1 Abstract

Oxidative stress and inflammation are interlinked processes that seem to play an important role in aging. The present work aimed to test the antioxidant and anti-inflammatory activity of ethanolic Mahonia aquifolium leaves and bark extracts in an experimental acute inflammation. Six polyphenols and four alkaloids were measured by HPLC. The radical scavenging activity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) test. Inflammation was induced in rat with turpentine oil. Anti-inflammatory activity was evaluated with serum nitric oxide (NOx) and tumour necrosis factor alpha (TNF-alpha), and oxidative stress with total oxidative status (TOS), total antioxidant reactivity (TAR), oxidative stress index (OSI), 3-nitrotyrosine (3NT), malondialdehyde (MDA) and total thiols (SH). Extracts were administrated orally (100%, 50%, 25%) for seven days prior to inflammation. The effects were compared to diclofenac. The most abundant polyphenol was chlorogenic acid, and alkaloids were identified only in the bark extract. The DPPH assay showed good results, except for the bark extract. All extracts decreased NOx, TOS, 3NT, and increased SH. TNF-alpha was reduced, and TAR was increased only by the leaves extract. MDA was not influenced. Our findings suggest that M. aquifolium leaves and bark extracts have anti-inflammatory and antioxidant effects that support the use in primary prevention of the “inflammaging” process.

2 Methods

2.1 Statistical analysis

Figure 1: Principal components 1 and 2 rotations for all variables. Together, PC1 and PC2 captured 52.4% of the variance of the data.

2.2 Results

2.2.1 Sample descriptions

Table 1: .

