research article hplc quantitative analysis of main...

Hindawi Publishing CorporationJournal of ChemistryVolume 2013 Article ID 452610 6 pageshttpdxdoiorg1011552013452610

Research ArticleHPLC Quantitative Analysis of Main Stilbenes and Flavones inDifferent Parts of Matteuccia struthiopteris

Shubei Li1 Dong Zhang1 Lan Yang1 Yujie Li1 Xiaoxin Zhu1

Eva KmoniacuteIkovaacute2 and Zdenjk Ziacutedek2

1 Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences Beijing 100700 China2 Institute of Experimental Medicine Academy of Sciences of the Czech Republic Vıdeoska 1083 142 20 Prague Czech Republic

Correspondence should be addressed to Lan Yang ylan 66163com and Xiaoxin Zhu zhuxx59yahoocomcn

Received 5 June 2013 Revised 29 September 2013 Accepted 11 October 2013

Academic Editor Alexander Kornienko

Copyright copy 2013 Shubei Li et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A simple and accurate HPLC-UVmethod was developed for the simultaneous quantitative analysis of main stilbenes and flavonesin different parts (fronds rhizomes and frond bases) of M struthiopteris The chromatographic separation was performed on aKromasil C18 column (46mmtimes 250mm 5 120583m) with the mobile phase of MeOH-H

2O (including 01 phosphoric acid) using a

gradient elution at the flow rate of 10mLminminus1 and UV detection at 295 nm The method was validated by specificity linearityaccuracy (recovery) and precision tests (repeatability intra- and interday) For all the six compounds the linear regressioncoefficients ranged from 09958 to 09998 within the test ranges intra- and interday precisions were lt2 and the mean recoveriesranged from 9809 to 10356 The amount of these compounds in the frond bases was almost the same as in the rhizomes butmuch higher than that in the fronds The results indicate that the HPLC method developed was appropriate for the analysis of thesix compounds in different parts (fronds rhizomes and frond bases) ofM struthiopteris

1 IntroductionMatteuccia struthiopteris (L) Todaro (ostrich fern) belong-ing to the family Onocleaceae is an edible and medicinalfern widely distributed in the temperate regions of the nor-thern hemisphereThe tightlywound immature fronds calledfiddleheads are used as a cooked vegetable and the youngshoots can also be eaten raw in saladThe rhizomes and frondbases ofM struthiopteris have been used as a traditional chi-nese medicine (TCM) for the treatment of pinworms influ-enza dysentery hematochezia and uterine hemorrhage forthousands of years The pharmacological activities of Mstruthiopteris include antivirus antiparasite and eliminatingbacterium [1 2] Several C-methyl flavanone derivativesfromM orientalis showed a very strong hypoglycemic effectin streptozotocin (STZ) induced diabetic rats [3 4] Aseries of flavonoids stilbenes and phenolics have beenpreviously identified fromM struthiopteris [5ndash8] whichwerevery similar to that from M orientalis [3 4] In searchingfor bioactive components of M struthiopteris six maincompounds (Figure 1) including three C-methyl flavones

demethoxymatteucinol (1) matteucinol (2) and matteuorien(3) and three stilbenes pinosylvin (4) pinosylvin 3-O-120573-D-glucopyranoside (5) and 5-120573-D-glucosyloxy-3-hydroxy-l-trans-stilbene-2-carboxylic acid (6) were separated andidentifiedMatteucinol could inhibit rat lens aldose reductase[9] and scavenging hydroxyl radical [10] Pinosylvin wasreported to have various biological activities including can-cer chemopreventiveanti-inflammatory [11ndash14] antioxidant[15] and antiproliferative effects in various cancer cells [1617]

