Chemical Profile and Biological Activities of Two Edible Plants: Chemical Investigation and Quantitative Analysis Using Liquid Chromatography Tandem Mass Spectrometry and Gas Chromatography Mass Spectrometry

Figures & data

Table 1 Analytical parameters of LC-MS/MS method

Analyte no	RT^a	Analytes	R^2b	Equation	RSD%^c	Linearity range (mg/L)	LOD/LOQ (µg/L)^d	Recovery (%)	U^e
1	3.36	Quinic acid	0.9927	f(x) = 33.6626 × x + 25132.9	0.0388	250–10000	22.3/74.5	103.3	4.8
2	3.60	Malic acid	0.9975	f(x) = 93.6102 × x – 5673.77	0.1214	250–10000	19.2/64.1	101.4	5.3
3	4.13	tr-Aconitic acid	0.9933	f(x) = 79.2908 × x – 28416.2	0.3908	250–10000	15.6/51.9	102.8	4.9
4	4.25	Gallic acid	0.9901	f(x) = 358.069 × x + 26417.5	0.4734	25–1000	4.8/15.9	102.3	5.1
5	5.29	Chlorogenic acid	0.9932	f(x) = 48.9828 × x + 26779.7	0.1882	250–10000	7.3/24.3	99.7	4.9
6	5.51	Protocatechuic acid	0.9991	f(x) = 36.8568 × x + 6197.38	0.5958	100–4000	25.8/85.9	100.2	5.1
7	6.30	Tannic acid	0.9955	f(x) = 90.2704 × x + 30233.2	0.9075	100–4000	10.2/34.2	97.8	5.1
8	7.11	tr- caffeic acid	0.9942	f(x) = 1585.16 × x + 83957.5	1.0080	25–1000	4.4/14.7	98.6	5.2
9	8.57	Vanillin	0.9995	f(x) = 44.5478 × x – 574.867	0.4094	250–10000	10.1/33.7	99.2	4.9
10	9.17	p-Coumaric acid	0.9909	f(x) = 73.5303 × x + 27064.3	1.1358	100–4000	15.2/50.8	98.4	5.1
11	9.19	Rosmarinic acid	0.9992	f(x) = 18.0298 × x – 1149.86	0.5220	250–10000	10.4/34.8	101.7	4.9
12	9.67	Rutin	0.9971	f(x) = 51.8835 × x + 3841.66	0.8146	250–10000	17.0/56.6	102.2	5.0
13	9.69	Hesperidin	0.9973	f(x) = 195.773 × x + 105641	0.1363	250–10000	21.6/71.9	100.2	4.9
14	9.96	Hyperoside	0.9549	f(x) = 0.978146 × x + 827.221	0.2135	100–4000	12.4/41.4	98.5	4.9
15	11.38	4-OH Benzoic acid	0.9925	f(x) = 635.003 × x + 54284.6	1.4013	25–1000	3.0/10.0	106.2	5.2
16	11.39	Salicylic acid	0.9904	f(x) = 915.178 × x + 72571.4	0.6619	25–1000	4/13.3	106.2	5.0
17	11.42	Myricetin	0.9991	f(x) = 54.2823 × x + 5414.67	2.8247	100–4000	9.9/32.9	106.0	5.9
18	12.10	Fisetin	0.9988	f(x) = 331.870 × x + 34409.0	2.4262	100–4000	10.7/35.6	96.9	5.5
19	12.18	Coumarin	0.9924	f(x) = 236.639 × x + 34370.3	0.4203	100–4000	9.1/30.4	104.4	4.9
20	13.93	Quercetin	0.9995	f(x) = 206.102 × x + 1693.14	4.3149	25–1000	2.0/6.8	98.9	7.1
21	14.15	Naringenin	0.9956	f(x) = 1100.55 × x + 39055.7	2.0200	25–1000	2.6/8.8	97.0	5.5
22	14.80	Hesperetin	0.9961	f(x) = 160.323 × x + 6545.07	1.0164	25–1000	3.3/11.0	102.4	5.3
23	14.84	Luteolin	0.9992	f(x) = 111.474 × x + 3057.10	3.9487	25–1000	5.8/19.4	105.4	6.9
24	14.85	Kaempferol	0.9917	f(x) = 20.9677 × x + 571.241	0.5885	25–1000	2.0/6.6	99.1	5.2
25	16.73	Apigenin	0.9954	f(x) = 543.793 × x + 18525.6	0.6782	25–1000	0.1/0.3	98.9	5.3
26	18.41	Rhamnetin	0.9994	f(x) = 110.091 × x + 632.444	2.5678	25–1000	0.2/0.7	100.8	6.1
27	20.60	Chrysin	0.9965	f(x) = 698.787 × x + 23531.7	1.5530	25–1000	0.05/0.17	102.2	5.3

^aRT: Retention time;

^bR²: Coefficient of determination;

^cRSD: Relative standard deviation;

^dLOD/LOQ(µg/L): Limit of detection/Limit of quantification;

^eU (%): Percent relative uncertainty at 95% confidence level (k = 2).

