Accepted Manuscript Chemical composition and antioxidant activity of seven cultivars of guava (Psidium guajava) fruits Gema Flores, Shi-Biao Wu, Adam Negrin, Edward J. Kennelly PII: DOI: Reference:
S0308-8146(14)01300-4 http://dx.doi.org/10.1016/j.foodchem.2014.08.076 FOCH 16299
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
28 April 2014 14 August 2014 14 August 2014
Please cite this article as: Flores, G., Wu, S-B., Negrin, A., Kennelly, E.J., Chemical composition and antioxidant activity of seven cultivars of guava (Psidium guajava) fruits, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/ j.foodchem.2014.08.076
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1
Chemical composition and antioxidant
2
activity of seven cultivars of guava
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(Psidium guajava) fruits
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Gema Floresa,b,†, Shi-Biao Wu a,†, Adam Negrina, and Edward J. Kennellya,*
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a
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b
Department of Biological Sciences, Lehman College and The Graduate Center, City University of New York, 250 Bedford Park Boulevard West, Bronx, NY 10468, United States of America Instituto de Fermentaciones Industriales, Consejo Superior de Investigaciones Científicas (CSIC), c/Juan de la Cierva 3, 28006 Madrid, Spain
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TITLE RUNNING HEAD: Phenolic profile and antioxidant activities of seven Psidium guajava cultivars †
Authors contributed equally to this manuscript.
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* Corresponding author. Tel.: +1 718 960 1105; fax.:
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[email protected] (E.J. Kennelly)
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1
+1 718 960 8236. E-mail:
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ABSTRACT
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The antioxidant activity and identification of phenolic compounds of seven edible guava
35
(Psidium guajava) cultivars that varied in color from white to pink were examined. In the
36
DPPH• assay all four pink-pulp guavas (Barbie Pink, Homestead, Sardina 1, Sardina 2)
37
included in the study showed higher activity than the white pulp cultivars (Yen 2 and
38
Sayla) and less than the red pulp guava cultivar (Thai Maroon). In the ABTS•+ assay this
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trend was the same up to 20 min, but from 20-40 min Barbie Pink showed lower activity
40
than the white guavas. Twenty one compounds were characterized in the cultivars, and
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ten of them are reported for the first time in this fruit. Principle component analysis was
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performed to identify differences in chemistry among these cultivars. Our results suggest
43
that the antioxidant activity and phytochemical composition of Psidium guajava vary
44
significantly according to the cultivar and pulp color.
45 46 47 48 49 50 51 52
Keywords: Psidium guajava, guava, cultivars, ABTS, DPPH, principle component
53
analysis
2
54
1. Introduction
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Psidium guajava L. is one of the most important crops belonging to the genus Psidium
56
and the Myrtaceae family (Joseph & Priya 2011). Psidium guajava is naturalized in
57
tropical and subtropical parts of the world, and is considered an invasive species in some
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areas. This plant is a small tree 10 m high with wide spreading branches, and leaves that
59
are oblong or oval, 5-15 cm long, with prominent pinnate veins. Flowers have four to six
60
white petals and white stamens with yellow anthers (Stone, 1970). The skin of the fruit
61
and flesh color varies between cultivars depending on the type and amount of pigments.
62 63
Psidium guajava is used as a traditional medicine in certain cultures. The fruits are known
64
to possess large amounts of vitamins and minerals, and have such high levels of
65
polyphenolic antioxidants (Hassimotto, Genoves & Lajolo, 2005).
66
literature, they have sometimes been referred to as “superfruits”, due to their high
67
antioxidant capacity (Sanda, Grema, Geidman, & Bukar-Kolo, 2011). Guava contains
68
four times more vitamin C than an orange (Hassimotto, Genovese & Lajolo, 2005).
69
Psidium guajava has been shown to contain flavonoids, triterpenoids, and other
70
biologically active secondary compounds. This may explain, in part, its long history of
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traditional use by people worldwide as it have many benefits for various ailments (Sanda,
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Grema, Geidman, & Bukar-Kolo, 2011; Flores et al., 2013). Different parts of this plant
73
have been used to treat diabetes, caries, wounds, diarrhoea, inflammation or hypertension
74
(Gutierrez, Mitchell & Solis, 2008). Guava has reported anti-plasmodial, anti-
75
inflammatory, hepatoprotective, anticancer and antioxidant activity (Ojowole, 2006; Roy,
76
Kamath, & Asad, 2006; Salib & Michael, 2004; Flores et al., 2013). The nutritional and
77
health-promoting properties of P. guajava, together with the increased interest in its
78
antioxidant properties, indicate the potential nutraceutical use of this fruit (Ho et al.,
3
In the popular
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2012). Therefore, there is a need for the proper selection of cultivars with the appropriate
80
polyphenol composition for the intended use of the fruit.
81
To our knowledge, there are two reports comparing the antioxidant activity and chemical
82
content of different P. guajava cultivars (Santos & Corrêa, 2012, Biegelmeyer, R. et al.
83
2011). As part of our ongoing studies on Myrtaceae fruits bioactivity and polyphenol
84
composition (Flores et al., 2012; Wu, Dastmalchi, Long, & Kennelly, 2012; Flores et al.,
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2013; Wu et al., 2013), this study focused on antioxidant activity and relationship to
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phytochemicals, including anthocyanins, flavonoids, proanthocyanins, sesquiterpenoids
87
and triterpenoids of fruit extract from seven P. guajava cultivars. The fruits were
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collected in Florida which, together with Hawaii and Puerto Rico, are the largest
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producers of guava in the United States. The study compared three groups of P. guajava
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cultivars: white, pink and red. We hypothesize that differences in the phytochemical
91
composition can be correlated with the color of the fruit cultivar.
92 93
2. Materials and methods
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2.1. Chemicals and reagents
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HPLC-grade CH3OH, formic acid and acetonitrile were obtained from J.T. Baker
96
(Phillipsburg, NJ, USA) and used as solvents for chromatography. GR-grade CH3OH,
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was supplied by VWR Inc. (Bridgeport, PA, USA). Ultrapure water was prepared using a
98
Millipore Milli-RO 12 plus system (Millipore Corp., Bedford, MA, USA). Trolox, 1,1-
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diphenyl-2-picrylhydrazyl, and potassium peroxosulfate were purchased from Sigma
100
Chemical-Aldrich (St. Louis, MO, USA). 2,2'-Azinobis (3-ethylbenzothiazoline-6-
101
sulphonate) diammonium salt (ABTS) was obtained from TCI-Ace (Tokyo, Japan).
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Abscisic acid was supplied by Sigma Chemical-Aldrich (St. Louis, MO, USA).
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Quercetin-3-O-glucoside and quercetin were purchased from Extrasynthese (Genay,
4
104
France). Delphinidin-3-O-glucoside and cyanidin-3-O-glucoside were obtained from
105
Chromadex (Irvine, CA, USA).
106 107
2.2. Plant material
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Seven P. guajava cultivars (each 10 g, the ratio of material to solvent 1:20, w/v) were
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included in this study. Three of them, Homestead, Barbie Pink, and Thai Maroon, were
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collected on July 2011 at the University of Florida, Institute of Food and Agricultural
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Sciences, Tropical Research and Education Center. Homestead, a pink guava produced
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by a cross between Ruby (red guava) x Supreme (white guava), was collected from Block
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10, Row 1, Tree 12. Barbie Pink, a pink guava, was collected from Block 10, Row 1, Tree
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15. Thai maroon, a maroon guava, was collected from Block 10, Row 1, Tree 21. The
115
other four, Sardina 1 (small pink guava), Sardina 2 (large pink guava), Yen 2 (white
116
guava), and Sayla (white guava) were shipped by overnight courier on dry ice to the
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laboratory from large commercial growers in Homestead, Florida on November 2011.
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Fruits were kept in cold (-20 °C) dark storage until processed.
119 120
2.3. Extraction
121
The freeze-dried pulp of the seven P. guajava cultivars was extracted three times with
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CH3OH/H2O/formic acid (70:25:5) at room temperature with a blender for 5 min per
123
extraction, and the combined extract was dried in vacuo. Samples were dissolved in
124
CH3OH at a final concentration of 20 mg/mL and 5 mg/mL for HPLC-PDA and for mass
125
spectrometry analysis, respectively. All samples were filtered through a 25 mm syringe
126
filter (0.45 µm PTFE membrane) prior to injection.
127 128
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129 130
2.4. HPLC-PDA
131
The chromatographic analysis was carried out on a Waters (Milford, MA, USA) liquid
132
chromatography system equipped with a 2695 Separation Module and a 2996 photodiode-
133
array detector (PDA). For data acquisition and processing Waters Empower software
134
(version 5.0) was used. The separation was performed on a 250 × 4.6 mm, 4 µm
135
Phenomenex Synergi Hydro-RP 80A column (Torrance, CA, USA) with a 3 × 4.0 mm
136
Phenomenex SecurityGuard guard column. Mobile phase consisted of solvent A (1%
137
aqueous formic acid solution) and B (acetonitrile) at different ratios and employed a
138
gradient profile starting with 95% A for 5 min, 85% A at 10 min, 75% A at 35 min, and
139
45 % A from 45 to 50 min. The composition was then returned to initial conditions in 5
140
min and maintained for 10 min. Flow rate and injection volume were 1.0 mL/min and 10
141
µL, respectively. The UV/vis spectra were recorded from 190 to 600 nm.
142 143
2.5. Mass spectrometry
144
High resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed
145
using a LCT premier XE TOF mass spectrometer (Waters, Manifold, MA) equipped with
146
an ESI interface and controlled by MassLynx V4.1 software. All the settings were carried
147
out using an ESI ion source type in the positive and the negative mode with the following
148
settings: capillary voltage, 3000 V (positive mode) and 2800 V (negative mode), cone
149
voltage, 20 V; nitrogen gas was used for both the nebulizer and in desolvation; the
150
desolvation and cone gas flow rates were 600 and 20L/h, respectively; the desolvation
151
temperature was 400ºC, and the source temperature was 120 ºC. Full scan spectra were
152
acquired in both the positive and negative mode over the range m/z 100-1000. The
153
analytical column used was a 250 × 4.6 mm, 4 µm Phenomenex Synergi Hydro-RP 80A
6
154
column (Torrance, CA, USA). The same elution solvent and method as the one described
155
above for HPLC-PDA were applied.
156 157
2.6. Principal component analysis (PCA)
158
The HPLC-TOF-MS data of samples from seven P. guajava cultivars was analyzed by
159
PCA to identify potential discriminate variables. Peak detection and alignment, and the
160
filtering of raw data were carried out using Markerlynx v4.1. The parameters used
161
included a retention time range of 5-30 min, a mass range of 100-1000 Da, and a mass
162
tolerance of 50 mDa. Isotopic peaks were excluded for analysis; noise elimination level
163
was set at 500; and retention time tolerance was set at 0.4 min. The retention time and m/z
164
data pair for each peak was determined by the software. The samples were labelled
165
numerically, corresponding to different cultivar, and alphabetically, corresponding to
166
different LC injections.
167 168
2.7. 1,1-Diphenyl-2-picrilhydrazyl Free Radical (DPPH•) Scavenging
169
The DPPH• assay was performed according to the method developed by Smith et al.
170
(1987) and modified slightly. To a 50 µL aliquot of the sample 150 µL of DPPH• (400
171
µM) was added. Decrease of absorbance was monitored at 517 nm after 30 min of
172
incubation at 37 ºC on a Molecular Devices Versamax microplate reader (Sunnyvale, CA).