The determination of the main bioactive compounds inM struthiopteris is important both for their characterizationand to facilitate more efficient uses of this important plantresource In the presentwork a specificmethod for the simul-taneous determination of demethoxymatteucinol (1)matteu-cinol (2) matteuorien (3) pinosylvin (4) pinosylvin 3-O-120573-D-glucopyranoside (5) and 5-120573-D-glucosyloxy-3-hydroxy-l-trans-stilbene-2-carboxylic acid (6) was developed andapplied to different parts (fronds rhizomes and frond bases)ofM struthiopteris

2 Journal of Chemistry

O

OOH

HO O

OOH

HO

O

OOH

HO

OH

HO

OGlc

HO

OGlc

HO

COOH

CH3 CH3

CH3

H3C H3C

H3C

OCH3

(1) Demethoxymatteucinol (2) Matteucinol

(3) Matteuorien (4) Pinosylvin

(5) Pinosylvin 3-O-120573-D-glucopyranoside (6) 5-120573-D-Glucosyloxy-3-hydroxy-l-trans-stilbene-2-carboxylic acid

Figure 1 Structures of standard compounds

2 Experimental

21 Plant Material and Chemicals The fronds rhizomes andfrond bases of M struthiopteris were collected from JinfoMountain (Chongqing China) and identified by SRYi (Ins-titute of Medicinal Plantation of Chongqing ChongqingChina) inMay 2010The samples were air-driedThe voucherspecimens were deposited in our laboratory Demethoxymat-teucinol (1) matteucinol (2) matteuorien (3) pinosylvin (4)and pinosylvin 3-O-120573-D-glucopyranoside (5) were isolatedand identified from the rhizomes of M struthiopteris inour lab [8] 5-120573-D-Glucosyloxy-3-hydroxy-l-trans-stilbene-2-carboxylic acid (6) was isolated from the genusMatteucciafor the first time Their structures are shown in Figure 1 Thepurity of the six compounds was determined to be more than98 by normalization of the peak areas detected by HPLC-DAD

HPLC grade methanol (MeOH) and acetonitrile (ACN)were purchased fromFisher Scientific (Geel Belgium) Ultra-pure water was prepared using Milli-Q purification systemfrom Millipore (Bedford MA USA) All other chemicalsand solvents were of analytical grade The silica gel (200ndash300mesh) for column chromatography (CC) was produced byQingdao Haiyang Chemical Co Ltd (Qingdao China)

22 Equipment and Apparatus Mass spectra were recordedon a Xevo G2 Q-Tof mass spectrometer (Waters) and Agilent6130 SQ spectrometers The 1H- and 13C-NMR spectra wererecorded on 600 and 150MHz (Bruker) The HPLC system

employed was a Shimadzu Prominence LC-20A (KyotoJapan) equipped with a binary pump a vacuum degasser anSPD-20A detector an SIL-20A autosampler and a CTO-20Acolumn oven Chromatographic separation was performedusing a Kromasil (Eka Chemicals Sweden) C

18column

(5 120583m 250mm times 46mm id)

23 Preparation of Standard Solutions Accurately weighedsix compounds were dissolved in methanol to prepare stocksolutions respectively A certain amount of each stock solu-tion was placed in a 25mL volumetric flask and diluted tovolume with methanol at the concentration of 43320 120583gmLDMN 40920120583gmL MN 2032120583gmL MR 4272120583gmLPV 2016120583gmL PVG and 7160 120583gmL SCG The mixturesolution was then diluted stepwise with methanol to givesix different concentrations for construction of calibrationcurves

24 Sample Preparation Accurately weight 03 g of the finelypowdered sample was placed in a round-bottom flask andthen 50mLmethanol was added accuratelyThen the samplewas heated under reflux for 20 h cooled and replenished theloss of the solvent with methanol filtered through a 045 120583mnylon membrane prior to analysis

25 HPLC Condition The mobile phase consisted of twosolutions (A and B solutions) and flowed with a programmedgradient elution The A solution was methanol and the B

