Research Article Fluorescent Properties of Hymecromone and ...Fluorescent Properties of Hymecromone and Fluorimetric ... Journal of Spectroscopy 250 300 350 400 450 500 550 0 500 1000

Hindawi Publishing CorporationJournal of SpectroscopyVolume 2013 Article ID 147128 9 pageshttpdxdoiorg1011552013147128

Research ArticleFluorescent Properties of Hymecromone and FluorimetricAnalysis of Hymecromone in Compound Dantong Capsule

Huanhuan Zhi12 Jingdan Wang2 Shujing Wang2 and Yongju Wei12

1 College of Life Science Hebei Normal University Shijiazhuang 050024 China2 College of Chemistry and Material Science Hebei Normal University Shijiazhuang 050024 China

Correspondence should be addressed to Yongju Wei weiyju126com

Received 5 September 2013 Accepted 21 October 2013

Academic Editor Yizhuang Xu

Copyright copy 2013 Huanhuan Zhi 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 properly cited

Fluorescence spectra of hymecromone (4MU) aqueous solutions are investigated at different pHs Two fluorescent species of 4MUneutral molecular form and anion form are considered to be the main fluorescent forms Quantum yields of the two forms aremeasured to be 074 at pH 598 and 095 at pH 975 respectivelyThe ionization constant of 7-hydroxyl proton of 4MU is determinedto be p119870

119886= 785 plusmn 003 by a pH-fluorescence method Addition of methanol into 4MU aqueous solution leads to a blue shift

of maximum emission wavelength from 445 nm to 380 nm and a decrease in fluorescence intensity 3D fluorescence spectra ofChinese patent drug Compound Dantong Capsule (CDC) and its four component herbal drugs are also investigated Based ontheir fluorescent properties a novel fluorimetric method is proposed for the selective determination of 4MU in CDC withoutpreseparation The new method is suitable for the routine quality evaluation of CDC

1 Introduction

Coumarins both natural and synthetic are of interestbecause of their multiple biological and photodynamic activ-ities They are extensively used as medicine and analyticalreagent [1ndash7] Hymecromone (4-Methyl-7-hydroxycouma-rin 7-Hydroxy-4-methylcoumarin 4-Methylumbelliferoneabbreviated as 4MU) is a synthetic coumarin compoundIt is claimed that 4MU and its derivatives or metal com-plexes possess diverse pharmacological properties such asantiviral [8] antifungal and antioxidative [9] anticancer [10ndash13] anticoagulant and spasmolytic activities [14 15] Thefluorescence of 4MU had been observed early [16] a water-soluble polymeric fluorescent probe for measurement ofnear-neutral pH was synthesized based on the fluorescentproperties of 4MU [17] and the effect of pH on fluorescencewas restudied recently [18] however no comprehensive anddetailed fluorescence spectra and quantum yield have beenfound in the literature

Compound Dantong Capsule (CDC also named FufangDantong Capsule) is a Chinese proprietary medicine [19]It is effective in cure cholecystitis cholangitis intercurrentinfection of gallbladder and biliary tract concretion [19 20]

The chemical composition of CDC includes 4MU (alsonamedDantong) and four kinds ofChinese herbalmedicinesincluding Herba Isodonis Lophanthoidis (Xihuangcao)Herba Artemisiae Scopariae (Yinchen) Herba Andrographis(Chuanxinlian) and Rhei Radix et Rhizoma (Dahuang)The content of 4MU is one of the main quality indexesof CDC [21] Earlier the spectrophotometric method wasapplied for the determination of 4MU in CDC accordingto the absorbance of 4MU at 372 nm [19] Later since theinterference of coexistent components high performanceliquid chromatographic methods (HPLC) were developedfor the determination of 4MU in CDC [21ndash23] So far nofluorimetric method was reported for the determination of4MU in CDC and other medicinal samples

Fluorimetry has been recognized as one of themost usefulanalyticalmethods owing to its high sensitivity good selectiv-ity simplicity speediness and low cost It can be suitable foranalysis of some complex samples such as traditional Chinesemedicine containing a fluorescent component especially inthe case of a routine analysisThree-dimensional fluorescencespectra (3D fluorescent fingerprint) have been used in ourlab for qualitative identification of Chinese herbal medicines

2 Journal of Spectroscopy

300 350 400 450 500 550

200

250

300

350

400120582

ex(n

m)

120582em (nm)

Rayleighscattering

Ramanscattering

Fluorescencecontour

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)

Figure 1 3D fluorescence spectra of 4MU aqueous solution at different pH 1198884MU 185 ngmLminus1 (a) pH 598 (b) pH 975 Contour interval

R

[24] Moreover a number of fluorimetric methods have beendeveloped for quantitative determination of active compo-nents in medicinal materials such as paeonol in CynanchiPaniculati Radix (Xuchangqing) [25] camptothecin in com-mon Camptotheca fruit (Xishuguo) [26] and arctiin inArctii Fructus (Niubangzi) [27] In this study the fluorescentproperties of 4MU are investigated and its quantum yieldand 7-hydroxyl proton ionization constant are measuredThefluorescent properties of CDC and its four consisting Chineseherbal medicines are also studied Based on the spectraldifferences between 4MU and other components in CDCa fluorimetric method is proposed for the determination of4MU in CDC sample without preseparation

