Bioactive compounds of pigmented rice extracts reveal by NMR-based metabolomics

Figures & data

Table 1. Biological activities of different varieties of pigmented rice extracted with different solvents.

Variety	Solvent	TPC (mg GAE/100 g sample)	DPPH scavenging activity IC50 (µg/mL)	α-glucosidase inhibitory activity, IC50 (µg/mL)	NO inhibition activity IC50 (µg/mL)
BR	Acetone	43.68 ± 9.03cB	ND	ND	198.95 ± 16.13^abA
	MeOH	3.83 ± 6.31bB	102.58 ± 10.31bC	101.98 ± 22.02^B	229.46 ± 22.48^aA
	Water	64.10 ± 1.34aA	68.69 ± 15.65aB	ND	246.69 ± 86.16^bB
PR	Acetone	16.24 ± 0.34aA	ND	ND	234.41 ± 6.53^abB
	MeOH	194.60 ± 4.08 bA	73.93 ± 7.56aB	149.93 ± 21.57^C	277.21 ± 3.21^bB
	Water	168.43 ± 3.53bB	164.21 ± 18.97bC	ND	176.28 ± 18.78^aA
RR	Acetone	934.06 ± 19.59bC	5.85 ± 0.05b	2.66 ± 1.60^a	225.91 ± 14.35^abB
	MeOH	878.47 ± 18.43aC	2.95 ± 1.09aA	0.43 ± 0.07^bA	204.83 ± 27.13^bA
	Water	1142.77 ± 23.97cC	7.25 ± 2.54 bA	2.90 ± 0.34^a	160.69 ± 14.83^aA
Standard			9.14 ± 1.34 (Quercetin)	0.94 ± 0.13 (Quercetin)	3.44 ± 0.23 (Curcumin)

Superscript lowercase letters within each column indicate differences of samples at same rice cultivars for different solvents.

Superscript capital letters within each column indicate differences of samples at same extraction solvent for different rice cultivars.

ND = not determined as the percent of inhibition was lower than 50%, BR; black rice, PR; purple rice, RR; red rice.

Figure 1. ¹H NMR spectra of different pigmented rice; black (BR), purple (PR) and red (RR) extracted using different extraction solvents.

Table 2. ¹H-NMR characteristic signals of pigmented rice extracts.

ID	Metabolites	^1H-NMR characteristic signals (δ)	Rice varieties/Extraction solvents
			BR			PR			RR
			ACE	MeOH	Water	ACE	MeOH	Water	ACE	MeOH	Water
1	Trigonelline	9.13 (s), 8.75 (m), 8.04 (m)	-	-	+	-	-	+	-	-	+
2	Nicotinic acid	8.96 (d), 8.57 (dd), 8.27 (m), 7.56 (m)	-	-	+	-	-	+	-	-	+
3	Histidine	8.33 (s), 7.27 (s)	-	+	+	-	+	+	-	+	+
4	Ƭ-methylhistidine	7.97 (s), 7.15 (s)	-	+	+	-	+	+	-	+	+
5	UDP glucose	7.92 (d), 5.97 (m), 5.57 (m)	+	+	+	+	+	+	+	+	+
6	Chlorogenic acid	5.28 (d), 2.2 (m), 2.1 (m), 2.03 (m), 7.60 (d), 7.20 (d), 7.10 (d), 6.94 (d), 6.35 (d), 5.28 (m)	-	+	+	-	+	+	-	+	+
7	Vanillic acid	7.53 (d), 7.46 (d), 6.95 (d), 3.90 (d)	-	+	+	-	+	+	-	+	+
8	Protocatechuic acid	7.40 (d), 7.36 (dd), 6.92 (d)	-	+	+	-	+	+	-	+	+
9	Tyrosine	7.16 (d), 6.86 (d)	-	+	+	-	+	+	-	+	+
10	Sucrose	5.40 (d), 4.16 (d), 4.01 (d), 3.88-3.76 (m), 3.65-3.51, 3.43 (m)	+	+	+	+	+	+	+	+	+
11	Maltose	5.21 (d)	+	+	+	+	+	+	+	+	+
12	Glucose	5.17 (d), 4.57 (d), 3.86-3.73 (m), 3.53 (t), 3,18 (t)	+	+	+	+	+	+	+	+	+
13	4-Aminobutyric acid (GABA)	2.98 (m), 2.26 (m), 1.87 (m)	-	+	+	-	+	+	-	+	+
14	Fructose	4.08 (m), 4.03 (m), 4.00 (m)	+	+	+	+	+	+	+	+	+
15	2-Dimethylamine	2.71 (s)	+	+	+	+	+	+	+	+	+
16	Choline	3.2 (s)	+	+	+	+	+	+	+	+	+
17	2-Oxoglutaric acid	2.99 (s), 2.44 (s)	-	+	+	-	+	+	-	+	+
18	Citric acid	2.70 (d), 2.55 (d)	+	+	+	+	+	+	+	+	+
19	Succinic acid	2.54 (s)	+	+	+	+	+	+	+	+	+
20	Pyruvic acid	2.3 (s)	-	+	+	-	+	+	-	+	+
21	Acetic acid	1.91 (s)	+	+	+	+	+	+	+	+	+
22	Alanine	1.47 (d)	-	+	+	-	+	+	-	+	+
23	Fatty acid derivatives	1.28 – 1.32 (m), 0.87 (s)	+	+	+	+	+	+	+	+	+
24	Propylene glycol	1.14 (s)	-	+	+	-	+	+	-	+	+
25	Valine	1.05 (d), 0.99 (d)	-	+	+	-	+	+	-	+	+
26	Isoleucine	1.00 (s), 0.98 (s)	-	+	+	-	+	+	-	+	+
27	2-Hydroxyisovaleric acid	0.81 (s), 0.98 (s), 2.00 (s), 3.8 (s), 2.00 (s)	-	+	+	-	+	+	-	+	+

d = doublet; dd = double of doublet; m = multiple; s = singlet; t = triplet; + = present; - = absent, BR = black rice, PR = purple rice, RR = red rice, ACE = acetone, MeOH = methanol.

Figure 2. PCA score plot (a) and loading plot (b) of ¹H NMR spectra of different pigmented rice extracts (black, purple, and red) with different extraction solvents (ACE: acetone, MeOH: methanol, H₂O: water). The represented numbers refer to metabolites listed in Table 2.

Figure 3. PLS biplot of ¹H NMR spectra of different varieties of the pigmented rice extracts (black, purple, and red) from different extraction solvents (ACE: acetone, MeOH: methanol, H₂O: water) describing the relationship between metabolites and bioactivities of pigmented rice extracts (1: trigonelline, 2: Nicotinic acid, 3: Histidine, 4: T-methylhistidine, 5: UDP glucose, 6: Chlorogenic acid, 7: Vanillic acid, 8: Protocatechuic acid, 9: Tyrosine, 10: Sucrose, 11: Maltose, 12: Glucose, 13: 4-Aminobutyric acid (GABA), 14: Fructose, 15: 2-Dimethylamine, 16: Choline, 17: 2-Oxoglutaric acid, 18: citric acid, 19: Succinic acid, 20: Pyruvic acid, 21: Acetic acid, 22: Alanine, 23: Fatty acid derivatives, 24: Propylene glycol, 25: Valine, 26: Isoleucine, 27: 2-Hydroxyisovaleric acid).

Figure 4. The variable importance in the projections (VIP) values of major contributing metabolites based on PLS model of pigmented rice from different cultivars and solvents.

Figure 5. Metabolite sets enrichment overview of pigmented rice presented in (A) pie chart and (B) over representation analysis (ORA) plot.