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Characterization of Free, Esterified and Bound phenolics in Custard apple (Annona squamosa L) fruit pulp by UPLC –ESI-MS/MS Revathy Baskaran, Dilshad Pullencheri, S.Rajarathnam PII: DOI: Reference:
S0963-9969(16)30042-4 doi: 10.1016/j.foodres.2016.02.001 FRIN 6167
To appear in:
Food Research International
Received date: Revised date: Accepted date:
27 November 2015 1 February 2016 2 February 2016
Please cite this article as: Baskaran, R., Pullencheri, D. & S.Rajarathnam, Characterization of Free, Esterified and Bound phenolics in Custard apple (Annona squamosa L) fruit pulp by UPLC –ESI-MS/MS, Food Research International (2016), doi: 10.1016/j.foodres.2016.02.001
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ACCEPTED MANUSCRIPT Characterization of Free, Esterified and Bound phenolics in Custard apple
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(Annona squamosa L) fruit pulp by UPLC –ESI-MS/MS
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Department of Fruit and Vegetable Technology, Central Food Technological Research Institute, Mysore – 570 020,
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Waters India Pvt. Ltd., Bangalore, Karnataka, INDIA
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Karnataka, INDIA
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Revathy Baskaran1*, Dilshad Pullencheri2 and S.Rajarathnam1
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*Corresponding author: Revathy Baskaran
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email:[email protected] Tel.: + 91 821-2515653 Fax: + 91 821 2517233
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Abstract:
Ultra High-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC-ESI-MS) was used
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to identify the free, bound and esterified phenolic acids in the extracts of Custard Apple (Annona squamosa L). In total around phenolic
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16, 15 and 13 free, bound and esterified phenolic compounds respectively were identified. Among these about 5 compounds like quinic acid, gallocatechin,
gallocatechin gallate, caffeoylhexoside, dihydroxyquercetin have and been
reported for the first time in A.squamosa. Also, compounds like 4-(β-D-glucopyranosyloxy) benzoic acid, procyanidin B1, procyanidin C1 in free form, 7 hydroxycoumarin 7 glucoside (skimmin), dihydroquercetin, xanthotaxol acetate, decycloxybenzoic acid in bound extract and Caffeoyl hexoside in esterified form have been tentatively identified. Apart from 2
ACCEPTED MANUSCRIPT phenolic compounds few organic acids like malic, citric, citramalic, adipic and acotinic acid have been found in the custard
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apple extracts. This study provides a newer insight into the phenolic profile of custard apple and their characterization by
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UPLC-ESI-MS/MS.
1.0 Introduction:
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Keywords: Annona squamosa, phenolic compounds, UPLC-ESI-MS, fragmentation
Custard apple or sugar apple ( Annona squamosa L) commonly known as Sitaphal in India, is a multiple fruit, consisting of many fruitlets each loaded with a seed having a shiny testa. Custard apple is a table fruit valued for its nutritional benefit and exotic taste. The fruit has a creamish white pulp and a gritty texture (Shravanthi et al., 2014). Apart from the 3
ACCEPTED MANUSCRIPT nutrients like carbohydrates, protein, vitamins etc., they are known to be contain secondary metabolites like phenolic
are secondary metabolites which are widely found in plant and plant derived foods. The
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Phenolic compounds
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compounds, alkaloids, saponins.
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predominant phenolic acids are hydroxylated derivatives of benzoic acid and cinnamic acid. Phenolic acids are mostly present
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in the plants in the bound form.
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Phenolics have received considerable attention as potentially protective factors against cancer and heart diseases mainly
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because of their potent antioxidative properties and their ubiquity in a range of commonly consumed foods of plant origin. Procyanidins are the most common classes of proanthocyanidins which are chains of catechin, epicatechin, and their Gallic acid esters. They are partly responsible for sensory properties such as bitterness, astringency, etc. They consist of
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oligomers and polymers of catechin units most frequently linked to either C4→C8 or C4→C6. Mostly in fruits they are
2001).
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present as monomers or in more complex polymeric forms when the monomers condense (Jiang et al., 2015; Lazarus et al.,
Several analytical methods are available for detection of phenolics. Most of the times, these phenolic compounds are analyzed by High Performance Liquid Chromatography (HPLC) Giusti et al., 2007; Vagiri et al., 2012, coupled with diode 4
ACCEPTED MANUSCRIPT array detector and mass spectrometer Revilla et al., 1999. Also, gas chromatography and capillary electrophoresis are used for
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this purpose (Harborne, 1998). To get structural information about the eluted compounds, these methods are coupled with
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Mass Spectrometry.
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Liquid chromatography Mass Spectrometry (LCMS/MS) is the most preferred method to determine plant metabolites
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because of their sensitivity and selectivity through mass fragmentation and enable structural identification. LC-MS analysis of
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phenolics is either done in APCI or ESI mode.
Studies on the antioxidant activity of A. muricata and A. crassiflora fruit extracts is reported (Singh et al., 2014;
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Roesler et al., 2007), while the total phenolic composition and antioxidant activity of A. cherimola
and A .diversifolia fruit
extracts also has been carried out Loizzo et al., 2012; Julian-Loaeza et al., 2010). Although there are reports on total phenolic
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esterified phenolics.
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composition and functional benefits of A. squamosa fruits, there are no studies that deals with the spectrum of free, bound and
Although A. squamosa is a good source of phenolics, there are very few studies on the phenolic profile. Few researchers have reported the presence of flavanols like catechin, epicatechin and procyanidins in custard apple pulp by LCMS (De-Pascual-Teresa et al., 2000), there are no reports on the profile of free, bound and esterified phenolic acids profile of
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ACCEPTED MANUSCRIPT Custard apple. Therefore, to fingerprint phenolic profile in A.squamosa, an attempt was made to characterize their profile
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along with chemical structure using UPLC-MS (ESI –ve) method.
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The aim of the present work was to fractionate the phenolic compounds of Custard apple pulp into free, bound and
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esterified forms and determine the profile of phenolic acids after hydrolysis by Ultra Performance Liquid Chromatography
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coupled with Electro Spray Ionization tandem mass spectrometry ( UPLC-ESI MS/MS).
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2.0 Materials and Methods:
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2.1 Samples:
The mature custard apple fruits were procured from local market. The fruits were allowed to ripen at room temperature (27-
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32°C). To separate the seeds from the fleshy portion, the scooped pulp along with the seeds were passed through a fruit pulper
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(M/s APV India, Calcutta, sieve size 1/80cm) to get a fine pulp. 2.1.1 Standards and Chemicals: Gallic acid, Epicatechin, Catechin, Protocatechuic acid, Caffeic acid, p-Coumaric acid, Sinapic acid, Epigallocatechin and Ferulic acids were purchased from Sigma- Aldrich, Bangalore. HPLC grade acetonitrile, methanol, and acetic acid were obtained from Company -E-pure water, Bangalore.
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ACCEPTED MANUSCRIPT 2.2 Extraction of free, soluble ester and insoluble bound phenolics:
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The free, soluble ester and insoluble bound phenolics were extracted according to previously reported method (Krygier et al.,
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1982), with slight modifications (Fig 1). Annona fruit fresh pulp (100 gms) was extracted with 70% ethanol ( 50ml x 3 times).
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The pooled supernatants were flash evaporated to remove all the ethanol, reduced the pH to 2.0 using 4 M HCl and phase
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separated with ethyl acetate ( ~ 6 times). The fraction of ethyl acetate soluble compounds was collected and flash evaporated to
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dryness and redissolved in HPLC grade methanol. The phenolic acids so extracted were labeled as free phenolics. The supernatant after the removal of ethyl acetate fraction which contains the esterified or free soluble conjugated phenolic
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compounds were treated with 2M NaOH for 2 hours at room temperature under nitrogen atmosphere. The resultant hydrolysate was then acidified to pH 2.0 using 4 M HCl and then extracted thrice with ethyl acetate. The ethyl acetate extracts were pooled
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and evaporated to dryness at 35°C under vacuum. The phenolics were the ones liberated from their esters and were labeled as
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esterified phenolics. The residue left over after extraction of fruit pulp with 70% ethanol initially, was treated with 2M NaOH for 2 hours at room temperature under nitrogen atmosphere. The samples were then acidified to pH 2.0 using 4M HCl followed by extraction with ethyl acetate ( ~ 6 times). The ethyl acetate fractions were pooled and evaporated to dryness under vacuum at 35°C. The phenolics so extracted were labeled as bound phenolics. All the free, esterified and bound phenolics were analyzed by UPLCMS/MS. 7
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2.2.1 UPLCMS/MS:
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Chromatographic analyzes of free, esterified and bound phenolics were performed on a Waters Acquity UPLC system
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with Waters acquity UPLC PDA detector for data collection. Phenolics were separated using Waters Atlantis T3 50 x 4.6 id, 3
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µm column at 30°C using a 5µl injection volume. The binary mobile phase consisted of solvent A composed of 0.2% acetic
system:
0.00
98
4.50
78
6.50
78
7.50
10
8.50
%B
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%A
Curves
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Initial
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Time (min)
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acid in water and solvent B, consisted of 0.2% acetic acid in acetonitrile. Separations were performed by the following gradient
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90
6
10
90
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9.50
98
02
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11.00
98
02
6
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ACCEPTED MANUSCRIPT Data were collected using the UV detector at 280 and 320nm. UPLC MS/MS analysis of the phenolics were performed using a
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Waters Synapt® G2 High Definition MS™ system interfaced through an electrospray interface (ESI- negative mode). LC/MS
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was optimized for annona phenolics with a capillary voltage of 2.50 KV, a cone voltage of 30V, source temperature of 140°C
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and desolvation gas temperature of 400°C. The cone and desolvation gas flows were 50 and 1100 L/hr. Nitrogen and argon were used as the cone and collision gasses. MS data were collected from 100 to 2500 m/z and processed with MassLynx™ 4.1
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Software with MassFragment™. The data were acquired using reference lock mass through Lock-Spray interface. Leucine
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enkephalin was used as the reference compound with M-H equal to 554.2615 and was introduced along with the LC stream
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for accurate mass calibration. Quantification of phenolics was done using phenolic standards. 3.0 Results and Discussion:
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Analysis of the free and bound phenolics of custard apple fruit pulp was performed by UPLC –DAD-ESI-MS/MS along with
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their retention times, detected mass, molecular formula and MS/MS fragment ions. Phenolic acids in custard apple pulp were monitored by a diode array detector set at 3 wavelengths 210nm, 280nm, and 320nm. The development of UPLC–MS/MS method was used for characterization of custard apple fruit phenolics for the first time. The partial characterization of free, bound and esterified phenolics in custard apple pulp is shown in Tables 1 to 3. The
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ACCEPTED MANUSCRIPT identified compounds can be classified into 3 groups namely, derivatives of hydroxycinnamic acid, hydroxybenzoic acid, and
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flavanols. Few non- phenolic compounds were also detected.
