HPLC–DAD–ESI-MS/MS screening of bioactive components from Rhus coriaria L. (Sumac) fruits Academic research paper on "Chemical sciences"

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Food Chemistry

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CHEMISTRY

HPLC-DAD-ESI-MS/MS screening of bioactive components from Rhus coriaria L. (Sumac) fruits

Ibrahim M. Abu-Reidah a,b,c, Mohammed S. Ali-Shtayeh a'*, Rana M. Jamousa, David Arráez-Román b,c, Antonio Segura-Carretero b,c'*

a Biodiversity & Environmental Research Center (BERC), Til, Nablus POB 696, Palestine

b Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avda. Fuentenueva, 18071 Granada, Spain c Functional Food Research and Development Centre (CIDAF), PTS Granada, Avda. del Conocimiento, Edificio Bioregión, 18016 Granada, Spain

ARTICLE INFO ABSTRACT

Rhus coriaria L. (sumac) is an important crop widely used in the Mediterranean basin as a food spice, and also in folk medicine, due to its health-promoting properties. Phytochemicals present in plant foods are in part responsible for these consequent health benefits. Nevertheless, detailed information on these bioactive compounds is still scarce. Therefore, the present work was aimed at investigating the phytochemical components of sumac fruit epicarp using HPLC-DAD-ESI-MS/MS in two different ionisation modes. The proposed method provided tentative identification of 211 phenolic and other phyto-constituents, most of which have not been described so far in R. coriaria fruits. More than 180 phytochemicals (tannins, (iso)flavonoids, terpenoids, etc.) are reported herein in sumac fruits for the first time. The obtained results highlight the importance of R. coriaria as a promising source of functional ingredients, and boost its potential use in the food and nutraceutical industries.

© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CCBY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/3XI/).

CrossMark

Article history:

Received 25 March 2014

Received in revised form 29 May 2014

Accepted 3 June 2014

Available online 12 June 2014

Keywords: Palestinian sumac Anacardiaceae Hydrolysable tannins Flavonoids Mediterranean diet HPLC-DAD-ESI-MS/MS

1. Introduction

Sumac, Rhus coriaria L. (Anacardiaceae), is a wild edible plant growing in the Mediterranean region, has long been used as a seasoning spice, either in pure form or in combination with other spices (Ali-Shtayeh, & Jamous, 2008), sauce, appetizer, drink, and as a souring agent in food recipes. R. coriaria L. is an important and the most widely used species of the genus Rhus in the Mediterranean region since antiquity. Recently, the consumption of sumac fruits has been increasing around the world as an important economic crop (Kizil, & Turk, 2010).

In folk medicine and traditional Arabic Palestinian herbal medicine, this plant has been used in the treatment of cancer, stroke, diarrhoea, hypertension, dysentery, haematemesis, ophthalmia, stomach ache, diuresis, diabetes, atherosclerosis, measles, smallpox, liver disease, aconuresis, teeth and gum ailments, headaches, animal bites, dermatitis, and liver disease (Ali-Shtayeh, & Jamous, 2008; Shafiei, Nobakht, & Moazzam, 2011). Furthermore, R. coriaria

* Corresponding authors. Tel.: +970 92536406 (M.S. Ali-Shtayeh). Address: Functional Food Research and Development Center (CIDAF), PTS Granada, Avda. del, Conocimiento, Edificio Bioregión, 18016 Granada, Spain. Tel.: +34 958248435 (A. Segura-Carretero).

E-mail addresses: msshtayeh@yahoo.com (M.S. Ali-Shtayeh), ansegura@ugr.es (A. Segura-Carretero).

is known to possess non-mutagenic, fever-reducing, DNA protective, antiseptic, antifungal, antibacterial, antioxidant, anti-ischae-mic, hypouricemic, hypoglycaemic, and hepatoprotective properties, which support its traditional uses (Anwer et al., 2013; Chakraborty et al., 2009; Madihi et al., 2013; Shafiei et al., 2011).

Among 56 Palestinian plants tested, sumac was found to have the greatest antimicrobial effect against Probionibacterium acnes (MIC 6 mg/ml, MBC 6 mg/ml), Staphylococcus aureus (MIC 4 mg/ ml, MBC 6 mg/ml), Escherichia coli (MIC 6 mg/ml, MBC 8 mg/ml) and Pseudomonas aeruginosa (MIC 4 mg/ml and MBC 6 mg/ml) (Ali-Shtayeh, Al-Assali, & Jamous, 2013).

The literature lacks detailed information on R. coriaria chemical composition. Previous works have reported sumac to contain phenolic compounds, such as hydrolysable tannins, anthocyanins and also organic acids such as malic and citric acids (Kosar, Bozan, Temelli, & Baser, 2007; Kossah, Nsabimana, Zhang, & Chen, 2010). Interestingly, the acidic and astringent tastes, may be due to indigenous organic acids (mainly, malic acid) and tannins. Many compounds have been identified from different parts of sumac, such as phenolics, organic acids, proteins, fibre, volatile oils, fatty acids, vitamins, and minerals (Anwer et al., 2013; Özcan, & Haciseferogullari, 2004). Only a few studies have been carried out on the chemical composition of R. coriaria leaves (Regazzoni et al., 2013; Van Loo, De Bruyn, & Verzele, 1988) and little is known about the phytochemical composition of the plant's fruit epicarps.

http://dx.doi.org/10.1016/j.foodchem.2014.06.011 0308-8146/© 2014 The Authors. Published by Elsevier Ltd.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Although R. coriaria is a particularly rich source of phenolic compounds (Kossah et al., 2010), the phenolic constituents of sumac fruit's epicarp remains so far incompletely investigated. Thus, detailed and extended profiling of the phytochemicals of sumac fruits using high sensitive tools is necessary. Consequently, suitable methods need to be established for the identification of phytochemicals in plant food matrices (Abu-Reidah, Contreras, Arráez-Román, Fernández-Gutiérrez, & Segura-Carretero, 2014). Mass spectrometry coupled to high-performance liquid chromatography (HPLC-MS) has been increasingly used in the structural characterisation of complex matrices and has proved to be the tool of choice to identify phenolic compounds (Abu-Reidah, Arráez-Román, Lozano-Sánchez, Segura-Carretero, & Fernández-Gutiérrez, 2013; Abu-Reidah, Arráez-Román, Segura-Carretero, & Fernández-Gutiérrez, 2013; Lee, Zweigenbaum, & Mitchell, 2013).

Therefore, the objective of the present study was to investigate the phytochemical composition of hydro-methanolic extracts of R. coriaria fruits cultivated in Palestine, by using high-performance liquid chromatography-diode array detector-hyphenated with tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) as a potent analytical technique.

2. Materials and methods

2.1. Chemicals

Acetonitrile and methanol of analytical or HPLC grade were purchased from Labscan (Dublin, Ireland). Acetic acid of analytical grade (assay >99.5%) was purchased from Fluka (Switzerland). Water was purified by using a Milli-Q system (Millipore, Bedford, USA).

2.2. Sample preparation

Sumac is commercially obtainable in local markets in ready-to-use ground form. In our present study, for quality control considerations, sumac samples were harvested in their mature stage from the wild habitat mountains of Nablus (Qusra village) in summer of 2012 and were identified by Prof. Mohammad S. Ali-Shtayeh from BERC. Collected sumac samples were dried, and then epicarps of R. coriaria L. fruits were liberated from kernels and ground into powder using a household mill and stored at room temperature until they were used for extraction.

2.3. Extraction of phenolic compounds

The extraction procedure was performed following Abu-Reidah, Arráez-Román, Segura-Carretero, and Fernández-Gutiérrez (2013), with some modifications. Portions of the dried and ground Sumac fruit epicarps (0.5 g) were extracted using methanol (80% v/v) and sonicated for 30 min at room temperature. The mixture was centri-fuged for 15 min at 3800g and the supernatant was collected into a round-bottom flask. The extraction process was repeated three times. To get rid of the non-polar fraction that could be extracted by 80% methanol, the supernatant was mixed twice with 5 mL of n-hexane. The solvent was evaporated using a rotary evaporator under vacuum at 40 °C and the dry residue was dissolved in aqueous methanol. Finally, the extract was centrifuged again and the supernatant was filtered through a 0.2-im syringe filter and stored at -20 °C until analysis.

