Metabolite Fingerprinting and Profiling of the Medicinal Grass Eleusine indica Based on HPLC-DAD, UPLC-DAD-MS/MS and NMR Analyses

Peñaloza, Evelyn M. C.; Casanova, Livia M.; Leal, Ivana C. R.; Aguiar, Paula F. de; Costa, Sônia S.

doi:10.21577/0103-5053.20180131

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Articles • J. Braz. Chem. Soc. 29 (12) • Dec 2018 • https://doi.org/10.21577/0103-5053.20180131 copy

Metabolite Fingerprinting and Profiling of the Medicinal Grass Eleusine indica Based on HPLC-DAD, UPLC-DAD-MS/MS and NMR Analyses

Authorship SCIMAGO INSTITUTIONS RANKINGS

Aqueous preparations of the grass Eleusine indica are used for treating malaria and lung infections. Despite its widespread occurrence and therapeutic potential, little is known about its chemical composition. This study reports a common chemical pattern for aqueous extracts of E. indica samples from four different localities, separated from each other by approximately 75 to 1340 km, in a wide variety of abiotic and biotic factors. High-performance liquid chromatography with diode array detection (HPLC-DAD), ultra-performance liquid chromatography with diode array detection and mass spectrometry (UPLC-DAD-MS/MS), and nuclear magnetic resonance (NMR) were the analytical techniques applied to characterize substances from E. indica, from each locality. Principal component analysis (PCA) confirmed that E. indica specimens came from four different localities. However, all of the four populations showed a common peaks pattern. This is the first chemical profile report of E. indica. Moreover, p-coumaric acid and isoschaftoside were characterized for the first time in this species.

Keywords:
Eleusine indica; aqueous extracts; fingerprinting and metabolite profiling; phenolic compounds; geographical variation

Locality (State)	Code	Altitude, a.s.l. / m	GPS coordinate / degree		Date of collection	Voucher specimennumber^a a All specimens were deposited in Herbarium at the Institute of Biology at the Federal University of Rio de Janeiro (UFRJ). a.s.l.: above sea level; MG: Minas Gerais State; RJ: Rio de Janeiro State; RS: Rio Grande do Sul State. Distance (km) between the four localities based on their GPS coordinates: PO - BH (1338.74 km), PO - BP (1113.80 km), PO - IF (1121.82 km), BH - IF (338.75 km), BH - BP (287.06 km) and IF - BP (74.87 km).
Locality (State)	Code	Altitude, a.s.l. / m	Latitude	Longitude	Date of collection
Belo Horizonte (MG)	BH	858	-19.894050	-44.004100	April, 2015	RFA 40.768
Barra do Piraí (RJ)	BP	363	-22.470278	-43.826111	December, 2015	RFA 40.765
Ilha do Fundão (RJ)	IF	5	-22.853056	-43.225833	January, 2016	RFA 40.766
Porto Alegre (RS)	PO	80	-30.083056	-51.096389	March, 2016	RFA 40.767