Variable	Details	control	inflammation	100% + inflammation	50% + inflammation	25% + inflammation	Diclofenac + inflammation	inflammation + 100%	inflammation + 50%	inflammation + 25%	inflammation + Diclofenac	Total	Statistics
Group		6 (10.0%)	6 (10.0%)	6 (10.0%)	6 (10.0%)	6 (10.0%)	6 (10.0%)	6 (10.0%)	6 (10.0%)	6 (10.0%)	6 (10.0%)	60
MDA	M (min:max)	2.56 (2.16:2.86)	4.13 (3.3:4.5)	3.76 (3.51:3.93)	3.24 (2.91:3.56)	2.91 (2.47:3.28)	3.41 (2.5:4.81)	3.51 (2.08:3.87)	3.51 (2.7:3.9)	4.03 (3.61:4.16)	3.28 (2.91:3.67)	3.33 (2.08:4.81)	KW: p=0.003
MDA	μ ±SD	2.50 ±0.258	3.91 ±0.559	3.74 ±0.187	3.25 ±0.245	2.89 ±0.354	3.54 ±1.0	3.17 ±0.764	3.36 ±0.522	3.93 ±0.215	3.28 ±0.262	3.34 ±0.63	KW: p=0.003
NO	M (min:max)	38.61 (33.91:45.4)	50.7 (48.64:61.3)	36.49 (34.5:43.33)	41.42 (31.11:45.69)	36.63 (24.63:43.04)	34.35 (29.34:49.52)	24.63 (21.68:37.16)	33.03 (24.48:37)	30.39 (22.86:33.47)	29.78 (22.86:42.45)	34.94 (21.68:61.3)	1-way ANOVA: p<0.001
NO	μ ±SD	39.35 ±4.61	53.56 ±5.35	37.70 ±3.87	39.82 ±6.2	35.23 ±8.15	36.09 ±7.86	27.11 ±6.55	31.79 ±5.16	29.46 ±4.08	31.55 ±7.28	36.14 ±9.04	1-way ANOVA: p<0.001
OSI	M (min:max)	6.08 (5.49:7.28)	5.46 (4.76:6.44)	4.22 (3.56:6.65)	5.35 (4.85:6.3)	5.27 (4.85:5.69)	5.34 (3.67:5.7)	5.83 (4.62:6.79)	3.81 (3.56:5.11)	3.68 (3.31:4.28)	4.98 (4.49:6.3)	5.11 (3.31:7.28)	1-way ANOVA: p<0.001
OSI	μ ±SD	6.16 ±0.626	5.57 ±0.621	4.66 ±1.43	5.46 ±0.604	5.27 ±0.374	4.82 ±0.948	5.63 ±0.856	4.13 ±0.662	3.75 ±0.405	5.27 ±0.724	5.11 ±0.988	1-way ANOVA: p<0.001
TAC	M (min:max)	1.09 (1.09:1.09)	1.09 (1.09:1.09)	1.09 (1.09:1.1)	1.09 (1.09:1.09)	1.09 (1.09:1.09)	1.09 (1.09:1.09)	1.09 (1.09:1.09)	1.09 (1.09:1.09)	1.09 (1.09:1.09)	1.09 (1.09:1.1)	1.09 (1.09:1.1)	1-way ANOVA: p<0.001
TAC	μ ±SD	1.09 ±0.00126	1.09 ±0.00118	1.09 ±0.00167	1.09 ±0.00145	1.09 ±0.00212	1.09 ±0.00146	1.09 ±0.00159	1.09 ±0.000763	1.09 ±0.00105	1.09 ±0.000771	1.09 ±0.00245	1-way ANOVA: p<0.001
TIOLI	M (min:max)	463 (379:485)	291 (213:299)	428 (355:471)	302 (137:367)	310 (273:359)	307 (255:315)	305 (287:375)	283 (255:345)	365 (313:387)	365 (299:415)	315 (137:485)	KW: p<0.001
TIOLI	μ ±SD	442.00 ±45.8	273.80 ±35.7	420.50 ±51.7	289.33 ±80.4	313.00 ±35.5	290.20 ±28.8	313.80 ±35.3	287.40 ±34.4	357.00 ±28.1	360.00 ±40.2	335.16 ±70.0	KW: p<0.001
TOS	M (min:max)	5.25 (4.01:6.49)	6.62 (5.96:7.92)	5.77 (4.92:6.62)	5.31 (4.01:6.88)	6.16 (5.83:7.4)	6.22 (5.18:7.01)	3.88 (3.62:4.66)	4.01 (3.88:5.18)	5.96 (5.31:7.27)	5.77 (5.31:6.62)	5.57 (3.62:7.92)	1-way ANOVA: p<0.001
TOS	μ ±SD	5.25 ±0.819	6.80 ±0.712	5.77 ±0.71	5.33 ±1.1	6.39 ±0.709	6.12 ±0.674	4.01 ±0.412	4.29 ±0.533	6.22 ±0.819	5.83 ±0.528	5.57 ±1.08	1-way ANOVA: p<0.001
PC1	M (min:max)	2.18 (1.67:2.87)	-2.19 (-2.94:-1.44)	0.34 (-0.28:0.68)	0.26 (-1.19:0.92)	0.15 (-0.82:0.71)	-0.67 (-2.65:0.65)	0.89 (-0.37:2.6)	-0.36 (-1.07:0.34)	-0.7 (-1.4:-0.62)	0.61 (-0.13:1.87)	0.15 (-2.94:2.87)	1-way ANOVA: p<0.001
PC1	μ ±SD	2.16 ±0.474	-2.09 ±0.643	0.268 ±0.399	0.073 ±0.704	0.0447 ±0.633	-1.01 ±1.47	0.867 ±1.12	-0.328 ±0.515	-0.929 ±0.388	0.824 ±0.786	0.00000000000000593 ±1.34	1-way ANOVA: p<0.001
PC2	M (min:max)	-0.7 (-1.95:0.07)	-1.41 (-2.49:-1.16)	1.48 (-0.56:1.89)	-0.59 (-1.12:0.07)	-0.38 (-1.11:-0.01)	-0.59 (-0.9:1.43)	-0.58 (-1.02:1.43)	0.33 (-0.05:1.31)	1.74 (1.03:2.35)	0.75 (-0.32:1.73)	-0.1 (-2.49:2.35)	1-way ANOVA: p<0.001
PC2	μ ±SD	-0.768 ±0.769	-1.71 ±0.6	1.07 ±1.13	-0.533 ±0.463	-0.474 ±0.522	-0.226 ±0.967	-0.0795 ±1.08	0.526 ±0.561	1.59 ±0.558	0.692 ±0.804	0.00000000000000557 ±1.16	1-way ANOVA: p<0.001
μ ±SD = Mean (standard deviation); M (min:max) = Median (min:max);

Figure 2: Box-plot of MDAby group (| median, ⋄ mean). Both experiments are depicted. Groups’ label annotations mean: 1 = vs. control, 2 = vs. infalmmation, 3 = vs. Diclofenac.