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Characterization of the phenolics, their derivatives and other compounds detected:
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The analytical method used in the present study allowed the identification of ~ 23 compounds. These included few of the previously reported compounds like flavonols (De-Pascual-Teresa et al., 2000), plus a number of new phenolic compounds and
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a few non-phenolic compounds. Peaks have been numbered in accordance with their retention times in the respective phenolic
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extracts.
Polyphenols belonging to hydroxybenzoic, hydroxycinnamic acid derivatives and flavanols were found in all the fractions extracted. Five of them namely, gallic acid, catechin, epicatechin, protocatechuic acid, caffeic acid, p-coumaric acid and
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sinapic acid were identified by comparing their retention times and characteristic MS spectral data with those of the reference
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standards (Tables 1). Accurate mass and fragmentation pattern confirmed their structural identification. The Total Ion Current (TIC) chromatogram of custard apple pulp extracts is shown in Figures 2 A,B and C. The major peaks indicated have been assigned in Tables 1 and Tables 2A-2C Identification of phenolic compounds was carried out by comparing retention times and masses with those of authentic standards. The compounds for which no standards were
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ACCEPTED MANUSCRIPT available, identification was based on accurate mass measurement of the pseudomolecular [M-H]- ions and CID fragmentation.
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Further, results of the accurate mass matched the elemental composition of the compounds analyzed.
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In the category of hydroxycinnamic acid derivatives, 3 different phenolic acids like p-coumaric, sinapic and caffeic acid were
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found to occur in all the extracts of custard apple pulp. Similarly, free, bound and esterified hydroxybenzoic acid derivatives like gallic acid and protocatechuic acid were also detected in all fractions i.e., free, bound and esterified. Further, flavanols
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procyanidin B2 were detected in only the bound fraction.
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such as catechin and epicatechin were identified in both free and bound form, whereas flavanols like epigallocatechin and
3.1Characterization of possible free phenolic compounds isolated from Custard apple fruit pulp (Table 1A): Seven phenolic compounds have been identified by comparing their retention times and mass spectra with respective
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standards. The ESI-MS signals of peaks 1,2,4 and 6 at m/z 169.01, m/z 153.01, m/z 179.03 and 163.04 were identified as gallic
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acid, protocatechuic acid, caffeic acid and p-coumaric acid respectively in comparison with their retention time and MS spectral data with those of authentic standards. Accurate mass measurement further confirmed their elemental composition (Table 1). MS/MS fragmentation of gallic acid, protocatechuic acid, caffeic acid and p-coumaric acid produced ions at m/z 125.02, m/z 109.02, m/z 135.04 and 119.05 respectively due to the loss of a CO2 molecule from their respective precursor ions. The fragmentation pattern has been found to be characteristic for hydroxybenzoic acid derivatives. In general, deprotonated 11
ACCEPTED MANUSCRIPT phenolic acids [M-H] produce a typical fragmentation pattern after collision induced dissociation (CID) by the loss of a CO 2
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(44u) from the carboxylic acid group, providing an anion of [ M-HCOO] Parejo et al., 2004; Hossain et al., 2010. Gallic acid
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and protocatechuic acid have been reported to be present in the wine prepared from custard apple fruits (Jagtap and Bapat,
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2015).
Peak 3 and 5 of deprotonated ion [M-H]- at m/z 289, were identified as catechin and epicatechin. These two being
resulted probably due to loss of a CO2
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peaks, the fragmentation at m/z 123(Hvattum, 2002)
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stereoisomers, gave same fragment ions since mass spectrometry cannot distinguish between stereoisomers. For both the molecule from B ring
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methylation. Also, fragment ions at m/z 245, 205 and 179, indicated that the compound was catechin/ epicatechin. The ion m/z 245 could be due to loss of CO2 group or divalent CH2-CHOH- group. Ion at m/z 205 might be due to the loss of A-ring and fragment m/z 179 due to the loss of water molecule (Savic et al., 2014). Also, the identification of catechin and epicatechin
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was confirmed by comparing the retention time and the spectrum of product ions of standard catechin and epicatechin.
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Peak 7, with m/z 223, produced a fragment ion at m/z 208 [M-H-15], due to loss of methyl group, indicating that the compound could be sinapic acid. Further, all the compounds from 1 to 7 were confirmed by comparing them with the MS and MS/MS spectra of respective standards. Apart from the above 7 phenolic acids, 9 peaks corresponding to phenolics and other organic compounds were tentatively identified based on their mass spectra and related data from the literature (Table 2A). 12
ACCEPTED MANUSCRIPT In addition, there were few dicarboxylic acids like malic, citramalic, acotinic and adipic acid, which were detected along with
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the phenolic acids (Table 2). Peak 8 with m/z at 133 which showed a major fragment at m/z 115 [M-H-18], due to the loss of
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water molecule and was identified as malic acid (Lay-Keow et al., 2004). Peak 9, identified as trans-acotinic acid, produced the
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base peak at m/z 111 [M-H-44-18], due to loss of a CO2 and H2O molecule. Similarly, peak 10 was identified as citric acid. A major fragment at m/z 111 [M-H-CO2.2H2O] corresponding to an additional loss of a water molecule was observed.
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Peak 11 was designated as citramalic acid ( m/z 147) due to the fragments at m/z 115 [M-H-OH-CH3] and 87 [M-H-COOH-
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[M-H-H2O-CO2], 101 [M-H-CH3-CO2] and 143 [M-H-H2O].
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CH3]. Peak 12 of free phenolics with the mass at 161 [M-H]- was identified as hydroxyadipic acid. The MS2 fragments were 99
Peak 13 of free phenolics with the ion at m/z 191 [M-H]- and fragmentations ions at 101 (M-CO2-H2O-CO), 115 (M-CO2H2O-CH2) and 129 (M-CO2-H2O) was characterized as quinic acid , Al-Rawahi et al., 2014 .
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Peak 14 (free phenolics), with m/z at 299 ([M-H]- and major fragment ion at m/z at 137 (M-H-162) with the loss of hexose
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moiety was identified as 4-(β-D-glucopyranosyloxy) benzoic acid. Peak 15 (m/z 577), was identified as procyanidin B2, with fragment ions at 425 (M-H-152), obtained probably due to rearrangement of Retro-Diels-Alder (RDA) heterocyclic ring (De Pascual-Teresa and Rivas-Gonzalo, 2003). Also, it gave a fragment ion at m/z at 407 (M-H-170) due to both RDA rearrangement and loss of water molecule. The fragment at m/z 451
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ACCEPTED MANUSCRIPT (M-H-126) may be because of cleavages between C4-C5 and O-C2 of one pyran ring leading to loss of ‘A’ ring and fragment at
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m/z at 289 (M-H-289) formed from the cleavage of the C-C linkage between 2 catechin units.
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Peak 16, with a mass of 865 (M-H) and empirical formula C45H37O18, was designated as type A procyanidin trimer, with
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typical fragmentation ions at m/z at 577 which may be due to cleavage of the type B interflavan bond and loss of neutral fragment (-288). Also, other fragments formed were similar to the fragmentation pattern earlier reported (Passos et al., 2007),
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where in the fragment 713 (- 152) is due to RDA fragmentation of m/z at 865, fragment 425 is due to RDA fragmentation of
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m/z 577. Further, the fragment ion at m/z 739 (-126) has been attributed to heterocyclic ring fission (HRF) Gu et al., 2003.