2.4. HPLC-DAD/QTOF-MS analysis

Separation of phenolic compounds from sumac extract was performed on an Agilent 1200 series Rapid Resolution LC (Agilent

Technologies, Santa Clara, CA) consisting of a vacuum degasser, an auto-sampler, a binary pump and diode-array detector (DAD). This instrument was equipped with an Agilent Zorbax C18 column (4.6 x 150 mm, 5 im) from Agilent Technologies. Acidified water (0.5% acetic acid, v/v) and acetonitrile were used as mobile phases A and B, respectively. The gradient was programmed as follows: 0 min, 0% B; 20 min, 20% B; 30 min, 30% B; 40 min, 50% B; 50 min, 75% B; 60 min, 100% B; 62 min 0% B, and finally, the initial conditions were held for 8 min as a re-equilibration step. The flow rate was set at 0.80 mL/min throughout the gradient. The flow from the HPLC system into the ESI-Q-TOF-MS detector was 0.2 mL/min. The injection volume was 10 iL and the column temperature was maintained at 25 °C.

The HPLC system was coupled to a quadrupole-time-of-flight (micrOTOF-Q™, Bruker Daltonik GmbH, Bremen, Germany) orthogonal accelerated Q-TOF mass spectrometer, equipped with an electrospray ionisation source (ESI). Parameters for analysis were set using negative and positive ion modes, with spectra acquired over a mass range from m/z 50 to 1100. The optimum values of the ESI-MS parameters were: capillary voltage, -3.5 and +4.0 kV; drying gas temperature, 190 °C; drying gas flow, 9.0 L/ min; nebulising gas pressure, 29 psi; collision RF, 150 Vpp; transfer time 70 is, and pre-pulse storage, 5 is. Moreover, automatic MS/ MS experiments were performed adjusting the collision energy values as follows: m/z 100, 20 eV; m/z 500, 30 eV; m/z 1000, 35 eV, using nitrogen as collision gas. The MS data were processed through Data Analysis 4.0 software (Bruker Daltonics, Bremen, Germany) which provided a list of possible elemental formulas by using the Generate Molecular Formula™ editor. The editor uses a CHNO algorithm, which provides standard functionalities, such as maximum/minimum elemental range, and a sophisticated comparison of the theoretical with the measured isotope pattern (mSigma value), for increasing the confidence in the suggested molecular formula. The widely accepted accuracy for confirmation of elemental compositions has been established as 5 ppm.

At some stage in the HPLC method development, an external apparatus calibration was performed using a Cole Palmer syringe pump (Vernon Hills, IL) directly linked to the interface, passing a solution of sodium acetate. Using this method, an exact calibration curve based on numerous cluster masses each differing by 82 Da (C2H3NaO2) was obtained. Due to the compensation of temperature drift in the Q-TOF, this external calibration provided accurate mass values for a complete run without the need for a dual sprayer set up for internal mass calibration.

3. Results and discussion

3.1. Characterisation of the phenolics and other phytochemical derivatives

3.1.1. General

Table 1 shows the list of 211 compounds tentatively identified through HPLC-DAD-ESI-MS/MS experiments along with their retention times (tR), detected accurate mass (ionisation modes either negative and/or positive, molecular formula, error in ppm (between the mass found and the accurate mass) of each phytochemical, as well as the MS/MS fragment ions and the bibliographic references used in the characterisation process.

In the present work, a qualitative analysis of the phenolic composition from the hydro-methanol extract of sumac fruits (epicarps) has been carried out using HPLC-DAD-ESI-MS/MS in negative and positive ionisation modes. The method was used to detect and characterise 211 phytochemical compounds, of which 188 were tentatively characterised for the first time in sumac (R. coriaria) fruits. Fig. 1A-C correspond to the base peak

Table 1

Phytochemical compounds detected and characterised in R. coriaria L. fruits by using HPLC-DAD/QTOF-MS in positive and negative ionisation modes.

Peak Tentative assignment tR [M+H]+ [M-H]- Error mSigma Molecular MS2/MS fragment ionsb Reference

No. (min.) (m/z) (m/z) (ppm) formula

1 Quinic acid I 2.35 193.0708 191.0566 -2.8 1.4 C7H,2O6 173.0442(4), 109.0302(4)a -

2 Malic acid I 2.69 - 133.0144 -1.2 1.8 C4H6O5 115.0034(100)a -

3 Malic acid hexoside I 2.91 - 295.0663 -1.3 7.6 C10H16O10 133.0140(100),115.0030(63)a Ley et al. (2006)

4 Malic acid hexoside II 3.16 - 295.0673 -0.8 7.3 C10H16O10 133.0137(100),115.0030(41)a Ley et al. (2006)

5 Malic acid hexoside III 3.36 - 295.0671 -0.2 0.9 C10H16°10 133.0136(100), 115.0044(48)a Ley et al. (2006)

6 Oxydisuccinic acid 4.32 251.0410 249.0262 -3.9 7.3 C8H10°9 133.0141(100),115.0036(52)a -

7 Malic acid II 4.37 135.0284 133.0143 -0.4 1.7 C4H6O5 115.0024(100)a -

8 Malic acid III 4.82 135.0281 133.0140 1.7 2.9 C4H6O5 115.0024(100)a -

9 Quinic acid II 5.71 193.0365 191.0555 3.5 1.8 C7H12O6 173.0409(100)a -

10 O-Succinoyl-di-O- 5.75 - 615.1383 -4.5 10 C29H28°15 307.0675(15), 191.0569(100)a -

caffeoylquinic acid

11 Malic acid derivative 6.52 - 289.0569 -1.5 10.3 C11H14O9 173.0466(26),155.0369(4), -

133.0141(100),115.0034(22)a

12 Caftaric acid 6.75 - 311.0354 8.5 21 C13H12O9 133.0135(100), 115.0031(37)a -

13 Galloylhexose I 7.44 - 331.0647 4.3 3.8 C13H16O10 169.0158(100)a Fröhlich et al. (2002)

14 Galloylhexose II 9.09 - 331.0669 0.6 14.8 C13H16O10 169.0148(100)a Fröhlich et al. (2002)

15 Levoglucosan gallate I 9.50 315.0717 - -2 1.3 C13H14O9 153.0196(100),109.0270(6) -

16 Galloylhexose III 9.86 - 331.0673 -0.8 13.4 C13H16O10 271.0470(100),211.0255(47), Fröhlich et al. (2002)

169.0142(55)a

17 Levoglucosan gallate II 10.68 315.0730 - -6.1 7.5 C13H14O9 153.0186(100),125.0219(6), -

109.0252(2)

18 Galloylhexose IV 11.00 - 331.0671 -0.1 0.3 C13H16O10 271.0462(100),211.0252(46), Fröhlich et al. (2002)

169.0144(38)a

19 O-galloylnorbergenin i 11.01 467.0803 - 3.7 7.2 C20H18O13 171.0278(2),153.0184(100) -

20 Digalloyl-hexoside I 11.40 - 483.0772 1.8 4.7 C20H20O14 331.067(25), 169.0143(56)a Fröhlich et al. (2002)

21 Galloylhexose 11.42 - 505.0606 3.5 10.3 C22H18O14 445.0404(6), 331.0665(6), -

derivative I 169.0102(10)a

22 O-galloylnorbergenin ii 11.54 467.0816 - 1 13 C20H18O13 171.0291(2),153.0181(100) -

23 Digalloyl-hexoside II 11.92 - 483.0773 1.2 1.8 C20H20O14 331.0671(20),313.0560(6), Fröhlich et al. (2002)

169.0144(52)a

24 Galloylhexose 11.94 - 505.0625 -0.2 13.4 C22H18O14 331.0650(9), 169.0134(11)a -

derivative II

25 Protocatechuic acid 12.21 - 315.0717 1.5 10.1 C13H16O9 153.0169(50), 152.0108(100), -

hexoside 109.0286(14), 108.0215(39)a

26 Gallic acid dihexose 12.56 - 493.1191 1.5 41.8 C19H26O15 313.0561(100)a -

27 Galloylhexose malic 12.73 - 447.0777 0.8 8.2 C17H20O14 331.0666(100),271.0481(10), -

acid I 169.0153(14)a

28 Galloylhexose V 12.86 - 331.0672 -0.5 7.1 C13H16O10 169.0146(100),125.0244(11)a Fröhlich et al. (2002)

29 Galloylhexose malic 13.00 - 447.0782 -0.4 4.8 C17H20O14 331.0673(100),169.0147(19), -

acid II 133.0146(6)a

30 Unknown 13.33 309.0632 307.0469 -3.3 7.5 C14H12O8 289.0339(50), 245.0457(35), -

201.0571(100)a

31 Protocatechoic acid 13.47 - 153.0194 -0.6 4.1 C7H6O4 109.0293(100)a Shabana et al. (2011)

32 Galloylshikimic acid I 13.49 - 325.0567 -0.6 2.4 C14H14O9 169.0145(100),153.0200(13), -

125.0244(20)a

33 Digalloyl-hexose-malic 13.55 - 599.0901 -2 14.4 C24H24O18 483.0794(48),465.0621(6), -

acid I 447.0773(8),313.0548(3),

169.0142(22)a

34 Gallic acid hexose 13.62 - 487.1082 2.2 27 C20H24O14 331.0618(28),169.0152(70)a -

derivative

35 Syringic acid hexoside 13.77 - 359.0977 1.7 12.8 C15H20O10 197.0425(7)a -

36 Gallic acid O-malic acid 13.80 - 285.0261 -3.1 1.8 C11H10O9 169.0153(5),133.0141(100)a Zhang et al. (2004)

37 Galloylshikimic acid II 13.94 - 325.0572 -2.2 5.4 C14H14O9 169.0152(100),125.0236(14)a -

38 Digalloyl-hexose malic 14.26 - 599.0891 -0.1 11.4 C24H24O18 483.0779(39),447.0756(21), -

acid II 313.0680(1),169.0146(19)a

39 Unknown 14.46 583.0937 - -1.3 14.1 C24H22O17 171.0332(3),154.0203(9), -

127.0371(13),109.0265(6),

97.0286(21)