2 Experimental

21 Apparatus Fluorescence measurements were performedon a Hitachi (Tokyo Japan) F-7000 spectrofluorimeterequipped with a xenon lamp 1 cm quartz cell and a UV-29 filter placed into the emission light path to removesecondary spectrum The excitation and emission slits (bandpass) 5 nm5 nmwere used throughout the work Absorptionspectra were recorded using a Shimadzu (Kyoto Japan) UV-2501PC recording spectrophotometer with 1 cm quartz cellAn Orion (Beverly USA) 868 pHISE meter was used for pHmeasurement

22 Chemicals and Materials Hymecromone (4MU serialno 100241-200503 a reagent for quantitative analysis molec-ular weight 17617) and Chinese herbal medicine (compar-ison drug for qualitative identification) Xihuangcao (serialno 121488-200501) Yinchen (serial no 120950-200305)Chuanxinlian (serial no 121082-200302) and Dahuang(serial no 120984-200301) were purchased from the NationalInstitute for the Control of Pharmaceuticals and BiologicalProducts of China (Beijing China) 4MU stock solution was

prepared by dissolving 361mg reagent in 100mL methanol(TEDIA HPLC grade) and diluted to appropriate concentra-tion with methanol as needed Compound Dantong Capsule(CDC product lot no 100601) was produced by NationalTsing Hua Pharmaceutical Co Ltd Hubei-day Saint ChinaQuinine bisulphate (HPLC grade) was acquired from JampKScientific Ltd (Beijing China) its solution was prepared bydissolving 39147mg reagent in 500mL 005mol Lminus1 H

2SO4

and diluted to appropriate concentration with 005mol Lminus1H2SO4when it was used Britton-Robinson buffer solution

was a mixture of phosphoric acid boric acid and acetic acid(each 002mol Lminus1) and adjusted to an appropriate pH byaddition of 01mol Lminus1 NaOH solution All the buffer chem-icals were of analytical grade The water used throughoutthe study was doubly-deionized and verified to be free fromfluorescence

23 General Procedure for Spectral Measurement A seriesof 10mL volumetric flasks was added appropriate amountof 4MU or CDC and buffer solutions The mixtures werediluted to the mark with water and mixed well Fluorescenceor absorption spectra were measured at room tempera-ture Meanwhile Raman scattering of water at excitationwavelength of 350 nm was measured (expressed as R forcalibration of 3D fluorescence spectra) [24]

24 Determination of Ionization Constant A number ofsolutions containing the same concentration of weak acidHB and different pH were prepared Fluorescence intensity 119865and pH of each solution were measured Then the ionizationconstant p119870

119886was calculated according to the following

equation [28]

p119870119886= pH minus lg119865HB minus 119865

119865 minus 119865B (1)

Journal of Spectroscopy 3

250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500Fl

uore

scen

ce in

tens

itypH672

631

576

504

240

225

215

197

120582 (nm)

(a)

5000

4000

3000

2000

1000

0

250 300 350 400 450 500 550

Fluo

resc

ence

inte

nsity

pH1043

996

975

944

742

728

722

712

120582 (nm)

(b)

5000

4000

3000

2000

1000

0

250 300 350 400 450 500 550

Fluo

resc

ence

inte

nsity

pH1080

1141

1185

1211

1311

1321

1330

1338

120582 (nm)

(c)

0 2 4 6 8 10 12 14pH

1

25000

4000

3000

2000

1000

0

Fluo

resc

ence

inte

nsity

(d)

Figure 2 (a) (b) and (c) Fluorescence excitation and emission spectra of 4MU at different pH and (d) relationship between fluorescenceintensity and pH c

4MU 185 ngmLminus1 120582ex120582em (a) and (d(1)) 320 nm445 nm (b) (c) and (d(2)) 360 nm445 nm

where 119865HB and 119865B were the fluorescence intensity of HB andits conjugate base B they can be measured at sufficient acidicpH where all HB species exist in the neutral molecular formand at sufficient alkaline pH where all HB species exist in theanion form respectively

25 Measurement of Fluorescence Quantum Yield Quininebisulphate was used as a reference (quantum yield 055 atexcitationwavelength of 313 nm) inmeasuring quantumyieldof 4MU For the measurement quinine bisulphate and 4MUsolutions were prepared in proper concentration so that theabsorbance (119860) of the two solutions was similar and notlarger than 005 Absorption and fluorescence spectra wererecorded and then quantum yields were calculated accordingto the following equation [28 29]

