development and validation of a hplc-uv method for the

Research ArticleDevelopment and Validation of a HPLC-UV Method forthe Evaluation of Ellagic Acid in Liquid Extracts of Eugeniauniflora L (Myrtaceae) Leaves and Its Ultrasound-AssistedExtraction Optimization

Paulo Isaac Dias Assunccedilatildeo1 Edemilson Cardoso da Conceiccedilatildeo2

Leonardo Luiz Borges1 and Joelma Abadia Marciano de Paula1

1Campus Anapolis de Ciencias Exatas e Tecnologicas Universidade Estadual de Goias Caixa Postal 45975132-903 Anapolis GO Brazil2Laboratorio de Pesquisa Desenvolvimento amp Inovacao de Bioprodutos Universidade Federal de Goias Caixa Postal 13174605-170 Goiania GO Brazil

Correspondence should be addressed to Joelma Abadia Marciano de Paula joelmapaulauolcombr

Received 6 March 2017 Revised 17 April 2017 Accepted 20 April 2017 Published 6 July 2017

Academic Editor Arpita Basu

Copyright copy 2017 Paulo Isaac Dias Assuncao et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

A simple HPLC-UV method has been developed and validated for the quantification of ellagic acid (EA) in ethanol extracts ofEugenia uniflora L (Myrtaceae) leavesThe ultrasound-assisted extraction (UAE) optimizationwas performed using a Box Behnkendesign (33) combinedwith response surfacemethodology to study the effects of the ethanol concentration ( ww) extraction time(minutes) and temperature (∘C) on the EA concentration The optimized results showed that the highest extraction yield of EA byUAE was 260 120583gmLminus1 when using 44 (ww) ethanol as the solvent 22 minutes as the extraction time and 59∘C as the extractiontemperature The concentration of EA in relation to the predicted value was 937 plusmn 04 UAE showed a strong potential for EAextraction

1 Introduction

Eugenia uniflora L popularly known as Surinam cherry isone of more than 5500 species from the Myrtaceae family[1]The species is native to South America despite being quitewidespread in other continents [2] According to popular usethe infusion of the leaves is used as a diuretic antirheumaticand antipyretic as well as in the control of hypotension bloodcholesterol and uric acid in the urine [3ndash6]

Some properties of E uniflora leaves such as theirhypotensive activity anti-inflammatory activity and reduc-tion of postprandial hyperglycemia have already been veri-fied by in vivo studies [4 7 8]

Many of the species of the genus Eugenia draw atten-tion for their essential oil and tannin contents In 1997 itwas reported for the first time that eugeniflorin D1 andeugeniflorinD2 as ellagitannins were present in themethanol

extract of the leaves of E uniflora [9] The authors also notedthe presence of oenothein B The antiviral and antiprolifer-ative activities of these compounds were later demonstrated[10 11]

From the hydrolysis of the ester bonds of ellagitanninshexaidroxidifenoil units are formed and these in turnundergo a process of spontaneous lactonization leading tothe formation of ellagic acid (EA) [12] EA is a phenoliccompound with poor water solubility whose presence hasalready been reported in human plasma [13]This compoundis often associated with several biological activities amongwhich the antioxidant anti-inflammatory and antiprolifera-tive activities stand out [13ndash18]

Various methods of chemical and biochemical analysishave been employed to determine tannins and their deriva-tives in different plant species [19ndash22] However the methodof preparation of the material to evaluate these compounds

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 1501038 9 pageshttpsdoiorg10115520171501038

2 Evidence-Based Complementary and Alternative Medicine

has shown potential for further study and optimizationtechniques

The method of ultrasound-assisted extraction (UAE) isapproached as a modern extraction alternative [23] Com-pared with the conventional extraction methods that involveheating and reflux UAE offers an alternative with lowerlosses due to phenolic oxidation hydrolysis and ionization[24] In view of other modern techniques such as extractionin supercritical fluids and assisted by microwaves UAE isconsidered to be a low-cost simple and efficient technique[25] Its extraction mechanism is attributed to the cavitationphenomenon the interaction of mechanical forces and theirthermal impact This provides the disruption of the cell wallthus reducing the particle size and increasing mass transferacross the cell membrane [26]

Improving the performance of a system process orproduct to obtain greater benefit can be understood by theconcept of optimization [27] In recent years tools for multi-variate analysis have been frequently used in the optimizationof analytical methods mainly due to the reduction in thenumber of experiments and consequently in the researchcosts [28ndash31] Moreover these tools allow the developmentof mathematical models in the evaluation of the interactionsbetween variables [32]

This work aimed to develop and validate a high-per-formance liquid chromatography (HPLC) analytical methodfor the quantification of EA in liquid extracts of E unifloraleaves and optimize the UAE of this chemical marker

2 Materials and Methods

21 Chemicals and Reagents Ethanol 95 (vv) (VetecDuque de Caxias SP Brazil) and water filtered througha Milli-Q apparatus (Millipore Bellerica MA USA) wereused to prepare the extraction solutions Methanol ace-tonitrile (J T Baker Center Valley PA USA) and Milli-Q water were used in standard sample and mobile phasepreparations Formic acid acetic acid orthophosphoric acidtrifluoroacetic acid and trichloroacetic acid (Sigma St LouisMO USA) were used as acidifying agents for the mobilephase Ellagic acid gallic acid catechin epicatechin rutinhesperidin and quercetin were of analytical grade (Sigma StLouis MO USA) and used as external standards

22 Equipment The ultrasound-assisted extractions (UAE)were performed with an ultrasonic device Unique USC2800A 40 kHz (Indaiatuba SP Brazil) A Varian HPLCProStar (Palo Alto CA USP) with a 240 pump 310 samplemanager 20599 UV detector and Star (version 62) softwarewas used Chromatographic separation was performed usinga Supelco C18 column (250mm times 46mm 5120583m) All of thesolutions were filtered through a 045 120583mmembrane (MerckBellerica MA USA) SPE was carried out on a C-18 Supelcocartridge (60mL capacity)