173
The percentage inhibition of the DPPH• at each concentration of sample was calculated
174
considering the percentage of the steady DPPH• in solution after reaction. Results were
175
expressed as the concentration of dry sample that leads to a 50% reduction in the DPPH•.
176 177 178
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179
2.8. 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) Free Radical (ABTS•+) Scavenging
180
The antioxidant activity of the seven P. guajava cultivars were measured by the ABTS•+
181
scavenging assay (Re et al., 1999). A Molecular Devices Versamax microplate reader
182
(Sunnyvale, CA, USA) was used. This assay is based on the formation of the free radical
183
cation ABTS•+ by reaction of ABTS aqueous solution (7mM) with K2S2O8 (2.45 mM,
184
final concentration) at ambient temperature in the dark for 12–16 h. Before use, this
185
solution was diluted with ethanol to an absorbance of 0.700 ± 0.020 at 734 nm. In a final
186
volume of 200 µL, the reaction mixture compromised 198 µL of ABTS•+ solution and 2
187
µL of the sample at different concentrations. Absorbances at 734 nm were measured at 5
188
min intervals during 40 min. Similarly, the reaction mixture of standard group was
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obtained by mixing 198 µL of ABTS•+ solution and 2 µL of Trolox. ABTS•+ scavenging
190
ability was expressed as the Trolox equivalent antioxidant capacity (TEAC, mmole
191
Trolox/ g of the sample) at different time intervals.
192 193
2.9. Statistical analysis
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Data are expressed as mean values ± 95 % confidence interval. Analysis of variance was
195
performed by one-way analysis of variance (ANOVA) with significant differences
196
between means determined by the Student’s t-test. JMP Statistics software package
197
version 8 (SAS Institute Inc., NC) was used for univariate statistical analysis.
198 199
3. Results and discussion
200
3.1. Chemical characterization of the P. guajava cultivars by LC-PDA and LC-TOF-MS
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The P. guajava cultivars were analyzed by HPLC-PDA and LC-TOF-MS. The peaks in
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the crude extract of each cultivar were detected by HPLC-PDA at 254 nm for phenolic
203
compounds and 520 nm for anthocyanins. They were identified by their elution order,
8
204
UV/vis spectra, and MS characteristics as compared with reported literature values, and
205
by coinjection with available standards. In this study negative and positive modes of ESI
206
mass detection were employed. TOF LC-MS (negative and positive modes) with ESI
207
mass detection was conducted. Fragmentation data, retention time and spectrum
208
information are displayed in Table 1 and their structures are represented in Figure 1. Ten
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of these compounds are reported for the first time in P. guajava.
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3.2. Anthocyanins
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These compounds have unique UV absorption maxima at around 278 and 520 nm.
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Considering this UV characteristic and MS profile compounds 1 and 2 were identified as
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delphinidin-3-O-glucoside (1) and cyanidin-3-O-glucoside (2), respectively. Their
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identification was confirmed by coinjection of the standard. Among all the cultivars P.
216
guajava Thai Maroon is the only cultivar purple in color, the rest of the cultivars are
217
yellow or light pink. As expected, anthocyanins were detected in Thai Maroon. There is
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one reference reporting anthocyanin pigments in guava cultivars (Siqueira, da Costa,
219
Afonso, and Clemente, 2011); however, this is the first time that delphinidin-3-O-
220
glucoside and cyanidin-3-O-glucoside are reported in P. guajava. We did not detect
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anthocyanins in the pink varieties, Barbie Pink, Homestead, Sardina 1, and Sardina 2.
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While it seems likely that these cultivars produce anthocyanins in their skins, we did not
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detect them, which may be a factor of levels of expression, limits of detection of the
224
detectors, or the chosen method of extraction.
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3.3. Flavonoids
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Ten flavonoids were characterized in the seven P. guajava cultivars. Among them
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myricetin-3-O-arabinoside (4), myricetin-3-O-xyloside (5), and isorhamnetin-3-O-
9
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galactopyranoside (11) are reported for the first time this plant. The characteristic UV
230
absorption maxima for the flavonoids are located between 350-370 nm (band I) and 240-
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260 nm (band II). The different UV absorbance profiles were helpful in the determination
232
of the aglycone moiety of the flavonoids. This information along with the MS data
233
allowed us to identify three aglycones: myricetin, quercetin, and isorhamnetin with
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positive fragmental ions at m/z 319, 303, and 317 respectively.
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Compounds 3, 4, and 5 were identified as myricetin glycosides. Compound 3 had [M +
236
Na]+ at m/z 503.0788 (C21H20O13Na, -2.8) which gave a fragment at m/z 319.0442 [M]+
237
(M − 162 amu) corresponding to loss of a hexose unit. On the basis of its UV/vis profile
238
and MS data, compound 3 was identified as myricetin-3-O-glucoside; this compound was
239
previously reported in a P. guajava species (Fu, Luo, & Zhang, 2009). Compounds 4 and
240
5 showed [M + H]+ at m/z 451. Both of them had one major fragment ion at m/z 319 (−
241
132 amu) denoting loss of a pentose unit. They were identified as myrcetin-3-O-
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pentosides. By reversed-phase HPLC, the glycosylation affects retention times differently
243
based on the nature of the sugar. For glycosylated flavonoids in the same bond position
244
arabinoside elutes before than xyloside According to this rule, compound 4 can be
245
identified as myricetin-3-O-arabinoside and compound 5 as myricetin-3-O-xyloside.
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The UV/vis absorption maxima of compounds 6, 7, 8, 9, and 13 at 250 and 360 nm and
247
the m/z fragment at 303 are characteristic of the quercetin aglycone. On the basis of the
248
similarity of MS and UV data, compounds 6 and 7 and 8 and 9 were considered isomers.
249
Compounds 6 and 7 showed [M + H]+ at m/z 465 and produced one major MS/MS
250
fragment at m/z 303 corresponding to the loss of 162 amu (a hexose unit) from a
251
quercetin backbone. The two common hexosides of flavonols are glucose and galactose
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(Dueñas, Hernández, Estrella & Muñoz, 2005; Chang & Wong, 2004). These two sugars
253
produce similar UV/vis profiles with flavonols; however, galactose typically elutes before
10
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glucose in reversed-phase chromatography (Prior, Lazarus, Cao, Muccitelli, &
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Hammerstone, 2001; Hong & Wrolstad, 1990). On the basis of the above reasoning, we
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assigned compound 6 as quercetin-3-O-galactoside and compound 7 as quercetin-3-O-
257
glucoside, and this was confirmed by co-injection with a standard. Both compounds were
258
identified before in P. guajava (Wang, Dub, Songa, 2010; Zhigang et al., 2012).
259
Compounds 8 and 9 had a parent ion [M + H]+ at m/z 405 and a loss of 132 amu
260
indicating that a pentoside sugar is attached to the quercetin aglycone. Compound 8 was
261
identified as quercetin-3-O-arabinopyranoside (guaijaverin) and compound 9 as
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quercetin-3-O-arabinoside (avicularin). Both compounds have been previously reported
263
in this plant (Wang, Dub, Songa, 2010).
264
Compound 13 had [M − H]− at m/z 301 and a fragmentation pattern corresponding to the
265
quasi molecular ion of quercetin in the negative ionization mode (Table 1). Its identity as
266
the aglycone quercetin was confirmed by matching its chromatographic and MS/MS
267
fragmentation profiles with an authentic standard. The free form of quercetin has been
268
reported before in P. guajava species (Wang, Dub, Songa, 2010).
269
The same molecular ions at m/z 479.1138 [M + H]+ /477.1024 [M − H]− of compound 10
270
and 479.1185 [M + H]+/477.1028 [M − H]− of compound 11 showed that they are
271
isomers. They had a fragmental ion at m/z 317 corresponding to a loss of 162 amu. Based
272
on the guidelines expressed above compound 10 was identified as isorhamnetin-3-O-
273
glucoside and 11 as isorharmentin-3-O-galactoside. Joseline et al. (2004) reported
274
isorhamnetin-3-O-glucoside in P. guajava species. Flavonols occur in P. guajava
275
primarily as glycosides. The majority of guava flavonoids (5 compounds) were quercetin
276
derivatives. Quercetin-glucoside and quercetin-galactoside were present in all the
277
cultivars except for P. guajava Sardina 2 whereas quercetin-arabinoside and quercetin-
278
xyloside were detected in all the cultivars except for P. guajava Sardina 1. Quercetin was
11
279
found in all the cultivars except for P. guajava Sayla. Myrcetin-glucoside was detected in
280
all the cultivars and the myrcetin pentosides were only characterized in P. guajava Thai
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Maroon and Sayla. Isorhamnetin derivatives were identified in P. guajava Thai Maroon.
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Isorhamnetin-glucoside was also found in P. guajava Barbie Pink and Homestead.
283 284
3.4. Proanthocyanidins
285
Two proanthocyanidins were identified in P. guajava. Compound 15 showed in the
286
positive mode m/z 633.1224 corresponding to [M + Na]+ (C30H26O14Na), and the
287
molecular ion [M + H − H2O]+ at 593.1255 (C30H25O13) (Table 1). A fragment with [M −
288
H]− at 609.1244 (C30H25O14) (Table 1) was found in the negative mode. The maximum
289
UV absorbance was registered at 356 and 265 nm. This compound was tentatively
290
determined as gallocatechin-(4α-8)-gallocatechol, which was previously identified in P.
291
guajava (F. Qa'dan, Petereit, F., & Nahrstedt, A., 2005).
292
The parent ion of compound 18 was obtained at m/z 617.1192 [M + Na]+ (C30H26O13Na).
293
It showed a deprotonated molecular ion in the negative mode at m/z 593.1314 (Table 1)
294
and had a corresponding formate adduct [M −H + HCOOH]− at m/z 639.1309
295
(C31H27O15). The UV spectra showed an absorption maxima at 356 and 265 nm. This
296
compound was determined to be gallocatechin-(4α-8)-catechin which was previously
297
identified in P. guajava by Qa'dan et al. (2005). Both compounds were identified in P.
298
guajava Thai Maroon and Barbie Pink. In addition we found compound 15 in the Sardina
299
2 cultivar and compound 18 in the Yen 2 and Sayla cultivars.
300 301
3.5. Triterpenes and Other Constituents
302
Compounds 12 and 16 were identified as sesquiterpenoids. Abscisic acid (12) was
303
characterized based on its MS fragmentation, UV profile and coinjection with standard.
12
304
Compound 16 showed a molecular ion at m/z 391.2314 [M + H]+ and 389.1269 [M − H]−
305
in the positive and negative mode respectively. In addition, an adduct was found in the
306
positive mode with m/z 413.2097 corresponding to [M + Na]+ (C19H34O8Na), and a
307
fragment at m/z 211.1705 that was associated with the loss of a glucose and a water
308
molecule [M + H − Glc − H2O]+ (C32H36O18). This compound was tentatively identified
309
as turpinionoside A.
310
Five compounds were characterized as triterpenes (14, 17, 19, 20, and 21). Compound 14
311
revealed an [M − H]− ion at m/z 609.1244 (C36H55O11). In the positive mode the parent
312
ion was found at m/z 687.3669 [M + Na]+ (C36H56O11Na) and the MS/MS spectrum
313
yielded an ion at m/z 503.3315 [M + H − Glc]+ (C30H47O6Na). Consequently this
314
component, which was present in P. guajava Thai Maroon and Homestead cultivars, was
315
tentatively assigned as pinfaensin.
