yang and Y H Wang ldquoRP-HPLCfingerprint of milk Stellera chamaejasmerdquo Zhong Yao Cai vol35 pp 1754ndash1757 2012

[11] W Yang M Ye M Liu et al ldquoA practical strategy for thecharacterization of coumarins in Radix Glehniae by liquidchromatography coupled with triple quadrupole-linear ion trapmass spectrometryrdquo Journal of Chromatography A vol 1217 no27 pp 4587ndash4600 2010

[12] M Yao LMaWGHumphreys andM Zhu ldquoRapid screeningand characterization of drug metabolites using a multiple ionmonitoring-dependentMSMS acquisitionmethod on a hybridtriple quadrupole-linear ion trapmass spectrometerrdquo Journal ofMass Spectrometry vol 43 no 10 pp 1364ndash1375 2008

[13] P Luo W Dai P Yin et al ldquoMultiple reaction monitoring-ion pair finder a systematic approach to transform nontar-geted mode to pseudotargeted mode for metabolomics studybased on liquid chromatographyndashmass spectrometryrdquo Analyt-ical Chemistry vol 87 no 10 pp 5050ndash5055 2015

[14] W Chen L Gong Z Guo et al ldquoA novel integrated methodfor large-scale detection identification and quantification ofwidely targeted metabolites Application in the study of ricemetabolomicsrdquo Molecular Plant vol 6 no 6 pp 1769ndash17802013

[15] L Sheng J Hu B Wang H Chen and Y Li ldquoDeterminationof bicyclol in dog plasma using liquid chromatographyndashtandemmass spectrometryrdquo Journal of Chromatography B vol 878 no23 pp 2106ndash2110 2010

[16] DV Rossetti CMartelli R Longhi F IavaroneMCastagnolaand C Desiderio ldquoQuantitative analysis of thymosin beta4 inwhole saliva by capillary electrophoresis-mass spectrometryusing multiple ions monitoring (CE-MIM-MS)rdquo Electrophore-sis vol 34 no 18 pp 2674ndash2682 2013

[17] C Jin R G Michetich and M Daneshtalab ldquoFlavonoids fromStellera chamaejasmerdquo Phytochemistry vol 50 no 3 pp 505ndash508 1998

[18] W Chen X Luo Z Wang Y Zhang L Liu and H WangldquoA new biflavone glucoside from the roots of Stellera chamae-jasmerdquo Chinese Journal of Natural Medicines vol 13 no 7 pp550ndash553 2015

[19] Z Yan H Guo J Yang et al ldquoPhytotoxic flavonoids from rootsof Stellera chamaejasme L (Thymelaeaceae)rdquo Phytochemistryvol 106 pp 61ndash68 2014

[20] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y ChaildquoCharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

International Journal of Analytical Chemistry 9

[21] Z Jiang T Tanaka T Sakamoto I Kouno J Duan and RZhou ldquoBiflavanones diterpenes and coumarins from the rootsof stellera chamaejasme lrdquoChemical amp Pharmaceutical Bulletinvol 50 no 1 pp 137ndash139

[22] Y Li C Li L Lv et al ldquoA UPLC-MSMSmethod for simultane-ous determination of five flavonoids from Stellera chamaejasmeL in rat plasma and its application to a pharmacokinetic studyrdquoBiomedical Chromatography vol 32 no 6 Article ID e41892018

[23] G Liu H Tatematsu M Kurokawa M Niwa and Y HirataldquoNovel C-3C-31015840rsquo-biflavanones from Stellera chamaejasme LrdquoChemical amp Pharmaceutical Bulletin vol 32 pp 362ndash365 1984

[24] X Li K Rahman J Zhu andH Zhang ldquoChemical Constituentsand Pharmacological Activities of Stellera chamaejasmerdquo Cur-rent Pharmaceutical Design vol 24 no 24 pp 2825ndash2838 2018

[25] X Su R Lin S Wong S Tsui and S Kwan ldquoIdentificationand characterisationof the Chinese herb Langdu by LC-MSMSanalysisrdquo Phytochemical Analysis vol 14 no 1 pp 40ndash47 2003