23 Plant Materials E uniflora leaves were collected in June2014 in the city of Anapolis Goias state Brazil (16∘17101584013810158401015840Sand 48∘57101584022710158401015840W altitude 1074m) The authenticity of

the material collected was verified by Dra Myrlei Lucienedos Santos and voucher specimens were deposited in theherbarium of the Universidade Estadual de Goias with theregistration number HUEG2090 The material was dried atroom temperature and ground in aWileymillThe packagingwas done in containers protected from light and moistureThe characterization of the powder obtained from the leavesof E uniflora [33] revealed a moisture content of 02 plusmn890 a total ash content of 914 plusmn 01 a swelling index of416 plusmn 003mLgminus1 in water and a particle size approximately250 120583m

24 Preliminary Tests for the HPLC-UVMethod DevelopmentFor the election of a chemical marker the evaluation ofthe chromatographic profile of the liquid extract from theleaves of E uniflora was carried out Extraction solutionswere prepared in 70 ethanol (ww) at a drugsolvent ratioof 15 (wv) and kept in an ultrasound bath for 30 minutes atroom temperature The chromatographic conditions for theevaluation of the profile were adapted from the work of Kimet al [34]

The identification of the constituents present in thechromatographic profile of the extract was carried out bycomparison with the retention times (RT) of peaks in thestandard solutions (gallic acid ellagic acid catechin epicat-echin hesperidin quercetin and rutin at a concentrationof 100120583gmLminus1) Among the compounds identified EA wasselected as the chemical marker to study the development ofthe analytical methodology

Once the chemical marker was selected three alcoholcontents were evaluated for the extraction solution 50 70and 90 ethanol (ww) The preparation of the extractswas performed in triplicate for each of the solvents Thedrugsolvent ratio was maintained at 15 (wv) and theprocess was conducted in an ultrasound bath for 30 minutesat room temperature The chromatographic evaluation of theextracts was performed using the samemethodology adaptedfrom Kim et al [34]

25 Sample Preparation Based on the data of the preliminarystudies the sample preparation for the development of themethodology used 50 ethanol (ww) as the extractionsolvent The drugsolvent mixture was maintained at a ratioof 10 (wv) to allow a better evaluation of the peak ofthe chemical marker and the process was conducted inultrasound equipment for 30 minutes at room temperature

26 Solid Phase Extraction (SPE) SPE was performedaccording to the method reported by Lopes et al [35]One milliliter of filtered extract was transferred to the SPEcartridge Elution was accomplished with a mobile phasecomposed of water acetonitrile (80 20) The eluted extractwas collected in a 10mL volumetric flask until the volumewascompleted

27 Chromatographic Conditions The chromatographic con-ditions for the evaluation of EA in the extracts were obtainedafter testing different methods reported in the literature

Evidence-Based Complementary and Alternative Medicine 3

and varying some of these conditions [34 36] We alsoevaluated isocratic elution with mobile phases constitutedof wateracetonitrile or methanol three different acidify-ing agents were tested (trichloroacetic acid trifluoroaceticacid and orthophosphoric acid) flows ranging from 05 to12mLminminus1 andwavelength of 254 and 280 nmThroughoutthe development of this study the injection volume of thechromatographic system was kept constant at 10 120583L

28 HPLC-UVMethod Validation Theparameters and spec-ifications for the method validation were determined basedon the International Conference on the Harmonization(ICH) of Technical Requirements for the Registration ofPharmaceuticals for Human Use and the Brazilian legislation[37ndash40]

The system suitability was evaluated by six consecutiveinjections of the same sample solutionThe following param-eters were considered number theoretical plates 1198961015840 tailingand relative standard deviation (RSD) of the peak areas of EA

The selectivity was determined by comparing the chro-matograms obtained with the standard solution samplesolution mobile phase and blank (water methanol 80 20vv) in the detection of interferents through coelution

Linearity was determined by the analytical curves of theEA standard at five concentration levels (145 293 242 290and 338 120583gmLminus1) in methanol The analysis was performedin triplicate for each concentration level and injected into

the chromatographic system The linear and determinationcoefficients were calculated mathematically

A calibration curve was prepared in triplicate with sevendifferent concentrations of a standard solution of EA (64 96128 160 192 224 and 256 120583gmLminus1) in methanolThe limitof detection (LOD) and limit of quantification (LOQ) werecalculated from the three standard curves using the standarddeviation (SD119886) of the intercept with the 119910-axis and the slopeof the calibration curve (119878) according to

LOD =SD119886 times 3119878

LOQ =SD119886 times 10119878

(1)

Repeatability (intraday) and intermediate precision(interday) were evaluated The relative standard deviation(RSD) was determined for twelve injections (two injectionsfor each preparation) The intermediate precision wasevaluated by this same process performed on a different day

The accuracy of the method was measured through theanalyte recovery test Volumetric liquid extract solutionswereprepared (80 100 and 120 of the standard concentration inthe linear range) in methanol with and without the additionof a known standard amount of EA (160 120583gmLminus1) Theaccuracy was calculated for every level according to

Accuracy = sample conc with standard minus sample conc without standardstandard theoretical concentration

times 100 (2)

The robustness of the method was evaluated by analyzingthe results of the EA peak area of sample preparationsobtained from both the original analysis conditions andmodified conditionsThe analysis was performed in triplicateand the results were evaluated by the RSD calculationThe following parameters were changed mobile phase flowinjection volume and column lot

29 Ultrasound-Assisted Extraction Process The optimiza-tion of the EA UAE process in leaves of E uniflora wasperformed from the experiments set out in the Box Behnkendesign (33) for the following parameters in the evaluationof the EA concentration ethanol concentration ( ww)extraction time (minutes) and temperature (∘C) The com-plete design including three replicates at the central point ispresented in Table 1

A second-order model was employed to express theeffects of the independent variables on the EA concentration(see (3))

119910 = 1205730 +119896

sum119894=1

120573119894119909119894 +119896

sum119894=1

1205731198941198941199092119894 +sumsum120573119894119895119909119894119909119895 (3)

where 119910 is the predicted response 1205730 is a constant 119909119894 and119909119895 are independent variables and 120573119894 120573119894119894 and 120573119894119895 are the

linear quadratic and interactive coefficients of the modelrespectively

The experimental results were analyzed using Statisticsoftware version 120 and the coefficients were interpretedusing the F test Three main tools were used in data analysisanalysis of variance (ANOVA) regression analysis and aresponse surface plot Effects were considered significant fora 119901 value lt 005