TABLE 2 Identification and quantification of compounds methanol extracts of T. latifolius var. angustifolius (TLAM) and L. orientalis (LOM) by LC-MS/MS

			Quantification (mg analyte/g extract)^c
Compound	Parent ion (m/z)^a	MS^2(Collision Energy)^b	TLAM	LOM
Quinic acid	190.95	85 (22),93 (22)	11.46 ± 0.55	2.98 ± 0.14
Malic acid	133.05	115 (14),71 (17)	0.470 ± 0.025	6.69 ± 0.35
tr-Aconitic acid	172.85	85 (12).129 (9)	0.305 ± 0.015	0.47 ± 0.02
Gallic acid	169.05	125 (14),79 (25)	D.^d	D.
Chlorogenic acid	353	191 (17)	2.12 ± 0.10	0.26 ± 0.13
Protocatechuic acid	152.95	109 (16),108 (26)	0.16 ± 0.008.	0.28 ± 0.014
Tannic acid	182.95	124 (22),78 (34)	N.D.^e	0.08 ± 0.004
tr-Caffeic acid	178.95	135 (15),134 (24),89 (31)	0.24 ± 0.012	0.295 ± 0.015
Vanillin	151.05	136 (17),92 (21)	N.D.	N.D.
p-Coumaric acid	162.95	119 (15),93 (31)	0.39 ± 0.02	0.21 ± 0.01
Rosmarinic acid	358.9	161 (17),133 (42)	N.D.	21.11 ± 1.03
Rutin	609.1	300 (37),271 (51),301 (38)	0.28 ± 0.01	4.26 ± 0.21
Hesperidin	611.1	303,465	0.29 ± 0.014	2.39 ± 0.12
Hyperoside	463.1	300,301	0.96 ± 0.05	332.37 ± 15.85
4-OH Benzoic acid	136.95	93,65	0.295 ± 0.015	0.39 ± 0.002
Salicylic acid	136.95	93,65,75	0.26 ± 0.013	0.35 ± 0.017
Myricetin	317	179,151,137	N.D.	N.D.
Fisetin	284.95	135,121	N.D.	N.D.
Coumarin	146.95	103,91,77	N.D.	D.
Quercetin	300.9	179,151,121	0.32 ± 0.002	N.D.
Naringenin	270.95	151,119,107	N.D.	N.D.
Hesperetin	300.95	164,136,108	N.D.	N.D.
Luteolin	284.95	175,151,133	0.52 ± 0.035	D.
Kaempferol	284.95	217,133,151	0.53 ± 0.027	N.D.
Apigenin	268.95	151,117	0.037 ± 0.002	0.005 ± 0.0003
Rhamnetin	314.95	165,121,300	N.D.	N.D.
Chrysin	253	143,119,107	N.D.	N.D.

^aParent ion (m/z): Molecular ions of the standard compounds (mass to charge ratio);

^bMS²(CE): MRM fragments for the related molecular ions (CE refers to related collision energies of the fragment ions);

^cValues in mg/g (w/w) of plant methanol extract;

^dD: peak observed, concentration is lower than the LOQ but higher than the LOD;

^eN.D.: not detected.

FIGURE 1 LC-MS/MS chromatogram of A: 250 ppb standard mix; B: T. latifolius var. angustifolius methanol extract; C: L. orientalis methnol extract. 1: Quinic acid; 2: Malic acid; 3: tr-Aconitic acid; 4: Gallic acid; 5: Chlorogenic acid; 6: Protocatechuic acid; 7: Tannic acid; 8: tr- caffeic acid; 9: Vanillin; 10: p-Coumaric acid; 11: Rosmarinic acid; 12: Rutin; 13: Hesperidin; 14: Hyperoside; 15: 4-OH Benzoic acid; 16: Salicylic acid; 17: Myricetin; 18: Fisetin;19: Coumarin; 20: Quercetin; 21: Naringenin; 22: Hesperetin; 23: Luteolin; 24: Kaempferol; 25: Apigenin; 26: Rhamnetin; 27: Chrysin.

TABLE 3 GC-MS Analysis of the petroleum ether of T. latifolius var. angustifolius (TLAP) and L. orientalis (LOP)

Rt (min)^a	Constituents^b	Composition (%)^c	Composition (%)
		TLAP	LOP
14.39	10-Undecenoic acid	0.8	0.6
18.60	Myristic acid	1.6	2.8
24.63	9-Hexadecenoic acid	1.8	—
24.94	Palmitoleic acid	—	0.2
25.27	Palmitic acid	28.7	17.6
29.75	Phytol	1.3	—
30.23	1-Octadecanol	—	8.2
30.64	Linoleic acid	20.6	17.2
30.77	Oleic acid	21.0	29.1
30.86	Linolenic acid	12.1	7.9
31.54	Stearic acid	6.3	5.1
36.23	Nonacosanol	—	0.5
36.55	Eicosane	0.9	—
36.69	11-Eicosenoic acid	—	2.5
37.38	Arachidic acid	1.9	6.3
38.19	6-Hexadecenoic acid	0.9	0.6
39.36	Docosane	0.6	—
42.65	Pentacosane	—	0.3
43.82	Behenic acid	1.3	1.0
	Total	99.8	99.9

^aRetention time (as minutes);

^bA non-polar Phenomenex DB-5 fused silica colum;

^cPercentage of relative weight.