Peak	t_R / min	UV λ_max / nm	Mean ± SE (RSD / %) / (mg per 100 g)
Peak	t_R / min	UV λ_max / nm	Belo Horizonte (BH)	Barra do Piraí (BP)	Ilha do Fundão (IF)	Porto Alegre (PO)
1^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	3.29	265, 270 sh	92.7 ± 0.6 (2.1)	n.q.	n.q.	54.6 ± 0.9 (4.6)
2^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	3.81	273	47.1 ± 0.5 (3.4)	n.q.	n.q.	53.6 ± 5.0 (26.6)
3^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	5.16	258	n.q.	49.4 ± 0.9 (5.7)	n.q.	177.9 ± 35.7 (56.8)
4^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	7.09	253, slope	n.q.	n.q.	46.8 ± 0.7 (4.6)	85.0 ± 10.2 (34.1)
5^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	7.61	274, slope	n.q.	40.0 ± 0.3 (2.5)	83.3 ± 1.8 (5.9)	40.1 ± 5.3 (37.6)
6^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	8.19	268	n.q.	37.2 ± 0.8 (5.9)	n.q.	100.5 ± 7.4 (20.7)
7 ^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	8.68	253, 273 sh	63.4 ± 1.2 (5.5)	50.8 ± 2.2 (12.3)	49.1 ± 0.6 (3.2)	75.7 ± 5.9 (22.4)
8 ^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	9.03	261	62.4 ± 0.1 (0.6)	164.9 ± 2.1 (3.6)	255.4 ± 1.3 (1.5)	121.4 ± 29.9 (69.7)
9 ^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	9.36	257	70.6 ± 2.2 (9.3)	43.8 ± 2.2 (14.4)	55.7 ± 0.7 (3.8)	103.2 ± 3.9 (10.8)
10 ^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	9.75	253, 272 sh	75.6 ± 0.8 (3.2)	101.4 ± 1.5 (4.2)	91.7 ± 0.8 (2.6)	104.4 ± 0.7 (1.8)
11^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	10.05	256	n.q.	55.4 ± 1.4 (7.2)	55.9 ± 0.3 (1.8)	91.1 ± 7.1 (22.0)
12^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	10.28	262	n.q.	46.1 ± 0.6 (3.5)	50.8 ± 0.6 (3.1)	49.1 ± 5.3 (30.6)
13^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	10.51	260	n.q.	n.q.	n.q.	52.0 ± 2.1 (11.5)
14 ^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	10.83	253, 275 sh	42.9 ± 0.4 (2.6)	94.4 ± 1.9 (5.6)	191.3 ± 3.6 (5.3)	174.9 ± 37.0 (59.8)
15^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	11.01	258	n.q.	41.5 ± 0.1 (0.6)	n.q.	46.9 ± 1.4 (8.4)
16 ^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	11.35	257	58.6 ± 0.8 (4.3)	121.7 ± 1.8 (4.2)	195.9 ± 0.6 (0.9)	121.2 ± 22.4 (52.3)
17^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	11.84	278	n.q.	52.8 ± 0.4 (2.2)	41.8 ± 0.2 (1.6)	n.q.
18^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	12.40	265	n.q.	37.9 ± 0.3 (2.0)	52.6 ± 0.2 (1.2)	40.8 ± 5.8 (40.4)
19^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	12.68	266	n.q.	67.7 ± 1.1 (4.6)	41.2 ± 0.1 (0.7)	n.q.
20 ^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	14.72	278	87.4 ± 0.9 (3.3)	57.8 ± 0.5 (2.6)	44.6 ± 0.1 (0.9)	38.1 ± 1.4 (10.8)
21 ^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	17.40	255, slope	61.4 ± 0.7 (3.5)	52.1 ± 0.4 (1.9)	42.9 ± 0.3 (1.9)	50.4 ± 0.8 (4.4)
22 ^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	17.98	266, 291	51.2 ± 0.3 (1.9)	53.4 ± 0.4 (2.2)	58.9 ± 0.2 (0.8)	72.3 ± 0.5 (1.8)
23 ^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	18.38	280	106.4 ± 0.7 (2.1)	60.5 ± 0.7 (3.3)	84.7 ± 0.1 (0.3)	110.6 ± 0.9 (2.3)
24 ^a a Peaks of group I: benzoic acid derivatives, amino acids and nucleosides (λmax 250-280 nm) were expressed as 1 mg of gallic acid per 100 g of lyophilized extract;	19.07	265, 285	41.5 ± 0.2 (1.7)	37.1 ± 0.3 (2.6)	39.0 ± 0.6 (4.3)	48.1 ± 1.0 (6.8)
25^c c peaks of group III: flavone derivatives (λmax 310-335 nm for band I and 260-280 nm for band II) were expressed as 1 mg of vitexin per 100 g of lyophilized extract. tR: retention time, each value is mean ± 0.05 (min). RSD: percentage of relative standard deviation are in parentheses; n.q.: not quantified; sh: shoulder. Each value content in the table is the mean of inter- and intra-day replicate analysis (n = 3 days, 3 replicates per day) ± standard error (SE). All 35 peaks were detected by HPLC-DAD, but only 15 (in bold: 7-10, 14, 16, 20-26, 30 and 32) were quantified for the four localities.	20.69	272, 334	42.8 ± 0.1 (0.3)	66.2 ± 1.4 (5.9)	110.0 ± 0.4 (1.1)	92.2 ± 0.4 (1.2)
26^c c peaks of group III: flavone derivatives (λmax 310-335 nm for band I and 260-280 nm for band II) were expressed as 1 mg of vitexin per 100 g of lyophilized extract. tR: retention time, each value is mean ± 0.05 (min). RSD: percentage of relative standard deviation are in parentheses; n.q.: not quantified; sh: shoulder. Each value content in the table is the mean of inter- and intra-day replicate analysis (n = 3 days, 3 replicates per day) ± standard error (SE). All 35 peaks were detected by HPLC-DAD, but only 15 (in bold: 7-10, 14, 16, 20-26, 30 and 32) were quantified for the four localities.	20.93	272, 330	11.8 ± 0.5 (13.6)	9.8 ± 0.6 (16.2)	25.7 ± 0.2 (2.2)	26.5 ± 0.2 (2.2)