3 Results and Discussion

31 Preliminary Tests for the HPLC-UVMethod DevelopmentAmong the seven standards tested gallic acid (626 minutes)and EA (2284 minutes) were identified in the ethanol extract(Figure 1)

The EA peak was selected as a chemical marker for theacquisition of quality parameters of the plant material Thepeak area was considered as a parameter in the markerelection and larger areas are related to higher concentrationsof the analyte Therefore the results are indicative that EAwould be one of the main constituents present in the extract

EA is known to present low solubility in water andespecially at lower pH the most phenolic compound is notionized therefore the use of alcoholic solutions to opti-mize the solubilization and extraction of this compounds is


252015105(Minutes)

minus43

0

100

200

300

400

(mAU

)

WI

8

WI

16A

Z

231

62

734

302

63

266

410

84

359

521

6 602

16

409

689

47

250

931

0

100

7310

485

109

9911

238

117

38

125

06

132

1113

712

138

3214

160

145

4814

921 15

681

159

95

166

2916

944

172

1917

696 17

997

181

86

189

5019

180

195

1319

873

200

1920

248

205

1520

862

212

8921

495

217

2322

196

226

0523

112

234

9423

996

246

8124

828

250

6325

278

259

1826

352

277

74

O OH

OH

OHHO

Gallic acid

Ellagic acid

O

O

O

OOH

OHHO

HO

Figure 1 Chromatographic profile of E uniflora leaves Chromatographic conditions Supelco Analytical C18 column (250 times 46mm 5120583m)mobile phase water (A)acetonitrile (B) (0min 98 A and 2 B 5min 95 A and 5 B 12min 80 A and 20 B 15min 75 A and 25 B18min 60 A and 40 B 25min 20 A and 80 B 28min 95 A and 5 B and 30min 98 A and 2 B) flow rate of 10mLminminus1 120582 =280 nm and injection volume of 10 120583L

Table 1 Levels of variables for the Box Behnken (33) experimentaldesign

Symbols Independent variables Levelsminus1 0 +1

1199091 Ethanol ( ww) 40 60 801199092 Temperature (∘C) 30 45 601199093 Extraction time (minutes) 20 30 401199091 = ethanol 1199092 = temperature 1199093 = extraction time

recommended [12 41] Bala et al found an aqueous solubilityvalue of 97120583gmLminus1 for EA the value for methanol solubilitywas higher (671120583gmLminus1) [42]

Preliminary tests indicated that higher intensities andpeak areas were obtained by using 50 ethanol (ww) Ilbayet al evaluated different alcohol contents for the extraction ofphenolic compounds from Rosa canina L leaves [29] Theyfound the optimal conditions for the extraction included anethanol content approximately 40 (vv) Similar data werereported for the extraction of tannins from Dipteryx alataVogel fruits [30] As EA is a product of the hydrolysis ofellagitannins it is believed that a lower alcohol content wouldfavor the extraction of these components

32 Chromatographic Conditions The detection wavelengthwas optimized to 254 nm according to the maximum absorp-tion wavelength of EA as reported in the literature [3442] Among the evaluated chromatographic conditions theincrease in the initial ratio of acetonitrile in the elutiongradient from 2 to 15 contributed to a higher elutioncapacity of the mobile phase and caused many unidentifiedpeaks to coelute in the dead volume of the methodologyThis change made the methodology more specific for theevaluation of the EA peak (Figures 2(a) and 2(b))

It was observed that prolonging the duration of thegradient which has the elution of the EA peak (60 40wateracetonitrile) in agreement with a reduced flow of 10to 05mLminminus1 showed significant improvement in theresolution between the peaks This balance reached betweenthe flow and mobile phase proportion was maintained at10mLminminus1 and 15 of acetonitrile As this is the initialproportion of the gradient methodology an isocratic methodwas proposed for 20 minutes in a proportion of 85 15(wateracetonitrile) and at a flow rate of 10mLminminus1 Theresolution between the peaks was maintained (Figure 2(b))

The use of acetonitrile in liquid chromatography asorganic solvent is mainly encouraged due to its lower absorp-tion interference at low wave-lengths compared to other


105 15 20(Minutes)

minus25

0

25

50

75

100

125

(mAU

)

X 199618 Minutes

AZ

II+

II+

IIminus FP+

FPminus

Ellagic acidRT = 1222 min

Y 0580 mAU

(a)

minus20

0

25

50

75

100

125

(mAU

)

AZ

105 15 20(Minutes)

II+

II+

IIminusFP

+FP

minus


(b)

Figure 2 HPLC-UV chromatograms from ellagic acid analytical standard (a) and E uniflora leaf extract (b) using the validated conditionsSupelco Analytical C18 column (250 times 46mm 5 120583m) mobile phase wateracetonitrile (85 15) flow rate of 10mLminminus1 120582 = 254 nm andinjection volume of 10 120583L RT = retention time

Table 2 Box Behnken (33) experimental design and concentrationsof ellagic acid (EA) for the UAE of E uniflora leaves

1199091 () 1199092 (∘C) 1199093 (min) EA (120583gmLminus1)

(1) 40 20 45 217(2) 80 20 45 92(3) 40 40 45 145(4) 80 40 45 81(5) 40 30 30 131(6) 80 30 30 87(7) 40 30 60 206(8) 80 30 60 145(9) 60 20 30 80(10) 60 40 30 111(11) 60 20 60 263(12) 60 40 60 173(13) 60 30 45 150(14) 60 30 45 146(15) 60 30 45 1641199091 = ethanol 1199092 = temperature 1199093 = extraction time

solvents asmethanol and in the EA elution it reacts similar tosolvent chosen for the plant extraction For chromatographicpurposes reaching an adequate analyte partition relation oranalyte distribution balance during the elution is neededThisconcept is brought in light by the retention factor also knownas capacity factor (1198961015840) In this study the adequate 1198961015840 value forEAwas acquired by the proportion of organic solvent and theacidification of the mobile phase [42 43]

To enhance the peak resolution and asymmetry (tailing)of the EA peak three different acidifying agents (orthophos-phoric acid trichloroacetic acid and trifluoroacetic acid)were tested in the mobile phase in the isocratic method

