Enzyme-assisted modification of flavonoids from Matricaria chamomilla: antioxidant activity and inhibitory effect on digestive enzymes

Figures & data

Figure 1. Boxplots of some selected statistically significant (p > .05) metabolites from chamomile infusion indicating normalised intensity differences before (red) and after (green) enzymatic treatment. Esculin (1.33_340.0800n), luteoloside (2.44_448.1009n), rutin (2.14_611.1610m/z), esculetin (1.71_179.0343m/z), luteolin (2.98_287.0552m/z) and umbelliferone (0.60_163.0394m/z). All features were observed in the positive ionisation mode.

Table 1. Putative identification of statistically significant secondary metabolites from chamomile infusion and the relative tendency after the enzymatic treatment.

Feature^a number	Feature label	p Value^b	FDR	Formula	Mass error^c (ppm)	ID^d	Mode	Trend^e
1	1.97_165.0548m/z	5.5999E-10	1.0685E-08	C9H8O3	1.02	2-Hydroxy Cinnamic Acid (o-coumaric acid)	ESI(+)	High
2	2.46_189.0549m/z	2.0257E-09	2.5899E-08	C11H12O5	1.03	4-Hydroxy-3,5-dimethoxycinnamic acid	ESI(+)	High
4	0.50_198.0524n	1.34E-03	1.02E-02	C9H10O5	−2.26	Alpha-(3,4-Dihydroxyphenyl) lactic acid	ESI(−)	High
5	2.67_180.0426n	1.683E-10	5.1599E-09	C9H8O4	1.65	3,4-dihydroxycinnamic acid (caffeic acid)	ESI(+)	High
6	5.13_271.0965m/z	1.09E-05	2.07E-05	C16H14O4	0.09	Dihydroformononetin	ESI(+)	High
7	1.26_155.0340m/z	1.30E-07	4.87E-07	C7H6O4	1.04	Dihydroxybenzoic acid	ESI(+)	High
8	4.00_182.1178m/z	1.3385E-12	5.5759E-10	C10H12O2	1.61	Eugenic acid	ESI(+)	High
9	3.51_301.0709m/z	2.8657E-07	9.3267E-07	C16H12O6	0.85	Hispidulin	ESI(+)	High
10	3.21_179.0706m/z	3.8202E-10	8.4129E-09	C10H12O4	1.81	Hydroferulic acid	ESI(+)	High
11	2.67_135.0442m/z	1.33E-11	1.35E-09	C8H8O3	1.24	Hydroxyphenylacetic acid	ESI(+)	High
12	0.56_169.0134m/z	5.37E-04	6.04E-03	C7H6O5	−5.04	Pyrogallolcarboxylic acid (triihydroxybenzoic acid)	ESI(−)	High
13	4.70_343.1175m/z	2.13E-06	5.07E-06	C19H18O6	−0.40	Tetramethyl-O-scutellarin	ESI(−)	High
14	3.52_447.1279m/z	4.53E-07	1.36E-06	C10H12O5	4.12	Vanillactic acid	ESI(+)	High
15	3.83_184.0370n	1.60E-10	5.08E-09	C8H8O5	−0.80	3-O-Methylgallic acid	ESI(+)	High
16	3.37_474.1166n	1.3749E-08	9.0792E-08	C23H22O11	0.73	6″-O-acetylgenistin	ESI(+)	Low
17	5.48_113.0383m/z	9.48E-07	2.55E-06	C9H8O2	−1.87	Cinnamic acid	ESI(+)	Low
18	3.61_303.0137m/z	1.14E-06	7.16E-05	C14H10O9	−3.05	Digallic acid	ESI(−)	Low
19	5.48_171.0806m/z	1.26E-07	4.76E-07	C12H14O3	0.70	Eugenol acetate	ESI(+)	Low
20	5.48_159.0441m/z	1.17E-07	4.50E-07	C10H10O4	0.18	Hesperetic acid (trans-cinnamic acid)	ESI(+)	Low
21	3.35_631.1660m/z	6.23E-07	1.77E-06	C28H32O15	4.30	Diosmin (diosmetin 7-rutinoside)	ESI(+)	Low
22	3.83_575.1397m/z	4.54E-07	1.36E-06	C27H30O16	0.20	Quercetin 3-O-neohesperidoside	ESI(+)	Low
23	2.84_725.1939m/z	1.16E-07	4.47E-07	C32H38O20	2.09	Quercetin 3-(2G-xylosylrutinoside)	ESI(+)	Low
24	1.78_338.1001n	1.07E-10	4.172E-09	C16H18O8	−0.18	4-p-Coumaroylquinic acid	ESI(+)	Low
25	2.33_317.0655m/z	1.35E-09	1.9489E-08	C16H12O7	−0.24	6-Methoxyluteolin	ESI(+)	Low
26	3.24_559.1449m/z	4.85E-06	1.02E-05	C27H30O15	0.53	Kaempferol-7-neohesperidoside	ESI(+)	Low
27	2.72_591.1348m/z	2.25E-09	2.75E-08	C27H30O17	0.63	6,8-Dihydroxykaempferol 3-rutinoside	ESI(+)	Low
28	2.40_535.1087m/z	1.01E-08	7.32E-08	C24H22O14	0.87	Luteolin 7-O-(6''-malonylglucoside)	ESI(+)	Low
29	3.65_317.0654m/z	9.87E-06	1.90E-05	C16H12O7	−0.61	Isorhamnetin	ESI(+)	Low
30	2.36_519.1142m/z	5.84E-08	2.62E-07	C24H22O13	1.63	Malonylgenistin	ESI(+)	Low