Table 2: [Prophylactic experiment] Pairwise comparrisons between groups for MDA (Holm p-values adjustment).

Pairwise	control	inflammation	100% + inflammation	50% + inflammation	25% + inflammation	Diclofenac + inflammation
control		0.002	0.015	0.205	>0.999	0.036
inflammation	0.002		>0.999	0.401	0.086	>0.999
100% + inflammation	0.015	>0.999		>0.999	0.294	>0.999
50% + inflammation	0.205	0.401	>0.999		>0.999	>0.999
25% + inflammation	>0.999	0.086	0.294	>0.999		0.602
Diclofenac + inflammation	0.036	>0.999	>0.999	>0.999	0.602

Table 3: [Curative experiment] Pairwise comparrisons between groups for MDA (Holm p-values adjustment).

Pairwise	control	inflammation	inflammation + 100%	inflammation + 50%	inflammation + 25%	inflammation + Diclofenac
control		<0.001	0.204	0.059	<0.001	0.078
inflammation	<0.001		0.179	0.378	>0.999	0.237
inflammation + 100%	0.204	0.179		>0.999	0.167	>0.999
inflammation + 50%	0.059	0.378	>0.999		0.378	>0.999
inflammation + 25%	<0.001	>0.999	0.167	0.378		0.231
inflammation + Diclofenac	0.078	0.237	>0.999	>0.999	0.231

Figure 3: Box-plot of NOby group (| median, ⋄ mean). Both experiments are depicted. Groups’ label annotations mean: 1 = vs. control, 2 = vs. infalmmation, 3 = vs. Diclofenac.

Table 4: [Prophylactic experiment] Pairwise comparrisons between groups for NO (Holm p-values adjustment).

Pairwise	control	inflammation	100% + inflammation	50% + inflammation	25% + inflammation	Diclofenac + inflammation
control		0.016	>0.999	>0.999	>0.999	>0.999
inflammation	0.016		0.012	0.016	0.003	0.003
100% + inflammation	>0.999	0.012		>0.999	>0.999	>0.999
50% + inflammation	>0.999	0.016	>0.999		>0.999	>0.999
25% + inflammation	>0.999	0.003	>0.999	>0.999		>0.999
Diclofenac + inflammation	>0.999	0.003	>0.999	>0.999	>0.999

Table 5: [Curative experiment] Pairwise comparrisons between groups for NO (Holm p-values adjustment).

Pairwise	control	inflammation	inflammation + 100%	inflammation + 50%	inflammation + 25%	inflammation + Diclofenac
control		0.006	0.022	0.320	0.099	0.259
inflammation	0.006		<0.001	<0.001	<0.001	<0.001
inflammation + 100%	0.022	<0.001		>0.999	>0.999	>0.999
inflammation + 50%	0.320	<0.001	>0.999		>0.999	>0.999
inflammation + 25%	0.099	<0.001	>0.999	>0.999		>0.999
inflammation + Diclofenac	0.259	<0.001	>0.999	>0.999	>0.999

Figure 4: Box-plot of OSIby group (| median, ⋄ mean). Both experiments are depicted. Groups’ label annotations mean: 1 = vs. control, 2 = vs. infalmmation, 3 = vs. Diclofenac.

Table 6: [Prophylactic experiment] Pairwise comparrisons between groups for OSI (Holm p-values adjustment).