316
Compound 17 had a precursor ion an [M − H]− (C36H57O10) at m/z 649.3928. The MS/MS
317
spectrum showed an adduct ion at m/z 695.3983 [M − H + HCOOH]− (C37H59O12). In the
318
positive mode the parent ion was found with m/z 651.4086 [M + H]+ (C36H59O10). The
319
fragment at m/z 489.3446 [M + H − Glc]+ (M − 162 amu) was attributed to the loss of a
320
glucose molecule, and was tentatively identified as pedunculoside. Compounds 16 and 17
321
were only detected in Thai Maroon and Sayla P. guajava cultivars. They are reported for
322
the first time in P. guajava species.
323
Compounds 19 and 20 showed successive losses in the positive mode of three molecules
324
of water from the molecular ion at m/z 485.3227, 467.3119, 449.3022 for compound 19
325
and 487.3428, 469.3293, 451.3226 for compound 20. Compound 19 was identified as
326
guavenoic acid, previously reported in P. guajava species (Begum, Hassan, & Siddiqui,
327
2002) and compound 20 as madecassic acid, reported for the first time in this plant. Both
328
of them were detected in all the cultivars.
13
329
Compound 21 had a parent ion at m/z 489.3591 [M + H]+ (C30H49O5). The MS/MS
330
fragments showed losses of one and two molecules of H2O. Compound 21 was
331
characterized as asiatic acid. This compound, reported in P. guajava species by Begum et
332
al. (2002), was found in all the cultivars except for Sayla.
333 334
3.6. PCA
335
PCA is a multivariate non-targeted metabolomics statistical analysis method (Wu,
336
Dastmalchi, Long, & Kennelly, 2012; Wu et al., 2013), and in our present study, we use it
337
to analyze the HPLC-TOF-MS total ion chromatograms (TIC) of these cultivars. Seven P.
338
guajava cultivars were compared by using PCA analysis to give an overview of the
339
influence of different cultivars on P. guajava composition. The retention times, m/z
340
accurate mass of fragmental ions obtained from the negative mode, and their mass
341
intensities were used to compare the differences in the composition of the seven P.
342
guajava cultivars. Each sample was injected in duplicate. The points in the plot are the
343
data observations, which when near each other are similar and when further apart are
344
dissimilar. The plot shows the possible presence of atypical observations, groups,
345
similarities, trends, and other patterns in the data. In our present plot, clear differences
346
between cultivars on the chemical composition of P. guajava were observed. In the score
347
plot (Figure 2), seven clusters can be differentiated, one of each cultivar. The close
348
proximity of the C-F clusters (Figure 2) corresponding to the cultivars with pink pulp,
349
Sardina 1, Sardina 2, Homestead, and Barbie Pink, indicates that they share certain
350
similar phytochemical profile. Also, the purple guava (P. guajava Thai Maroon) is
351
located in the upper left corner of the plot, owing to its unique anthocyanin components.
352
One white pulp cultivar (P. guajava Yen 2) is in the middle of the plot with a second
14
353
white cultivar (P. guajava Sayla) in the lower right corner of the plot. This is the first
354
report using PCA to separate different-colored guava cultivars based on LC-MS data.
355 356
3.7. Antioxidant activity
357
In order to measure antioxidant activities of the P. guajava cultivars, DPPH• and ABTS•+
358
radical scavenging assays were used. The order of DPPH• scavenging activity of the P.
359
guajava cultivars was Thai Maroon > Barbie Pink, Homestead, and Sardina 2 (not
360
significantly different, P > 0.05) > Sardina 1 > Yen 2 > Sayla (Figure 3).
361
All the cultivars demonstrated a wide range of ABTS•+ scavenging activities. At time 0
362
min the order of activity was Sardina 2 > Thai Maroon > Sardina 1 > Sayla > Homestead
363
and Yen 2 (not significantly different, P > 0.05) > Barbie Pink (Figure 4). The order of
364
activity changed over time and after 20 min remained constant (Thai Maroon > Sardina 2
365
and Sardina 1 (not significantly different, P > 0.05) > Homestead > Sayla > Yen 2 >
366
Barbie Pink).
367
It is well established that the DPPH• radical is used to evaluate the free radical scavenging
368
activity of hydrogen donating antioxidants. ABTS•+ in addition measures chain breaking
369
antioxidants (Choi, Jeong, & Lee, 2007). Based on the above considerations, our results
370
suggest that the extracts of the P. guajava cultivars are potent free radical scavengers and
371
may be utilized as a good source of natural antioxidants for food, pharmaceutical,
372
medical, and commercial uses. Thai Maroon, which exhibited the highest antioxidant
373
activity in both assays, also contained all of the compounds identified in this study and
374
was the only one in which we could identify anthocyanins. In the DPPH• and ABTS•+
375
assays Yen and Sayla exerted the lowest activity through 20 min. Some studies have
376
demonstrated a linear correlation between total phenolic content and antioxidant activity
377
in fruits and vegetables (Jayaprakasha, Girennavar, & Patil, 2008). Mahattanatawee et al.
15
378
(2006) reported higher antioxidant activity of red guava over white guava in a study
379
comparing fourteen tropical fruits from south Florida. In the same study the Red Dragon
380
cultivar showed higher antioxidant activity compared to white.
381 382
4. Conclusions
383
Even though there are major compounds common to all P. guajava cultivars, important
384
differences exist in the accumulation of a significant number of compounds between
385
these cultivars. Differences in these profiles may subsequently result in changes in
386
antioxidant activity or other bioactivities. This study provides a good foundation upon
387
which future studies linking nutritional properties of P. guajava with specific cultivars
388
can be built.
389 390
Acknowledgments
391
Support for this study was provided by NIH-NHLBI grant 5SC1HL096016, and by the
392
Spanish Ministry of Science and Innovation postdoctoral fellowship (G.F.). We express
393
our gratitude to Dr. Jonathan Crane and Ms. Wanda Montas (University of Florida, IFAS,
394
Tropical Research and Education Center) for providing the guavas included in this
395
manuscript. We would also like to acknowledge Mr. Rogelio Sardina for
396
developing/selecting the ‘Sardina 1’ and ‘Sardina 2’ cultivars, and Mr. Ernie Sardina for
397
providing the samples for this research. Additionally, we are grateful to Mr. Sayla Pith, a
398
green-guava producer in Florida, for developing the ‘Sayla’ cultivar and providing these
399
and the ‘Yen 2’ cultivar used in the study. The authors acknowledge Dr. Kurt Reynertson
400
for his research on Myrtaceae family fruits. We also thank Dr. Keyvan Dastmalchi, Dr.
401
Dan Kulakowski, and Ms. Vanya Petrova (Lehman College, CUNY) for their technical
402
assistance.
16
403
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404
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491
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493
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494
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495
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20
496
Figure Captions
497
Fig. 1. Chemical structures of compounds identified in the seven P. guajava cultivars.
498
Delphinidin 3-O-glucoside (1), cyanidin-3-O-glucoside (2), myricetin-3-O-β-D-glucoside
499
(3), myricetin-3-O-arabinoside (4), myricetin-3-O-xyloside (5), quercetin-3-O-galactoside
500
(6), quercetin-3-O-glucoside (7) quercetin-3-O-α-L-arabinopyranoside (guaijaverin) (8),
501
quercetin-3-O-arabinoside
502
isorhamnetin-3-O-galactoside (11), abscisic acid (12), quercetin (13), pinfaensin (14),
503
gallocatechin-(4α-8)-gallocatechol (15), turpinionosides A (16), pedunculoside (17),
504
gallocatechin-(4α-8)-catechin (18), guavenoic acid (19), madecassic acid (20), and asiatic
505
acid (21).
506
Fig. 2. PCA (scores plots) of the seven P. guajava cultivars (negative mode).
507
Fig. 3. DPPH• scavenging activity of the seven P. guajava cultivars. Values are expressed
508
as means ± SD (n=8). Bars with different letters (a-g) are significantly different (P >
509
0.05). Analysis of variance was performed by ANOVA procedures, with significant
510
differences between means determined by t- Student’s t-test comparisons.
511
Fig. 4. ABTS•+ scavenging activity of the seven P. guajava guava cultivars. Values are
512
expressed as means ± 95% confidence intervals (n=8) of Trolox equivalent antioxidant
513
capacity (TEAC) (milimoles of Trolox per gram of dry extract)
(avicularin)
(9),
21
isorhamnetin-3-O-glucoside
(10),
Figure 1.
R2
OH
R3
OH HO
HO
O
R4
R OGlc OH
R1 1 : R = OH 2:R=H
OH
O
3 : R1 = OGlc; R2 = R3 = R4 = OH 4 : R1 = OAra; R2 = R3 = R4 = OH 5 : R1 = OXyl; R2 = R3 = R4 = OH 6 : R1 = OGal; R2 = R3 = OH; R4 = H 7 : R1 = OGlc; R2 = R3 = OH; R4 = H 8 : R1 = OAra; R2 = R3 = OH; R4 = H 9 : R1 = OXyl; R2 = R3 = OH; R4 = H 10: R1 = OGlc; R2 = OCH3; R3 = OH; R4 = H 11: R1 = OGal; R2 = OCH3; R3 = OH; R4 = H 13: R1 = R2 = R3 = OH; R4 = H
OH OH HO
O OH
15 : R = OH 18 : R = H
OH
12
COOH O
R OH OH OH
OGlc HO
O OH 16
OH OH HO OH
OH R2 HO OH OGlc
HO O
R1
O HO
HO R2
14 : R1 = OH, R2 = CHO 17 : R1 = H, R 2 = CH2OH
R1 HO
22
19 : R1 = OH; R2 = CH2 20 : R1 = H; R2 = CH3 21 : R1 = OH; R2 = CH3
Figure 2.
A: B: C: D: E: F: G:
Red Guava
White Guava
P. guajava 'Thai Maroon' P. guajava 'Yen 2' P. guajava 'Sardina 1' P. guajava 'Sardina 2' P. guajava 'Barbie Pink' P. guajava 'Homestead' P. guajava 'Sayla'
Pink Guava
White Guava
23
Figure 3.
Cultivar sample
24
Figure 4.
