Research ArticleDetermination of Polar Compounds in Guava Leaves Infusionsand Ultrasound Aqueous Extract by HPLC-ESI-MS
Elixabet Diacuteaz-de-Cerio12 Vito Verardo34 Ana Mariacutea Goacutemez-Caravaca12
Alberto Fernaacutendez-Gutieacuterrez12 and Antonio Segura-Carretero12
1Department of Analytical Chemistry Faculty of Sciences University of Granada Avenida Fuentenueva sn 18071 Granada Spain2Functional Food Research and Development Center Health Science Technological Park Avenida del ConocimientoBioregion Building 18100 Granada Spain3Department of Chemistry and Physics (Analytical Chemistry Area) University of Almerıa Carretera de Sacramento sn04120 Almerıa Spain4Research Centre for Agricultural and Food Biotechnology (BITAL) Agrifood Campus of International Excellence ceiA3Carretera de Sacramento sn 04120 Almerıa Spain
Correspondence should be addressed to Vito Verardo vitoverardouniboit
Received 10 December 2014 Revised 4 March 2015 Accepted 7 March 2015
Academic Editor Serkos A Haroutounian
Copyright copy 2015 Elixabet Dıaz-de-Cerio et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Literature lacks publications about polar compounds content in infusion or guava leaves tea Because of that a comparison betweendifferent times of infusion and a conventional ultrasound aqueous extract was carried out Several polar compounds have beenidentified by HPLC-ESI-MS and their antioxidant activity was evaluated by FRAP and ABTS assays Four different classes ofphenolic compounds (gallic and ellagic acid derivatives flavonols flavanones and flavan-3-ols) and some benzophenones weredetermined The quantification results reported that the order in terms of concentration of the classes of polar compounds in allsamples was flavonols gt flavan-3-ols gt gallic and ellagic acid derivatives gt benzophenones gt flavanones As expected the aqueousextract obtained by sonication showed the highest content in the compounds studied Significative differences were noticed aboutthe different times of infusion and five minutes was the optimal time to obtain the highest content in polar compounds using thisculinary method All the identified compounds except HHDP isomers and naringenin were positively correlated with antioxidantactivity
1 Introduction
The studies on antioxidant activity of plants have increaseddramatically in recent years because they are identifiedas natural antioxidant resources by traditional Chinesemedicine [1] Medicinal plants have usually been applied tocontrol the blood glucose or reduce the diabetic complica-tions they have the potential to increase the life span andquality of life in these patients [2] The increasing prevalenceof type 2 diabetes mellitus and the negative clinical outcomesobserved with the commercially available antidiabetic drugshave led to the investigation of new therapeutic approachesfocused on controlling postprandial glucose levels The useof carbohydrate digestive enzyme inhibitors from natural
resources could be a possible strategy to block dietary car-bohydrate absorption with less adverse effects than syntheticdrugs In fact some authors [3] reported in vitro and in vivostudies in relation to pancreatic alpha-amylase inhibitors ofplant origin and presented bioactive compounds of phenolicnature that exhibit anti-amylase activity
Guava leaves (Psidium guajava L) are considered nativeto Mexico but today they are extended throughout SouthAmerica Europe Africa and Asia Different studies consid-ered these leaves as a promising source of phenolic com-pounds for diabetes treatment [4] Several authors noticedthat oral administration of capsules containing aqueous leafextract from Psidium guajava L showed hypoglycemic effect[5]
Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 250919 9 pageshttpdxdoiorg1011552015250919
2 Journal of Chemistry
Recently Eidenberger and coworkers [6] investigated theeffect of extracts from Psidium guajava L leaves particu-larly the effects of main flavonol-glycoside components ondipeptidyl-peptidase IV (DP-IV) a key enzyme of blood glu-cose homoeostasis and finally indicated that guava extracthas a potential to exert the effect observed in vitro also inhumans after oral administration
In vivo experiments carried out by Cheng et al [7]reported that quercetin in the aqueous extract of guava leavespromotes glucose uptake in liver cells and as a consequencecontributes to the alleviation of hypoglycemia in diabetes
Usually guava leaf tea was consumed after infusion how-ever different infusion times were advice from productioncompany Because of that in the presentwork the antioxidantactivities of infusions obtained at different infusion timesand conventional ultrasound aqueous extracts of guava leaveswere evaluated and compared in terms of their compositionin polar compounds
2 Material and Methods
21 Chemicals Double-deionised water with conductivitylower than 182MΩ was obtained with a Milli-Q system(Millipore Bedford MA USA) Methanol LC-MS ldquooptimardquograde and acetonitrile were obtained from Fisher Scien-tific (Leicestershire UK) Acetic acid and the standardsgallic acid catechin ellagic acid naringenin quercetinand rutin were all from Sigma-Aldrich (Steinheim Ger-many) The reagents used to measure the antioxidant capac-ity TPTZ (246-tripyridyl-S-triazine) Trolox (6-hydroxy-2578-tetramethylchroman-2-carboxylic acid) ABTS (221015840-azinobis (3-ethylbenzothiazoline-6-sulfonate)) potassiumpersulfate and ferric sulfate were purchased from Sigma-Aldrich (St Louis MO USA) Sodium acetate ferric chlo-ride and hydrochloric acid were obtained from Panreac(Barcelona Spain)
22 Plant Material and Sample Preparation Fresh guavaleaves were harvested in Motril Spain (36∘4410158404310158401015840N3∘3110158401410158401015840O) They were middle age intense green leaves andthey were collected in February 2014 The environmentalconditions had mean maxmin temperature of 1810∘Cprecipitation of 0mm and saturated light duration thatranged from 955 to 1050 h dayminus1
The samples were air-dried and ground before theanalyses Two different extraction methodologies such asultrasound extraction and infusion were carried out
Conventional Ultrasound Extraction 05 g of dry guava leaveswas extracted with 15mL of water (times3) using a sonicatorBransonB3510 for 10min at room temperatureThen sampleswere centrifuged for 15min at 6000 rpm using a centrifugeto remove solids The supernatants were pooled evaporatedand dissolved in 2mL of 50 methanol This solution wasfiltered through a 020 120583m syringe filter and kept at minus20∘C inamber bottles to avoid degradation until analysis
Infusion Extraction For the infusion 1 g of dried guava leavesand 50mL of boiling water were used The extract was
prepared according to the method previously described byChen and Yen [8] where they prepared 5min infusion andcompared with infusion for 3 and 7min After the extractionby infusion for 3 5 and 7min the solution was raised to50mL with water filtered through a 020120583m syringe filterand kept at minus20∘C in amber bottles to avoid degradation untilanalysis
All extractions were made in triplicate
23 Trolox Equivalent Antioxidant Capacity (ABTS) AssayTheABTS assay which measures the reduction of the radicalcation of 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonate)(ABTS) by antioxidants was performed by using a methodpreviously described by Laporta and coworkers [9] Con-cisely ABTS radical cation was produced by reacting ABTSstock solution with 245mMpotassium persulfate in the darkat room temperature for 12ndash24 h before use The absorbanceof ABTS radical cation was adjusted to 070 (plusmn002) at734 nm A calibration curve was prepared with differentconcentrations of Trolox (0ndash20120583M)
24 Ferric-Reducing Antioxidant Power (FRAP) The reduc-ing power was evaluated according to the method validatedby Benzie and Strain [10] Briefly 300mM acetate buffer(pH 36) 10mM TPTZ in 40mM HCl and 20mM aqueousFeCl3were prepared andmixed (10 1 1) to obtain the FRAP
reagent The FRAP reagent was warmed to 37∘C beforereading its absorbance Then the samples were added Thechange in absorbance (593 nm) between the samples and theblank was related to the absorbance of an aqueous solution ofknown Fe(II) concentration prepared for calibration
25 HPLC-ESI-MS Analysis Phenolic and other polar com-pounds in the extracts obtained from guava leaves were iden-tified using a method introduced by Chang et al [11] withslight modifications Briefly HPLC analyses were performedusing a HP 1100 Series instrument (Hewlett Packard Wilm-ington DE USA) equipped with a binary pump deliverysystem a degasser (model G1322A) an autosampler (Auto-matic Liquid Sampler ALSmodel G1312A) aHPdiode-arrayUV-VIS detector (DAD model G1315A) and a quadrupoleHP-Mass Spectrometer Detector (MSD model G1946A)integration and data elaboration were performed usingChemstation software (Hewlett Packard) A PhenomenexLuna C18 analytical column (150mm times 20mm particle size3 120583m) (Phenomenex Inc Torrance CA USA) was used forpolar compounds separation All analyses were carried outat room temperature using the gradient proposed by Changet al [11] MS analysis was carried out using an electrosprayionization (ESI) interface in negative ionization mode atthe following conditions drying gas flow (N
2) 90 Lmin
nebulizer pressure 50 psi gas drying temperature 350∘Ccapillary voltage 3500V and fragmentor voltage and scanrange 100V andmz 50ndash1000 respectively
Phenolic standards of interest such as gallic acid catechinellagic acid naringenin and rutin were used for quantifi-cation of phenolic compounds in guava leaf extracts Theidentified compounds were quantified on the basis of theirpeak area and compared with calibration curves obtained
Journal of Chemistry 3
with the corresponding standards and then expressed as 120583ggof extract
26 Statistical Analysis The results reported in this studyare the averages of three repetitions (119899 = 3) Fisherrsquosleast significance difference (LSD) test and Pearsonrsquos linearcorrelations both at 119875 lt 005 were evaluated using Statistica60 (2001 StatSoft Tulsa OK)
3 Results and Discussion
31 Identification of Polar Compounds Phenolic and otherpolar compounds were identified by their elution orderUVvis spectra and MS characteristics compared withreported literature values and by coinjection with availablestandards (Table 1)
About phenolic compounds four different classes identi-fied as gallic and ellagic acid derivatives flavonols flavanonesand flavan-3-ols were determined
Thirteen gallic and ellagic acid derivatives were deter-mined Three compounds (1 2 and 3) with molecular ionat mz 481 and fragment at mz 301 were identified as hex-ahydroxydiphenic acid (HHDP) glucose and its presence inguava was previously reported by Okuda and coworkers [12]Gallic acid (compound 4) was identified according to its MSdata (mz 169 andmz 125) and by coelution with a chemicalstandard Two compounds (6 and 11) with [MndashH]minus at mz783 and two fragments at mz 481 and 301 corresponding toloss of ellagic acid were detected This fragmentation patternwas assigned to pedunculagincasuariin compounds thesecompounds were described in guava leaves by Okuda et al[12]
Two compounds (10 and 12) with molecular ion at mz951 and fragments at mz 783 and 481 were also determinedThese compounds were identified as geraniin isomers [13]
Two compounds (15 and 18) showing significant [MndashH]minussignals at mz 785 with fragment ions at mz 615 and at mz301were foundThis fragmentation pattern corresponded to adigalloyl-HHDP-glucose structure probably tellimagrandinI isomer This compound was previously detected in guavaleaves by Okuda et al [12] Compound 17 atmz 935 reporteda fragment ion at mz 783 this fragmentation pattern wasassigned to casuarinincasuarictin and this compound wasdescribed in guava tea by Yamanaka and coworkers [13]Compound 30 was identified as ellagic acid due its coelutionwith commercial standard Finally guavin B (compound 46)that reported a molecular ion at mz 693 was identifiedaccording to Okuda et al [14]
Moreover ten flavan-3-ol derivatives were determinedCompounds 5 and 42 showed amolecular ion atmz 609 andthree fragments atmz 441 423 and 305 (gallocatechin unit)these compounds were identified as prodelphinidin B2 andits isomer and their presence in guava leaves was noticed byQarsquoDan et al [15]
Compounds 7 and 9 showed a molecular ion at mz 593and two fragments atmz 425 and 407 According to QarsquoDanet al [15] these compounds were identified as prodelphinidindimer (4120572-8)
Two compounds (8 and 19) with [MndashH]minus at mz 305and fragment ions at mz 221 and 179 were identified asgallocatechin isomers and their presence in guava leaves wasreported by QarsquoDan et al [15] Three procyanidin dimers([MndashH]minus atmz 577) were also described (compounds 13 14and 21)
Catechin compound (16) was identified by mass spectradata elaboration and coelution with a commercial standard
The flavonols were themost representative phenolic com-pounds in fact eighteen flavonol-derivatives were identifiedFour compounds (compounds 20 24 27 and 29) reported amolecular ion atmz 449 and twomajor fragments atmz 316and 317 According to Chang et al [11] these compounds wereidentified as myricetin-arabinosidexylopyranoside isomers
Two flavonol compounds (compounds 22 and 23) withmolecular ion at mz 479 and fragments at mz 317 and316 were identified as myricetin-hexoside isomers [11] Twocompounds (25 and 26) corresponding to [MndashH]minus signals atmz 615 were also detected Based on their molecular weightand the presence of two fragments at mz 463 and 301 theywere assigned to quercetin-galloylhexoside isomers and theirpresence in guava leaves was reported by Park et al [16]
Two compounds at mz 301 were detected (28 and 47)moreover they showed the same fragment ion at mz 151Quercetin standard solution was injected and because of thatcompound 28 was assigned to morin and compound 47 wasassigned to quercetin their presence in guava leaves wasreported by several authors [11 17]
Compounds detected at mz 463 (31 and 33) withfragment ion at mz 301 corresponded to hyperin andisoquercitrin respectively They have previously been foundin leaves of guava by Eidenberger et al [6]
Quercetin glucuronide (32) withmolecular ion atmz 477and fragment ions atmz 433 and 301was identified accordingto Chang et al [11]
Three compounds (34 35 and 37) reported the samemolecular ion (mz 433) and a fragment ion atmz 301 (corre-sponding to quercetin aglycone) according to their retentiontimes they were identified as reynoutrin guajaverin andavicularin as reported by Chang et al [11]
Quercitrin (38) was identified at mz 447 and fragmentionmz 300 according to Park et al [16]
A flavanone compound namely naringenin (mz 271)was detected and identified by analyzing themass spectra andby coelution with a chemical standard [18] Compound 39withmz 585 was identified as guavinoside C [19]
Six benzophenone compounds were also determined inguava leaves sampleThe [MndashH]minus ion atmz 543 revealed thepresence of two compounds namely guavinoside A isomerstheir presence in guava leaves was noticed by Matsuzaki andcoworkers [19]
Finally four compounds with mz 571 (40 43 44 and45) were identified as guavinoside B isomers according toMatsuzaki et al [19]
32 Quantification of Polar Compounds Comparisonbetween different times of infusion and a conventionalultrasound aqueous extract was carried out due to several
4 Journal of Chemistry
Table 1 Identification of polar compounds in guava leaves by HPLC-DAD-ESI-MS
publications about phenolic and other polar compoundscontent in infusion or guava leaves tea
Quantification of polar compounds was performed bypreparing five calibration curves with the standards availablegallic acid catechin ellagic acid naringenin and rutin Forthose with no commercial standard available quantificationwas carried out comparing with compounds bearing similarstructures
It is important to underline that the quantification resultsreported that the order in terms of concentration of thefamilies of polar compounds in all samples decreased in thefollowing order flavonols gt flavan-3-ols gt gallic and ellagicderivatives gt benzophenones gt flavanones
In general the results given in Table 2 show that theconcentration of each compound is greater in the ultrasoundaqueous extract (AE) except the compounds identified asHHDP glucose that was higher in the infusion of 3min(I3) and in the 5min (I5) samples and naringenin whichpresented the largest concentration in I3 Similar results wereobtained by Nantitanon and coworkers [20] using ethanol asextraction solvent In fact they extracted the guava leavesby maceration and ultrasounds and the highest recovery ofphenolic compounds was obtained by sonication