Pairwise	control	inflammation	100% + inflammation	50% + inflammation	25% + inflammation	Diclofenac + inflammation
control		>0.999	0.123	>0.999	>0.999	0.154
inflammation	>0.999		>0.999	>0.999	>0.999	>0.999
100% + inflammation	0.123	>0.999		>0.999	>0.999	>0.999
50% + inflammation	>0.999	>0.999	>0.999		>0.999	>0.999
25% + inflammation	>0.999	>0.999	>0.999	>0.999		>0.999
Diclofenac + inflammation	0.154	>0.999	>0.999	>0.999	>0.999

Table 7: [Curative experiment] Pairwise comparrisons between groups for OSI (Holm p-values adjustment).

Pairwise	control	inflammation	inflammation + 100%	inflammation + 50%	inflammation + 25%	inflammation + Diclofenac
control		0.938	>0.999	<0.001	<0.001	0.204
inflammation	0.938		>0.999	0.018	0.002	>0.999
inflammation + 100%	>0.999	>0.999		0.014	0.002	>0.999
inflammation + 50%	<0.001	0.018	0.014		>0.999	0.068
inflammation + 25%	<0.001	0.002	0.002	>0.999		0.009
inflammation + Diclofenac	0.204	>0.999	>0.999	0.068	0.009

Figure 5: Box-plot of TACby group (| median, ⋄ mean). Both experiments are depicted. Groups’ label annotations mean: 1 = vs. control, 2 = vs. infalmmation, 3 = vs. Diclofenac.

Table 8: [Prophylactic experiment] Pairwise comparrisons between groups for TAC (Holm p-values adjustment).

Pairwise	control	inflammation	100% + inflammation	50% + inflammation	25% + inflammation	Diclofenac + inflammation
control		<0.001	0.517	0.768	0.494	0.016
inflammation	<0.001		<0.001	<0.001	0.077	0.541
100% + inflammation	0.517	<0.001		0.432	0.035	<0.001
50% + inflammation	0.768	<0.001	0.432		0.541	0.030
25% + inflammation	0.494	0.077	0.035	0.541		0.541
Diclofenac + inflammation	0.016	0.541	<0.001	0.030	0.541

Table 9: [Curative experiment] Pairwise comparrisons between groups for TAC (Holm p-values adjustment).

Pairwise	control	inflammation	inflammation + 100%	inflammation + 50%	inflammation + 25%	inflammation + Diclofenac
control		<0.001	0.001	<0.001	0.179	0.029
inflammation	<0.001		0.179	0.179	0.001	<0.001
inflammation + 100%	0.001	0.179		0.842	0.179	<0.001
inflammation + 50%	<0.001	0.179	0.842		0.139	<0.001
inflammation + 25%	0.179	0.001	0.179	0.139		<0.001
inflammation + Diclofenac	0.029	<0.001	<0.001	<0.001	<0.001

Figure 6: Box-plot of TIOLIby group (| median, ⋄ mean). Both experiments are depicted. Groups’ label annotations mean: 1 = vs. control, 2 = vs. infalmmation, 3 = vs. Diclofenac.

Table 10: [Prophylactic experiment] Pairwise comparrisons between groups for TIOLI (Holm p-values adjustment).

Pairwise	control	inflammation	100% + inflammation	50% + inflammation	25% + inflammation	Diclofenac + inflammation
control		<0.001	>0.999	<0.001	0.007	<0.001
inflammation	<0.001		0.003	>0.999	>0.999	>0.999
100% + inflammation	>0.999	0.003		0.006	0.054	0.008
50% + inflammation	<0.001	>0.999	0.006		>0.999	>0.999
25% + inflammation	0.007	>0.999	0.054	>0.999		>0.999
Diclofenac + inflammation	<0.001	>0.999	0.008	>0.999	>0.999

Table 11: [Curative experiment] Pairwise comparrisons between groups for TIOLI (Holm p-values adjustment).

