Journal of Functional Foods 22 (2016) 376388

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Determination of guava (Psidium guajava L.) leaf

phenolic compounds using HPLC-DAD-QTOF-MS

Elixabet Daz-de-Cerio a,b, Ana Mara Gmez-Caravaca a,b,

Vito Verardo c,*, Alberto Fernndez-Gutirrez a,b,
Antonio Segura-Carretero a,b
a
Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avd. Fuentenueva s/n, 18071,
Granada, Spain
b
Functional Food Research and Development Center, Health Science Technological Park, Avd. del Conocimiento,
Bioregion building, 18100, Granada, Spain
c
Department of Chemistry and Physics (Analytical Chemistry Area) and Research Centre for Agricultural and
Food Biotechnology (BITAL), Agrifood Campus of International Excellence (ceiA3), University of Almera,
Carretera de Sacramento s/n, E-04120 Almera, Spain

A R T I C L E I N F O A B S T R A C T

Article history: Markets of different countries have proposed guava tea infusions as a drink that can modu-
Received 19 September 2015 late the glycaemic index in blood. This property has been attributed to the phenolic compounds
Received in revised form 20 January contained in guava leaves. However, phenolic profile of guava leaves is still not well-
2016 known. Based on this information, different ethanol/water mixtures were used to extract
Accepted 29 January 2016 the phenolic compounds in guava leaves. Phenolic identification was carried out by HPLC-
Available online 12 February 2016 ESI-QTOF-MS in guava leaves from pomifera and pyrifera varieties; moreover, the antioxidant
activities of the ethanolic extracts were determined by TEAC and FRAP methods. To sum
Keywords: up, seventy-two phenolic compounds were identified. To our knowledge, twelve of them were
Guava (Psidium guajava L.) leaves determined for the first time in guava leaves. The highest amount of phenolic compounds
Flavan-3-ols was found in EtOH/H2O 80:20 (v/v) mixture. Furthermore, pyrifera var. showed higher con-
Flavonols centration of phenolic compounds than pomifera var. (113.34 vs. 86.12 mg/g leaf d.w.) and
Gallic and ellagic derivatives also greater antioxidant capacity.
Phenolic compounds 2016 Elsevier Ltd. All rights reserved.
HPLC-DAD-QTOF-MS

* Corresponding author. Department of Chemistry and Physics (Analytical Chemistry Area) and Research Centre for Agricultural and Food
Biotechnology (BITAL), Agrifood Campus of International Excellence (ceiA3), University of Almera, Carretera de Sacramento s/n, E-04120
Almera, Spain. Tel.: +34 958 637 206; fax: +34 958 637 083.
E-mail address: vito.verardo@unibo.it (V. Verardo)
Chemical compounds: Casuarinin (PubChem CID: 157395); Castalagin (PubChem CID: 168165); Gallocatechin (PubChem CID: 65084); Gallic
acid (PubChem CID: 370); Morin (PubChem CID: 5281670); Quercetin (PubChem CID: 5280343); Hyperin (PubChem CID: 5281643); Reynoutrin
(PubChem CID: 5320863); Avicularin (PubChem CID: 5490064); Quercitrin (PubChem CID: 5280459); Guajaverin (PubChem CID: 5481224);
Myrciaphenone B (PubChem CID: 183139).
http://dx.doi.org/10.1016/j.jff.2016.01.040
1756-4646/ 2016 Elsevier Ltd. All rights reserved.
 Journal of Functional Foods 22 (2016) 376388 377

TPTZ (2,4,6-tripyridyl-S-triazine), Trolox (6-hydroxy-2,5,7,8-

1. Introduction tetramethylchroman-2-carboxylic acid), ABTS [2,2-azinobis (3-
ethylbenzothiazoline-6-sulphonate)], potassium persulphate,
Diabetic complications are now a global health problem without ferric sulphate and the standards namely gallic acid, cat-
effective therapeutic approach. Hyperglycaemia and oxida- echin, ellagic acid, naringenin, quercetin and rutin were all from
tive stress are important components for the development of Sigma-Aldrich (Steinheim, Germany). Ethanol, sodium acetate,
diabetic complications due to an excessive production of free ferric chloride, and hydrochloric acid were obtained from
radicals (Singh, Kaur, Kishore, & Gupta, 2013). It is known that Panreac (Barcelona, Spain).
plants are a rich source of secondary metabolites that have been
implicated in several therapeutic methodologies like flavo- 2.2. Plant material
noids, alkaloids, terpenoids and tannins. Thus, for diabetic
complications, an antioxidant treatment coupled with other Commercial P. guajava L. leaves were used for the optimiza-
approaches could be effective in ameliorating these compli- tion of solvent extraction. Then, P. guajava L. var. pyrifera and
cations (Scott & King, 2004). pomifera harvested in Motril (Spain) (364443N 33114W) were
Psidium guajava L. is a small tree native to Central America collected. They were middle age intense green leaves and they
from Southern Mexico to Northern South America. It is popu- were collected in February 2015. The environmental condi-
larly known as guava and belongs to the myrtle family tions had mean max/min temperature of 23/8 C, precipitation
(Myrtaceae). Today, guava tree has been distributed through of 00.8 mm, and saturated light duration that ranged from 9.45
many countries as a result of its capacity to grow in tropical to 10.40 h per day.
and subtropical conditions (Morton, 1987).
Extracts from the leaves of this plant have traditionally been 2.3. Guava leaves extraction
used in folk medicine around the world. They are mainly known
for their antispasmodic and antimicrobial properties in the The phenolic compounds extraction was performed using an
treatment of diarrhoea and dysentery, although they also exhibit ultrasound bath and a mixture of ethanol:water 80/20 (v/v) as
antioxidant, hepatoprotection, anti-allergy, antimicrobial, extractant solvent. Briefly, 0.5 g of air-dried and crushed guava
antigenotoxic, antiplasmodial, cytotoxic, antispasmodic, leaves were extracted with 15 mL of solvent (3) using a soni-
cardioactive, anticough, anti-inflammatory and antinociceptive cator Branson B3510 for 10 min at room temperature. Then,
properties. Besides, they have extensively been used as a samples were centrifuged for 15 min at 6000 rpm using a cen-
hypoglycaemic agent for diabetes, due to their high concen- trifuge to remove solids. The supernatants were pooled,
tration of total phenolic compounds (Gutirrez, Mitchell, & Solis, evaporated and dissolved in 2 mL of methanol/water 1/1 (v/v).
2008). This solution was filtered through a 0.20-m RC syringe filter
Several authors (Haida, Baron, Haida, de Faci, & Haas, 2011; and kept at 20 C in amber vials until analysis to avoid deg-
Wang, Jiao, Liu, & Hong, 2007) reported that the leaves of white radation. The analysis were run in triplicate (n = 3) and results
(P. guajava L. var. pyrifera) and red guava (P. guajava L. var. pomifera) expressed as mg of phenolic content/g leaf dry weight (d.w.).
presented higher amounts of phenolic compounds with an-
tioxidant activity compared with other vegetable species. 2.4. Preparation of standards
In the last years, traditional and advanced techniques have
been applied to extract phenolic compounds from natural Phenolic standards of interest such as gallic acid, catechin,
product matrices (Stalikas, 2007). Nantitanon, Yotsawimonwat, ellagic acid, naringenin, and rutin were used for quantifica-
and Okonogi (2010) found that ultrasonication was the best tion of phenolic compounds in guava leaf extracts. The standard
method to extract phenolic compounds from guava leaves, fol- stock solutions were prepared at 250 mg/L in methanol, except
lowed by Soxhlet extraction and maceration. The same authors for ellagic acid, which was solved in water. Then, each solu-
affirmed that ultrasound assisted extraction is also simpler, tion was diluted from 50 mg/L to 0.01 mg/L.
faster and cheaper than conventional extraction methods. Thus,
in this study, pure ethanol and different ethanol:water mix- 2.5. HPLC-DAD-ESI-QTOF-MS analysis
tures were used to extract commercial guava leaves. The extracts
were analysed and evaluated by HPLC-DAD-ESI-QTOF. More- Chromatographic analyses were performed using an HPLC
over, the phenolic content and the antioxidant capacity of Agilent 1260 series (Agilent Technologies, Santa Clara, CA, USA)
P. guajava L. var. pyrifera leaf extracts were compared with those equipped with a binary pump, an online degasser, an
of P. guajava L. var. pomifera. autosampler and a thermostatically controlled column com-
partment, and a UVVis Diode Array Detector (DAD). The column
was maintained at 25 C. Phenolic compounds from P. guajava
2. Experimental L. leaves were separated at room temperature using a method
previously reported by Lpez-Cobo, Gmez-Caravaca,
2.1. Chemicals varc-Gajic, Segura-Carretero, and Fernndez-Gutirrez (2015)
with slight modifications. Briefly, a Poroshell 120 EC-C18
Double-deionized water with conductivity lower than 18.2 M (4.6 mm 100 mm, particle size 2.7 m) (Agilent Technolo-
was obtained with a Milli-Q system (Millipore, Bedford, MA, gies) was used to separate the compounds. The gradient elution
USA). Methanol LCMS optima grade and acetonitrile were was carried out using water containing 1% acetic acid as solvent
obtained from Fisher Scientific (Leicestershire, UK). Acetic acid, system A and acetonitrile as solvent system B, and applied as
378 Journal of Functional Foods 22 (2016) 376388