3.2 Characterization of Bound phenolics from custard apple fruit pulp (Table 2A): In the analysis of bound fraction, 8 compounds were detected and corresponded with the reference phenolic standards with
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respect to retention time and mass spectra. It was found that phenolic compounds (peak 3,4,8,11 and 13 – Table 2A),
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corresponding to gallic acid, protocatechuic acid, caffeic acid, p-coumaric and sinapic acid were also found in the bound form. Similarly, the flavanols catechin, epicatechin and epigallocatechin were detected. Peak no. 10 of bound phenolics at m/z at 305 (M-H) with fragment ions at m/z at 125 and at m/z 179, was identified as epigallocatechin (Sun et al., 2007).
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ACCEPTED MANUSCRIPT Based on MS spectra and by comparing with the data in the literature, 7 peaks were identified (Table 2B). Peak 1 and 2 with
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m/z at 133 and 191 were identified as malic and citric acid respectively. They exhibited similar fragmentation pattern as
due to the loss of 2 hydroxyl groups was named as 7
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Peak 5 at m/z at 323 with a major fragment ion m/z at 289
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observed in the free phenolic fraction.
hydroxycoumarin 7 glucoside (skimmin), which is also known to be a phenolic compound present in few species of the genus
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Berberis (Vereskovskii and Shapiro, 1986). Peak 7 with [M-H] at m/z 303 gave a characteristic fragment ion m/z at 151, due
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to the cleavage of the C ring and another fragment ion m/z at 125 (A-CO), indicative of the compound being dihydroquercetin
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(Yi-Long et al., 2015).
Peak 9 was identified as the flavanol epicatechin, as found in the free form. Whereas Peak 10, with m/z 305 [M-H] was designated as epigallocatechin. This flavanol was found in bound fraction only. The fragments m/z at 137 [M-168] may be due
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epigallocatechin (Callemien and Collin, 2008).
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to RDA and m/z at 125 [M-180] by HRF. Further, the fragment m/z at 179 due to loss of 126 Da were all typical of
Peak 12 m/z at 243 [M-H] which gave a major fragment ion m/z at 199 [M-44], might be due to the loss of a CO2 molecule and has been tentatively looked upon as xanthotoxol acetate. Xanthotoxol has been found to be present in fruits like Aegle marmelos L (Lim, 2012).
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ACCEPTED MANUSCRIPT Peak no. 15 with m/z at 577 [M-H] was identified as procyanidin B2 and it gave similar fragmentation pattern as in the free
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form.
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Peak 16 with m/z at 277 that gave a fragment ion at m/z 205 due to loss of CH3(CH2)2COOH has been tentatively named as
3.3 Phenolic compounds from soluble esters (Table 1 and 2C):
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decycloxybenzoic acid.
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The esterified phenolic acids were also identified using reference standards, MS fragmentation pattern, and literature. In the
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case of esterified phenolics, peaks 3, 5, 8, 11 and 12 were identified as gallic, protocatechuic, caffeic, p-coumaric and sinapic
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acid and had similar fragmentation pattern as found in free and bound phenolics. Similarly, Peak 7 and 9 were identified as flavanols catechin and epicatechin. Mass spectra of peak 6 displayed a parent ion at m/z 341 and two fragment ions, one at m/z 179 for caffeic acid by the loss of a hexose moiety and the other at m/z at 135 for decarboxylated caffeic acid after the loss
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of both hexose and CO2, indicating that the compound may be caffeoyl hexoside. Peak 10 of esterified phenolics with the mass
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at 457 was designated as epigallocatechin gallate, had major ions at m/z 169 and m/z 305. The m/z 169 ion must have resulted from the loss of an intact gallic acid anion and the ion at m/z 305 may be due to neutral loss of gallic acid. A 14 Da shift of m/z169 to m/z 183 is characteristic of methylated gallic acid moiety. The absence of this ion i.e., m/z 183, indicates 4’ position methylation, because the presence of a methyl group at the 4’ position of gallic acid would have blocked the formation of m/z 183 ion. 16
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Peak no. 13 of esterified phenolics with m/z at 193 [M-H]- was identified as ferulic acid by comparison with the reference
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standard. Further MS2 fragmentation revealed fragment ion at m/z 178 [M-H-CH3], due to the loss of methyl group (Sanz et
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al., 2012).
Compounds 1,2 and 4 were identified as malic, citric and quinic acid, as the mass and fragmentation were similar to that found
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in the free and bound extracts.
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It is well reported that same phenolic acids can occur in free, esterified and bound forms, but their relative abundance might be
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different in various forms (Djurdjevic et al., 2005; Nicoletti et al., 2013). Also in wine prepared from custard apple, Jagpat et al., 2015, have reported the presence of 3 hydroxybenzoic acids like gallic, protocatechuic and gentisic acid and 2 hydroxycinnamic acids like caffeic and p-coumaric acid by reverse phase HPLC and
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have identified the compounds with reference standards.
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Similar to our results, De Pascual –Teresa et al., 2000, have also reported the presence of catechin, epicatechin, procyanidin dimer B2 and procyanidin trimer C1 in custard apple pulp. Apart from these they have also reported procyanidin dimmers B1, B3, B4, B5, B7 and trimer EEC in custard apple pulp, which were not detected in our samples. The fruit juice custard apple was found to contain phenolic acids like caffeic, ferulic, p-coumaric and sinapic acid in free form (Lee et al., 2003).
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ACCEPTED MANUSCRIPT 4.0 Conclusion:
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Using UPLC-ESI-MS, around 16 free, 15 bound and 13 esterified phenolic acids respectively have been identified in fresh
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custard apple pulp. Among these few phenolic acids like gallic, protocatechuic , sinapic, ferulic, p-coumaric acid, catechin and
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epicatechin have already been reported, while phenolic acids like quinic acid, gallocatechin, gallocatechin gallate,
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caffeoylhexoside, dihydroxyquercetin etc. are reported for the first time in custard apple. The data provided is one of its first
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kind which gives an idea on the phenolics present in the free, bound and esterified form in custard apple, which in turn can contribute to the dietary polyphenols intake.
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5.0 Acknowledgement:
6.0 References:
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The authors wish to thank Director, CSIR-CFTRI for his constant support and encouragement.
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ACCEPTED MANUSCRIPT 14. Krygier, K., Sosulski, F., & Hogge, L.(1982). Free, esterified, and insoluble-bound phenolic acids. 1. Extraction
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and purification procedure. Journal of Agricultural Food Chemistry, 30, 330.
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15. Lay-Keow ng., Lafontaine, P., & Vanier, M. (2004). Characterization of Cigarette Tobacco by Direct Electrospray
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Ionization-Ion Trap Mass Spectrometry (ESI-ITMS) Analysis of the Aqueous Extracts: A Novel and Simple Approach
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Journal of Agricultural Food Chemistry, 52, 7251-7257.
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16. Lazarus, S.A., Hammerstone, J.F., Adamson, G.E., & Schmitz, H.H. (2001). High performance liquid chromatography/mass spectrometry analysis of proanthocyanidins in food and beverages In: Flavonoids and other
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polyphenols Ed. Lester Packer volume 335: 46, Academic Press, USA.. 17. Lee, P.R., Tan, R.M., Yu, B., Curran, P., & Liu, S.Q. (2003). Sugars, organic acids and phenolic acids of exotic
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seasonable tropical fruits. Nutrition Food Science, 43, 267-276.
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18. Lim, T.K. (2012). Edible Medicinal and Non-Medicinal Plants; Vol. 4, Fruits. Chapter Aegle Marmelos L, pp 594, DOI 10.1007/978-94-007-4053.2-70© Springer Science of Business Media B.V. 19. Loizzo, M.R., Tundis, R., Bonesi, M., Menichini, F., Mastellone, V., Avallone, L., & Menichini, F.(2012). Radical scavenging, antioxidant and metal chelating activities of Annona cherimola Mill. (cherimoya) peel and pulp in relation to their total phenolic and total flavonoid contents. Journal of Food Composition and Analysis, 25, 179-184. 21
ACCEPTED MANUSCRIPT D’Egidio, M.G., & Corradini,D. (2013). Identification and
20. Nicoletti, I., Martini, D., De Rossi,A., Taddei, F.,
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Quantification of Soluble Free, Soluble Conjugated, and Insoluble Bound Phenolic Acids in Durum Wheat (Triticum
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turgidum L. var. durum) and Derived Products by RP-HPLC on a Semimicro Separation Scale. Journal of Agricultural
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Food Chemistry, 61, 11800-11807.
21. Parejo, I., Jauregui, O., Rabaneda, S., Viladomat, F., Bastida, J., & Codina,C.(2004). Separation and characterization
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of phenolic compounds in Fennel (Foeniculum vulgare) using liquid chromatography-negative electrospray ionization
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tandem mass spectrometry. Journal of Agricultural Food Chemistry, 52, 3679-3687.
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22. Passos, C.P., Susana, M.C., Rosario, M.D., Pedro, D., Carlos, M.S., & Manuel, A.C. (2007). Evidence for galloylated type-A procyanidins in grape seeds. Food Chemistry, 105,1457-1467. 23. Revilla, I., Magarino, S.P., Gonzalez-SanJose, M.L., & Beltran,S. Identification of anthocyanin derivatives in grape
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Chromatography A9, 847,83-90
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skin extracts and red wines by liquid chromatography with diode array and mass spectrometric detection. Journal of
24. Roesler, R., Catharino, R.R., & Malta,L.G. (2007). Antioxidant activity of Annona crassiflora: Characterization of major components by electrospray ionization mass spectrometry, Food Chemistry, 104, 1048–1054.