Table 3 Summary of the effects of factors and their significance(119901 value)

Factor 119901 value Effect1199091 0008167lowast minus0007351199092 0074161lowastlowast 00017041199093 0033663lowast minus00035511990912 065681 000025411990922 0004965lowast 00094511990932 0349469lowast minus000059611990911199092 0084095lowastlowast 00030511990911199093 0463396 minus00008511990921199093 0023548lowast minus0006051199091 = ethanol 1199092 = temperature 1199093 = extraction time lowast119901 lt 005 lowastlowast119901 lt 01

Among them trichloroacetic acid (005 wv) showedgreater impact on the peak resolution and asymmetry param-eters

33 Solid Phase Extraction (SPE) SPE allowed the extractionof the vegetable matrix components without influencing theevaluation of EA A difference in extract color indicating agreater purification was observed Because the SPE processallows the same EA quantification in a purified extractand may improve the analytical column utilization it wasincorporated into the methodology for the preparation of thesample of liquid extract

The chromatogram obtained from the E uniflora leafextract after SPE using the HPLC-UV developed conditionsis presented in Figure 2(b)

34 HPLC-UV Method Validation The validation method-ology followed the sample preparation with a drugsolvent


EA co

nc (120583

gmLminus1)

28262422201816141210864

Extraction time (min)

40

3638

3230

34

282624

2220

18 Ethanol ( ww)

3540 45

5055

6065 70

7580

85

(a)

EA co

nc (120583

gmLminus1)

Ethanol ( ww)

Temperature ( ∘C) 3540 45

5055

6065

7075

8085

30

25

20

15

10

5

0

6560

5550

4540

3530

25

(b)

EA co

nc (120583

gmLminus1

)


Temperature ( ∘C)

30

25

20

15

10

5

35

6560

5550

4540

3530

25

4240

383634

3230

2826

2422

2018

(c)

Figure 3 Response surface for the EA concentration fromUAE experiments of E uniflora leaves (a) Effects of the extraction time (min) andethanol content ( ww) (b) Effects of the temperature (∘C) and ethanol content ( ww) (c) Effects of the temperature (∘C) and extractiontime (min)

ratio of 10 (wv) using 50 ethanol (vv) as the extractionsolvent The extract was subjected to an ultrasound bathfor 30 minutes at room temperature and SPE has beenincorporated into the end of the process The followingchromatographic conditions were validated Supelco Ana-lytical C18 column (250 times 46mm 5 120583m) mobile phasewateracetonitrile (85 15) acidified to 005 (wv) withtrichloroacetic acid flow rate of 10mLminminus1 wavelength of254 nm and injection volume of 10 120583L

No interference was observed by coelution of the diluentor mobile phase at the same retention time of EA at 254 nmdemonstrating the selectivity of this methodology

The method was linear in the range of 145 to338 120583gmLminus1 The analytical curve presented a linearcorrelation coefficient (119903) of 0994 and a determinationcoefficient (1199032) of 0988 These data are in accordance withthe specifications adopted [38]

The limits found based on three analytical curves ofthe EA standard were 066 120583gmLminus1 for the LOD and222120583gmLminus1 for the LOQ The LOD determined from thelinear regression calculation is considered as the lowestdetectable concentration but it is not necessarily quantifiableThe lowest measurable concentration of the analyte in asolution is translated in the value of the LOQ [44]


In this study the precision data were expressed bydetermining the relative standard deviation (RSD) obtainedby the ratio between the standard deviation of the dataand the average The RSD value for the areas of EA in therepeatability test was 351The RSD between the data of twodays was 446 These results were also in accordance withthe specifications (RSD lt 15) [39]

Themethodology showed accuracy represented in recov-ery values between 10513 and 11061 for the test Thesedata are in agreement with the specification adopted for testsin complex matrices (80ndash120) [37]

In the evaluation of the robustness all of the RSDsremained below 5 and the percentage of the assessmentof the averages in relation to the normal conditions variedbetween 7330 and 10732 whereas for a variation between90 and 110 compared to the normal conditions themethod-ology showed no robustness to a variation of minus10 in itsinjection volume

35 Effects of the Ultrasound-Assisted Extraction Parameterson the EA Concentration The measurement of EA wasdetermined based on a calibration curve (119903 = 099841 119910 =64680119909 minus 23970) obtained with the validated methodologyThe EA concentration data of the 15 experiments generatedby the Box Behnken (33) design are presented in Table 2 Theconcentrations of EA varied between 80 and 263 expressedin 120583gmLminus1

The model presented a 119901 value of 0000358 showing nosignificance to the lack of fit (119901 = 0097575) A summaryof the effects is shown in Table 3 The central idea of theanalysis of variance (ANOVA) is to compare the variances ofthe different treatments due to the variance of experimentalerror and thereby determine the significance of the regressionadopted to provide responses [27]

As the extraction of phenolic compounds depends largelyon the polarity of the solvent it is possible that a single solventis not efficient for the extraction of bioactive compounds[25 42ndash45] In this work the alcohol content of the extractionsolution showed a negative linear effect on the concentrationof EA indicating that lower degrees of alcohol would providegreater extraction of the marker for the leaves of E unifloraThis reaffirms the preliminary assessment of the behaviorof the alcohol content in the development of the analyticalmethodology and also showed agreement with the data ofother studies [27ndash30]

The linear effects of the independent variables timeand extraction temperature were significant as well as theinteraction between them (Figure 3(c))

With the exception of the extraction temperature theother variables showed negative effects on the concentrationof EA as shown in the response surface graphics illustratedin Figure 3 The temperature can impact the extractiveprocesses leading to an increase in the substances extractionwhile causing losses of those that are thermosensitive [31]It is possible that there is a higher release of EA amongother phenolic compounds from the hydrolysis of the ellag-itannins accentuated by temperature which may explain theeffect of this variable on the concentration of the marker[28]

Based on the analysis of response surface data usinggeneral function optimization the UAE conditions for max-imizing EA extraction were determined Thus the followingextraction parameters have been suggested alcohol contentof 44 (ww) 22minutes of extraction time and temperatureof 59∘C for a predicted value of 260 120583gmLminus1 of EA Theseconditions were confirmed from extractions conducted intriplicate and the concentration of EA in relation to thepredicted value was 937 plusmn 04