27^c c peaks of group III: flavone derivatives (λmax 310-335 nm for band I and 260-280 nm for band II) were expressed as 1 mg of vitexin per 100 g of lyophilized extract. tR: retention time, each value is mean ± 0.05 (min). RSD: percentage of relative standard deviation are in parentheses; n.q.: not quantified; sh: shoulder. Each value content in the table is the mean of inter- and intra-day replicate analysis (n = 3 days, 3 replicates per day) ± standard error (SE). All 35 peaks were detected by HPLC-DAD, but only 15 (in bold: 7-10, 14, 16, 20-26, 30 and 32) were quantified for the four localities.	22.18	269, 323	n.q.	15.7 ± 0.4 (7.8)	n.q.	n.q.
28^b b peaks of group II: cinnamic acid derivatives (λmax 295-330 nm) were expressed as 1 mg of p-coumaric acid per 100 g of lyophilized extract;	22.61	312	n.q.	37.5 ± 0.5 (4.0)	38.1 ± 0.4 (2.9)	n.q.
29^b b peaks of group II: cinnamic acid derivatives (λmax 295-330 nm) were expressed as 1 mg of p-coumaric acid per 100 g of lyophilized extract;	22.94	307	n.q.	24.2 ± 0.3 (3.6)	25.4 ± 0.1 (0.6)	n.q.
30 ^b b peaks of group II: cinnamic acid derivatives (λmax 295-330 nm) were expressed as 1 mg of p-coumaric acid per 100 g of lyophilized extract;	23.71	309	60.6 ± 1.2 (5.9)	39.5 ± 0.2 (1.6)	53.5 ± 0.9 (5.2)	63.1 ± 7.7 (34.5)
31^c c peaks of group III: flavone derivatives (λmax 310-335 nm for band I and 260-280 nm for band II) were expressed as 1 mg of vitexin per 100 g of lyophilized extract. tR: retention time, each value is mean ± 0.05 (min). RSD: percentage of relative standard deviation are in parentheses; n.q.: not quantified; sh: shoulder. Each value content in the table is the mean of inter- and intra-day replicate analysis (n = 3 days, 3 replicates per day) ± standard error (SE). All 35 peaks were detected by HPLC-DAD, but only 15 (in bold: 7-10, 14, 16, 20-26, 30 and 32) were quantified for the four localities.	24.42	285, 320 sh	6.5 ± 0.3 (15.9)	12.5 ± 0.1 (2.6)	6.0 ± 0.1 (2.6)	n.q.
32^c c peaks of group III: flavone derivatives (λmax 310-335 nm for band I and 260-280 nm for band II) were expressed as 1 mg of vitexin per 100 g of lyophilized extract. tR: retention time, each value is mean ± 0.05 (min). RSD: percentage of relative standard deviation are in parentheses; n.q.: not quantified; sh: shoulder. Each value content in the table is the mean of inter- and intra-day replicate analysis (n = 3 days, 3 replicates per day) ± standard error (SE). All 35 peaks were detected by HPLC-DAD, but only 15 (in bold: 7-10, 14, 16, 20-26, 30 and 32) were quantified for the four localities.	24.78	267, 324	8.4 ± 0.2 (8.2)	34.6 ± 1.5 (12.5)	42.7 ± 0.3 (2.3)	41.6 ± 1.4 (9.6)
33^c c peaks of group III: flavone derivatives (λmax 310-335 nm for band I and 260-280 nm for band II) were expressed as 1 mg of vitexin per 100 g of lyophilized extract. tR: retention time, each value is mean ± 0.05 (min). RSD: percentage of relative standard deviation are in parentheses; n.q.: not quantified; sh: shoulder. Each value content in the table is the mean of inter- and intra-day replicate analysis (n = 3 days, 3 replicates per day) ± standard error (SE). All 35 peaks were detected by HPLC-DAD, but only 15 (in bold: 7-10, 14, 16, 20-26, 30 and 32) were quantified for the four localities.	26.16	290 sh, 322	18.0 ± 0.2 (2.9)	n.q.	12.4 ± 0.7 (15.8)	25.0 ± 1.7 (19.8)
34^c c peaks of group III: flavone derivatives (λmax 310-335 nm for band I and 260-280 nm for band II) were expressed as 1 mg of vitexin per 100 g of lyophilized extract. tR: retention time, each value is mean ± 0.05 (min). RSD: percentage of relative standard deviation are in parentheses; n.q.: not quantified; sh: shoulder. Each value content in the table is the mean of inter- and intra-day replicate analysis (n = 3 days, 3 replicates per day) ± standard error (SE). All 35 peaks were detected by HPLC-DAD, but only 15 (in bold: 7-10, 14, 16, 20-26, 30 and 32) were quantified for the four localities.	28.10	268, 330	n.q.	13.0 ± 0.3 (5.5)	19.1 ± 0.1 (0.8)	n.q.
35^c c peaks of group III: flavone derivatives (λmax 310-335 nm for band I and 260-280 nm for band II) were expressed as 1 mg of vitexin per 100 g of lyophilized extract. tR: retention time, each value is mean ± 0.05 (min). RSD: percentage of relative standard deviation are in parentheses; n.q.: not quantified; sh: shoulder. Each value content in the table is the mean of inter- and intra-day replicate analysis (n = 3 days, 3 replicates per day) ± standard error (SE). All 35 peaks were detected by HPLC-DAD, but only 15 (in bold: 7-10, 14, 16, 20-26, 30 and 32) were quantified for the four localities.	29.52	269, 330	n.q.	n.q.	25.7 ± 0.1 (1.1)	n.q.