Journal of Chemistry 3

solution was 01 phosphoric acid aqueous solution Gra-dient elution was programmed as follows AB = 4555(0min hold for 18min) 7030 (28min hold for 22min) rarr4555 (55min hold for 15min to equilibrate the column) rarrdetermining next sample until the last one rarr washing thecolumn Recording of the chromatogram was set from 0minto 50min The column temperature was 40∘C and the flowrate was 10mLmin The detection wavelength was set at295 nm Five 120583L of the sample solutionwas injected forHPLCanalysis

26 Calibration Curve Limits of Detection and Quantifica-tion The standard mixture solutions at six different concen-trations were injected in triplicate The calibration curve wasconstructed by plotting six points of peak area (119910 axis) versusconcentration (119909 axis) for each compound The standardmixture solution was further diluted to serial concentrationsto explore the limits of detection (LOD) and quantification(LOQ) The LOD and LOQ were determined at a signal-to-noise (SN) ratio of 3 and 10 respectively

27 Precision Stability and Accuracy Studies The intra- andinterday precisions were determined by continuously inje-cting the sample solution for six replicates on the same dayand by measuring it once a day for six consecutive daysrespectively Stability was determined on one sample solutionsix times in 24 h The recovery tests were performed toexamine the accuracy of the extraction methodThe accurateamounts of the six compounds were spiked to a certainamount of the rhizomes powder and were then extractedand analyzed as described above The spiked amount of eachstandardwas adjusted so as to provide a concentration similarto that present in the sample The mean recovery rate ()was measured from the spiked sample solution versus thenonspiked solution

3 Results and Discussion

31 Structure Elucidation of Isolated Compounds Structureelucidation of demethoxymatteucinol (1) matteucinol (2)matteuorien (3) pinosylvin (4) and pinosylvin 3-O-120573-D-glucopyranoside (5)was reported previously by us [8] 5-120573-D-Glucosyloxy-3-hydroxyl-trans-stilbene-2-carboxylic acid (6)was isolated from 119872 struthiopteris for the first time andidentified by comparing with the reported data [18]

32 5-120573-D-Glucosyloxy-3-hydroxyl-trans-stilbene-2-carboxylic Acid (6)

White Powder HRESI-MS mz 4171174 [M-H]minus (Calcd forC21H22O9 4171185) 1H-NMR (600MHz CD

3OD) 120575 339 sim

395 (6H m 210158401015840 sim 610158401015840-H) 503 (1H d 119869 71 Hz 110158401015840-H) 659(1H d 119869 23Hz 4-H) 691 (1H d 119869 23Hz 6-H) 695 (1Hd 119869 16Hz 1205721015840-H) 726 (1H t 119869 76Hz 41015840-H) 735 (2H t119869 76Hz 3101584051015840-H) 752 (2H d 119869 76Hz 2101584061015840-H) and 789(1H d 119869 16Hz 120572-H) 13C-NMR (150MHz CD

3OD) 120575 611

(C-610158401015840) 700 (C-410158401015840) 734 (C-210158401015840) 765 (C-310158401015840) 770 (C-510158401015840)1002 (C-110158401015840) 1029 (C-4) 1063 (C-2) 1074 (C-6) 1264

(C-2101584061015840) 1274 (C-41015840) 1283 (C-3101584051015840) 1291 (C-120572) 1308 (C-1205721015840) 1376 (C-11015840) 1429 (C-1) 1616 (C-5) 1641 (C-3) and 1728

(COOH)

33 Optimization of Chromatographic Conditions Due to thedifficulty in separating the six compounds by one isocraticelution a gradient method was developed to determineall the constituents in one analysis run Various mixturesof acetonitrile and water were tried as mobile phases butseparation was not satisfactory After replacing acetonitrileby methanol the situation was greatly improved In RP-HPLC the elution ability of acetonitrile is better than ofmethanol Separation could be achieved in less time usingacetonitrile compared to using methanol [19] but theremay not be ideal separation in the reduced time usingacetonitrile owing to similar polarities of the compoundsAlso the compoundswithmoderate polaritiesmayhave goodsolubility and separation in the system containing methanoland H