119884119906= 119884119903sdot119865119906

119865119903

sdot119860119903

119860119906

(2)

where 119884119906and 119884

119903were the fluorescence quantum yield of

unknown and the reference 119865119906and 119865

119903were the integral flu-

orescence intensity of unknown and reference solutions and119860119906and 119860

119903were the absorbance of unknown and reference

solutions at their excitation wavelengths respectively

26 Sample Preparation Dissolve 921mg CDC powder in100mL methanol and dilute to 0921 120583gmLminus1 with methanolas sample solution

3 Results and Discussion

31 Fluorescence Properties of 4MU

311 3D Fluorescence Spectra of 4MU 3D (three-dimension-al) fluorescence spectra of solvent blank and 4MU aqueoussolutions at pH 598 and at pH 975 were measured as shownin Figure 1 The spectra indicated that the solvent used in


OH+ HO OO O O

O

Ominus

Ominus

CH3 CH3 CH3CH3

O minusOminusOHO

H+H+H+

OHminusOHminusOHminus

I II III IV

Figure 3 Proton ionization and hydrolysis process of 4MU

this study was basically no fluorescence 4MU can producestrong fluorescence in near neutral condition with maximumexcitation wavelength (120582ex) of 320 nm and maximum emis-sion wavelength (120582em) of 445 nm while in weak alkaline con-dition the fluorescence intensity enhanced 120582ex red shiftedfrom 320 nm to 360 nm but 120582em remained constant Figure 1revealed that pH has an important influence on fluorescence

312 Effect of pH on Fluorescence As shown in Figure 2effect of pH on fluorescence excitation and emission spectraof 4MU at various pHswere studied In acidic conditions (pH197ndash672) along with the decrease in pH fluorescence inten-sity declined 120582ex centered at 320 nm and 120582em red shiftedslightly from 445 nm to 455 nm In the range of pH 712ndash1043 along with the increase in pH fluorescence emissionat 445 nm enhanced but the excitation band centered at320 nm declined meanwhile a new excitation band centeredat 360 nmemerged and an iso-fluorescence point [30] formedat 330 nm In the range of pH 1080ndash1338 along with theincrease in pH fluorescence emission at 445 nm graduallyquenched while 120582ex and 120582em remained constant

The relationship between pH and fluorescence intensitywas summarized in Figure 2(d)

The variation of fluorescence spectra implies that thesolution equilibrium ormolecular structure of 4MU changedalong with the change in pH According to Figure 2 wesuppose that the proton dissociation and hydrolysis processof 4MU is as shown in Figure 3

The structure of 4MU contains a benzene ring (linkedwith 7-OH) and a lactonic ring (include lactone bond and2-C=O) In strong acid conditions the 2-carbonyl oxygenprotonated [28 30] to form a cationic species (type I) leadingto a decrease in fluorescence intensity and a red shift inemissionwavelength In near neutral conditions (pH40ndash70)4MU exist mainly as molecular form (type II) which havestrong fluorescence with 120582ex of 320 nm and 120582em of 445 nmIn weak alkaline conditions (pH 90ndash120) 7-hydroxyl protondissociated and 4MU exist mainly as anion form (type III)which have stronger fluorescence with 120582ex of 360 nm and120582em of 445 nm In strong alkaline conditions (pH gt 120) thehydrolysis of lactone bond take place [28 31] leading to afluorescence quenching

313 Ionization Constant of 7-Hydroxyl Proton With theabove discussion ionization constant of 7-OH proton can bedetermined in suitable conditionsUsing the pH-fluorescencemethod described in Section 24 the ionization constant of7-hydroxyl proton was determined to be p119870

119886= 785 plusmn 003

(Table 1)

Table 1 Determination of ionization constant of 4MU

pH F lg[(119865HB minus 119865)(119865 minus 119865B)]lowast p119870

119886Average p119870

119886

752 1601 minus030 782

785 plusmn 003777 2195 minus007 784798 2672 011 787812 3074 026 786828 3427 041 787lowast

119865HB = 2118 119865B = 4753

250 300 350 400 450 500 550

0

500

1000

1500

2000

2500Fl

uore

scen

ce in

tens

ity

120582 (nm)

Methanol6915

9095100

Figure 4 Fluorescence spectra of 4MU in aqueous solutionscontaining different amounts of methanol 119888

4MU 185 ngmLminus1 pH598 120582ex120582em 320 nm445 nm

In addition according to the relationship between flu-orescence intensity and pH we can graphically estimatethe ionization constant In Figure 2(d) the abscissa of theintersection of curve (a) and curve (b) is about 785 whichis just the p119870

119886

The ionization constant determined above is close to theionization constant of umbelliferone p119870

119886= 761 plusmn 003 [28]

The little difference between the two constants reflects theimpact of 4-methyl on the acidity of 7-hydroxyl proton

314 Effect of Solvent on Fluorescence Solvent polarity usu-ally has a profound effect on the emission spectral propertiesof fluorophores and further influences the sensitivity of afluorimetric method Figure 4 shows the fluorescence spectraof 4MU in aqueous solutions containing different amounts ofmethanol Along with the increase percentage of methanolfluorescence emission band centered at 445 nm gradually


300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(c)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(d)