Plant organ (Eleusine indica)	Geographic origin	Peaks grouped by PCA (designation by UV spectrum)^a a Roman numerals designated (I, II and III) for both aerial and underground parts are according to classification of their UV-spectrum (I: benzoic acid derivatives, aromatic amino acids and nucleosides (270 nm); II: cinnamic acid derivatives (300 nm); III: flavone derivatives (330 nm)). Localities: BH: Belo Horizonte, Minas Gerais State; BP: Barra do Piraí and IF: Ilha do Fundão, Rio de Janeiro State; and PO: Porto Alegre, Rio Grande do Sul State, Brazil. Numbers in bold were common to the four localities and were quantified
Aerial parts	BH	20 (I), 21 (I), 1 (I)
	BP	31 (III), 19 (I), 17 (I)
	IF	5 (I), 8 (I), 14 (I), 18 (I), 16 (I), 25 (III), 26 (III), 35 (III)
	PO	2 (I), 3 (I), 4 (I), 6 (I), 7 (I), 9 (I), 23 (III), 24 (I), 30 (II), 33 (III)
Underground parts	BH	19 (I), 20 (I), 21 (II), 22 (III)
	BP	2 (I), 3 (I), 6 (I), 8 (I), 11 (I), 13 (I)
	IF	4 (I), 7 (I), 16 (I)
	PO	17 (I), 18 (I)