TEAC (umol Trolox/g dry sample)
600
Barbie Pink 'Barbie Pink' Ruby Supreme 'Homestead' Sardina 11' 'Sardina Sardina 22' 'Sardina Sayla 'Sayla' Thai Maroon 'Thai Maroon' Yen 22' 'Yen
500
400
300
200
0
10
20
30
Time (min)
25
40
Table 1. Chemical profile of the identified compounds in the Psidium guajava cultivars1 N o. 1
2
R.T. (min ) 14.3
15.1
UV
[M+H]+ or [M-H](M.F., ppm)
Adduct and fragmental ion exact masses [M-X]+ or [M-X]- (M.F., ppm)
Identification
Detected from species1
Note
520, 274
465.1033 [M]+ (C21 H21O12, 0.0) 463.0875 [M – 2H]– (C21 H19O12, -0.4) 449.1070 [M]+ (C21 H21O11, –2.8) 447.0934[M – 2H]– (C21 H19O11, 1.7) 481.0987 [M + H]+ (C21 H21O13, 1.0) 479.0820 [M – H]– (C21 H19O13, –1.3) 451.0887 [M + H]+ (C20 H19O12, 2.2) 449.0730 [M – H]– (C20 H17O12, 2.2) 451.0878 [M + H]+ (C20 H19O12, 0.2); 449.0709 [M – H]– (C20 H17O12, –2.4) 465.1047 [M + H]+ (C21 H21O12, 3.0) 463.0889 [M – H]– (C21 H19O12, 2.6) 465.1032 [M + H]+ (C21 H21O12, –0.2) 463.0865 [M – H]– (C21 H19O12, –2.6) 435.0930 [M + H]+ (C20 H19O11, 0.7) 433.0779 [M – H]– (C20 H17O11, 1.8) 435.0947 [M + H]+ (C20 H19O11, 4.6) 433.0782 [M – H]– (C20 H17O11, 2.5) 479.1138 [M + H]+ (C22 H23O12, –10.9) 477.1024 [M – H]– (C22 H21O12, –1.9) 479.1185 [M + H]+ (C22 H23O12, –1.0)
303.0496 [M – Glc]+ (C15H11 O7 , –3.0);
Delphinidin 3-O-glucoside (co-injection)
a
detected for first time in this genus
Cyanidin-3-O-glucoside (coinjection)
a
detected for first time in this genus
503.0788 [M + Na]+ (C21 H20O13Na, –2.8); 319.0442 [M + H – Glc]+ (C15H11 O8 , 1.0); 983.1730 [2M + H]+ (C42 H39O26, 2.4) 959.1770 [2M – H]– (C42H39O26, 4.2)
Myricetin-3-O-β-D-glucoside
a-g
reported earlier in P.guajava(Fu, Luo, & Zhang, 2009)
319.0427 [M + H – Glc]+ (C15H11O8, –8.5); 923.1470 [2M + H]+ (C42 H36O24Na, –2.6)
Myricetin-3-O-arabinoside
a and g
detected for first time in this genus
319.0463 [M + H – Glc]+ (C15 H11O8, 2.8); 473.0710 [M + Na]+ (C20H18O12Na, 3.0); 923.1459 [2M + H]+ (C42 H36O24Na, –3.8) 899.1510 [2M – H]- (C40H35O24 , –0.9)
Myricetin-3-O-xyloside
a and g
detected for first time in this genus
487.0873 [M + Na]+ (C21H20O12 Na, –0.8); 303.0514 [M + H – Glc]+ (C15H11O7, –3.0)
Quercetin-3-O-galactoside (Hyperin)
a-c, e-g
reported earlier in P.guajava(Wang, 2010)
487.0842 [M + Na]+ (C21H20O12 Na, –1.8); 303.0504 [M + H – Glc]+ (C15H11 O7 , –0.3); 951.1800 [2M + Na]+ (C42H40O24 Na, –0.7) 509.0944 [M – H + HCOOH]- (C22H21O14, –2.6); 927.1852 [2M – H]- (C42 H39O24, 2.3)
Quercetin-3-O-glucoside (Isoquercitrin) (co-injection)
a-c, e-g
reported earlier P.guajava(Zhigang, 2012)
457.0774 [M + Na]+ (C20H18 O11Na, 5.9); 303.0512 [M + H – Glc]+ (C15H11O7, 2.3); 891.1585 [2M + Na]+ (C40H36O22 Na, –1.2) 479.0815 [M – H + HCOOH]– (C23 H19O13, –2.3); 867.1547 [2M – H]– (C40H35 O22, –8.4)
Quercetin-3-O-α-Larabinoside (Guaijaverin)
a, b, d-g
reported earlier in P.guajava(Wang, 2010)
457.0733 [M + Na]+ (C20 H18O11Na, –3.1); 303.0513 [M + H – Glc]+ (C15H11 O7 , 2.6); 891.1654 [2M + Na]+ (C40H36O22 Na, 3.8) 479.0830 [M – H + HCOOH]– (C23 H19O13, 0.8); 867.1572 [2M – H]– (C40H35O22, –5.5)
Avicularin
a, b, d-g
reported earlier in P.guajava(Wang, 2010)
501.1012 [M + Na]+ (C22H22O12 Na, 0.6); 317.0653 [M + H – Glc]+ (C16H13O7, –2.5)
Isorhamnetin-3-O-glucoside
a, e, f
reported earlier P.guajava(Josline, 2004)
501.1014 [M + Na]+ (C22H22O12 Na, 1.0); 979.2115 [2M + H]+ (C44 H44O24Na, –0.5)
Isorhamnetin-3-Ogalactoside (Cacticin)
a
detected for first time in this genus
516, 279
3
16.0
243, 356
4
17.1
240, 356
5
18.3
240, 356
6
19.0
240, 365
7
19.5
240, 365
8
20.0
250, 360
9
20.6
250, 360
10
21.0
254, 365
11
21.9
254, 364
509.0931 [M – 2H + HCOOH]– (C22H21O14 , –12.8); 481.0945 [M – 2H+H2O]– (C21 H21O13, –7.7) 287.0515 [M – Glc]+ (C15H11 O6 , –3.2); 465.1036 [M – 2H + H2O]– (C21H21 O12, 0.6)
–
899.1478 [2M – H] (C40H35O24, –4.4)
509.0907 [M – H + HCOOH]– (C22 H21O14, –4.7)
26
in
in
12
13
14
15
16
17
18
19
477.1028 [M – H]– (C22 H21O12, -1.0) 265.1414 [M + H]+ (C15 H21O4, –9.1)
24.0
26.1
254, 365
Abscisic acid (co-injection)
a-g
detected for first time in this genus
Quercetin (co-injection)
a-f
reported earlier P.guajava(Josline, 2004; Wang, 2010)
Pinfaensin
a-f
detected for first time in this genus
H]–
687.3669 [M + Na]+ (C36 H56O11Na, –7.4); 503.3315 [M + H – Glc]+ (C30H47O6Na, – 11.5); 485.3217 [M + H – Glc – H2 O]+ (C30H45O5, –10.3); 467.3152 [M + H – Glc – 2H2O]+ (C30H43 O4 , –1.9) 709.3747 [M – H + HCOOH]– (C37 H57O13, –7.3); 699.3534 [M + Cl]– (C36 H56O11Cl, 3.3)
H]+
633.1224 [M + Na]+ (C30H26O14 Na, 0.6); 593.1255 [M + H – H2O]+ (C30H25O13, –6.7)
Gallocatechin-(4α-8)gallocatechol
a, d, e,
413.2097 [M + Na]+ (C19 H34O8Na, –13.1); 211.1705 [M + H – Glc – H2O]+ (C32 H36O18, 3.3) 779.4515 [2M – H]– (C38H67O16, 11.0)
Turpinionosides A
a-g
reported earlier in P.guajava(F. Qa'dan, Petereit, F., Nahrstedt, A. , 2005) detected for first time in this genus
673.3901 [M + Na]+ (C36H58O10 Na, –4.0); 489.3446 [M + H – Glc]+ (C36H49 O5 , –6.9); 471.3456 [M + H – Glc – H2O]+ (C36 H47 O4 , –3.8); 453.3348 [M + H – Glc – 2H2 O]+ (C36 H45O3, –4.6) 695.3983 [M – H + HCOOH]– (C37 H59O12, 5.4); 685.3698 [M + Cl]- (C36 H58O10Cl, 3.3)
Pedunculoside
a-g
detected for first time in this genus
617.1192 [M + Na]+ (C30H26O13 Na, –12.8)
Gallocatechin-(4α-8)catechin
a, b, e, g
485.3227 [M + H – H2 O]+ (C30H45O5, –8.2); 467.3119 [M + H – 2H2O]+ (C30 H43O4, – 9.0); 449.3022 [M + H – 3H2 O]+ (C30H41O3, –7.6) 547.3253 [M – H + HCOOH]– (C31 H47O8, –3.3); 537.2928 [M + Cl]– (C30H46O6 Cl, 3.3)
Guavenoic acid
a-g
487.3428 [M + H – H2 O]+ (C30H47O5, –0.8); 469.3293 [M + H – 2H2O]+ (C30 H45O4, – 5.3); 451.3226 [M + H – 3H2 O]+ (C30H43O3, –3.1) 549.3430 [M – H + HCOOH]– (C31 H49O8, –0.5); 539.3128 [M + Cl]– (C30H48O6 Cl, –2.0)
Madecassic acid
a-g
reported earlier in P.guajava(F. Qa'dan, Petereit, F., Nahrstedt, A. , 2005) reported earlier in P.guajava(Begum, Hassan, & Siddiqui, 2002) detected for first time in this genus
471.3441 [M + H – H2O]+ (C30H47 O4 , –7.0); 453.3354 [M + H – 2H2O]+ (C30H45O3, –3.3)
Asiatic acid
a-f
263.1286 [M-H]– (C15 H19O4, 1.1) 303.0500 [M + H]+ (C15 H11O7, –1.6) 301.0348[M – H]– (C15 H9 O7 , 4.0)
26.4
27.8
265 356
28.6
29.1
30.6
266 356
663.3792 [M – (C36 H55O11, 7.2) 611.1385 [M + (C30 H27O14, –2.6) 609.1244 [M – (C30 H25O14, 0.2) 391.2314 [M + (C19 H35O8, -4.6) 389.1269 [M – (C19 H33O8, 10.5) 651.4086 [M + (C36 H59O10, –3.4)
H]+ H]– H]+
649.3928 [M – H]– (C36 H57O10, –3.7); 595.1452 [M + H]+ (C30 H27O13, –1.8); 593.1314 [M – H]– (C30 H25O13, 3.2) –
38.5
21
45.2
1
309.1338 [M – H + HCOOH]– (C16 H21O6, 2.6); 527.2656 [2M – H]– (C30 H39O8, 2.1) 605.0982 [2M + H]+ (C30 H21O14, 8.4) 347.0381 [M – H + HCOOH]– (C16 H11O9, –6.3); 603.0770 [2M – H]– (C30 H19O14, –0.8)
H]–
35.6
20
287.1259 [M + Na]+ (C15H20O4Na, –7.3); 247.1312 [M + H – H2O]+ (C15H19 O3 , –8.9); 529.2801 [2M + H]+ (C30 H41O8, –12.8); 551.2576 [2M + Na]+ (C30 H40O8Na, –8.2)
501.3185 [M – H] (C30 H45O6, –6.2) 505.3517 [M + H]+ (C30 H49O6, –2.4) 503.3343 [M – H]– (C30 H47O6, –6.0) 489.3591 [M + H]+ (C30 H49O5, 2.2) 503.3343 [M – H]– (C30 H47O6, –6.0)
–
639.1309 [M – H + HCOOH] (C31 H27O15, –6.4)
in
reported earlier in P.guajava(Begum, Hassan, Siddiqui, Shaheen, Ghayur, & Gilani, 2002)
a: P. guajava Thai Maroon; b: P. guajava Yen 2; c: P. guajava Sardina 1; d: P. guajava Sardina 2; e: P. guajava Barbie pink; f: P. guajava Homestead; g: P. guajava Sayla
27
Highlights - Chemical composition of Psidium guajava cultivars assessed by LC-TOF-MS - Antioxidant activity was evaluated by ABTS and DPPH assays - Twenty one compounds were identified - Ten compounds are reported for the first time in this fruit - Antioxidant activity and chemical profile differs depending on the cultivar
28
S0308-8146(14)01300-4 http://dx.doi.org/10.1016/j.foodchem.2014.08.076 FOCH 16299
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
28 April 2014 14 August 2014 14 August 2014
Please cite this article as: Flores, G., Wu, S-B., Negrin, A., Kennelly, E.J., Chemical composition and antioxidant activity of seven cultivars of guava (Psidium guajava) fruits, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/ j.foodchem.2014.08.076
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1
Chemical composition and antioxidant
2
activity of seven cultivars of guava
3
(Psidium guajava) fruits
4 5
Gema Floresa,b,†, Shi-Biao Wu a,†, Adam Negrina, and Edward J. Kennellya,*
6 7 8 9
a
10 11 12 13 14
b
Department of Biological Sciences, Lehman College and The Graduate Center, City University of New York, 250 Bedford Park Boulevard West, Bronx, NY 10468, United States of America Instituto de Fermentaciones Industriales, Consejo Superior de Investigaciones Científicas (CSIC), c/Juan de la Cierva 3, 28006 Madrid, Spain
15 16 17 18 19 20 21 22 23 24 25 26
TITLE RUNNING HEAD: Phenolic profile and antioxidant activities of seven Psidium guajava cultivars †
Authors contributed equally to this manuscript.
