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: S0308-8146(14)01300-4
DOI: http://dx.doi.org/10.1016/j.foodchem.2014.08.076
Reference: FOCH 16299

To appear in: Food Chemistry

Received Date: 28 April 2014

Revised Date: 14 August 2014
Accepted Date: 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
a
7 Department of Biological Sciences, Lehman College and The Graduate Center, City
8 University of New York, 250 Bedford Park Boulevard West, Bronx, NY 10468, United
9 States of America
b
10 Instituto de Fermentaciones Industriales, Consejo Superior de Investigaciones
11 Científicas (CSIC), c/Juan de la Cierva 3, 28006 Madrid, Spain
12
13
14
15
16
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19
20
21
22 TITLE RUNNING HEAD: Phenolic profile and antioxidant activities of seven Psidium
23 guajava cultivars

24
†
25 Authors contributed equally to this manuscript.
26

27 * Corresponding author. Tel.: +1 718 960 1105; fax.: +1 718 960 8236. E-mail:

28 Edward.kennelly@lehman.cuny.edu (E.J. Kennelly)

29

30
31

32

1
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

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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). In the popular

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
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
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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.

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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.

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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
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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 (avicularin) (9), isorhamnetin-3-O-glucoside (10),

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)

21
Figure 1.

R2 3 : R1 = OGlc; R2 = R3 = R4 = OH
OH
4 : R1 = OAra; R2 = R3 = R4 = OH
OH R3 5 : R1 = OXyl; R2 = R3 = R4 = OH
6 : R1 = OGal; R2 = R3 = OH; R4 = H
HO O HO 7 : R1 = OGlc; R2 = R3 = OH; R4 = H
R R4
8 : R1 = OAra; R2 = R3 = OH; R4 = H
9 : R1 = OXyl; R2 = R3 = OH; R4 = H
OGlc R1 10: R1 = OGlc; R2 = OCH3; R3 = OH; R4 = H
1 : R = OH
OH 2:R=H OH O 11: R1 = OGal; R2 = OCH3; R3 = OH; R4 = H
13: R1 = R2 = R3 = OH; R4 = H

OH
OH

HO O 15 : R = OH
18 : R = H OH 12
OH COOH
O
R
OH
OH
OH
OGlc
HO O
OH
OH 16
OH
HO
OH OH
R2

HO
OH
OGlc HO
R1 O
O
HO 19 : R1 = OH; R2 = CH2
HO 14 : R1 = OH, R2 = CHO 20 : R1 = H; R2 = CH3
R1
R2 17 : R1 = H, R 2 = CH2OH 21 : R1 = OH; R2 = CH3
HO

22
Figure 2.

A: P. guajava 'Thai Maroon'

Red Guava B: P. guajava 'Yen 2'
C: P. guajava 'Sardina 1'
D: P. guajava 'Sardina 2'
E: P. guajava 'Barbie Pink'
F: P. guajava 'Homestead'
White Guava G: P. guajava 'Sayla'

Pink Guava

White Guava

23
Figure 3.

Cultivar sample

24
Figure 4.

600 Barbie Pink

'Barbie Pink'
Ruby Supreme
'Homestead'
Sardina 11'
'Sardina
Sardina 22'
'Sardina
500 Sayla
'Sayla'
TEAC (umol Trolox/g dry sample)

Thai Maroon
'Thai Maroon'
Yen 22'
'Yen

400

300

200

0 10 20 30 40

Time (min)