follows: 0 min, 0.8% B; 2.5 min, 0.8% B; 5.5 min, 6.8% B; 11 min, Gmez-Caravaca, Fernndez-Gutirrez, and Segura-Carretero
14.4% B; 17 min, 24% B; 22 min, 40% B; 26 min, 100% B, 30 min, (2015) by HPLC-ESI-Q-MS. These compounds were also veri-
100% B; 32 min, 0.8% B; 34 min, 0.8% B. The sample volume in- fied by HPLC-ESI-QTOF-MS as is shown in Table 1.
jected was 5 L and the flow rate used was 0.8 mL min1. However, HPLC-ESI-QTOF-MS platform allowed the identi-
MS analysis was carried out using a 6540 Agilent Ultra- fication of several more phenolic compounds in the same
High-Definition Accurate-Mass Q-TOF-MS coupled to the HPLC, analysis. Six of the identified compounds were noticed by
equipped with an Agilent Dual Jet Stream electrospray ion- several authors in guava leaves before. Compounds number 9
ization (Dual AJS ESI) interface in negative ionization mode at and 40, with molecular ion at m/z 933.0649 and two frag-
the following conditions: drying gas flow (N2), 12.0 L/min; nebu- ments at m/z 466.0299 and 300.9968, corresponding to the
lizer pressure, 50 psi; gas drying temperature, 370 C; capillary [M-2H]2 and to a loss of ellagic acid, respectively, were iden-
voltage, 3500 V; fragmentor voltage, 3500 V; and scan range, m/z tified as vescalagin/castalagin isomers. Compound 33, also with
501500. Automatic MS/MS experiments were carried out using molecular ion at m/z 933.0649 and based on the fragment at
the followings collision energy values: m/z 100, 30 eV; m/z 500, m/z 457.0781, due to [M-2H-H 2 O] 2 , was also described as
35 eV; m/z 1000, 40 eV; and m/z 1500, 45 eV. vescalagin. These compounds had previously been described
Integration and data elaboration were performed using in fresh guava leaves using HPLC-DAD and 1 H NMR by
MassHunter Workstation software (Agilent Technologies, Santa Yamanaka et al. (2008) and via HPLC-MicroTOF-Q by Moilanen,
Clara, CA, USA). Sinkkonen, and Salminen (2013).
Two compounds (21 and 34) were identified as
2.6. Antioxidant assays stenophyllanin A isomers at m/z 1207.1472. MS/MS spectra
yielded ions at m/z 917.0763 (MH-289, loss of catechin), 603.0735
Two different antioxidant assays were carried out to evaluate ([M-2H]2). These compounds have previously been reported in
the antioxidant capacity of guava leaf extracts. The reducing guava leaves by Yamanaka et al. (2008).
power (FRAP) was evaluated according to the method vali- Guavin A (compound 26) that reported a molecular ion at
dated by Benzie and Strain (1996) using a Fe (II) solution as a m/z 1223.1423 was identified according to Okuda (1987) by NMR.
standard. As far as we are concerned, twelve compounds were iden-
The TEAC assay, was performed by using a method previ- tified in P. guajava L. leaves for the first time using this
ously described by Laporta, Perezfons, Mallavia, Caturla, and methodology. Compound 11, with a molecular ion at m/z
Micol (2007), using the radical cation of 2,2-azinobis-(3- 285.0624, and fragments at m/z 153.0193, due to the loss of
ethylbenzothiazoline-6-sulphonate) (ABTS) and Trolox as hydroxybenzoic acid unit, and m/z 109.0279, because of the loss
standard. of a CO2 group from the carboxylic acid moiety, was assigned
as uralenneoside according to the fragmentation pattern re-
ported by Yu, Chen, and Liang (2000).
2.7. Statistical analysis
Two isomers were detected at m/z 745.1420 (compounds 16
and 28). According to the MS/MS fragmentation pattern pro-
The results reported in this study are the averages of three rep-
posed by Jaiswal, Jayasinghe, and Kuhnert (2012), the isomers
etitions (n = 3). Fishers least significant difference (LSD) test
were identified as galloyl(epi)catechin-(epi)gallocatechin.
and Pearsons linear correlations, both at p < 0.05, were evalu-
Isomers 19 and 20 (m/z 1067.1220) provided four frag-
ated using Statistica 6.0 (2001, StatSoft, Tulsa, OK).
ments at m/z 533.0585 ([M-2H] 2 ), 377.0313, 301.0330, and
249.0377, which matched with the MS/MS fragments identi-
fied by Regueiro et al. (2014) for pterocarinin A. This compound
3. Results and discussion has been identified in Myrtaceae family by several authors
(Nonaka, Ishimaru, Azuma, Ishimatsu, & Nishioka, 1989; Tanaka,
3.1. Identification of phenolic compounds Orii, Nonaka, & Nishioka, 1993); however, it has not been de-
scribed in guava leaves previously.
As first step, to identify the phenolic compounds, a conven- Procyanidin trimer isomers (22 and 25) with [M-H] at m/z
tional extraction described by Seo and coworkers (2014) using 865.1998 provided a fragmentation pattern in accordance with
an ethanol:water mixture (70:30, v/v) was carried out in com- Regueiro et al. (2014). Procyanidin tetramer (24) and procyanidin
mercial guava leaves. After that, the extracts were analysed by pentamer (29) showed significant [M2H]2 signals at m/z
HPLC-DAD-QTOF-MS and seventy two phenolic compounds 576.1291 and 720.1604, respectively (Regueiro et al., 2014).
could be identified. An overview of all the compounds tenta- Two isomers (30 and 35) were identified as galloyl-
tively identified in the extract is given in Table 1. Peak (epi)catechin trimer isomers based on their molecular formula,
identification was performed on the basis of their retention on their molecular ion at m/z 1017.2097, and on their [M2H]2
times, their UVVis and mass spectra together with the infor- fragment at m/z 508.1040 (De Freitas, Glories, Bourgeois, & Vitry,
mation previously reported in the literature. Table 1 summarizes 1998).
the information about identification: retention times, experi- Compound 37 provided a molecular ion at m/z 1225.1587,
mental and calculated m/z, maximum absorbance, fragments, an [M2H]2 at m/z 612.0779 and a molecular formula (C56H42O32).
molecular formula, score and error (ppm). Forty eight of Thus, it was identified as stachyuranin A.
these compounds (numbers 18; 10;1215; 17; 18; 23; 27; Three isomers (compounds 38, 43 and 58), at m/z 729.1476,
31; 32; 36; 39; 41; 42; 4448; 5057; 5963; 6568; and 7072) corresponded to procyanidin gallate isomers. The isomers pro-
had previously been identified by Daz-de-Cerio, Verardo, duced fragments at m/z 577.1356 due to the loss of a galloyl
 Journal of Functional Foods 22 (2016) 376388 379

Table 1 Identification of phenolic compounds in Psidium guajava L. leaves by HPLC-DAD-ESI-QTOF-MS.