The higher extraction of HHDP and naringenin in someinfusions than ultrasound extraction could be justified by thetemperature that has been reached during the two extractionmethodologies As reported by Zhang and coworkers [21] thesolubility of naringenin gradually increases as the tempera-ture increases based on these results it is expected to obtainlower extraction of these compounds during ultrasoundextraction instead of that of infusion This hypothesis canbe confirmed with the results obtained by Wen et al [22]that noticed that naringenin is insoluble in water at roomtemperature
However naringenin content in infusion samplesreported a decreasing trend when increasing the time ofinfusion these results should be attributed to a degradation ofthis compound when the thermal treatment was prolonged
To the best of our knowledge there is no literature aboutthe water solubility and the effect of temperature on HHDPcompound Nevertheless taking into account the resultsreported in Table 2 a similar trend to the one reported fornaringenin compound could be supposed for HHDP
Flavan-3-ols gallic and ellagic acid derivatives ben-zophenones and flavonols in the ultrasound aqueous extractwere from 3 to 5 times more concentrated than leavesinfusions Compared to the ultrasound aqueous extract (AE)and infusion of 7min (I7) samples naringenin was 15 and 17times higher in the infusion of 3min (I3) and in the 5min (I5)samples respectively
Flavonols represent about 50 percent of total polar com-pounds in each sample Avicularin and guajaverin were themajor flavonol components and their concentrations variedfrom 137 to 32mgg and from 128 to 27mgg respectivelySimilar trend was showed by Chang et al [11] Morin was alsofound in high concentration with a range that varies from 30to 84mgg in I7 and AE sample respectively Other flavonolcompounds presented in all samples in higher quantities andin the same order of magnitude were hyperin quercitrin
reynoutrin and isoquercitrin Myricetin-arabinoside wasdetected in all samples but it was quantified only in AEsample instead quercetin was only detected and quantifiedin the AE sample These data could promote the use of guavaleaves extract for nutraceutical scopes because as reported byWang et al [23]myricetin and quercetin have high inhibitoryactivities against some enzymes that are involved in diabetesGuavinoside C was quantified in ultrasound aqueous extractit was identified in infusion samples but its content was lowerthan LOQ
The second class of polar compounds was representedby flavan-3-ols which correspond to 26ndash30 of total polarcompounds Procyanidin was the first polar compound andits amounts ranged from 61 to 177mgg Catechin was thesecond flavan-3-ol ranging between 51 and 129mgg
Two epigallocatechin isomers and prodelphinidin dimerwere the third flavan-3-ols and their amounts were about 54ndash59mgg
Gallic and ellagic acid derivatives account for 20 ofthe total concentration of polar compounds in each sam-ple In this case ultrasound aqueous extract and infusionsreported different extraction power Effectively ultrasoundaqueous extract showed casuarinincasuarictin as first ellagicacid derivative (87mgg) on the contrary infusion samplesreported HHDP glucose compounds in the highest amounts(20ndash23mgg) Benzophenones were 2ndash4 of total polarcompounds Guavinoside A was the first benzophenone andit was represented by two isomers Finally four guavinosideB isomers were also detected in the extract but only one wasquantified their content in infusion samples was less thanLOQ or in some cases they were not detected
At last a flavanone namely naringenin was presentedin all samples I3 sample showed the higher content on thecontrary aqueous extract and I7 samples reported the lowestquantities
33 Comparison between Phenolic Content and AntioxidantActivity As shown in Figure 1 the amount of total polarcompounds is significantly higher in the ultrasound aqueousextract than in the infusions Comparing the results obtainedfor the infusions the quantity of these compounds is quitehigher for I5 than for the others I3 and I7 In fact I3 samplereported a lower content probably due to an incompleteextraction of polar compounds instead I7 sample showedlower amounts probably due to a degradation of thesecompounds during maceration
To evaluate the antioxidant activity of the extract andto corroborate the correlation between phenolic content andantioxidant activity two different assays were developedTEAC evaluated by ABTS∙+ test and FRAP
The choice of these two methods was assessed based ontheir different mechanisms the radical scavenging capacitydemonstrated by ABTS and ferric reducing capacities evalu-able by FRAP method Moreover the results obtained byThaipong et al [24] demonstrated that ABTS and FRAPreported higher correlation with total phenolic content inguava fruit compared to other antioxidant activity assays
Total polar compounds by HPLC are in concordancewith the values obtained for the FRAP and ABTS assays
6 Journal of Chemistry
Table 2 Quantification (mean plusmn SD n = 3) of the compounds identified in guava leaves infusions and ultrasound aqueous extract
Number Compounds Quantification (120583g analyteg leaves)AE I3 I5 I7
nd not detected AE aqueous extract obtained by ultrasound I3 I5 and I7 infusion obtained at 3 5 and 7 minutes of infusion time respectivelyThe different letter in the same line means that the compounds are significantly different (P le 005)
Journal of Chemistry 7
AE I3 I5 I7Samples
20
40
60
80
100
120
140
160
180
Tota
l pol
ar co
mpo
unds
(mg
g)
Median Nonoutlier range
OutliersExtremes
Figure 1 Total content (mgg) of total polar compounds by HPLC in analysed samples
Table 3 Comparison between total polar compound (mgg) determined by HPLC and antioxidant activity evaluated by FRAP (120583Mof FeSO4equivalentsmg) and ABTS (120583M of Trolox equivalentsmg)
Means in the same column with different letter are significantly different (P lt 005)
(Table 3) Besides the reducing power and radical scavengingcapacity displayed a significative difference between thesamples obtained by infusion and the ultrasound aqueousextract Positive correlations with R = 09883 and 119875 lt 0001and R = 09973 and 119875 lt 0001 were noticed between totalpolar compounds content and FRAP and ABTS respectively
FRAP and ABTS did not report significative differences(119875 lt 005) among infusion samples however ultrasoundaqueous extract values were higher than infusions values
Moreover a simple linear regression analysis was carriedout to compare the correlation between all compoundsidentified and the antioxidant activity (Table 4)
Most of the polar compounds were highly correlated withFRAP assay (R = 098 119875 lt 0001) except compounds 10 30and 32 that reported an119877 value ranging between 096 and 097(119875 lt 0001) Compound 23 showed a lower correlation (R =076 119875 lt 005) HHDP glucose isomers resulted in inversecorrelation with FRAP assay Moreover naringenin did notshow any correlation
ABTS assay confirmed data reported by FRAP assay infact the two antioxidant assays showed a good correlation
between them that reported an R value of 09916 and 119875 lt00001 These results agreed with the data reported byThaipong et al [24]
4 Conclusions
Several polar compounds have been identified and quantifiedin guava leaves extracts (ultrasound aqueous extract andinfusions) According to the amount of polar compoundsand also the FRAP and ABTS assays the water ultrasoundassisted extraction provided better results than the infusionSignificative positive correlations R gt 098 and 119875 lt 0001were detected between total polar content and antioxi-dant activity assays Moreover positive correlation was alsodetected for single compounds except for HHDP and narin-genin
The results suggested that aqueous ultrasound extract canrepresent a valuable strategy to obtain nutraceuticals using agreen technology About infusions the 5-minute infusion isadvisable for guava leaves culinary uses because of reportedhigher polar compounds content
8 Journal of Chemistry
Table 4 Correlation between the antioxidant activity and polar compounds
Compounds FRAP ABTS119877 value 119875 value 119877 value 119875 value
lowastlowastlowast119875 lt 0001 lowast119875 lt 005 NC not correlated
Journal of Chemistry 9
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was funded by the Project cofinanced by FEDER-Andalucıa 2007ndash2013 (Cod 461100) andAndalusian RegionalGovernment Council of Innovation and Science (P11-CTS-7625) The author Elixabet Dıaz-de-Cerio also would liketo thank the CEIBiotic for the ldquoAyudas a la EnsenanzaPracticardquo Grant (CADP2-71) Ana Marıa Gomez-Caravacaand Vito Verardo thank the Spanish Ministry of Economyand Competitiveness (MINECO) for ldquoJuan de la Ciervardquopostdoctoral contract
References
[1] Q-M Liu and J-G Jiang ldquoAntioxidative activities of medicinalplants from TCMrdquo Mini-Reviews in Medicinal Chemistry vol12 no 11 pp 1154ndash1172 2012
[2] S Z Bathaie N Mokarizade and S Shirali ldquoAn overview of themechanisms of plant ingredients in the treatment of diabetesmellitusrdquo Journal of Medicinal Plants vol 11 no 44 pp 1ndash242012
[3] U Etxeberria A L de la Garza J Campin J A Martnez andF I Milagro ldquoAntidiabetic effects of natural plant extracts viainhibition of carbohydrate hydrolysis enzymes with emphasison pancreatic alpha amylaserdquo Expert Opinion on TherapeuticTargets vol 16 no 3 pp 269ndash297 2012
[4] R M P Gutierrez S Mitchell and R V Solis ldquoPsidiumguajava a review of its traditional uses phytochemistry andpharmacologyrdquo Journal of Ethnopharmacology vol 117 no 1 pp1ndash27 2008
[5] J T Cheng and R S Yang ldquoHypoglycemic effect of guava juicein mice and human subjectsrdquo The American Journal of ChineseMedicine vol 11 no 1-4 pp 74ndash76 1983
[6] T Eidenberger M Selg and K Krennhuber ldquoInhibition ofdipeptidyl peptidase activity by flavonol glycosides of guava(Psidium guajava L) a key to the beneficial effects of guava intype II diabetes mellitusrdquo Fitoterapia vol 89 no 1 pp 74ndash792013
[7] F-C Cheng S-C Shen and J S-B Wu ldquoEffect of guava(Psidium guajava L) leaf extract on glucose uptake in rathepatocytesrdquo Journal of Food Science vol 74 no 5 pp H132ndashH138 2009
[8] H-Y Chen and G-C Yen ldquoAntioxidant activity and freeradical-scavenging capacity of extracts from guava (Psidiumguajava L) leavesrdquo Food Chemistry vol 101 no 2 pp 686ndash6942007
[9] O Laporta L Perez-Fons R Mallavia N Caturla and VMicol ldquoIsolation characterization and antioxidant capacityassessment of the bioactive compounds derived from Hypoxisrooperi corm extract (African potato)rdquo Food Chemistry vol 101no 4 pp 1425ndash1437 2007
[10] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[11] C H Chang C L Hsieh H E Wang C C Peng C CChyau and R Y Peng ldquoUnique bioactive polyphenolic profile
of guava (Psidium guajava) budding leaf tea is related to plantbiochemistry of budding leaves in early dawnrdquo Journal of theScience of Food andAgriculture vol 93 no 4 pp 944ndash954 2013
[12] T Okuda T Yoshida T Hatano K Yazaki and M AshidaldquoEllagitannins of the casuarinaceae stachyuraceae and myr-taceaerdquo Phytochemistry vol 21 no 12 pp 2871ndash2874 1980
[13] F A Yamanaka T A Hatano H A Ito S B Taniguchi E CTakahashi and K C Okamoto ldquoAntibacterial effects of guavatannins and related polyphenols on Vibrio and AeromonasspeciesrdquoNatural Product Communications vol 3 no 5 pp 711ndash720 2008
[14] T Okuda T Hatano and K Yazaki ldquoGuavin B an ellagitanninof novel typerdquo Chemical amp Pharmaceutical Bulletin vol 32 no9 pp 3787ndash3788 1984
[15] F QarsquoDan F Petereit and A Nahrstedt ldquoPolymeric proantho-cyanidins from Psidium guajavardquo Scientia Pharmaceutica vol73 no 3 pp 113ndash125 2005
[16] B-J Park T Matsuta T Kanazawa C-H Park K-J Changand M Onjo ldquoPhenolic compounds from the leaves of Psidiumguajava II quercetin and its glycosidesrdquo Chemistry of NaturalCompounds vol 48 no 3 pp 477ndash479 2012
[17] S Tachakittirungrod F Ikegami and S Okonogi ldquoAntioxidantactive principles isolated from Psidium guajava grown inThailandrdquo Scientia Pharmaceutica vol 75 no 4 pp 179ndash1932007
[18] K-C Chen C-M Chuang L-Y Lin et al ldquoThe polyphenolicsin the aqueous extract of Psidium guajava kinetically reveal aninhibition model on LDL glycationrdquo Pharmaceutical Biologyvol 48 no 1 pp 23ndash31 2010
[19] K Matsuzaki R Ishii K Kobiyama and S Kitanaka ldquoNewbenzophenone and quercetin galloyl glycosides from Psidiumguajava Lrdquo Journal of Natural Medicines vol 64 no 3 pp 252ndash256 2010
[20] W Nantitanon S Yotsawimonwat and S Okonogi ldquoFactorsinfluencing antioxidant activities and total phenolic content ofguava leaf extractrdquo LWTmdashFood Science and Technology vol 43no 7 pp 1095ndash1103 2010
[21] P Zhang R Lin G Yang J Zhang L Zhou and T Liu ldquoSol-ubility of naringenin in ethanol and water mixturesrdquo Journal ofChemical and Engineering Data vol 58 no 9 pp 2402ndash24042013
[22] J Wen B Liu E Yuan Y Ma and Y Zhu ldquoPreparation andphysicochemical properties of the complex of naringenin withhydroxypropyl-120573-cyclodextrinrdquo Molecules vol 15 no 6 pp4401ndash4407 2010
[23] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010
[24] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006
Recently Eidenberger and coworkers [6] investigated theeffect of extracts from Psidium guajava L leaves particu-larly the effects of main flavonol-glycoside components ondipeptidyl-peptidase IV (DP-IV) a key enzyme of blood glu-cose homoeostasis and finally indicated that guava extracthas a potential to exert the effect observed in vitro also inhumans after oral administration
In vivo experiments carried out by Cheng et al [7]reported that quercetin in the aqueous extract of guava leavespromotes glucose uptake in liver cells and as a consequencecontributes to the alleviation of hypoglycemia in diabetes
Usually guava leaf tea was consumed after infusion how-ever different infusion times were advice from productioncompany Because of that in the presentwork the antioxidantactivities of infusions obtained at different infusion timesand conventional ultrasound aqueous extracts of guava leaveswere evaluated and compared in terms of their compositionin polar compounds
2 Material and Methods
21 Chemicals Double-deionised water with conductivitylower than 182MΩ was obtained with a Milli-Q system(Millipore Bedford MA USA) Methanol LC-MS ldquooptimardquograde and acetonitrile were obtained from Fisher Scien-tific (Leicestershire UK) Acetic acid and the standardsgallic acid catechin ellagic acid naringenin quercetinand rutin were all from Sigma-Aldrich (Steinheim Ger-many) The reagents used to measure the antioxidant capac-ity TPTZ (246-tripyridyl-S-triazine) Trolox (6-hydroxy-2578-tetramethylchroman-2-carboxylic acid) ABTS (221015840-azinobis (3-ethylbenzothiazoline-6-sulfonate)) potassiumpersulfate and ferric sulfate were purchased from Sigma-Aldrich (St Louis MO USA) Sodium acetate ferric chlo-ride and hydrochloric acid were obtained from Panreac(Barcelona Spain)
22 Plant Material and Sample Preparation Fresh guavaleaves were harvested in Motril Spain (36∘4410158404310158401015840N3∘3110158401410158401015840O) They were middle age intense green leaves andthey were collected in February 2014 The environmentalconditions had mean maxmin temperature of 1810∘Cprecipitation of 0mm and saturated light duration thatranged from 955 to 1050 h dayminus1
The samples were air-dried and ground before theanalyses Two different extraction methodologies such asultrasound extraction and infusion were carried out
Conventional Ultrasound Extraction 05 g of dry guava leaveswas extracted with 15mL of water (times3) using a sonicatorBransonB3510 for 10min at room temperatureThen sampleswere centrifuged for 15min at 6000 rpm using a centrifugeto remove solids The supernatants were pooled evaporatedand dissolved in 2mL of 50 methanol This solution wasfiltered through a 020 120583m syringe filter and kept at minus20∘C inamber bottles to avoid degradation until analysis
Infusion Extraction For the infusion 1 g of dried guava leavesand 50mL of boiling water were used The extract was