40 Galloylquinic acid I 14.71 - 343.0691 -5.8 45.8 C14H16O10 191.0626(12), 169.0156(83)a -

41 O-galloylnorbergenin iii 15.04 467.0828 - -1.7 31.4 C20H18O13 153.0191(100) -

42 Digalloyl-hexose malic 15.35 - 599.0881 1.5 5.2 C24H24O18 599.0875(100),483.0771(12), -

acid III 447.0772(14),169.0143(11)a

43 Coumaryl-hexoside 15.77 - 325.0924 1.5 10.8 C15H18O8 163.0398(100), 119.0491(60)a -

44 Digalloyl-hexoside III 16.10 485.0949 483.0793 -2.6 12 C20H20O14 423.0570(37), 331.0665(12), Fröhlich et al. (2002)

169.0143(17)a

45 O-galloylnorbergenin iv 16.25 476.0837 - -3.6 11.8 C20H18O13 303.0561(20),153.0193(100) -

46 Digalloyl-hexoside IV 16.49 485.0809 483.0774 1.2 4.9 C20H20O14 423.0581(3), 331.0699(6), Fröhlich et al. (2002)

169.0149(25)a

47 Galloylquinic acid II 16.62 - 343.0675 -1.3 16.6 C14H16O10 191.0570(33), 169.0139(100)a -

48 Trigalloyllevoglucosan I 16.67 619.0961 - -5 2.2 C27H22O17 153.0183(100),109.0309(1) Chen, and Bergmeier

(2011)

(continued on next

Peak Tentative assignment tR [M+H]+ [M-H]- Error mSigma Molecular MS2/MS fragment ionsb Reference

No. (min.) (m/z) (m/z) (ppm) formula

49 Digalloyl-hexose malic 16.68 - 599.0884 1 5.5 C24H24O18 483.0784(40), 447.0757(6), -

acid IV 331.0664(5), 313.0537(2),

169.0138(18)a

50 Kaempferol hexoside or 16.92 449.1048 - 6.8 65 C21H20O11 287.0571(100) Buziashvili,

Luteolin hexoside I Komissarenko, and

Kolesnikov (1970) and

Shrestha, et al. (2012)

51 Tri-galloyl-hexoside I 16.94 637.1110 635.0896 -0.9 4.1 C27H24O18 483.0759(23), 465.0699(9), Regazzoni et al. (2013)

169.0128(9)a

52 Penstemide 17.16 - 443.1917 1.3 7.1 C21H32O10 101.0229(2)a Rodríguez-Pérez et al.

(2013)

53 Digallic acid I 17.18 323.0403 321.0260 -2.4 6.4 C14H10O9 169.0139(100), 125.0240(18)a El Sissi et al. (1972)

54 Digalloyl-hexoside V 17.50 - 483.0775 1.1 3.5 C20H20O14 331.0681(4), 169.0144(19)a Fröhlich et al. (2002)

55 Kaempferol hexoside or 17.55 449.1082 - -0.9 4.7 C21H20O12 287.0576(100) Buziashvili,

Luteolin hexoside II Komissarenko, and

Kolesnikov(1970) and

Shrestha et al. (2012)

56 O-galloylnorbergenin v 17.75 467.0826 - -1.1 15.8 C20H18O13 153.0187(100) -

57 Methyl gallate 18.24 185.0441 183.0302 -1.5 1.7 C8H8O5 168.0076(28), 140.0112(64), Shabana et al. (2011)

124.0170(39)a

58 Trigalloyllevoglucosan 18.40 619.0945 - -2.4 3.3 C27H22O17 303.0531(3),153.0180(100) Chen, and Bergmeier

II (2011)

59 Tri-galloyl-hexoside II 18.57 637.1106 635.0886 0.6 2.7 C27H24O18 331.0699(1), 169.0128(8)a Regazzoni et al. (2013)

60 Digallic acid II 18.71 323.0408 321.0257 -1.1 2.7 C14H10O9 169.0164(100),125.0243(18)a El Sissi et al. (1972)

61 Coumaric acid 18.72 - 163.0403 -1.3 3.9 C9H8O3 119.0507(100)a Min-Young, Ill-Min,

Deog-Cheon, and Hee-

Juhn (2009)

62 Trigalloyllevoglucosan 18.81 619.0950 - -2.4 3.3 C27H22O17 153.0186(100) Chen, and Bergmeier

III (2011)

63 Galloylpyrogallol 18.82 279.0512 - -4.5 5.8 C13H10O7 153.0190(100) -

64 Isorhamnetin hexoside I 18.94 479.1167 - 3.5 13 C22H22O12 317.0671(100) -

65 Apigenin glucoside I 18.96 433.1149 - -4.6 13.4 C21H20O10 271.0617(100) Shabana et al. (2011)

66 Tri-galloyl-hexoside III 19.04 637.1100 635.0882 1.3 2 C27H24O18 483.0774(7), 465.0658(4), Regazzoni et al. (2013)

169.0147(3)a

67 Isorhamnetin hexoside it 19.14 479.1189 - -1.1 7.6 C22H22O12 317.0664(100) -

68 II Kaempferol-hexose 19.16 565.1194 - -1.1 14 C25H24O15 287.0558(100) Perestrelo et al. (2012)

malic acid I

69 Hydroxy- 19.45 - 453.1053 -3.1 40.4 C20H22O12 313.0573(15), 179.0414(9), -

methoxyphenyl-O-(O- 169.0153(13)a

galloyl)-hexose

70 Cyanidin-3-O- 19.65 601.1186 599.1039 0.6 30 C28H24O15 285.0405(100)a Kirby et al. (2013)

(2"galloyl)-galactoside

71 Trigalloyllevoglucosan 20.23 619.0935 - -0.9 3.4 C27H22O17 153.0183(100) Chen, and Bergmeier

IV (2011)

72 Tri-galloyl-hexoside IV 20.37 - 635.0895 -0.8 4.4 C27H24O18 483.0777(7), 465.0675(4), Regazzoni et al. (2013)

169.0147(3)a

73 7-O-Methyl- 20.38 631.1301 - -1.3 17 C29H26O16 317.0650(100), 233.0448(3), Kirby et al. (2013)

delphinidin-3-O-(2" 153.0195(27)

galloyl)-galactoside I

74 Kaempferol-hexose 20.39 565.1193 - -0.8 41 C25H24O15 287.0549(100) Perestrelo et al. (2012)

malic acid II

75 7-O-Methyl- 20.57 631.1304 - -1.3 17 C29H26O16 317.0665(100), 233.0425(2), Kirby et al. (2013)

delphinidin-3-O-(2" 153.0183(10)

galloyl)-galactoside II

76 Spinochrome A 20.92 265.1465 263.0217 -7.4 13.2 C12H8O7 245.0085(30), 235.0277(30), -

219.0267(24), 207.0309(22),

191.0391(19)a

77 Apigenin-7-O-(6"-O- 20.97 585.1241 - 6 20.6 C28H24O14 271.0618(100), 153.0187(10) Tian et al., 2010

galloyl)-p-D-

glucopyranoside

78 O-Galloyl arbutin 21.04 425.1066 - 2.8 30.5 C19H20O11 273.0707(4) Shi & Zuo, (1992)

79 Coumaryl-hexose malic 21.06 - 441.1037 0.3 8.5 C19H22O12 325.0926(13), 163.0405(100), -

acid 119.0509(5)a

80 Methyl- 21.64 - 479.1190 1 4.2 C22H24O12 317.0701(26), 299.0574(100)a -

dihydroquercetin

hexoside

81 7-O-Methyl-cyanidin- 21.66 463.1231 461.1090 -0.1 11.8 C22H22O11 299.0562(61), 298.0480(100)a Kirby et al. (2013)

3-O-galactoside

82 Caffeoylquinic acid 21.88 355.1040 - -4.6 48 C16H18O9 193.0494(100) -

83 Trigalloyllevoglucosan 22.03 619.0959 - -4.7 12 C27H22O17 153.0183(100) Chen, and Bergmeier

V (2011)

