Phytochemical Composition and in Vitro Analysis of Nopal (O. Ficus-Indica) Cladodes at Different Stages of Maturity

Figures & data

Table 1. Total phytochemicals content of O. ficus-indica cladodes at different stages of maturity.

Compound	Maturity stage
	Young	Medium	Old
Phenols*	28.5 ± 0.6^b	32.6 ± 0.8^a	19.6 ± 0.5^c
Flavonoids**	12.5 ± 0.4^b	18.6 ± 0.6^a	11.2 ± 0.2^b
Condensed tannins^§	3.2 ± 0.1^b	1.5 ± 0.0^a	1.2 ± 0.0^a
Hydrolyzable tannins^€	6.2 ± 0.1^a	5.2 ± 0.2^ab	4.3 ± 0 .4^b
Saponins^©	29.8 ± 1.2^a	32.1 ± 0.9^a	22.5 ± 1.1^c
Phytosterols^¥	24.3 ± 0.9^b	33.2 ± 1.3^a	18.7 ± 0.6^c

Results are the average of three independent determinations ± SE.

^a,b,cDifferent letters in each row indicate significant differences (p < 0.05, Tukey’s test). *mg GAE/g; **mg CE/g; ^§mg PAE/g; mg ^€MGE/g; ^©mg equivalents/g; ^¥mM equivalents/g.

Table 2. Phytochemical profile of O. ficus-indica cladodes at different stages of maturity assessed by HPLC-DAD-MSD.

Proposed compound	Retention time (min)	m/z	Maturity stage
			Young	Medium	Old
Phenolic acids
Chlorogenic acid*	3.8	100	1.5 ± 0.0^a	2.1 ± 0.1^a	LDL
Gallic acid*	5.5	169	4.0 ± 0.2^b	5.6 ± 0.3^b	8.1 ± 0.5^a
Protocatechuic acid*	12.6	153	3.5 ± 0.2^a	3.4 ± 0.1^a	LDL
Caftaric acid	14.0	311	1.1 ± 0.0^b	LDL	1.9 ± 0.1^a
p-Hydroxybenzoic acid*	17.7	137	19.9 ± 1.2^a	17.1 ± 1.1^a	11.0 ± 0.7^b
Coutaric acid	20.9	295	2.7 ± 0.1^b	4.1 ± 0.3^a	3.7 ± 0.3^a
Caffeic acid*	23.5	179	6.9 ± 0.4^b	8.9 ± 0.3^b	13.5 ± 0.6^a
Syringic acid	25.5	197	5.1 ± 0.3^a	6.1 ± 0.2^a	6.3 ± 0.3^a
p-Coumaric acid*	31.4	163	28.5 ± 1.2^b	39.3 ± 1.5^a	14.1 ± 0.7^c
Sinapic acid*	32.6	223	8.1 ± 0.4^b	12.3 ± 0.6^a	LDL
Ferulic acid*	33.0	193	41.8 ± 2.0^b	50.3 ± 2.4^a	33.9 ± 2.1^c