prepared according to the method previously described byChen and Yen [8] where they prepared 5min infusion andcompared with infusion for 3 and 7min After the extractionby infusion for 3 5 and 7min the solution was raised to50mL with water filtered through a 020120583m syringe filterand kept at minus20∘C in amber bottles to avoid degradation untilanalysis
All extractions were made in triplicate
23 Trolox Equivalent Antioxidant Capacity (ABTS) AssayTheABTS assay which measures the reduction of the radicalcation of 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonate)(ABTS) by antioxidants was performed by using a methodpreviously described by Laporta and coworkers [9] Con-cisely ABTS radical cation was produced by reacting ABTSstock solution with 245mMpotassium persulfate in the darkat room temperature for 12ndash24 h before use The absorbanceof ABTS radical cation was adjusted to 070 (plusmn002) at734 nm A calibration curve was prepared with differentconcentrations of Trolox (0ndash20120583M)
24 Ferric-Reducing Antioxidant Power (FRAP) The reduc-ing power was evaluated according to the method validatedby Benzie and Strain [10] Briefly 300mM acetate buffer(pH 36) 10mM TPTZ in 40mM HCl and 20mM aqueousFeCl3were prepared andmixed (10 1 1) to obtain the FRAP
reagent The FRAP reagent was warmed to 37∘C beforereading its absorbance Then the samples were added Thechange in absorbance (593 nm) between the samples and theblank was related to the absorbance of an aqueous solution ofknown Fe(II) concentration prepared for calibration
25 HPLC-ESI-MS Analysis Phenolic and other polar com-pounds in the extracts obtained from guava leaves were iden-tified using a method introduced by Chang et al [11] withslight modifications Briefly HPLC analyses were performedusing a HP 1100 Series instrument (Hewlett Packard Wilm-ington DE USA) equipped with a binary pump deliverysystem a degasser (model G1322A) an autosampler (Auto-matic Liquid Sampler ALSmodel G1312A) aHPdiode-arrayUV-VIS detector (DAD model G1315A) and a quadrupoleHP-Mass Spectrometer Detector (MSD model G1946A)integration and data elaboration were performed usingChemstation software (Hewlett Packard) A PhenomenexLuna C18 analytical column (150mm times 20mm particle size3 120583m) (Phenomenex Inc Torrance CA USA) was used forpolar compounds separation All analyses were carried outat room temperature using the gradient proposed by Changet al [11] MS analysis was carried out using an electrosprayionization (ESI) interface in negative ionization mode atthe following conditions drying gas flow (N
2) 90 Lmin
nebulizer pressure 50 psi gas drying temperature 350∘Ccapillary voltage 3500V and fragmentor voltage and scanrange 100V andmz 50ndash1000 respectively
Phenolic standards of interest such as gallic acid catechinellagic acid naringenin and rutin were used for quantifi-cation of phenolic compounds in guava leaf extracts Theidentified compounds were quantified on the basis of theirpeak area and compared with calibration curves obtained
Journal of Chemistry 3
with the corresponding standards and then expressed as 120583ggof extract
26 Statistical Analysis The results reported in this studyare the averages of three repetitions (119899 = 3) Fisherrsquosleast significance difference (LSD) test and Pearsonrsquos linearcorrelations both at 119875 lt 005 were evaluated using Statistica60 (2001 StatSoft Tulsa OK)
3 Results and Discussion
31 Identification of Polar Compounds Phenolic and otherpolar compounds were identified by their elution orderUVvis spectra and MS characteristics compared withreported literature values and by coinjection with availablestandards (Table 1)
About phenolic compounds four different classes identi-fied as gallic and ellagic acid derivatives flavonols flavanonesand flavan-3-ols were determined
Thirteen gallic and ellagic acid derivatives were deter-mined Three compounds (1 2 and 3) with molecular ionat mz 481 and fragment at mz 301 were identified as hex-ahydroxydiphenic acid (HHDP) glucose and its presence inguava was previously reported by Okuda and coworkers [12]Gallic acid (compound 4) was identified according to its MSdata (mz 169 andmz 125) and by coelution with a chemicalstandard Two compounds (6 and 11) with [MndashH]minus at mz783 and two fragments at mz 481 and 301 corresponding toloss of ellagic acid were detected This fragmentation patternwas assigned to pedunculagincasuariin compounds thesecompounds were described in guava leaves by Okuda et al[12]
Two compounds (10 and 12) with molecular ion at mz951 and fragments at mz 783 and 481 were also determinedThese compounds were identified as geraniin isomers [13]
Two compounds (15 and 18) showing significant [MndashH]minussignals at mz 785 with fragment ions at mz 615 and at mz301were foundThis fragmentation pattern corresponded to adigalloyl-HHDP-glucose structure probably tellimagrandinI isomer This compound was previously detected in guavaleaves by Okuda et al [12] Compound 17 atmz 935 reporteda fragment ion at mz 783 this fragmentation pattern wasassigned to casuarinincasuarictin and this compound wasdescribed in guava tea by Yamanaka and coworkers [13]Compound 30 was identified as ellagic acid due its coelutionwith commercial standard Finally guavin B (compound 46)that reported a molecular ion at mz 693 was identifiedaccording to Okuda et al [14]
Moreover ten flavan-3-ol derivatives were determinedCompounds 5 and 42 showed amolecular ion atmz 609 andthree fragments atmz 441 423 and 305 (gallocatechin unit)these compounds were identified as prodelphinidin B2 andits isomer and their presence in guava leaves was noticed byQarsquoDan et al [15]
Compounds 7 and 9 showed a molecular ion at mz 593and two fragments atmz 425 and 407 According to QarsquoDanet al [15] these compounds were identified as prodelphinidindimer (4120572-8)
Two compounds (8 and 19) with [MndashH]minus at mz 305and fragment ions at mz 221 and 179 were identified asgallocatechin isomers and their presence in guava leaves wasreported by QarsquoDan et al [15] Three procyanidin dimers([MndashH]minus atmz 577) were also described (compounds 13 14and 21)
Catechin compound (16) was identified by mass spectradata elaboration and coelution with a commercial standard
The flavonols were themost representative phenolic com-pounds in fact eighteen flavonol-derivatives were identifiedFour compounds (compounds 20 24 27 and 29) reported amolecular ion atmz 449 and twomajor fragments atmz 316and 317 According to Chang et al [11] these compounds wereidentified as myricetin-arabinosidexylopyranoside isomers
Two flavonol compounds (compounds 22 and 23) withmolecular ion at mz 479 and fragments at mz 317 and316 were identified as myricetin-hexoside isomers [11] Twocompounds (25 and 26) corresponding to [MndashH]minus signals atmz 615 were also detected Based on their molecular weightand the presence of two fragments at mz 463 and 301 theywere assigned to quercetin-galloylhexoside isomers and theirpresence in guava leaves was reported by Park et al [16]
Two compounds at mz 301 were detected (28 and 47)moreover they showed the same fragment ion at mz 151Quercetin standard solution was injected and because of thatcompound 28 was assigned to morin and compound 47 wasassigned to quercetin their presence in guava leaves wasreported by several authors [11 17]
Compounds detected at mz 463 (31 and 33) withfragment ion at mz 301 corresponded to hyperin andisoquercitrin respectively They have previously been foundin leaves of guava by Eidenberger et al [6]
Quercetin glucuronide (32) withmolecular ion atmz 477and fragment ions atmz 433 and 301was identified accordingto Chang et al [11]
Three compounds (34 35 and 37) reported the samemolecular ion (mz 433) and a fragment ion atmz 301 (corre-sponding to quercetin aglycone) according to their retentiontimes they were identified as reynoutrin guajaverin andavicularin as reported by Chang et al [11]
Quercitrin (38) was identified at mz 447 and fragmentionmz 300 according to Park et al [16]
A flavanone compound namely naringenin (mz 271)was detected and identified by analyzing themass spectra andby coelution with a chemical standard [18] Compound 39withmz 585 was identified as guavinoside C [19]
Six benzophenone compounds were also determined inguava leaves sampleThe [MndashH]minus ion atmz 543 revealed thepresence of two compounds namely guavinoside A isomerstheir presence in guava leaves was noticed by Matsuzaki andcoworkers [19]
Finally four compounds with mz 571 (40 43 44 and45) were identified as guavinoside B isomers according toMatsuzaki et al [19]
32 Quantification of Polar Compounds Comparisonbetween different times of infusion and a conventionalultrasound aqueous extract was carried out due to several
4 Journal of Chemistry
Table 1 Identification of polar compounds in guava leaves by HPLC-DAD-ESI-MS
publications about phenolic and other polar compoundscontent in infusion or guava leaves tea
Quantification of polar compounds was performed bypreparing five calibration curves with the standards availablegallic acid catechin ellagic acid naringenin and rutin Forthose with no commercial standard available quantificationwas carried out comparing with compounds bearing similarstructures
It is important to underline that the quantification resultsreported that the order in terms of concentration of thefamilies of polar compounds in all samples decreased in thefollowing order flavonols gt flavan-3-ols gt gallic and ellagicderivatives gt benzophenones gt flavanones
In general the results given in Table 2 show that theconcentration of each compound is greater in the ultrasoundaqueous extract (AE) except the compounds identified asHHDP glucose that was higher in the infusion of 3min(I3) and in the 5min (I5) samples and naringenin whichpresented the largest concentration in I3 Similar results wereobtained by Nantitanon and coworkers [20] using ethanol asextraction solvent In fact they extracted the guava leavesby maceration and ultrasounds and the highest recovery ofphenolic compounds was obtained by sonication
The higher extraction of HHDP and naringenin in someinfusions than ultrasound extraction could be justified by thetemperature that has been reached during the two extractionmethodologies As reported by Zhang and coworkers [21] thesolubility of naringenin gradually increases as the tempera-ture increases based on these results it is expected to obtainlower extraction of these compounds during ultrasoundextraction instead of that of infusion This hypothesis canbe confirmed with the results obtained by Wen et al [22]that noticed that naringenin is insoluble in water at roomtemperature
However naringenin content in infusion samplesreported a decreasing trend when increasing the time ofinfusion these results should be attributed to a degradation ofthis compound when the thermal treatment was prolonged
To the best of our knowledge there is no literature aboutthe water solubility and the effect of temperature on HHDPcompound Nevertheless taking into account the resultsreported in Table 2 a similar trend to the one reported fornaringenin compound could be supposed for HHDP
Flavan-3-ols gallic and ellagic acid derivatives ben-zophenones and flavonols in the ultrasound aqueous extractwere from 3 to 5 times more concentrated than leavesinfusions Compared to the ultrasound aqueous extract (AE)and infusion of 7min (I7) samples naringenin was 15 and 17times higher in the infusion of 3min (I3) and in the 5min (I5)samples respectively
Flavonols represent about 50 percent of total polar com-pounds in each sample Avicularin and guajaverin were themajor flavonol components and their concentrations variedfrom 137 to 32mgg and from 128 to 27mgg respectivelySimilar trend was showed by Chang et al [11] Morin was alsofound in high concentration with a range that varies from 30to 84mgg in I7 and AE sample respectively Other flavonolcompounds presented in all samples in higher quantities andin the same order of magnitude were hyperin quercitrin
reynoutrin and isoquercitrin Myricetin-arabinoside wasdetected in all samples but it was quantified only in AEsample instead quercetin was only detected and quantifiedin the AE sample These data could promote the use of guavaleaves extract for nutraceutical scopes because as reported byWang et al [23]myricetin and quercetin have high inhibitoryactivities against some enzymes that are involved in diabetesGuavinoside C was quantified in ultrasound aqueous extractit was identified in infusion samples but its content was lowerthan LOQ
The second class of polar compounds was representedby flavan-3-ols which correspond to 26ndash30 of total polarcompounds Procyanidin was the first polar compound andits amounts ranged from 61 to 177mgg Catechin was thesecond flavan-3-ol ranging between 51 and 129mgg
Two epigallocatechin isomers and prodelphinidin dimerwere the third flavan-3-ols and their amounts were about 54ndash59mgg
Gallic and ellagic acid derivatives account for 20 ofthe total concentration of polar compounds in each sam-ple In this case ultrasound aqueous extract and infusionsreported different extraction power Effectively ultrasoundaqueous extract showed casuarinincasuarictin as first ellagicacid derivative (87mgg) on the contrary infusion samplesreported HHDP glucose compounds in the highest amounts(20ndash23mgg) Benzophenones were 2ndash4 of total polarcompounds Guavinoside A was the first benzophenone andit was represented by two isomers Finally four guavinosideB isomers were also detected in the extract but only one wasquantified their content in infusion samples was less thanLOQ or in some cases they were not detected
At last a flavanone namely naringenin was presentedin all samples I3 sample showed the higher content on thecontrary aqueous extract and I7 samples reported the lowestquantities
33 Comparison between Phenolic Content and AntioxidantActivity As shown in Figure 1 the amount of total polarcompounds is significantly higher in the ultrasound aqueousextract than in the infusions Comparing the results obtainedfor the infusions the quantity of these compounds is quitehigher for I5 than for the others I3 and I7 In fact I3 samplereported a lower content probably due to an incompleteextraction of polar compounds instead I7 sample showedlower amounts probably due to a degradation of thesecompounds during maceration
To evaluate the antioxidant activity of the extract andto corroborate the correlation between phenolic content andantioxidant activity two different assays were developedTEAC evaluated by ABTS∙+ test and FRAP
The choice of these two methods was assessed based ontheir different mechanisms the radical scavenging capacitydemonstrated by ABTS and ferric reducing capacities evalu-able by FRAP method Moreover the results obtained byThaipong et al [24] demonstrated that ABTS and FRAPreported higher correlation with total phenolic content inguava fruit compared to other antioxidant activity assays
Total polar compounds by HPLC are in concordancewith the values obtained for the FRAP and ABTS assays
6 Journal of Chemistry
Table 2 Quantification (mean plusmn SD n = 3) of the compounds identified in guava leaves infusions and ultrasound aqueous extract
Number Compounds Quantification (120583g analyteg leaves)AE I3 I5 I7
nd not detected AE aqueous extract obtained by ultrasound I3 I5 and I7 infusion obtained at 3 5 and 7 minutes of infusion time respectivelyThe different letter in the same line means that the compounds are significantly different (P le 005)
Journal of Chemistry 7
AE I3 I5 I7Samples
20
40
60
80
100
120
140
160
180
Tota
l pol
ar co
mpo
unds
(mg
g)
Median Nonoutlier range
OutliersExtremes
Figure 1 Total content (mgg) of total polar compounds by HPLC in analysed samples
Table 3 Comparison between total polar compound (mgg) determined by HPLC and antioxidant activity evaluated by FRAP (120583Mof FeSO4equivalentsmg) and ABTS (120583M of Trolox equivalentsmg)
Means in the same column with different letter are significantly different (P lt 005)
(Table 3) Besides the reducing power and radical scavengingcapacity displayed a significative difference between thesamples obtained by infusion and the ultrasound aqueousextract Positive correlations with R = 09883 and 119875 lt 0001and R = 09973 and 119875 lt 0001 were noticed between totalpolar compounds content and FRAP and ABTS respectively
FRAP and ABTS did not report significative differences(119875 lt 005) among infusion samples however ultrasoundaqueous extract values were higher than infusions values
Moreover a simple linear regression analysis was carriedout to compare the correlation between all compoundsidentified and the antioxidant activity (Table 4)