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ACCEPTED MANUSCRIPT 25. Sanz, M., de Simon, B.F., Cadahia, E., Esteruelas, E., Munoz, A.M., Hernandez, T., Estrella, I.,& Pinto, E. (2012).
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LC-DAD/ESI-MS/MS study of phenolic compounds in ash (Fraxinus excelsior L and F.americana L) heartwood. Effect
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of toasting intensity at cooperage. Journal of Mass Spectrometry, 47, 905-918.
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26. Savic, I.M., Nikolic, V.D., Savic,I.M., Nikolic, L.B., Jovic, M.D., & Jovic, M.D. (2014). The qualitative analysis of the green tea extract using ESI-MS method. Advanced Technologies, 3, 30-37. Singh, D.R., Singh, S., & Banu.S. (2014). Phytochemical composition, antioxidant activity and Sensory evaluation of
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27.
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potential underutilized fruit Soursop (Annona muricata L.) in Bay Islands. Journal of Andaman Science Association,19,
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30-37.
28. Sun, J., Liang, F., Bin, Y., Li, P., & Duan, C. (2007). Screening Non-colored Phenolics in Red Wines using Liquid
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Chromatography/Ultraviolet and Mass Spectrometry/Mass Spectrometry Libraries. Molecules, 12, 679-693.
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29. Vagiri, M., Ekholm, A., Andersson, SC., Johansson, E., & Rumpunen, K. (2012). An optimized method for analysis of phenolic compounds in buds, leaves, and fruits of black currant ( Ribes nigrum L.). Journal of Agricultural Food Chemistry, 60,10501-10.
30. Vereskovskii, V.V., & Shapiro, D.K. (1986). Flavonoids, phenolic acids and hydroxycoumarins from the fruit of various species of the genus Berberis. Chemistry of Natural Compounds, 2, 482-483. 23
ACCEPTED MANUSCRIPT 31. Yi-Long, W., Ye, Z., Ping, W., Wai-Zhen, X., Jian-Zhong, W., Hua Chong, L., & Hui-Qiong, L. (2015). Rapid
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separation and identification of multiple constituents in vine tea by UFLC system coupled with QTOF-MS/MS. Journal
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of Pharmaceutical and Scientific Innovation, DOI No. 10.7897/2277-4572-04228.
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Figure 1 : Flow chart depicting the extraction and analytical procedure adopted for phenolic compounds in custard apple Figure 2 : Chromatogram of free phenolics of custard apple pulp a) Diode array detection at 320nm; b) Diode array detection
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at 280nm; c) ESI negative –MS in full scan mode
Peak No. 1,3,3
Polyphenols Gallic Acid
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Table 1: UPLC ESI MS/MS characterization of Free, Bound and Esterified phenolics (Identified with reference standards)
Empirical formula
RT (min)
[M-H](m/z)
C7H5O5
2.87
169.0137
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MS/MS fragment ions 125.02 (100)
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Catechin
3.86
C15H13O6
153.0188
4.92
Caffeic acid
C9H7O4
5.36
5,9,9
Epicatechin
C15H13O6
5.41
-, 10,10
Epigallocatechin
C22H17O11
6,11,11
gallate p-Coumaric acid
C9H7O3
7,13,12 -, -, 13
Sinapic Acid Ferulic acid
179.0344
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123.0 (100); 109.02(72); 151.04 (60);245.08(10); 203.07 (50); 205.07(20); 179.07 (9) 135.04 (100)
289.0712
5.55
457.0771
123.04 (100); 109.02 (75); 205.07(20); 245.08(10); 151.04 179.07 (54); (9)) 203.07 (52); 289.06 (40); 169.01 (28);
6.50
163.0395
305.02 119.05 (15) (100)
6.93 6.98
223.0615 193.0505
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C11H11O5 C10H9O4
109.02 (100)
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4,8,8
289.0712
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3,6,7
Protocatechuic acid
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2,4,5
208.03 (100); 149.02 (95); 134.03 (100); 178.02 (30); 164.04(19); (82);149.05 179.07(5)(10) 137.02
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Empirical formula C4H5O5
[M-H](m/z) 133.0137
C6H5O6
173.0086
1.63
C6H7O7
191.0192
1.98
C6H7O5
147.0293
2.29
C6H9O5
161.0450
Quinic Acid
3.10
C7H11O6
191.0556
4-(β-Dglucopyranosyloxy)benzoic acid Procyanidin B2
3.69
C13H15O8
299.0767
4.26
C30H25O12
577.1346
Tentative assignment
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Malic Acid
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Trans-Acotinic Acid
10
Citric Acid
11
(R)-(-)-Citramalic acid
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HydroxyAdipic Acid
13 14
RT (min) 0.89
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Peak No
15
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Table 2A: Free phenolics and other compounds (identified from MS fragmentation and literature)
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1.55
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MS/MS fragment ions 115.00 (100); 71.0136 (35); 72.98 (12); 89.02 (8) 111.00 (100); 154.99 (25); 129.01 (18); 85.02 (11); 101.02 (7) 111.00 (100; 87.00 (30); 85.02 (20); 129.01 (5) 115.00 (100); 71.01 (27); 103.04 (26) 99.04 (100); 101.02 (38); 57.03 (7) 101.06 (100; 115.03 (88); 129.05 (50); 85.06 (7) 137.02 (100); 179.03 (75); 239.05 (35); 151.04 (15) 289.07 (100); 407.07 (85); 425.08 (32); 451.10 (25); 125.02 (20);
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Procyanidin trimer
5.51
C45H37O18
865.1980
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161.02 (11) 577.13 (100); 287.05 (69); 695.13 (60); 713.15 (50); 425.08 (48); 451.10 (35); 125.02 (20); 543.02 (20)
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Table 2B: Bound Phenolics and other compounds ( identified from MS fragmentation and literature)
Malic acid
RT (min) 1.36
Empirical [M-H]formula (m/z) C4H5O5 133.0137
2
Citric acid
1.70
C6H7O7
191.0193
5
7 Hydroxycoumarin 7 glucoside (Skimmin) Dihydroxyquercetin
4.37
C15H15O8
323.0767
5.14
C15H11O7
303.0505
Peak No.
Tentative assignment
1
7
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MS/MS fragment ions 115.00 (100); 71.01 (22); 89.02 (8);72.9912 (5) 111.60 (100; 87.00 (15); 85.02 (14); 129.01 (10) 289.06 (100); 125.02 (90); 96.96 (50); 245.04 (48); 198.91 (47); 137.02 (47) 216.04 (100); 191.03 (85); 233.04 (80); 151.03 (52); 111.0 (35)
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Xanthotoxol acetate
6.76
C13H7O5
243.0293
199.04 (100)
14
Procyanidin B2
7.26
C30H25O12
577.1346
15
p-Decycloxybenzoic acid
8.94
C17H25O3
277.1804
245.04 (100); 289.07 (85); 331.08 (65); 425.00 (30); 451.10 (28); 125.0244 (20); 161.0232 (11) 205.16 (100); 233.19 (23)
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Unidentified
9.08
C16H23O6
311.1495
183.01 9(100); 225.14 935); 116.92 (20)
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Unidentified
9.23
C14H21O2
221.1542
205.12 (100); 148.05 (13)
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Unidentified
9.52
C18H35O2
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183.01(100); 269.08 (20)
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283.2637
Table 2C: Esterified phenolics and other compounds (identified from MS fragmentation and literature) Peak No.
Tentative
RT
Empirical 28
[M-H]-
MS/MS fragment ions
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(min) 1.40
formula C4H5O5
(m/z) 133.0137
2
Citric acid
1.70
C6H7O7
191.0193
4
Quinic Acid
3.10
C7H11O6
191.0556
6
Caffeoylhexoside
4.60
C15H17O9
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341.0873
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115.00 (100); 71.01 (22); 89.02 (8);72.99 (5) 111.60 (100; 87.00 (15); 85.02 (14); 129.01 (10) 101.06 (100%; 115.03 (88); 129.05 (50); 85.06 (7) 179, 13(100)
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assignment Malic acid
ACCEPTED MANUSCRIPT Annona fruit (Optimum mature)
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Scooped out pulp and seeds
Passed through pulper
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Fruit pulp
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Extracted with 70% ethanol ( 3 times)
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Pooled extract (supernatant), evaporated the ethanol, adjusted pH to 1.5 with 4 N HCl
Redissloved in methanol
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Extracted with 2M NaOH, 0.5% sodium borohydride under N2 atmosphere (3 times)
Extracted with 2M NaOH, 0.5% sodium borohydride under N2 atmosphere (3 times)
Ethyl acetate phase evaporated to dryness
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Aqueous phase
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Phase separated with ethyl acetate (6 times)
Residue
Free Phenolics
Pooled the extracts and reduced pH to 1.5 with 4N HCl
Phase separated with ethyl acetate
Pooled the extracts and reduced pH to 1.5 with 4N HCl
Ethyl acetate phase evaporated to dryness
Phase separated with ethyl acetate Redissloved in HPLC grade Methanol Ethyl acetate phase evaporated to dryness Bound phenolics Redissloved in HPLC grade Methanol
Esterified phenolics
Figure 1 30
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7
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4 5
10 11
12
13
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3 16
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Figure 2 31
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Highlights:
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Custard apple (Annona squamosa L) fruits are good source of phenolic compounds. Phenolic profile in free, bound and esterified form has been fingerprinted using UPLC-ESI-MSMS. The phenolics identified fall into benzoic acid and cinnamic acid derivatives and flavanol group. Some of the phenolics like quinic acid, gallocatechin are reported for the first time in this fruit.