Pairwise	control	inflammation	inflammation + 100%	inflammation + 50%	inflammation + 25%	inflammation + Diclofenac
control		<0.001	<0.001	<0.001	0.009	0.009
inflammation	<0.001		0.414	>0.999	0.015	0.009
inflammation + 100%	<0.001	0.414		0.827	0.400	0.313
inflammation + 50%	<0.001	>0.999	0.827		0.048	0.029
inflammation + 25%	0.009	0.015	0.400	0.048		>0.999
inflammation + Diclofenac	0.009	0.009	0.313	0.029	>0.999

Figure 7: Box-plot of TOSby group (| median, ⋄ mean). Both experiments are depicted. Groups’ label annotations mean: 1 = vs. control, 2 = vs. infalmmation, 3 = vs. Diclofenac.

Table 12: [Prophylactic experiment] Pairwise comparrisons between groups for TOS (Holm p-values adjustment).

Pairwise	control	inflammation	100% + inflammation	50% + inflammation	25% + inflammation	Diclofenac + inflammation
control		0.072	>0.999	>0.999	0.549	0.927
inflammation	0.072		0.821	0.101	>0.999	>0.999
100% + inflammation	>0.999	0.821		>0.999	>0.999	>0.999
50% + inflammation	>0.999	0.101	>0.999		0.709	>0.999
25% + inflammation	0.549	>0.999	>0.999	0.709		>0.999
Diclofenac + inflammation	0.927	>0.999	>0.999	>0.999	>0.999

Table 13: [Curative experiment] Pairwise comparrisons between groups for TOS (Holm p-values adjustment).

Pairwise	control	inflammation	inflammation + 100%	inflammation + 50%	inflammation + 25%	inflammation + Diclofenac
control		0.006	0.036	0.149	0.149	0.540
inflammation	0.006		<0.001	<0.001	0.541	0.149
inflammation + 100%	0.036	<0.001		0.672	<0.001	0.001
inflammation + 50%	0.149	<0.001	0.672		0.001	0.006
inflammation + 25%	0.149	0.541	<0.001	0.001		0.672
inflammation + Diclofenac	0.540	0.149	0.001	0.006	0.672

Figure 8: Box-plot of PC1by group (| median, ⋄ mean). Both experiments are depicted. Groups’ label annotations mean: 1 = vs. control, 2 = vs. infalmmation, 3 = vs. Diclofenac.

Table 14: [Prophylactic experiment] Pairwise comparrisons between groups for PC1 (Holm p-values adjustment).

Pairwise	control	inflammation	100% + inflammation	50% + inflammation	25% + inflammation	Diclofenac + inflammation
control		<0.001	0.017	0.004	0.007	<0.001
inflammation	<0.001		0.003	0.003	0.007	0.244
100% + inflammation	0.017	0.003		>0.999	>0.999	0.200
50% + inflammation	0.004	0.003	>0.999		>0.999	0.232
25% + inflammation	0.007	0.007	>0.999	>0.999		0.265
Diclofenac + inflammation	<0.001	0.244	0.200	0.232	0.265

Table 15: [Curative experiment] Pairwise comparrisons between groups for PC1 (Holm p-values adjustment).

Pairwise	control	inflammation	inflammation + 100%	inflammation + 50%	inflammation + 25%	inflammation + Diclofenac
control		<0.001	0.044	<0.001	<0.001	0.029
inflammation	<0.001		<0.001	0.004	0.060	<0.001
inflammation + 100%	0.044	<0.001		0.060	0.004	0.920
inflammation + 50%	<0.001	0.004	0.060		0.376	0.060
inflammation + 25%	<0.001	0.060	0.004	0.376		0.004
inflammation + Diclofenac	0.029	<0.001	0.920	0.060	0.004

Figure 9: Box-plot of PC2by group (| median, ⋄ mean). Both experiments are depicted. Groups’ label annotations mean: 1 = vs. control, 2 = vs. infalmmation, 3 = vs. Diclofenac.

Table 16: [Prophylactic experiment] Pairwise comparrisons between groups for PC2 (Holm p-values adjustment).