Most of the polar compounds were highly correlated withFRAP assay (R = 098 119875 lt 0001) except compounds 10 30and 32 that reported an119877 value ranging between 096 and 097(119875 lt 0001) Compound 23 showed a lower correlation (R =076 119875 lt 005) HHDP glucose isomers resulted in inversecorrelation with FRAP assay Moreover naringenin did notshow any correlation
ABTS assay confirmed data reported by FRAP assay infact the two antioxidant assays showed a good correlation
between them that reported an R value of 09916 and 119875 lt00001 These results agreed with the data reported byThaipong et al [24]
4 Conclusions
Several polar compounds have been identified and quantifiedin guava leaves extracts (ultrasound aqueous extract andinfusions) According to the amount of polar compoundsand also the FRAP and ABTS assays the water ultrasoundassisted extraction provided better results than the infusionSignificative positive correlations R gt 098 and 119875 lt 0001were detected between total polar content and antioxi-dant activity assays Moreover positive correlation was alsodetected for single compounds except for HHDP and narin-genin
The results suggested that aqueous ultrasound extract canrepresent a valuable strategy to obtain nutraceuticals using agreen technology About infusions the 5-minute infusion isadvisable for guava leaves culinary uses because of reportedhigher polar compounds content
8 Journal of Chemistry
Table 4 Correlation between the antioxidant activity and polar compounds
Compounds FRAP ABTS119877 value 119875 value 119877 value 119875 value
lowastlowastlowast119875 lt 0001 lowast119875 lt 005 NC not correlated
Journal of Chemistry 9
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was funded by the Project cofinanced by FEDER-Andalucıa 2007ndash2013 (Cod 461100) andAndalusian RegionalGovernment Council of Innovation and Science (P11-CTS-7625) The author Elixabet Dıaz-de-Cerio also would liketo thank the CEIBiotic for the ldquoAyudas a la EnsenanzaPracticardquo Grant (CADP2-71) Ana Marıa Gomez-Caravacaand Vito Verardo thank the Spanish Ministry of Economyand Competitiveness (MINECO) for ldquoJuan de la Ciervardquopostdoctoral contract
References
[1] Q-M Liu and J-G Jiang ldquoAntioxidative activities of medicinalplants from TCMrdquo Mini-Reviews in Medicinal Chemistry vol12 no 11 pp 1154ndash1172 2012
[2] S Z Bathaie N Mokarizade and S Shirali ldquoAn overview of themechanisms of plant ingredients in the treatment of diabetesmellitusrdquo Journal of Medicinal Plants vol 11 no 44 pp 1ndash242012
[3] U Etxeberria A L de la Garza J Campin J A Martnez andF I Milagro ldquoAntidiabetic effects of natural plant extracts viainhibition of carbohydrate hydrolysis enzymes with emphasison pancreatic alpha amylaserdquo Expert Opinion on TherapeuticTargets vol 16 no 3 pp 269ndash297 2012
[4] R M P Gutierrez S Mitchell and R V Solis ldquoPsidiumguajava a review of its traditional uses phytochemistry andpharmacologyrdquo Journal of Ethnopharmacology vol 117 no 1 pp1ndash27 2008
[5] J T Cheng and R S Yang ldquoHypoglycemic effect of guava juicein mice and human subjectsrdquo The American Journal of ChineseMedicine vol 11 no 1-4 pp 74ndash76 1983
[6] T Eidenberger M Selg and K Krennhuber ldquoInhibition ofdipeptidyl peptidase activity by flavonol glycosides of guava(Psidium guajava L) a key to the beneficial effects of guava intype II diabetes mellitusrdquo Fitoterapia vol 89 no 1 pp 74ndash792013
[7] F-C Cheng S-C Shen and J S-B Wu ldquoEffect of guava(Psidium guajava L) leaf extract on glucose uptake in rathepatocytesrdquo Journal of Food Science vol 74 no 5 pp H132ndashH138 2009
[8] H-Y Chen and G-C Yen ldquoAntioxidant activity and freeradical-scavenging capacity of extracts from guava (Psidiumguajava L) leavesrdquo Food Chemistry vol 101 no 2 pp 686ndash6942007
[9] O Laporta L Perez-Fons R Mallavia N Caturla and VMicol ldquoIsolation characterization and antioxidant capacityassessment of the bioactive compounds derived from Hypoxisrooperi corm extract (African potato)rdquo Food Chemistry vol 101no 4 pp 1425ndash1437 2007
[10] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[11] C H Chang C L Hsieh H E Wang C C Peng C CChyau and R Y Peng ldquoUnique bioactive polyphenolic profile
of guava (Psidium guajava) budding leaf tea is related to plantbiochemistry of budding leaves in early dawnrdquo Journal of theScience of Food andAgriculture vol 93 no 4 pp 944ndash954 2013
[12] T Okuda T Yoshida T Hatano K Yazaki and M AshidaldquoEllagitannins of the casuarinaceae stachyuraceae and myr-taceaerdquo Phytochemistry vol 21 no 12 pp 2871ndash2874 1980
[13] F A Yamanaka T A Hatano H A Ito S B Taniguchi E CTakahashi and K C Okamoto ldquoAntibacterial effects of guavatannins and related polyphenols on Vibrio and AeromonasspeciesrdquoNatural Product Communications vol 3 no 5 pp 711ndash720 2008
[14] T Okuda T Hatano and K Yazaki ldquoGuavin B an ellagitanninof novel typerdquo Chemical amp Pharmaceutical Bulletin vol 32 no9 pp 3787ndash3788 1984
[15] F QarsquoDan F Petereit and A Nahrstedt ldquoPolymeric proantho-cyanidins from Psidium guajavardquo Scientia Pharmaceutica vol73 no 3 pp 113ndash125 2005
[16] B-J Park T Matsuta T Kanazawa C-H Park K-J Changand M Onjo ldquoPhenolic compounds from the leaves of Psidiumguajava II quercetin and its glycosidesrdquo Chemistry of NaturalCompounds vol 48 no 3 pp 477ndash479 2012
[17] S Tachakittirungrod F Ikegami and S Okonogi ldquoAntioxidantactive principles isolated from Psidium guajava grown inThailandrdquo Scientia Pharmaceutica vol 75 no 4 pp 179ndash1932007
[18] K-C Chen C-M Chuang L-Y Lin et al ldquoThe polyphenolicsin the aqueous extract of Psidium guajava kinetically reveal aninhibition model on LDL glycationrdquo Pharmaceutical Biologyvol 48 no 1 pp 23ndash31 2010
[19] K Matsuzaki R Ishii K Kobiyama and S Kitanaka ldquoNewbenzophenone and quercetin galloyl glycosides from Psidiumguajava Lrdquo Journal of Natural Medicines vol 64 no 3 pp 252ndash256 2010
[20] W Nantitanon S Yotsawimonwat and S Okonogi ldquoFactorsinfluencing antioxidant activities and total phenolic content ofguava leaf extractrdquo LWTmdashFood Science and Technology vol 43no 7 pp 1095ndash1103 2010
[21] P Zhang R Lin G Yang J Zhang L Zhou and T Liu ldquoSol-ubility of naringenin in ethanol and water mixturesrdquo Journal ofChemical and Engineering Data vol 58 no 9 pp 2402ndash24042013
[22] J Wen B Liu E Yuan Y Ma and Y Zhu ldquoPreparation andphysicochemical properties of the complex of naringenin withhydroxypropyl-120573-cyclodextrinrdquo Molecules vol 15 no 6 pp4401ndash4407 2010
[23] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010
[24] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006
with the corresponding standards and then expressed as 120583ggof extract
26 Statistical Analysis The results reported in this studyare the averages of three repetitions (119899 = 3) Fisherrsquosleast significance difference (LSD) test and Pearsonrsquos linearcorrelations both at 119875 lt 005 were evaluated using Statistica60 (2001 StatSoft Tulsa OK)
3 Results and Discussion
31 Identification of Polar Compounds Phenolic and otherpolar compounds were identified by their elution orderUVvis spectra and MS characteristics compared withreported literature values and by coinjection with availablestandards (Table 1)
About phenolic compounds four different classes identi-fied as gallic and ellagic acid derivatives flavonols flavanonesand flavan-3-ols were determined
Thirteen gallic and ellagic acid derivatives were deter-mined Three compounds (1 2 and 3) with molecular ionat mz 481 and fragment at mz 301 were identified as hex-ahydroxydiphenic acid (HHDP) glucose and its presence inguava was previously reported by Okuda and coworkers [12]Gallic acid (compound 4) was identified according to its MSdata (mz 169 andmz 125) and by coelution with a chemicalstandard Two compounds (6 and 11) with [MndashH]minus at mz783 and two fragments at mz 481 and 301 corresponding toloss of ellagic acid were detected This fragmentation patternwas assigned to pedunculagincasuariin compounds thesecompounds were described in guava leaves by Okuda et al[12]
Two compounds (10 and 12) with molecular ion at mz951 and fragments at mz 783 and 481 were also determinedThese compounds were identified as geraniin isomers [13]
Two compounds (15 and 18) showing significant [MndashH]minussignals at mz 785 with fragment ions at mz 615 and at mz301were foundThis fragmentation pattern corresponded to adigalloyl-HHDP-glucose structure probably tellimagrandinI isomer This compound was previously detected in guavaleaves by Okuda et al [12] Compound 17 atmz 935 reporteda fragment ion at mz 783 this fragmentation pattern wasassigned to casuarinincasuarictin and this compound wasdescribed in guava tea by Yamanaka and coworkers [13]Compound 30 was identified as ellagic acid due its coelutionwith commercial standard Finally guavin B (compound 46)that reported a molecular ion at mz 693 was identifiedaccording to Okuda et al [14]
Moreover ten flavan-3-ol derivatives were determinedCompounds 5 and 42 showed amolecular ion atmz 609 andthree fragments atmz 441 423 and 305 (gallocatechin unit)these compounds were identified as prodelphinidin B2 andits isomer and their presence in guava leaves was noticed byQarsquoDan et al [15]
Compounds 7 and 9 showed a molecular ion at mz 593and two fragments atmz 425 and 407 According to QarsquoDanet al [15] these compounds were identified as prodelphinidindimer (4120572-8)
Two compounds (8 and 19) with [MndashH]minus at mz 305and fragment ions at mz 221 and 179 were identified asgallocatechin isomers and their presence in guava leaves wasreported by QarsquoDan et al [15] Three procyanidin dimers([MndashH]minus atmz 577) were also described (compounds 13 14and 21)
Catechin compound (16) was identified by mass spectradata elaboration and coelution with a commercial standard
The flavonols were themost representative phenolic com-pounds in fact eighteen flavonol-derivatives were identifiedFour compounds (compounds 20 24 27 and 29) reported amolecular ion atmz 449 and twomajor fragments atmz 316and 317 According to Chang et al [11] these compounds wereidentified as myricetin-arabinosidexylopyranoside isomers
Two flavonol compounds (compounds 22 and 23) withmolecular ion at mz 479 and fragments at mz 317 and316 were identified as myricetin-hexoside isomers [11] Twocompounds (25 and 26) corresponding to [MndashH]minus signals atmz 615 were also detected Based on their molecular weightand the presence of two fragments at mz 463 and 301 theywere assigned to quercetin-galloylhexoside isomers and theirpresence in guava leaves was reported by Park et al [16]
Two compounds at mz 301 were detected (28 and 47)moreover they showed the same fragment ion at mz 151Quercetin standard solution was injected and because of thatcompound 28 was assigned to morin and compound 47 wasassigned to quercetin their presence in guava leaves wasreported by several authors [11 17]
Compounds detected at mz 463 (31 and 33) withfragment ion at mz 301 corresponded to hyperin andisoquercitrin respectively They have previously been foundin leaves of guava by Eidenberger et al [6]
Quercetin glucuronide (32) withmolecular ion atmz 477and fragment ions atmz 433 and 301was identified accordingto Chang et al [11]
Three compounds (34 35 and 37) reported the samemolecular ion (mz 433) and a fragment ion atmz 301 (corre-sponding to quercetin aglycone) according to their retentiontimes they were identified as reynoutrin guajaverin andavicularin as reported by Chang et al [11]
Quercitrin (38) was identified at mz 447 and fragmentionmz 300 according to Park et al [16]
A flavanone compound namely naringenin (mz 271)was detected and identified by analyzing themass spectra andby coelution with a chemical standard [18] Compound 39withmz 585 was identified as guavinoside C [19]
Six benzophenone compounds were also determined inguava leaves sampleThe [MndashH]minus ion atmz 543 revealed thepresence of two compounds namely guavinoside A isomerstheir presence in guava leaves was noticed by Matsuzaki andcoworkers [19]
Finally four compounds with mz 571 (40 43 44 and45) were identified as guavinoside B isomers according toMatsuzaki et al [19]
32 Quantification of Polar Compounds Comparisonbetween different times of infusion and a conventionalultrasound aqueous extract was carried out due to several
4 Journal of Chemistry
Table 1 Identification of polar compounds in guava leaves by HPLC-DAD-ESI-MS
publications about phenolic and other polar compoundscontent in infusion or guava leaves tea
Quantification of polar compounds was performed bypreparing five calibration curves with the standards availablegallic acid catechin ellagic acid naringenin and rutin Forthose with no commercial standard available quantificationwas carried out comparing with compounds bearing similarstructures
It is important to underline that the quantification resultsreported that the order in terms of concentration of thefamilies of polar compounds in all samples decreased in thefollowing order flavonols gt flavan-3-ols gt gallic and ellagicderivatives gt benzophenones gt flavanones
In general the results given in Table 2 show that theconcentration of each compound is greater in the ultrasoundaqueous extract (AE) except the compounds identified asHHDP glucose that was higher in the infusion of 3min(I3) and in the 5min (I5) samples and naringenin whichpresented the largest concentration in I3 Similar results wereobtained by Nantitanon and coworkers [20] using ethanol asextraction solvent In fact they extracted the guava leavesby maceration and ultrasounds and the highest recovery ofphenolic compounds was obtained by sonication
The higher extraction of HHDP and naringenin in someinfusions than ultrasound extraction could be justified by thetemperature that has been reached during the two extractionmethodologies As reported by Zhang and coworkers [21] thesolubility of naringenin gradually increases as the tempera-ture increases based on these results it is expected to obtainlower extraction of these compounds during ultrasoundextraction instead of that of infusion This hypothesis canbe confirmed with the results obtained by Wen et al [22]that noticed that naringenin is insoluble in water at roomtemperature
However naringenin content in infusion samplesreported a decreasing trend when increasing the time ofinfusion these results should be attributed to a degradation ofthis compound when the thermal treatment was prolonged
To the best of our knowledge there is no literature aboutthe water solubility and the effect of temperature on HHDPcompound Nevertheless taking into account the resultsreported in Table 2 a similar trend to the one reported fornaringenin compound could be supposed for HHDP
Flavan-3-ols gallic and ellagic acid derivatives ben-zophenones and flavonols in the ultrasound aqueous extractwere from 3 to 5 times more concentrated than leavesinfusions Compared to the ultrasound aqueous extract (AE)and infusion of 7min (I7) samples naringenin was 15 and 17times higher in the infusion of 3min (I3) and in the 5min (I5)samples respectively
Flavonols represent about 50 percent of total polar com-pounds in each sample Avicularin and guajaverin were themajor flavonol components and their concentrations variedfrom 137 to 32mgg and from 128 to 27mgg respectivelySimilar trend was showed by Chang et al [11] Morin was alsofound in high concentration with a range that varies from 30to 84mgg in I7 and AE sample respectively Other flavonolcompounds presented in all samples in higher quantities andin the same order of magnitude were hyperin quercitrin
reynoutrin and isoquercitrin Myricetin-arabinoside wasdetected in all samples but it was quantified only in AEsample instead quercetin was only detected and quantifiedin the AE sample These data could promote the use of guavaleaves extract for nutraceutical scopes because as reported byWang et al [23]myricetin and quercetin have high inhibitoryactivities against some enzymes that are involved in diabetesGuavinoside C was quantified in ultrasound aqueous extractit was identified in infusion samples but its content was lowerthan LOQ
The second class of polar compounds was representedby flavan-3-ols which correspond to 26ndash30 of total polarcompounds Procyanidin was the first polar compound andits amounts ranged from 61 to 177mgg Catechin was thesecond flavan-3-ol ranging between 51 and 129mgg