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S0963-9969(16)30042-4 doi: 10.1016/j.foodres.2016.02.001 FRIN 6167
To appear in:
Food Research International
Received date: Revised date: Accepted date:
27 November 2015 1 February 2016 2 February 2016
Please cite this article as: Baskaran, R., Pullencheri, D. & S.Rajarathnam, Characterization of Free, Esterified and Bound phenolics in Custard apple (Annona squamosa L) fruit pulp by UPLC –ESI-MS/MS, Food Research International (2016), doi: 10.1016/j.foodres.2016.02.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Characterization of Free, Esterified and Bound phenolics in Custard apple
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(Annona squamosa L) fruit pulp by UPLC –ESI-MS/MS
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Department of Fruit and Vegetable Technology, Central Food Technological Research Institute, Mysore – 570 020,
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Waters India Pvt. Ltd., Bangalore, Karnataka, INDIA
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2
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Karnataka, INDIA
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1
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Revathy Baskaran1*, Dilshad Pullencheri2 and S.Rajarathnam1
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*Corresponding author: Revathy Baskaran
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email:[email protected] Tel.: + 91 821-2515653 Fax: + 91 821 2517233
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Abstract:
Ultra High-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC-ESI-MS) was used
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to identify the free, bound and esterified phenolic acids in the extracts of Custard Apple (Annona squamosa L). In total around phenolic
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16, 15 and 13 free, bound and esterified phenolic compounds respectively were identified. Among these about 5 compounds like quinic acid, gallocatechin,
gallocatechin gallate, caffeoylhexoside, dihydroxyquercetin have and been
reported for the first time in A.squamosa. Also, compounds like 4-(β-D-glucopyranosyloxy) benzoic acid, procyanidin B1, procyanidin C1 in free form, 7 hydroxycoumarin 7 glucoside (skimmin), dihydroquercetin, xanthotaxol acetate, decycloxybenzoic acid in bound extract and Caffeoyl hexoside in esterified form have been tentatively identified. Apart from 2
ACCEPTED MANUSCRIPT phenolic compounds few organic acids like malic, citric, citramalic, adipic and acotinic acid have been found in the custard
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apple extracts. This study provides a newer insight into the phenolic profile of custard apple and their characterization by
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UPLC-ESI-MS/MS.
1.0 Introduction:
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Keywords: Annona squamosa, phenolic compounds, UPLC-ESI-MS, fragmentation
Custard apple or sugar apple ( Annona squamosa L) commonly known as Sitaphal in India, is a multiple fruit, consisting of many fruitlets each loaded with a seed having a shiny testa. Custard apple is a table fruit valued for its nutritional benefit and exotic taste. The fruit has a creamish white pulp and a gritty texture (Shravanthi et al., 2014). Apart from the 3
ACCEPTED MANUSCRIPT nutrients like carbohydrates, protein, vitamins etc., they are known to be contain secondary metabolites like phenolic
are secondary metabolites which are widely found in plant and plant derived foods. The
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Phenolic compounds
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compounds, alkaloids, saponins.
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predominant phenolic acids are hydroxylated derivatives of benzoic acid and cinnamic acid. Phenolic acids are mostly present
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in the plants in the bound form.
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Phenolics have received considerable attention as potentially protective factors against cancer and heart diseases mainly
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because of their potent antioxidative properties and their ubiquity in a range of commonly consumed foods of plant origin. Procyanidins are the most common classes of proanthocyanidins which are chains of catechin, epicatechin, and their Gallic acid esters. They are partly responsible for sensory properties such as bitterness, astringency, etc. They consist of
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oligomers and polymers of catechin units most frequently linked to either C4→C8 or C4→C6. Mostly in fruits they are
2001).
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present as monomers or in more complex polymeric forms when the monomers condense (Jiang et al., 2015; Lazarus et al.,
Several analytical methods are available for detection of phenolics. Most of the times, these phenolic compounds are analyzed by High Performance Liquid Chromatography (HPLC) Giusti et al., 2007; Vagiri et al., 2012, coupled with diode 4
ACCEPTED MANUSCRIPT array detector and mass spectrometer Revilla et al., 1999. Also, gas chromatography and capillary electrophoresis are used for
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this purpose (Harborne, 1998). To get structural information about the eluted compounds, these methods are coupled with
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Mass Spectrometry.
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Liquid chromatography Mass Spectrometry (LCMS/MS) is the most preferred method to determine plant metabolites
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because of their sensitivity and selectivity through mass fragmentation and enable structural identification. LC-MS analysis of
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phenolics is either done in APCI or ESI mode.
Studies on the antioxidant activity of A. muricata and A. crassiflora fruit extracts is reported (Singh et al., 2014;
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Roesler et al., 2007), while the total phenolic composition and antioxidant activity of A. cherimola
and A .diversifolia fruit
extracts also has been carried out Loizzo et al., 2012; Julian-Loaeza et al., 2010). Although there are reports on total phenolic
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esterified phenolics.
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composition and functional benefits of A. squamosa fruits, there are no studies that deals with the spectrum of free, bound and
Although A. squamosa is a good source of phenolics, there are very few studies on the phenolic profile. Few researchers have reported the presence of flavanols like catechin, epicatechin and procyanidins in custard apple pulp by LCMS (De-Pascual-Teresa et al., 2000), there are no reports on the profile of free, bound and esterified phenolic acids profile of
5
ACCEPTED MANUSCRIPT Custard apple. Therefore, to fingerprint phenolic profile in A.squamosa, an attempt was made to characterize their profile
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along with chemical structure using UPLC-MS (ESI –ve) method.
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The aim of the present work was to fractionate the phenolic compounds of Custard apple pulp into free, bound and
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esterified forms and determine the profile of phenolic acids after hydrolysis by Ultra Performance Liquid Chromatography
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coupled with Electro Spray Ionization tandem mass spectrometry ( UPLC-ESI MS/MS).
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2.0 Materials and Methods:
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2.1 Samples:
The mature custard apple fruits were procured from local market. The fruits were allowed to ripen at room temperature (27-
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32°C). To separate the seeds from the fleshy portion, the scooped pulp along with the seeds were passed through a fruit pulper
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(M/s APV India, Calcutta, sieve size 1/80cm) to get a fine pulp. 2.1.1 Standards and Chemicals: Gallic acid, Epicatechin, Catechin, Protocatechuic acid, Caffeic acid, p-Coumaric acid, Sinapic acid, Epigallocatechin and Ferulic acids were purchased from Sigma- Aldrich, Bangalore. HPLC grade acetonitrile, methanol, and acetic acid were obtained from Company -E-pure water, Bangalore.
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ACCEPTED MANUSCRIPT 2.2 Extraction of free, soluble ester and insoluble bound phenolics:
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The free, soluble ester and insoluble bound phenolics were extracted according to previously reported method (Krygier et al.,
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1982), with slight modifications (Fig 1). Annona fruit fresh pulp (100 gms) was extracted with 70% ethanol ( 50ml x 3 times).