No. Compound tr (min) m/z exp m/z Molecular (nm) Fragments Score Error
calculated Formula (ppm)
1 HHDP glucose Isomer 1 1.861 481.0640 481.3406 C20H18O14 290 421.0406, 300.9991, 275.0202 96.51 2.55
2 HHDP glucose Isomer 2 2.061 481.0638 481.3406 C20H18O14 290 421.0406, 300.9991, 275.0202 99.09 0.19
3 HHDP glucose Isomer 3 2.393 481.0639 481.3406 C20H18O14 290 421.0406, 300.9991, 275.0202 97.21 2.24
4 Prodelphinidin B2 Isomer 3.506 609.1276 609.5111 C30H26O14 272, 225 441.0838, 423.0701, 97.84 1.7
305.0687, 125.0226
5 Gallic acid 3.749 169.0142 169.1116 C7H6O5 280, 360 125.0243 99.27 0.37
6 Pedunculagin/Casuariin 6.733 783.0699 783.5332 C34H24O22 253 481.0606, 391.0307, 98.57 1.29
Isomer 300.9999, 275.0191
7 Prodelphinidin Dimer 6.955 593.1311 593.5117 C30H26O13 280, 340 425.0875, 289.0715, 96.51 2.35
Isomer
8 Gallocatechin 7.497 305.0698 305.2595 C15H14O7 270 125.0241, 179.0347, 95.55 3.32
219.0661, 261.0774
9 Vescalagin/Castalagin 7.52 933.0649 933.6216 C41H26O26 260, 280 466.0299, 300.9968 99.19 0.79
Isomer
10 Prodelphinidin Dimer 7.863 593.1316 593.5117 C30H26O13 280, 340 305.0667, 423.0719, 441.0841 96.51 2.35
Isomer
11 Uralenneoside 9.203 285.0624 285.2268 C12H14O8 270 153.0193, 109.0279 97.8 2.69
12 Geraniin Isomer 9.214 951.0749 951.6369 C41H28O27 270 907.0825, 783.0785, 99.56 0.2
481.0606, 300.9999
13 Pedunculagin/Casuariin 9.241 783.0699 783.5332 C34H24O22 253 481.0606, 391.0307, 98.39 1.36
Isomer 300.9999, 275.0191
14 Geraniin Isomer 9.391 951.0752 951.6369 C41H28O27 270 907.0825, 783.0785, 99.56 0.2
481.0606, 300.9999
15 Procyanidin B Isomer 9.784 577.1367 577.5123 C30H26O12 278 425.0881, 407.0777, 95.68 2.55
289.0718, 125.0243
16 Galloyl(epi)catechin- 10.051 745.142 745.6160 C37H30O17 280, 340 593.1302, 575.1214, 96.9 0.62
(epi)gallocatechin 423.0694, 305.0688
17 Procyanidin B Isomer 10.066 577.1367 577.5123 C30H26O13 278 425.0881, 407.0777, 99.41 0.61
289.0718, 125.0243
18 Tellimagrandin I Isomer 10.432 785.0851 785.5491 C34H26O22 279, 340 615.0674, 392.0396, 99.13 0.96
300.9985, 169.0144
19 Pterocarinin A 10.703 1067.122 1067.7521 C46H36O30 280 533.0585, 377.0313, 99.82 0.11
301.0330, 249.0377
20 Pterocarinin A Isomer 10.913 1067.122 1067.7521 C46H36O30 280 533.0585, 377.0313, 98.39 1.26
301.0330, 249.0377
21 Stenophyllanin A 10.941 1207.1495 1207.8903 C56H40O31 278 917.0763, 603.0735 98.64 1.08
22 Procyanidin trimer Isomer 10.958 865.1998 865.7645 C45H38O18 278 739.1593, 577.1337, 97.53 1.59
449.0888, 289.0745
23 Catechin 10.974 289.0727 289.2601 C15H14O6 281 245.0821, 203.0718, 96.76 3.18
179.0349, 125.0242
24 Procyanidin tetramer 11.041 1153.2612 1155.0246 C60H50O24 280 576.1291 99.6 0.5
25 Procyanidin trimer Isomer 11.129 865.1998 865.7645 C45H38O18 278 739.1593, 577.1337, 97.53 1.59
449.0888, 289.0745
26 Guavin A 11.201 1223.1423 1223.8897 C56H40O32 277 611.0724 99.05 0.85
27 Casuarinin/Casuarictin 11.578 935.081 935.6375 C41H28O26 275 783.0637, 633.0735, 97.67 1.43
Isomer 300.9979, 275.0189
28 Galloyl(epi)catechin- 11.773 745.142 745.6160 C37H30O17 280, 340 593.1302, 575.1214, 96.9 0.62
(epi)gallocatechin 423.0694, 305.0688
29 Procyanidin pentamer 11.845 1441.3234 1442.2688 C75H62O30 280 720.1604 95.66 1.97
30 Galloyl-(epi)catechin trimer 11.855 1017.2097 1017.8687 C52H42O22 280 508.104 99.72 0.01
Isomer
31 Gallocatechin 12.087 305.0702 305.2595 C15H14O7 270 125.0241, 179.0347, 95.55 3.32
219.0661, 261.0774
32 Tellimagrandin I Isomer 12.198 785.0855 785.5491 C34H26O22 277, 338 615.0674, 392.0396, 98.44 1.38
300.9985, 169.0144
33 Vescalagin 12.425 933.0649 933.6216 C41H26O26 260, 280 466.0295, 457.0781, 300.9968 96.33 0.8
34 Stenophyllanin A Isomer 12.635 1207.1472 1207.8903 C56H40O31 280 917.0763, 603.0735 98.37 0.89
35 Galloyl-(epi)catechin trimer 12.985 1017.2097 1017.8687 C52H42O22 280 508.104 98.17 1.35
Isomer
36 Myricetin hexoside Isomer 13.067 479.0836 479.3678 C21H20O13 261, 358 317.0294, 316.0226, 271.0255 98.36 0.92
37 Stachyuranin A 13.073 1225.1587 1225.9055 C56H42O32 276 612.0779 95.54 1.35
38 Procyanidin gallate Isomer 13.25 729.1476 729.6166 C37H30O16 280 577.1356, 559.1226, 96.89 1.91
425.0874, 407.0798, 298.0716
(continued on next page)
380 Journal of Functional Foods 22 (2016) 376388

Table 1(continued)
No. Compound tr (min) m/z exp m/z Molecular (nm) Fragments Score Error
calculated Formula (ppm)
39 Myricetin hexoside Isomer 13.46 479.0835 479.3678 C21H20O13 261, 358 317.0294, 316.0226, 271.0255 97.89 0.08
40 Vescalagin/castalagin 13.538 933.0645 933.6216 C41H26O26 260 466.0299, 300.9968 88.32 1.57
Isomer
41 Myricetin-arabinoside/ 13.759 449.0728 449.3418 C20H18O12 264, 356 317.0291, 316.0241, 271.0249 98.39 1.65
xylopyranoside Isomer
42 Myricetin-arabinoside/ 13.992 449.0726 449.3418 C20H18O12 264, 357 317.0291, 316.0241, 271.0249 98.02 1.65
xylopyranoside Isomer
43 Procyanidin gallate Isomer 14.263 729.6356 577.5123 C30H26O12 280 577.1356, 559.1226, 98.17 1.73
425.0874, 407.0798, 298.0716
44 Myricetin-arabinoside/ 14.761 449.0726 449.3418 C20H18O12 264, 356 317.0291, 316.0241, 271.0249 98.66 1.65
xylopyranoside Isomer
45 Myricetin hexoside Isomer 14.806 479.0839 479.3678 C21H20O13 261, 358 317.0294, 316.0226, 271.0255 97.08 1.92
46 Myricetin hexoside Isomer 14.999 479.0841 479.3678 C21H20O13 264, 356 317.0288, 316.0241, 271.0253 97.08 1.92
47 Myricetin-arabinoside/ 15.376 449.0743 449.3418 C20H18O12 264, 356 317.0291, 316.0241, 271.0249 98.39 1.65
xylopyranoside Isomer
48 Quercetin-galloylhexoside 15.42 615.1008 615.4726 C28H24O16 268, 350 463.0886, 300.0283 99.16 0.98
Isomer
49 Ellagic acid deoxyhexoside 15.653 447.0578 447.3259 C20H16O12 265, 350 300.9974, 91.25 3.19
50 Quercetin-galloylhexoside 15.83 615.0999 615.4726 C28H24O16 280, 345 463.0886, 300.0283 99.16 0.98
Isomer
51 Myricetin-arabinoside/ 15.968 449.0736 449.3418 C20H18O12 256, 356 317.0291, 316.0241, 271.0249 98.39 1.65
xylopyranoside Isomer
52 Morin 16.024 301.0362 301.2278 C15H10O7 257, 374 178.9978, 151.0032 97.46 2.5
53 Myricetin-arabinoside/ 16.24 449.0735 449.3418 C20H18O12 257, 356 317.0291, 316.0241, 271.0249 98.39 1.65
xylopyranoside Isomer
54 Ellagic acid 16.262 300.9996 301.1847 C14H6O8 254, 360 283.9921, 257.0088, 98.88 1.71
229.0169, 185.0233
55 Hyperin 16.522 463.0895 463.3684 C21H20O12 259, 355 301.0350, 300.0279, 96.41 2.65
178.9980, 151.0032
56 Quercetin glucuronide 16.616 477.0659 477.3519 C21H18O13 265, 355 301.0359, 151.0026 98.1 1.83
57 Isoquercitrin 16.76 463.0893 463.3684 C21H20O12 258, 355 301.0353, 300.0281, 97.04 2.33
178.9983, 151.0090
58 Procyanidin gallate Isomer 16.777 729.1476 729.6166 C37H30O16 280 577.1356, 559.1226, 96.89 1.91
425.0874, 407.0798, 298.0716
59 Reynoutrin 17.303 433.0792 433.3424 C20H18O11 258, 356 301.0356 95.94 2.9
60 Guajaverin 17.607 433.0795 433.3424 C20H18O11 257, 356 301.0352 97.99 1.91
61 Guavinoside A 17.801 543.1159 544.4610 C26H24O13 218, 288 313.0568, 229.0503, 169.0148 98.1 1.77
62 Avicularin 18.000 433.0803 433.3424 C20H18O11 257, 355 301.0359 96.7 2.2
63 Quercitrin 18.244 447.0947 447.3690 C21H20O11 264, 353 301.0348, 271.0247, 95.23 3.02
178.9988, 151.0028
64 Myrciaphenone B 18.991 481.0999 481.3836 C21H22O13 280, 340 313.0570, 169.0141 97.2 2.23
65 Guavinoside C 19.556 585.0898 585.4466 C27H22O15 265, 355 433.0757, 301.0351, 97.19 1.92
283.0449, 169.0142
66 Guavinoside B 20.658 571.1470 571.5062 C28H28O13 218, 283 313.057, 257.0829, 169.0142 97.26 2.05
67 Guavinoside A Isomer 20.702 543.1159 543.4530 C26H24O13 218, 288 313.0568, 229.0503, 169.0148 98.1 1.77
68 Guavinoside B Isomer 21.549 571.1470 571.5062 C28H28O13 218, 283 313.057, 257.0829, 169.0142 97.26 2.05
69 2,6-dihydroxy-3-methyl-4- 21.881 557.1318 557.4796 C27H26O13 280 313.0575, 243.0670, 169.0146 96.93 2.12
O-(6-O-galloyl--D-
glucopyranosyl)-
benzophenone
70 Guavin B 22.103 693.1110 693.5414 C33H26O17 283 391.1468 97.82 1.67
71 Quercetin 22.147 301.0358 301.2278 C15H10O7 257, 374 178.9985, 151.0036 98.9 1.34
72 Naringenin 26.726 271.0622 271.2448 C15H12O5 280 118.6395, 150.5022 96.09 3.67