84 Chrysoriol-hexose 22.08 579.1361 - -2.8 4.2 C26H26O15 301.0705(100) -

Peak Tentative assignment tR [M+H]+ [M-H]- Error mSigma Molecular MS2/MS fragment ionsb Reference

No. (min.) (m/z) (m/z) (ppm) formula

malic acid

85 Myricetin hexose-malic 22.13 - 595.1297 1.3 12.6 C26H28O16 479.1180(100), 369.0832(29), -

acid I 317.0687(7), 299.0570(34)a

86 Tri-galloyl-hexoside V 22.15 - 635.0888 0.2 12 C27H24O18 465.0620(21), 483.0748(12), Regazzoni et al. (2013)

169.0147(4)a

87 Eriodictyol hexoside or 22.18 - 449.1087 0.5 21 C21H22O11 287.0570(86), 269.0448(54), -

Dihydrokaempferol 259.0603(66)a

hexoside I

88 Ampeloptin 22.27 - 319.0470 -3.4 13.6 C15H12O8 193.0153(100), 179.0005(35), -

153.0181(45), 125.0251(68)a

89 Myricetin galloyl- 22.72 - 631.1306 -0.2 6.8 C29H28O16 317.0675(100)a -

hexoside

90 7-O-Methyl-cyanidin- 22.74 615.1358 613.1196 0.5 2.5 C29H26O15 299.0568(100)a Kirby et al. (2013)

3-O-(2"galloyl)-

galactoside

91 Myricetin-hexose malic 22.85 - 595.1303 0.2 16.5 C26H28O16 479.1181(100), 369.0824(28), -

acid II 317.0683(35), 299.0572(42)a

92 Di-O-galloyl-3,4-(S)- 23.00 619.0950 - -3.3 8.1 C27H22O17 301.0716(100) Nishimura, Nonaka, and

hexahydroxydiphenoyl Nishioka (1984)

protoquercitol I

93 Di-O-galloyl-2,3-(S)- 23.05 771.1092 - -6.8 4.8 C34H26O21 153.0177(100) Nishimura et al. (1984)

hexahydroxydiphenoyl-

scyllo-quercitol II

94 Tetra-O-galloylhexoside I 23.07 789.1208 787.1008 -1 4.9 C34H28O22 635.0872(8), 169.0109(1)a Regazzoni et al. (2013)

95 Eriodictyol xyloyl- 23.41 - 565.1197 0.4 18.7 C25H26O15 287.0553(76)a -

deoxyhexose

96 Umbelliferone 23.46 163.0391 161.0241 2.2 9.4 C9H6O3 133.0299(100), 117.0341(61), -

105.0332(10)a

97 Trigalloyllevoglucosan 23.62 619.0945 - -2.4 33 C27H22O17 301.0713(37), 153.0182(100) Chen, and Bergmeier

VI (2011)

98 Isorhamnetin hexoside III 23.66 - 477.1030 1.7 32 C22H22O12 314.0576(8),313.0561(50)a -

99 Tetra-O-galloyl-scyllo- 23.74 731.1477 - -3.2 2.8 C33H30O19 301.0716(100),153.0179(7) Nishimura et al. (1984)

quercitol

100 Glycitein 7-O-glucoside 23.76 447.1282 - 0.8 23.7 C22H22O10 285.0768(100) -

101 Myricetin O- 23.86 627.1577 625.1409 0.3 6.1 C27H30O17 317.0311(3), 316.0198(5)a Regazzoni et al. (2013)

rhamnosylglucose

102 Ampelopsin glucoside 23.88 - 481.0995 -1.6 16.7 C21H22O13 319.0460(65), 301.0360(40), Yeom et al. (2003)

193.0144(100)a

103 Quercetin glucoside I 24.09 465.1017 - 2.3 13.8 C21H20O12 303.0512(100) Regazzoni et al. (2013)

104 Myricetin-hexose malic 24.11 597.1081 - 0.9 18.8 C25H24O17 319.0454(100) -

acid III

105 Myricetin-3-O- 24.20 495.0766 493.0625 -0.2 3.2 C21H18O14 317.0308(100)a Regazzoni et al. (2013)

glucuronide

106 Myricitin derivative 24.21 - 515.0451 3.2 11 C23H16O14 339.0125(23), 317.0307(100)a -

107 Myricitin derivative 24.23 657.1317 - 5.9 19.8 C27H28O19 319.0478(100) -

108 Myricetin-3-O- 24.40 481.0970 479.0826 1.1 6.6 C21H20O13 317.0291(28), 316.0243(76), Regazzoni et al. (2013)

glucoside 169.0144(26)a

109 Trigallic acid 24.43 - 473.0362 -0.2 2.4 C21H14O13 321.0262(22), 169.0147(100)a Nishimura et al. (1984)

110 Myricetin-hexose malic 24.48 597.1077 - 1.5 18.9 C25H24O17 319.0466(100) -

acid IV

111 Trigalloyllevoglucosan 25.12 619.0945 - -2.4 33 C27H22O17 301.0707(3), 153.0185(100) Chen, and Bergmeier

VII (2011)

112 Benzoic acid, 3,4,5- 25.14 - 393.0449 3.6 41.9 C17H14O11 317.0402(49), 241.0355(100), -

trihydroxy-2-oxo-1,3- 169.0144(76), 125.0240(9)a

propanediyl ester

113 Tetra-O-galloylhexoside II 25.15 789.1224 787.0992 0.9 2.3 C34H28O22 635.0871(5), 169.0130(1)a Regazzoni et al. (2013)

114 Horridin 25.25 595.1669 - -2 38 C27H30O15 433.1152(48), 301.0714(100) -

115 Pentagalloyl-hexoside I 25.39 941.1328 939.1081 3 9.5 C41H32O26 787.1001(4), 617.0767(6), Regazzoni et al. (2013)

465.0660(4), 393.0444(81),

317.0402(100), 241.0367(24),

169.0148(27)a

116 Trigalloyllevoglucosan 25.47 619.0973 617.0833 -7.9 41.6 C27H22O17 465.0710(6), 393.0458(73), Chen, and Bergmeier

VIII 317.0407(100), 241.0356(22), (2011)

169.0150(33)a

117 Mingjinianuronide B 25.55 563.1402 - -1.1 25.8 C26H26O14 301.0720(100) Tan and Zuo (1994)

118 Apiin I 25.74 565.1577 563.1385 3.7 13.0 C26H28O14 443.1033(8),413.0890(100)a Abu-Reidah et al. (2013)

119 Trigalloyllevoglucosan 25.77 619.0961 - -5.1 6.7 C27H22O17 301.0698(14),237.0422(4), -

IX 153.0186(100)

120 Apigenin 25.82 579.1710 - -0.2 45.6 C27H30O14 433.1151(100),271.0606(4) Matsuda (1966)

neohesperidoside I

Peak Tentative assignment No.

tR [M+H]+

(min.) (m/z)

[M-H]-

Error (ppm)

mSigma Molecular formula

MS2/MS fragment ionsb

Reference

121 Quercetin-3-O-(6"-3-hydroxy-3-methylglutaroyl)-â-galactoside

122 Spicoside E

25.84 593.1552 -

25.86 615.1353 -

45.0 C27H28O15

301.0721(100)

126 Isovitexin

26.23 433.1116 26.30 -26.38 465.1027

26.44 303.0158 -

Chrysoeriol-6-O-acetyl- 26.51 4'-ß-d-glucoside

Trigalloyllevoglucosan 26.53 IX

Quercetin-hexose malic 26.56 acid I

Eriodictyol hexoside or Dihydrokaempferol hexoside II

Quercetin glucoside II Quercetin glucuronide Kaempferol hexoside or Luteolin hexoside I Quercetin-hexose malic acid II

Quercetin glucoside III Pentagalloyl-hexoside

Kaempferol rutinoside I Kaempferol-hexose malic acid III Chrysoriol derivative Mangiferitin Pentagalloyl-hexoside

1,5-di-O-galloyl-3,4-(S)-

hexahydroxydiphenoyl

protoquercitol

Myricetin-rhamnose

malic acid

Dihydroxybenzoic

acetate-digallate I

Pentagalloyl-hexoside

Kaempferol rutinoside II

Methyl digallate I Kaempferol hexoside or Luteolin hexoside II

Quercetin arabinoside Apigenin

neohesperidoside II Methyl digallate II Kaempferol-hexose malic acid IV Kaempferol 3-glucuronide