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The pooled supernatants were flash evaporated to remove all the ethanol, reduced the pH to 2.0 using 4 M HCl and phase
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separated with ethyl acetate ( ~ 6 times). The fraction of ethyl acetate soluble compounds was collected and flash evaporated to
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dryness and redissolved in HPLC grade methanol. The phenolic acids so extracted were labeled as free phenolics. The supernatant after the removal of ethyl acetate fraction which contains the esterified or free soluble conjugated phenolic
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compounds were treated with 2M NaOH for 2 hours at room temperature under nitrogen atmosphere. The resultant hydrolysate was then acidified to pH 2.0 using 4 M HCl and then extracted thrice with ethyl acetate. The ethyl acetate extracts were pooled
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and evaporated to dryness at 35°C under vacuum. The phenolics were the ones liberated from their esters and were labeled as
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esterified phenolics. The residue left over after extraction of fruit pulp with 70% ethanol initially, was treated with 2M NaOH for 2 hours at room temperature under nitrogen atmosphere. The samples were then acidified to pH 2.0 using 4M HCl followed by extraction with ethyl acetate ( ~ 6 times). The ethyl acetate fractions were pooled and evaporated to dryness under vacuum at 35°C. The phenolics so extracted were labeled as bound phenolics. All the free, esterified and bound phenolics were analyzed by UPLCMS/MS. 7
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2.2.1 UPLCMS/MS:
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Chromatographic analyzes of free, esterified and bound phenolics were performed on a Waters Acquity UPLC system
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with Waters acquity UPLC PDA detector for data collection. Phenolics were separated using Waters Atlantis T3 50 x 4.6 id, 3
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µm column at 30°C using a 5µl injection volume. The binary mobile phase consisted of solvent A composed of 0.2% acetic
system:
0.00
98
4.50
78
6.50
78
7.50
10
8.50
%B
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%A
Curves
02
Initial
22
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Time (min)
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acid in water and solvent B, consisted of 0.2% acetic acid in acetonitrile. Separations were performed by the following gradient
6
90
6
10
90
6
9.50
98
02
6
11.00
98
02
6
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ACCEPTED MANUSCRIPT Data were collected using the UV detector at 280 and 320nm. UPLC MS/MS analysis of the phenolics were performed using a
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Waters Synapt® G2 High Definition MS™ system interfaced through an electrospray interface (ESI- negative mode). LC/MS
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was optimized for annona phenolics with a capillary voltage of 2.50 KV, a cone voltage of 30V, source temperature of 140°C
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and desolvation gas temperature of 400°C. The cone and desolvation gas flows were 50 and 1100 L/hr. Nitrogen and argon were used as the cone and collision gasses. MS data were collected from 100 to 2500 m/z and processed with MassLynx™ 4.1
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Software with MassFragment™. The data were acquired using reference lock mass through Lock-Spray interface. Leucine
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enkephalin was used as the reference compound with M-H equal to 554.2615 and was introduced along with the LC stream
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for accurate mass calibration. Quantification of phenolics was done using phenolic standards. 3.0 Results and Discussion:
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Analysis of the free and bound phenolics of custard apple fruit pulp was performed by UPLC –DAD-ESI-MS/MS along with
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their retention times, detected mass, molecular formula and MS/MS fragment ions. Phenolic acids in custard apple pulp were monitored by a diode array detector set at 3 wavelengths 210nm, 280nm, and 320nm. The development of UPLC–MS/MS method was used for characterization of custard apple fruit phenolics for the first time. The partial characterization of free, bound and esterified phenolics in custard apple pulp is shown in Tables 1 to 3. The
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ACCEPTED MANUSCRIPT identified compounds can be classified into 3 groups namely, derivatives of hydroxycinnamic acid, hydroxybenzoic acid, and
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flavanols. Few non- phenolic compounds were also detected.
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Characterization of the phenolics, their derivatives and other compounds detected:
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The analytical method used in the present study allowed the identification of ~ 23 compounds. These included few of the previously reported compounds like flavonols (De-Pascual-Teresa et al., 2000), plus a number of new phenolic compounds and
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a few non-phenolic compounds. Peaks have been numbered in accordance with their retention times in the respective phenolic
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extracts.
Polyphenols belonging to hydroxybenzoic, hydroxycinnamic acid derivatives and flavanols were found in all the fractions extracted. Five of them namely, gallic acid, catechin, epicatechin, protocatechuic acid, caffeic acid, p-coumaric acid and
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sinapic acid were identified by comparing their retention times and characteristic MS spectral data with those of the reference
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standards (Tables 1). Accurate mass and fragmentation pattern confirmed their structural identification. The Total Ion Current (TIC) chromatogram of custard apple pulp extracts is shown in Figures 2 A,B and C. The major peaks indicated have been assigned in Tables 1 and Tables 2A-2C Identification of phenolic compounds was carried out by comparing retention times and masses with those of authentic standards. The compounds for which no standards were
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Further, results of the accurate mass matched the elemental composition of the compounds analyzed.
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In the category of hydroxycinnamic acid derivatives, 3 different phenolic acids like p-coumaric, sinapic and caffeic acid were
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found to occur in all the extracts of custard apple pulp. Similarly, free, bound and esterified hydroxybenzoic acid derivatives like gallic acid and protocatechuic acid were also detected in all fractions i.e., free, bound and esterified. Further, flavanols
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procyanidin B2 were detected in only the bound fraction.
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such as catechin and epicatechin were identified in both free and bound form, whereas flavanols like epigallocatechin and
3.1Characterization of possible free phenolic compounds isolated from Custard apple fruit pulp (Table 1A): Seven phenolic compounds have been identified by comparing their retention times and mass spectra with respective
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standards. The ESI-MS signals of peaks 1,2,4 and 6 at m/z 169.01, m/z 153.01, m/z 179.03 and 163.04 were identified as gallic
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acid, protocatechuic acid, caffeic acid and p-coumaric acid respectively in comparison with their retention time and MS spectral data with those of authentic standards. Accurate mass measurement further confirmed their elemental composition (Table 1). MS/MS fragmentation of gallic acid, protocatechuic acid, caffeic acid and p-coumaric acid produced ions at m/z 125.02, m/z 109.02, m/z 135.04 and 119.05 respectively due to the loss of a CO2 molecule from their respective precursor ions. The fragmentation pattern has been found to be characteristic for hydroxybenzoic acid derivatives. In general, deprotonated 11
ACCEPTED MANUSCRIPT phenolic acids [M-H] produce a typical fragmentation pattern after collision induced dissociation (CID) by the loss of a CO 2
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(44u) from the carboxylic acid group, providing an anion of [ M-HCOO] Parejo et al., 2004; Hossain et al., 2010. Gallic acid
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and protocatechuic acid have been reported to be present in the wine prepared from custard apple fruits (Jagtap and Bapat,
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2015).
Peak 3 and 5 of deprotonated ion [M-H]- at m/z 289, were identified as catechin and epicatechin. These two being
resulted probably due to loss of a CO2
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peaks, the fragmentation at m/z 123(Hvattum, 2002)
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stereoisomers, gave same fragment ions since mass spectrometry cannot distinguish between stereoisomers. For both the molecule from B ring
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methylation. Also, fragment ions at m/z 245, 205 and 179, indicated that the compound was catechin/ epicatechin. The ion m/z 245 could be due to loss of CO2 group or divalent CH2-CHOH- group. Ion at m/z 205 might be due to the loss of A-ring and fragment m/z 179 due to the loss of water molecule (Savic et al., 2014). Also, the identification of catechin and epicatechin
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was confirmed by comparing the retention time and the spectrum of product ions of standard catechin and epicatechin.
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Peak 7, with m/z 223, produced a fragment ion at m/z 208 [M-H-15], due to loss of methyl group, indicating that the compound could be sinapic acid. Further, all the compounds from 1 to 7 were confirmed by comparing them with the MS and MS/MS spectra of respective standards. Apart from the above 7 phenolic acids, 9 peaks corresponding to phenolics and other organic compounds were tentatively identified based on their mass spectra and related data from the literature (Table 2A). 12
ACCEPTED MANUSCRIPT In addition, there were few dicarboxylic acids like malic, citramalic, acotinic and adipic acid, which were detected along with
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the phenolic acids (Table 2). Peak 8 with m/z at 133 which showed a major fragment at m/z 115 [M-H-18], due to the loss of
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water molecule and was identified as malic acid (Lay-Keow et al., 2004). Peak 9, identified as trans-acotinic acid, produced the
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base peak at m/z 111 [M-H-44-18], due to loss of a CO2 and H2O molecule. Similarly, peak 10 was identified as citric acid. A major fragment at m/z 111 [M-H-CO2.2H2O] corresponding to an additional loss of a water molecule was observed.
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Peak 11 was designated as citramalic acid ( m/z 147) due to the fragments at m/z 115 [M-H-OH-CH3] and 87 [M-H-COOH-
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[M-H-H2O-CO2], 101 [M-H-CH3-CO2] and 143 [M-H-H2O].
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CH3]. Peak 12 of free phenolics with the mass at 161 [M-H]- was identified as hydroxyadipic acid. The MS2 fragments were 99
Peak 13 of free phenolics with the ion at m/z 191 [M-H]- and fragmentations ions at 101 (M-CO2-H2O-CO), 115 (M-CO2H2O-CH2) and 129 (M-CO2-H2O) was characterized as quinic acid , Al-Rawahi et al., 2014 .
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Peak 14 (free phenolics), with m/z at 299 ([M-H]- and major fragment ion at m/z at 137 (M-H-162) with the loss of hexose
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moiety was identified as 4-(β-D-glucopyranosyloxy) benzoic acid. Peak 15 (m/z 577), was identified as procyanidin B2, with fragment ions at 425 (M-H-152), obtained probably due to rearrangement of Retro-Diels-Alder (RDA) heterocyclic ring (De Pascual-Teresa and Rivas-Gonzalo, 2003). Also, it gave a fragment ion at m/z at 407 (M-H-170) due to both RDA rearrangement and loss of water molecule. The fragment at m/z 451
13
ACCEPTED MANUSCRIPT (M-H-126) may be because of cleavages between C4-C5 and O-C2 of one pyran ring leading to loss of ‘A’ ring and fragment at
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m/z at 289 (M-H-289) formed from the cleavage of the C-C linkage between 2 catechin units.
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Peak 16, with a mass of 865 (M-H) and empirical formula C45H37O18, was designated as type A procyanidin trimer, with
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typical fragmentation ions at m/z at 577 which may be due to cleavage of the type B interflavan bond and loss of neutral fragment (-288). Also, other fragments formed were similar to the fragmentation pattern earlier reported (Passos et al., 2007),
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where in the fragment 713 (- 152) is due to RDA fragmentation of m/z at 865, fragment 425 is due to RDA fragmentation of
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m/z 577. Further, the fragment ion at m/z 739 (-126) has been attributed to heterocyclic ring fission (HRF) Gu et al., 2003.