Pairwise	control	inflammation	100% + inflammation	50% + inflammation	25% + inflammation	Diclofenac + inflammation
control		0.435	0.020	>0.999	>0.999	>0.999
inflammation	0.435		<0.001	0.176	0.188	0.062
100% + inflammation	0.020	<0.001		0.043	0.093	0.176
50% + inflammation	>0.999	0.176	0.043		>0.999	>0.999
25% + inflammation	>0.999	0.188	0.093	>0.999		>0.999
Diclofenac + inflammation	>0.999	0.062	0.176	>0.999	>0.999

Table 17: [Curative experiment] Pairwise comparrisons between groups for PC2 (Holm p-values adjustment).

Pairwise	control	inflammation	inflammation + 100%	inflammation + 50%	inflammation + 25%	inflammation + Diclofenac
control		0.357	0.484	0.095	<0.001	0.034
inflammation	0.357		0.022	0.001	<0.001	<0.001
inflammation + 100%	0.484	0.022		0.484	0.020	0.415
inflammation + 50%	0.095	0.001	0.484		0.248	0.719
inflammation + 25%	<0.001	<0.001	0.020	0.248		0.357
inflammation + Diclofenac	0.034	<0.001	0.415	0.719	0.357

Table 18: .

Subset	N	Mean ±SD	Med (min:max)
MDA (Shapiro-Wilk normality test: p=0.928)
(total)	51 (100.0%)	3.339 ±0.63	3.33 (2.08:4.8)
Group (ANOVA: p<0.001)
control	6 (11.8%)	2.496 ±0.26	2.56 (2.16:2.9)
inflammation	5 (9.8%)	3.910 ±0.56	4.13 (3.30:4.5)
100% + inflammation	4 (7.8%)	3.737 ±0.19	3.76 (3.51:3.9)
50% + inflammation	6 (11.8%)	3.248 ±0.24	3.24 (2.91:3.6)
25% + inflammation	4 (7.8%)	2.892 ±0.35	2.91 (2.47:3.3)
Diclofenac + inflammation	5 (9.8%)	3.536 ±1.00	3.41 (2.50:4.8)
inflammation + 100%	5 (9.8%)	3.173 ±0.76	3.51 (2.08:3.9)
inflammation + 50%	5 (9.8%)	3.364 ±0.52	3.51 (2.70:3.9)
inflammation + 25%	5 (9.8%)	3.931 ±0.21	4.03 (3.61:4.2)
inflammation + Diclofenac	6 (11.8%)	3.285 ±0.26	3.28 (2.91:3.7)

Table 19: .

Subset	N	Mean ±SD	Med (min:max)
NO (Shapiro-Wilk normality test: p=0.176)
(total)	50 (100.0%)	36.135 ±9.04	34.94 (21.68:61.3)
Group (ANOVA: p<0.001)
control	5 (10.0%)	39.355 ±4.61	38.61 (33.91:45.4)
inflammation	5 (10.0%)	53.555 ±5.35	50.70 (48.64:61.3)
100% + inflammation	4 (8.0%)	37.701 ±3.87	36.49 (34.50:43.3)
50% + inflammation	6 (12.0%)	39.824 ±6.20	41.42 (31.11:45.7)
25% + inflammation	4 (8.0%)	35.234 ±8.15	36.63 (24.63:43.0)
Diclofenac + inflammation	5 (10.0%)	36.088 ±7.86	34.35 (29.34:49.5)
inflammation + 100%	5 (10.0%)	27.108 ±6.55	24.63 (21.68:37.2)
inflammation + 50%	5 (10.0%)	31.788 ±5.16	33.03 (24.48:37.0)
inflammation + 25%	5 (10.0%)	29.464 ±4.08	30.39 (22.86:33.5)
inflammation + Diclofenac	6 (12.0%)	31.550 ±7.28	29.78 (22.86:42.5)

Table 20: .