26.71 26.88 27.03

505.1331 619.0966 581.1153

465.1026 479.0825 449.1086

27.45 27.49

27.64 27.84 27.86

465.1028 941.1320

595.1660 565.1208

657.1482 261.0394 939.1098

27.89 771.1085 -

28.16 28.18

28.33 28.38

28.40 28.43

28.75 28.96

581.1149

941.1317 595.1640

449.1086

435.0942 579.1717

337.0578 565.1210

-1.3 168.3 C29H26O15 303.0516(100)1153.0196(70)

123 Apiin II 25.97 565.1577 -

124 Rutin 26.01 611.1627 609.1441

125 Pentagalloyl-hexoside II 26.19 941.1325 939.1095

545.0892

127 Petunidin-3-O-glucoside pyruvate

128 Myricetin-3-O-rhamnoside

129 Digalloyl-hexoyl-ellagic 26.43 767.1437 765.0955 acid

130 Ellagic acid

3 8 0.1 -1.3 7.6

579.0984

449.1076

477.0670 447.0928

27.05 581.1151 579.0982

939.1096

563.1031

259.0240

579.0990 545.0544 939.1088

335.0403 447.0930

433.0760 577.1534

335.0412 563.1010

8.9 C26H28O14

6.1 C27H30O17

37.1 C41H32O26

37 C21H20O10

30 C21H20O12

5.6 C21H20O12

11.1 C35H26O20

5.1 C14H6O8

1.8 30.7 C24H24O12

3.7 24.8 C20H26O22

1.3 7.2 C25H24O16

2.9 59.3 C21H22O11

0.4 4.7 C21H20O12

0.9 6.6 C21H18O13

1.1 16.2 C21H20O11

1.7 10

C25H24O1

-0.2 21.4 C21H20O12 1.4 34.5 C41H32O26

-0.4 52.6 C27H30O15

2.1 11.1 C25H24O15

-4.8 11.5 C31H28O16

3.3 82.1 C13H8O6

1.2 9.8 C41H30O26

-5.9 4.8 C34H26O21

2.6 19 C25H24O16

5.3 42.7 C24H18O15

2.3 9.5 C41H32O26

2.9 15.7 C27H30O15

-0.4 7.2 C15H12O9

0.5 9.6 C21H20O11

3.6 18.8 C20H18O11

5.1 37.5 C27H30O14

-1 1.8 C15H12O9

5.8 21.2 C25H24O15

433.1116(99)1271.0643(6) 303.0512(100) 787.1003(5)1393.0445(42)1 169.0154(2)a

415.1022(6)1343.0762(10)1 313.0719(l00) 463.08781 316.0227(100)a

319.0460(100)

463.0869(25)1 300.9994(100)a

303.0149(42)1285.0055(39)1 275.0207(69)1257.0087(100)1 247.0288(35)1 229.0161(51)1 201.0187(33)1 173.0241(12) 301.0732(100)

301.0692(2)1 153.0187(100)

463.0864(100)1 301.0339(6)a

287.0560(100)1 151.0029(30)a

303.0511(100) 301.0358(100)a 285.0415(50)a

463.0879(100)1 301.0360(9)a

303.0514(100) 769.0887(6)1 617.0777(11)1 447.0572(7)1 393.0444(22)1 317.0402(25)1 169.0142(100)a 287.0567(100) 447.0930(100)1 285.0409(4)a

301.0726(100) 191.0312(30)a 393.0376(1)1 169.0142(100)

153.0186(100)

463.0873(100)1 316.0223(3)1 301.0345(1)a

393.0454(l00)1 317.0408(11)1 169.0136(3)a

393.0443(22)1 169.0135(3)a

287.0581(100)1 153.0223(8)

183.0302(100)a 285.0381(29)1 284.0318(77)a

301.0324(39)1 300.0261(100)a 269.0452(44)a

183.0303(100)a 447.0904(100)1285.0426(12)a

29.25 463.0902 -

-6.7 10.0 C2lHl8Ol2 287.0574(100)

Sari1 Heikki1 Sampo1 and

Ari (2006)

Albach1 Grayer1 Kite1 and

Jensen (2005) Abu-Reidah et al. (2013) Olchowik et al. (2012)

Sáenz-navajas et al. (2010)

Regazzoni et al. (2013) Wu et al. (2013) El Sissi et al. (1972)

Chandrashekar et al. (2005)

Shabana et al. (2011) and Regazzoni et al. (2013)

Regazzoni et al. (2013) Al Sayed et al. (2010) Buziashvili et al. (1970)

Shabana et al. (2011) and Regazzoni et al. (2013)

Regazzoni et al. (2013) Regazzoni et al. (2013)

Ding et al. (2009) Perestrelo et al. (2012)

Regazzoni et al. (2013) Nishimura et al. (1984)

Hahn and Fekete, 1954

Regazzoni et al. (2013)

Ding et al. (2009)

Shabana et al. (2011) Buziashvili et al. 1970 and Shrestha et al. (2012)

Buziashvili et al. (1970) Matsuda (1966)

Shabana et al. (2011) Perestrelo et al. (2012)

Al Sayed et al. (2010)

Peak No. Tentative assignment tR (min.) [M+H]+ (m/z) [M-H]-(m/z) Error (ppm) mSigma Molecular formula MS2/MS fragment ionsb Reference

158 Quercetin rhamnoside 29.30 449.1097 447.0925 1.9 3.5 C21H20O11 301.0350(100)a Regazzoni et al. (2013)

159 Dihydroxybenzoic 29.32 - 545.0546 5 32.1 C24H18O15 469.0489(100), 393.5454(21), Hahn and Fekete, 1954

acetate-digallate II 169.0144(44)a

160 Hexagalloyl-hexoside 29.42 - 1091.1192 2.4 3.9 Q8H36O30 939.0980(1), 769.0780(12), Regazzoni et al. (2013)

617.0649, 393.0443(39),

169.0140(34)a

161 Kaempferol-hexose 29.58 565.1152 - -3.7 11.0 C25H24O15 287.0549(100) Perestrelo et al. (2012)

malic acid V

162 Dihydroxybenzoic 29.62 - 545.0556 3.2 38.7 C24H18O15 469.0493(100), 393.5466(15), Hahn and Fekete, 1954

acetate-digallate III 169.0147(34)a

163 Apigenin glucuronide 29.90 447.0928 445.0765 -1.5 143.0 C21H20O11 271.0613(100)a -

164 Apigenin glucoside II 29.92 433.1143 431.0953 -3.2 62.8 C21H22O10 271.0618(100)a Shabana et al. (2011)

165 Camellianin A 30.81 621.1855 - -6.7 28 C29H32O15 433.1153(100), 313.0726(63), -

271.0648(8)

166 Genistein-hexose malic acid 31.08 549.1265 - 1.7 177.0 C25H24O14 271.0605(100) -

167 Galloyl-valoneic acid 31.11 623.1887 621.0596 -2.4 26.6 C22H22O21 469.5507(46), 393.5454(2), Sanz et al. (2010)

bilactone 169.0139(3)a

168 Quercetin-rhamnose 31.13 565.1089 563.1024 3.3 4.2 C25H24O15 447.0917(100), 301.0354(10)a -

malic acid I

169 Quercetin-rhamnose 31.40 565.0903 - -4.4 9.0 C28H20O13 303.0520(100) -

malic acid II

170 Myricetin 31.41 319.0457 317.0300 0.8 28.3 C15H10O8 287.0218(38), 271.0222(4), Regazzoni et al. (2013)

178.9985(85), 151.0036(87),

137.0240(34)a

171 Dihydroxybenzoic 31.42 - 545.0542 5.7 46.3 C24H18O15 393.0465(100), 169.0151(94)a Hahn and Fekete, 1954

acetate-digallate IV

172 Quercetin glucoside IV 31.48 465.1026 - 0.3 9.3 C21H20O12 303.0520(100), 129.0545(32) Regazzoni et al. (2013)

173 Quercetin-hexose malic 31.62 581.1151 - -2.4 45.4 C25H24O16 303.0691(100) -

acid III

174 Myricitrin O-gallate 31.80 617.1164 615.0988 0.6 30.5 C28H24O16 469.5507(33), 393.0439(10), Moharram et al. (2006)

317.0299(2), 169.0134(3)a

175 Kaempherol 31.92 433.1153 - -5.6 15.7 C21H20O10 287.0571(100) Shabana et al. (2011)

rhamnoside

176 Quercetin I 32.14 - 301.0346 2.5 12.8 C15H10O7 217.0060(2), 191.0389(1), Shabana et al. (2011)

151.0054(2)a and Kosar et al. (2007)

177 Quercetin-hexose malic 32.20 581.1132 - 0.8 46.4 C25H24O16 303.0524(100) -

acid IV

178 Isorhamentin hexose- 33.60 595.1376 - -13 49 C26H26O16 317.0700(100) -

malic acid

179 Kaempferol rhamnose- 33.80 - 547.1060 6.1 31.0 C25H24O14 431.0974(100), 285.0396(43)a -

malic acid

180 Homoprotocatechuic 34.15 169.0497 - -1.2 6.0 C8H8O4 141.0615(36), 126.0261(56), -

acid 108.0218(100), 95.0393(50)