residue, at m/z 559.1226 by the loss of a gallic acid and at Compound 64 corresponding to [M-H] signal at m/z 481.0999
425.0874 and 407.0798 from a retro-DielsAlder fragmenta- was detected. Based on its molecular formula and its frag-
tion (Jaiswal et al., 2012). Moreover, they provided a fragment ments at m/z 313.0570, due to a loss of a dihydroxybenzoic acid
at m/z 289.0716 corresponded to an (epi)-catechin unit. unit, and 169.0141, due to a gallic acid unit, was assigned as
Compound 49 was identified as ellagic acid deoxyhexoside. Myrciaphenone B. This compound was previously reported in
It showed [M-H] ion at m/z 447.0578 and yielded a fragment Myrcia multiflora (Myrtaceae) by Yoshikawa and coworkers (1998).
at m/z 300.9974 from an ellagic acid unit. It was described in Compound 69 was identified as 2,6-dihydroxy-3-methyl-4-
Psidium fruits by several authors (Gordon, Jungfer, da Silva, O-(6-O-galloyl--D-glucopyranosyl)-benzophenone according
Maia, & Marx, 2011; Ribeiro et al., 2014). to its molecular ion at m/z 557.1318, and to its MS/MS spectrum,
 Journal of Functional Foods 22 (2016) 376388 381

Table 2 Analytical parameters of the method.

Standard Equation R2 Linear range (mg/L) LOD (mg/L) LOQ (mg/L)
Gallic acid y = 2.20E5X + 3.85E5 0.9855 LOQ-50 0.062 0.21
Catechin y = 2.82E5X + 4.71E5 0.9986 LOQ-50 0.049 0.16
Rutin y = 4.24E5X 5.33E5 0.9996 LOQ-50 0.032 0.11
Ellagic acid y = 4.30E5X 6.95E5 0.9975 LOQ-50 0.032 0.11
Naringenin y = 6.09E5X + 2.46E6 0.9664 LOQ-50 0.023 0.08

which showed fragments at m/z 313.0575 (loss of C14H12O4), no standard available, quantification was done using com-
243.0670 (loss of C13H15O10), and 169.0146 (loss of C7H5O5). It was pounds with similar structure.
described in P. guajava L. fruit (Shu, Chou, & Wang, 2010).
3.3. Effect of solvent mixture on phenolic extraction
3.2. Method parameters
Extraction is the most important step to obtain extracts en-
Table 2 summarizes the parameters of the method for each riched in phenolic compounds. Ethanol and water are the most
standard: calibration curve (y = a + bx), R2, linear range and limits widely applied extraction solvents in food systems because of
of detection (LOD) and quantification (LOQ). Standard calibra- the hygiene, low cost and abundance in addition to being com-
tion curves were done in the range of concentration from the patible with health (Moure et al., 2001). In this study, pure
quantification limit (LOQ) to 50 mg/L, and five calibration points ethanol and different hydroethanolic mixtures, such as
for each standard were run in triplicate (n = 3). The values of ethanol:water in the ratios of 90:10, 80:20, 70:30, 60:40, and 50:50
the regression coefficients varied from 0.9964 to 0.9996. The (v/v), were evaluated with the same extraction procedure de-
limits of detection (LOD) and quantification (LOQ) were cal- scribed in the Guava leaves extraction section. Total phenolic
culated as the concentration corresponding to 3 and 10 times, compounds quantified by HPLC-MS using the different mix-
respectively, the standard deviation of the background noise. tures are shown in Fig. 1. The phenolic content of the pure
The LOD values ranged from 0.023 mg/L for naringenin to ethanol extract (201 5 mg/g leaf d.w.) was lower than the
0.062 mg/L for gallic acid. LOQs ranged from 0.08 mg/L for content of the extracts obtained with the different mixtures,
naringenin to 0.21 mg/L for gallic acid. due to a less solubility of polar compounds in pure organic sol-
Quantification of phenolic compounds was done with the vents. The best recoveries of phenolic compounds, without
calibration curves shown in Table 2. For the compounds with significant differences (P < 0.05), were provided by EtOH/H2O

350

300

250
TPC (mg/g leaf d.w.)

200

150

100

50

0
100% EtOH 90% EtOH 80% EtOH 70% EtOH 60% EtOH 50% EtOH

Fig. 1 Total phenolic content (mean SD, n = 3) by HPLC-DAD-QTOF-MS (mg/g leaf d.w.) present in each extract.
382 Journal of Functional Foods 22 (2016) 376388

80:20 v/v (304 7 mg/g leaf d.w.), 70:30 v/v (301 1 mg/g leaf flavonols > flavan-3-ols > gallic and ellagic derivatives > flava-
d.w.) and 50:50 v/v (298 4 mg/g leaf d.w.) mixtures. EtOH/ nones. The same order was noticed by Chang et al. (2013) for
H 2 O 60:40 (v/v) (284 9 mg/g leaf d.w.) and 90:10 (v/v) a methanolic extract of guava budding leaves, because these
(255 5 mg/g leaf d.w.) mixtures provided significant lower con- compounds emerge at the early stage of budding. Seo et al.
tents of phenolic compounds than the other mixtures (P < 0.05). (2014) also noticed that the content of flavonoids was higher
As can be seen, water content enhances the extraction due to in 70% hydroethanolic extract than in 50 and 90%.
a better solubility of the target compounds in the solvent This trend could also be applied to the amount of each
systems (Seo et al., 2014). TPC value increased from 100 to 80% family of phenolic compounds that increased when the content
ethanol, where the highest content was reached, a fact also of water increased between 0 and 20%, due to a higher solu-
observed for palm kernel cake (Kua et al., 2015). Then, the ex- bility of the compounds in hydroalcoholic mixtures rather than
traction of phenolic compounds remained nearly constant. Zhu, in pure ethanol, and remained with slight variations when the
Lian, Guo, Peng, and Zhou (2011) compared the total pheno- presence of water is higher. These results are in concordance
lic content, using FolinCiocalteu assay, of 30, 50, 70 and 100% with Fatiha et al. (2012) who found that the highest recovery
ethanol, and aqueous extracts of defatted wheat germ, and of phenolic compounds can be achieved at 75% ethanol com-
found that the 70% and the 50% ethanol extract exhibited rela- pared with pure ethanol or with an aqueous mixture at 50%
tively higher antioxidant capacity than the others. In contrast ethanol. This is because both the polar and less polar com-
to these results, Taha, Mohamed, Mohamed, Mohamed, and pounds are co-extracted together (Wu, Hsieh, Wang, & Chen,
Kamil (2011) examined different ethanol concentrations (80, 2009).
70, 60, and 50%) and their results indicated that 60% was the Moreover, flavonols, the main class of phenolic com-
most effective solvent to extract total phenolic compounds from pounds, varied between 53.5 and 76% of total polar compounds
sunflower meal. In general, ethanolwater mixtures, particu- in each extraction mixture. The second class of polar com-
larly from 40 to 80% ethanol, had greater efficiency in the pounds was represented by flavan-3-ols, which correspond to
extraction of phenolic compounds compared to water or pure 2345% of total polar compounds. Gallic and ellagic acid de-
ethanol or methanol (Fatiha et al., 2012). rivatives ranged from 17 to 35% of the total concentration of
Fig. 2 indicates the variability of the different families of phe- polar compounds, and flavanones accounted for a percent-
nolic compounds quantified with all solvent systems tested. age lower than 1%.
These results showed that the concentration of the families Among the better extraction mixtures, EtOH/H2O (80:20, v/v)
of phenolic compounds decreased in the following order: was chosen as the best solvent mixture to extract the target