Figure 5 3D fluorescence spectra of four kinds of Chinese herbal medicines in CDC (a) Xihuangcao c 400 120583gmLminus1 (b) Yinchen c400 120583gmLminus1 (c) Chuanxinlian c 400 120583gmLminus1 (d) Dahuang c 100120583gmLminus1 Contour interval R

declined while a new emission band centered at 380 nmemerged When the percentage of methanol reached 100the 120582em blue shifted to 380 nm however the fluorescenceintensity was less than that in aqueous solutions Thereforewe choose water as the appropriate solvent in establishing afluorimetric method in the following study and control themethanol percentage in aqueous solution no more than 20to obtain sensitive and stable fluorescence

315 Measurement of Fluorescence Quantum Yield of 4MUUsing the method described in Section 25 quantum yieldsof 4MU in near neutral (pH 598) and weak alkaline (pH975) conditions were measured as shown in Table 2 Innear neutral solution 4MU exist as molecular form itsquantumyield at excitationwavelength 320 nmwasmeasuredto be 074 In weak alkaline solution 4MU exist as anionform its quantum yield at excitation wavelength 360 nmwas measured to be 095 These results indicate that 4MU is

an excellent fluorophore A fluorimetric method for deter-mination of 4MU in Chinese patent drug should be verysensitive either in near neutral or in weak alkaline conditions

32 Determination of 4MU in Compound Dantong Capsule

321 3D Fluorescence Spectra of Four Kinds of Chinese HerbalMedicines in CDC The key point for developing a fluorimet-ric method is to understand the fluorescent properties of theanalyte and other components in the sample then find a wayto avoid interference of the coexistent components on thefluorescence of the analyte For these reason 3D fluorescencespectra of four kinds ofChinese herbalmaterials inCDCweremeasured and shown in Figure 5

From Figure 5 we showed that all the four kinds ofChinese herbal medicines contain fluorescent componentsThe interference of these components on the fluorescenceof 4MU seems to be a serious problem However we noted


300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)

Figure 6 3D fluorescence spectra of CDC solution at different pH 119888CDC 553 ngmLminus1 (a) pH 585 (b) pH 922 Contour interval R

Table 2 Measurement of fluorescence quantum yield of 4MU in different condition

120582exnmQuinine bisulphate 4MU (pH 598) 4MU (pH 975)

119860 119865lowast

119884 119860 119865lowast

119884 119860 119865lowast

119884

313 00365 105566 055 00440 178748 077320 00391 104878 051 00488 190213 074350 00411 123457 057 00440 220646 095360 00320 95986 057 00481 239780 095119865lowast wavelength range of integral fluorescence intensity 380 nmsim600 nm

that the concentrations of these herbal medicines in Figure 5(400 120583gmLminus1 sim 100120583gmLminus1) were much higher than theconcentration of 4MU in Figure 1 (185 ngmLminus1) So whetherthese herbal medicines in CDC interfere with the fluores-cence of 4MU should be considered further from the viewpoint of concentration

322 3D Fluorescence Spectra of CDC Figure 6 shows the 3Dfluorescence spectra of CDC dilute solutions at pH 585 andat pH 922 Comparing Figure 6 to Figure 1 we figured outthat the two pictures are basically the same The fluorescencepeaks presented in Figure 5 are not emerged in Figure 6Thisresult indicated that the fluorescence of those four herbalmedicines in CDC is too weak to be seen either because oftheir low concentration or due to their poor luminous abilitySo we can conclude that the coexistent components in CDCdo not interfere with the fluorescence of 4MU The contentof 4MU in CDC can be determined simply by a fluorimetricmethod

From Figures 6 and 2 we also know that the fluorimetricmethodmay be performed at near neutral or at weak alkalinepH For simplicity we prefer the method to be performed atnear neutral pH so that the fluorescence intensity of CDCwater solution can be measured directly without using abuffer solution

323 Determination of 4MU in CDC by a Standard CurveMethod A series of standard aqueous solutions containingdifferent amounts of 4MU from 155 to 310 ngmLminus1 wereprepared Fluorescence spectra as shown in Figure 7(a) andfluorescence intensity at measuring wavelength of 120582ex120582em =320 nm445 nm of each solution were measured A linear cal-ibration curve of fluorescence intensity against concentrationwas plotted as shown in Figure 7(b)The regression equationobtained was 119868

119865= 55 + 1041119888 (ngmLminus1) with correlation

coefficient 119877 = 09998 (119899 = 13)A number of solutions containing different amounts of

CDC were prepared and their fluorescence intensities atwavelength of 120582ex120582em = 320 nm445 nm were recordedUsing the calibration curve the 4MU content in CDC samplewas determined to be 3335 plusmn 010 as shown in Table 3

324 Determination of 4MU in CDC by a Standard AdditionMethod As shown in Figure 8 a standard addition methodwas employed to verify the result obtained aboveThe regres-sion equation obtained was 119868