2O Meanwhile phosphoric acid was also added to the

eluent to improve the imperfect peak shapes and resolution ofthe three flavones Although the UV absorption maxima ofthe target compounds were somewhat different all showedmaximum absorption around 295 nm and therefore thedetection wavelength was set at 295 nm

Typical chromatograms of samples and mixed standardsare shown in Figure 2 in which all target constituents werecompletely separated within 50min

34 Validation on the HPLC Method The analytical methodwas validated with respect to the linearity limits of detection(LOD) limits of quantification (LOQ) precision and stabil-ity The linear ranges regression equations and correlationcoefficients obtained from typical calibration curves andLOD and LOQ are shown in Table 1 All standard curvesexhibited good linearity and the correlation coefficients werehigher than 09958 The LODs (119878119873 = 3) and LOQs(119878119873 = 10) obtained from each standard compound were inthe range of 002ndash010 and 006ndash033 120583gmL respectively Asshown in Table 2 the method gave good precision with therelative standard deviations (RSD) of intra-day precision andinter-day precision for the six compounds being less than140 and 170 respectively The compounds proved to bestable in sample solution within 24 h at room temperaturewith their RSD values below 151 The extraction recoveriesdetermined for all compounds are shown in Table 3 Themean recoveries were in the range of 9809 and 10356with RSD less than 130 for all the six compounds Ingeneral themethod is precise accurate and sensitive enoughfor the simultaneous quantitative evaluation of the six majorcomponents inM struthiopteris

35 Optimization of Method of Sample Preparation In orderto achieve a complete extraction of the six compounds com-mon extraction methods and solvents were studied Refluxand ultrasonic-assisted were tried as extraction methodsIt was found that the extraction at reflux temperature wasmore efficient than the ultrasonic-assisted method Thenthe other factors as extracting solvent (60 70 and 80


Table 1 Calibration curves detection limits and quantification limits of the compounds

Compound no Calibration equationa Linear range (120583gmL) 1198772b LODc (120583gmL) LOQd (120583gmL)

1 119910 = 14800119909 minus 21814 866ndash433 times 102 09998 007 0232 119910 = 14947119909 minus 26564 818ndash409 times 102 09997 007 0223 119910 = 10685119909 + 5656 041ndash2032 09958 009 0324 119910 = 39250119909 minus 14643 085ndash4272 09991 002 0065 119910 = 16973119909 minus 33584 040ndash2016 09993 008 0276 119910 = 95451119909 minus 14385 143ndash7160 09998 010 033a119910 peak area at 295 nm 119909 concentration of the compounds (120583gmL)

b1198772 correlation coefficient for 6 data points in the calibration curves (n = 2)

cLOD limit of detection (SN = 3)dLOQ limit of quantification (SN = 10)

Table 2 Analytical results of intraday and interday precision and stability for the compounds in the rhizome ofM struthiopteris (119899 = 6)

Compound no Intraday precision Interday precision StabilityContent (mgg) RSD () Content (mgg) RSD () Content (mgg) RSD ()

1 4848 plusmn 006 013 4749 plusmn 030 063 4676 plusmn 010 0212 3055 plusmn 004 014 2989 plusmn 013 044 2947 plusmn 007 0233 219 plusmn 003 135 215 plusmn 003 128 212 plusmn 003 1514 420 plusmn 001 015 414 plusmn 004 091 417 plusmn 001 0215 282 plusmn 001 039 276 plusmn 002 070 281 plusmn 001 0426 729 plusmn 001 019 727 plusmn 012 162 751 plusmn 002 030

Table 3 Recovery of each compound as determined by standard addition method (in rhizome) (119899 = 6)

Compound no Initial concentration(120583gmL)