181 Unknown 34.52 - 593.1327 -4.4 31.3 C30H26O13 513.1687(18), 441.1239(36)a -

182 Quercitrin 2" O-gallate 34.77 - 599.1008 5.8 25.0 C28H24O15 301.0358(100)a Moharram et al. (2006)

183 Isorhamnetin hexoside 34.81 - 477.1012 5.6 14.7 C22H22O12 315.0506(58), 314.0438(80)a -

184 IV Di-benzopyrano- 35.31 - 515.0429 7.4 52.0 C23H16O14 469.0477(34), 384.0422(42), -

furanacetic acid deriv. 303.0118(38), 169.0129(100)a

185 Luteolin 36.30 287.0562 285.0406 -0.6 7.1 C15H10O6 217.0486(2), 199.0418(2), Kim, Chung, Choi, and

175.0387(1), 151.0038(3), Park (2009)

133.0288(3)a

186 Quercetin II 36.57 303.0520 301.0352 0.6 2.3 C15H10O7 273.0399(13), 229.0504(3), Shabana et al. (2011)

178.9983(48), 151.0029(100), and Kosar et al. (2007)

121.0292(15)a

187 Quercetin dimer 36.59 - 603.0760 3.4 25 C30H20O14 301.0354(100)a -

188 Isorhamnetin hexoside V 36.60 - 477.1030 1.8 22.4 C22H22O12 315.0517(100), 271.0590(26)a -

189 Afzelin O-gallate 37.11 585.1265 583.1072 3.7 17.2 C28H24O14 297.0596(40), 285.0411(100), Moharram et al. (2006)

169.0108(7)a

190 Butein 38.91 273.0773 - -5.7 13.0 C15H12O5 142.9542(28), 163.0369(16), Lee et al. (2008)

137.0232(100)

191 Chrysoriol 40.16 301.0692 - 3.0 49.2 C16H12O6 286.0470(100), 258.0545(81) -

192 Kaempferol 40.22 287.0556 285.0404 0.3 10.0 C15H10O6 257.0437(1), 229.0526(1), Shabana et al. (2011)

213.0525(1), 201.0348(1),

151.0027(2)a

193 Hinokiflavone or 41.46 539.0992 537.0822 1.1 4.7 C30H18O10 541.2242(13), 425.2128(14), Van Loo et al. (1988)

Amenthoflavone or 417.0566(3), 375.0507(13)a

Agathisflavone I

194 Ascorbyl 41.60 387.2393 - -4 5.8 C20H34O7 121.1006(100) -

monomyristate

195 Dihydroxypalmitic acid 41.92 289.2393 287.2231 -6.7 11.1 C16H34O4 147.1175(49), 133.1016(73), -

121.1025(67), 109.1001(100)a

Peak Tentative assignment tR [M+H]+ [M-H]- Error mSigma Molecular MS2/MS fragment ionsb Reference

No. (min.) (m/z) (m/z) (ppm) formula

196 Hexadecadienoic acid 41.94 253.2180 - -7 1.6 C16H28O2 142.9508(100), 132.9601(58), -

109.1001(45), 95.0848(88)

197 Deacetylforskolin 42.12 369.2284 - -3.3 1.3 C20H32O6 253.2123(12), 235.2088(14), Zhang et al. (2009)

217.1924(18)

198 Hinokiflavone or 42.33 539.0996 537.0818 1.7 12 C30H18O10 425.2064(13)a Van Loo et al. (1988)

Amenthoflavone or

Agathisflavone II

199 Rhamnetin I 42.43 - 315.0505 0.5 17 C16H12O7 179.0352(100),164.0099(32)a Wollenweber (1974)

200 Unknown 42.54 405.2497 403.2315 -3.4 5.8 C20H38O8 323.2266(13), 305.2146(8), -

253.2189(100), 235.2055(87),

217.1956(53)a

201 Rhamnetin II 43.29 317.0675 315.0511 -0.1 6.1 C16H12O7 300.0279(27), 193.0141(17), Wollenweber (1974)

165.0195(100), 121.0285(17)a

202 Hinokiflavone or 46.86 539.0998 - -4.8 30.5 C30H18O10 - Van Loo et al. (1988)

Amenthoflavone or

Agathisflavone III

203 Vapiprost 50.57 478.2952 - 0.0 35 C30H39NO4 337.2748(100),306.2805(29) -

204 Sespendole 50.77 520.3416 - 0.9 36.4 C33H45NO4 184.0743(100),104.1077(31) -

205 Linoleic acid amide 51.17 280.2647 - -4.4 10.4 C18H33NO 109.1001(59),95.0837(100) -

206 Unknown 52.67 522.3587 - -0.7 33 C33H47NO4 184.0736(100) -

207 Linoleylhydroxamate I 53.04 296.2598 - -4.7 3.2 C18H33NO2 169.1235(100),95.0840(75) -

208 Unknown 53.17 522.3581 - -0.7 33 C18H33NO4 184.0743(100),104.1076(29) -

209 Linoleylhydroxamate II 53.44 296.2584 - -4.7 3.4 C18H33NO2 169.1235(100),95.0840(75) -

210 Betunolic acid I 55.12 455.3518 - 0.4 27.8 C30H46O3 437.3483(12), 419.3347(17), Shabana et al. (2011)

295.2454(12), 189.1606(45),

139.1118(100),121.0998(54)

211 Triterpenoid derivative 55.44 663.4616 - 0.5 51.7 C42H62O6 551.3333(80), 495.2626(100), -

439.2103(35)

212 Moroctic acid 55.66 277.2177 - -5.4 50.8 C18H28O2 149.0229(100) -

213 Vebonol 57.17 453.3384 - -4.7 9.1 C30H44O3 435.3301(32), 213.1652(27), -

201.1641(100)

214 Betunolic acid II 57.97 455.3535 - -3.3 2.5 C30H46O3 201.1633(100),187.1465(66), Shabana et al. (2011)

161.1301(87), 133.1010(81),

121.1001(79), 109.1015(55)

215 Deoxycorticosterone 58.73 493.2809 -- -2.7 5.3 C27H40O8 337.2781(43), 263.2339(31), -

glucoside 109.0987(70), 95.0850(100)

216 Dihydroisovaltrate 59.17 425.2170 - 0.0 15.6 C22H32O8 425.2103(38), 365.1975(64), -

281.1337(24)

217 Oxoglycyrrhetinic acid 59.70 469.3320 - -1.7 13.6 C30H44O4 337.2849(3), 221.1595(3), -

137.0970(100), 175.1419(8)

Rt: retention time. I, II, III... stand for isomers. a Fragmentation pattern in negative ionization mode. b Between parenthesis (relative intensity %).

chromatogram (BPC) in positive and negative ionisation modes together with the UV chromatogram at 280 nm in aqueous methanol extract of R. coriaria L.

The compounds detected in this work were tentatively characterised by means of MS data, together with the interpretation of the observed MS/MS spectra in comparison with those found in the literature. The formerly identified phytochemicals from the same botanical family or species have been also utilised in the identification when applicable. In the identification process, the following public databases were consulted: ChemSpider (http:// www.chemspider.com), SciFinder Scholar (https://scifind-er.cas.org), Kegg Ligand Database (http://www.genome.jp/kegg/ ligand.html), and Phenol-Explorer (www.phenol-explorer.eu). Commercial standards were not available for all the sumac pheno-lics and phytochemical compounds detected in this work.

3.1.2. Organic acids

At the beginning of analysis, several very polar compounds such as malic acid isomers and derivatives have been detected, in accordance with the literature; malic acid was reported to be the most abundant organic acid in R. coriaria (Kossah, Nsabimana, Zhang, Chen, 2010). Thus, compounds 2, 7 and 8 were proposed as malic acid isomers, while 3, 4, and 5 were suggested as glycosides of malic acid (Ley et al., 2006).

3.1.3. Phenolic acids and derivatives

In the present work we were able to characterise 9 phenolic acid derivatives, 3 of which (25, 35, 43) were detected in negative ionisation mode and show the neutral loss of a hexose moiety. Based on QTOF-MS analysis and MS/MS fragmentation pattern, these compounds were proposed as protocatechuic acid hexoside, syringic acid hexoside and coumaryl-hexoside, respectively. In positive ionisation mode a compound with a major fragment at m/z 355.1040 was assigned as caffeoylquinic acid (Fig. 2a), relying on the neutral loss of caffeic acid moiety (-162 Da) and the a product ion at m/z 193.0494 (quinic acid). Compound 12 (tR 6.75 min), is suggested as caftaric acid.