119865= 6369 + 1053119888 (ngmLminus1)

with correlation coefficient R = 09998 (119899 = 5) The contentof 4MU in CDC sample was calculated to be 333 whichwas coincident with the result obtained by standard curvemethod


250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

0 5 10 15 20 25 30 35

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)

Figure 7 (a) Fluorescence spectra of 4MU in different concentrations (b) plot of fluorescence intensity versus concentration pH 598120582ex120582em 320 nm445 nm

250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

minus10 minus5 0 5 10 15 20 25

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)

Figure 8 (a) Fluorescence spectra and (b) plot of fluorescence intensity versus concentration of a standard addition method for thedetermination of 4MU in CDC sample pH 598 120582ex120582em 320 nm445 nm

Table 3 Determination of 4MU inCDC by standard curvemethod

No 1 2 3 4 5CDC added (VmL) 020 040 060 080 100Fluorescence intensity (119868

119865) 6435 1285 1924 2561 3217

4MU determined (ngmLminus1) 613 123 184 245 3084MU content in CDC () 333 334 333 333 335Average content () 334 plusmn 01

According to the CDC drug label the content of 4MUin CDC is 100mg per grain (03 g) It is evident that theanalytical result of this method agrees with the drug label

4 Conclusion

Hymecromone (4MU) is an excellent fluorophore Acidityand solvent have important influence on its fluorescence Innear neutral aqueous solutions 4MU exist mainly as neutralmolecular form which can be produced strong fluorescenceat 445 nm In weak alkaline solutions 4MU exist mainly asanion form which can be produced stronger fluorescenceat 445 nm In methanol solution the fluorescence peak at445 nm blue shifted to 380 nm and the fluorescence intensitydeclined in a certain extentThere is a good linear relationshipbetween fluorescent intensity and 4MU concentration The3D fluorescence spectra of Chinese patent drug CDC dilutesolutions are very similar to the 3D fluorescence spectra of4MU indicating that the main fluorescent component in


CDC is 4MU The coexistent components in CDC do notinterfere with the fluorescence of 4MU because of their lowconcentration or poor luminous ability So the content of4MU in CDC can be determined simply by fluorimetricmethod without preseparation

Abbreviations

CDC Compound Dantong Capsule (FufangDantong Capsule)

120582ex Maximum excitation wavelength120582em Maximum emission wavelength

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

This work was supported by the National Natural Sci-ence Foundation of China (nos 20675025 20975029 and81173496)

References

[1] Z Zhou N Li and A Tong ldquoA new coumarin-based fluores-cence turn-on chemodosimeter for Cu2+ in waterrdquo AnalyticaChimica Acta vol 702 no 1 pp 81ndash86 2011

[2] L Y Wang H H Li and D R Cao ldquoA new photoresponsivecoumarin-derived Schiff base chemosensor selectively for Al3+and Fe3+ and fluorescence ldquo turn-onrdquo under room lightrdquo Sensorsand Actuators B vol 181 pp 749ndash755 2013

[3] K Li N Li X Chen and A Tong ldquoA ratiometric fluorescentchemodosimeter for Cu(II) in water with high selectivity andsensitivityrdquo Analytica Chimica Acta vol 712 pp 115ndash119 2012

[4] H X Xu X Q Wang C L Zhang Y P Wu and Z PLiu ldquoCoumarin-hydrazone based high selective fluorescencesensor for copper(II) detection in aqueous solutionrdquo InorganicChemistry Communications vol 34 pp 8ndash11 2013

[5] X B Huang Y Dong Q W Huang and Y X ChengldquoHydrogen bond induced fluorescence recovery of coumarin-based sensor systemrdquo Tetrahedron Letters vol 54 pp 3822ndash3825 2013

[6] N Chattopadhyay A Mallick and S Sengupta ldquoPhotophysi-cal studies of 7-hydroxy-4-methyl-8- (41015840-methylpiperazin-11015840-yl)methylcoumarin a new fluorescent chemosensor for zinc andnickel ions inwaterrdquo Journal of Photochemistry andPhotobiologyA vol 177 no 1 pp 55ndash60 2006

[7] P Mladenka K Macakova L Zatloukalova et al ldquoIn vitrointeractions of coumarins with ironrdquo Biochimie vol 92 no 9pp 1108ndash1114 2010

[8] M Mazzei E Nieddu M Miele et al ldquoActivity of Mannichbases of 7-hydroxycoumarin against Flaviviridaerdquo Bioorganicand Medicinal Chemistry vol 16 no 5 pp 2591ndash2605 2008

[9] B Sarkanj M Molnar M Cacic and L Gille ldquo4-Methyl-7-hydroxycoumarin antifungal and antioxidant activity enhance-ment by substitution with thiosemicarbazide and thiazolidi-none moietiesrdquo Food Chemistry vol 139 pp 488ndash495 2013

[10] H Morohashi A Kon M Nakai et al ldquoStudy of hyaluro-nan synthase inhibitor 4-methylumbelliferone derivatives on

human pancreatic cancer cell (KP1-NL)rdquo Biochemical and Bio-physical Research Communications vol 345 no 4 pp 1454ndash1459 2006

