A multidisciplinary approach to the antioxidant and hepatoprotective activities of Arbutus pavarii Pampan fruit; in vitro and in Vivo biological evaluations, and in silico investigations

Figures & data

Table 4. LC-MS-MS analysis of Arbutus pavarii Pampan fruits extract.

No	RT	Compounds	MS (m/z): [M-H]-	%^b	MS/MS, m/z
1	3.94	Gallic acid glucoside	331.0640	0.063	169, 211, 271^26
2	9.66	Gallic acid	169.0137	0.784	125^27
3	10.03	Galloyl quinic acid	343.0652	0.046	191^28
4	10.15	Protocatecuic acid	153.0194	0.009	109^27
5	10.60	Protocatechuic acid O-hexoside	315.0698	0.046	108, 153^26
6	10.68	Vanillic acid-O-glucoside ester	329.0855	0.031	108, 123, 167, 219^26
7	10.83	Galloyl shikimic acid^a	325.0561	0.118	125, 169, 170^29
8	10.90	Epigallocatechin	305.0649	0.244	125, 137, 165^27
9	11.28	2,4-Dihydroxybenzoic acid	153.0198	0.118	109^30
10	11.66	Procyanidin dimer B3^a	577.1272	3.215	125, 161, 289, 407, 425, 577^31
11	11.66	β-Type procyanidin trimer C^a	865.1884	0.991	287, 575, 577, 695^31
12	11.74	Vanillin	151.0403	0.006	123, 137^30
13	12.08	Trigalloyl glucoside	635.0840	0.052	169^26
14	12.12	Cyanidin-3-O-glucoside	449.1037	0.571	259, 287^26
15	12.23	Methyl gallate	183.0285	0.679	78, 124, 168^32
16	12.30	Digalloyl shikmic acid^a	477.1514	0.002	169, 297, 196
17	12.38	3-Hydroxy benzoic acid	137.0236	0.003	93, 108^27
18	12.38	Myricetin glucoside	479.0793	0.380	316
19	12.49	Quercetin 3-O- rutinoside (Rutin)^a	609.1396	0.239	151, 300
20	12.61	Quercetin-O-galloyl-glucoside	615.0919	0.507	169, 300, 302, 463^26
21	12.76	Procyanidin A1	575.1158	0.014	259, 452, 509
22	12.80	Luteolin-7-glucoside	447.0904	0.024	152, 285^33
23	12.83	Quercetin-3-O-glucoside (Isoquercitin)^a	463.0833	1.207	151, 179, 301^33
24	13.06	Ellagic acid	300.9983	0.301	145, 201, 229, 299^32
25	13.21	Quercetin-3-O-(arabinoside/xyloside)	433.0743	0.182	271, 300^26
26	13.29	Isorhamnetin 3-O-glucoside	477.1003	0.063	243, 271, 315, 399^26
27	13.33	Quercetin 3-O-rhamnoside (Quercitrin)^a	447.0884	0.628	151, 255, 302
28	13.55	Myricetin rhamnoside^a	463.0821	0.027	179, 301, 316, 317, 463^34
29	13.82	Vitexin	431.0963	0.035	171, 285, 401, 431
30	14.08	Myricetin	317.0288	0.177	151, 178, 271^26
31	14.12	Catechin^a	289.0817	0.001	125, 178, 259, 287
32	14.80	Chrysin	253.0492	0.001	152^32
33	15.03	Quercetin	301.0344	0.219	151, 179^30
34	15.22	Luteolin	285.0420	0.278	133, 149, 175, 285^35
35	15.98	Kaempferol	285.0396	0.137	211, 239, 285^30
Total relative percentages				11.397

^a Compounds identified by matching with retention time (RT) of standard authentic;

^b Relative abundance of the compounds in the plant extract based on peak areas of the identified compounds compared to the total peaks’ areas in the LC-chromatogram.

Figure 1. The timeline and design of the study.

Table 1. The specifications of commercial ELIZA kits used in the biochemical parameters.

Kit	Catalog#	Function	Source
AST	AS 10 61 (45)	Liver function biomarkers	Biodiagnostic, Giza, Egypt
ALT	AL 10 31 (45)
Total bilirubin	BR 1111
MDA	MD2529	Oxidative marker
GSH	GR2511	Antioxidant markers
SOD	SD2521
mtROS	LS-F9759		LS Bio, Seattle, USA
NO	MBS723386		MyBioSource, USA
TNF-α	MBS2507393	Proinflammatory markers
IL-6	MBS269892

Table 2. Primary antibodies applied for immunohistochemistry.

Antibody	Caspase 3	IL-1B	NF-KB
Catalog#	PDR172	Ab283818	E-AB-32232
Host and type	Rabbit, polyclonal, conjugated	Rabbit, recombinant multiclonal	Rabbit, polyclonal, unconjugated
Optimal dilution	Optimized for use	1:500	1:200
Cellular localisation	Cytoplasmic	Cytoplasmic	Nuclear
Function	Proapoptotic	Proinflammatory	Proinflammatory, Proapoptotic

Table 3. Quantitative constituents and antioxidant measurements of A. pavarii fruit extract.