3.1.4. Phenolic compounds conjugated with malic acid derivatives

For the first time, in the present work, the methodology used

allowed us to identify 26 unusual phenolics conjugated with glyco-side-malic acid. This fragmentation pattern was previously described by Perestrelo et al. (2012). From MS and MS/MS fragmentation pattern data, a dominant neutral loss of 287 Da was observed, which may be attributed to the loss of hexose-malic acid moiety in all 26 detected compounds in both positive and negative ionisation modes. Compounds 27 and 29, with a precursor ion [M-H]- at m/z 447.0777 and with the identical formula C17H19O14, have been assigned as galloyl-hexose-malic acid

Fig. 1. HPLC-DAD/QTOF-MS base peak chromatograms (BPC) of: (A) MS in positive ion mode, (B) MS in negative ion mode, and (C) UV at 280 nm, for the hydro-methanol extract of sumac fruits.

Ints. ' 4000:

3000: 2000: 1000: 0

Ints. 3000' 2500 2000 1500' 1000' 500 0

[M-H-162]-319.0461

b. (102) Ampelopsin glucoside

[M-H]-481.0968

Ints. j-4000

[M+H-308]+

c. (120, 154) Kaempferol rutinoside

[M+H]+ 595.1641

x10 2.5 2.0 1.5 1.0 0.5 0.0

[M+H-308]+

d. (124) Rutin

[M+H]+ 611.1584

Ints. 6000

[M-H-152]-183.0302

e. (151, 155) Methyl digallate

[M-H]-335.0403

Fig. 2. MS2 spectra and structure of new phenolics detected in R. coriaria by QTOF-MS in NIM and PIM.

450 m/z

600 m/z

isomers. QTOF-MS analysis showed a product ion at m/z 331.0666, [M-H-116]~, implying the loss of malic acid (C4H4O4) to give a galloylhexose moiety, and a product ion at m/z 169.0153 representing gallic acid. Four digalloyl-hexose malic acid isomers (tR 13.55, 14.26, 15.35, and 16.68 min) were detected in ESI- mode. Loss of malic acid [M-H-116]~ from the precursor ion at m/z 483.0794 occurred giving a product ion at m/z 169.0142 (gallic acid).

The QTOF-MS analysis revealed the presence of five isomers of kaempferol hexose-malic acid in the ESI- and ESI + modes with ions at m/z 563.1010 and 565.1210, respectively. The appearance of fragment ions at m/z 447.0904, [M-H-116]~ and a product

ion at m/z 285.0426 corresponded to kaempferol (Perestrelo et al., 2012). Four isomers of myricetin-hexose malic acid (C25H24O17) were observed, as shown by the appearance of product ions at m/z 319.0466/317.0687, and corresponded to myricetin in structure after the neutral loss of 287 Da (hexose-malic acid moiety loss).

At 26.56, 27.05, 31.62 and 32.20 min pseudomolecular ions at m/z 581.1151/579.0982 were observed. In the MS/MS spectra, product ions at m/z 301.0360/303.0520 (quercetin) were observed. These isomers were assigned as quercetin-hexose malic acid. The product ion at m/z 463.0879 was proposed as quercetin hexose, in keeping with a previous report on sumac (Regazzoni et al.,

2013). Isorhamnetin hexose-malic acid was tentatively identified as compound 178, which showed a product ion at m/z 317.0700, which corresponds to neutral loss of hexose-malic acid moiety [M+H-278]+, giving the isorhamnetin aglycone. Compound 179 was suggested as kaempferol rhamnose-malic acid.

3.1.5. Flavonoids derivatives

A total of 61 flavonoid derivatives were detected and characterised in sumac.

Five isomers showed a molecular ion at m/z 479.1167/477.1030, with a product ion at 317.0671/315.0506 (corresponding to isorhamnetin in structure) in the MS/MS spectra. Based on the MS and MS/MS spectra, compounds 64, 67, 98,183, and 188 are suggested as isorhamnetin hexosides. These compound are being suggested as components of sumac for the first time.

Apigenin-7-O-(6"-O-galloyl)-ß-D-glucopyranoside is proposed for compound 77 (m/z 585.1241, [M+H]+). In the MS/MS spectra, the loss of hexose and galloyl (-314 Da) moieties gave a fragment ion at m/z 271.0618, which corresponds to apigenin in structure. This compound was reported as an active compound in Euphorbia humifusa (Tian et al., 2010). In the same manner, compound 89 was tentatively proposed as dihydrotamarixetin galloyl-hexoside.

Compound 102 ([M - H]- at m/z 481.0995) has been tentatively assigned as ampelopsin glucoside (Yeom et al., 2003). MS/MS spectrum of this compound has shown the characteristic product ion at m/z 319.0460 (Fig. 2b).

Two compounds (118 and 123) had pseudomolecular ions at m/ z 565.1577/563.1385. Based on QTOF-MS data and the previous literature (Abu-Reidah et al., 2013a), these compounds have been characterised as apiin isomers, apigenin glycoside derivatives. These isomers were not observed previously in sumac. Compound 126 is suggested as isovitexin, identified for the first time in sumac; the [M+H]+ ion at m/z 433.1116 produced fragment ions at m/z 415.1022, 343.0762, 313.0719, corresponding to the C-glycoside fragmentation pattern (Abu-Reidah et al., 2013).

The glucuronated form of quercetin at 26.88 min, has molecular ions at m/z 479.0825/477.067 and had an MS/MS fragment ion at m/z 301.0358, which is due to the loss of glucoronic acid [M-H-176]- and the presence of quercetin; it is reported for the first time in sumac. A main ion at m/z 505.1331 was detected by ESI-. Furthermore, MS/MS revealed a product ion at m/z 301, corresponding to chrysoeriol in structure. Thus, compound 131 was characterised as chrysoeriol-6-O-acetyl-40-ß-D-glucoside (Chandrashekar, Arun, & Satyanarayana, 2005). Compounds 87 and 134 with the same MS and MS/MS data were tentatively assigned as eriodictyol hexoside or dihydrokaempferol hexoside isomers. Two compounds (tR 27.54 and 28.31 min) with [M+H]+ at m/z 595.1640, (C27H31O15), gave a fragment ion at m/z 287.0581, corresponding to kaempferol aglycone in structure. Thus, 141 and 150 were identified as kaempferol rutinosides (Fig. 2c). These compounds were previously identified in leaves of R. sylvestris (Ding, Nguyen, Choi, Bae, & Kim, 2009).

A precursor ion of m/z 579.1717/577.1534 at retention times of 25.82 and 28.43 min, gave fragment ions at m/z 269.0452 (apige-nin). Compounds 120 and 154 have been proposed as isomers of apigenin neohesperidoside, a compound already found in leaves of other species of Rhus (Matsuda, 1966). Rutin was suggested for the precursor ion at m/z 611.1627/609.1441. The MS and MS/MS spectra showed a product ion [M+H]+ at m/z 303.0512 (quercetin) (Fig. 2d). This compound has been already described in R. typhina leaves (Olchowik et al., 2012). Compound 163 was proposed as apigenin glucuronide. In the same manner, apigenin glucoside has been suggested for compounds 65 and 164. In the MS/MS spectra, both compounds had the fragment ion at m/z 271.0613, indicating the existence of apigenin in the structure.

An [M-H]- ion at m/z 599.1008 gave a product ion at m/z 301.0358 with 100% relative intensity. This compound was assigned as quercitrin 200-O-gallate (Fig. 3). Similarly, compounds 189 and 174 were proposed as afzelin O-gallate and myricitrin O-gallate, respectively. These three compounds were described in Calliandra haematocephala (Moharram, Marzouk, Ibrahim, & Mabry, 2006). As far as we know, these compounds are reported herein in sumac for the first time. Two isomers (199 and 201) with the precursor ion at m/z 317.0675/315.0511 have been assigned as rhamnetin (Wollenweber, 1974).

3.1.6. Hydrolysable tannins derivatives

In this work, it was found that hydrolysable tannins derivatives are the most abundant compounds in sumac. Thus, 74 compounds have been characterised in this class.

Five isomers had a pseudomolecular ion at m/z 331.0647 in the ESI- mode. Compounds 13,14,16,18, and 28, have been characterised as galloylhexose, based on the data obtained by MS and MS/ MS data, and literature already cited (Fröhlich, Niemetz, & Gross, 2002). To the best of our knowledge, this is the first characterisation of these compounds in R. coriaria. Compound 112 had a molecular ion at m/z 393.0449, and was proposed as benzoic acid, 3,4, 5-trihydroxy-, 2-oxo-1,3-propanediyl ester. Five isomers (19, 22, 41, 45, and 56) were tentatively characterised as O-galloylnor-bergenin isomers.