Two epigallocatechin isomers and prodelphinidin dimerwere the third flavan-3-ols and their amounts were about 54ndash59mgg
Gallic and ellagic acid derivatives account for 20 ofthe total concentration of polar compounds in each sam-ple In this case ultrasound aqueous extract and infusionsreported different extraction power Effectively ultrasoundaqueous extract showed casuarinincasuarictin as first ellagicacid derivative (87mgg) on the contrary infusion samplesreported HHDP glucose compounds in the highest amounts(20ndash23mgg) Benzophenones were 2ndash4 of total polarcompounds Guavinoside A was the first benzophenone andit was represented by two isomers Finally four guavinosideB isomers were also detected in the extract but only one wasquantified their content in infusion samples was less thanLOQ or in some cases they were not detected
At last a flavanone namely naringenin was presentedin all samples I3 sample showed the higher content on thecontrary aqueous extract and I7 samples reported the lowestquantities
33 Comparison between Phenolic Content and AntioxidantActivity As shown in Figure 1 the amount of total polarcompounds is significantly higher in the ultrasound aqueousextract than in the infusions Comparing the results obtainedfor the infusions the quantity of these compounds is quitehigher for I5 than for the others I3 and I7 In fact I3 samplereported a lower content probably due to an incompleteextraction of polar compounds instead I7 sample showedlower amounts probably due to a degradation of thesecompounds during maceration
To evaluate the antioxidant activity of the extract andto corroborate the correlation between phenolic content andantioxidant activity two different assays were developedTEAC evaluated by ABTS∙+ test and FRAP
The choice of these two methods was assessed based ontheir different mechanisms the radical scavenging capacitydemonstrated by ABTS and ferric reducing capacities evalu-able by FRAP method Moreover the results obtained byThaipong et al [24] demonstrated that ABTS and FRAPreported higher correlation with total phenolic content inguava fruit compared to other antioxidant activity assays
Total polar compounds by HPLC are in concordancewith the values obtained for the FRAP and ABTS assays
6 Journal of Chemistry
Table 2 Quantification (mean plusmn SD n = 3) of the compounds identified in guava leaves infusions and ultrasound aqueous extract
Number Compounds Quantification (120583g analyteg leaves)AE I3 I5 I7
nd not detected AE aqueous extract obtained by ultrasound I3 I5 and I7 infusion obtained at 3 5 and 7 minutes of infusion time respectivelyThe different letter in the same line means that the compounds are significantly different (P le 005)
Journal of Chemistry 7
AE I3 I5 I7Samples
20
40
60
80
100
120
140
160
180
Tota
l pol
ar co
mpo
unds
(mg
g)
Median Nonoutlier range
OutliersExtremes
Figure 1 Total content (mgg) of total polar compounds by HPLC in analysed samples
Table 3 Comparison between total polar compound (mgg) determined by HPLC and antioxidant activity evaluated by FRAP (120583Mof FeSO4equivalentsmg) and ABTS (120583M of Trolox equivalentsmg)
Means in the same column with different letter are significantly different (P lt 005)
(Table 3) Besides the reducing power and radical scavengingcapacity displayed a significative difference between thesamples obtained by infusion and the ultrasound aqueousextract Positive correlations with R = 09883 and 119875 lt 0001and R = 09973 and 119875 lt 0001 were noticed between totalpolar compounds content and FRAP and ABTS respectively
FRAP and ABTS did not report significative differences(119875 lt 005) among infusion samples however ultrasoundaqueous extract values were higher than infusions values
Moreover a simple linear regression analysis was carriedout to compare the correlation between all compoundsidentified and the antioxidant activity (Table 4)
Most of the polar compounds were highly correlated withFRAP assay (R = 098 119875 lt 0001) except compounds 10 30and 32 that reported an119877 value ranging between 096 and 097(119875 lt 0001) Compound 23 showed a lower correlation (R =076 119875 lt 005) HHDP glucose isomers resulted in inversecorrelation with FRAP assay Moreover naringenin did notshow any correlation
ABTS assay confirmed data reported by FRAP assay infact the two antioxidant assays showed a good correlation
between them that reported an R value of 09916 and 119875 lt00001 These results agreed with the data reported byThaipong et al [24]
4 Conclusions
Several polar compounds have been identified and quantifiedin guava leaves extracts (ultrasound aqueous extract andinfusions) According to the amount of polar compoundsand also the FRAP and ABTS assays the water ultrasoundassisted extraction provided better results than the infusionSignificative positive correlations R gt 098 and 119875 lt 0001were detected between total polar content and antioxi-dant activity assays Moreover positive correlation was alsodetected for single compounds except for HHDP and narin-genin
The results suggested that aqueous ultrasound extract canrepresent a valuable strategy to obtain nutraceuticals using agreen technology About infusions the 5-minute infusion isadvisable for guava leaves culinary uses because of reportedhigher polar compounds content
8 Journal of Chemistry
Table 4 Correlation between the antioxidant activity and polar compounds
Compounds FRAP ABTS119877 value 119875 value 119877 value 119875 value
lowastlowastlowast119875 lt 0001 lowast119875 lt 005 NC not correlated
Journal of Chemistry 9
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was funded by the Project cofinanced by FEDER-Andalucıa 2007ndash2013 (Cod 461100) andAndalusian RegionalGovernment Council of Innovation and Science (P11-CTS-7625) The author Elixabet Dıaz-de-Cerio also would liketo thank the CEIBiotic for the ldquoAyudas a la EnsenanzaPracticardquo Grant (CADP2-71) Ana Marıa Gomez-Caravacaand Vito Verardo thank the Spanish Ministry of Economyand Competitiveness (MINECO) for ldquoJuan de la Ciervardquopostdoctoral contract
References
[1] Q-M Liu and J-G Jiang ldquoAntioxidative activities of medicinalplants from TCMrdquo Mini-Reviews in Medicinal Chemistry vol12 no 11 pp 1154ndash1172 2012
[2] S Z Bathaie N Mokarizade and S Shirali ldquoAn overview of themechanisms of plant ingredients in the treatment of diabetesmellitusrdquo Journal of Medicinal Plants vol 11 no 44 pp 1ndash242012
[3] U Etxeberria A L de la Garza J Campin J A Martnez andF I Milagro ldquoAntidiabetic effects of natural plant extracts viainhibition of carbohydrate hydrolysis enzymes with emphasison pancreatic alpha amylaserdquo Expert Opinion on TherapeuticTargets vol 16 no 3 pp 269ndash297 2012
[4] R M P Gutierrez S Mitchell and R V Solis ldquoPsidiumguajava a review of its traditional uses phytochemistry andpharmacologyrdquo Journal of Ethnopharmacology vol 117 no 1 pp1ndash27 2008
[5] J T Cheng and R S Yang ldquoHypoglycemic effect of guava juicein mice and human subjectsrdquo The American Journal of ChineseMedicine vol 11 no 1-4 pp 74ndash76 1983
[6] T Eidenberger M Selg and K Krennhuber ldquoInhibition ofdipeptidyl peptidase activity by flavonol glycosides of guava(Psidium guajava L) a key to the beneficial effects of guava intype II diabetes mellitusrdquo Fitoterapia vol 89 no 1 pp 74ndash792013
[7] F-C Cheng S-C Shen and J S-B Wu ldquoEffect of guava(Psidium guajava L) leaf extract on glucose uptake in rathepatocytesrdquo Journal of Food Science vol 74 no 5 pp H132ndashH138 2009
[8] H-Y Chen and G-C Yen ldquoAntioxidant activity and freeradical-scavenging capacity of extracts from guava (Psidiumguajava L) leavesrdquo Food Chemistry vol 101 no 2 pp 686ndash6942007
[9] O Laporta L Perez-Fons R Mallavia N Caturla and VMicol ldquoIsolation characterization and antioxidant capacityassessment of the bioactive compounds derived from Hypoxisrooperi corm extract (African potato)rdquo Food Chemistry vol 101no 4 pp 1425ndash1437 2007
[10] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[11] C H Chang C L Hsieh H E Wang C C Peng C CChyau and R Y Peng ldquoUnique bioactive polyphenolic profile
of guava (Psidium guajava) budding leaf tea is related to plantbiochemistry of budding leaves in early dawnrdquo Journal of theScience of Food andAgriculture vol 93 no 4 pp 944ndash954 2013
[12] T Okuda T Yoshida T Hatano K Yazaki and M AshidaldquoEllagitannins of the casuarinaceae stachyuraceae and myr-taceaerdquo Phytochemistry vol 21 no 12 pp 2871ndash2874 1980
[13] F A Yamanaka T A Hatano H A Ito S B Taniguchi E CTakahashi and K C Okamoto ldquoAntibacterial effects of guavatannins and related polyphenols on Vibrio and AeromonasspeciesrdquoNatural Product Communications vol 3 no 5 pp 711ndash720 2008
[14] T Okuda T Hatano and K Yazaki ldquoGuavin B an ellagitanninof novel typerdquo Chemical amp Pharmaceutical Bulletin vol 32 no9 pp 3787ndash3788 1984
[15] F QarsquoDan F Petereit and A Nahrstedt ldquoPolymeric proantho-cyanidins from Psidium guajavardquo Scientia Pharmaceutica vol73 no 3 pp 113ndash125 2005
[16] B-J Park T Matsuta T Kanazawa C-H Park K-J Changand M Onjo ldquoPhenolic compounds from the leaves of Psidiumguajava II quercetin and its glycosidesrdquo Chemistry of NaturalCompounds vol 48 no 3 pp 477ndash479 2012
[17] S Tachakittirungrod F Ikegami and S Okonogi ldquoAntioxidantactive principles isolated from Psidium guajava grown inThailandrdquo Scientia Pharmaceutica vol 75 no 4 pp 179ndash1932007
[18] K-C Chen C-M Chuang L-Y Lin et al ldquoThe polyphenolicsin the aqueous extract of Psidium guajava kinetically reveal aninhibition model on LDL glycationrdquo Pharmaceutical Biologyvol 48 no 1 pp 23ndash31 2010
[19] K Matsuzaki R Ishii K Kobiyama and S Kitanaka ldquoNewbenzophenone and quercetin galloyl glycosides from Psidiumguajava Lrdquo Journal of Natural Medicines vol 64 no 3 pp 252ndash256 2010
[20] W Nantitanon S Yotsawimonwat and S Okonogi ldquoFactorsinfluencing antioxidant activities and total phenolic content ofguava leaf extractrdquo LWTmdashFood Science and Technology vol 43no 7 pp 1095ndash1103 2010
[21] P Zhang R Lin G Yang J Zhang L Zhou and T Liu ldquoSol-ubility of naringenin in ethanol and water mixturesrdquo Journal ofChemical and Engineering Data vol 58 no 9 pp 2402ndash24042013
[22] J Wen B Liu E Yuan Y Ma and Y Zhu ldquoPreparation andphysicochemical properties of the complex of naringenin withhydroxypropyl-120573-cyclodextrinrdquo Molecules vol 15 no 6 pp4401ndash4407 2010
[23] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010
[24] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006
publications about phenolic and other polar compoundscontent in infusion or guava leaves tea
Quantification of polar compounds was performed bypreparing five calibration curves with the standards availablegallic acid catechin ellagic acid naringenin and rutin Forthose with no commercial standard available quantificationwas carried out comparing with compounds bearing similarstructures
It is important to underline that the quantification resultsreported that the order in terms of concentration of thefamilies of polar compounds in all samples decreased in thefollowing order flavonols gt flavan-3-ols gt gallic and ellagicderivatives gt benzophenones gt flavanones
In general the results given in Table 2 show that theconcentration of each compound is greater in the ultrasoundaqueous extract (AE) except the compounds identified asHHDP glucose that was higher in the infusion of 3min(I3) and in the 5min (I5) samples and naringenin whichpresented the largest concentration in I3 Similar results wereobtained by Nantitanon and coworkers [20] using ethanol asextraction solvent In fact they extracted the guava leavesby maceration and ultrasounds and the highest recovery ofphenolic compounds was obtained by sonication
The higher extraction of HHDP and naringenin in someinfusions than ultrasound extraction could be justified by thetemperature that has been reached during the two extractionmethodologies As reported by Zhang and coworkers [21] thesolubility of naringenin gradually increases as the tempera-ture increases based on these results it is expected to obtainlower extraction of these compounds during ultrasoundextraction instead of that of infusion This hypothesis canbe confirmed with the results obtained by Wen et al [22]that noticed that naringenin is insoluble in water at roomtemperature
However naringenin content in infusion samplesreported a decreasing trend when increasing the time ofinfusion these results should be attributed to a degradation ofthis compound when the thermal treatment was prolonged
To the best of our knowledge there is no literature aboutthe water solubility and the effect of temperature on HHDPcompound Nevertheless taking into account the resultsreported in Table 2 a similar trend to the one reported fornaringenin compound could be supposed for HHDP
Flavan-3-ols gallic and ellagic acid derivatives ben-zophenones and flavonols in the ultrasound aqueous extractwere from 3 to 5 times more concentrated than leavesinfusions Compared to the ultrasound aqueous extract (AE)and infusion of 7min (I7) samples naringenin was 15 and 17times higher in the infusion of 3min (I3) and in the 5min (I5)samples respectively
Flavonols represent about 50 percent of total polar com-pounds in each sample Avicularin and guajaverin were themajor flavonol components and their concentrations variedfrom 137 to 32mgg and from 128 to 27mgg respectivelySimilar trend was showed by Chang et al [11] Morin was alsofound in high concentration with a range that varies from 30to 84mgg in I7 and AE sample respectively Other flavonolcompounds presented in all samples in higher quantities andin the same order of magnitude were hyperin quercitrin
reynoutrin and isoquercitrin Myricetin-arabinoside wasdetected in all samples but it was quantified only in AEsample instead quercetin was only detected and quantifiedin the AE sample These data could promote the use of guavaleaves extract for nutraceutical scopes because as reported byWang et al [23]myricetin and quercetin have high inhibitoryactivities against some enzymes that are involved in diabetesGuavinoside C was quantified in ultrasound aqueous extractit was identified in infusion samples but its content was lowerthan LOQ
The second class of polar compounds was representedby flavan-3-ols which correspond to 26ndash30 of total polarcompounds Procyanidin was the first polar compound andits amounts ranged from 61 to 177mgg Catechin was thesecond flavan-3-ol ranging between 51 and 129mgg
Two epigallocatechin isomers and prodelphinidin dimerwere the third flavan-3-ols and their amounts were about 54ndash59mgg
Gallic and ellagic acid derivatives account for 20 ofthe total concentration of polar compounds in each sam-ple In this case ultrasound aqueous extract and infusionsreported different extraction power Effectively ultrasoundaqueous extract showed casuarinincasuarictin as first ellagicacid derivative (87mgg) on the contrary infusion samplesreported HHDP glucose compounds in the highest amounts(20ndash23mgg) Benzophenones were 2ndash4 of total polarcompounds Guavinoside A was the first benzophenone andit was represented by two isomers Finally four guavinosideB isomers were also detected in the extract but only one wasquantified their content in infusion samples was less thanLOQ or in some cases they were not detected
At last a flavanone namely naringenin was presentedin all samples I3 sample showed the higher content on thecontrary aqueous extract and I7 samples reported the lowestquantities
33 Comparison between Phenolic Content and AntioxidantActivity As shown in Figure 1 the amount of total polarcompounds is significantly higher in the ultrasound aqueousextract than in the infusions Comparing the results obtainedfor the infusions the quantity of these compounds is quitehigher for I5 than for the others I3 and I7 In fact I3 samplereported a lower content probably due to an incompleteextraction of polar compounds instead I7 sample showedlower amounts probably due to a degradation of thesecompounds during maceration
To evaluate the antioxidant activity of the extract andto corroborate the correlation between phenolic content andantioxidant activity two different assays were developedTEAC evaluated by ABTS∙+ test and FRAP
The choice of these two methods was assessed based ontheir different mechanisms the radical scavenging capacitydemonstrated by ABTS and ferric reducing capacities evalu-able by FRAP method Moreover the results obtained byThaipong et al [24] demonstrated that ABTS and FRAPreported higher correlation with total phenolic content inguava fruit compared to other antioxidant activity assays