25
Table 1. Chemical profile of the identified compounds in the Psidium guajava cultivars1

N R.T. UV [M+H]+ or [M-H]- Adduct and fragmental ion exact masses [M-X]+ or [M-X]- (M.F., ppm) Identification Detected Note
o. (min (M.F., ppm) from species1
)
1 14.3 520, 465.1033 [M]+ 303.0496 [M – Glc]+ (C15H11 O7 , –3.0); Delphinidin 3-O-glucoside a detected for first time in
274 (C21 H21O12, 0.0) (co-injection) this genus
463.0875 [M – 2H]– 509.0931 [M – 2H + HCOOH]– (C22H21O14 , –12.8); 481.0945 [M – 2H+H2O]–
(C21 H19O12, -0.4) (C21 H21O13, –7.7)
2 15.1 516, 449.1070 [M]+ 287.0515 [M – Glc]+ (C15H11 O6 , –3.2); Cyanidin-3-O-glucoside (co- a detected for first time in
279 (C21 H21O11, –2.8) injection) this genus
447.0934[M – 2H]– 465.1036 [M – 2H + H2O]– (C21H21 O12, 0.6)
(C21 H19O11, 1.7)
3 16.0 243, 481.0987 [M + H]+ 503.0788 [M + Na]+ (C21 H20O13Na, –2.8); 319.0442 [M + H – Glc]+ (C15H11 O8 , 1.0); Myricetin-3-O-β-D-glucoside a-g reported earlier in
356 (C21 H21O13, 1.0) 983.1730 [2M + H]+ (C42 H39O26, 2.4) P.guajava(Fu, Luo, &
479.0820 [M – H]– 959.1770 [2M – H]– (C42H39O26, 4.2) Zhang, 2009)
(C21 H19O13, –1.3)
4 17.1 240, 451.0887 [M + H]+ 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
356 (C20 H19O12, 2.2) this genus
449.0730 [M – H]– –
899.1478 [2M – H] (C40H35O24, –4.4)
(C20 H17O12, 2.2)
5 18.3 240, 451.0878 [M + H]+ 319.0463 [M + H – Glc]+ (C15 H11O8, 2.8); 473.0710 [M + Na]+ (C20H18O12Na, 3.0); Myricetin-3-O-xyloside a and g detected for first time in
356 (C20 H19O12, 0.2); 923.1459 [2M + H]+ (C42 H36O24Na, –3.8) this genus
449.0709 [M – H]– 899.1510 [2M – H]- (C40H35O24 , –0.9)
(C20 H17O12, –2.4)
6 19.0 240, 465.1047 [M + H]+ 487.0873 [M + Na]+ (C21H20O12 Na, –0.8); 303.0514 [M + H – Glc]+ (C15H11O7, –3.0) Quercetin-3-O-galactoside a-c, e-g reported earlier in
365 (C21 H21O12, 3.0) (Hyperin) P.guajava(Wang, 2010)
463.0889 [M – H]– 509.0907 [M – H + HCOOH]– (C22 H21O14, –4.7)
(C21 H19O12, 2.6)
7 19.5 240, 465.1032 [M + H]+ 487.0842 [M + Na]+ (C21H20O12 Na, –1.8); 303.0504 [M + H – Glc]+ (C15H11 O7 , –0.3); Quercetin-3-O-glucoside a-c, e-g reported earlier in
365 (C21 H21O12, –0.2) 951.1800 [2M + Na]+ (C42H40O24 Na, –0.7) (Isoquercitrin) P.guajava(Zhigang,
463.0865 [M – H]– 509.0944 [M – H + HCOOH]- (C22H21O14, –2.6); 927.1852 [2M – H]- (C42 H39O24, 2.3) (co-injection) 2012)
(C21 H19O12, –2.6)
8 20.0 250, 435.0930 [M + H]+ 457.0774 [M + Na]+ (C20H18 O11Na, 5.9); 303.0512 [M + H – Glc]+ (C15H11O7, 2.3); Quercetin-3-O-α-L- a, b, d-g reported earlier in
360 (C20 H19O11, 0.7) 891.1585 [2M + Na]+ (C40H36O22 Na, –1.2) arabinoside P.guajava(Wang, 2010)
433.0779 [M – H]– 479.0815 [M – H + HCOOH]– (C23 H19O13, –2.3); 867.1547 [2M – H]– (C40H35 O22, –8.4) (Guaijaverin)
(C20 H17O11, 1.8)
9 20.6 250, 435.0947 [M + H]+ 457.0733 [M + Na]+ (C20 H18O11Na, –3.1); 303.0513 [M + H – Glc]+ (C15H11 O7 , 2.6); Avicularin a, b, d-g reported earlier in
360 (C20 H19O11, 4.6) 891.1654 [2M + Na]+ (C40H36O22 Na, 3.8) P.guajava(Wang, 2010)
433.0782 [M – H]– 479.0830 [M – H + HCOOH]– (C23 H19O13, 0.8); 867.1572 [2M – H]– (C40H35O22, –5.5)
(C20 H17O11, 2.5)
10 21.0 254, 479.1138 [M + H]+ 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 in
365 (C22 H23O12, –10.9) P.guajava(Josline,
477.1024 [M – H]– 2004)
(C22 H21O12, –1.9)
11 21.9 254, 479.1185 [M + H]+ 501.1014 [M + Na]+ (C22H22O12 Na, 1.0); 979.2115 [2M + H]+ (C44 H44O24Na, –0.5) Isorhamnetin-3-O- a detected for first time in
364 (C22 H23O12, –1.0) galactoside (Cacticin) this genus