180

gallic and ellagic derivatives flavonols flavanones flavan-3-ols

160

140

120
Concentration (mg/g leaf d.w.)

100

80

60

40

20

0
EtOH 90% EtOH 80% EtOH 70% EtOH 60% EtOH 50% EtOH

Fig. 2 Variability of the different families of phenolic compounds (mg/g leaf d.w.) quantified (mean SD, n = 3) in each
solvent system.
 Journal of Functional Foods 22 (2016) 376388 383

compounds. Despite the fact that there were no significant dif- Higher amounts of gallic acid (0.06 and 0.22 mg/g d.w.),
ferences in the extraction of total phenolic compounds among gallocatechin (5 and 5.6 mg/g d.w.) and catechin (7.2 and
80:20 (v/v), 70:30 (v/v) and 50:50 (v/v), EtOH/H2O (80:20, v/v) ex- 7.1 mg/g d.w.) were noticed compared to the data obtained by
traction mixture showed better recoveries for flavonols and Jang et al. (2014), who reported 0.09, 2.88 and 0.72 mg/g d.w.,
flavan-3-ols. respectively. This discordance could be due to the different s
Several works demonstrate beneficial effects of com- of samples and/or the method of extraction.
pounds that are also present in guava leaves in the control of Haida et al. (2011) also compared the phenolic content of
diabetes mellitus. Flavonol-glycosides, such as quercetin- P. guajava L. var. pomifera and pyrifera leaves. Contrary to the
glycosides, exert a dose-dependent inhibition of dipeptidyl present results, where concentrations obtained for pomifera and
peptidase in vitro (Eidenberger, Selg, & Krennhuber, 2013). Cat- pyrifera were 86.1 and 113.3 mg/g d.w., respectively, they noticed
echins protect type 2 diabetic erythrocytes from t-BHP- that total phenolic content of P. guajava L. var. pomifera was
induced oxidative stress, providing some protection against the higher than TPC of pyrifera variety, between 161175 and 159
development of long-term diabetic complications (Rizvi, Zaid, 164 mg/g extract, respectively; however, they analysed total
Anis, & Mishra, 2005). Myrciaphenone B exhibited inhibitory phenolic content by FolinCiocalteu assay.
activity on aldose reductase and -glucosidase (Yoshikawa et al., Chen and Yen (2007) also studied leaves from different guava
1998). Geraniin also possesses in vitro hypoglycaemic activity cultivars (cv. Hong Ba, Shi Ji Ba, Shui Jing Ba and Tu Ba) and no
and has the ability to prevent the formation of advanced concordances were noticed with the data shown in this work.
glycation end-products (AGE) at more significant levels than This could be justified by the different determination methods
the positive controls acarbose, quercetin and green tea used to quantify the phenolic compounds; in fact, they analysed
(Palanisamy, Ling, Manaharan, & Appleton, 2011). Wu et al. different varieties of guava leaves and fruit by FolinCiocalteu
(2009) compared the potential of guava leaves, with Polyphe- assay and HPLC-UV. Because of that, not all the phenolic com-
nol 60 and with an antiglycation agent, as inhibitor of glycation pounds could be identified. As a matter of fact, they identified
of proteins. They found that guava extract presented higher only gallic and ferulic acid because there were no other avail-
activity than the supplements due to a greater concentration able commercial standards. As reported in the literature (Table 5),
of phenolic compounds such as quercetin, catechin and gallic the concentration of gallic acid in guava leaves varied from 0.8
acid, and suggested their daily consumption for prevention of to 1.6 mg/g leaf and total phenolic contents ranged between
diabetes complication. Moreover, in Japan, Guava Leaf Tea con- 267 and 313 mg/g leaf, expressed as (+)-catechin equivalents
taining the aqueous leaf extract from guava has been approved and from 414 to 483 mg/g leaf expressed as gallic acid equiva-
as one of the Foods for Specified Health Uses and is now com- lents. In contrast, in the present work, the concentration of gallic
mercially available (Deguchi & Miyazaki, 2010). acid recovered was lower than those shown in Table 5.
The differences noticed among the different works in the lit-
erature could be explained by the fact that flavonoids vary
3.4. Comparison between P. guajava L. leaves phenolic substantially among genotypes, seasons, age and level of damaged
content and antioxidant capacity leaves, and location (Salazar et al., 2006; Vargas-Alvarez,
Soto-Hernndez, Gonzlez-Hernndez, Engleman, &
The phenolic content (Table 3) and antioxidant capacity (Table 4) Martnez-Garza, 2006). Moreover, the guava tree (P. guajava L.)
of two guava leaf varieties were determined; moreover, the shows different phenological stages throughout its vegetative
guava leaf phenolic content was compared with guava fruit phe- period in response to environmental conditions (Eidenberger et al.,
nolic content (Table 5). 2013). In addition, the FolinCiocalteu method represents a none
Guava leaves var. pyrifera and pomifera were extracted with selective spectrophotometric method for the estimation of total
a hydroethanolic mixture (80:20, v/v), and then analysed by phenolic compounds, because other substances (such as sugars
HPLC-DAD-QTOF-MS. The comparison of the concentration of and proteins) can react with FolinCiocalteu reagent and can
the different phenolic families present in both leaves is shown overestimate the phenolic content.
in Fig. 3 and for each compound in Table 3. The quantifica- Briefly, comparing the results obtained in this work for guava
tion results reported that significant differences (P < 0.05) for leaves with data reported by several authors for guava fruits
each family were found when both varieties were compared, (Luximon-Ramma, Bahorun, & Crozier, 2003; Thaipong,
being higher in pyrifera than in pomifera variety. The compari- Boonprakob, Crosby, Cisneros-Zevallos, & Hawkins Byrne, 2006;
son in terms of single phenolic compounds and also total Thuaytong & Anprung, 2011), we confirm using the present data
phenolic content showed that pyrifera variety concentrations that guava leaves have higher content of phenolic com-
were higher than pomifera variety concentrations (Table 4). Total pounds than guava fruits.
phenolic content in the two varieties ranged between 86.1 and Moreover, Chen and Yen (2007) found that all the leaves had
113.3 mg/g leaf d.w. for P. guajava L. var. pomifera and pyrifera, higher amounts of phenolic compounds than the fruit. Values
respectively. These data are in the same order of magnitude exhibited for the dried fruit were 69.9 mg/g d.w., expressed as
as those obtained in a previous work (Daz-de-Cerio et al., 2015). (+)-catechin equivalents, and 115 mg/g d.w., expressed as gallic
For both varieties, the most abundant phenolic family was acid equivalents.
flavonols that represented 49.5% for pomifera var. and 55.3% for The same phenolic classes detected in guava leaves were
pyrifera, followed by flavan-3-ols, which exhibited a percent- also detected in guava fruits and guava juice by-products and
age of 27.3 and 23.8%, respectively. The sum of gallic and ellagic they demonstrate anti-inflammatory and antiproliferative ac-
acid derivates supposed 19.6 and 22.4%, and flavanones 0.8 and tivity, and reduced hepatic steatosis (Amaya-Cruz et al., 2015;
1.3% for pomifera and pyrifera varieties, respectively. Chen & Yen, 2007; Li et al., 2013). In fact, Li et al. (2013) only
384 Journal of Functional Foods 22 (2016) 376388

