Bioactive Compounds Responsible for Antioxidant Activity of Different Varieties of Date (Phoenix dactylifera L.) Elucidated by ¹H- NMR Based Metabolomics

Figures & data

Table 1. Percentage of extraction yield from different date varieties

Date	Yield (%)
Ajwa	20.13 ± 1.02 ^b
Rabbi	21.20 ± 0.75 ^b
Piyarom	23.80 ± 0.95 ^a
Deglet Nour	16.07 ± 1.15 ^c
Anbara	19.90 ± 1.00 ^b

Mean of with the same letters are not significantly different at P > 0.05.

Figure 1. Total phenolic content of different date varieties

Mean of with the same letters are not significantly different at p > 0.05.

Figure 2. Total flavonoid content of different date varieties

Mean of with the same letters are not significantly different at p > 0.05.

Figure 3. DPPH radical scavenging activity of different date varieties

Mean of different letters are significantly different at p < 0.05.

Figure 4. FRAP ferric reducing antioxidant power of different date varieties

Mean of with the same letters are not significantly different at p > 0.05.

Figure 5. ABTS antioxidant capacity of different date varieties

Mean of different letters are significantly different at p < 0.05.

Figure 6. ¹H-NMR spectra of different date varieties. (A) Full Spectra of δ 1.0 to 8.5 ppm. (B) Spectra of δ 1.0 to 3.0 ppm. (C) Spectra of δ 3.0 to 6.0 ppm. (D) Spectra of δ 6.0 to 8.0 ppm

Identified ¹H-NMR signals:: 1, Alanine; 2, Arginine; 3, Glutamine; 4, citric acid; 5, Malic acid; 6, Glycine; 7, Ascorbic acid; 8, Fructose; 9, Beta Glucose; 10, Alpha Glucose. 11, Sucrose; 12, Fumaric acid 13, Gallic acid. 14, Epicatechin.

Table 2. Assignments of ¹H-NMR spectral signals attained from different date varieties

Compound	Chemical shift	Date varieties
		Ajwa	Anbara	Rabbi	Piyarom	Deglet nour
Valine	δ 2.30 (m)	$\times$	$\surd$	$\surd$	$\surd$	$\surd$
Isoleucine	δ 0.89 (t, J = 7,414Hz)	$\surd$	$\surd$	$\surd$	$\surd$	$\times$
Leucine	δ 1.7 (m)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Inosine	δ 4.28 (m), δ 4.74 (m)	$\surd$	$\surd$	$\surd$	$\surd$	$\times$
Lysine	δ 3.73 (t, J = 6.09Hz)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Alanine	δ 1.50 (d, J= 7.2Hz)	$\times$	$\surd$	$\surd$	$\surd$	$\surd$
Arginine	δ 1.61(m)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Acetate	δ 1.91(s)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Glutamine	δ 2.36 (m), δ 2.08 (m)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Succinic acid	δ 2.42 (s)	$\times$	$\surd$	$\times$	$\surd$	$\surd$
Malic acid	δ 2.69 (dd, J = 15.32,3.10Hz), δ 2.38 (m)	$\surd$	$\surd$	$\surd$	$\surd$	$\times$
Asparagine	δ 2.96 (dd, J= 4.0,16.8Hz), δ 2.83 (m, overlapped)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Proline	δ 1.98 (m)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Glycine	δ 3.57 (s)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Fructose	δ 3.86 (dd, J = 9.91Hz), δ 4.08 (dd, J = 12.88)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Lactic acid	δ 1.31 (d, J = 6.5Hz)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Sucrose	δ 5.41 (d, J = 3.81Hz), δ 4.18 (d, J = 8.66Hz)	$\times$	$\times$	$\surd$	$\surd$	$\surd$
Ascorbic acid	δ 3.72 (m)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Alpha Glucose	δ 5.19 (d, J= 3.72Hz)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Beta Glucose	δ 4.56 (d, J = 7.90Hz)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
xylose	δ 4.59(d, J = 7.9Hz)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Fumaric acid	δ 6.53 (s)	$\surd$	$\times$	$\surd$	$\surd$	$\times$
Iso-butyrate	δ 2.31 (m)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Citric acid	δ 2.661(d, J = 3.113Hz)	$\times$	$\surd$	$\surd$	$\times$	$\times$
Epicatechin	δ 5.04(d), δ 6.89–6.91 (m)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Cysteine	δ 3.074(m)	$\times$	$\times$	$\surd$	$\surd$	$\surd$
Betaine	δ 3.876 (s), 3.220 (s)	$\times$	$\times$	$\surd$	$\surd$	$\surd$
Choline	δ 3.186(s)	$\surd$	$\surd$	$\surd$	$\surd$	$\surd$
Pyruvic acid	δ 2.38 (s)	$\times$	$\surd$	$\surd$	$\surd$	$\surd$
Gallic acid	δ 7.03(s)	$\times$	$\surd$	$\surd$	$\surd$	$\times$

NMR spectra: s, singlet; d, doublet; t, triplet; q, quartet; m, multiple.

Figure 7. Analysis for the date spectra obtained using ¹H-NMR. (A) PCA (PC1 vs. PC2), (B) PLS-DA score plot, (C) Loading column plots of PC1 and (D) PC2

Assignments in b and c: 1, valine; 2,isoleucine; 3, leucine; 4, inosine; 5,lysine; 6,alanine; 7,arginine; 8, acetic acid; 9,glutamine; 10,succinic acid; 11, malic acid; 12,asparagine; 14,proline; 15,fructose; 16,lactic acid; 17,Sucrose; 18,ascorbic Acid; 19,α-glucose; 20,β-glucose; 21,xylose; 22,fumaric acid; 23,iso-butyrate; 24,citric acid;25,epicatechin; 26,cysteine; 27,Betaine; 28, choline; 29, pyruvic acid; and 30,gallic acid.

Figure 8. Relative quantities of the metabolites identified in all five varieties of date extracts based on the mean peak area of the ¹H-NMR signals

A, Ajwa: AN, Anbara; R, Rabbi; P, Piyarom; DN, Deglet Nour. Values with different letters are significantly different (p < 0.05).

Figure 9. The biplot obtained from PLS describing the relation between the metabolites with antioxidant activities in extract of different date varieties

A, Ajwa: AN, Anbara; R, Rabbi; P, Piyarom; DN, Deglet Nour.

Table 3. VIP values of the major contributing compounds in the PLS

Chemical Shifts (ppm)	Compound	VIP values
5.42	Sucrose	2.60
3.87	Betaine	2.57
3.54	Glycine	2.50
3.74	Lysine	2.41
4.58	Beta glucose	2.21
3.18	Choline	2.20
4.06	Fructose	1.71
2.38	Glutamine	1.54
2.66	citric acid	1.30
5.22	Alpha Glucose	1.29
1.91	Arginine	1.08
7.03	Epicatechin	0.84
7.05	Gallic acid	0.72
3.72	Ascorbic acid	0.70