Quantitative tests	TPC^a	TFC^b	DPPH^c	FRAB^c	ORAC^c
Results	354.54 ± 0.79	36.2 ± 1.90	37.7 ± 1.23	27.10 ± 0.12	13.32 ± 1.45

^a Total phenolic content in mg/g gallic acid equivalent of the fruits extract;

^b Total flavonoid content in mg/g rutin equivalent of the fruits extract;

^c Results of the DPPH (2,2-Diphenyl-1-picrylhydrazyl), FRAB (ferric ion reducing antioxidant power), and ORAC (oxygen radical absorbance capacity) in mg Trolox equivalents. All the results were displayed as the mean ± standard deviation of three independent experiments.

Figure 2. Effects of ARB extract on serum liver function markers in rats subjected to PAR-induced liver toxicity. Data are represented as mean ± SD (n = 6) using one-way ANOVA followed by Tukey’s multiple comparison test at ****P < 0.0001.

Figure 3. Effects of ARB extract on oxidative stress markers in rats subjected to PAR-induced liver toxicity. Data are represented as mean ± SD (n = 6) using one-way ANOVA followed by Tukey’s multiple comparison test at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4. Effects of ARB extract on proinflammatory biomarkers in rats subjected to PAR-induced liver toxicity. Data are represented as mean ± SD (n = 6) using one-way ANOVA followed by Tukey’s multiple comparison test at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5. Histopathological changes in liver samples from all study groups. Haematoxylin and eosin (magnification ×200, scale bar = 500 mm). (A and B) Control and ARB groups, respectively, showed normal hepatic lobular architecture with regular cords of eosinophilic hepatocytes with vesicular nuclei (arrow) radiating from the central vein (C.V) separated by blood sinusoids (S). (C and D) PAR group revealed severely disturbed hepatic architecture with massive lobular necrosis and rarified areas (N), markedly congested central vein (C.V), focal hyaline deposits (H), heavy inflammatory infiltrates (star), and degenerated ballooned hepatocytes with pyknotic nuclei (arrowheads). (E) NAC + PAR group showed relatively improved liver histology, few inflammatory infiltrates (arrow), and mild central vein congestion (C.V). (F) ARB + PAR group revealed almost restored normal liver architecture with regularly arranged hepatocytes’ cords and mildly congested central vein (C.V).

Table 5. The criteria of the histopathologic scoring of PAR-induced liver injury.

Character	Score
Lobular disarray	0 = no disarray, 1 = disarray
Lobular inflammation	1 = mild, 2 = moderate, 3 = severe
Steatosis area%	1 = mild, 2 = moderate, 3 = severe
Apoptosis	0 = less than 1/HPF 1 = 1–3/HPF 2 = more than 3/HPF

Figure 6. Immunohistochemical analysis of Caspase 3, IL-1B, and NF-KB in liver tissue from all study groups (magnification ×400, scale bar = 50 μm). (A-C) Control group; (D-F) ARB group; (G-I) PAR group; (J-L) NAC + PAR group; (M-O) ARB + PAR group. Control and ARB groups showed negative cytoplasmic expression of Caspase 3 and IL-1B (A,B,D,E) and negative nuclear expression of NF-KB (C,F). PAR group exhibited intense positive cytoplasmic Caspase 3 and IL-1B staining (arrows) (G,H respectively) and strong positive NF-KB nuclear expression (arrows) (I). NAC + PAR group showed mild Caspase 3 and moderate IL-1B cytoplasmic expression (arrow) (J,K respectively) and mild nuclear expression for NF-KB (arrow) (L). ARB + PAR group exhibited negative Caspase 3 and mild IL-1B cytoplasmic staining (M,N respectively) and negative nuclear expression for NF-KB (O). Data are represented as mean ± SD (n = 10) using one-way ANOVA followed by Tukey’s multiple comparison test at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7. Chemical structure of the docked compounds.

Table 6. Binding energy scores (kcal./mol.) for the tested compounds, the highlighted cells correspond to the two highest scores for each target.

Compound	XO	COX-1	5-LOX	PI3K
PDB ID	3ETR	2OYE	6NCF	1E7U
Co-crystallized ligand	−6.3	−8.4	−11.8	−6.5
1. Catechin	−8.4	−6.1	−6.2	−6.3
2. Procyanidin C2	−8.6	−12.4	−9.8	−12.3
3. Myricetin rhamnoside	−10.3	−9.2	−9.9	−8.3
4. Quercetin rhamnoside	−9.4	−10.2	−8.1	−7.2
5. Quercetin glucoside	−8.1	−10.1	−7.2	−7.9
6. Procyanidin B3	−11.4	−9.3	−10.5	−10.6
7. 3-Galloylshikimic acid	−8.7	−6.4	−6.5	−6.7
8. Digalloyl shikimic acid	−8.3	−7.4	−8.9	−8.6
9. Quercetin 3-O-rutinoside	−6.5	−11.6	−10.6	−9.8

Figure 8. 2D interactions of myricetin rhamnoside (3) and procyanidin B3 (6) in the binding pocket of XO enzyme.

Figure 9. 2D interactions of procyanidin C2 (2) and quercetin 3-O-rutinoside (9) within COX-1 binding pocket.

Figure 10. 2D interactions of procyanidin B3 (6) and quercetin 3-O-rutinoside (9) within 5-LOX binding pocket.

Figure 11. 2D interactions of procyanidin C2 (2) and procyanidin B3 (6) within PI3K binding pocket.

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