Table 3 Quantification (mg/g leaf d.w.) by HPLC of the phenolic compounds identified in guava leaves var. pomifera and
pyrifera. Means in the same line with different superscript letters are significantly different (P < 0.05).
Compound var. pomifera var. pyrifera
a 0.771 0.027b
HHDP glucose Isomer 1 1.270 0.011
HHDP glucose Isomer 2 1.422 0.035a 0.941 0.014b
HHDP glucose Isomer 3 1.204 0.014a 0.726 0.053b
Prodelphinidin B2 Isomer 0.347 0.002b 0.417 0.005a
Gallic acid 0.223 0.003a 0.060 0.008b
Pedunculagin/Casuariin Isomer 0.999 0.003a 0.807 0.006b
Prodelphinidin Dimer Isomer 0.502 0.001a 0.593 0.010a
Gallocatechin 3.894 0.017a 3.624 0.011b
Vescalagin/castalagin 0.529 0.020a 0.191 0.001b
Prodelphinidin Dimer Isomer 1.450 0.004b 1.598 0.013a
Uralenneoside 0.368 0.005b 0.973 0.012a
Geraniin Isomer 1.044 0.020a 0.459 0.006b
Pedunculagin/Casuariin Isomer 1.424 0.006a 1.161 0.028b
Geraniin Isomer 1.150 0.012a 0.567 0.005b
Procyanidin B Isomer 5.111 0.003b 7.672 0.033a
Galloyl(epi)catechin-(epi)gallocatechin <LOQ <LOQ
Procyanidin B Isomer 0.575 0.002b 0.633 0.013a
Tellimagrandin I Isomer 0.641 0.006a 0.444 0.001b
Pterocarinin A <LOQ 0.646 0.011a
Pterocarinin A Isomer <LOQ 0.371 0.016a
Stenophyllanin A 1.201 0.040b 1.677 0.142a
Procyanidin trimer Isomer 0.399 0.014b 0.978 0.005a
Catechin 7.216 0.045a 7.142 0.010a
Procyanidin tetramer <LOQ <LOQ
Procyanidin trimer Isomer <LOQ 0.233 0.003a
Guavin A 0.430 0.028b 0.634 0.017a
Casuarinin/Casuarictin Isomer 0.368 0.001b 4.147 0.055a
Galloyl(epi)catechin-(epi)gallocatechin 0.494 0.004a 0.225 0.005b
Procyanidin pentamer <LOQ <LOQ
Galloyl(epi)catechin trimer Isomer <LOQ 0.007 0.001a
Gallocatechin 0.930 0.013b 1.944 0.004a
Tellimagrandin I Isomer 1.008 0.014a 0.682 0.011b
Vescalagin/castalagin 0.191 0.004a 0.192 0.003a
Stenophyllanin A Isomer 0.320 0.010b 0.770 0.030a
Galloyl(epi)catechin trimer Isomer <LOQ <LOQ
Stachyuranin A 0.252 0.011b 0.312 0.010a
Myricetin hexoside Isomer 0.127 0.001b 0.182 0.007a
Myricetin hexoside Isomer 0.130 0.001b 0.184 0.004a
Procyanidin gallate 2.353 0.046a 1.808 0.033b
Vescalagin/castalagin 0.194 0.001a 0.199 0.003a
Myricetin-arabinoside/xylopyranoside Isomer 0.152 0.004b 0.225 0.010a
Myricetin-arabinoside/xylopyranoside Isomer 0.231 0.005b 0.503 0.016a
Procyanidin gallate <LOQ 0.060 0.003
Myricetin-arabinoside/xylopyranoside Isomer 0.198 0.002b 0.512 0.005a
Myricetin hexoside Isomer 0.746 0.010b 0.819 0.010a
Myricetin hexoside Isomer 0.251 0.003a 0.216 0.001b
Myricetin-arabinoside/xylopyranoside Isomer 0.500 0.003a 0.412 0.001b
Quercetin- galloylhexoside Isomer 0.335 0.001b 0.391 0.004a
Ellagic acid deoxyhexoside 0.974 0.017a 0.697 0.005b
Quercetin-galloylhexoside Isomer 0.258 0.002a 0.309 0.008a
Myricetin-arabinoside/xylopyranoside Isomer 0.780 0.017a 0.617 0.008b
Morin 3.788 0.126b 5.505 0.055a
Myricetin-arabinoside/xylopyranoside Isomer 0.711 0.009b 0.762 0.010a
Ellagic acid 2.151 0.060a 2.081 0.028b
Hyperin 2.858 0.011b 7.192 0.056a
Quercetin glucuronide <LOQ 1.864 0.024a
Isoquercitrin 2.109 0.019b 3.274 0.031a
Procyanidin gallate 0.219 0.010a 0.111 0.002b
Reynoutrin 3.126 0.048b 4.730 0.057a
Guajaverin 8.027 0.105b 11.924 0.049a
Guavinoside A 0.543 0.010b 1.080 0.014a
Avicularin 6.250 0.106b 12.396 0.034a
Quercitrin 6.522 0.118a 5.154 0.051b
Myrciaphenone B 0.616 0.001b 0.978 0.013a
Guavinoside C 2.461 0.068a 2.626 0.019a
Guavinoside B 1.450 0.026a 0.875 0.003b
Guavinoside A Isomer 0.147 0.001b 0.398 0.004a
Guavinoside B Isomer 0.652 0.008a 0.154 0.001a
2,6-dihydroxy-3-methyl-4-O-(6-O-galloyl--D-glucopyranosyl)-benzophenone 0.982 0.022b 1.348 0.020a
Guavin B 0.357 0.008a 0.362 0.004a
Quercetin 0.320 0.009b 0.460 0.003a
Naringenin 0.647 0.020b 1.509 0.021a
 Journal of Functional Foods 22 (2016) 376388 385

present data are in the same order of magnitude as those ob-

Table 4 Comparison (mean SD, n = 3) between TPC by
tained for cv. Shui Jing Ba (0.79 mg/g leaf) by Chen and Yen (2007)
HPLC, FRAP and ABTS of Psidium guajava L. leaves var.
pyrifera and pomifera. Means in the same column with in guava leaves and higher if they are compared with guava
different superscript letters are significantly different juice, where the concentration was lower than the detection
(P < 0.05). limit (Amaya-Cruz et al., 2015). In addition, Amaya-Cruz et al.
Sample TPC FRAP ABTS (2015) identified and quantified several phenolic compounds
in guava juice by-products, and some of them such as ellagic
var. pyrifera 113.34 0.48a 5.82 0.12a 3.27 0.09a
var. pomifera 86.12 0.94b 5.55 0.02a 2.66 0.06b
acid, quercetin and catechin were also found in leaves. However,
the content of these phenolic compounds in guava juice by-
Expressed in mg of polar compound/g leaf d.w.

products was lower than the amounts of the same compounds
Expressed in mM of FeSO4 equivalents /mg leaf d.w.

Expressed in mM of Trolox equivalents /mg leaf d.w. shown in the studied guava leaves. It is important to high-
light that several phenolic compounds identified in guava fruits
and fruit by-products (Amaya-Cruz et al., 2015; Flores, Wu,
Negrin, & Kennelly, 2015) are not described in guava leaves.
detected and quantified gallic acid in pulp and peel of guava About guava fruits, several authors reported that red guava
fruits; its content varied from 1.2 to 1.6 mg/100 g in fresh peel (P. guajava L.) cv. Samsi has higher antioxidant activity, higher
and pulp, respectively. The obtained data in this work for guava total phenolics and also higher total flavonoid contents than
leaves showed higher content of gallic acid (0.060 and 0.223 mg/g white guava cv. Pansithong (Thuaytong & Anprung, 2011). In
dry weight) compared to the peel and the pulp. Moreover, the contrast, other authors (Thaipong et al., 2006) analysed one

Table 5 Comparison between guava leaves and fruits phenolic content.

Origin (type) Quantification Identified Content Reference
method compounds
FRUIT
Mexico (white guava FolinCiocalteu TPC 7.5 0.3 GAE mg/g d.w. (Amaya-Cruz et al., 2015)
by-products) AlCl3 Flavonoids 2.3 0.1 CE mg/g d.w.
HPLC-DAD-MS Gallic acid <LOD
Ellagic acid 0.6 0.1 ng/g d.w.
Catechin 0.3 0.0 ng/g d.w.
Quercetin 0.1 0.0 ng/g d.w.
Taiwan FolinCiocalteu TPC 69.6 2.8 CE mg/g d.w. (Chen & Yen, 2007)
FolinCiocalteu TPC 115 4.2 GAE mg/g d.w.
China (peel and pulp) HPLC-DAD Gallic acid 1.241.58 mg/100 g Li et al., 2013
Brazil (pulp) FolinCiocalteu TPC 17.2 1.1 mg/g d.w (Ribeiro da Silva et al., 2014)
Thailand (white cv. Pansithong) FolinCiocalteu TPC 145.52 0.08 GAE mg/g d.w. (Thuaytong & Anprung, 2011)
AlCl3 Flavonoids 19.06 0.18 CE mg/g d.w.
Thailand (red cv. Samsi) FolinCiocalteu TPC 163.36 0.05 GAE mg/g f.w.
AlCl3 Flavonoids 35.85 0.13 CE mg/g f.w.
USA (white cv. Allahabad Safeda) FolinCiocalteu TPC 344.97 33.6 GAE mg/100 g f.w. (Thaipong et al., 2006)
USA (red cv. Fan Retief, Ruby Supreme FolinCiocalteu TPC 170300 GAE mg/100 g f.w.
and an advanced selection)
Mauritius (white) FolinCiocalteu TPC 2473 45 GAE g/g f.w. (Luximon-Ramma et al., 2003)
AlCl3 Flavonoids 209 10 Q g/g f.w.
Mauritius (pink) FolinCiocalteu TPC 1264 60 GAE g/g f.w.
AlCl3 Flavonoids 110 21 Q g/g f.w.
LEAVES
Spain (young leaf) HPLC-MS TPC 157 6 mg/g d.w. (Daz-de-Cerio et al., 2015)
Mexico (young leaf) HPLC-DAD Quercetin 0.620 mg/g d.w. (Vargas-Alvarez et al., 2006)
Thailand (young leaf) HPLC-DAD Gallic acid 3.67 0.12 mg/g e (Nantitanon et al., 2010)
Quercetin 26.12 0.98 mg/g e
Ellagic acid 13.82 0.34 mg/g e
Taiwan (Shi Ji Ba, Tu Ba) HPLC-DAD Gallic acid 1122 mg/g e (Wu et al., 2009)
Quercetin 911 mg/g e
Catechin 45 mg/g e
Korea HPLC-DAD Gallic acid 0.09 0.00 mg/g d.w. (Jang et al., 2014)
Gallocatechin 2.88 0.02 mg/g d.w.
Catechin 0.72 0.04 mg/g d.w.
Paran (Pomifera var.) FolinCiocalteu TPC 161175 GAE mg/g e (Haida et al., 2011)
Paran (Pyrifera var.) 159164 GAE mg/g e
Taiwan (Hong Ba, Shi Ji Ba, Shui Jing Ba, FolinCiocalteu TPC 267313 CE mg/g l (Chen & Yen, 2007)
Tu Ba) 414483 GAE mg/g l
GAE, gallic acid equivalent; CE, catechin equivalent; Q, quercetin; LOD, detection limit; f.w., fresh weight; d.w., dry weight; e, extract; l, leaf.
386 Journal of Functional Foods 22 (2016) 376388