Amount added(120583g)

Concentration after addition (120583gmL) Recovery () RSD ()Expected Measured

1 14388 16130 30518 30597 10026 0752 9084 9223 18307 18294 9993 0783 667 640 1307 1282 9809 1264 1291 1173 2464 2511 10191 1015 852 809 1661 1718 10343 0726 2328 2787 5115 5297 10356 100The data was present as average of six determinations

methanol aqueous solution and pure methanol) reflux time(10 h 15 h 20 h 25 h 3 h) and sample-solvent ratio (25mL50mL and 100mL per 03 gram of sample) were investigatedindependently The results showed that pure methanol 2 h ofreflux time and 50mL per 03 gram of sample emerged as theconditions at which efficient extraction was attained

36 Sample Analysis Three parts (fronds rhizomes andfrond bases) of M struthiopteris were collected from thesame area at the same timeThe established method has beensuccessfully applied for the simultaneous determination ofthe three flavonoids and three stilbenes in different parts ofM struthiopteris as shown in Figure 2 and Table 4 In eachsingle part the content of the flavones was much higher thanof the stilbenes The contents of all targeted compounds in

the rhizomes and the frond bases were very similar but muchhigher than that in the fronds

4 Conclusion

The six compounds isolated from the rhizomes and frondbases of M struthiopteris could be regarded as the maineffective components according to their characteristics TheHPLC-UV method established for the quantitative analysisof these compounds in different parts (fronds rhizomes andfrond bases) of M struthiopteris had high selectivity highsensitivity and good reproducibility The results showed thatthe amount of all the six compounds in the frond bases wasalmost the same as in the rhizomes but much higher thanthat in the fronds which might explain the reason that the


(Q) (A)

(B) (C)

42 1

3

100

75

50

25

0

0

66

55

10 20 30 40(min)

6 5 6 5

6 5

2 1

34

0 10 20 30 40(min)

3025201510

50

6 5

21

3

4

0 10 20 30 40(min)

300250200150100

500

minus50

6 5

12

3

4

0 10 20 30 40(min)

300250200150100

500

(mAU

)

(mAU

)(m

AU)

(mAU

)

Figure 2 (Q) Chromatogram of mixed standard compounds (A) Chromatogram of fronds of M struthiopteris (B) Chromatogram ofrhizomes of M struthiopteris (C) Chromatogram of frond bases of M struthiopteris (1) Demethoxymatteucinol (2) matteucinol (3)matteuorien (4) pinosylvin (5) pinosylvin 3-O-120573-D-glucopyranoside and (6) 5-120573-D-glucosyloxy-3-hydroxyl-trans-stilbene-2-carboxylicacid

Table 4 Content of the compounds (119899 = 3)

Position Compound no Content ofcompounds (mgg) RSD ()

Fronds

1 363 plusmn 009 2542 496 plusmn 011 2143 107 plusmn 002 2004 026 plusmn 001 2775 007 plusmn 001 2026 066 plusmn 001 108

Rhizomes


Frond bases


rhizomes and frond bases were used as the medicinal parts inTCM AsM struthiopteris is a perennial fern overcollectingthe rhizomes could lead to resource exhaustion The similarcontents of the main effective components in the rhizomes

and the frond bases implied that we can use the frond basesas medicinal parts other than the whole frond bases andrhizomes so as to ensure the fern sustainable usage

Conflict of Interests

The authors declared that they have no commercial conflictof interests related to this work

Acknowledgments

The work was supported by the International Science andTechnology Cooperation Program of the Peoplersquos Republicof China no 2011DFA30870 and by the bilateral grant fromthe Ministry of Education Youth and Sports of the CzechRepublic no ME10116

References

[1] Z C LouVariety Arrangement and Quality Research of ChineseMateria Medica Commonly Used vol 2 Peking Union MedicalCollege Press Beijing China 1995