27
* Corresponding author. Tel.: +1 718 960 1105; fax.:
28
[email protected] (E.J. Kennelly)
29 30 31 32
1
+1 718 960 8236. E-mail:
33
ABSTRACT
34
The antioxidant activity and identification of phenolic compounds of seven edible guava
35
(Psidium guajava) cultivars that varied in color from white to pink were examined. In the
36
DPPH• assay all four pink-pulp guavas (Barbie Pink, Homestead, Sardina 1, Sardina 2)
37
included in the study showed higher activity than the white pulp cultivars (Yen 2 and
38
Sayla) and less than the red pulp guava cultivar (Thai Maroon). In the ABTS•+ assay this
39
trend was the same up to 20 min, but from 20-40 min Barbie Pink showed lower activity
40
than the white guavas. Twenty one compounds were characterized in the cultivars, and
41
ten of them are reported for the first time in this fruit. Principle component analysis was
42
performed to identify differences in chemistry among these cultivars. Our results suggest
43
that the antioxidant activity and phytochemical composition of Psidium guajava vary
44
significantly according to the cultivar and pulp color.
45 46 47 48 49 50 51 52
Keywords: Psidium guajava, guava, cultivars, ABTS, DPPH, principle component
53
analysis
2
54
1. Introduction
55
Psidium guajava L. is one of the most important crops belonging to the genus Psidium
56
and the Myrtaceae family (Joseph & Priya 2011). Psidium guajava is naturalized in
57
tropical and subtropical parts of the world, and is considered an invasive species in some
58
areas. This plant is a small tree 10 m high with wide spreading branches, and leaves that
59
are oblong or oval, 5-15 cm long, with prominent pinnate veins. Flowers have four to six
60
white petals and white stamens with yellow anthers (Stone, 1970). The skin of the fruit
61
and flesh color varies between cultivars depending on the type and amount of pigments.
62 63
Psidium guajava is used as a traditional medicine in certain cultures. The fruits are known
64
to possess large amounts of vitamins and minerals, and have such high levels of
65
polyphenolic antioxidants (Hassimotto, Genoves & Lajolo, 2005).
66
literature, they have sometimes been referred to as “superfruits”, due to their high
67
antioxidant capacity (Sanda, Grema, Geidman, & Bukar-Kolo, 2011). Guava contains
68
four times more vitamin C than an orange (Hassimotto, Genovese & Lajolo, 2005).
69
Psidium guajava has been shown to contain flavonoids, triterpenoids, and other
70
biologically active secondary compounds. This may explain, in part, its long history of
71
traditional use by people worldwide as it have many benefits for various ailments (Sanda,
72
Grema, Geidman, & Bukar-Kolo, 2011; Flores et al., 2013). Different parts of this plant
73
have been used to treat diabetes, caries, wounds, diarrhoea, inflammation or hypertension
74
(Gutierrez, Mitchell & Solis, 2008). Guava has reported anti-plasmodial, anti-
75
inflammatory, hepatoprotective, anticancer and antioxidant activity (Ojowole, 2006; Roy,
76
Kamath, & Asad, 2006; Salib & Michael, 2004; Flores et al., 2013). The nutritional and
77
health-promoting properties of P. guajava, together with the increased interest in its
78
antioxidant properties, indicate the potential nutraceutical use of this fruit (Ho et al.,
3
In the popular
79
2012). Therefore, there is a need for the proper selection of cultivars with the appropriate
80
polyphenol composition for the intended use of the fruit.
81
To our knowledge, there are two reports comparing the antioxidant activity and chemical
82
content of different P. guajava cultivars (Santos & Corrêa, 2012, Biegelmeyer, R. et al.
83
2011). As part of our ongoing studies on Myrtaceae fruits bioactivity and polyphenol
84
composition (Flores et al., 2012; Wu, Dastmalchi, Long, & Kennelly, 2012; Flores et al.,
85
2013; Wu et al., 2013), this study focused on antioxidant activity and relationship to
86
phytochemicals, including anthocyanins, flavonoids, proanthocyanins, sesquiterpenoids
87
and triterpenoids of fruit extract from seven P. guajava cultivars. The fruits were
88
collected in Florida which, together with Hawaii and Puerto Rico, are the largest
89
producers of guava in the United States. The study compared three groups of P. guajava
90
cultivars: white, pink and red. We hypothesize that differences in the phytochemical
91
composition can be correlated with the color of the fruit cultivar.
92 93
2. Materials and methods
94
2.1. Chemicals and reagents
95
HPLC-grade CH3OH, formic acid and acetonitrile were obtained from J.T. Baker
96
(Phillipsburg, NJ, USA) and used as solvents for chromatography. GR-grade CH3OH,
97
was supplied by VWR Inc. (Bridgeport, PA, USA). Ultrapure water was prepared using a
98
Millipore Milli-RO 12 plus system (Millipore Corp., Bedford, MA, USA). Trolox, 1,1-
99
diphenyl-2-picrylhydrazyl, and potassium peroxosulfate were purchased from Sigma
100
Chemical-Aldrich (St. Louis, MO, USA). 2,2'-Azinobis (3-ethylbenzothiazoline-6-
101
sulphonate) diammonium salt (ABTS) was obtained from TCI-Ace (Tokyo, Japan).
102
Abscisic acid was supplied by Sigma Chemical-Aldrich (St. Louis, MO, USA).
103
Quercetin-3-O-glucoside and quercetin were purchased from Extrasynthese (Genay,
4
104
France). Delphinidin-3-O-glucoside and cyanidin-3-O-glucoside were obtained from
105
Chromadex (Irvine, CA, USA).
106 107
2.2. Plant material
108
Seven P. guajava cultivars (each 10 g, the ratio of material to solvent 1:20, w/v) were
109
included in this study. Three of them, Homestead, Barbie Pink, and Thai Maroon, were
110
collected on July 2011 at the University of Florida, Institute of Food and Agricultural
111
Sciences, Tropical Research and Education Center. Homestead, a pink guava produced
112
by a cross between Ruby (red guava) x Supreme (white guava), was collected from Block
113
10, Row 1, Tree 12. Barbie Pink, a pink guava, was collected from Block 10, Row 1, Tree
114
15. Thai maroon, a maroon guava, was collected from Block 10, Row 1, Tree 21. The
115
other four, Sardina 1 (small pink guava), Sardina 2 (large pink guava), Yen 2 (white
116
guava), and Sayla (white guava) were shipped by overnight courier on dry ice to the
117
laboratory from large commercial growers in Homestead, Florida on November 2011.
118
Fruits were kept in cold (-20 °C) dark storage until processed.
119 120
2.3. Extraction
121
The freeze-dried pulp of the seven P. guajava cultivars was extracted three times with
122
CH3OH/H2O/formic acid (70:25:5) at room temperature with a blender for 5 min per
123
extraction, and the combined extract was dried in vacuo. Samples were dissolved in
124
CH3OH at a final concentration of 20 mg/mL and 5 mg/mL for HPLC-PDA and for mass
125
spectrometry analysis, respectively. All samples were filtered through a 25 mm syringe
126
filter (0.45 µm PTFE membrane) prior to injection.
127 128
5
129 130
2.4. HPLC-PDA
131
The chromatographic analysis was carried out on a Waters (Milford, MA, USA) liquid
132
chromatography system equipped with a 2695 Separation Module and a 2996 photodiode-
133
array detector (PDA). For data acquisition and processing Waters Empower software
134
(version 5.0) was used. The separation was performed on a 250 × 4.6 mm, 4 µm
135
Phenomenex Synergi Hydro-RP 80A column (Torrance, CA, USA) with a 3 × 4.0 mm
136
Phenomenex SecurityGuard guard column. Mobile phase consisted of solvent A (1%
137
aqueous formic acid solution) and B (acetonitrile) at different ratios and employed a
138
gradient profile starting with 95% A for 5 min, 85% A at 10 min, 75% A at 35 min, and
139
45 % A from 45 to 50 min. The composition was then returned to initial conditions in 5
140
min and maintained for 10 min. Flow rate and injection volume were 1.0 mL/min and 10
141
µL, respectively. The UV/vis spectra were recorded from 190 to 600 nm.
142 143
2.5. Mass spectrometry
144
High resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed
145
using a LCT premier XE TOF mass spectrometer (Waters, Manifold, MA) equipped with
146
an ESI interface and controlled by MassLynx V4.1 software. All the settings were carried
147
out using an ESI ion source type in the positive and the negative mode with the following
148
settings: capillary voltage, 3000 V (positive mode) and 2800 V (negative mode), cone
149
voltage, 20 V; nitrogen gas was used for both the nebulizer and in desolvation; the
150
desolvation and cone gas flow rates were 600 and 20L/h, respectively; the desolvation
151
temperature was 400ºC, and the source temperature was 120 ºC. Full scan spectra were
152
acquired in both the positive and negative mode over the range m/z 100-1000. The
153
analytical column used was a 250 × 4.6 mm, 4 µm Phenomenex Synergi Hydro-RP 80A
6
154
column (Torrance, CA, USA). The same elution solvent and method as the one described
155
above for HPLC-PDA were applied.
156 157
2.6. Principal component analysis (PCA)
158
The HPLC-TOF-MS data of samples from seven P. guajava cultivars was analyzed by
159
PCA to identify potential discriminate variables. Peak detection and alignment, and the
160
filtering of raw data were carried out using Markerlynx v4.1. The parameters used
161
included a retention time range of 5-30 min, a mass range of 100-1000 Da, and a mass
162
tolerance of 50 mDa. Isotopic peaks were excluded for analysis; noise elimination level
163
was set at 500; and retention time tolerance was set at 0.4 min. The retention time and m/z
164
data pair for each peak was determined by the software. The samples were labelled
165
numerically, corresponding to different cultivar, and alphabetically, corresponding to
166
different LC injections.
167 168
2.7. 1,1-Diphenyl-2-picrilhydrazyl Free Radical (DPPH•) Scavenging
169
The DPPH• assay was performed according to the method developed by Smith et al.
170
(1987) and modified slightly. To a 50 µL aliquot of the sample 150 µL of DPPH• (400
171
µM) was added. Decrease of absorbance was monitored at 517 nm after 30 min of
172
incubation at 37 ºC on a Molecular Devices Versamax microplate reader (Sunnyvale, CA).