[26] L Qiao L Yang D Zhang J Zou and J Dai ldquoStudies on chem-ical constitutes from callus cultures of Stellera chamaejasmerdquoZhongguo Zhongyao Zazhi vol 36 no 24 pp 3457ndash3462 2011httpswwwncbinlmnihgovpubmed22368856

[27] F M Ursache I O Ghinea M Turturica I Aprodu GRapeanu andN Stanciuc ldquoPhytochemicals content and antiox-idant properties of sea buckthorn (Hippophae rhamnoides L)as affected by heat treatment - Quantitative spectroscopic andkinetic approachesrdquo FoodChemistry vol 233 pp 442ndash449 2017

[28] Q Lu Y Peng C Zhu and S Pan ldquoEffect of thermal treatmenton carotenoids flavonoids and ascorbic acid in juice of orangecv Cara Carardquo Food Chemistry vol 265 pp 39ndash48 2018

[29] S Maini H L Hodgson and E S Krol ldquoTheUVA and aqueousstability of flavonoids is dependent on b-ring substitutionrdquoJournal of Agricultural and Food Chemistry vol 60 no 28 pp6966ndash6976 2012

[30] Z Yan L Zeng H Jin and B Qin ldquoPotential ecological rolesof flavonoids from Stellera chamaejasmerdquo Plant Signaling andBehavior vol 10 Article ID e1001225 2015

[31] Y Lou J Zheng B Wang X Zhang X Zhang and SZeng ldquoMetabolites characterization of chamaechromone invivo and in vitro by using ultra-performance liquid chro-matographyXevo G2 quadrupole time-of-flight tandem massspectrometryrdquo Journal of Ethnopharmacology vol 151 no 1 pp242ndash252 2014

[32] Y H Wang X Yang L J Sun and L M Yang ldquoStudies onthe cytotoxicity of Stellera chamaejasme L in vitrordquo ChineseMedicinal Biotechnology vol 7 pp 9ndash13 2012

[33] M M Badawi S S Handa A D Kinghorn G A Cordelland N R Farnsworth ldquoPlant anticancer agents XXVIIAntileukemic and cytotoxic constituents of Dirca occiden-talis (thymelaeaceae)rdquo Journal of Pharmaceutical Sciences vol72 no 11 pp 1285ndash1287 1983 httpswwwncbinlmnihgovpubmed6644588

[34] J F Yang W J Dong J C Zong et al ldquoToxicity detectionof Stella Chamaejasme in zebrafish embryo with differentextraction methodsrdquo Journal of Inner Mongolia University forNationalities vol 31 pp 236ndash239 2016

8 International Journal of Analytical Chemistry

4 Conclusions

With the help of MIM-based metabolomics this studyillustrated the chemical basis of toxicity reducing for pro-cessed ruixianglangdu Flavonoids especially biflavononescould be the important chemical basis of toxicity reducingIn addition some coumarins and lignanoids that decreasedafter processing could also play a role in reducing toxicityCompared with vinegar processing processing method withmilk could be better for toxicity reduction of ruixianglangduIt is believed that MIM-based metabolomics method wasvaluable for exploring chemical basis of toxicity reductionfor processed TCM The data is critical to further study thetoxicity reduction mechanism of processed ruixianglangdu

Data Availability

The data supporting the findings of this study are availablewithin the article Rawdata and additional information of thisstudy are available from the corresponding author on request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Wei Yang and Xiaoli Ma contributed equally to this work

Acknowledgments

The study has been financially supported by Natural Sci-ence Foundation of Beijing (7174325) National Science andTechnology Major Project (2015ZX09501004-003-005) andNational Natural Science Foundation Youth Science Founda-tion (81803722)

References

[1] Z Z Zhao Z T Liang K Chan et al ldquoA unique issue in thestandardization of Chinese materia medica processingrdquo PlantaMedica vol 76 no 17 pp 1975ndash1986 2010

[2] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y Chaildquoharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

[3] G Yang andD Chen ldquoBiflavanones flavonoids and coumarinsfrom the roots of Stellera chamaejasme and their antiviral effecton hepatitis B virusrdquo Chemistry amp Biodiversity vol 5 no 7 pp1419ndash1424 2008