[11] S S Bhattacharyya S Paul S K Mandal A Banerjee NBoujedaini and A R Khuda-Bukhsh ldquoA synthetic coumarin(4-Methyl-7 hydroxy coumarin) has anti-cancer potentialsagainst DMBA-induced skin cancer in micerdquo European Journalof Pharmacology vol 614 no 1ndash3 pp 128ndash136 2009

[12] K Harada H Kubo Y Tomigahara et al ldquoCoumarins as novel17120573-hydroxysteroid dehydrogenase type 3 inhibitors for poten-tial treatment of prostate cancerrdquo Bioorganic and MedicinalChemistry Letters vol 20 no 1 pp 272ndash275 2010

[13] A Kultti S Pasonen-Seppanen M Jauhiainen et al ldquo4-Methylumbelliferone inhibits hyaluronan synthesis by deple-tion of cellular UDP-glucuronic acid and downregulation ofhyaluronan synthase 2 and 3rdquo Experimental Cell Research vol315 no 11 pp 1914ndash1923 2009

[14] I P Kostova I I Manolov I N Nicolova and N D DanchevldquoNew metal complexes of 4-methyl-7-hydroxycoumarin sodi-um salt and their pharmacological activityrdquo Il Farmaco vol 56no 9 pp 707ndash713 2001

[15] I Kostova I Manolov I Nicolova S Konstantinov andM Karaivanova ldquoNew lanthanide complexes of 4-methyl-7-hydroxycoumarin and their pharmacological activityrdquo Euro-pean Journal of Medicinal Chemistry vol 36 no 4 pp 339ndash3472001

[16] L D Li and G Z Jin ldquoLuminescence properties of 4-Methyl-7-hydroxy-coumarinrdquo Chinese Journal of Analytical Chemistryvol 22 no 5 pp 440ndash444 1994

[17] S Dong HMa X Li M Sun and X Duan ldquoSynthesis of a newwater-soluble polymeric probe and its fluorescent propertiesfor ratiometric measurement of near-neutral pHrdquo AnalyticalLetters vol 37 no 14 pp 2937ndash2948 2004

[18] I Jones J Hamilton R Srivastava and P Galloway ldquoEffect ofneutral and acid pH on the fluorescence of 4-methylumbellifer-one and the implications for dry blood spot assaysrdquo MolecularGenetics and Metabolism vol 108 no 2 article S51 2013

[19] Pharmacopoeia Committee of Chinese Ministry of HealthTraditional Chinese Medicine Drugs Preparation vol 5 1991

[20] A Abate V Dimartino P Spina et al ldquoHymecromone in thetreatment of motor disorders of the bile ducts a multicenterdouble-blind placebo-controlled clinical studyrdquo Drugs underExperimental and Clinical Research vol 27 no 5-6 pp 223ndash2312001

[21] L Zhang B Y Li and A L Yang ldquoStudies on the qualitystandard for Compound Dantong Capsulerdquo Tianjin Pharmacyvol 16 no 5 pp 15ndash17 2004

[22] L Zhang and J Zhu ldquoDetermination of the content anddissolution of hymecromone in Compound Dantong Capsulesby HPLCrdquo Traditional Chinese Drug Research amp Clinical Phar-macology vol 17 no 3 pp 222ndash224 2006

[23] L R Zheng D Liang and Y Chen ldquoDetermination of 7-hydroxy-4-methylcoumarin in Compound Dantong Capsuleby HPLCrdquo Pharmaceutical and Clinical Research vol 16 no 4pp 333ndash334 2008

[24] Y JWei 3D Fluorescent Fingerprint of Chinese Herbal MedicineScience Press Beijing China 2012

[25] F Yang C G Liu and Y J Wei ldquoFluorimetric analysis ofpaeonol in Chinese herbal medicine Cynanchi Paniculati Radixby aluminum ion-sensitized fluorescencerdquo Acta PharmaceuticaSinica B vol 2 no 3 pp 294ndash299 2012


[26] L PWang and Y JWei ldquoFluorimetric analysis of camptothecinin Chinese herbal medicine common Camptotheca fruitrdquo ActaPharmaceutica Sinica vol 47 no 10 pp 1370ndash1374 2012

[27] S J Wang Q Zhang L P Wang and Y J Wei ldquoFluorescentproperties of arctiin and arctigenin and fluorimetric analysis ofarctiin in traditional Chinese medicine Arctii Fructusrdquo Journalof Instrumental Analysis vol 33 2014

[28] J Zhang C Liu and Y Wei ldquoFluorescence quantum yield andionization constant of umbelliferonerdquoHuaxue Tongbao vol 74no 10 pp 957ndash960 2011

[29] J G Xu and Z B Wang Fluorimetry Science Press BeijingChina 3rd edition 2007

[30] C G Liu Y Z Xu Y J Wei et al ldquoFluorescence spectra andprotonation of ofloxacinrdquo Spectroscopy and Spectral Analysisvol 25 no 4 pp 584ndash587 2005