4 Conclusions

A HPLC-UV method has been developed and validated forquantifying EA in E uniflora ethanol extracts In obtainingthe liquid extract by UAE the optimization study has beenshown to be able to predict responses to the levels of the vari-ables studied An EA concentration of 937 plusmn 04 comparedto the predicted value under the optimized conditions (44alcohol content (ww) 22 minutes of extraction time andtemperature of 59∘C) was foundThe UAEmethod presenteda strong potential as a methodology for the extraction of EAfrom the leaves of E uniflora

Conflicts of Interest

The authors declare no competing financial interest

Acknowledgments

This work was carried out with the support of the Universi-dade Estadual de Goias (UEG) and Fundacao de Amparo aPesquisa do Estado de Goias (FAPEG)

References

[1] A H Thornhill L W Popple R J Carter S Y W Ho andM D Crisp ldquoAre pollen fossils useful for calibrating relaxedmolecular clock dating of phylogenies A comparative studyusing Myrtaceaerdquo Molecular Phylogenetics and Evolution vol63 no 1 pp 15ndash27 2012

[2] H Lorenzi and F J A Matos Plantas Medicinais no BrasilNativas e Exoticas Nova Odessa Sao Paulo Brazil 2nd edition2008

[3] C B Alice V M F Vargas G A A B Silva et al ldquoScreeningof plants used in south Brazilian folk medicinerdquo Journal ofEthnopharmacology vol 35 no 2 pp 165ndash171 1991

[4] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[5] A T Henriques M E Sobral A D Cauduro et al ldquoAromaticplants from Brazil II The chemical composition of someEugenia essential oilsrdquo Journal of Essential Oil Research vol 5no 5 pp 501ndash505 1993

[6] G Schmeda-Hirschmann C Theoduloz L Franco E Ferro Band A R De Arias ldquoPreliminary pharmacological studies onEugenia uniflora leaves xanthine oxidase inhibitory activityrdquoJournal of Ethnopharmacology vol 21 no 2 pp 183ndash186 1987

[7] E E S Schapoval S M Silveira M LMiranda C B Alice andA THenriques ldquoEvaluation of some pharmacological activities


of Eugenia uniflora Lrdquo Journal of Ethnopharmacology vol 44no 3 pp 137ndash142 1994

[8] I Arai S Amagaya Y Komatsu et al ldquoImproving effectsof the extracts from Eugenia uniflora on hyperglycemia andhypertriglyceridemia in micerdquo Journal of Ethnopharmacologyvol 68 no 1-3 pp 307ndash314 1999

[9] M-H Lee S Nishimoto L-L Yang et al ldquoTwo macrocyclichydrolysable tannin dimers fromEugenia uniflorardquo Phytochem-istry vol 44 no 7 pp 1343ndash1349 1997

[10] M-H Lee J-F Chiou K-Y Yen and L-L Yang ldquoEBV DNApolymerase inhibition of tannins fromEugenia uniflorardquoCancerLetters vol 154 no 2 pp 131ndash136 2000

[11] A Vitalone J McColl D Thome L G Costa and B TitaldquoCharacterization of the effect of Epilobium extracts on humancell proliferationrdquo Pharmacology vol 69 no 2 pp 79ndash87 2003

[12] J M Landete ldquoEllagitannins ellagic acid and their derivedmetabolites a review about source metabolism functions andhealthrdquoFoodResearch International vol 44 no 5 pp 1150ndash11602011

[13] N P Seeram L S Adams S M Henning et al ldquoIn vitroantiproliferative apoptotic and antioxidant activities of puni-calagin ellagic acid and a total pomegranate tannin extract areenhanced in combination with other polyphenols as found inpomegranate juicerdquo Journal of Nutritional Biochemistry vol 16no 6 pp 360ndash367 2005

[14] F Festa T Aglitti G Duranti R Ricordy P Perticone and RCozzi ldquoStrong antioxidant activity of ellagic acid inmammaliancells in vitro revealed by the Comet assayrdquo Anticancer Researchvol 21 no 6 pp 3903ndash3908 2001

[15] J N Losso R R Bansode A Trappey II H A Bawadi andR Truax ldquoIn vitro anti-proliferative activities of ellagic acidrdquoJournal of Nutritional Biochemistry vol 15 no 11 pp 672ndash6782004

[16] N P Seeram R Lee and D Heber ldquoBioavailability of ellagicacid in human plasma after consumption of ellagitannins frompomegranate (Punica granatum L) juicerdquoClinica Chimica Actavol 348 no 1-2 pp 63ndash68 2004

[17] L Zhang YWang andMXu ldquoAcid hydrolysis of crude tanninsfrom infructescence of Platycarya strobilacea Sieb et Zucc toproduce ellagic acidrdquo Natural Product Research vol 28 no 19pp 1637ndash1640 2014

[18] J Beltran-Heredia J Sanchez-Martın andM A Davila-AcedoldquoOptimization of the synthesis of a new coagulant from a tanninextractrdquo Journal of Hazardous Materials vol 186 no 2-3 pp1704ndash1712 2011

[19] J Lee S Kim H Namgung et al ldquoEllagic acid identi-fied through metabolomic analysis is an active metabolitein strawberry (ldquoSeolhyangrdquo) regulating lipopolysaccharide-induced inflammationrdquo Journal of Agricultural and Food Chem-istry vol 62 no 18 pp 3954ndash3962 2014

[20] C A O Pengfei ldquoStudy on the extraction technology of tanninfrom Mountain Walnut Shellsrdquo Agricultural Biothecnology vol3 no 5 pp 21ndash23 2014

[21] L-L Zhang M Xu Y-M Wang D-M Wu and J-H ChenldquoOptimizing ultrasonic ellagic acid extraction conditions frominfructescence of platycarya strobilacea using response surfacemethodologyrdquoMolecules vol 15 no 11 pp 7923ndash7932 2010

[22] M Gasperotti D Masuero U Vrhovsek G Guella and FMattivi ldquoProfiling and accurate quantification of Rubus ellag-itannins and ellagic acid conjugates using direct uplc-q-tofhdms and hplc-dad analysisrdquo Journal of Agricultural and FoodChemistry vol 58 no 8 pp 4602ndash4616 2010