3.2 Characterization of Bound phenolics from custard apple fruit pulp (Table 2A): In the analysis of bound fraction, 8 compounds were detected and corresponded with the reference phenolic standards with
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respect to retention time and mass spectra. It was found that phenolic compounds (peak 3,4,8,11 and 13 – Table 2A),
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corresponding to gallic acid, protocatechuic acid, caffeic acid, p-coumaric and sinapic acid were also found in the bound form. Similarly, the flavanols catechin, epicatechin and epigallocatechin were detected. Peak no. 10 of bound phenolics at m/z at 305 (M-H) with fragment ions at m/z at 125 and at m/z 179, was identified as epigallocatechin (Sun et al., 2007).
14
ACCEPTED MANUSCRIPT Based on MS spectra and by comparing with the data in the literature, 7 peaks were identified (Table 2B). Peak 1 and 2 with
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m/z at 133 and 191 were identified as malic and citric acid respectively. They exhibited similar fragmentation pattern as
due to the loss of 2 hydroxyl groups was named as 7
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Peak 5 at m/z at 323 with a major fragment ion m/z at 289
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observed in the free phenolic fraction.
hydroxycoumarin 7 glucoside (skimmin), which is also known to be a phenolic compound present in few species of the genus
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Berberis (Vereskovskii and Shapiro, 1986). Peak 7 with [M-H] at m/z 303 gave a characteristic fragment ion m/z at 151, due
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to the cleavage of the C ring and another fragment ion m/z at 125 (A-CO), indicative of the compound being dihydroquercetin
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(Yi-Long et al., 2015).
Peak 9 was identified as the flavanol epicatechin, as found in the free form. Whereas Peak 10, with m/z 305 [M-H] was designated as epigallocatechin. This flavanol was found in bound fraction only. The fragments m/z at 137 [M-168] may be due
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epigallocatechin (Callemien and Collin, 2008).
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to RDA and m/z at 125 [M-180] by HRF. Further, the fragment m/z at 179 due to loss of 126 Da were all typical of
Peak 12 m/z at 243 [M-H] which gave a major fragment ion m/z at 199 [M-44], might be due to the loss of a CO2 molecule and has been tentatively looked upon as xanthotoxol acetate. Xanthotoxol has been found to be present in fruits like Aegle marmelos L (Lim, 2012).
15
ACCEPTED MANUSCRIPT Peak no. 15 with m/z at 577 [M-H] was identified as procyanidin B2 and it gave similar fragmentation pattern as in the free
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form.
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Peak 16 with m/z at 277 that gave a fragment ion at m/z 205 due to loss of CH3(CH2)2COOH has been tentatively named as
3.3 Phenolic compounds from soluble esters (Table 1 and 2C):
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decycloxybenzoic acid.
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The esterified phenolic acids were also identified using reference standards, MS fragmentation pattern, and literature. In the
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case of esterified phenolics, peaks 3, 5, 8, 11 and 12 were identified as gallic, protocatechuic, caffeic, p-coumaric and sinapic
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acid and had similar fragmentation pattern as found in free and bound phenolics. Similarly, Peak 7 and 9 were identified as flavanols catechin and epicatechin. Mass spectra of peak 6 displayed a parent ion at m/z 341 and two fragment ions, one at m/z 179 for caffeic acid by the loss of a hexose moiety and the other at m/z at 135 for decarboxylated caffeic acid after the loss
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of both hexose and CO2, indicating that the compound may be caffeoyl hexoside. Peak 10 of esterified phenolics with the mass
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at 457 was designated as epigallocatechin gallate, had major ions at m/z 169 and m/z 305. The m/z 169 ion must have resulted from the loss of an intact gallic acid anion and the ion at m/z 305 may be due to neutral loss of gallic acid. A 14 Da shift of m/z169 to m/z 183 is characteristic of methylated gallic acid moiety. The absence of this ion i.e., m/z 183, indicates 4’ position methylation, because the presence of a methyl group at the 4’ position of gallic acid would have blocked the formation of m/z 183 ion. 16
ACCEPTED MANUSCRIPT
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Peak no. 13 of esterified phenolics with m/z at 193 [M-H]- was identified as ferulic acid by comparison with the reference
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standard. Further MS2 fragmentation revealed fragment ion at m/z 178 [M-H-CH3], due to the loss of methyl group (Sanz et
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al., 2012).
Compounds 1,2 and 4 were identified as malic, citric and quinic acid, as the mass and fragmentation were similar to that found
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in the free and bound extracts.
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It is well reported that same phenolic acids can occur in free, esterified and bound forms, but their relative abundance might be
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different in various forms (Djurdjevic et al., 2005; Nicoletti et al., 2013). Also in wine prepared from custard apple, Jagpat et al., 2015, have reported the presence of 3 hydroxybenzoic acids like gallic, protocatechuic and gentisic acid and 2 hydroxycinnamic acids like caffeic and p-coumaric acid by reverse phase HPLC and
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have identified the compounds with reference standards.
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Similar to our results, De Pascual –Teresa et al., 2000, have also reported the presence of catechin, epicatechin, procyanidin dimer B2 and procyanidin trimer C1 in custard apple pulp. Apart from these they have also reported procyanidin dimmers B1, B3, B4, B5, B7 and trimer EEC in custard apple pulp, which were not detected in our samples. The fruit juice custard apple was found to contain phenolic acids like caffeic, ferulic, p-coumaric and sinapic acid in free form (Lee et al., 2003).
17
ACCEPTED MANUSCRIPT 4.0 Conclusion:
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Using UPLC-ESI-MS, around 16 free, 15 bound and 13 esterified phenolic acids respectively have been identified in fresh
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custard apple pulp. Among these few phenolic acids like gallic, protocatechuic , sinapic, ferulic, p-coumaric acid, catechin and
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epicatechin have already been reported, while phenolic acids like quinic acid, gallocatechin, gallocatechin gallate,
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caffeoylhexoside, dihydroxyquercetin etc. are reported for the first time in custard apple. The data provided is one of its first
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kind which gives an idea on the phenolics present in the free, bound and esterified form in custard apple, which in turn can contribute to the dietary polyphenols intake.
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5.0 Acknowledgement:
6.0 References:
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The authors wish to thank Director, CSIR-CFTRI for his constant support and encouragement.
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Isomers in Lager Beer Extracts. Journal of American Society. of Brewing Chemists, 66, 109-115.
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Methods in Polyphenol Analysis, Santos-Buelga, C.; Williamson, G (eds) , Cambridge, The Royal Society of Chemistry, 48-62.
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4. De-Pascual-Teresa, S., Santos-Buelga, C., & Rivas-Gonzalo, J.C. (2000). Quantitative analysis of flavan-3-ols in
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composition in Lamiaceae spices by LC-ESI-MS/MS. Journal of Agricultural Food Chemistry, 58, 10576-10581. 10. Hvattum, E. (2002). Determination of phenolic compounds in rose hip ( Rosa canina) using liquid chromatography
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ACCEPTED MANUSCRIPT 14. Krygier, K., Sosulski, F., & Hogge, L.(1982). Free, esterified, and insoluble-bound phenolic acids. 1. Extraction
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15. Lay-Keow ng., Lafontaine, P., & Vanier, M. (2004). Characterization of Cigarette Tobacco by Direct Electrospray
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Ionization-Ion Trap Mass Spectrometry (ESI-ITMS) Analysis of the Aqueous Extracts: A Novel and Simple Approach
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16. Lazarus, S.A., Hammerstone, J.F., Adamson, G.E., & Schmitz, H.H. (2001). High performance liquid chromatography/mass spectrometry analysis of proanthocyanidins in food and beverages In: Flavonoids and other
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polyphenols Ed. Lester Packer volume 335: 46, Academic Press, USA.. 17. Lee, P.R., Tan, R.M., Yu, B., Curran, P., & Liu, S.Q. (2003). Sugars, organic acids and phenolic acids of exotic
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18. Lim, T.K. (2012). Edible Medicinal and Non-Medicinal Plants; Vol. 4, Fruits. Chapter Aegle Marmelos L, pp 594, DOI 10.1007/978-94-007-4053.2-70© Springer Science of Business Media B.V. 19. Loizzo, M.R., Tundis, R., Bonesi, M., Menichini, F., Mastellone, V., Avallone, L., & Menichini, F.(2012). Radical scavenging, antioxidant and metal chelating activities of Annona cherimola Mill. (cherimoya) peel and pulp in relation to their total phenolic and total flavonoid contents. Journal of Food Composition and Analysis, 25, 179-184. 21
ACCEPTED MANUSCRIPT D’Egidio, M.G., & Corradini,D. (2013). Identification and
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Quantification of Soluble Free, Soluble Conjugated, and Insoluble Bound Phenolic Acids in Durum Wheat (Triticum
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Food Chemistry, 61, 11800-11807.
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Chromatography A9, 847,83-90
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skin extracts and red wines by liquid chromatography with diode array and mass spectrometric detection. Journal of
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ACCEPTED MANUSCRIPT 25. Sanz, M., de Simon, B.F., Cadahia, E., Esteruelas, E., Munoz, A.M., Hernandez, T., Estrella, I.,& Pinto, E. (2012).