[4] M Kim H J Lee A Randy J H Yun S Oh and CW Nho ldquoStellera chamaejasme and its constituents inducecutaneous wound healing and anti-inflammatory activitiesrdquoScientific Reports vol 7 Article ID 42490 2017

[5] X Liu Q Yang G Zhang et al ldquoAnti-tumor pharmacologicalevaluation of extracts from stellera chamaejasme L basedon hollow fiber assayrdquo BMC Complementary and AlternativeMedicine vol 14 p 116 2014

[6] Y Wang J Li M Han W Wang and Y Li ldquoPrevention andtreatment effect of total flavonoids in Stellera chamaejasme Lon nonalcoholic fatty liver in ratsrdquo Lipids in Health and Diseasevol 14 p 85 2015

[7] T Bai and D J L H Wang ldquoProcessing difference betweenmongolian medicine and traditional chinese medicinerdquo WorldLatest Medicine Information vol 18 p 182 2018

[8] P E C o M O Health Chinese Pharmacopoeia PeoplersquosMedical PublishingHouse Beijing China Edition 1977 edition1979

[9] Z F Jiang X L Ma B Han S Y Cao and Y Y Gen ldquoInitialstudy on effection of different processing methods on theStellera chamaejasme chemical constituentsrdquoMedical Researchand Education vol 32 pp 1ndash6 2015

[10] X W Ji M L Hao X Yang and Y H Wang ldquoRP-HPLCfingerprint of milk Stellera chamaejasmerdquo Zhong Yao Cai vol35 pp 1754ndash1757 2012

[11] W Yang M Ye M Liu et al ldquoA practical strategy for thecharacterization of coumarins in Radix Glehniae by liquidchromatography coupled with triple quadrupole-linear ion trapmass spectrometryrdquo Journal of Chromatography A vol 1217 no27 pp 4587ndash4600 2010

[12] M Yao LMaWGHumphreys andM Zhu ldquoRapid screeningand characterization of drug metabolites using a multiple ionmonitoring-dependentMSMS acquisitionmethod on a hybridtriple quadrupole-linear ion trapmass spectrometerrdquo Journal ofMass Spectrometry vol 43 no 10 pp 1364ndash1375 2008

[13] P Luo W Dai P Yin et al ldquoMultiple reaction monitoring-ion pair finder a systematic approach to transform nontar-geted mode to pseudotargeted mode for metabolomics studybased on liquid chromatographyndashmass spectrometryrdquo Analyt-ical Chemistry vol 87 no 10 pp 5050ndash5055 2015

[14] W Chen L Gong Z Guo et al ldquoA novel integrated methodfor large-scale detection identification and quantification ofwidely targeted metabolites Application in the study of ricemetabolomicsrdquo Molecular Plant vol 6 no 6 pp 1769ndash17802013

[15] L Sheng J Hu B Wang H Chen and Y Li ldquoDeterminationof bicyclol in dog plasma using liquid chromatographyndashtandemmass spectrometryrdquo Journal of Chromatography B vol 878 no23 pp 2106ndash2110 2010

[16] DV Rossetti CMartelli R Longhi F IavaroneMCastagnolaand C Desiderio ldquoQuantitative analysis of thymosin beta4 inwhole saliva by capillary electrophoresis-mass spectrometryusing multiple ions monitoring (CE-MIM-MS)rdquo Electrophore-sis vol 34 no 18 pp 2674ndash2682 2013

[17] C Jin R G Michetich and M Daneshtalab ldquoFlavonoids fromStellera chamaejasmerdquo Phytochemistry vol 50 no 3 pp 505ndash508 1998

[18] W Chen X Luo Z Wang Y Zhang L Liu and H WangldquoA new biflavone glucoside from the roots of Stellera chamae-jasmerdquo Chinese Journal of Natural Medicines vol 13 no 7 pp550ndash553 2015

[19] Z Yan H Guo J Yang et al ldquoPhytotoxic flavonoids from rootsof Stellera chamaejasme L (Thymelaeaceae)rdquo Phytochemistryvol 106 pp 61ndash68 2014