[23] M Yolmeh M B Habibi Najafi and R Farhoosh ldquoOptimisa-tion of ultrasound-assisted extraction of natural pigment fromannatto seeds by response surface methodology (RSM)rdquo FoodChemistry vol 155 pp 319ndash324 2014

[24] H Li B Chen and S Yao ldquoApplication of ultrasonic techniquefor extracting chlorogenic acid from Eucommia ulmodies Oliv(E ulmodies)rdquoUltrasonics Sonochemistry vol 12 no 4 pp 295ndash300 2005

[25] X S Wang Y F Wu G Y Chen W Yue Q L Liang andQ N Wu ldquoOptimisation of ultrasound assisted extraction ofphenolic compounds from Sparganii rhizoma with responsesurface methodologyrdquo Ultrasonics Sonochemistry vol 20 no 3pp 846ndash854 2013

[26] M B Hossain N P Brunton A Patras et al ldquoOptimizationof ultrasound assisted extraction of antioxidant compoundsfrommarjoram (Origanummajorana L) using response surfacemethodologyrdquoUltrasonics Sonochemistry vol 19 no 3 pp 582ndash590 2012

[27] M A Bezerra R E Santelli E P Oliveira L S Villar and L AEscaleira ldquoResponse surface methodology (RSM) as a tool foroptimization in analytical chemistryrdquo Talanta vol 76 no 5 pp965ndash977 2008

[28] L L Borges F S Martins E C Conceicao and D SilveiraldquoOptimization of the spray-drying process for developing jabu-ticaba waste powder employing response surfacemethodologyrdquoJournal of Food Process Engineering vol 4 no 1 pp 1ndash9 2015

[29] Z Ilbay S Sahin and S I Kirbaslar ldquoOptimisation ofultrasound-assisted extraction of rosehip (Rosa canina L) withresponse surface methodologyrdquo Journal of the Science of Foodand Agriculture vol 93 no 11 pp 2804ndash2809 2013

[30] F S Martins L L Borges J R Paula and E C ConceicaoldquoImpact of different extraction methods on the quality ofDipteryx alata extractsrdquo Brazilian Journal of Pharmacognosyvol 23 no 3 pp 521ndash526 2013

[31] J AM Paula L F Brito K L F N Caetano et al ldquoUltrasound-assisted extraction of azadirachtin from dried entire fruits ofAzadirachta indica A Juss (Meliaceae) and its determinationby a validated HPLC-PDA methodrdquo Talanta vol 149 pp 77ndash84 2016

[32] S L C Ferreira R E Bruns H S Ferreira et al ldquoBox-Behnkendesign an alternative for the optimization of analyticalmethodsrdquo Analytica Chimica Acta vol 597 no 2 pp 179ndash1862007

[33] Farmacopeia Brasileira Brasılia Brazil 5th edition 2010[34] J-H Kim C-S Seo S-S Kim andHHa ldquoSimultaneous deter-

mination of gallic acid ellagic acid and eugenol in Syzygiumaromaticum and verification of chemical antagonistic effectby the combination with Curcuma aromatica using regressionanalysisrdquo Journal of Analytical Methods in Chemistry vol 2013Article ID 375294 7 pages 2013

[35] G C Lopes A C Sanches C E Toledo A C Isler andJ C Mello ldquoDeterminacao quantitativa de taninos em tresespecies de Stryphnodendron por cromatografia lıquida de altaeficienciardquo Brazilian Journal of Pharmaceutical Sciences vol 45no 1 pp 135ndash143 2009

[36] A L Oliveira E Destandau L Fougere and M Lafosse ldquoIso-lation by pressurised fluid extraction (PFE) and identificationusing CPC and HPLCESIMS of phenolic compounds fromBrazilian cherry seeds (Eugenia uniflora L)rdquo Food Chemistryvol 145 pp 522ndash529 2014


[37] International Conference on Harmonization ldquoTopic Q2 (R1)Validation of analytical procedures text and methodologyrdquo2005 httpwwwichorg

[38] Brazil Agencia Nacional de Vigilancia Sanitaria RE 8992003-ANVISA 2003

[39] Brazil Agencia Nacional de Vigilancia Sanitaria ldquoGuia deorientacao para registro deMedicamento Fitoterapico e registroe notificacao de Produto Tradicional Fitoterapicordquo ANVISA2014


[41] A Gonzalez-Sarrıas R Garcıa-Villalba M A Nunez-Sanchezet al ldquoIdentifying the limits for ellagic acid bioavailability acrossover pharmacokinetic study in healthy volunteers afterconsumption of pomegranate extractsrdquo Journal of FunctionalFoods vol 19 no 2015 pp 225ndash235 2015

[42] I Bala V Bhardwaj S Hariharan and M N V R KumarldquoAnalytical methods for assay of ellagic acid and its solubilitystudiesrdquo Journal of Pharmaceutical and Biomedical Analysis vol40 no 1 pp 206ndash210 2006

[43] United States Pharmacopeia (USP) 39 (NF34) Chromatogra-phy chapter 621 2016

[44] T Klein R Longhini and J C P Mello ldquoDevelopment ofan analytical method using reversed-phase HPLC-PDA for asemipurified extract of Paullinia cupana var sorbilis (guarana)rdquoTalanta vol 88 pp 502ndash506 2012

[45] C Gan and A A Latiff ldquoOptimisation of the solvent extrac-tion of bioactive compounds from Parkia speciosa pod usingresponse surface methodologyrdquo Food Chemistry vol 124 no 3pp 1277ndash1283 2011

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


has shown potential for further study and optimizationtechniques

The method of ultrasound-assisted extraction (UAE) isapproached as a modern extraction alternative [23] Com-pared with the conventional extraction methods that involveheating and reflux UAE offers an alternative with lowerlosses due to phenolic oxidation hydrolysis and ionization[24] In view of other modern techniques such as extractionin supercritical fluids and assisted by microwaves UAE isconsidered to be a low-cost simple and efficient technique[25] Its extraction mechanism is attributed to the cavitationphenomenon the interaction of mechanical forces and theirthermal impact This provides the disruption of the cell wallthus reducing the particle size and increasing mass transferacross the cell membrane [26]