Total polar compounds by HPLC are in concordancewith the values obtained for the FRAP and ABTS assays
6 Journal of Chemistry
Table 2 Quantification (mean plusmn SD n = 3) of the compounds identified in guava leaves infusions and ultrasound aqueous extract
Number Compounds Quantification (120583g analyteg leaves)AE I3 I5 I7
nd not detected AE aqueous extract obtained by ultrasound I3 I5 and I7 infusion obtained at 3 5 and 7 minutes of infusion time respectivelyThe different letter in the same line means that the compounds are significantly different (P le 005)
Journal of Chemistry 7
AE I3 I5 I7Samples
20
40
60
80
100
120
140
160
180
Tota
l pol
ar co
mpo
unds
(mg
g)
Median Nonoutlier range
OutliersExtremes
Figure 1 Total content (mgg) of total polar compounds by HPLC in analysed samples
Table 3 Comparison between total polar compound (mgg) determined by HPLC and antioxidant activity evaluated by FRAP (120583Mof FeSO4equivalentsmg) and ABTS (120583M of Trolox equivalentsmg)
Means in the same column with different letter are significantly different (P lt 005)
(Table 3) Besides the reducing power and radical scavengingcapacity displayed a significative difference between thesamples obtained by infusion and the ultrasound aqueousextract Positive correlations with R = 09883 and 119875 lt 0001and R = 09973 and 119875 lt 0001 were noticed between totalpolar compounds content and FRAP and ABTS respectively
FRAP and ABTS did not report significative differences(119875 lt 005) among infusion samples however ultrasoundaqueous extract values were higher than infusions values
Moreover a simple linear regression analysis was carriedout to compare the correlation between all compoundsidentified and the antioxidant activity (Table 4)
Most of the polar compounds were highly correlated withFRAP assay (R = 098 119875 lt 0001) except compounds 10 30and 32 that reported an119877 value ranging between 096 and 097(119875 lt 0001) Compound 23 showed a lower correlation (R =076 119875 lt 005) HHDP glucose isomers resulted in inversecorrelation with FRAP assay Moreover naringenin did notshow any correlation
ABTS assay confirmed data reported by FRAP assay infact the two antioxidant assays showed a good correlation
between them that reported an R value of 09916 and 119875 lt00001 These results agreed with the data reported byThaipong et al [24]
4 Conclusions
Several polar compounds have been identified and quantifiedin guava leaves extracts (ultrasound aqueous extract andinfusions) According to the amount of polar compoundsand also the FRAP and ABTS assays the water ultrasoundassisted extraction provided better results than the infusionSignificative positive correlations R gt 098 and 119875 lt 0001were detected between total polar content and antioxi-dant activity assays Moreover positive correlation was alsodetected for single compounds except for HHDP and narin-genin
The results suggested that aqueous ultrasound extract canrepresent a valuable strategy to obtain nutraceuticals using agreen technology About infusions the 5-minute infusion isadvisable for guava leaves culinary uses because of reportedhigher polar compounds content
8 Journal of Chemistry
Table 4 Correlation between the antioxidant activity and polar compounds
Compounds FRAP ABTS119877 value 119875 value 119877 value 119875 value
lowastlowastlowast119875 lt 0001 lowast119875 lt 005 NC not correlated
Journal of Chemistry 9
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was funded by the Project cofinanced by FEDER-Andalucıa 2007ndash2013 (Cod 461100) andAndalusian RegionalGovernment Council of Innovation and Science (P11-CTS-7625) The author Elixabet Dıaz-de-Cerio also would liketo thank the CEIBiotic for the ldquoAyudas a la EnsenanzaPracticardquo Grant (CADP2-71) Ana Marıa Gomez-Caravacaand Vito Verardo thank the Spanish Ministry of Economyand Competitiveness (MINECO) for ldquoJuan de la Ciervardquopostdoctoral contract
References
[1] Q-M Liu and J-G Jiang ldquoAntioxidative activities of medicinalplants from TCMrdquo Mini-Reviews in Medicinal Chemistry vol12 no 11 pp 1154ndash1172 2012
[2] S Z Bathaie N Mokarizade and S Shirali ldquoAn overview of themechanisms of plant ingredients in the treatment of diabetesmellitusrdquo Journal of Medicinal Plants vol 11 no 44 pp 1ndash242012
[3] U Etxeberria A L de la Garza J Campin J A Martnez andF I Milagro ldquoAntidiabetic effects of natural plant extracts viainhibition of carbohydrate hydrolysis enzymes with emphasison pancreatic alpha amylaserdquo Expert Opinion on TherapeuticTargets vol 16 no 3 pp 269ndash297 2012
[4] R M P Gutierrez S Mitchell and R V Solis ldquoPsidiumguajava a review of its traditional uses phytochemistry andpharmacologyrdquo Journal of Ethnopharmacology vol 117 no 1 pp1ndash27 2008
[5] J T Cheng and R S Yang ldquoHypoglycemic effect of guava juicein mice and human subjectsrdquo The American Journal of ChineseMedicine vol 11 no 1-4 pp 74ndash76 1983
[6] T Eidenberger M Selg and K Krennhuber ldquoInhibition ofdipeptidyl peptidase activity by flavonol glycosides of guava(Psidium guajava L) a key to the beneficial effects of guava intype II diabetes mellitusrdquo Fitoterapia vol 89 no 1 pp 74ndash792013
[7] F-C Cheng S-C Shen and J S-B Wu ldquoEffect of guava(Psidium guajava L) leaf extract on glucose uptake in rathepatocytesrdquo Journal of Food Science vol 74 no 5 pp H132ndashH138 2009
[8] H-Y Chen and G-C Yen ldquoAntioxidant activity and freeradical-scavenging capacity of extracts from guava (Psidiumguajava L) leavesrdquo Food Chemistry vol 101 no 2 pp 686ndash6942007
[9] O Laporta L Perez-Fons R Mallavia N Caturla and VMicol ldquoIsolation characterization and antioxidant capacityassessment of the bioactive compounds derived from Hypoxisrooperi corm extract (African potato)rdquo Food Chemistry vol 101no 4 pp 1425ndash1437 2007
[10] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[11] C H Chang C L Hsieh H E Wang C C Peng C CChyau and R Y Peng ldquoUnique bioactive polyphenolic profile
of guava (Psidium guajava) budding leaf tea is related to plantbiochemistry of budding leaves in early dawnrdquo Journal of theScience of Food andAgriculture vol 93 no 4 pp 944ndash954 2013
[12] T Okuda T Yoshida T Hatano K Yazaki and M AshidaldquoEllagitannins of the casuarinaceae stachyuraceae and myr-taceaerdquo Phytochemistry vol 21 no 12 pp 2871ndash2874 1980
[13] F A Yamanaka T A Hatano H A Ito S B Taniguchi E CTakahashi and K C Okamoto ldquoAntibacterial effects of guavatannins and related polyphenols on Vibrio and AeromonasspeciesrdquoNatural Product Communications vol 3 no 5 pp 711ndash720 2008
[14] T Okuda T Hatano and K Yazaki ldquoGuavin B an ellagitanninof novel typerdquo Chemical amp Pharmaceutical Bulletin vol 32 no9 pp 3787ndash3788 1984
[15] F QarsquoDan F Petereit and A Nahrstedt ldquoPolymeric proantho-cyanidins from Psidium guajavardquo Scientia Pharmaceutica vol73 no 3 pp 113ndash125 2005
[16] B-J Park T Matsuta T Kanazawa C-H Park K-J Changand M Onjo ldquoPhenolic compounds from the leaves of Psidiumguajava II quercetin and its glycosidesrdquo Chemistry of NaturalCompounds vol 48 no 3 pp 477ndash479 2012
[17] S Tachakittirungrod F Ikegami and S Okonogi ldquoAntioxidantactive principles isolated from Psidium guajava grown inThailandrdquo Scientia Pharmaceutica vol 75 no 4 pp 179ndash1932007
[18] K-C Chen C-M Chuang L-Y Lin et al ldquoThe polyphenolicsin the aqueous extract of Psidium guajava kinetically reveal aninhibition model on LDL glycationrdquo Pharmaceutical Biologyvol 48 no 1 pp 23ndash31 2010
[19] K Matsuzaki R Ishii K Kobiyama and S Kitanaka ldquoNewbenzophenone and quercetin galloyl glycosides from Psidiumguajava Lrdquo Journal of Natural Medicines vol 64 no 3 pp 252ndash256 2010
[20] W Nantitanon S Yotsawimonwat and S Okonogi ldquoFactorsinfluencing antioxidant activities and total phenolic content ofguava leaf extractrdquo LWTmdashFood Science and Technology vol 43no 7 pp 1095ndash1103 2010
[21] P Zhang R Lin G Yang J Zhang L Zhou and T Liu ldquoSol-ubility of naringenin in ethanol and water mixturesrdquo Journal ofChemical and Engineering Data vol 58 no 9 pp 2402ndash24042013
[22] J Wen B Liu E Yuan Y Ma and Y Zhu ldquoPreparation andphysicochemical properties of the complex of naringenin withhydroxypropyl-120573-cyclodextrinrdquo Molecules vol 15 no 6 pp4401ndash4407 2010
[23] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010
[24] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006
publications about phenolic and other polar compoundscontent in infusion or guava leaves tea
Quantification of polar compounds was performed bypreparing five calibration curves with the standards availablegallic acid catechin ellagic acid naringenin and rutin Forthose with no commercial standard available quantificationwas carried out comparing with compounds bearing similarstructures
It is important to underline that the quantification resultsreported that the order in terms of concentration of thefamilies of polar compounds in all samples decreased in thefollowing order flavonols gt flavan-3-ols gt gallic and ellagicderivatives gt benzophenones gt flavanones
In general the results given in Table 2 show that theconcentration of each compound is greater in the ultrasoundaqueous extract (AE) except the compounds identified asHHDP glucose that was higher in the infusion of 3min(I3) and in the 5min (I5) samples and naringenin whichpresented the largest concentration in I3 Similar results wereobtained by Nantitanon and coworkers [20] using ethanol asextraction solvent In fact they extracted the guava leavesby maceration and ultrasounds and the highest recovery ofphenolic compounds was obtained by sonication
The higher extraction of HHDP and naringenin in someinfusions than ultrasound extraction could be justified by thetemperature that has been reached during the two extractionmethodologies As reported by Zhang and coworkers [21] thesolubility of naringenin gradually increases as the tempera-ture increases based on these results it is expected to obtainlower extraction of these compounds during ultrasoundextraction instead of that of infusion This hypothesis canbe confirmed with the results obtained by Wen et al [22]that noticed that naringenin is insoluble in water at roomtemperature
However naringenin content in infusion samplesreported a decreasing trend when increasing the time ofinfusion these results should be attributed to a degradation ofthis compound when the thermal treatment was prolonged
To the best of our knowledge there is no literature aboutthe water solubility and the effect of temperature on HHDPcompound Nevertheless taking into account the resultsreported in Table 2 a similar trend to the one reported fornaringenin compound could be supposed for HHDP
Flavan-3-ols gallic and ellagic acid derivatives ben-zophenones and flavonols in the ultrasound aqueous extractwere from 3 to 5 times more concentrated than leavesinfusions Compared to the ultrasound aqueous extract (AE)and infusion of 7min (I7) samples naringenin was 15 and 17times higher in the infusion of 3min (I3) and in the 5min (I5)samples respectively
Flavonols represent about 50 percent of total polar com-pounds in each sample Avicularin and guajaverin were themajor flavonol components and their concentrations variedfrom 137 to 32mgg and from 128 to 27mgg respectivelySimilar trend was showed by Chang et al [11] Morin was alsofound in high concentration with a range that varies from 30to 84mgg in I7 and AE sample respectively Other flavonolcompounds presented in all samples in higher quantities andin the same order of magnitude were hyperin quercitrin
reynoutrin and isoquercitrin Myricetin-arabinoside wasdetected in all samples but it was quantified only in AEsample instead quercetin was only detected and quantifiedin the AE sample These data could promote the use of guavaleaves extract for nutraceutical scopes because as reported byWang et al [23]myricetin and quercetin have high inhibitoryactivities against some enzymes that are involved in diabetesGuavinoside C was quantified in ultrasound aqueous extractit was identified in infusion samples but its content was lowerthan LOQ
The second class of polar compounds was representedby flavan-3-ols which correspond to 26ndash30 of total polarcompounds Procyanidin was the first polar compound andits amounts ranged from 61 to 177mgg Catechin was thesecond flavan-3-ol ranging between 51 and 129mgg
Two epigallocatechin isomers and prodelphinidin dimerwere the third flavan-3-ols and their amounts were about 54ndash59mgg
Gallic and ellagic acid derivatives account for 20 ofthe total concentration of polar compounds in each sam-ple In this case ultrasound aqueous extract and infusionsreported different extraction power Effectively ultrasoundaqueous extract showed casuarinincasuarictin as first ellagicacid derivative (87mgg) on the contrary infusion samplesreported HHDP glucose compounds in the highest amounts(20ndash23mgg) Benzophenones were 2ndash4 of total polarcompounds Guavinoside A was the first benzophenone andit was represented by two isomers Finally four guavinosideB isomers were also detected in the extract but only one wasquantified their content in infusion samples was less thanLOQ or in some cases they were not detected
At last a flavanone namely naringenin was presentedin all samples I3 sample showed the higher content on thecontrary aqueous extract and I7 samples reported the lowestquantities
33 Comparison between Phenolic Content and AntioxidantActivity As shown in Figure 1 the amount of total polarcompounds is significantly higher in the ultrasound aqueousextract than in the infusions Comparing the results obtainedfor the infusions the quantity of these compounds is quitehigher for I5 than for the others I3 and I7 In fact I3 samplereported a lower content probably due to an incompleteextraction of polar compounds instead I7 sample showedlower amounts probably due to a degradation of thesecompounds during maceration
To evaluate the antioxidant activity of the extract andto corroborate the correlation between phenolic content andantioxidant activity two different assays were developedTEAC evaluated by ABTS∙+ test and FRAP
The choice of these two methods was assessed based ontheir different mechanisms the radical scavenging capacitydemonstrated by ABTS and ferric reducing capacities evalu-able by FRAP method Moreover the results obtained byThaipong et al [24] demonstrated that ABTS and FRAPreported higher correlation with total phenolic content inguava fruit compared to other antioxidant activity assays
Total polar compounds by HPLC are in concordancewith the values obtained for the FRAP and ABTS assays
6 Journal of Chemistry
Table 2 Quantification (mean plusmn SD n = 3) of the compounds identified in guava leaves infusions and ultrasound aqueous extract
Number Compounds Quantification (120583g analyteg leaves)AE I3 I5 I7
nd not detected AE aqueous extract obtained by ultrasound I3 I5 and I7 infusion obtained at 3 5 and 7 minutes of infusion time respectivelyThe different letter in the same line means that the compounds are significantly different (P le 005)
Journal of Chemistry 7
AE I3 I5 I7Samples
20
40
60
80
100
120
140
160
180
Tota
l pol
ar co
mpo
unds
(mg
g)
Median Nonoutlier range
OutliersExtremes
Figure 1 Total content (mgg) of total polar compounds by HPLC in analysed samples
Table 3 Comparison between total polar compound (mgg) determined by HPLC and antioxidant activity evaluated by FRAP (120583Mof FeSO4equivalentsmg) and ABTS (120583M of Trolox equivalentsmg)
Means in the same column with different letter are significantly different (P lt 005)
(Table 3) Besides the reducing power and radical scavengingcapacity displayed a significative difference between thesamples obtained by infusion and the ultrasound aqueousextract Positive correlations with R = 09883 and 119875 lt 0001and R = 09973 and 119875 lt 0001 were noticed between totalpolar compounds content and FRAP and ABTS respectively
FRAP and ABTS did not report significative differences(119875 lt 005) among infusion samples however ultrasoundaqueous extract values were higher than infusions values
Moreover a simple linear regression analysis was carriedout to compare the correlation between all compoundsidentified and the antioxidant activity (Table 4)