[2] P G XiaoModern Chinese Materia Medica Chemical IndustryPress Beijing China 2002

[3] P Basnet S Kadota M Shimizu H-X Xu and T Nambaldquo21015840-Hydroxymatteucinol a new C-methyl flavanone deriva-tive from Matteccia orientalis potent hypoglycemic activityin streptozotocin (STZ)-induced diabetic ratrdquo Chemical andPharmaceutical Bulletin vol 41 no 10 pp 1790ndash1795 1993

[4] P Basnet S Kadota K Hase and T Namba ldquoFive new C-methyl flavonoids the potent aldose reductase inhibitors from


Matteuccia orientalis TREVrdquo Chemical and PharmaceuticalBulletin vol 43 no 9 pp 1558ndash1564 1995

[5] L Yang M-Y Wang Y-Y Zhao and Y-Y Tu ldquoStudies on che-mical constituents in rhizome of Matteuccia struthiopterisrdquoChina Journal of ChineseMateriaMedica vol 29 no 7 pp 648ndash649 2004

[6] L Yang M-Y Wang Y-Y Zhao and Y-Y Tu ldquoChemicalconstituents of the rhizome of Matteuccia struthiopterisrdquo ActaPharmaceutica Sinica vol 40 no 3 pp 252ndash254 2005

[7] L Yang Y Y Zhao and Y Y Tu ldquoChemical constituents of therhizome ofMatteuccia struthiopterisrdquo China Journal of ChineseMateria Medica vol 28 pp 278ndash279 2003

[8] D Zhang L YangM-H Fu and Y-Y Tu ldquoStudies on chemicalconstituents of rhizome of Matteuccia struthiopteris IIIrdquo ChinaJournal of ChineseMateriaMedica vol 33 no 14 pp 1703ndash17052008

[9] S Z Li W S Mao X Y Du S W Liang B R Hu and YQ Ma ldquoInhibition of rat lens aldose reductase by flavonoids-matteucinol and baicaleinrdquo Yan Ke Xue Bao vol 3 pp 93ndash941987

[10] J-W Chen Z-Q Zhu T-X Hu and D-Y Zhu ldquoStructure-activity relationship of natural flavonoids in hydroxyl radical-scavenging effectsrdquo Acta Pharmacologica Sinica vol 23 no 7pp 667ndash672 2002

[11] E-J Park H-Y Min H-J Chung Y-H Ahn J-H Pyee and SK Lee ldquoPinosylvin suppresses LPS-stimulated inducible nitricoxide synthase expression via the MyD88-independent butTRIF-dependent downregulation of IRF-3 signaling pathway inmouse macrophage cellsrdquo Cellular Physiology and Biochemistryvol 27 no 3-4 pp 353ndash362 2011

[12] E-J Park H-Y Min H J Park et al ldquoNuclear factor E2-relatedfactor 2-mediated induction of NAD(P)Hquinone oxidore-ductase 1 by 35-dimethoxy-trans-stilbenerdquo Journal of Pharma-cological Sciences vol 116 no 1 pp 89ndash96 2011

[13] E J Park H Y Min Y H Ahn C M Bae J H Pyee and S KLee ldquoSynthesis and inhibitory effects of pinosylvin derivativeson prostaglandin E2 production in lipopolysaccharide-inducedmouse macrophage cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 14 no 23 pp 5895ndash5898 2004

[14] S K Lee H J Lee H YMin et al ldquoAntibacterial and antifungalactivity of pinosylvin a constituent of pinerdquo Fitoterapia vol 76no 2 pp 258ndash260 2005

[15] J G Fang M Lu Z H Chen et al ldquoAntioxidant effects ofresveratrol and its analogues against the free-radicalinducedperoxidation of linoleic acid in micellesrdquo Chemistry vol 8 pp4191ndash4198 2002