Improving the performance of a system process orproduct to obtain greater benefit can be understood by theconcept of optimization [27] In recent years tools for multi-variate analysis have been frequently used in the optimizationof analytical methods mainly due to the reduction in thenumber of experiments and consequently in the researchcosts [28ndash31] Moreover these tools allow the developmentof mathematical models in the evaluation of the interactionsbetween variables [32]

This work aimed to develop and validate a high-per-formance liquid chromatography (HPLC) analytical methodfor the quantification of EA in liquid extracts of E unifloraleaves and optimize the UAE of this chemical marker

2 Materials and Methods

21 Chemicals and Reagents Ethanol 95 (vv) (VetecDuque de Caxias SP Brazil) and water filtered througha Milli-Q apparatus (Millipore Bellerica MA USA) wereused to prepare the extraction solutions Methanol ace-tonitrile (J T Baker Center Valley PA USA) and Milli-Q water were used in standard sample and mobile phasepreparations Formic acid acetic acid orthophosphoric acidtrifluoroacetic acid and trichloroacetic acid (Sigma St LouisMO USA) were used as acidifying agents for the mobilephase Ellagic acid gallic acid catechin epicatechin rutinhesperidin and quercetin were of analytical grade (Sigma StLouis MO USA) and used as external standards

22 Equipment The ultrasound-assisted extractions (UAE)were performed with an ultrasonic device Unique USC2800A 40 kHz (Indaiatuba SP Brazil) A Varian HPLCProStar (Palo Alto CA USP) with a 240 pump 310 samplemanager 20599 UV detector and Star (version 62) softwarewas used Chromatographic separation was performed usinga Supelco C18 column (250mm times 46mm 5120583m) All of thesolutions were filtered through a 045 120583mmembrane (MerckBellerica MA USA) SPE was carried out on a C-18 Supelcocartridge (60mL capacity)

23 Plant Materials E uniflora leaves were collected in June2014 in the city of Anapolis Goias state Brazil (16∘17101584013810158401015840Sand 48∘57101584022710158401015840W altitude 1074m) The authenticity of

the material collected was verified by Dra Myrlei Lucienedos Santos and voucher specimens were deposited in theherbarium of the Universidade Estadual de Goias with theregistration number HUEG2090 The material was dried atroom temperature and ground in aWileymillThe packagingwas done in containers protected from light and moistureThe characterization of the powder obtained from the leavesof E uniflora [33] revealed a moisture content of 02 plusmn890 a total ash content of 914 plusmn 01 a swelling index of416 plusmn 003mLgminus1 in water and a particle size approximately250 120583m

24 Preliminary Tests for the HPLC-UVMethod DevelopmentFor the election of a chemical marker the evaluation ofthe chromatographic profile of the liquid extract from theleaves of E uniflora was carried out Extraction solutionswere prepared in 70 ethanol (ww) at a drugsolvent ratioof 15 (wv) and kept in an ultrasound bath for 30 minutes atroom temperature The chromatographic conditions for theevaluation of the profile were adapted from the work of Kimet al [34]

The identification of the constituents present in thechromatographic profile of the extract was carried out bycomparison with the retention times (RT) of peaks in thestandard solutions (gallic acid ellagic acid catechin epicat-echin hesperidin quercetin and rutin at a concentrationof 100120583gmLminus1) Among the compounds identified EA wasselected as the chemical marker to study the development ofthe analytical methodology

Once the chemical marker was selected three alcoholcontents were evaluated for the extraction solution 50 70and 90 ethanol (ww) The preparation of the extractswas performed in triplicate for each of the solvents Thedrugsolvent ratio was maintained at 15 (wv) and theprocess was conducted in an ultrasound bath for 30 minutesat room temperature The chromatographic evaluation of theextracts was performed using the samemethodology adaptedfrom Kim et al [34]

25 Sample Preparation Based on the data of the preliminarystudies the sample preparation for the development of themethodology used 50 ethanol (ww) as the extractionsolvent The drugsolvent mixture was maintained at a ratioof 10 (wv) to allow a better evaluation of the peak ofthe chemical marker and the process was conducted inultrasound equipment for 30 minutes at room temperature

26 Solid Phase Extraction (SPE) SPE was performedaccording to the method reported by Lopes et al [35]One milliliter of filtered extract was transferred to the SPEcartridge Elution was accomplished with a mobile phasecomposed of water acetonitrile (80 20) The eluted extractwas collected in a 10mL volumetric flask until the volumewascompleted

27 Chromatographic Conditions The chromatographic con-ditions for the evaluation of EA in the extracts were obtainedafter testing different methods reported in the literature









LOD =SD119886 times 3119878

LOQ =SD119886 times 10119878

(1)




times 100 (2)




119910 = 1205730 +119896

sum119894=1

120573119894119909119894 +119896

sum119894=1

1205731198941198941199092119894 +sumsum120573119894119895119909119894119909119895 (3)









252015105(Minutes)

minus43

0

100

200

300

400

(mAU

)

WI

8

WI

16A

Z

231

62

734

302

63

266

410

84

359

521

6 602

16

409

689

47

250

931

0

100

7310

485

109

9911

238

117

38

125

06

132

1113

712

138

3214

160

145

4814

921 15

681

159

95

166

2916

944

172

1917

696 17

997

181

86

189

5019

180

195

1319

873

200

1920

248

205

1520

862

212

8921

495

217

2322

196

226

0523

112

234

9423

996

246

8124

828

250

6325

278

259

1826

352

277

74

O OH

OH

OHHO

Gallic acid

Ellagic acid

O

O

O

OOH

OHHO

HO











105 15 20(Minutes)

minus25

0

25

50

75

100

125

(mAU

)

X 199618 Minutes

AZ

II+

II+

IIminus FP+

FPminus


Y 0580 mAU

(a)

minus20

0

25

50

75

100

125

(mAU

)

AZ

105 15 20(Minutes)

II+

II+

IIminusFP

+FP

minus


(b)



1199091 () 1199092 (∘C) 1199093 (min) EA (120583gmLminus1)











EA co

nc (120583

gmLminus1)