Rosmarinic acid*	34.3	360	10.3 ± 0.8^b	15.1 ± 0.7^a	LDL
Cinammic acid	35.1	148	5.6 ± 0.3^b	6.4 ± 0.4^ab	7.3 ± 0.4^a
Ellagic acid*	37.4	302	8.1 ± 0.7^a	8.9 ± 0.5^a	LDL
Vanillic acid	38.5	167	LDL	1.7 ± 0.1^b	3.0 ± 0.1^a
Flavonoids
Procyanidin B1	13.4	577	3.7 ± 0.2^b	5.6 ± 0.3^a	LDL
Catechin*	17.6	289	8.1 ± 0.4^c	12.1 ± 0.5^b	16.5 ± 0.5^a
Procyanidin B2	21.2	577	1.4 ± 0.1^a	0.9 ± 0.1^a	LDL
Epicatechin*	25.6	289	LDL	3.8 ± 0.3^a	2.8 ± 0.2^b
Rutin*	35.1	609	15.4 ± 0.6^b	19.2 ± 0.8^a	10.4 ± 0.5^c
Gallocatechin gallate*	38.5	457	9.4 ± 0.4^a	8.9 ± 0.5^a	LDL
Epigallocatechin gallate*	39.9	457	12.3 ± 0.7^ab	15.4 ± 0.7^a	9.9 ± 0.4^b
Epicatechin gallate	41.5	441	6.9 ± 0.3^a	7.2 ± 0.3^a	LDL
Quercetin-3-O-galactoside	49.6	463	12.0 ± 0.7^b	15.3 ± 0.8^b	24.1 ± 1.2^a
Quercetin-3-O-glucoside*	50.2	463	27.1 ± 1.3^b	37.1 ± 1.8^a	34.5 ± 1.5^a
Coumestrol	55.7	268	3.5 ± 0.1^a	2.9 ± 0.2^a	3.5 ± 0.2^a
Narcissin	60.3	525	45.9 ± 2.0^a	53.1 ± 2.2^a	31.9 ± 1.6^b
Nicotiflorin	64.5	595	35.1 ± 1.3^a	30.4 ± 1.5^a	22.4 ± 1.0^b
Phytosterols
β-Sitosterol	3.6	414	25.1 ± 1.2^b	36.9 ± 1.8^a	12.7 ± 0.9^c
β-Campesterol	4.2	400	5.6 ± 0.3^a	5.8 ± 0.4^a	LDL
Campestanol	4.6	402	9.1 ± 0.5^a	7.8 ± 0.4^a	2.3 ± 0.1^b
Δ5-Avenasterol	5.3	412	8.8 ± 0.4^a	9.3 ± 0.6^a	3.7 ± 0.2^b
Δ7-Stigmasterol	5.8	412	12.0 ± 0.7^a	15.4 ± 1.0^a	7.4 ± 0.4^b
Stigmastanol	8.1	416	19.3 ± 1.2^a	17.9 ± 1.4^a	4.9 ± 0.3^b
Saponins
Campesteryl 3β-D-glucopyranoside	85.5	562	23.9 ± 1.7^a	19.5 ± 1.3^a	10.3 ± 0.6^b
Stigmasteryl 3β-D-glucopyranoside	88.7	574	25.6 ± 1.9^a	21.8 ± 1.4^a	15.6 ±1.0^b
Sitosteryl 3β-D-glucopyranoside	92.9	576	39.8 ± 2.3^b	48.1 ± 2.6^a	29.4 ± 1.9^c
Isorhamnetin 3-O-rutinoside	98.9	625	45.2 ± 2.0^a	40.7 ± 1.7^a	21.8 ± 1.3^b