[31] L Y Kong Coumarin Chemistry Chemical Industry PressBeijing China 1st edition 2008

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Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


300 350 400 450 500 550

200

250

300

350

400120582

ex(n

m)

120582em (nm)

Rayleighscattering

Ramanscattering

Fluorescencecontour

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)

Figure 1 3D fluorescence spectra of 4MU aqueous solution at different pH 1198884MU 185 ngmLminus1 (a) pH 598 (b) pH 975 Contour interval

R

[24] Moreover a number of fluorimetric methods have beendeveloped for quantitative determination of active compo-nents in medicinal materials such as paeonol in CynanchiPaniculati Radix (Xuchangqing) [25] camptothecin in com-mon Camptotheca fruit (Xishuguo) [26] and arctiin inArctii Fructus (Niubangzi) [27] In this study the fluorescentproperties of 4MU are investigated and its quantum yieldand 7-hydroxyl proton ionization constant are measuredThefluorescent properties of CDC and its four consisting Chineseherbal medicines are also studied Based on the spectraldifferences between 4MU and other components in CDCa fluorimetric method is proposed for the determination of4MU in CDC sample without preseparation

2 Experimental

21 Apparatus Fluorescence measurements were performedon a Hitachi (Tokyo Japan) F-7000 spectrofluorimeterequipped with a xenon lamp 1 cm quartz cell and a UV-29 filter placed into the emission light path to removesecondary spectrum The excitation and emission slits (bandpass) 5 nm5 nmwere used throughout the work Absorptionspectra were recorded using a Shimadzu (Kyoto Japan) UV-2501PC recording spectrophotometer with 1 cm quartz cellAn Orion (Beverly USA) 868 pHISE meter was used for pHmeasurement

22 Chemicals and Materials Hymecromone (4MU serialno 100241-200503 a reagent for quantitative analysis molec-ular weight 17617) and Chinese herbal medicine (compar-ison drug for qualitative identification) Xihuangcao (serialno 121488-200501) Yinchen (serial no 120950-200305)Chuanxinlian (serial no 121082-200302) and Dahuang(serial no 120984-200301) were purchased from the NationalInstitute for the Control of Pharmaceuticals and BiologicalProducts of China (Beijing China) 4MU stock solution was

prepared by dissolving 361mg reagent in 100mL methanol(TEDIA HPLC grade) and diluted to appropriate concentra-tion with methanol as needed Compound Dantong Capsule(CDC product lot no 100601) was produced by NationalTsing Hua Pharmaceutical Co Ltd Hubei-day Saint ChinaQuinine bisulphate (HPLC grade) was acquired from JampKScientific Ltd (Beijing China) its solution was prepared bydissolving 39147mg reagent in 500mL 005mol Lminus1 H

2SO4

and diluted to appropriate concentration with 005mol Lminus1H2SO4when it was used Britton-Robinson buffer solution

was a mixture of phosphoric acid boric acid and acetic acid(each 002mol Lminus1) and adjusted to an appropriate pH byaddition of 01mol Lminus1 NaOH solution All the buffer chem-icals were of analytical grade The water used throughoutthe study was doubly-deionized and verified to be free fromfluorescence

23 General Procedure for Spectral Measurement A seriesof 10mL volumetric flasks was added appropriate amountof 4MU or CDC and buffer solutions The mixtures werediluted to the mark with water and mixed well Fluorescenceor absorption spectra were measured at room tempera-ture Meanwhile Raman scattering of water at excitationwavelength of 350 nm was measured (expressed as R forcalibration of 3D fluorescence spectra) [24]

24 Determination of Ionization Constant A number ofsolutions containing the same concentration of weak acidHB and different pH were prepared Fluorescence intensity 119865and pH of each solution were measured Then the ionizationconstant p119870

119886was calculated according to the following

equation [28]

p119870119886= pH minus lg119865HB minus 119865

119865 minus 119865B (1)


250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500Fl

uore

scen

ce in

tens

itypH672

631

576

504

240

225

215

197

120582 (nm)

(a)

5000

4000

3000

2000

1000

0

250 300 350 400 450 500 550

Fluo

resc

ence

inte

nsity

pH1043

996

975

944

742

728

722

712

120582 (nm)

(b)

5000

4000

3000

2000

1000

0

250 300 350 400 450 500 550

Fluo

resc

ence

inte

nsity

pH1080

1141

1185

1211

1311

1321

1330

1338

120582 (nm)

(c)

0 2 4 6 8 10 12 14pH

1

25000

4000

3000

2000

1000

0

Fluo

resc

ence

inte

nsity

(d)





119884119906= 119884119903sdot119865119906

119865119903

sdot119860119903

119860119906

(2)

where 119884119906and 119884












OH+ HO OO O O

O

Ominus

Ominus

CH3 CH3 CH3CH3

O minusOminusOHO

H+H+H+


I II III IV








119886= 785 plusmn 003

(Table 1)