28262422201816141210864


40

3638

3230

34

282624

2220

18 Ethanol ( ww)

3540 45

5055

6065 70

7580

85

(a)

EA co

nc (120583

gmLminus1)

Ethanol ( ww)


5055

6065

7075

8085

30

25

20

15

10

5

0

6560

5550

4540

3530

25

(b)

EA co

nc (120583

gmLminus1

)


Temperature ( ∘C)

30

25

20

15

10

5

35

6560

5550

4540

3530

25

4240

383634

3230

2826

2422

2018

(c)
















4 Conclusions




Acknowledgments


References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























LOD =SD119886 times 3119878

LOQ =SD119886 times 10119878

(1)




times 100 (2)




119910 = 1205730 +119896

sum119894=1

120573119894119909119894 +119896

sum119894=1

1205731198941198941199092119894 +sumsum120573119894119895119909119894119909119895 (3)









252015105(Minutes)

minus43

0

100

200

300

400

(mAU

)

WI

8

WI

16A

Z

231

62

734

302

63

266

410

84

359

521

6 602

16

409

689

47

250

931

0

100

7310

485

109

9911

238

117

38

125

06

132

1113

712

138

3214

160

145

4814

921 15

681

159

95

166

2916

944

172

1917

696 17

997

181

86

189

5019

180

195

1319

873

200

1920

248

205

1520

862

212

8921

495

217

2322

196

226

0523

112

234

9423

996

246

8124

828

250

6325

278

259

1826

352

277

74

O OH

OH

OHHO

Gallic acid

Ellagic acid

O

O

O

OOH

OHHO

HO











105 15 20(Minutes)

minus25

0

25

50

75

100

125

(mAU

)

X 199618 Minutes

AZ

II+

II+

IIminus FP+

FPminus


Y 0580 mAU

(a)

minus20

0

25

50

75

100

125

(mAU

)

AZ

105 15 20(Minutes)

II+

II+

IIminusFP

+FP

minus


(b)



1199091 () 1199092 (∘C) 1199093 (min) EA (120583gmLminus1)











EA co

nc (120583

gmLminus1)

28262422201816141210864


40

3638

3230

34

282624

2220

18 Ethanol ( ww)

3540 45

5055

6065 70

7580

85

(a)

EA co

nc (120583

gmLminus1)

Ethanol ( ww)


5055

6065

7075

8085

30

25

20

15

10

5

0

6560

5550

4540

3530

25

(b)

EA co

nc (120583

gmLminus1

)


Temperature ( ∘C)

30

25

20

15

10

5

35

6560

5550

4540

3530

25

4240

383634

3230

2826

2422

2018

(c)
















4 Conclusions




Acknowledgments


References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















252015105(Minutes)

minus43

0

100

200

300

400

(mAU

)

WI

8

WI

16A

Z

231

62

734

302

63

266

410

84

359

521

6 602

16

409

689

47

250

931

0

100

7310

485

109

9911

238

117

38

125

06

132

1113

712

138

3214

160

145

4814

921 15

681

159

95

166

2916

944

172

1917

696 17

997

181

86

189

5019

180

195

1319

873

200

1920

248

205

1520

862

212

8921

495

217

2322

196

226

0523

112

234

9423

996

246

8124

828

250

6325

278

259

1826

352

277

74

O OH

OH

OHHO

Gallic acid

Ellagic acid

O

O

O

OOH

OHHO

HO











105 15 20(Minutes)

minus25

0

25

50

75

100

125

(mAU

)

X 199618 Minutes

AZ

II+

II+

IIminus FP+

FPminus


Y 0580 mAU

(a)

minus20

0

25

50

75

100

125

(mAU

)

AZ

105 15 20(Minutes)

II+

II+

IIminusFP

+FP

minus


(b)



1199091 () 1199092 (∘C) 1199093 (min) EA (120583gmLminus1)











EA co

nc (120583

gmLminus1)

28262422201816141210864


40

3638

3230

34

282624

2220

18 Ethanol ( ww)

3540 45

5055

6065 70

7580

85

(a)

EA co

nc (120583

gmLminus1)

Ethanol ( ww)


5055

6065

7075

8085

30

25

20

15

10

5

0

6560

5550

4540

3530

25

(b)

EA co

nc (120583

gmLminus1

)


Temperature ( ∘C)

30

25

20

15

10

5

35

6560

5550

4540

3530

25

4240

383634

3230

2826

2422

2018

(c)
















4 Conclusions




Acknowledgments


References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















105 15 20(Minutes)

minus25

0

25

50

75

100

125

(mAU

)

X 199618 Minutes

AZ

II+

II+

IIminus FP+

FPminus


Y 0580 mAU

(a)

minus20

0

25

50

75

100

125

(mAU

)

AZ

105 15 20(Minutes)

II+

II+

IIminusFP

+FP

minus


(b)



1199091 () 1199092 (∘C) 1199093 (min) EA (120583gmLminus1)











EA co

nc (120583

gmLminus1)

28262422201816141210864


40

3638

3230

34

282624

2220

18 Ethanol ( ww)

3540 45

5055

6065 70

7580

85

(a)

EA co

nc (120583

gmLminus1)

Ethanol ( ww)


5055

6065

7075

8085

30

25

20

15

10

5

0

6560

5550

4540

3530

25

(b)

EA co

nc (120583

gmLminus1

)


Temperature ( ∘C)

30

25

20

15

10

5

35

6560

5550

4540

3530

25

4240

383634

3230

2826

2422

2018

(c)
















4 Conclusions




Acknowledgments


References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















EA co

nc (120583

gmLminus1)

28262422201816141210864


40

3638

3230

34

282624

2220

18 Ethanol ( ww)

3540 45

5055

6065 70

7580

85

(a)

EA co

nc (120583

gmLminus1)

Ethanol ( ww)


5055

6065

7075

8085

30

25

20

15

10

5

0

6560

5550

4540

3530

25

(b)

EA co

nc (120583

gmLminus1

)


Temperature ( ∘C)

30

25

20

15

10

5

35

6560

5550

4540

3530

25

4240

383634

3230

2826

2422

2018

(c)
















4 Conclusions




Acknowledgments


References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























4 Conclusions




Acknowledgments


References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















development and validation of a hplc-uv method for the

Documents