[20] L Zhao Z Lou Z Zhu Hai-Zhang G Zhang and Y ChaildquoCharacterization of constituents in Stellera chamaejasme L byrapid-resolution liquid chromatography-diode array detectionand electrospray ionization time-of-flight mass spectrometryrdquoBiomedical Chromatography vol 22 no 1 pp 64ndash72 2008

International Journal of Analytical Chemistry 9

[21] Z Jiang T Tanaka T Sakamoto I Kouno J Duan and RZhou ldquoBiflavanones diterpenes and coumarins from the rootsof stellera chamaejasme lrdquoChemical amp Pharmaceutical Bulletinvol 50 no 1 pp 137ndash139

[22] Y Li C Li L Lv et al ldquoA UPLC-MSMSmethod for simultane-ous determination of five flavonoids from Stellera chamaejasmeL in rat plasma and its application to a pharmacokinetic studyrdquoBiomedical Chromatography vol 32 no 6 Article ID e41892018

[23] G Liu H Tatematsu M Kurokawa M Niwa and Y HirataldquoNovel C-3C-31015840rsquo-biflavanones from Stellera chamaejasme LrdquoChemical amp Pharmaceutical Bulletin vol 32 pp 362ndash365 1984

[24] X Li K Rahman J Zhu andH Zhang ldquoChemical Constituentsand Pharmacological Activities of Stellera chamaejasmerdquo Cur-rent Pharmaceutical Design vol 24 no 24 pp 2825ndash2838 2018

[25] X Su R Lin S Wong S Tsui and S Kwan ldquoIdentificationand characterisationof the Chinese herb Langdu by LC-MSMSanalysisrdquo Phytochemical Analysis vol 14 no 1 pp 40ndash47 2003

[26] L Qiao L Yang D Zhang J Zou and J Dai ldquoStudies on chem-ical constitutes from callus cultures of Stellera chamaejasmerdquoZhongguo Zhongyao Zazhi vol 36 no 24 pp 3457ndash3462 2011httpswwwncbinlmnihgovpubmed22368856

[27] F M Ursache I O Ghinea M Turturica I Aprodu GRapeanu andN Stanciuc ldquoPhytochemicals content and antiox-idant properties of sea buckthorn (Hippophae rhamnoides L)as affected by heat treatment - Quantitative spectroscopic andkinetic approachesrdquo FoodChemistry vol 233 pp 442ndash449 2017

[28] Q Lu Y Peng C Zhu and S Pan ldquoEffect of thermal treatmenton carotenoids flavonoids and ascorbic acid in juice of orangecv Cara Carardquo Food Chemistry vol 265 pp 39ndash48 2018

[29] S Maini H L Hodgson and E S Krol ldquoTheUVA and aqueousstability of flavonoids is dependent on b-ring substitutionrdquoJournal of Agricultural and Food Chemistry vol 60 no 28 pp6966ndash6976 2012

[30] Z Yan L Zeng H Jin and B Qin ldquoPotential ecological rolesof flavonoids from Stellera chamaejasmerdquo Plant Signaling andBehavior vol 10 Article ID e1001225 2015

[31] Y Lou J Zheng B Wang X Zhang X Zhang and SZeng ldquoMetabolites characterization of chamaechromone invivo and in vitro by using ultra-performance liquid chro-matographyXevo G2 quadrupole time-of-flight tandem massspectrometryrdquo Journal of Ethnopharmacology vol 151 no 1 pp242ndash252 2014

[32] Y H Wang X Yang L J Sun and L M Yang ldquoStudies onthe cytotoxicity of Stellera chamaejasme L in vitrordquo ChineseMedicinal Biotechnology vol 7 pp 9ndash13 2012

[33] M M Badawi S S Handa A D Kinghorn G A Cordelland N R Farnsworth ldquoPlant anticancer agents XXVIIAntileukemic and cytotoxic constituents of Dirca occiden-talis (thymelaeaceae)rdquo Journal of Pharmaceutical Sciences vol72 no 11 pp 1285ndash1287 1983 httpswwwncbinlmnihgovpubmed6644588