Most of the polar compounds were highly correlated withFRAP assay (R = 098 119875 lt 0001) except compounds 10 30and 32 that reported an119877 value ranging between 096 and 097(119875 lt 0001) Compound 23 showed a lower correlation (R =076 119875 lt 005) HHDP glucose isomers resulted in inversecorrelation with FRAP assay Moreover naringenin did notshow any correlation
ABTS assay confirmed data reported by FRAP assay infact the two antioxidant assays showed a good correlation
between them that reported an R value of 09916 and 119875 lt00001 These results agreed with the data reported byThaipong et al [24]
4 Conclusions
Several polar compounds have been identified and quantifiedin guava leaves extracts (ultrasound aqueous extract andinfusions) According to the amount of polar compoundsand also the FRAP and ABTS assays the water ultrasoundassisted extraction provided better results than the infusionSignificative positive correlations R gt 098 and 119875 lt 0001were detected between total polar content and antioxi-dant activity assays Moreover positive correlation was alsodetected for single compounds except for HHDP and narin-genin
The results suggested that aqueous ultrasound extract canrepresent a valuable strategy to obtain nutraceuticals using agreen technology About infusions the 5-minute infusion isadvisable for guava leaves culinary uses because of reportedhigher polar compounds content
8 Journal of Chemistry
Table 4 Correlation between the antioxidant activity and polar compounds
Compounds FRAP ABTS119877 value 119875 value 119877 value 119875 value
lowastlowastlowast119875 lt 0001 lowast119875 lt 005 NC not correlated
Journal of Chemistry 9
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was funded by the Project cofinanced by FEDER-Andalucıa 2007ndash2013 (Cod 461100) andAndalusian RegionalGovernment Council of Innovation and Science (P11-CTS-7625) The author Elixabet Dıaz-de-Cerio also would liketo thank the CEIBiotic for the ldquoAyudas a la EnsenanzaPracticardquo Grant (CADP2-71) Ana Marıa Gomez-Caravacaand Vito Verardo thank the Spanish Ministry of Economyand Competitiveness (MINECO) for ldquoJuan de la Ciervardquopostdoctoral contract
References
[1] Q-M Liu and J-G Jiang ldquoAntioxidative activities of medicinalplants from TCMrdquo Mini-Reviews in Medicinal Chemistry vol12 no 11 pp 1154ndash1172 2012
[2] S Z Bathaie N Mokarizade and S Shirali ldquoAn overview of themechanisms of plant ingredients in the treatment of diabetesmellitusrdquo Journal of Medicinal Plants vol 11 no 44 pp 1ndash242012
[3] U Etxeberria A L de la Garza J Campin J A Martnez andF I Milagro ldquoAntidiabetic effects of natural plant extracts viainhibition of carbohydrate hydrolysis enzymes with emphasison pancreatic alpha amylaserdquo Expert Opinion on TherapeuticTargets vol 16 no 3 pp 269ndash297 2012
[4] R M P Gutierrez S Mitchell and R V Solis ldquoPsidiumguajava a review of its traditional uses phytochemistry andpharmacologyrdquo Journal of Ethnopharmacology vol 117 no 1 pp1ndash27 2008
[5] J T Cheng and R S Yang ldquoHypoglycemic effect of guava juicein mice and human subjectsrdquo The American Journal of ChineseMedicine vol 11 no 1-4 pp 74ndash76 1983
[6] T Eidenberger M Selg and K Krennhuber ldquoInhibition ofdipeptidyl peptidase activity by flavonol glycosides of guava(Psidium guajava L) a key to the beneficial effects of guava intype II diabetes mellitusrdquo Fitoterapia vol 89 no 1 pp 74ndash792013
[7] F-C Cheng S-C Shen and J S-B Wu ldquoEffect of guava(Psidium guajava L) leaf extract on glucose uptake in rathepatocytesrdquo Journal of Food Science vol 74 no 5 pp H132ndashH138 2009
[8] H-Y Chen and G-C Yen ldquoAntioxidant activity and freeradical-scavenging capacity of extracts from guava (Psidiumguajava L) leavesrdquo Food Chemistry vol 101 no 2 pp 686ndash6942007
[9] O Laporta L Perez-Fons R Mallavia N Caturla and VMicol ldquoIsolation characterization and antioxidant capacityassessment of the bioactive compounds derived from Hypoxisrooperi corm extract (African potato)rdquo Food Chemistry vol 101no 4 pp 1425ndash1437 2007
[10] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[11] C H Chang C L Hsieh H E Wang C C Peng C CChyau and R Y Peng ldquoUnique bioactive polyphenolic profile
of guava (Psidium guajava) budding leaf tea is related to plantbiochemistry of budding leaves in early dawnrdquo Journal of theScience of Food andAgriculture vol 93 no 4 pp 944ndash954 2013
[12] T Okuda T Yoshida T Hatano K Yazaki and M AshidaldquoEllagitannins of the casuarinaceae stachyuraceae and myr-taceaerdquo Phytochemistry vol 21 no 12 pp 2871ndash2874 1980
[13] F A Yamanaka T A Hatano H A Ito S B Taniguchi E CTakahashi and K C Okamoto ldquoAntibacterial effects of guavatannins and related polyphenols on Vibrio and AeromonasspeciesrdquoNatural Product Communications vol 3 no 5 pp 711ndash720 2008
[14] T Okuda T Hatano and K Yazaki ldquoGuavin B an ellagitanninof novel typerdquo Chemical amp Pharmaceutical Bulletin vol 32 no9 pp 3787ndash3788 1984
[15] F QarsquoDan F Petereit and A Nahrstedt ldquoPolymeric proantho-cyanidins from Psidium guajavardquo Scientia Pharmaceutica vol73 no 3 pp 113ndash125 2005
[16] B-J Park T Matsuta T Kanazawa C-H Park K-J Changand M Onjo ldquoPhenolic compounds from the leaves of Psidiumguajava II quercetin and its glycosidesrdquo Chemistry of NaturalCompounds vol 48 no 3 pp 477ndash479 2012
[17] S Tachakittirungrod F Ikegami and S Okonogi ldquoAntioxidantactive principles isolated from Psidium guajava grown inThailandrdquo Scientia Pharmaceutica vol 75 no 4 pp 179ndash1932007
[18] K-C Chen C-M Chuang L-Y Lin et al ldquoThe polyphenolicsin the aqueous extract of Psidium guajava kinetically reveal aninhibition model on LDL glycationrdquo Pharmaceutical Biologyvol 48 no 1 pp 23ndash31 2010
[19] K Matsuzaki R Ishii K Kobiyama and S Kitanaka ldquoNewbenzophenone and quercetin galloyl glycosides from Psidiumguajava Lrdquo Journal of Natural Medicines vol 64 no 3 pp 252ndash256 2010
[20] W Nantitanon S Yotsawimonwat and S Okonogi ldquoFactorsinfluencing antioxidant activities and total phenolic content ofguava leaf extractrdquo LWTmdashFood Science and Technology vol 43no 7 pp 1095ndash1103 2010
[21] P Zhang R Lin G Yang J Zhang L Zhou and T Liu ldquoSol-ubility of naringenin in ethanol and water mixturesrdquo Journal ofChemical and Engineering Data vol 58 no 9 pp 2402ndash24042013
[22] J Wen B Liu E Yuan Y Ma and Y Zhu ldquoPreparation andphysicochemical properties of the complex of naringenin withhydroxypropyl-120573-cyclodextrinrdquo Molecules vol 15 no 6 pp4401ndash4407 2010
[23] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010
[24] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006
nd not detected AE aqueous extract obtained by ultrasound I3 I5 and I7 infusion obtained at 3 5 and 7 minutes of infusion time respectivelyThe different letter in the same line means that the compounds are significantly different (P le 005)
Journal of Chemistry 7
AE I3 I5 I7Samples
20
40
60
80
100
120
140
160
180
Tota
l pol
ar co
mpo
unds
(mg
g)
Median Nonoutlier range
OutliersExtremes
Figure 1 Total content (mgg) of total polar compounds by HPLC in analysed samples
Table 3 Comparison between total polar compound (mgg) determined by HPLC and antioxidant activity evaluated by FRAP (120583Mof FeSO4equivalentsmg) and ABTS (120583M of Trolox equivalentsmg)
Means in the same column with different letter are significantly different (P lt 005)
(Table 3) Besides the reducing power and radical scavengingcapacity displayed a significative difference between thesamples obtained by infusion and the ultrasound aqueousextract Positive correlations with R = 09883 and 119875 lt 0001and R = 09973 and 119875 lt 0001 were noticed between totalpolar compounds content and FRAP and ABTS respectively
FRAP and ABTS did not report significative differences(119875 lt 005) among infusion samples however ultrasoundaqueous extract values were higher than infusions values
Moreover a simple linear regression analysis was carriedout to compare the correlation between all compoundsidentified and the antioxidant activity (Table 4)
Most of the polar compounds were highly correlated withFRAP assay (R = 098 119875 lt 0001) except compounds 10 30and 32 that reported an119877 value ranging between 096 and 097(119875 lt 0001) Compound 23 showed a lower correlation (R =076 119875 lt 005) HHDP glucose isomers resulted in inversecorrelation with FRAP assay Moreover naringenin did notshow any correlation
ABTS assay confirmed data reported by FRAP assay infact the two antioxidant assays showed a good correlation
between them that reported an R value of 09916 and 119875 lt00001 These results agreed with the data reported byThaipong et al [24]
4 Conclusions
Several polar compounds have been identified and quantifiedin guava leaves extracts (ultrasound aqueous extract andinfusions) According to the amount of polar compoundsand also the FRAP and ABTS assays the water ultrasoundassisted extraction provided better results than the infusionSignificative positive correlations R gt 098 and 119875 lt 0001were detected between total polar content and antioxi-dant activity assays Moreover positive correlation was alsodetected for single compounds except for HHDP and narin-genin
The results suggested that aqueous ultrasound extract canrepresent a valuable strategy to obtain nutraceuticals using agreen technology About infusions the 5-minute infusion isadvisable for guava leaves culinary uses because of reportedhigher polar compounds content
8 Journal of Chemistry
Table 4 Correlation between the antioxidant activity and polar compounds
Compounds FRAP ABTS119877 value 119875 value 119877 value 119875 value
lowastlowastlowast119875 lt 0001 lowast119875 lt 005 NC not correlated
Journal of Chemistry 9
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was funded by the Project cofinanced by FEDER-Andalucıa 2007ndash2013 (Cod 461100) andAndalusian RegionalGovernment Council of Innovation and Science (P11-CTS-7625) The author Elixabet Dıaz-de-Cerio also would liketo thank the CEIBiotic for the ldquoAyudas a la EnsenanzaPracticardquo Grant (CADP2-71) Ana Marıa Gomez-Caravacaand Vito Verardo thank the Spanish Ministry of Economyand Competitiveness (MINECO) for ldquoJuan de la Ciervardquopostdoctoral contract
References
[1] Q-M Liu and J-G Jiang ldquoAntioxidative activities of medicinalplants from TCMrdquo Mini-Reviews in Medicinal Chemistry vol12 no 11 pp 1154ndash1172 2012
[2] S Z Bathaie N Mokarizade and S Shirali ldquoAn overview of themechanisms of plant ingredients in the treatment of diabetesmellitusrdquo Journal of Medicinal Plants vol 11 no 44 pp 1ndash242012
[3] U Etxeberria A L de la Garza J Campin J A Martnez andF I Milagro ldquoAntidiabetic effects of natural plant extracts viainhibition of carbohydrate hydrolysis enzymes with emphasison pancreatic alpha amylaserdquo Expert Opinion on TherapeuticTargets vol 16 no 3 pp 269ndash297 2012
[4] R M P Gutierrez S Mitchell and R V Solis ldquoPsidiumguajava a review of its traditional uses phytochemistry andpharmacologyrdquo Journal of Ethnopharmacology vol 117 no 1 pp1ndash27 2008
[5] J T Cheng and R S Yang ldquoHypoglycemic effect of guava juicein mice and human subjectsrdquo The American Journal of ChineseMedicine vol 11 no 1-4 pp 74ndash76 1983
[6] T Eidenberger M Selg and K Krennhuber ldquoInhibition ofdipeptidyl peptidase activity by flavonol glycosides of guava(Psidium guajava L) a key to the beneficial effects of guava intype II diabetes mellitusrdquo Fitoterapia vol 89 no 1 pp 74ndash792013
[7] F-C Cheng S-C Shen and J S-B Wu ldquoEffect of guava(Psidium guajava L) leaf extract on glucose uptake in rathepatocytesrdquo Journal of Food Science vol 74 no 5 pp H132ndashH138 2009
[8] H-Y Chen and G-C Yen ldquoAntioxidant activity and freeradical-scavenging capacity of extracts from guava (Psidiumguajava L) leavesrdquo Food Chemistry vol 101 no 2 pp 686ndash6942007
[9] O Laporta L Perez-Fons R Mallavia N Caturla and VMicol ldquoIsolation characterization and antioxidant capacityassessment of the bioactive compounds derived from Hypoxisrooperi corm extract (African potato)rdquo Food Chemistry vol 101no 4 pp 1425ndash1437 2007
[10] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[11] C H Chang C L Hsieh H E Wang C C Peng C CChyau and R Y Peng ldquoUnique bioactive polyphenolic profile
of guava (Psidium guajava) budding leaf tea is related to plantbiochemistry of budding leaves in early dawnrdquo Journal of theScience of Food andAgriculture vol 93 no 4 pp 944ndash954 2013
[12] T Okuda T Yoshida T Hatano K Yazaki and M AshidaldquoEllagitannins of the casuarinaceae stachyuraceae and myr-taceaerdquo Phytochemistry vol 21 no 12 pp 2871ndash2874 1980
[13] F A Yamanaka T A Hatano H A Ito S B Taniguchi E CTakahashi and K C Okamoto ldquoAntibacterial effects of guavatannins and related polyphenols on Vibrio and AeromonasspeciesrdquoNatural Product Communications vol 3 no 5 pp 711ndash720 2008
[14] T Okuda T Hatano and K Yazaki ldquoGuavin B an ellagitanninof novel typerdquo Chemical amp Pharmaceutical Bulletin vol 32 no9 pp 3787ndash3788 1984
[15] F QarsquoDan F Petereit and A Nahrstedt ldquoPolymeric proantho-cyanidins from Psidium guajavardquo Scientia Pharmaceutica vol73 no 3 pp 113ndash125 2005
[16] B-J Park T Matsuta T Kanazawa C-H Park K-J Changand M Onjo ldquoPhenolic compounds from the leaves of Psidiumguajava II quercetin and its glycosidesrdquo Chemistry of NaturalCompounds vol 48 no 3 pp 477ndash479 2012
[17] S Tachakittirungrod F Ikegami and S Okonogi ldquoAntioxidantactive principles isolated from Psidium guajava grown inThailandrdquo Scientia Pharmaceutica vol 75 no 4 pp 179ndash1932007
[18] K-C Chen C-M Chuang L-Y Lin et al ldquoThe polyphenolicsin the aqueous extract of Psidium guajava kinetically reveal aninhibition model on LDL glycationrdquo Pharmaceutical Biologyvol 48 no 1 pp 23ndash31 2010
[19] K Matsuzaki R Ishii K Kobiyama and S Kitanaka ldquoNewbenzophenone and quercetin galloyl glycosides from Psidiumguajava Lrdquo Journal of Natural Medicines vol 64 no 3 pp 252ndash256 2010
[20] W Nantitanon S Yotsawimonwat and S Okonogi ldquoFactorsinfluencing antioxidant activities and total phenolic content ofguava leaf extractrdquo LWTmdashFood Science and Technology vol 43no 7 pp 1095ndash1103 2010
[21] P Zhang R Lin G Yang J Zhang L Zhou and T Liu ldquoSol-ubility of naringenin in ethanol and water mixturesrdquo Journal ofChemical and Engineering Data vol 58 no 9 pp 2402ndash24042013
[22] J Wen B Liu E Yuan Y Ma and Y Zhu ldquoPreparation andphysicochemical properties of the complex of naringenin withhydroxypropyl-120573-cyclodextrinrdquo Molecules vol 15 no 6 pp4401ndash4407 2010
[23] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010
[24] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006
Figure 1 Total content (mgg) of total polar compounds by HPLC in analysed samples
Table 3 Comparison between total polar compound (mgg) determined by HPLC and antioxidant activity evaluated by FRAP (120583Mof FeSO4equivalentsmg) and ABTS (120583M of Trolox equivalentsmg)
Means in the same column with different letter are significantly different (P lt 005)
(Table 3) Besides the reducing power and radical scavengingcapacity displayed a significative difference between thesamples obtained by infusion and the ultrasound aqueousextract Positive correlations with R = 09883 and 119875 lt 0001and R = 09973 and 119875 lt 0001 were noticed between totalpolar compounds content and FRAP and ABTS respectively
FRAP and ABTS did not report significative differences(119875 lt 005) among infusion samples however ultrasoundaqueous extract values were higher than infusions values
Moreover a simple linear regression analysis was carriedout to compare the correlation between all compoundsidentified and the antioxidant activity (Table 4)
Most of the polar compounds were highly correlated withFRAP assay (R = 098 119875 lt 0001) except compounds 10 30and 32 that reported an119877 value ranging between 096 and 097(119875 lt 0001) Compound 23 showed a lower correlation (R =076 119875 lt 005) HHDP glucose isomers resulted in inversecorrelation with FRAP assay Moreover naringenin did notshow any correlation
ABTS assay confirmed data reported by FRAP assay infact the two antioxidant assays showed a good correlation
between them that reported an R value of 09916 and 119875 lt00001 These results agreed with the data reported byThaipong et al [24]
4 Conclusions