[16] A Ludwiczuk A Saha T Kuzuhara and Y Asakawa ldquoBioac-tivity guided isolation of anticancer constituents from leaves ofAlnus sieboldiana (Betulaceae)rdquo Phytomedicine vol 18 no 6pp 491ndash498 2011

[17] F Simard J Legault S Lavoie V Mshvildadze and A PichetteldquoIsolation and identification of cytotoxic compounds from thewood of Pinus resinosardquo Phytotherapy Research vol 22 no 7pp 919ndash922 2008

[18] MYukinori I Yoshitaka KHaruhisa andT Tsuyoshi ldquoStudieson the Nepalese crude drugs (X) On the flavonoid and the stil-bene constituents of the leaves of Scutellaria scandens Buch-Ham ex D Donrdquo Shoyakugaku Zasshi vol 42 pp 204ndash2071988

[19] N Manchon M DrsquoArrigo A Garcıa-Lafuente et al ldquoCompar-ison of different types of stationary phase for the analysis of soy

isoflavones by HPLCrdquo Analytical and Bioanalytical Chemistryvol 400 pp 1251ndash1261 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

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Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

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Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


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Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


O

OOH

HO O

OOH

HO

O

OOH

HO

OH

HO

OGlc

HO

OGlc

HO

COOH

CH3 CH3

CH3

H3C H3C

H3C

OCH3

(1) Demethoxymatteucinol (2) Matteucinol

(3) Matteuorien (4) Pinosylvin

(5) Pinosylvin 3-O-120573-D-glucopyranoside (6) 5-120573-D-Glucosyloxy-3-hydroxy-l-trans-stilbene-2-carboxylic acid

Figure 1 Structures of standard compounds

2 Experimental

21 Plant Material and Chemicals The fronds rhizomes andfrond bases of M struthiopteris were collected from JinfoMountain (Chongqing China) and identified by SRYi (Ins-titute of Medicinal Plantation of Chongqing ChongqingChina) inMay 2010The samples were air-driedThe voucherspecimens were deposited in our laboratory Demethoxymat-teucinol (1) matteucinol (2) matteuorien (3) pinosylvin (4)and pinosylvin 3-O-120573-D-glucopyranoside (5) were isolatedand identified from the rhizomes of M struthiopteris inour lab [8] 5-120573-D-Glucosyloxy-3-hydroxy-l-trans-stilbene-2-carboxylic acid (6) was isolated from the genusMatteucciafor the first time Their structures are shown in Figure 1 Thepurity of the six compounds was determined to be more than98 by normalization of the peak areas detected by HPLC-DAD

HPLC grade methanol (MeOH) and acetonitrile (ACN)were purchased fromFisher Scientific (Geel Belgium) Ultra-pure water was prepared using Milli-Q purification systemfrom Millipore (Bedford MA USA) All other chemicalsand solvents were of analytical grade The silica gel (200ndash300mesh) for column chromatography (CC) was produced byQingdao Haiyang Chemical Co Ltd (Qingdao China)

22 Equipment and Apparatus Mass spectra were recordedon a Xevo G2 Q-Tof mass spectrometer (Waters) and Agilent6130 SQ spectrometers The 1H- and 13C-NMR spectra wererecorded on 600 and 150MHz (Bruker) The HPLC system

employed was a Shimadzu Prominence LC-20A (KyotoJapan) equipped with a binary pump a vacuum degasser anSPD-20A detector an SIL-20A autosampler and a CTO-20Acolumn oven Chromatographic separation was performedusing a Kromasil (Eka Chemicals Sweden) C

18column

(5 120583m 250mm times 46mm id)

23 Preparation of Standard Solutions Accurately weighedsix compounds were dissolved in methanol to prepare stocksolutions respectively A certain amount of each stock solu-tion was placed in a 25mL volumetric flask and diluted tovolume with methanol at the concentration of 43320 120583gmLDMN 40920120583gmL MN 2032120583gmL MR 4272120583gmLPV 2016120583gmL PVG and 7160 120583gmL SCG The mixturesolution was then diluted stepwise with methanol to givesix different concentrations for construction of calibrationcurves