^aFeature: mass-to charge ratio and retention time pairs, ^bFalse Discovered Ratio (FDR) adjusted, ^cCalculated in comparison with theoretical value, ^dPutative Identity (ID) All listed compounds reached level 2 identification, except for feature numbers 4, 16, 17 and 22 that reached level 3. ^eIn product of hydrolysis when compared to native chamomilla infusion.

Table 2. DPPH• scavenging activity (%) and kinetic parameters of inhibitory effect of chamomile infusion toward digestive enzymes activities in the absence (control) and presence of the inhibitors (40 and 60 µM).

	Control	Native chamomile infusion		Enzymatically modified chamomile infusion
DPPH• scavenging activity (%)	–	41.3 ± 3.1^a		49.5 ± 1.5^b
Kinetic parameters		40 µM^1	60 µM^2	40 µM^3	60 µM^4
α-glucosidase
Inhibition (%)	0	36.7 ± 8.5^a	37.4 ± 5.6^a	45.2 ± 3.1^a	56.9 ± 3.7^b
Vmax (µmol/min)	0.79 ± 0.15^a	0.60 ± 0.29^a	0.29 ± 0.23 ^a,b	0.32 ± 0.25^a,b	0.21 ± 0.08^b
Km (mM)	354.4^a	332.4^a	265.2^a	332.4^a	295.2^a
Pancreatic lipase
Inhibition (%)	0	32.1 ± 6.3^a	39.2 ± 5.7^a	60.4 ± 4.9^b	73.6 ± 3.2^c
Vmax (µmol/min)	3.19 ± 0.42^a	3.29 ± 0.63^a	2.08 ± 0.25^b	1.97 ± 0.17^c	1.03 ± 0.04^d
Km (mM)	341.6^a	756.7^b	724.5^b	924.5^c	1107.3^d
α-amylase
Inhibition (%)	0	12.7 ± 4.1^a	10.8 ± 3.5^a	9.1 ± 4.8^a	11.6 ± 6.0^a
Vmax (µmol/min)	0.43 ± 0.27^a	0.38 ± 0.14^a	0.63 ± 0.28^a	0.45 ± 0.26^a	0.54 ± 0.14^a
Km (mM)	357.4^a	312.4^a	327.9^a	298.4^a	350.9^a

Results are expressed as the mean ± standard deviation (SD) of three determinations. Means within a line with different superscript letters are significantly different p < .05.

¹R²=0.8294; ²R²=0.9555; ³R²=0.8787; ⁴R²=0.9025.

Figure 2. Lineweaver–Burk plot of glucosidase (A) and pancreatic lipase (B) activities. V is initial velocity and [S] is the concentration of substrate. The values were shown in absence (■) and presence of enzymatically modified chamomile infusion at 40 µM (●) and 60 µM (▲).The values are means of triplicate determinations, and the error bars indicate SD (n = 3).