173
The percentage inhibition of the DPPH• at each concentration of sample was calculated
174
considering the percentage of the steady DPPH• in solution after reaction. Results were
175
expressed as the concentration of dry sample that leads to a 50% reduction in the DPPH•.
176 177 178
7
179
2.8. 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) Free Radical (ABTS•+) Scavenging
180
The antioxidant activity of the seven P. guajava cultivars were measured by the ABTS•+
181
scavenging assay (Re et al., 1999). A Molecular Devices Versamax microplate reader
182
(Sunnyvale, CA, USA) was used. This assay is based on the formation of the free radical
183
cation ABTS•+ by reaction of ABTS aqueous solution (7mM) with K2S2O8 (2.45 mM,
184
final concentration) at ambient temperature in the dark for 12–16 h. Before use, this
185
solution was diluted with ethanol to an absorbance of 0.700 ± 0.020 at 734 nm. In a final
186
volume of 200 µL, the reaction mixture compromised 198 µL of ABTS•+ solution and 2
187
µL of the sample at different concentrations. Absorbances at 734 nm were measured at 5
188
min intervals during 40 min. Similarly, the reaction mixture of standard group was
189
obtained by mixing 198 µL of ABTS•+ solution and 2 µL of Trolox. ABTS•+ scavenging
190
ability was expressed as the Trolox equivalent antioxidant capacity (TEAC, mmole
191
Trolox/ g of the sample) at different time intervals.
192 193
2.9. Statistical analysis
194
Data are expressed as mean values ± 95 % confidence interval. Analysis of variance was
195
performed by one-way analysis of variance (ANOVA) with significant differences
196
between means determined by the Student’s t-test. JMP Statistics software package
197
version 8 (SAS Institute Inc., NC) was used for univariate statistical analysis.
198 199
3. Results and discussion
200
3.1. Chemical characterization of the P. guajava cultivars by LC-PDA and LC-TOF-MS
201
The P. guajava cultivars were analyzed by HPLC-PDA and LC-TOF-MS. The peaks in
202
the crude extract of each cultivar were detected by HPLC-PDA at 254 nm for phenolic
203
compounds and 520 nm for anthocyanins. They were identified by their elution order,
8
204
UV/vis spectra, and MS characteristics as compared with reported literature values, and
205
by coinjection with available standards. In this study negative and positive modes of ESI
206
mass detection were employed. TOF LC-MS (negative and positive modes) with ESI
207
mass detection was conducted. Fragmentation data, retention time and spectrum
208
information are displayed in Table 1 and their structures are represented in Figure 1. Ten
209
of these compounds are reported for the first time in P. guajava.
210 211
3.2. Anthocyanins
212
These compounds have unique UV absorption maxima at around 278 and 520 nm.
213
Considering this UV characteristic and MS profile compounds 1 and 2 were identified as
214
delphinidin-3-O-glucoside (1) and cyanidin-3-O-glucoside (2), respectively. Their
215
identification was confirmed by coinjection of the standard. Among all the cultivars P.
216
guajava Thai Maroon is the only cultivar purple in color, the rest of the cultivars are
217
yellow or light pink. As expected, anthocyanins were detected in Thai Maroon. There is
218
one reference reporting anthocyanin pigments in guava cultivars (Siqueira, da Costa,
219
Afonso, and Clemente, 2011); however, this is the first time that delphinidin-3-O-
220
glucoside and cyanidin-3-O-glucoside are reported in P. guajava. We did not detect
221
anthocyanins in the pink varieties, Barbie Pink, Homestead, Sardina 1, and Sardina 2.
222
While it seems likely that these cultivars produce anthocyanins in their skins, we did not
223
detect them, which may be a factor of levels of expression, limits of detection of the
224
detectors, or the chosen method of extraction.
225 226
3.3. Flavonoids
227
Ten flavonoids were characterized in the seven P. guajava cultivars. Among them
228
myricetin-3-O-arabinoside (4), myricetin-3-O-xyloside (5), and isorhamnetin-3-O-
9
229
galactopyranoside (11) are reported for the first time this plant. The characteristic UV
230
absorption maxima for the flavonoids are located between 350-370 nm (band I) and 240-
231
260 nm (band II). The different UV absorbance profiles were helpful in the determination
232
of the aglycone moiety of the flavonoids. This information along with the MS data
233
allowed us to identify three aglycones: myricetin, quercetin, and isorhamnetin with
234
positive fragmental ions at m/z 319, 303, and 317 respectively.
235
Compounds 3, 4, and 5 were identified as myricetin glycosides. Compound 3 had [M +
236
Na]+ at m/z 503.0788 (C21H20O13Na, -2.8) which gave a fragment at m/z 319.0442 [M]+
237
(M − 162 amu) corresponding to loss of a hexose unit. On the basis of its UV/vis profile
238
and MS data, compound 3 was identified as myricetin-3-O-glucoside; this compound was
239
previously reported in a P. guajava species (Fu, Luo, & Zhang, 2009). Compounds 4 and
240
5 showed [M + H]+ at m/z 451. Both of them had one major fragment ion at m/z 319 (−
241
132 amu) denoting loss of a pentose unit. They were identified as myrcetin-3-O-
242
pentosides. By reversed-phase HPLC, the glycosylation affects retention times differently
243
based on the nature of the sugar. For glycosylated flavonoids in the same bond position
244
arabinoside elutes before than xyloside According to this rule, compound 4 can be
245
identified as myricetin-3-O-arabinoside and compound 5 as myricetin-3-O-xyloside.
246
The UV/vis absorption maxima of compounds 6, 7, 8, 9, and 13 at 250 and 360 nm and
247
the m/z fragment at 303 are characteristic of the quercetin aglycone. On the basis of the
248
similarity of MS and UV data, compounds 6 and 7 and 8 and 9 were considered isomers.
249
Compounds 6 and 7 showed [M + H]+ at m/z 465 and produced one major MS/MS
250
fragment at m/z 303 corresponding to the loss of 162 amu (a hexose unit) from a
251
quercetin backbone. The two common hexosides of flavonols are glucose and galactose
252
(Dueñas, Hernández, Estrella & Muñoz, 2005; Chang & Wong, 2004). These two sugars
253
produce similar UV/vis profiles with flavonols; however, galactose typically elutes before
10
254
glucose in reversed-phase chromatography (Prior, Lazarus, Cao, Muccitelli, &
255
Hammerstone, 2001; Hong & Wrolstad, 1990). On the basis of the above reasoning, we
256
assigned compound 6 as quercetin-3-O-galactoside and compound 7 as quercetin-3-O-
257
glucoside, and this was confirmed by co-injection with a standard. Both compounds were
258
identified before in P. guajava (Wang, Dub, Songa, 2010; Zhigang et al., 2012).
259
Compounds 8 and 9 had a parent ion [M + H]+ at m/z 405 and a loss of 132 amu
260
indicating that a pentoside sugar is attached to the quercetin aglycone. Compound 8 was
261
identified as quercetin-3-O-arabinopyranoside (guaijaverin) and compound 9 as
262
quercetin-3-O-arabinoside (avicularin). Both compounds have been previously reported
263
in this plant (Wang, Dub, Songa, 2010).
264
Compound 13 had [M − H]− at m/z 301 and a fragmentation pattern corresponding to the
265
quasi molecular ion of quercetin in the negative ionization mode (Table 1). Its identity as
266
the aglycone quercetin was confirmed by matching its chromatographic and MS/MS
267
fragmentation profiles with an authentic standard. The free form of quercetin has been
268
reported before in P. guajava species (Wang, Dub, Songa, 2010).
269
The same molecular ions at m/z 479.1138 [M + H]+ /477.1024 [M − H]− of compound 10
270
and 479.1185 [M + H]+/477.1028 [M − H]− of compound 11 showed that they are
271
isomers. They had a fragmental ion at m/z 317 corresponding to a loss of 162 amu. Based
272
on the guidelines expressed above compound 10 was identified as isorhamnetin-3-O-
273
glucoside and 11 as isorharmentin-3-O-galactoside. Joseline et al. (2004) reported
274
isorhamnetin-3-O-glucoside in P. guajava species. Flavonols occur in P. guajava
275
primarily as glycosides. The majority of guava flavonoids (5 compounds) were quercetin
276
derivatives. Quercetin-glucoside and quercetin-galactoside were present in all the
277
cultivars except for P. guajava Sardina 2 whereas quercetin-arabinoside and quercetin-
278
xyloside were detected in all the cultivars except for P. guajava Sardina 1. Quercetin was
11
279
found in all the cultivars except for P. guajava Sayla. Myrcetin-glucoside was detected in
280
all the cultivars and the myrcetin pentosides were only characterized in P. guajava Thai
281
Maroon and Sayla. Isorhamnetin derivatives were identified in P. guajava Thai Maroon.
282
Isorhamnetin-glucoside was also found in P. guajava Barbie Pink and Homestead.
283 284
3.4. Proanthocyanidins
285
Two proanthocyanidins were identified in P. guajava. Compound 15 showed in the
286
positive mode m/z 633.1224 corresponding to [M + Na]+ (C30H26O14Na), and the
287
molecular ion [M + H − H2O]+ at 593.1255 (C30H25O13) (Table 1). A fragment with [M −
288
H]− at 609.1244 (C30H25O14) (Table 1) was found in the negative mode. The maximum
289
UV absorbance was registered at 356 and 265 nm. This compound was tentatively
290
determined as gallocatechin-(4α-8)-gallocatechol, which was previously identified in P.
291
guajava (F. Qa'dan, Petereit, F., & Nahrstedt, A., 2005).
292
The parent ion of compound 18 was obtained at m/z 617.1192 [M + Na]+ (C30H26O13Na).
293
It showed a deprotonated molecular ion in the negative mode at m/z 593.1314 (Table 1)
294
and had a corresponding formate adduct [M −H + HCOOH]− at m/z 639.1309
295
(C31H27O15). The UV spectra showed an absorption maxima at 356 and 265 nm. This
296
compound was determined to be gallocatechin-(4α-8)-catechin which was previously
297
identified in P. guajava by Qa'dan et al. (2005). Both compounds were identified in P.
298
guajava Thai Maroon and Barbie Pink. In addition we found compound 15 in the Sardina
299
2 cultivar and compound 18 in the Yen 2 and Sayla cultivars.
300 301
3.5. Triterpenes and Other Constituents
302
Compounds 12 and 16 were identified as sesquiterpenoids. Abscisic acid (12) was
303
characterized based on its MS fragmentation, UV profile and coinjection with standard.
12
304
Compound 16 showed a molecular ion at m/z 391.2314 [M + H]+ and 389.1269 [M − H]−
305
in the positive and negative mode respectively. In addition, an adduct was found in the
306
positive mode with m/z 413.2097 corresponding to [M + Na]+ (C19H34O8Na), and a
307
fragment at m/z 211.1705 that was associated with the loss of a glucose and a water
308
molecule [M + H − Glc − H2O]+ (C32H36O18). This compound was tentatively identified
309
as turpinionoside A.
310
Five compounds were characterized as triterpenes (14, 17, 19, 20, and 21). Compound 14
311
revealed an [M − H]− ion at m/z 609.1244 (C36H55O11). In the positive mode the parent
312
ion was found at m/z 687.3669 [M + Na]+ (C36H56O11Na) and the MS/MS spectrum
313
yielded an ion at m/z 503.3315 [M + H − Glc]+ (C30H47O6Na). Consequently this
314
component, which was present in P. guajava Thai Maroon and Homestead cultivars, was
315
tentatively assigned as pinfaensin.