Results are the average of three independent determinations ± SE.

Data is presented as areas under the curve (mA).

*Confirmation in comparison with authentic standards.

LDL: lower than detection limit.

Figure 1. Representative chromatogram of A: Low molecular weight metabolites assessed by GC-MSD; B: phenolic compounds; C: saponins and phytosterols evaluated by HPLC-DAD-MSD of O. ficus-indica cladodes.

Table 3. Low molecular weight metabolites of O. ficus-indica cladodes at different stages of maturity assessed by GC-MSD.

Proposed compound	Retention time (min)	Representative ions in peaks (m/z)	Maturity stage
			Young	Medium	Old
Amino acids
L-Alanine	4.1	218, 190,172, 116, 94	24.6 ± 1.1^a	16.8 ± 0.8^b	LDL
L-Leucine	6.8	260, 232, 203, 176, 158	44.0 ± 2.3^b	51.8 ± 2.5^a	45.3 ± 2.2^b
L-Isoleucine	7.2	260, 218, 158,149	36.2 ± 1.7^b	55.7 ± 2.7^a	LDL
L-Proline	7.3	244, 216, 170,142,119	117.8 ± 3.5^a	104.9 ± 3.8^b	96.7 ± 3.7^b
L-Serine	8.5	306, 278, 260, 222, 204	16.8 ± 1.1^a	6.5 ± 0.3^b	3.9 ± 0.2^b
L-Threonine	9.0	320, 291, 248, 218,117	LDL	LDL	32.4 ± 1.4^a
γ-Aminobutiric acid	11.7	304, 246, 216, 174	125.6 ± 4.0^ab	111.3 ± 3.7^b	147.6 ± 4.2^a
L-Glutamine	13.4	363, 348, 320, 246, 218	6.5 ± 0.4^b	5.2 ± 0.2^b	22.0 ± 1.1^a
L-Phenylalanine	13.5	294, 266, 218, 192,174	22.6 ± 1.4^a	19.4 ± 1.3^a	6.5 ± 0.4^b
L-Asparagine	14.4	351,333, 258, 188,169	29.8 ± 1.6^b	36.2 ± 2.0^a	LDL
L-Tyrosine	19.0	382, 354, 280, 218,179	32.4 ± 2.1^a	27.2 ± 1.5^a	31.6 ± 2.5^a
L-Tryptophan	23.8	357, 325, 291, 251, 202	29.8 ± 1.3^a	19.4 ± 1.4^b	22.0 ± 0.8^b
Carbohydrates
Arabinose	18.7	333, 305, 219, 204,187	97.1 ± 3.5^b	88.0 ± 2.8^c	121.7 ± 4.4^a
D-glucose	20.0	435, 393, 345, 305, 231	75.1 ± 2.8^a	60.8 ± 2.5^b	22.0 ± 0.7^c
Organic acids
Glycolic acid	3.8	205, 177, 121	31.1 ± 1.4^b	41.4 ± 1.2^a	23.3 ± 0.9^c
Malonic acid	5.6	233, 204, 117, 99	23.3 ± 0.8^a	LDL	LDL
Succinic acid	7.5	247, 218, 190, 172	86.7 ± 3.1^b	104.9 ± 4.1^a	107.4 ± 3.9^a
Fumaric acid	8.1	245, 217, 191, 171,115	16.8 ± 0.9^a	18.1 ± 1.0^a	LDL
Malic acid	11.0	355, 307, 265, 233,189	262.8 ± 5.3^b	334.6 ± 6.1^a	337.9 ± 6.4^a
Threonic acid	12.2	409, 379, 319, 292, 263	28.5 ± 0.9^b	35.0 ± 1.2^a	16.8 ± 0.5^c
Lauric acid	13.8	257, 229, 201, 171,145	18.1 ± 0.5^a	LDL	LDL
Tartaric acid	14.0	423, 395, 367, 333, 292	14.2 ± 0.3^a	LDL	LDL
Citric acid	17.2	465, 375, 305, 273, 217	42.7 ± 1.8^a	22.0 ± 0.8^b	7.8 ± 0.2^c
Fatty acids
Myristic acid	17.3	303, 285, 257, 201, 171	5.2 ± 0.2^b	19.4 ± 0.7^a	18.1 ± 0.7^a
Palmitic acid	20.5	331, 313, 269, 171,145	25.7 ± 0.8^ab	27.2 ± 1.1^a	20.1 ± 1.5^b
Linoleic acid	23.3	337, 309, 262, 220,199	27.2 ± 1.0^a	19.5 ± 0.8^b	LDL
Oleic acid	23.4	339, 264, 222, 185,145	24.6 ± 0.9^b	41.4 ± 1.7^a	44.2 ± 2.0^a
Stearic acid	24.0	341, 315, 297, 257,227	32.0 ± 0.8^a	35.7 ± 1.1^a	38.8 ± 1.6^a
α-linolenic acid	23.5	335, 291, 260, 213	31.1 ± 1.3^a	14.2 ± 0.7^b	LDL

Results are the average of three independent determinations ± SE.

Data is presented as areas under the curve (A × 10⁵).

LDL: lower than detection limit.

Table 4. Indigestible fraction, antioxidant capacity, and inhibitory activity of digestive enzymes of O. ficus-indica cladodes at different stages of maturity.

Parameter	TSH Maturity stage
	Young	Medium	Old
Indigestible fraction (g/Kg)
Insoluble indigestible fraction	325.6 ± 9.2 ^b	365.3 ± 0.4^a	291.3 ± 0.0^c
Soluble indigestible fraction	54.6 ± 0.2 ^ab	55.4 ± 0.4^a	48.3 ± 0.0^b
Antioxidant capacity (IC50, µg/mL)
DPPH•	48.1 ± 1.5 ^a	42.3 ± 2.5^b	46.1 ± 3.1^a
ABTS•+	24.8 ± 1.2 ^ab	22.7 ± 2.0^b	25.4 ± 0.9^a
Digestive enzymes inhibitory activity (IC50, µg/mL)
α-amylase	18.5 ± 1.0 ^a	13.5 ± 0.8^b	14.7 ± 1.2^ab
α-Glucosidase	28.7 ± 1.6 ^a	21.3 ± 0.9^b	22.1 ± 0.6^b
Pancreatic lipase	16.5 ± 1.2^b	18.4 ± 1.1^b	25.4 ± 1.5^a

Results are the average of three independent determinations ± SE. ^a,b,cDifferent letters in each row indicate significant differences (p < 0.05, Tukey’s test).