26
 477.1028 [M – H]–
(C22 H21O12, -1.0)
12 24.0 265.1414 [M + H]+ 287.1259 [M + Na]+ (C15H20O4Na, –7.3); 247.1312 [M + H – H2O]+ (C15H19 O3 , –8.9); Abscisic acid (co-injection) a-g detected for first time in
(C15 H21O4, –9.1) 529.2801 [2M + H]+ (C30 H41O8, –12.8); 551.2576 [2M + Na]+ (C30 H40O8Na, –8.2) this genus
263.1286 [M-H]– 309.1338 [M – H + HCOOH]– (C16 H21O6, 2.6); 527.2656 [2M – H]– (C30 H39O8, 2.1)
(C15 H19O4, 1.1)
13 26.1 254, 303.0500 [M + H]+ 605.0982 [2M + H]+ (C30 H21O14, 8.4) Quercetin (co-injection) a-f reported earlier in
365 (C15 H11O7, –1.6) P.guajava(Josline,
301.0348[M – H]– 347.0381 [M – H + HCOOH]– (C16 H11O9, –6.3); 603.0770 [2M – H]– (C30 H19O14, –0.8) 2004; Wang, 2010)
(C15 H9 O7 , 4.0)
14 26.4 687.3669 [M + Na]+ (C36 H56O11Na, –7.4); 503.3315 [M + H – Glc]+ (C30H47O6Na, – Pinfaensin a-f detected for first time in
11.5); 485.3217 [M + H – Glc – H2 O]+ (C30H45O5, –10.3); 467.3152 [M + H – Glc – this genus
2H2O]+ (C30H43 O4 , –1.9)
663.3792 [M – H]– 709.3747 [M – H + HCOOH]– (C37 H57O13, –7.3); 699.3534 [M + Cl]– (C36 H56O11Cl, 3.3)
(C36 H55O11, 7.2)
15 27.8 265 611.1385 [M + H]+ 633.1224 [M + Na]+ (C30H26O14 Na, 0.6); 593.1255 [M + H – H2O]+ (C30H25O13, –6.7) Gallocatechin-(4α-8)- a, d, e, reported earlier in
356 (C30 H27O14, –2.6) gallocatechol P.guajava(F. Qa'dan,
609.1244 [M – H]– Petereit, F., Nahrstedt,
(C30 H25O14, 0.2) A. , 2005)
16 28.6 391.2314 [M + H]+ 413.2097 [M + Na]+ (C19 H34O8Na, –13.1); 211.1705 [M + H – Glc – H2O]+ (C32 H36O18, Turpinionosides A a-g detected for first time in
(C19 H35O8, -4.6) 3.3) this genus
389.1269 [M – H]– 779.4515 [2M – H]– (C38H67O16, 11.0)
(C19 H33O8, 10.5)
17 29.1 651.4086 [M + H]+ 673.3901 [M + Na]+ (C36H58O10 Na, –4.0); 489.3446 [M + H – Glc]+ (C36H49 O5 , –6.9); Pedunculoside a-g detected for first time in
(C36 H59O10, –3.4) 471.3456 [M + H – Glc – H2O]+ (C36 H47 O4 , –3.8); 453.3348 [M + H – Glc – 2H2 O]+ this genus
(C36 H45O3, –4.6)
649.3928 [M – H]– 695.3983 [M – H + HCOOH]– (C37 H59O12, 5.4); 685.3698 [M + Cl]- (C36 H58O10Cl, 3.3)
(C36 H57O10, –3.7);
18 30.6 266 595.1452 [M + H]+ 617.1192 [M + Na]+ (C30H26O13 Na, –12.8) Gallocatechin-(4α-8)- a, b, e, g reported earlier in
356 (C30 H27O13, –1.8); catechin P.guajava(F. Qa'dan,
593.1314 [M – H]– –
639.1309 [M – H + HCOOH] (C31 H27O15, –6.4) Petereit, F., Nahrstedt,
(C30 H25O13, 3.2) A. , 2005)
19 35.6 485.3227 [M + H – H2 O]+ (C30H45O5, –8.2); 467.3119 [M + H – 2H2O]+ (C30 H43O4, – Guavenoic acid a-g reported earlier in
9.0); 449.3022 [M + H – 3H2 O]+ (C30H41O3, –7.6) P.guajava(Begum,
–
501.3185 [M – H] 547.3253 [M – H + HCOOH]– (C31 H47O8, –3.3); 537.2928 [M + Cl]– (C30H46O6 Cl, 3.3) Hassan, & Siddiqui,
(C30 H45O6, –6.2) 2002)
20 38.5 505.3517 [M + H]+ 487.3428 [M + H – H2 O]+ (C30H47O5, –0.8); 469.3293 [M + H – 2H2O]+ (C30 H45O4, – Madecassic acid a-g detected for first time in
(C30 H49O6, –2.4) 5.3); 451.3226 [M + H – 3H2 O]+ (C30H43O3, –3.1) this genus
503.3343 [M – H]– 549.3430 [M – H + HCOOH]– (C31 H49O8, –0.5); 539.3128 [M + Cl]– (C30H48O6 Cl, –2.0)
(C30 H47O6, –6.0)
21 45.2 489.3591 [M + H]+ 471.3441 [M + H – H2O]+ (C30H47 O4 , –7.0); 453.3354 [M + H – 2H2O]+ (C30H45O3, –3.3) Asiatic acid a-f reported earlier in
(C30 H49O5, 2.2) P.guajava(Begum,
503.3343 [M – H]– Hassan, Siddiqui,
(C30 H47O6, –6.0) Shaheen, Ghayur, &
Gilani, 2002)
1
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