Several polar compounds have been identified and quantifiedin guava leaves extracts (ultrasound aqueous extract andinfusions) According to the amount of polar compoundsand also the FRAP and ABTS assays the water ultrasoundassisted extraction provided better results than the infusionSignificative positive correlations R gt 098 and 119875 lt 0001were detected between total polar content and antioxi-dant activity assays Moreover positive correlation was alsodetected for single compounds except for HHDP and narin-genin
The results suggested that aqueous ultrasound extract canrepresent a valuable strategy to obtain nutraceuticals using agreen technology About infusions the 5-minute infusion isadvisable for guava leaves culinary uses because of reportedhigher polar compounds content
8 Journal of Chemistry
Table 4 Correlation between the antioxidant activity and polar compounds
Compounds FRAP ABTS119877 value 119875 value 119877 value 119875 value
lowastlowastlowast119875 lt 0001 lowast119875 lt 005 NC not correlated
Journal of Chemistry 9
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was funded by the Project cofinanced by FEDER-Andalucıa 2007ndash2013 (Cod 461100) andAndalusian RegionalGovernment Council of Innovation and Science (P11-CTS-7625) The author Elixabet Dıaz-de-Cerio also would liketo thank the CEIBiotic for the ldquoAyudas a la EnsenanzaPracticardquo Grant (CADP2-71) Ana Marıa Gomez-Caravacaand Vito Verardo thank the Spanish Ministry of Economyand Competitiveness (MINECO) for ldquoJuan de la Ciervardquopostdoctoral contract
References
[1] Q-M Liu and J-G Jiang ldquoAntioxidative activities of medicinalplants from TCMrdquo Mini-Reviews in Medicinal Chemistry vol12 no 11 pp 1154ndash1172 2012
[2] S Z Bathaie N Mokarizade and S Shirali ldquoAn overview of themechanisms of plant ingredients in the treatment of diabetesmellitusrdquo Journal of Medicinal Plants vol 11 no 44 pp 1ndash242012
[3] U Etxeberria A L de la Garza J Campin J A Martnez andF I Milagro ldquoAntidiabetic effects of natural plant extracts viainhibition of carbohydrate hydrolysis enzymes with emphasison pancreatic alpha amylaserdquo Expert Opinion on TherapeuticTargets vol 16 no 3 pp 269ndash297 2012
[4] R M P Gutierrez S Mitchell and R V Solis ldquoPsidiumguajava a review of its traditional uses phytochemistry andpharmacologyrdquo Journal of Ethnopharmacology vol 117 no 1 pp1ndash27 2008
[5] J T Cheng and R S Yang ldquoHypoglycemic effect of guava juicein mice and human subjectsrdquo The American Journal of ChineseMedicine vol 11 no 1-4 pp 74ndash76 1983
[6] T Eidenberger M Selg and K Krennhuber ldquoInhibition ofdipeptidyl peptidase activity by flavonol glycosides of guava(Psidium guajava L) a key to the beneficial effects of guava intype II diabetes mellitusrdquo Fitoterapia vol 89 no 1 pp 74ndash792013
[7] F-C Cheng S-C Shen and J S-B Wu ldquoEffect of guava(Psidium guajava L) leaf extract on glucose uptake in rathepatocytesrdquo Journal of Food Science vol 74 no 5 pp H132ndashH138 2009
[8] H-Y Chen and G-C Yen ldquoAntioxidant activity and freeradical-scavenging capacity of extracts from guava (Psidiumguajava L) leavesrdquo Food Chemistry vol 101 no 2 pp 686ndash6942007
[9] O Laporta L Perez-Fons R Mallavia N Caturla and VMicol ldquoIsolation characterization and antioxidant capacityassessment of the bioactive compounds derived from Hypoxisrooperi corm extract (African potato)rdquo Food Chemistry vol 101no 4 pp 1425ndash1437 2007
[10] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[11] C H Chang C L Hsieh H E Wang C C Peng C CChyau and R Y Peng ldquoUnique bioactive polyphenolic profile
of guava (Psidium guajava) budding leaf tea is related to plantbiochemistry of budding leaves in early dawnrdquo Journal of theScience of Food andAgriculture vol 93 no 4 pp 944ndash954 2013
[12] T Okuda T Yoshida T Hatano K Yazaki and M AshidaldquoEllagitannins of the casuarinaceae stachyuraceae and myr-taceaerdquo Phytochemistry vol 21 no 12 pp 2871ndash2874 1980
[13] F A Yamanaka T A Hatano H A Ito S B Taniguchi E CTakahashi and K C Okamoto ldquoAntibacterial effects of guavatannins and related polyphenols on Vibrio and AeromonasspeciesrdquoNatural Product Communications vol 3 no 5 pp 711ndash720 2008
[14] T Okuda T Hatano and K Yazaki ldquoGuavin B an ellagitanninof novel typerdquo Chemical amp Pharmaceutical Bulletin vol 32 no9 pp 3787ndash3788 1984
[15] F QarsquoDan F Petereit and A Nahrstedt ldquoPolymeric proantho-cyanidins from Psidium guajavardquo Scientia Pharmaceutica vol73 no 3 pp 113ndash125 2005
[16] B-J Park T Matsuta T Kanazawa C-H Park K-J Changand M Onjo ldquoPhenolic compounds from the leaves of Psidiumguajava II quercetin and its glycosidesrdquo Chemistry of NaturalCompounds vol 48 no 3 pp 477ndash479 2012
[17] S Tachakittirungrod F Ikegami and S Okonogi ldquoAntioxidantactive principles isolated from Psidium guajava grown inThailandrdquo Scientia Pharmaceutica vol 75 no 4 pp 179ndash1932007
[18] K-C Chen C-M Chuang L-Y Lin et al ldquoThe polyphenolicsin the aqueous extract of Psidium guajava kinetically reveal aninhibition model on LDL glycationrdquo Pharmaceutical Biologyvol 48 no 1 pp 23ndash31 2010
[19] K Matsuzaki R Ishii K Kobiyama and S Kitanaka ldquoNewbenzophenone and quercetin galloyl glycosides from Psidiumguajava Lrdquo Journal of Natural Medicines vol 64 no 3 pp 252ndash256 2010
[20] W Nantitanon S Yotsawimonwat and S Okonogi ldquoFactorsinfluencing antioxidant activities and total phenolic content ofguava leaf extractrdquo LWTmdashFood Science and Technology vol 43no 7 pp 1095ndash1103 2010
[21] P Zhang R Lin G Yang J Zhang L Zhou and T Liu ldquoSol-ubility of naringenin in ethanol and water mixturesrdquo Journal ofChemical and Engineering Data vol 58 no 9 pp 2402ndash24042013
[22] J Wen B Liu E Yuan Y Ma and Y Zhu ldquoPreparation andphysicochemical properties of the complex of naringenin withhydroxypropyl-120573-cyclodextrinrdquo Molecules vol 15 no 6 pp4401ndash4407 2010
[23] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010
[24] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006
lowastlowastlowast119875 lt 0001 lowast119875 lt 005 NC not correlated
Journal of Chemistry 9
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was funded by the Project cofinanced by FEDER-Andalucıa 2007ndash2013 (Cod 461100) andAndalusian RegionalGovernment Council of Innovation and Science (P11-CTS-7625) The author Elixabet Dıaz-de-Cerio also would liketo thank the CEIBiotic for the ldquoAyudas a la EnsenanzaPracticardquo Grant (CADP2-71) Ana Marıa Gomez-Caravacaand Vito Verardo thank the Spanish Ministry of Economyand Competitiveness (MINECO) for ldquoJuan de la Ciervardquopostdoctoral contract
References
[1] Q-M Liu and J-G Jiang ldquoAntioxidative activities of medicinalplants from TCMrdquo Mini-Reviews in Medicinal Chemistry vol12 no 11 pp 1154ndash1172 2012
[2] S Z Bathaie N Mokarizade and S Shirali ldquoAn overview of themechanisms of plant ingredients in the treatment of diabetesmellitusrdquo Journal of Medicinal Plants vol 11 no 44 pp 1ndash242012
[3] U Etxeberria A L de la Garza J Campin J A Martnez andF I Milagro ldquoAntidiabetic effects of natural plant extracts viainhibition of carbohydrate hydrolysis enzymes with emphasison pancreatic alpha amylaserdquo Expert Opinion on TherapeuticTargets vol 16 no 3 pp 269ndash297 2012
[4] R M P Gutierrez S Mitchell and R V Solis ldquoPsidiumguajava a review of its traditional uses phytochemistry andpharmacologyrdquo Journal of Ethnopharmacology vol 117 no 1 pp1ndash27 2008
[5] J T Cheng and R S Yang ldquoHypoglycemic effect of guava juicein mice and human subjectsrdquo The American Journal of ChineseMedicine vol 11 no 1-4 pp 74ndash76 1983
[6] T Eidenberger M Selg and K Krennhuber ldquoInhibition ofdipeptidyl peptidase activity by flavonol glycosides of guava(Psidium guajava L) a key to the beneficial effects of guava intype II diabetes mellitusrdquo Fitoterapia vol 89 no 1 pp 74ndash792013
[7] F-C Cheng S-C Shen and J S-B Wu ldquoEffect of guava(Psidium guajava L) leaf extract on glucose uptake in rathepatocytesrdquo Journal of Food Science vol 74 no 5 pp H132ndashH138 2009
[8] H-Y Chen and G-C Yen ldquoAntioxidant activity and freeradical-scavenging capacity of extracts from guava (Psidiumguajava L) leavesrdquo Food Chemistry vol 101 no 2 pp 686ndash6942007
[9] O Laporta L Perez-Fons R Mallavia N Caturla and VMicol ldquoIsolation characterization and antioxidant capacityassessment of the bioactive compounds derived from Hypoxisrooperi corm extract (African potato)rdquo Food Chemistry vol 101no 4 pp 1425ndash1437 2007
[10] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[11] C H Chang C L Hsieh H E Wang C C Peng C CChyau and R Y Peng ldquoUnique bioactive polyphenolic profile
of guava (Psidium guajava) budding leaf tea is related to plantbiochemistry of budding leaves in early dawnrdquo Journal of theScience of Food andAgriculture vol 93 no 4 pp 944ndash954 2013
[12] T Okuda T Yoshida T Hatano K Yazaki and M AshidaldquoEllagitannins of the casuarinaceae stachyuraceae and myr-taceaerdquo Phytochemistry vol 21 no 12 pp 2871ndash2874 1980
[13] F A Yamanaka T A Hatano H A Ito S B Taniguchi E CTakahashi and K C Okamoto ldquoAntibacterial effects of guavatannins and related polyphenols on Vibrio and AeromonasspeciesrdquoNatural Product Communications vol 3 no 5 pp 711ndash720 2008
[14] T Okuda T Hatano and K Yazaki ldquoGuavin B an ellagitanninof novel typerdquo Chemical amp Pharmaceutical Bulletin vol 32 no9 pp 3787ndash3788 1984
[15] F QarsquoDan F Petereit and A Nahrstedt ldquoPolymeric proantho-cyanidins from Psidium guajavardquo Scientia Pharmaceutica vol73 no 3 pp 113ndash125 2005
[16] B-J Park T Matsuta T Kanazawa C-H Park K-J Changand M Onjo ldquoPhenolic compounds from the leaves of Psidiumguajava II quercetin and its glycosidesrdquo Chemistry of NaturalCompounds vol 48 no 3 pp 477ndash479 2012
[17] S Tachakittirungrod F Ikegami and S Okonogi ldquoAntioxidantactive principles isolated from Psidium guajava grown inThailandrdquo Scientia Pharmaceutica vol 75 no 4 pp 179ndash1932007
[18] K-C Chen C-M Chuang L-Y Lin et al ldquoThe polyphenolicsin the aqueous extract of Psidium guajava kinetically reveal aninhibition model on LDL glycationrdquo Pharmaceutical Biologyvol 48 no 1 pp 23ndash31 2010
[19] K Matsuzaki R Ishii K Kobiyama and S Kitanaka ldquoNewbenzophenone and quercetin galloyl glycosides from Psidiumguajava Lrdquo Journal of Natural Medicines vol 64 no 3 pp 252ndash256 2010
[20] W Nantitanon S Yotsawimonwat and S Okonogi ldquoFactorsinfluencing antioxidant activities and total phenolic content ofguava leaf extractrdquo LWTmdashFood Science and Technology vol 43no 7 pp 1095ndash1103 2010
[21] P Zhang R Lin G Yang J Zhang L Zhou and T Liu ldquoSol-ubility of naringenin in ethanol and water mixturesrdquo Journal ofChemical and Engineering Data vol 58 no 9 pp 2402ndash24042013
[22] J Wen B Liu E Yuan Y Ma and Y Zhu ldquoPreparation andphysicochemical properties of the complex of naringenin withhydroxypropyl-120573-cyclodextrinrdquo Molecules vol 15 no 6 pp4401ndash4407 2010
[23] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010
[24] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was funded by the Project cofinanced by FEDER-Andalucıa 2007ndash2013 (Cod 461100) andAndalusian RegionalGovernment Council of Innovation and Science (P11-CTS-7625) The author Elixabet Dıaz-de-Cerio also would liketo thank the CEIBiotic for the ldquoAyudas a la EnsenanzaPracticardquo Grant (CADP2-71) Ana Marıa Gomez-Caravacaand Vito Verardo thank the Spanish Ministry of Economyand Competitiveness (MINECO) for ldquoJuan de la Ciervardquopostdoctoral contract
References
[1] Q-M Liu and J-G Jiang ldquoAntioxidative activities of medicinalplants from TCMrdquo Mini-Reviews in Medicinal Chemistry vol12 no 11 pp 1154ndash1172 2012
[2] S Z Bathaie N Mokarizade and S Shirali ldquoAn overview of themechanisms of plant ingredients in the treatment of diabetesmellitusrdquo Journal of Medicinal Plants vol 11 no 44 pp 1ndash242012
[3] U Etxeberria A L de la Garza J Campin J A Martnez andF I Milagro ldquoAntidiabetic effects of natural plant extracts viainhibition of carbohydrate hydrolysis enzymes with emphasison pancreatic alpha amylaserdquo Expert Opinion on TherapeuticTargets vol 16 no 3 pp 269ndash297 2012
[4] R M P Gutierrez S Mitchell and R V Solis ldquoPsidiumguajava a review of its traditional uses phytochemistry andpharmacologyrdquo Journal of Ethnopharmacology vol 117 no 1 pp1ndash27 2008
[5] J T Cheng and R S Yang ldquoHypoglycemic effect of guava juicein mice and human subjectsrdquo The American Journal of ChineseMedicine vol 11 no 1-4 pp 74ndash76 1983
[6] T Eidenberger M Selg and K Krennhuber ldquoInhibition ofdipeptidyl peptidase activity by flavonol glycosides of guava(Psidium guajava L) a key to the beneficial effects of guava intype II diabetes mellitusrdquo Fitoterapia vol 89 no 1 pp 74ndash792013
[7] F-C Cheng S-C Shen and J S-B Wu ldquoEffect of guava(Psidium guajava L) leaf extract on glucose uptake in rathepatocytesrdquo Journal of Food Science vol 74 no 5 pp H132ndashH138 2009
[8] H-Y Chen and G-C Yen ldquoAntioxidant activity and freeradical-scavenging capacity of extracts from guava (Psidiumguajava L) leavesrdquo Food Chemistry vol 101 no 2 pp 686ndash6942007
[9] O Laporta L Perez-Fons R Mallavia N Caturla and VMicol ldquoIsolation characterization and antioxidant capacityassessment of the bioactive compounds derived from Hypoxisrooperi corm extract (African potato)rdquo Food Chemistry vol 101no 4 pp 1425ndash1437 2007
[10] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996
[11] C H Chang C L Hsieh H E Wang C C Peng C CChyau and R Y Peng ldquoUnique bioactive polyphenolic profile
of guava (Psidium guajava) budding leaf tea is related to plantbiochemistry of budding leaves in early dawnrdquo Journal of theScience of Food andAgriculture vol 93 no 4 pp 944ndash954 2013
[12] T Okuda T Yoshida T Hatano K Yazaki and M AshidaldquoEllagitannins of the casuarinaceae stachyuraceae and myr-taceaerdquo Phytochemistry vol 21 no 12 pp 2871ndash2874 1980
[13] F A Yamanaka T A Hatano H A Ito S B Taniguchi E CTakahashi and K C Okamoto ldquoAntibacterial effects of guavatannins and related polyphenols on Vibrio and AeromonasspeciesrdquoNatural Product Communications vol 3 no 5 pp 711ndash720 2008
[14] T Okuda T Hatano and K Yazaki ldquoGuavin B an ellagitanninof novel typerdquo Chemical amp Pharmaceutical Bulletin vol 32 no9 pp 3787ndash3788 1984
[15] F QarsquoDan F Petereit and A Nahrstedt ldquoPolymeric proantho-cyanidins from Psidium guajavardquo Scientia Pharmaceutica vol73 no 3 pp 113ndash125 2005
[16] B-J Park T Matsuta T Kanazawa C-H Park K-J Changand M Onjo ldquoPhenolic compounds from the leaves of Psidiumguajava II quercetin and its glycosidesrdquo Chemistry of NaturalCompounds vol 48 no 3 pp 477ndash479 2012
[17] S Tachakittirungrod F Ikegami and S Okonogi ldquoAntioxidantactive principles isolated from Psidium guajava grown inThailandrdquo Scientia Pharmaceutica vol 75 no 4 pp 179ndash1932007
[18] K-C Chen C-M Chuang L-Y Lin et al ldquoThe polyphenolicsin the aqueous extract of Psidium guajava kinetically reveal aninhibition model on LDL glycationrdquo Pharmaceutical Biologyvol 48 no 1 pp 23ndash31 2010
[19] K Matsuzaki R Ishii K Kobiyama and S Kitanaka ldquoNewbenzophenone and quercetin galloyl glycosides from Psidiumguajava Lrdquo Journal of Natural Medicines vol 64 no 3 pp 252ndash256 2010
[20] W Nantitanon S Yotsawimonwat and S Okonogi ldquoFactorsinfluencing antioxidant activities and total phenolic content ofguava leaf extractrdquo LWTmdashFood Science and Technology vol 43no 7 pp 1095ndash1103 2010
[21] P Zhang R Lin G Yang J Zhang L Zhou and T Liu ldquoSol-ubility of naringenin in ethanol and water mixturesrdquo Journal ofChemical and Engineering Data vol 58 no 9 pp 2402ndash24042013
[22] J Wen B Liu E Yuan Y Ma and Y Zhu ldquoPreparation andphysicochemical properties of the complex of naringenin withhydroxypropyl-120573-cyclodextrinrdquo Molecules vol 15 no 6 pp4401ndash4407 2010
[23] H Wang Y-J Du and H-C Song ldquo120572-Glucosidase and 120572-amylase inhibitory activities of guava leavesrdquo Food Chemistryvol 123 no 1 pp 6ndash13 2010
[24] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006
![Determination of Polar Compounds in Guava Leaves …digibug.ugr.es/bitstream/10481/35876/1/DiazdeCerio_PolarCompounds.… · Fresh guava leaves were ... [12]. Gallicacid ... The second](https://static.documents.pub/doc/80x56/5b15c2c97f8b9a45448e0285/determination-of-polar-compounds-in-guava-leaves-fresh-guava-leaves-were-.jpg)