Subset	N	Mean ±SD	Med (min:max)
OSI (Shapiro-Wilk normality test: p=0.229)
(total)	51 (100.0%)	5.109 ±0.99	5.11 (3.31:7.3)
Group (ANOVA: p<0.001)
control	6 (11.8%)	6.157 ±0.63	6.08 (5.49:7.3)
inflammation	5 (9.8%)	5.570 ±0.62	5.46 (4.76:6.4)
100% + inflammation	4 (7.8%)	4.662 ±1.43	4.22 (3.56:6.7)
50% + inflammation	6 (11.8%)	5.461 ±0.60	5.35 (4.85:6.3)
25% + inflammation	4 (7.8%)	5.269 ±0.37	5.27 (4.85:5.7)
Diclofenac + inflammation	5 (9.8%)	4.817 ±0.95	5.34 (3.67:5.7)
inflammation + 100%	5 (9.8%)	5.633 ±0.86	5.83 (4.62:6.8)
inflammation + 50%	5 (9.8%)	4.128 ±0.66	3.81 (3.56:5.1)
inflammation + 25%	5 (9.8%)	3.749 ±0.40	3.68 (3.31:4.3)
inflammation + Diclofenac	6 (11.8%)	5.272 ±0.72	4.98 (4.49:6.3)

Table 21: .

Subset	N	Mean ±SD	Med (min:max)
TAC (Shapiro-Wilk normality test: p=0.269)
(total)	51 (100.0%)	1.091 ±0.00	1.09 (1.09:1.1)
Group (ANOVA: p<0.001)
control	6 (11.8%)	1.093 ±0.00	1.09 (1.09:1.1)
inflammation	5 (9.8%)	1.088 ±0.00	1.09 (1.09:1.1)
100% + inflammation	4 (7.8%)	1.094 ±0.00	1.09 (1.09:1.1)
50% + inflammation	6 (11.8%)	1.092 ±0.00	1.09 (1.09:1.1)
25% + inflammation	4 (7.8%)	1.091 ±0.00	1.09 (1.09:1.1)
Diclofenac + inflammation	5 (9.8%)	1.089 ±0.00	1.09 (1.09:1.1)
inflammation + 100%	5 (9.8%)	1.090 ±0.00	1.09 (1.09:1.1)
inflammation + 50%	5 (9.8%)	1.089 ±0.00	1.09 (1.09:1.1)
inflammation + 25%	5 (9.8%)	1.091 ±0.00	1.09 (1.09:1.1)
inflammation + Diclofenac	6 (11.8%)	1.095 ±0.00	1.09 (1.09:1.1)

Table 22: .

Subset	N	Mean ±SD	Med (min:max)
TIOLI (Shapiro-Wilk normality test: p=0.057)
(total)	51 (100.0%)	335.157 ±69.97	315.00 (137.00:485.0)
Group (ANOVA: p<0.001)
control	6 (11.8%)	442.000 ±45.78	463.00 (379.00:485.0)
inflammation	5 (9.8%)	273.800 ±35.65	291.00 (213.00:299.0)
100% + inflammation	4 (7.8%)	420.500 ±51.68	428.00 (355.00:471.0)
50% + inflammation	6 (11.8%)	289.333 ±80.35	302.00 (137.00:367.0)
25% + inflammation	4 (7.8%)	313.000 ±35.52	310.00 (273.00:359.0)
Diclofenac + inflammation	5 (9.8%)	290.200 ±28.76	307.00 (255.00:315.0)
inflammation + 100%	5 (9.8%)	313.800 ±35.34	305.00 (287.00:375.0)
inflammation + 50%	5 (9.8%)	287.400 ±34.39	283.00 (255.00:345.0)
inflammation + 25%	5 (9.8%)	357.000 ±28.11	365.00 (313.00:387.0)
inflammation + Diclofenac	6 (11.8%)	360.000 ±40.21	365.00 (299.00:415.0)

Table 23: .