Table 5. Correlations between phytochemicals, antioxidant capacity, and digestive enzymes inhibitory activity O. ficus-indica cladodes at different stages of maturity.

Phytochemicals	Correlation coefficient (R^2)
	DPPH	ABTS	Amylase	Glucosidase	Lipase
Total phenols	–0.471	–0.868	–0.022	0.111	–0.869
Total flavonoids	–0.872	–0.999	–0.557	–0.441	–0.466
Condensed tannins	0.667	0.170	0.932	0.972	–0.763
Hydrolizable tannins	0.368	–0.182	0.748	0.830	–0.939
Chlorogenic acid	–0.442	–0.851	0.011	0.143	–0.885
Gallic acid	–0.218	0.333	–0.636	–0.733	0.981
Protocatechuic acid	–0.152	–0.653	0.311	0.435	–0.984
Caftaric acid	0.712	0.976	0.317	0.188	0.683
p-Hydroxybenzoic acid	0.135	–0.412	0.568	0.673	–0.994
Coutaric acid	–0.913	–0.558	–0.999	–0.983	0.432
Caffeic acid	–0.122	0.423	–0.558	–0.663	0.995
Syringic acid	–0.655	–0.154	–0.926	–0.968	0.773
p-Coumaric acid	–0.580	–0.924	–0.149	–0.016	–0.799
Sinapic acid	–0.497	–0.882	–0.051	0.082	–0.854
Ferulic acid	–0.661	–0.958	–0.250	–0.119	–0.732
Rosmarinic acid	–0.474	–0.869	–0.025	0.108	–0.868
Cinammic acid	–0.307	0.245	–0.704	–0.792	0.960
Ellagic acid	–0.256	–0.730	0.209	0.337	–0.960
Vanillic acid	–0.411	0.136	–0.778	–0.855	0.922
Procyanidin B1	–0.495	–0.881	–0.048	0.085	–0.856
Catechin	–0.313	0.238	–0.709	–0.796	0.958
Procyanidin B2	0.182	–0.368	0.607	0.707	–0.988
Epicatechin	–0.903	–0.537	–0.996	–0.988	0.454
Rutin	–0.583	–0.925	–0.153	–0.020	–0.797
Gallocatechin gallate	–0.130	–0.636	0.332	0.455	–0.988
Epigallocatechin gallate	–0.699	–0.972	–0.301	–0.171	–0.696
Epicatechin gallate	–0.212	–0.699	0.252	0.378	–0.971
Quercetin-3-O-galactoside	–0.090	0.453	–0.530	–0.638	0.998
Quercetin-3-O-glucoside	–0.901	–0.534	–0.995	–0.988	0.457
Coumestrol	0.941	0.977	0.686	0.583	0.314
Narcissin	–0.495	–0.881	–0.049	0.084	–0.855
Nicotiflorin	0.196	–0.354	0.618	0.717	–0.985
Total phytosterols	–0.739	–0.984	–0.355	–0.227	–0.654
β-Sitosterol	–0.634	–0.948	–0.216	–0.084	–0.756
β-Campesterol	–0.206	–0.694	0.258	0.384	–0.973
Campestanol	0.004	–0.528	0.455	0.570	–0.995
Δ5-Avenasterol	–0.255	–0.729	0.209	0.337	–0.960
Δ7-Stigmasterol	–0.577	–0.922	–0.145	–0.012	–0.801
Stigmastanol	–0.089	–0.604	0.371	0.491	–0.993
Total saponins	–0.398	–0.824	0.060	0.192	–0.907
Campesteryl 3β-D glucopyranoside	0.145	–0.402	0.576	0.680	–0.993
Stigmasteryl 3β-D-glucopyranoside	0.207	–0.344	0.627	0.725	–0.984
Sitosteryl 3β-D-glucopyranoside	–0.594	–0.930	–0.166	–0.034	–0.788
Isorhamnetin 3-O-rutinoside	0.005	–0.527	0.456	0.571	–0.995

Bold values indicate significant correlation by Pearson’s test.