119886Average p119870

119886

752 1601 minus030 782


119865HB = 2118 119865B = 4753

250 300 350 400 450 500 550

0

500

1000

1500

2000

2500Fl

uore

scen

ce in

tens

ity

120582 (nm)

Methanol6915

9095100




119886


119886= 761 plusmn 003 [28]




300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(c)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(d)









300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)




119860 119865lowast

119884 119860 119865lowast

119884 119860 119865lowast

119884










119865= 6369 + 1053119888 (ngmLminus1)



250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

0 5 10 15 20 25 30 35

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)


250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

minus10 minus5 0 5 10 15 20 25

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)




119865) 6435 1285 1924 2561 3217



4 Conclusion




Abbreviations





Acknowledgment


References











































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500Fl

uore

scen

ce in

tens

itypH672

631

576

504

240

225

215

197

120582 (nm)

(a)

5000

4000

3000

2000

1000

0

250 300 350 400 450 500 550

Fluo

resc

ence

inte

nsity

pH1043

996

975

944

742

728

722

712

120582 (nm)

(b)

5000

4000

3000

2000

1000

0

250 300 350 400 450 500 550

Fluo

resc

ence

inte

nsity

pH1080

1141

1185

1211

1311

1321

1330

1338

120582 (nm)

(c)

0 2 4 6 8 10 12 14pH

1

25000

4000

3000

2000

1000

0

Fluo

resc

ence

inte

nsity

(d)





119884119906= 119884119903sdot119865119906

119865119903

sdot119860119903

119860119906

(2)

where 119884119906and 119884












OH+ HO OO O O

O

Ominus

Ominus

CH3 CH3 CH3CH3

O minusOminusOHO

H+H+H+


I II III IV








119886= 785 plusmn 003

(Table 1)



119886Average p119870

119886

752 1601 minus030 782


119865HB = 2118 119865B = 4753

250 300 350 400 450 500 550

0

500

1000

1500

2000

2500Fl

uore

scen

ce in

tens

ity

120582 (nm)

Methanol6915

9095100




119886


119886= 761 plusmn 003 [28]




300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(c)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(d)









300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)




119860 119865lowast

119884 119860 119865lowast

119884 119860 119865lowast

119884










119865= 6369 + 1053119888 (ngmLminus1)



250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

0 5 10 15 20 25 30 35

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)


250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

minus10 minus5 0 5 10 15 20 25

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)




119865) 6435 1285 1924 2561 3217



4 Conclusion




Abbreviations





Acknowledgment


References











































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


OH+ HO OO O O

O

Ominus

Ominus

CH3 CH3 CH3CH3

O minusOminusOHO

H+H+H+


I II III IV








119886= 785 plusmn 003

(Table 1)



119886Average p119870

119886

752 1601 minus030 782


119865HB = 2118 119865B = 4753

250 300 350 400 450 500 550

0

500

1000

1500

2000

2500Fl

uore

scen

ce in

tens

ity

120582 (nm)

Methanol6915

9095100




119886


119886= 761 plusmn 003 [28]




300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(c)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(d)









300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)




119860 119865lowast

119884 119860 119865lowast

119884 119860 119865lowast

119884










119865= 6369 + 1053119888 (ngmLminus1)



250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

0 5 10 15 20 25 30 35

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)


250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

minus10 minus5 0 5 10 15 20 25

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)




119865) 6435 1285 1924 2561 3217



4 Conclusion




Abbreviations





Acknowledgment


References











































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(c)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(d)









300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)




119860 119865lowast

119884 119860 119865lowast

119884 119860 119865lowast

119884










119865= 6369 + 1053119888 (ngmLminus1)



250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

0 5 10 15 20 25 30 35

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)


250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

minus10 minus5 0 5 10 15 20 25

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)




119865) 6435 1285 1924 2561 3217



4 Conclusion




Abbreviations





Acknowledgment


References











































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(a)

300 350 400 450 500 550

200

250

300

350

400

120582ex

(nm

)

120582em (nm)

(b)




119860 119865lowast

119884 119860 119865lowast

119884 119860 119865lowast

119884










119865= 6369 + 1053119888 (ngmLminus1)



250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

0 5 10 15 20 25 30 35

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)


250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

minus10 minus5 0 5 10 15 20 25

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)




119865) 6435 1285 1924 2561 3217



4 Conclusion




Abbreviations





Acknowledgment


References











































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

0 5 10 15 20 25 30 35

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)


250 300 350 400 450 500 550

0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

inte

nsity

120582 (nm)

(a)

minus10 minus5 0 5 10 15 20 25

0

500

1000

1500

2000

2500

3000

3500

IF

4MU (ng mLminus1)

(b)




119865) 6435 1285 1924 2561 3217



4 Conclusion




Abbreviations





Acknowledgment


References











































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



Abbreviations





Acknowledgment


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










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article Fluorescent Properties of Hymecromone and ...Fluorescent Properties of Hymecromone and Fluorimetric ... Journal of Spectroscopy 250 300 350 400 450 500 550 0 500 1000