70

var. pomifera var. pyrifera

60

50
Concentration (mg/g leaf d.w.)

40

30

20

10

0
gallic and ellagic derivatives flavonols flavanones flavan-3-ols

Fig. 3 Comparison between the quantification (mean SD, n = 3) of the different families of compounds (mg/g leaf d.w.)
identified in Psidium guajava L. leaves var. pyrifera and pomifera.

white guava (cv. Allahabad Safeda) and three pink guava fruits compared by FRAP and TEAC. Table 4 shows the data ob-
(cv. Fan Retief, Ruby Supreme and an advanced selection) tained by TEAC, FRAP and TPC (total phenolic compounds
clones of guava fruits and reported that white pulp guava had expressed as the sum of total compounds determined by HPLC-
higher antioxidant capacity and total phenolic content than MS) for the two varieties of guava leaves. P. guajava L. leaves
pink pulp guavas; Luximon-Ramma et al. (2003) found the same var. pyrifera provided higher amount of total phenolic com-
trend in their analysis. Data from these works are summa- pounds, and consequently, a higher antioxidant capacity than
rized in Table 5. As it can be seen, a wide range is displayed pomifera variety. Correlation analysis was carried out between
for TPC. In this case, TPC varies from 1.3 to 163 GAE mg/g f.w., TPC, TEAC and FRAP assay, and the results showed the highest
and from 7.5 to 115 GAE mg/g d.w. in the fruit. Instead, guava correlation between TPC and TEAC (R > 0.97 P < 0.03). More-
leaf phenolic content showed less variation (from 157 mg/g in over, positive correlation was also detected for and each family
d.w. and 159 GAE mg/g extract to 483 GAE mg/g leaf). This trend of phenolic compounds (R > 0.97 P < 0.03).
could be justified not only by genotypes, seasonal changes, and Flores et al. (2015) studied seven P. guajava cultivars that varied
location, but also by the determination method that the authors in colour from white to pink.They found that pink-pulp guavas
used for phenolic determination. In fact, FolinCiocalteu reagent had higher antioxidant capacity than white-pulp ones, and even
can react with sugars that are naturally present in fruits and though the major compounds were common to all cultivars,
the phenolic content could be overestimated. important differences existed in the accumulation of a sig-
The health benefits of phenolic composition of guava fruits nificant number of compounds. Briefly, the antioxidant capacity
and leaves have been studied by several authors. Singh et al. and the phytochemical composition of P. guajava vary signifi-
(2013) reported that flavonoids are one of major chemical con- cantly according to the cultivar and pulp colour. ABTS data
stituents of plant species used in the management of diabetic obtained in this work for guava fruits reported lower TEAC values
complications. Eidenberger and coworkers (2013) evaluated the compared to guava leaves.
inhibition of dipeptidyl peptidase activity by flavonol glyco-
sides of guava (P. guajava L.) and found that these compounds
have a potential to exert the effect observed in vitro. More-
over, species like Murraya koenigii L. and Mentha piperitae L. show 4. Conclusions
antidiabetic properties (Narendhirakannan, Subramanian, &
Kandaswamy, 2006). The results of phytochemical analysis of Several compounds were verified and twelve new compounds
the leaves of these species (Samarth & Samarth, 2009; Uraku were identified in P. guajava L. leaves by HPLC-DAD-ESI-QTOF-
& Nwankwo, 2015) revealed a lower content of flavonoids than MS. Moreover, the hydroethanolic mixture (80:20, v/v) was
guava leaves, thus, P. guajava L. leaves (var. pyrifera) could serve selected as the best solvent to extract phenolic compounds,
as an effective extract for the amelioration of certain disease especially for flavonols and flavan-3-ols. Significant differ-
complications. ences (P < 0.05) between varieties pyrifera and pomifera were
Furthermore, the antioxidant capacity was tested. To our found in the quantification of total phenolic compounds and
knowledge, this is the first time that these varieties are their families by HPLC-MS. FRAP and TEAC assays were carried
 Journal of Functional Foods 22 (2016) 376388 387

out for the first time to compare the varieties of guava leaves. Flores, G., Wu, S.-B., Negrin, A., & Kennelly, E. J. (2015). Chemical
High correlation was noticed among TPC, FRAP and TEAC. Due composition and antioxidant activity of seven cultivars of
to its composition, P. guajava L. var. pyrifera extract is a good guava (Psidium guajava) fruits. Food Chemistry, 170, 327
335.
source of antioxidants that could be employed in diabetic thera-
Gordon, A., Jungfer, E., da Silva, B. A., Maia, J. G. S., & Marx, F.
peutic approaches. Further studies will be carried out to obtain (2011). Phenolic constituents and antioxidant capacity of four
flavonols- and flavan-3-ols-enriched extracts by green underutilized fruits from the Amazon Region. Journal of
technologies. Agricultural and Food Chemistry, 59, 76887699.
Gutirrez, R. M. P., Mitchell, S., & Solis, R. V. (2008). Psidium
guajava: A review of its traditional uses, phytochemistry
and pharmacology. Journal of Ethnopharmacology, 117(1), 1
Conflicts of interest 27.
Haida, K. S., Baron, ., Haida, K. S., de Faci, D., & Haas, J. (2011).
Phenolic compounds and antioxidant activity of two varieties
The authors declare no competing financial interest.
of guava and rue. Revista Brasileira de Cincias Da Sade, 28, 11
19.
Jaiswal, R., Jayasinghe, L., & Kuhnert, N. (2012). Identification and
Acknowledgments characterization of proanthocyanidins of 16 members of the
Rhododendron genus (Ericaceae) by tandem LC-MS. Journal of
Mass Spectrometry, 47, 502515.
This work was funded by the project co-financed by FEDER- Jang, M., Jeong, S.-W., Cho, S. K., Yang, H. J., Yoon, D.-S., Kim, J.-C.,
Andaluca 20072013 (Cod. 461100) and Andalusian Regional & Park, K.-H. (2014). Improvement in the anti-inflammatory
Government Council of Innovation and Science (P11-CTS- activity of guava (Psidium guajava L.) leaf extracts through
7625). The authors Ana Mara Gmez-Caravaca and Vito Verardo optimization of extraction conditions. Journal of Functional
Foods, 10, 161168.
thank the Spanish Ministry of Economy and Competitive-
Kua, S. F., Ibrahim, J., Ooi, C. K. W., Nan, K. I., Hashim, N., & Mohd
ness (MINECO) for Juan de la Cierva post-doctoral contracts.
Yusof, H. (2015). Optimisation of phenolic extraction and
quantification of phenolics in palm kernel cake. Renewable
Bioresources, 3(1), 2.
REFERENCES
Laporta, O., Perezfons, L., Mallavia, R., Caturla, N., & Micol, V.
(2007). Isolation, characterization and antioxidant capacity
assessment of the bioactive compounds derived from
Amaya-Cruz, D. M., Rodrguez-Gonzlez, S., Prez-Ramrez, I. F., Hypoxis rooperi corm extract (African potato). Food Chemistry,
Loarca-Pia, G., Amaya-Llano, S., Gallegos-Corona, M. A., & 101(4), 14251437.
Reynoso-Camacho, R. (2015). Juice by-products as a source of Li, F., Li, S., Li, H. B., Deng, G. F., Ling, W. H., Wu, S., & Chen, F.
dietary fibre and antioxidants and their effect on hepatic (2013). Antiproliferative activity of peels, pulps and seeds of
steatosis. Journal of Functional Foods, 17, 93102. 61 fruits. Journal of Functional Foods, 5(3), 12981309.
Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of Lpez-Cobo, A., Gmez-Caravaca, A. M., varc-Gajic, J.,
plasma (FRAP) as a measure of antioxidant power: the FRAP Segura-Carretero, A., & Fernndez-Gutirrez, A. (2015).
assay. Analytical Biochemistry, 239(1), 7076. Determination of phenolic compounds and antioxidant
Chang, C.-H., Hsieh, C.-L., Wang, H.-E., Peng, C.-C., Chyau, C.-C., & activity of a Mediterranean plant: the case of Satureja
Peng, R. Y. (2013). Unique bioactive polyphenolic profile of montana subsp. kitaibelii. Journal of Functional Foods, 18, 1167
guava (Psidium guajava) budding leaf tea is related to plant 1178.
biochemistry of budding leaves in early dawn. Journal of the Luximon-Ramma, A., Bahorun, T., & Crozier, A. (2003).
Science of Food and Agriculture, 93(4), 944954. Antioxidant actions and phenolic and vitamin C contents of
Chen, H.-Y., & Yen, G.-C. (2007). Antioxidant activity and free common Mauritian exotic fruits. Journal of the Science of Food
radical-scavenging capacity of extracts from guava (Psidium and Agriculture, 83(5), 496502.
guajava L.) leaves. Food Chemistry, 101(2), 686694. Moilanen, J., Sinkkonen, J., & Salminen, J.-P. (2013).
De Freitas, V. A. P., Glories, Y., Bourgeois, G., & Vitry, C. (1998). Characterization of bioactive plant ellagitannins by
Characterisation of oligomeric and polymeric procyanidins chromatographic, spectroscopic and mass spectrometric
from grape seeds by liquid secondary ion mass spectrometry. methods. Chemoecology, 23, 165179.
Phytochemistry, 49(5), 14351441. Morton, J. F. (1987). Guava. Fruits of warm climates (pp. 356363).
Deguchi, Y., & Miyazaki, K. (2010). Anti-hyperglycemic and anti- Miami, FL.
hyperlipidemic effects of guava leaf extract. Nutrition & Moure, A., Cruz, J. M., Franco, D., Domnguez, J. M., Sineiro, J.,
Metabolism, 7(9), 110. Domnguez, H., & Paraj, J. C. (2001). Natural antioxidants
Daz-de-Cerio, E., Verardo, V., Gmez-Caravaca, A. M., Fernndez- from residual sources. Food Chemistry, 72, 145171.
Gutirrez, A., & Segura-Carretero, A. (2015). Determination of Nantitanon, W., Yotsawimonwat, S., & Okonogi, S. (2010). Factors
polar compounds in guava leaves infusions and ultrasound influencing antioxidant activities and total phenolic content
aqueous extract by HPLC-ESI-MS. Journal of Chemistry, 2015, of guava leaf extract. LWT Food Science and Technology, 43(7),
19. 10951103.
Eidenberger, T., Selg, M., & Krennhuber, K. (2013). Inhibition of Narendhirakannan, R. T., Subramanian, S., & Kandaswamy, M.
dipeptidyl peptidase activity by flavonol glycosides of guava (2006). Biochemical evaluation of antidiabetogenic properties
(Psidium guajava L.): a key to the beneficial effects of guava in of some commonly used Indian plants on
type II diabetes mellitus. Fitoterapia, 89, 7479. streptozotocin-induced diabetes in experimental rats.
Fatiha, B., Khodir, M., Farid, D., Tiziri, R., Karima, B., Sonia, O., & Clinical and Experimental Pharmacology & Physiology, 33(12),
Mohamed, C. (2012). Optimisation of solvent extraction of 11501157.
antioxidants (phenolic compounds) from Algerian Mint Nonaka, G., Ishimaru, K., Azuma, R., Ishimatsu, M., & Nishioka, I.
(Mentha spicata L.). Pharmacognosy Communications, 2(4), 7286. (1989). Tannins and related compounds. LXXXV. Structures of
388 Journal of Functional Foods 22 (2016) 376388