Peak (plant organ)^a a Plant organs of Eleusine indica are abbreviated by letter code: AP: aerial parts and UGP: underground parts;	t_R / min	UV / nm	Molecular ion [M - H]-	MS/MS fragment ion^d d numbers in parentheses show the relative abundance of each MS/MS fragment expressed in percentage. tR: retention time.	Compound	Reference
25 (AP)	18.89	214, 272, 333	563.25^b b confirmation by MS/MS using electrospray ionization (ESI);	545.17 (7.38), 473.08 (60.86), 443.08 (100), 383.17 (65.28), 353.17 (66.88), 325.17 (2.80)	schaftoside	standard
26 (AP)	19.19	213, 274, 320	563.17^b b confirmation by MS/MS using electrospray ionization (ESI);	545.17 (9.50), 503.08 (28.33), 473.08 (65.02), 443.17 (61.63), 383.08 (86.47), 353.08 (100)	isoschaftoside	Picariello et al.²⁶26 Picariello, G.; Sciammaro, L.; Siano, F.; Volpe, M. G.; Puppo, M. C.; Mamone, G.; Food Res. Int. 2017, 99, 730.
30 (AP), 21 (UGP)	22.61	219, 309	163.00^c c confirmation by MS/MS using atmospheric pressure chemical ionization (APCI);	119.0	p-coumaric acid	standard
32 (AP)	23.39	214, 269, 324	431.08^b b confirmation by MS/MS using electrospray ionization (ESI);	341.00 (11.49), 311.08 (100), 283.08 (8.52)	vitexin	standard