Subset	N	Mean ±SD	Med (min:max)
TOS (Shapiro-Wilk normality test: p=0.195)
(total)	51 (100.0%)	5.575 ±1.08	5.57 (3.62:7.9)
Group (ANOVA: p<0.001)
control	6 (11.8%)	5.246 ±0.82	5.25 (4.01:6.5)
inflammation	5 (9.8%)	6.798 ±0.71	6.62 (5.96:7.9)
100% + inflammation	4 (7.8%)	5.768 ±0.71	5.77 (4.92:6.6)
50% + inflammation	6 (11.8%)	5.333 ±1.10	5.31 (4.01:6.9)
25% + inflammation	4 (7.8%)	6.387 ±0.71	6.16 (5.83:7.4)
Diclofenac + inflammation	5 (9.8%)	6.120 ±0.67	6.22 (5.18:7.0)
inflammation + 100%	5 (9.8%)	4.008 ±0.41	3.88 (3.62:4.7)
inflammation + 50%	5 (9.8%)	4.295 ±0.53	4.01 (3.88:5.2)
inflammation + 25%	5 (9.8%)	6.224 ±0.82	5.96 (5.31:7.3)
inflammation + Diclofenac	6 (11.8%)	5.833 ±0.53	5.77 (5.31:6.6)

Table 24: .

Subset	N	Mean ±SD	Med (min:max)
PC1 (Shapiro-Wilk normality test: p=0.609)
(total)	50 (100.0%)	0.000 ±1.34	0.15 (-2.94:2.9)
Group (ANOVA: p<0.001)
control	5 (10.0%)	2.164 ±0.47	2.18 (1.67:2.9)
inflammation	5 (10.0%)	-2.087 ±0.64	-2.19 (-2.94:-1.4)
100% + inflammation	4 (8.0%)	0.268 ±0.40	0.34 (-0.28:0.7)
50% + inflammation	6 (12.0%)	0.073 ±0.70	0.26 (-1.19:0.9)
25% + inflammation	4 (8.0%)	0.045 ±0.63	0.15 (-0.82:0.7)
Diclofenac + inflammation	5 (10.0%)	-1.012 ±1.47	-0.67 (-2.65:0.7)
inflammation + 100%	5 (10.0%)	0.867 ±1.12	0.89 (-0.37:2.6)
inflammation + 50%	5 (10.0%)	-0.328 ±0.51	-0.36 (-1.07:0.3)
inflammation + 25%	5 (10.0%)	-0.929 ±0.39	-0.70 (-1.40:-0.6)
inflammation + Diclofenac	6 (12.0%)	0.824 ±0.79	0.61 (-0.13:1.9)

Table 25: .

Subset	N	Mean ±SD	Med (min:max)
PC2 (Shapiro-Wilk normality test: p=0.207)
(total)	50 (100.0%)	0.000 ±1.16	-0.10 (-2.49:2.3)
Group (ANOVA: p<0.001)
control	5 (10.0%)	-0.768 ±0.77	-0.70 (-1.95:0.1)
inflammation	5 (10.0%)	-1.711 ±0.60	-1.41 (-2.49:-1.2)
100% + inflammation	4 (8.0%)	1.074 ±1.13	1.48 (-0.56:1.9)
50% + inflammation	6 (12.0%)	-0.533 ±0.46	-0.59 (-1.12:0.1)
25% + inflammation	4 (8.0%)	-0.474 ±0.52	-0.38 (-1.11:0.0)
Diclofenac + inflammation	5 (10.0%)	-0.226 ±0.97	-0.59 (-0.90:1.4)
inflammation + 100%	5 (10.0%)	-0.079 ±1.08	-0.58 (-1.02:1.4)
inflammation + 50%	5 (10.0%)	0.526 ±0.56	0.33 (-0.05:1.3)
inflammation + 25%	5 (10.0%)	1.587 ±0.56	1.74 (1.03:2.3)
inflammation + Diclofenac	6 (12.0%)	0.692 ±0.80	0.75 (-0.32:1.7)

2.3 Correlations

2.3.1 MDA

Figure 10: .

2.4 Intra-group correlations

Figure 11: .

Table 26: .

Figure 12: .

Table 27: .

Figure 13: .

Table 28: .

Figure 14: .

Table 29: .

Figure 15: .

Table 30: .

Figure 16: .

Table 31: .

Figure 17: .

Table 32: .

Figure 18: .

Table 33: .

Figure 19: .

Table 34: .

Figure 20: .

Table 35: .

Title

Subtitle

Elisabeta Chera