[34] J F Yang W J Dong J C Zong et al ldquoToxicity detectionof Stella Chamaejasme in zebrafish embryo with differentextraction methodsrdquo Journal of Inner Mongolia University forNationalities vol 31 pp 236ndash239 2016

International Journal of Analytical Chemistry 9

[21] Z Jiang T Tanaka T Sakamoto I Kouno J Duan and RZhou ldquoBiflavanones diterpenes and coumarins from the rootsof stellera chamaejasme lrdquoChemical amp Pharmaceutical Bulletinvol 50 no 1 pp 137ndash139

[22] Y Li C Li L Lv et al ldquoA UPLC-MSMSmethod for simultane-ous determination of five flavonoids from Stellera chamaejasmeL in rat plasma and its application to a pharmacokinetic studyrdquoBiomedical Chromatography vol 32 no 6 Article ID e41892018

[23] G Liu H Tatematsu M Kurokawa M Niwa and Y HirataldquoNovel C-3C-31015840rsquo-biflavanones from Stellera chamaejasme LrdquoChemical amp Pharmaceutical Bulletin vol 32 pp 362ndash365 1984

[24] X Li K Rahman J Zhu andH Zhang ldquoChemical Constituentsand Pharmacological Activities of Stellera chamaejasmerdquo Cur-rent Pharmaceutical Design vol 24 no 24 pp 2825ndash2838 2018

[25] X Su R Lin S Wong S Tsui and S Kwan ldquoIdentificationand characterisationof the Chinese herb Langdu by LC-MSMSanalysisrdquo Phytochemical Analysis vol 14 no 1 pp 40ndash47 2003

[26] L Qiao L Yang D Zhang J Zou and J Dai ldquoStudies on chem-ical constitutes from callus cultures of Stellera chamaejasmerdquoZhongguo Zhongyao Zazhi vol 36 no 24 pp 3457ndash3462 2011httpswwwncbinlmnihgovpubmed22368856

[27] F M Ursache I O Ghinea M Turturica I Aprodu GRapeanu andN Stanciuc ldquoPhytochemicals content and antiox-idant properties of sea buckthorn (Hippophae rhamnoides L)as affected by heat treatment - Quantitative spectroscopic andkinetic approachesrdquo FoodChemistry vol 233 pp 442ndash449 2017

[28] Q Lu Y Peng C Zhu and S Pan ldquoEffect of thermal treatmenton carotenoids flavonoids and ascorbic acid in juice of orangecv Cara Carardquo Food Chemistry vol 265 pp 39ndash48 2018

[29] S Maini H L Hodgson and E S Krol ldquoTheUVA and aqueousstability of flavonoids is dependent on b-ring substitutionrdquoJournal of Agricultural and Food Chemistry vol 60 no 28 pp6966ndash6976 2012

[30] Z Yan L Zeng H Jin and B Qin ldquoPotential ecological rolesof flavonoids from Stellera chamaejasmerdquo Plant Signaling andBehavior vol 10 Article ID e1001225 2015

[31] Y Lou J Zheng B Wang X Zhang X Zhang and SZeng ldquoMetabolites characterization of chamaechromone invivo and in vitro by using ultra-performance liquid chro-matographyXevo G2 quadrupole time-of-flight tandem massspectrometryrdquo Journal of Ethnopharmacology vol 151 no 1 pp242ndash252 2014

[32] Y H Wang X Yang L J Sun and L M Yang ldquoStudies onthe cytotoxicity of Stellera chamaejasme L in vitrordquo ChineseMedicinal Biotechnology vol 7 pp 9ndash13 2012

[33] M M Badawi S S Handa A D Kinghorn G A Cordelland N R Farnsworth ldquoPlant anticancer agents XXVIIAntileukemic and cytotoxic constituents of Dirca occiden-talis (thymelaeaceae)rdquo Journal of Pharmaceutical Sciences vol72 no 11 pp 1285ndash1287 1983 httpswwwncbinlmnihgovpubmed6644588

[34] J F Yang W J Dong J C Zong et al ldquoToxicity detectionof Stella Chamaejasme in zebrafish embryo with differentextraction methodsrdquo Journal of Inner Mongolia University forNationalities vol 31 pp 236ndash239 2016

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