Five compounds (tR11.40, 11.92, 16.10, 16.49, and 17.50 min) with the precursor ion at m/z 485.0949/483.0793 have been assigned to digalloyl-hexoside relying on the MS and MS/MS spectra that showed product ions at m/z 331.067[M-H-162]-, and 169.0143[M-H-162-152]- corresponding to the neutral losses of hexose and galloyl moieties, respectively. These compounds have been noted in R. typhina leaves (Fröhlich et al., 2002), but for the first time in R. coriaria.

QTOF-MS revealed two isomers at m/z 325.0567 having the same molecular formula C14H13O9. MS/MS spectral data showed a product ion at m/z 169.0145, which is due to the neutral loss of shikimate moiety [M-H-156]-, and the appearance of gallic acid. Based on these data, compounds (32 and 37) were proposed for the first time in sumac, as galloylshikimic acid. Two compounds (151 and 155) had a precursor ion at m/z 337.0578/335.0412 and a fragment ion at m/z 183.0303 (Shabana, El Sayed, Yousif, El Sayed, & Sleem, 2011). These isomers have been suggested to be methyl digallate isomers (Fig. 2e).

Two compounds at 14.71 and 16.62 min exhibited molecular ions at m/z 343.0691 and were assigned to galloylquinic acid. The product ion in the MS/MS spectrum was at m/z 191.0570 corresponding to quinic acid in structure, a fragment ion at m/z 169.0139 suggested gallic acid. Compounds (48, 58, 62, 71, 83, 97, 111, 116, and 119) are proposed to be isomers of trigalloyllev-oglucosan. Two hydrolysable tannin isomers (53 and 60) showed a molecular ion at m/z 323.0403/321.0260. Based on the MS and MS/ MS data and previous literature (El Sissi, Ishak, & Abd El Wahid, 1972), these compounds were assigned to digallic acid.

The compound (tR 22.72 min) with the molecular formula C29H27O16 and having the precursor ion at m/z 631.1306 in the ESI- mode, was been tentatively proposed as myricetin galloyl-hexoside. In the MS/MS spectrum, this compound produced a fragment ion at m/z 317.0675 [M-H-314]-; (314 Da) is referred to gallic acid + hexose moiety loss. Fragment ions at m/z 321.0262 and 169.0147 resulted after the successive loss of gallic acid moieties from a main ion at m/z 473.0362 in the QTOF-MS analysis. This compound was assigned as trigallic acid, not previously reported in R. coriaria. Notably, this compound was discussed in Toona sinensis (Wang, Yang, & Zhang, 2007).

A compound (78) with molecular ion [M+H]+ at m/z 425.1066 was proposed to be O-galloyl arbutin. The fragment ion at m/z

Ints. x104

Intens. x104 ■

2.0 ■ 1.5 ■ 1.0 ■ 0.5 ■ 0.0 ■

-MS2(599.1017)

-i-1—I-1—I-1-r-

Jl/'Vn___A<JAA

EIC 599.100

-1-1-1—r

1—'—

-1-1-1—

—I—

—I-1-1-1-1-1-r

—I—I—I—Ï—I—\—I

40 Time [min]

Fig. 3. Extracted ion chromatogram (EIC) together with the fragmentation pathway for the ion separated by HPLC/QTOF-MS at tR 34.77 min, m/z 599.1008.

273.0707 was characteristic of arbutin. This compound has been described in the Anacardiaceae family (Shi, & Zuo, 1992). Compound 129 was tentatively suggested as digalloyl-hexoyl-ellagic acid (Wu, McCallum, Wang, Liu, Zhu, & Tsao, 2013). The precursor ion found at m/z 941.1328/939.1081 was assigned to pentagalloyl-hexoside for five isomers 115, 125, 140, 145, and 149. Similarly, compound 160 (tR 29.42 min) was suggested as hexagalloyl-hexo-side. The characterisation was based on the acceptable MS and MS/ MS data, in addition to the literature cited on sumac leaves (Regazzoni, Arlandini, Garzon, Santagati, Beretta, & Facino, 2013).

Four isomers with a molecular ion at m/z 545.0556 in ESI- mode were tentatively identified as dihydroxybenzoic acetate-digallate (Hahn and Fekete, 1954). Compound 167 gave a precursor ion at m/z 623.1887/621.0596 in the MS spectrum. However, in MS/MS spectrum, we observed a neutral loss of galloyl moiety [M-H-152]- which yielded the product ion at m/z 469.5507, indicating valoneic acid bilactone in structure (Sanz et al., 2010). Therefore, the compound has been assigned to galloyl-valoneic acid bilactone.

Compounds 193, 198, and 202 showed a precursor ion at m/z 539.0996/537.0818 in ESI+ and ESI- modes. These compounds have been already noticed in R. coriaria leaves and they are being reported herein in the fruits for the first time. By the method used, it was possible to characterise the compounds by their acceptable data from MS and MS/MS together with the literature cited (Van Loo et al., 1988) as isomers of hinokiflavone or amenthoflavone or agathisflavone.

3.1.7. Anthocyanins and derivatives

A total of six anthocyanin derivatives have been detected in R. coriaria fruits. Thus, compound 70 (tR 19.65 min) with product ions

at m/z 601.1186/599.1039, had a fragment ion at m/z 287.0557/ 285.0405, indicating cyanidin in structure. So, this compound was proposed as cyanidin-3-O-(2"galloyl)-galactoside (Kirby, Wu, Tsao, & McCallum, 2013). Two isomers (73 and 75) with the precursor ion at m/z 631.1301 had the molecular formula C29H27O16. These compounds were assigned to 7-O-methyl-delphinidin-3-O-(2"galloyl)-galactoside (Kirby et al., 2013), the product ion at m/z 317.0650 indicates methyl-delphinidin aglycone yielded after the neutral loss of galloyl-galactoside moiety.

The compounds 81 and 90 possessed a fragment ion at m/z 299.0568 and were characterised as 7-O-methyl-cyanidin-3-O-galactoside and 7-O-methyl-cyanidin-3-O-(2"-galloyl)-galactoside, respectively; both of them were described in R. typhina (Kirby et al., 2013).

3.1.8. Isoflavonoid derivatives

Two isoflavonid derivatives have been detected in the sumac sample analysed. Compound 100 was proposed as glycitein-O-glu-coside on the basis of its MS spectra, which showed the main ion [M+H]+ at m/z 447.1282 and an MS/MS fragment ion at m/z 285.0768 (glycitein), this latter ion was obtained after a neutral loss of glucose moiety. This compound has never been reported previously in sumac. At 33.80 min, one molecular ion [M-H]~ at m/z 547.1060 was detected and characterised as oxoglycyrrhetinic acid.

3.1.9. Terpenoid derivatives

A couple of isomers (tR 55.12 and 57.97 min) showed a precursor ion [M+H]+ at m/z 455.3518. These compounds were assigned to betunolic acid, an already identified compound in sumac leaves (Shabana et al., 2011). This compound was discussed in other

sumac species to have antiviral activity (anti-HIV) (Wang et al., 2008).

One diterpene derivative showed a molecular ion [M+H]+ at m/z 369.2284. This compound was postulated as deacetylforskolin (Zhang, Luo, Wang, Lu, & Kong, 2009). Oxoglycyrrhetinic acid was tentatively identified as the compound detected at 59.70 min with [M+H]+ at m/z 469.3320.

3.1.10. Other compounds

Other compounds were also characterised in sumac, like butein (compound 190), a bioactive chalcone which was found in other species of Rhus (Lee et al., 2008), but we report it in this work for the first time in R. coriaria. Iridoid and coumarin derivatives (52 and 96) were detected and tentatively characterised as penstemide and umbelliferone, respectively.

4. Conclusion

It has been established in this work that HPLC-DAD/QTOF-MS is a powerful analytical technique for the separation and detection of phenolics and other phytochemicals in R. coriaria L. Consequently, by using this method, a total of 211 compounds were tentatively identified in sumac, based on accurate mass determination of the deprotonated/protonated ions which were obtained from the MS data and MS/MS fragmentation pattern, besides other relevant bibliographic information. To our knowledge, this work marks the first extensive study of the phenolic and other phytochemical components from sumac fruit (epicarps) extract. In this context, the obtained data indicate qualitatively that sumac is an abundant source of bioactive phytochemicals. The obtained results could explain the past and current usage of R. coriaria L. as a food spice, as well as support the widespread uses of sumac in health, nutrition and pharmacology and as a source of functional ingredients.

Acknowledgments

This research was partly funded by the European Union under the ENPI CBC MED Program and is a collaborative international project ref. no. I-B/1.1/288. This work was also supported by the project AGL2011-29857-C03-02 (Spanish Ministry of Science and Innovation), as well as P10-FQM-6563 and P11-CTS-7625 (Andalu-sian Regional Government Council of Innovation and Science), and A1/041035/11 (Spanish Agency for International Development Cooperation).

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