TABLE 4 Antioxidant activity*, total phenolic-flavonoid contents* and anticholinesterase activity* of T. latifolius var. angustifolius and L. orientalis extracts, BHT, α-TOC, and galantamine

Samples	Inhibition % against AChE	Inhibition % against BChE	Phenolic content (mg PEs/g extract)^α	Flavonoid content (mg QEs/g extract) ^β	Lipid peroxidation	IC50 (µg/mL) DPPH Free Radical	ABTS cation radical
TLAP	NA	NA	—	—	>200^a	>200^a	>200^a
TLAA	NA	19.45 ± 0.30^a	—	—	105.3 ± 0.98^b	>200^a	92.61 ± 0.37^b
TLAM	NA	35.61 ± 0.76^b	84.38 ± 2.98^a	49.04 ± 0.09^a	112.61 ± 0.67^c	117.72 ± 0.91^b	55.51 ± 0.62^c
TLAW	NA	NA	—	—	>200^a	102.60 ± 1.26^c	16.61 ± 0.69^d
LOP	NA	11.59 ± 0.29^c	—	—	109.39 ± 0.31^c	>200^a	>200^a
LOA	17.41 ± 0.84^a	22.27 ± 0.49^a	—	—	89.49 ± 1.38^d	89.27 ± 1.36^d	27.81 ± 0.17^e
LOM	46.59 ± 1.45^b	47.82 ± 0.52^d	124.70 ± 1.74^b	15.74 ± 0.32^b	38.46 ± 1.30^e	50.82 ± 0.33^e	7.62 ± 0.09^f
LOW	NA	12.50 ± 0.39^c	—	—	172.33 ± 2.12^f	59.31 ± 1.18^f	8.51 ± 0.12^g
Galantamine^†	85.07 ± 0.07^c	84.83 ± 0.17^e	—	—	>200^a	>200^a	>200^a
α-TOC^†	—	—	—	—	15.21 ± 0.09^g	17.41 ± 0.39^g	9.54 ± 0.09^h
BHT^†	—	—	—	—	9.48 ± 0.12^h	49.91 ± 0.17^e	10.31 ± 0.74^h

*Values expressed are means ± SEM of three parallel measurements (p < 0.05);

^†Standard drug;

^αPEs, pyrocatechol equivalents (y = 0.0164x + 0.0266 R² = 0.9969);

^βQEs, quercetin equivalents (y = 0.1519x – 0.1294 R² = 0.9986);

NA: Not active.

FIGURE 2 Cupric reducing antioxidant capacity of the T. latifolius var. angustifolius, L. orientalis, α-tocopherol and BHT. Values are means ± S.D., n = 3, p < 0.05, significantly different with student’s t-test.

TABLE 5 Zones of growth inhibition (mm) and MIC values showing the antimicrobial activities of the extracts compared to positive controls

Microorganisms
		Gram positive		Gram negative		Yeast
			S. aureus	S.pyogenes	E.coli	P. aeruginosa	C. albicans
Positive controls	TLAA	^aDD	10 ± 0.5	11 ± 0.3	11 ± 0.1	12 ± 0.3	14±0.5
		MIC	>1000	40 ± 0.4	80 ± 0.2	200 ± 0.2	110±0.5
	TLAM	^aDD	9 ± 0.4	10 ± 0.2	10 ± 0.6	10 ± 0.4	11±0.5
		MIC	90 ± 0.5	>1000	30 ± 0.1	50 ± 0.7	35±0.1
	LOA	^aDD	12 ± 0.2	12 ± 0.1	12 ± 0.2	11 ± 0.1	10±0.1
		MIC	30 ± 0.2	>1000	45 ± 0.2	>1000	>1000
	LOM	^aDD	11 ± 0.3	10 ± 0.1	11 ± 0.2	9 ± 0.3	12±0.4
		MIC	42 ± 0.3	35 ± 0.2	20 ± 0.7	50 ± 0.3	25±0.4
		^bDD	35 ± 0.2	19 ± 0.2	20 ± 0.1	—	30 ± 0.3
		MIC	1.95 ± 0.3	7.815 ± 0.1	7.815 ± 0.4	—	3.125 ± 0.2

—: Not active;

^aDD: Inhibition zone in diameter (mm) around the discs (6 mm) impregnated with 30 mg mL^–1 of plant extracts;

^bDD: Inhibition zone in diameter (mm) of positive controls that are ampicillin for bacteria and fluconazole for yeast. Minimum inhibitory concentration (MIC) values are given as μg mL^–1.