Metabolite		¹H NMR d (multiplicity, J (in Hz))^a a The chemical shifts of the signals are given in bold types. Multiplicity: s, singlet; d, doublet; t, triplet; q; quintet; dd, doublet of doublets; dt, doublet of triplets; m, multiplet. Samples of aerial and underground parts of E. indica used: 20 mg (Ei)/0.7 mL D2O at 25 ºC / d ¹³C
Metabolite		Aerial parts	Underground parts
Lipid
1	fatty acid derivatives	0.85 / 13.22; 0.88 / 20.75; 0.92 (m) / 10.98 / 18.49; 0.94 (m) / 13.44 / 20.73; 1.28 / 21.26 / 30.55; 1.30 / 25.43; 2.34 (d, 7.1) / 28.87 / 35.54	0.87 / 14.32; 0.86 / 13.06; 0.88 / 20.50; 0.93 / 10.85 / 13.13; 1.28 / 21.7 / 30.60; 1.30 / 24.12 / 28.18
Amino acid
2	alanine	1.47 (d, 7.2) / 16.02; 3.76 (m) / 54.29	1.47 (d, 7.0) / 15.87; 3.77 (m) / 55.49
3	arginine	1.70 (dt, 13.7, 7.7) / 26.1; 1.89 (t, 7.3) / 29.75; 3.24-3.22 (m) / 40.36; 3.77 (dt, 8.6, 3.5) / 54.29	1.67; 1.9 / 29.6; 3.23 / 40.22; 3.76 / 54.73
4	aspartate / aspartic acid	2.73-2.69 (m) / 56.73; 2.74 (s) / 36.29; 2.78 / 39.11	-
5	asparagine	2.85 (dd, 17.0, 7.4) / 34.37; 2.94 (m) / 34.32; 4.00 (m) / 51.16	-
6	γ-aminobutyrate (gaba)	1.89 (t, 7.3) / 29.75; 2.29 (t, 7.4) / 34.12; 3.02-2.98 (m) / 39.13	1.90 (q, 7.2) / 27.37; 2.29 (t, 7.4) / 34.17; 3.00 (t, 7.3) / 38.96
7	isoleucine	0.97-0.92 (m) / 11.02; 1.00 (d. 6.9) / 14.53; 1.23 (dd, 6.7, 3.3) / 18.55; 1.46 / 25.14; 1.97 (d, 3.4) / 35.79; 3.64 / 62.22	0.94 (m) / 13.13; 1.00 (d, 6.8) / 14.33; 1.45 / 24.2; 1.97 (d, 3.4) / 35.69; 3.64 / 62.31
8	leucine	0.97-0.92 (m) / 20.72 / 21.89; 1.70 (dt, 13.7, 7.7) / 26.26 / 39.67; 3.72 / 53.11	0.94 (m) / 20.59; 1.70 (dt, 13.7, 7.7) / 26.26 / 39.67; 3.72 / 58.25
9	glutamine	2.13 (d, 6.4) / 25.96; 2.44 (d, 4.4) / 30.7; 3.77 (m) / 54.29	2.13 (m) / 26.00; 2.48-2.42 (m) / 30.56; 3.77 (d, 8.7) / 55.49
10	glutamic acid	2.09-2.03 (m) / 28.69; 2.16 (d, 7.9) / 27.07; 2.34 (d, 7.1) / 37.90; 3.75 / 54.39	2.06 (m) / 28.72; 2.21-2.16 (m); 2.30 (t, 7.3) / 34.17; 3.76 / 54.33
11	glycine	3.55 / 41.17	-
12	threonine	1.32 (d, 6.6) / 24.18; 3.57-3.55 (m) / 63.69; 4.25 / 65.67	1.32 (d, 6.3) / 19.16; 3.57 / 62.3; 4.24 / 65.6
13	L-serine	3.83 / 60.71; 4.00 (d, 6.8) / 63.09	-
14	valine	0.98 (d, 7.0) / 16.55; 1.03 (d, 7.0) / 17.65; 2.26 / 28.74; 3.59 / 60.97	0.98 (d, 7.0) / 16.37; 1.03 (d, 6.9) / 17.68; 2.27 / 28.78; 3.58 / 61.16
15	tyrosine	3.08 / 37.4; 3.92 (q, 3.0) / 56.03; 6.88 (d, 8.2) / 115.61; 7.18 (d, 8.1) / 130.65	6.88 (d, 7.9) /115.42; 7.18 (d, 8.0) / 130.46
16	tryptophan	3.47 (dd, 9.9, 4.2); 4.05 (m) / 55.48; 7.18 (d, 8.1); 7.27 (d, 8.1); 7.32 (d, 7.5) / 129.20; 7.52 (d, 8.4) / 115.68; 7.72 (d, 7.8)	3.46 (m); 4.05 (d, 2.7) / 55.3; 7.32 (d, 7.7) / 129.32; 7.52 (d, 7.8) / 111.34; 7.72 (d, 7.7)
17	phenylalanine	3.13 / 41.11; 3.95 / 59.89; 7.32 (d, 7.5) / 129.2; 7.38-7.34 (m) / 127.38; 7.44-7.39 (m) / 128.97	3.13 / 40.96; 3.23 / 40.22; 7.32 (d, 7.7) / 129.26; 7.37 (d, 7.0) / 127.5; 7.41 (d, 7.2) / 128.76
Sugar
18	β-glucose	4.63 (d, 7.9) / 95.77; 3.46 (m) / 75.75; 3.39 (td, 9.5, 5.4) / 69.46	4.63 (d, 7.9) / 95.58; 3.46 (m) / 75.75; 3.39 / 69.31; 3.45 (d, 5.3); 3.90 (m) / 60.44
19	α-glucose	5.22 (d, 3.7) / 91.95; 3.82 (m) / 71.25; 3.51 (d, 3.9); 3.39 (td, 9.5, 5.4) / 69.72	5.22 (d, 3.7) / 91.78; 3.88 / 69.7; 3.51 (m) / 67.12; 3.39 / 69.31
20	sucrose	5.40 (d, 3.9) / 92.01; 4.21 (d, 8.8) / 76.2; 4.04 (m) / 71.93; 3.81-3.80 (m) / 62.19 / 3.74 (m) / 70.83; 3.57-3.55 (m) / 69.08	5.40 (d, 5.4) / 91.83; 4.21 (d, 8.7) / 76.2
21	fructose	4.10 / 75.26; 3.98 / 69.08; 3.88 / 69.51; 3.81-3.80 (m) / 80.5; 3.79 / 69.7; 3.69 / 63.32; 3.54 / 63.06	4.11 / 75.08; 4.03; 4.00 / 70.74; 3.82 / 80.36; 3.76 / 70.89; 3.69 / 67.22; 3.54 / 63.55
Organic acid
22	lactic acid	1.32 (d, 6.6) / 24.02; 4.1 (t, 3.7) / 74.21	1.32 (d, 6.3) / 19.16; 4.07 / 70.21
23	formic acid	8.46 (s)	-
24	fumaric acid	6.51 (s) / 135.07	-
Phenolic compound
25	p-coumaric acid	7.51 / 129.40; 6.88 / 115.61; 6.23 / 109.76	7.44 / 141; 7.52 / 129.26; 6.85 / 115.02; 6.32 / 114.76
Miscellaneous
26	choline	3.19 (s) / 53.73; 3.50 (m) / 67.13; 4.04 (m) / 55.48	3.19 (s) / 53.56; 3.5; 4.05 / 55.3
27	betaine	3.25 (s) / 53.23; 3.88 / 69.51	3.25 (s) / 53.05; 3.88 / 67.22
28	pyruvic acid	2.34 / 28.87	-
29	tyrosol	2.78 (d, 9.8) / 39.11; 3.77 (m) / 65.41; 6.88 (d, 8.2) / 115.61; 7.18 (d, 8.1) / 130.49	-

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