novel C-glycosidic ellagitannins, grandinin and Taha, F. S., Mohamed, G. F., Mohamed, S. H., Mohamed, S. S., &
pterocarinins A and B. Chemical & Pharmaceutical Bulletin, 37(8), Kamil, M. M. (2011). Optimization of the extraction of total
20712077. phenolic compounds from sunflower meal and evaluation of
Okuda, T. (1987). Guavins A, C and D, complex tannins from the bioactivities of chosen extracts. American Journal of Food
Psidium guajava. Chemical & Pharmaceutical Bulletin, 35(1), 443 Technology, 6, 10021020.
446. Tanaka, T., Orii, Y., Nonaka, G., & Nishioka, I. (1993). Tannins and
Palanisamy, U. D., Ling, L. T., Manaharan, T., & Appleton, D. (2011). related compounds. CXXIII. Chromone, acetophenone and
Rapid isolation of geraniin from Nephelium lappaceum rind phenylpropanoid glycosides and their galloyl and/or
waste and its anti-hyperglycemic activity. Food Chemistry, hexahydroxydiphenoyl esters from the leaves of Syzygium
127(1), 2127. aromaticum Merr. et Perry. Chemical & Pharmaceutical Bulletin,
Regueiro, J., Snchez-Gonzlez, C., Vallverd-Queralt, A., 41(7), 12321237.
Simal-Gndara, J., Lamuela-Ravents, R., & Izquierdo-Pulido, Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., &
M. (2014). Comprehensive identification of walnut Hawkins Byrne, D. (2006). Comparison of ABTS, DPPH, FRAP,
polyphenols by liquid chromatography coupled to linear ion and ORAC assays for estimating antioxidant activity from
trap-Orbitrap mass spectrometry. Food Chemistry, 152, 340 guava fruit extracts. Journal of Food Composition and Analysis,
348. 19(67), 669675.
Ribeiro, A. B., Chist, R. C., Freitas, M., da Silva, A. F., Visentainer, Thuaytong, W., & Anprung, P. (2011). Bioactive compounds and
J. V., & Fernandes, E. (2014). Psidium cattleianum fruit extracts prebiotic activity in Thailand-grown red and white guava fruit
are efficient in vitro scavengers of physiologically relevant (Psidium guajava L.). Food Science and Technology International
reactive oxygen and nitrogen species. Food Chemistry, 165, = Ciencia Y Tecnologa de Los Alimentos Internacional, 17(3), 205
140148. 212.
Ribeiro da Silva, L. M., Teixeira de Figueiredo, E. A., Silva Ricardo, Uraku, A., & Nwankwo, V. (2015). Phytochemical and nutritional
N. M. P., Pinto Vieira, I. G., Wilane de Figueiredo, R., Brasil, I. composition analysis of murraya koenigii linn leaves. British
M., & Gomes, C. L. (2014). Quantification of bioactive Journal of Pharmaceutical Research, 6(3), 174180.
compounds in pulps and by-products of tropical fruits from Vargas-Alvarez, D., Soto-Hernndez, M., Gonzlez-Hernndez, V.
Brazil. Food Chemistry, 143, 398404. A., Engleman, E. M., & Martnez-Garza, . (2006). Kinetics of
Rizvi, S. I., Zaid, M. A., Anis, R., & Mishra, N. (2005). Protective role accumulation and distribution of flavonoids in guava
of tea catechins against oxidation-induced damage of type 2 (Psidium guajava L.). Agrociencia, 40, 109115.
diabetic erythrocytes. Clinical and Experimental Pharmacology Wang, B., Jiao, S., Liu, H., & Hong, J. (2007). Study on antioxidative
and Physiology, 32, 7075. activities of Psidium guajava Linn leaves extracts. Wei Sheng
Salazar, D. M., Melgarejo, P., Martnez, R., Martnez, J. J., Yan Jiu, 36, 298300.
Hernndez, F., & Burguera, M. (2006). Phenological stages of Wu, J.-W., Hsieh, C.-L., Wang, H.-Y., & Chen, H.-Y. (2009).
the guava tree (Psidium guajava L.). Scientia Horticulturae, 108, Inhibitory effects of guava (Psidium guajava L.) leaf extracts
157161. and its active compounds on the glycation process of protein.
Samarth, R. M., & Samarth, M. (2009). Protection against Food Chemistry, 113(1), 7884.
radiation-induced testicular damage in Swiss albino mice by Yamanaka, F., Hatano, T., Ito, H., Taniguch, S., Takahashi, E., &
Mentha piperita (Linn.). Basic & Clinical Pharmacology & Okamoto, K. (2008). Antibacterial effects of guava tannins and
Toxicology, 104(4), 329334. related polyphenols on Vibrio and Aeromonas species. Natural
Scott, J. A., & King, G. L. (2004). Oxidative stress and antioxidant Product Communications, 3(5), 711720.
treatment in diabetes. Annals of the New York Academy of Yoshikawa, M., Shimada, H., Nishida, N., Li, Y., Toguchida, I.,
Sciences, 1031, 204213. Yamahara, J., & Matsuda, H. (1998). Antidiabetic principles of
Seo, J., Lee, S., Elam, M. L., Johnson, S. A., Kang, J., & Arjmandi, B. natural medicines. II. Aldose reductase and -glucosidase
H. (2014). Study to find the best extraction solvent for use inhibitors from Brazilian natural medicine, the leaves of
with guava leaves (Psidium guajava L.) for high antioxidant Myrcia multiflora DC. (Myrtaceae): structures of myrciacitrins
efficacy. Food Science & Nutrition, 2(2), 174180. I and II and myrciaphenones A and B. Chemical &
Shu, J., Chou, G., & Wang, Z. (2010). Two new benzophenone Pharmaceutical Bulletin, 46(1), 113119.
glycosides from the fruit of Psidium guajava L. Fitoterapia, Yu, D. Q., Chen, Y., & Liang, X. T. (2000). Structural chemistry and
81(6), 532535. biological activities of natural products from Chinese herbal
Singh, R., Kaur, N., Kishore, L., & Gupta, G. K. (2013). Management medicines Part II. Research Communications in Molecular
of diabetic complications: a chemical constituents based Pathology and Pharmacology, 108, 393436.
approach. Journal of Ethnopharmacology, 150(1), 5170. Zhu, K.-X., Lian, C.-X., Guo, X.-N., Peng, W., & Zhou, H.-M. (2011).
Stalikas, C. D. (2007). Extraction, separation, and detection Antioxidant activities and total phenolic contents of various
methods for phenolic acids and flavonoids. Journal of extracts from defatted wheat germ. Food Chemistry, 126(3),
Separation Science, 30(18), 32683295. 11221126.