316
Compound 17 had a precursor ion an [M − H]− (C36H57O10) at m/z 649.3928. The MS/MS
317
spectrum showed an adduct ion at m/z 695.3983 [M − H + HCOOH]− (C37H59O12). In the
318
positive mode the parent ion was found with m/z 651.4086 [M + H]+ (C36H59O10). The
319
fragment at m/z 489.3446 [M + H − Glc]+ (M − 162 amu) was attributed to the loss of a
320
glucose molecule, and was tentatively identified as pedunculoside. Compounds 16 and 17
321
were only detected in Thai Maroon and Sayla P. guajava cultivars. They are reported for
322
the first time in P. guajava species.
323
Compounds 19 and 20 showed successive losses in the positive mode of three molecules
324
of water from the molecular ion at m/z 485.3227, 467.3119, 449.3022 for compound 19
325
and 487.3428, 469.3293, 451.3226 for compound 20. Compound 19 was identified as
326
guavenoic acid, previously reported in P. guajava species (Begum, Hassan, & Siddiqui,
327
2002) and compound 20 as madecassic acid, reported for the first time in this plant. Both
328
of them were detected in all the cultivars.
13
329
Compound 21 had a parent ion at m/z 489.3591 [M + H]+ (C30H49O5). The MS/MS
330
fragments showed losses of one and two molecules of H2O. Compound 21 was
331
characterized as asiatic acid. This compound, reported in P. guajava species by Begum et
332
al. (2002), was found in all the cultivars except for Sayla.
333 334
3.6. PCA
335
PCA is a multivariate non-targeted metabolomics statistical analysis method (Wu,
336
Dastmalchi, Long, & Kennelly, 2012; Wu et al., 2013), and in our present study, we use it
337
to analyze the HPLC-TOF-MS total ion chromatograms (TIC) of these cultivars. Seven P.
338
guajava cultivars were compared by using PCA analysis to give an overview of the
339
influence of different cultivars on P. guajava composition. The retention times, m/z
340
accurate mass of fragmental ions obtained from the negative mode, and their mass
341
intensities were used to compare the differences in the composition of the seven P.
342
guajava cultivars. Each sample was injected in duplicate. The points in the plot are the
343
data observations, which when near each other are similar and when further apart are
344
dissimilar. The plot shows the possible presence of atypical observations, groups,
345
similarities, trends, and other patterns in the data. In our present plot, clear differences
346
between cultivars on the chemical composition of P. guajava were observed. In the score
347
plot (Figure 2), seven clusters can be differentiated, one of each cultivar. The close
348
proximity of the C-F clusters (Figure 2) corresponding to the cultivars with pink pulp,
349
Sardina 1, Sardina 2, Homestead, and Barbie Pink, indicates that they share certain
350
similar phytochemical profile. Also, the purple guava (P. guajava Thai Maroon) is
351
located in the upper left corner of the plot, owing to its unique anthocyanin components.
352
One white pulp cultivar (P. guajava Yen 2) is in the middle of the plot with a second
14
353
white cultivar (P. guajava Sayla) in the lower right corner of the plot. This is the first
354
report using PCA to separate different-colored guava cultivars based on LC-MS data.
355 356
3.7. Antioxidant activity
357
In order to measure antioxidant activities of the P. guajava cultivars, DPPH• and ABTS•+
358
radical scavenging assays were used. The order of DPPH• scavenging activity of the P.
359
guajava cultivars was Thai Maroon > Barbie Pink, Homestead, and Sardina 2 (not
360
significantly different, P > 0.05) > Sardina 1 > Yen 2 > Sayla (Figure 3).
361
All the cultivars demonstrated a wide range of ABTS•+ scavenging activities. At time 0
362
min the order of activity was Sardina 2 > Thai Maroon > Sardina 1 > Sayla > Homestead
363
and Yen 2 (not significantly different, P > 0.05) > Barbie Pink (Figure 4). The order of
364
activity changed over time and after 20 min remained constant (Thai Maroon > Sardina 2
365
and Sardina 1 (not significantly different, P > 0.05) > Homestead > Sayla > Yen 2 >
366
Barbie Pink).
367
It is well established that the DPPH• radical is used to evaluate the free radical scavenging
368
activity of hydrogen donating antioxidants. ABTS•+ in addition measures chain breaking
369
antioxidants (Choi, Jeong, & Lee, 2007). Based on the above considerations, our results
370
suggest that the extracts of the P. guajava cultivars are potent free radical scavengers and
371
may be utilized as a good source of natural antioxidants for food, pharmaceutical,
372
medical, and commercial uses. Thai Maroon, which exhibited the highest antioxidant
373
activity in both assays, also contained all of the compounds identified in this study and
374
was the only one in which we could identify anthocyanins. In the DPPH• and ABTS•+
375
assays Yen and Sayla exerted the lowest activity through 20 min. Some studies have
376
demonstrated a linear correlation between total phenolic content and antioxidant activity
377
in fruits and vegetables (Jayaprakasha, Girennavar, & Patil, 2008). Mahattanatawee et al.
15
378
(2006) reported higher antioxidant activity of red guava over white guava in a study
379
comparing fourteen tropical fruits from south Florida. In the same study the Red Dragon
380
cultivar showed higher antioxidant activity compared to white.
381 382
4. Conclusions
383
Even though there are major compounds common to all P. guajava cultivars, important
384
differences exist in the accumulation of a significant number of compounds between
385
these cultivars. Differences in these profiles may subsequently result in changes in
386
antioxidant activity or other bioactivities. This study provides a good foundation upon
387
which future studies linking nutritional properties of P. guajava with specific cultivars
388
can be built.
389 390
Acknowledgments
391
Support for this study was provided by NIH-NHLBI grant 5SC1HL096016, and by the
392
Spanish Ministry of Science and Innovation postdoctoral fellowship (G.F.). We express
393
our gratitude to Dr. Jonathan Crane and Ms. Wanda Montas (University of Florida, IFAS,
394
Tropical Research and Education Center) for providing the guavas included in this
395
manuscript. We would also like to acknowledge Mr. Rogelio Sardina for
396
developing/selecting the ‘Sardina 1’ and ‘Sardina 2’ cultivars, and Mr. Ernie Sardina for
397
providing the samples for this research. Additionally, we are grateful to Mr. Sayla Pith, a
398
green-guava producer in Florida, for developing the ‘Sayla’ cultivar and providing these
399
and the ‘Yen 2’ cultivar used in the study. The authors acknowledge Dr. Kurt Reynertson
400
for his research on Myrtaceae family fruits. We also thank Dr. Keyvan Dastmalchi, Dr.
401
Dan Kulakowski, and Ms. Vanya Petrova (Lehman College, CUNY) for their technical
402
assistance.
16
403
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404
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491
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493
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494
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495
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20
496
Figure Captions
497
Fig. 1. Chemical structures of compounds identified in the seven P. guajava cultivars.
498
Delphinidin 3-O-glucoside (1), cyanidin-3-O-glucoside (2), myricetin-3-O-β-D-glucoside
499
(3), myricetin-3-O-arabinoside (4), myricetin-3-O-xyloside (5), quercetin-3-O-galactoside
500
(6), quercetin-3-O-glucoside (7) quercetin-3-O-α-L-arabinopyranoside (guaijaverin) (8),
501
quercetin-3-O-arabinoside
502
isorhamnetin-3-O-galactoside (11), abscisic acid (12), quercetin (13), pinfaensin (14),
503
gallocatechin-(4α-8)-gallocatechol (15), turpinionosides A (16), pedunculoside (17),
504
gallocatechin-(4α-8)-catechin (18), guavenoic acid (19), madecassic acid (20), and asiatic
505
acid (21).
506
Fig. 2. PCA (scores plots) of the seven P. guajava cultivars (negative mode).
507
Fig. 3. DPPH• scavenging activity of the seven P. guajava cultivars. Values are expressed
508
as means ± SD (n=8). Bars with different letters (a-g) are significantly different (P >
509
0.05). Analysis of variance was performed by ANOVA procedures, with significant
510
differences between means determined by t- Student’s t-test comparisons.
511
Fig. 4. ABTS•+ scavenging activity of the seven P. guajava guava cultivars. Values are
512
expressed as means ± 95% confidence intervals (n=8) of Trolox equivalent antioxidant
513
capacity (TEAC) (milimoles of Trolox per gram of dry extract)
(avicularin)
(9),
21
isorhamnetin-3-O-glucoside
(10),
Figure 1.
R2
OH
R3
OH HO
HO
O
R4
R OGlc OH
R1 1 : R = OH 2:R=H
OH
O
3 : R1 = OGlc; R2 = R3 = R4 = OH 4 : R1 = OAra; R2 = R3 = R4 = OH 5 : R1 = OXyl; R2 = R3 = R4 = OH 6 : R1 = OGal; R2 = R3 = OH; R4 = H 7 : R1 = OGlc; R2 = R3 = OH; R4 = H 8 : R1 = OAra; R2 = R3 = OH; R4 = H 9 : R1 = OXyl; R2 = R3 = OH; R4 = H 10: R1 = OGlc; R2 = OCH3; R3 = OH; R4 = H 11: R1 = OGal; R2 = OCH3; R3 = OH; R4 = H 13: R1 = R2 = R3 = OH; R4 = H
OH OH HO
O OH
15 : R = OH 18 : R = H
OH
12
COOH O
R OH OH OH
OGlc HO
O OH 16
OH OH HO OH
OH R2 HO OH OGlc
HO O
R1
O HO
HO R2
14 : R1 = OH, R2 = CHO 17 : R1 = H, R 2 = CH2OH
R1 HO
22
19 : R1 = OH; R2 = CH2 20 : R1 = H; R2 = CH3 21 : R1 = OH; R2 = CH3
Figure 2.
A: B: C: D: E: F: G:
Red Guava
White Guava
P. guajava 'Thai Maroon' P. guajava 'Yen 2' P. guajava 'Sardina 1' P. guajava 'Sardina 2' P. guajava 'Barbie Pink' P. guajava 'Homestead' P. guajava 'Sayla'
Pink Guava
White Guava
23
Figure 3.
Cultivar sample
24
Figure 4.