24 Sample Preparation Accurately weight 03 g of the finelypowdered sample was placed in a round-bottom flask andthen 50mLmethanol was added accuratelyThen the samplewas heated under reflux for 20 h cooled and replenished theloss of the solvent with methanol filtered through a 045 120583mnylon membrane prior to analysis

25 HPLC Condition The mobile phase consisted of twosolutions (A and B solutions) and flowed with a programmedgradient elution The A solution was methanol and the B









3OD) 120575 339 sim


3OD) 120575 611

(C-610158401015840) 700 (C-410158401015840) 734 (C-210158401015840) 765 (C-310158401015840) 770 (C-510158401015840)1002 (C-110158401015840) 1029 (C-4) 1063 (C-2) 1074 (C-6) 1264

(C-2101584061015840) 1274 (C-41015840) 1283 (C-3101584051015840) 1291 (C-120572) 1308 (C-1205721015840) 1376 (C-11015840) 1429 (C-1) 1616 (C-5) 1641 (C-3) and 1728

(COOH)
























4 Conclusion



(Q) (A)

(B) (C)

42 1

3

100

75

50

25

0

0

66

55

10 20 30 40(min)

6 5 6 5

6 5

2 1

34

0 10 20 30 40(min)

3025201510

50

6 5

21

3

4

0 10 20 30 40(min)

300250200150100

500

minus50

6 5

12

3

4

0 10 20 30 40(min)

300250200150100

500

(mAU

)

(mAU

)(m

AU)

(mAU

)




Fronds


Rhizomes


Frond bases






Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of









3OD) 120575 339 sim


3OD) 120575 611

(C-610158401015840) 700 (C-410158401015840) 734 (C-210158401015840) 765 (C-310158401015840) 770 (C-510158401015840)1002 (C-110158401015840) 1029 (C-4) 1063 (C-2) 1074 (C-6) 1264

(C-2101584061015840) 1274 (C-41015840) 1283 (C-3101584051015840) 1291 (C-120572) 1308 (C-1205721015840) 1376 (C-11015840) 1429 (C-1) 1616 (C-5) 1641 (C-3) and 1728

(COOH)
























4 Conclusion



(Q) (A)

(B) (C)

42 1

3

100

75

50

25

0

0

66

55

10 20 30 40(min)

6 5 6 5

6 5

2 1

34

0 10 20 30 40(min)

3025201510

50

6 5

21

3

4

0 10 20 30 40(min)

300250200150100

500

minus50

6 5

12

3

4

0 10 20 30 40(min)

300250200150100

500

(mAU

)

(mAU

)(m

AU)

(mAU

)




Fronds


Rhizomes


Frond bases






Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


















4 Conclusion



(Q) (A)

(B) (C)

42 1

3

100

75

50

25

0

0

66

55

10 20 30 40(min)

6 5 6 5

6 5

2 1

34

0 10 20 30 40(min)

3025201510

50

6 5

21

3

4

0 10 20 30 40(min)

300250200150100

500

minus50

6 5

12

3

4

0 10 20 30 40(min)

300250200150100

500

(mAU

)

(mAU

)(m

AU)

(mAU

)




Fronds


Rhizomes


Frond bases






Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(Q) (A)

(B) (C)

42 1

3

100

75

50

25

0

0

66

55

10 20 30 40(min)

6 5 6 5

6 5

2 1

34

0 10 20 30 40(min)

3025201510

50

6 5

21

3

4

0 10 20 30 40(min)

300250200150100

500

minus50

6 5

12

3

4

0 10 20 30 40(min)

300250200150100

500

(mAU

)

(mAU

)(m

AU)

(mAU

)




Fronds


Rhizomes


Frond bases






Acknowledgments


References
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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