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LC-DAD/ESI-MS/MS study of phenolic compounds in ash (Fraxinus excelsior L and F.americana L) heartwood. Effect
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26. Savic, I.M., Nikolic, V.D., Savic,I.M., Nikolic, L.B., Jovic, M.D., & Jovic, M.D. (2014). The qualitative analysis of the green tea extract using ESI-MS method. Advanced Technologies, 3, 30-37. Singh, D.R., Singh, S., & Banu.S. (2014). Phytochemical composition, antioxidant activity and Sensory evaluation of
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30. Vereskovskii, V.V., & Shapiro, D.K. (1986). Flavonoids, phenolic acids and hydroxycoumarins from the fruit of various species of the genus Berberis. Chemistry of Natural Compounds, 2, 482-483. 23
ACCEPTED MANUSCRIPT 31. Yi-Long, W., Ye, Z., Ping, W., Wai-Zhen, X., Jian-Zhong, W., Hua Chong, L., & Hui-Qiong, L. (2015). Rapid
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separation and identification of multiple constituents in vine tea by UFLC system coupled with QTOF-MS/MS. Journal
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of Pharmaceutical and Scientific Innovation, DOI No. 10.7897/2277-4572-04228.
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Figure 1 : Flow chart depicting the extraction and analytical procedure adopted for phenolic compounds in custard apple Figure 2 : Chromatogram of free phenolics of custard apple pulp a) Diode array detection at 320nm; b) Diode array detection
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at 280nm; c) ESI negative –MS in full scan mode
Peak No. 1,3,3
Polyphenols Gallic Acid
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Table 1: UPLC ESI MS/MS characterization of Free, Bound and Esterified phenolics (Identified with reference standards)
Empirical formula
RT (min)
[M-H](m/z)
C7H5O5
2.87
169.0137
24
MS/MS fragment ions 125.02 (100)
ACCEPTED MANUSCRIPT C7H5O4
Catechin
3.86
C15H13O6
153.0188
4.92
Caffeic acid
C9H7O4
5.36
5,9,9
Epicatechin
C15H13O6
5.41
-, 10,10
Epigallocatechin
C22H17O11
6,11,11
gallate p-Coumaric acid
C9H7O3
7,13,12 -, -, 13
Sinapic Acid Ferulic acid
179.0344
25
123.0 (100); 109.02(72); 151.04 (60);245.08(10); 203.07 (50); 205.07(20); 179.07 (9) 135.04 (100)
289.0712
5.55
457.0771
123.04 (100); 109.02 (75); 205.07(20); 245.08(10); 151.04 179.07 (54); (9)) 203.07 (52); 289.06 (40); 169.01 (28);
6.50
163.0395
305.02 119.05 (15) (100)
6.93 6.98
223.0615 193.0505
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AC
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C11H11O5 C10H9O4
109.02 (100)
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4,8,8
289.0712
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3,6,7
Protocatechuic acid
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2,4,5
208.03 (100); 149.02 (95); 134.03 (100); 178.02 (30); 164.04(19); (82);149.05 179.07(5)(10) 137.02
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SC
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PT
ACCEPTED MANUSCRIPT
Empirical formula C4H5O5
[M-H](m/z) 133.0137
C6H5O6
173.0086
1.63
C6H7O7
191.0192
1.98
C6H7O5
147.0293
2.29
C6H9O5
161.0450
Quinic Acid
3.10
C7H11O6
191.0556
4-(β-Dglucopyranosyloxy)benzoic acid Procyanidin B2
3.69
C13H15O8
299.0767
4.26
C30H25O12
577.1346
Tentative assignment
8
Malic Acid
9
Trans-Acotinic Acid
10
Citric Acid
11
(R)-(-)-Citramalic acid
12
HydroxyAdipic Acid
13 14
RT (min) 0.89
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Peak No
15
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Table 2A: Free phenolics and other compounds (identified from MS fragmentation and literature)
AC
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1.55
26
MS/MS fragment ions 115.00 (100); 71.0136 (35); 72.98 (12); 89.02 (8) 111.00 (100); 154.99 (25); 129.01 (18); 85.02 (11); 101.02 (7) 111.00 (100; 87.00 (30); 85.02 (20); 129.01 (5) 115.00 (100); 71.01 (27); 103.04 (26) 99.04 (100); 101.02 (38); 57.03 (7) 101.06 (100; 115.03 (88); 129.05 (50); 85.06 (7) 137.02 (100); 179.03 (75); 239.05 (35); 151.04 (15) 289.07 (100); 407.07 (85); 425.08 (32); 451.10 (25); 125.02 (20);
ACCEPTED MANUSCRIPT
Procyanidin trimer
5.51
C45H37O18
865.1980
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SC
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16
161.02 (11) 577.13 (100); 287.05 (69); 695.13 (60); 713.15 (50); 425.08 (48); 451.10 (35); 125.02 (20); 543.02 (20)
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Table 2B: Bound Phenolics and other compounds ( identified from MS fragmentation and literature)
Malic acid
RT (min) 1.36
Empirical [M-H]formula (m/z) C4H5O5 133.0137
2
Citric acid
1.70
C6H7O7
191.0193
5
7 Hydroxycoumarin 7 glucoside (Skimmin) Dihydroxyquercetin
4.37
C15H15O8
323.0767
5.14
C15H11O7
303.0505
Peak No.
Tentative assignment
1
7
27
MS/MS fragment ions 115.00 (100); 71.01 (22); 89.02 (8);72.9912 (5) 111.60 (100; 87.00 (15); 85.02 (14); 129.01 (10) 289.06 (100); 125.02 (90); 96.96 (50); 245.04 (48); 198.91 (47); 137.02 (47) 216.04 (100); 191.03 (85); 233.04 (80); 151.03 (52); 111.0 (35)
ACCEPTED MANUSCRIPT
Xanthotoxol acetate
6.76
C13H7O5
243.0293
199.04 (100)
14
Procyanidin B2
7.26
C30H25O12
577.1346
15
p-Decycloxybenzoic acid
8.94
C17H25O3
277.1804
245.04 (100); 289.07 (85); 331.08 (65); 425.00 (30); 451.10 (28); 125.0244 (20); 161.0232 (11) 205.16 (100); 233.19 (23)
16
Unidentified
9.08
C16H23O6
311.1495
183.01 9(100); 225.14 935); 116.92 (20)
17
Unidentified
9.23
C14H21O2
221.1542
205.12 (100); 148.05 (13)
18
Unidentified
9.52
C18H35O2
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12
183.01(100); 269.08 (20)
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283.2637
Table 2C: Esterified phenolics and other compounds (identified from MS fragmentation and literature) Peak No.
Tentative
RT
Empirical 28
[M-H]-
MS/MS fragment ions
ACCEPTED MANUSCRIPT
(min) 1.40
formula C4H5O5
(m/z) 133.0137
2
Citric acid
1.70
C6H7O7
191.0193
4
Quinic Acid
3.10
C7H11O6
191.0556
6
Caffeoylhexoside
4.60
C15H17O9
RI
SC
341.0873
NU MA PT ED CE AC
29
115.00 (100); 71.01 (22); 89.02 (8);72.99 (5) 111.60 (100; 87.00 (15); 85.02 (14); 129.01 (10) 101.06 (100%; 115.03 (88); 129.05 (50); 85.06 (7) 179, 13(100)
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1
assignment Malic acid
ACCEPTED MANUSCRIPT Annona fruit (Optimum mature)
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Scooped out pulp and seeds
Passed through pulper
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Fruit pulp
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Extracted with 70% ethanol ( 3 times)
MA
Pooled extract (supernatant), evaporated the ethanol, adjusted pH to 1.5 with 4 N HCl
Redissloved in methanol
AC CE P
Extracted with 2M NaOH, 0.5% sodium borohydride under N2 atmosphere (3 times)
Extracted with 2M NaOH, 0.5% sodium borohydride under N2 atmosphere (3 times)
Ethyl acetate phase evaporated to dryness
TE
Aqueous phase
D
Phase separated with ethyl acetate (6 times)
Residue
Free Phenolics
Pooled the extracts and reduced pH to 1.5 with 4N HCl
Phase separated with ethyl acetate
Pooled the extracts and reduced pH to 1.5 with 4N HCl
Ethyl acetate phase evaporated to dryness
Phase separated with ethyl acetate Redissloved in HPLC grade Methanol Ethyl acetate phase evaporated to dryness Bound phenolics Redissloved in HPLC grade Methanol
Esterified phenolics
Figure 1 30
PT
ACCEPTED MANUSCRIPT
9
7
NU
SC
RI
14
4 5
10 11
12
13
AC
8
CE
PT ED
MA
1
15
3 16
2
Figure 2 31
6
ACCEPTED MANUSCRIPT
Highlights:
CE
PT ED
MA
NU
SC
RI
PT
Custard apple (Annona squamosa L) fruits are good source of phenolic compounds. Phenolic profile in free, bound and esterified form has been fingerprinted using UPLC-ESI-MSMS. The phenolics identified fall into benzoic acid and cinnamic acid derivatives and flavanol group. Some of the phenolics like quinic acid, gallocatechin are reported for the first time in this fruit.
AC
32