TEAC (umol Trolox/g dry sample)
600
Barbie Pink 'Barbie Pink' Ruby Supreme 'Homestead' Sardina 11' 'Sardina Sardina 22' 'Sardina Sayla 'Sayla' Thai Maroon 'Thai Maroon' Yen 22' 'Yen
500
400
300
200
0
10
20
30
Time (min)
25
40
Table 1. Chemical profile of the identified compounds in the Psidium guajava cultivars1 N o. 1
2
R.T. (min ) 14.3
15.1
UV
[M+H]+ or [M-H](M.F., ppm)
Adduct and fragmental ion exact masses [M-X]+ or [M-X]- (M.F., ppm)
Identification
Detected from species1
Note
520, 274
465.1033 [M]+ (C21 H21O12, 0.0) 463.0875 [M – 2H]– (C21 H19O12, -0.4) 449.1070 [M]+ (C21 H21O11, –2.8) 447.0934[M – 2H]– (C21 H19O11, 1.7) 481.0987 [M + H]+ (C21 H21O13, 1.0) 479.0820 [M – H]– (C21 H19O13, –1.3) 451.0887 [M + H]+ (C20 H19O12, 2.2) 449.0730 [M – H]– (C20 H17O12, 2.2) 451.0878 [M + H]+ (C20 H19O12, 0.2); 449.0709 [M – H]– (C20 H17O12, –2.4) 465.1047 [M + H]+ (C21 H21O12, 3.0) 463.0889 [M – H]– (C21 H19O12, 2.6) 465.1032 [M + H]+ (C21 H21O12, –0.2) 463.0865 [M – H]– (C21 H19O12, –2.6) 435.0930 [M + H]+ (C20 H19O11, 0.7) 433.0779 [M – H]– (C20 H17O11, 1.8) 435.0947 [M + H]+ (C20 H19O11, 4.6) 433.0782 [M – H]– (C20 H17O11, 2.5) 479.1138 [M + H]+ (C22 H23O12, –10.9) 477.1024 [M – H]– (C22 H21O12, –1.9) 479.1185 [M + H]+ (C22 H23O12, –1.0)
303.0496 [M – Glc]+ (C15H11 O7 , –3.0);
Delphinidin 3-O-glucoside (co-injection)
a
detected for first time in this genus
Cyanidin-3-O-glucoside (coinjection)
a
detected for first time in this genus
503.0788 [M + Na]+ (C21 H20O13Na, –2.8); 319.0442 [M + H – Glc]+ (C15H11 O8 , 1.0); 983.1730 [2M + H]+ (C42 H39O26, 2.4) 959.1770 [2M – H]– (C42H39O26, 4.2)
Myricetin-3-O-β-D-glucoside
a-g
reported earlier in P.guajava(Fu, Luo, & Zhang, 2009)
319.0427 [M + H – Glc]+ (C15H11O8, –8.5); 923.1470 [2M + H]+ (C42 H36O24Na, –2.6)
Myricetin-3-O-arabinoside
a and g
detected for first time in this genus
319.0463 [M + H – Glc]+ (C15 H11O8, 2.8); 473.0710 [M + Na]+ (C20H18O12Na, 3.0); 923.1459 [2M + H]+ (C42 H36O24Na, –3.8) 899.1510 [2M – H]- (C40H35O24 , –0.9)
Myricetin-3-O-xyloside
a and g
detected for first time in this genus
487.0873 [M + Na]+ (C21H20O12 Na, –0.8); 303.0514 [M + H – Glc]+ (C15H11O7, –3.0)
Quercetin-3-O-galactoside (Hyperin)
a-c, e-g
reported earlier in P.guajava(Wang, 2010)
487.0842 [M + Na]+ (C21H20O12 Na, –1.8); 303.0504 [M + H – Glc]+ (C15H11 O7 , –0.3); 951.1800 [2M + Na]+ (C42H40O24 Na, –0.7) 509.0944 [M – H + HCOOH]- (C22H21O14, –2.6); 927.1852 [2M – H]- (C42 H39O24, 2.3)
Quercetin-3-O-glucoside (Isoquercitrin) (co-injection)
a-c, e-g
reported earlier P.guajava(Zhigang, 2012)
457.0774 [M + Na]+ (C20H18 O11Na, 5.9); 303.0512 [M + H – Glc]+ (C15H11O7, 2.3); 891.1585 [2M + Na]+ (C40H36O22 Na, –1.2) 479.0815 [M – H + HCOOH]– (C23 H19O13, –2.3); 867.1547 [2M – H]– (C40H35 O22, –8.4)
Quercetin-3-O-α-Larabinoside (Guaijaverin)
a, b, d-g
reported earlier in P.guajava(Wang, 2010)
457.0733 [M + Na]+ (C20 H18O11Na, –3.1); 303.0513 [M + H – Glc]+ (C15H11 O7 , 2.6); 891.1654 [2M + Na]+ (C40H36O22 Na, 3.8) 479.0830 [M – H + HCOOH]– (C23 H19O13, 0.8); 867.1572 [2M – H]– (C40H35O22, –5.5)
Avicularin
a, b, d-g
reported earlier in P.guajava(Wang, 2010)
501.1012 [M + Na]+ (C22H22O12 Na, 0.6); 317.0653 [M + H – Glc]+ (C16H13O7, –2.5)
Isorhamnetin-3-O-glucoside
a, e, f
reported earlier P.guajava(Josline, 2004)
501.1014 [M + Na]+ (C22H22O12 Na, 1.0); 979.2115 [2M + H]+ (C44 H44O24Na, –0.5)
Isorhamnetin-3-Ogalactoside (Cacticin)
a
detected for first time in this genus
516, 279
3
16.0
243, 356
4
17.1
240, 356
5
18.3
240, 356
6
19.0
240, 365
7
19.5
240, 365
8
20.0
250, 360
9
20.6
250, 360
10
21.0
254, 365
11
21.9
254, 364
509.0931 [M – 2H + HCOOH]– (C22H21O14 , –12.8); 481.0945 [M – 2H+H2O]– (C21 H21O13, –7.7) 287.0515 [M – Glc]+ (C15H11 O6 , –3.2); 465.1036 [M – 2H + H2O]– (C21H21 O12, 0.6)
–
899.1478 [2M – H] (C40H35O24, –4.4)
509.0907 [M – H + HCOOH]– (C22 H21O14, –4.7)
26
in
in
12
13
14
15
16
17
18
19
477.1028 [M – H]– (C22 H21O12, -1.0) 265.1414 [M + H]+ (C15 H21O4, –9.1)
24.0
26.1
254, 365
Abscisic acid (co-injection)
a-g
detected for first time in this genus
Quercetin (co-injection)
a-f
reported earlier P.guajava(Josline, 2004; Wang, 2010)
Pinfaensin
a-f
detected for first time in this genus
H]–
687.3669 [M + Na]+ (C36 H56O11Na, –7.4); 503.3315 [M + H – Glc]+ (C30H47O6Na, – 11.5); 485.3217 [M + H – Glc – H2 O]+ (C30H45O5, –10.3); 467.3152 [M + H – Glc – 2H2O]+ (C30H43 O4 , –1.9) 709.3747 [M – H + HCOOH]– (C37 H57O13, –7.3); 699.3534 [M + Cl]– (C36 H56O11Cl, 3.3)
H]+
633.1224 [M + Na]+ (C30H26O14 Na, 0.6); 593.1255 [M + H – H2O]+ (C30H25O13, –6.7)
Gallocatechin-(4α-8)gallocatechol
a, d, e,
413.2097 [M + Na]+ (C19 H34O8Na, –13.1); 211.1705 [M + H – Glc – H2O]+ (C32 H36O18, 3.3) 779.4515 [2M – H]– (C38H67O16, 11.0)
Turpinionosides A
a-g
reported earlier in P.guajava(F. Qa'dan, Petereit, F., Nahrstedt, A. , 2005) detected for first time in this genus
673.3901 [M + Na]+ (C36H58O10 Na, –4.0); 489.3446 [M + H – Glc]+ (C36H49 O5 , –6.9); 471.3456 [M + H – Glc – H2O]+ (C36 H47 O4 , –3.8); 453.3348 [M + H – Glc – 2H2 O]+ (C36 H45O3, –4.6) 695.3983 [M – H + HCOOH]– (C37 H59O12, 5.4); 685.3698 [M + Cl]- (C36 H58O10Cl, 3.3)
Pedunculoside
a-g
detected for first time in this genus
617.1192 [M + Na]+ (C30H26O13 Na, –12.8)
Gallocatechin-(4α-8)catechin
a, b, e, g
485.3227 [M + H – H2 O]+ (C30H45O5, –8.2); 467.3119 [M + H – 2H2O]+ (C30 H43O4, – 9.0); 449.3022 [M + H – 3H2 O]+ (C30H41O3, –7.6) 547.3253 [M – H + HCOOH]– (C31 H47O8, –3.3); 537.2928 [M + Cl]– (C30H46O6 Cl, 3.3)
Guavenoic acid
a-g
487.3428 [M + H – H2 O]+ (C30H47O5, –0.8); 469.3293 [M + H – 2H2O]+ (C30 H45O4, – 5.3); 451.3226 [M + H – 3H2 O]+ (C30H43O3, –3.1) 549.3430 [M – H + HCOOH]– (C31 H49O8, –0.5); 539.3128 [M + Cl]– (C30H48O6 Cl, –2.0)
Madecassic acid
a-g
reported earlier in P.guajava(F. Qa'dan, Petereit, F., Nahrstedt, A. , 2005) reported earlier in P.guajava(Begum, Hassan, & Siddiqui, 2002) detected for first time in this genus
471.3441 [M + H – H2O]+ (C30H47 O4 , –7.0); 453.3354 [M + H – 2H2O]+ (C30H45O3, –3.3)
Asiatic acid
a-f
263.1286 [M-H]– (C15 H19O4, 1.1) 303.0500 [M + H]+ (C15 H11O7, –1.6) 301.0348[M – H]– (C15 H9 O7 , 4.0)
26.4
27.8
265 356
28.6
29.1
30.6
266 356
663.3792 [M – (C36 H55O11, 7.2) 611.1385 [M + (C30 H27O14, –2.6) 609.1244 [M – (C30 H25O14, 0.2) 391.2314 [M + (C19 H35O8, -4.6) 389.1269 [M – (C19 H33O8, 10.5) 651.4086 [M + (C36 H59O10, –3.4)
H]+ H]– H]+
649.3928 [M – H]– (C36 H57O10, –3.7); 595.1452 [M + H]+ (C30 H27O13, –1.8); 593.1314 [M – H]– (C30 H25O13, 3.2) –
38.5
21
45.2
1
309.1338 [M – H + HCOOH]– (C16 H21O6, 2.6); 527.2656 [2M – H]– (C30 H39O8, 2.1) 605.0982 [2M + H]+ (C30 H21O14, 8.4) 347.0381 [M – H + HCOOH]– (C16 H11O9, –6.3); 603.0770 [2M – H]– (C30 H19O14, –0.8)
H]–
35.6
20
287.1259 [M + Na]+ (C15H20O4Na, –7.3); 247.1312 [M + H – H2O]+ (C15H19 O3 , –8.9); 529.2801 [2M + H]+ (C30 H41O8, –12.8); 551.2576 [2M + Na]+ (C30 H40O8Na, –8.2)
501.3185 [M – H] (C30 H45O6, –6.2) 505.3517 [M + H]+ (C30 H49O6, –2.4) 503.3343 [M – H]– (C30 H47O6, –6.0) 489.3591 [M + H]+ (C30 H49O5, 2.2) 503.3343 [M – H]– (C30 H47O6, –6.0)
–
639.1309 [M – H + HCOOH] (C31 H27O15, –6.4)
in
reported earlier in P.guajava(Begum, Hassan, Siddiqui, Shaheen, Ghayur, & Gilani, 2002)
a: P. guajava Thai Maroon; b: P. guajava Yen 2; c: P. guajava Sardina 1; d: P. guajava Sardina 2; e: P. guajava Barbie pink; f: P. guajava Homestead; g: P. guajava Sayla
27
Highlights - Chemical composition of Psidium guajava cultivars assessed by LC-TOF-MS - Antioxidant activity was evaluated by ABTS and DPPH assays - Twenty one compounds were identified - Ten compounds are reported for the first time in this fruit - Antioxidant activity and chemical profile differs depending on the cultivar
28
