Phytochemical profiling, antimicrobial, antibiofilm, insecticidal, and anti-leishmanial properties of aqueous extract from Juglans regia L. root bark: In vitro and in silico approaches

Figures & data

Table 1. Qualitative phytochemical tests of the JRRAE.

Phytochemical tests		Results
Secondary metabolites	Reagents	J. regiaextract	Coloration
Alkaloids	Mayer	+	White precipitate
	Wagner	+	Brown precipitate
Flavonoids	Mg^2+	+++	Pink or red or yellow
Phenol	FeCl3	+++	Dark green color
Triterpenes	Liebermann–Burchard test	+	Purplish red
Sterols	Salkowski test	-	Red
Steroids	Chloroform/concentrated sulfuric acid	-	The upper layer: red Sulfuric acid layer: yellow with green fluorescence
Coumarins	UV at 366 nm	+	Intense fluorescence
Resins	CuSO4	+	Green color precipitate
Tannins	FeCl3	+++	Blackish blue
Quinones	Alc. KOH	+	Color ranging from red to blue
Carboxylic acid	NaHCO₃	-	Effervescence
Oxylates	Acetic acid	++	Brown ring at interface
Reducing compounds	Fehling’s solution	+++	Red brick precipitate
Inulins	Fehling’s solution	+++	Black precipitate
Proteins	Ninhydrin	+++	Blue color
Free-anthraquinones	Borntrager’s reaction	++	Red
Saponins	Froth test	+	Height>1 cm of froth
Organic acids	Bromothymol Blue	+	Yellow canary
Starch	Iodine (I2)	+	Blue
Anthocyanins	HCl + NH4OH	+	Pink-red/purplish blue
Condensed tannins	HCl	-	Red

(+): presence, (++): abundance, (-): absence

Table 2. Physicochemical characteristics of JRRAE.

Characteristics	Amounts
Yield (%)	11.3
Total phenols (mg of GAE/100 g DW)	668.38 ± 42.21
Flavonoids (mg QE/100 g DW)	267.75 ± 12.31 mg/l
Flavonols (mg RE/100 g DW)	68 ± 3.23
Anthocyanins (mg/L)	29 ± 1.45
Proteins (mg/mL)	31.39 ± 1.173
Reducing sugars (g/L)	17.986 ± 1.318

Figure 1b. UV-VIS spectra of JRRAE, HPLC chromatogram of JRRAE: (1) gallic acid, (2) juglone, (3) catechin, (4) vanillic acid, (5) epicatechin, (6) coumarin.IR spectrum of JRRAE.

Figure 1c. IRspectrum of JRRAE.

Figure 1a. UV-VISspectra of JRRAE.

Table 3. Antibacterial activity and effect of JRRAE on pathogenic bacterial adhesion evaluated by the crystal violet test.

	Extract (µg/mL)		Extract (100 µg/mL)
	MIC	MBC	Inhibition biofilm (%)
Bacillus cereus ATCC 14,579	32	64	25
Escherichia coli ATCC 35,218	256	512	60
Enterococcus faecalis ATCC 29,212	128	256	37
Salmonella enterica serovar typhimurium ATCC 1408	256	512	40
Staphylococcus aureus ATCC 25,923	8	16	23
Staphylococcus epidermidis CIP 106510	64	128	55
Listeria monocytogenes ATCC 19,115	16	32	30
Micrococcus luteus NCIMB 8166	8	16	75
Pseudomonas aeruginosa ATCC 27,853	128	>128	80
Vibrio alginolyticus ATCC 33,787	256	512	90
Vibrio paraheamolyticus ATCC 1780	512	>512	87

Table 4. Bacterial count in the small intestine and feces in control and treated rats for 10 days by JRRAE at 200 mg/kg.

	Bacteria in the small intestine of rats/cm^2		Bacteria/gram of feces in the large intestine
	Control	Treated	Control	Treated
Hectoen medium	99 × 10^3 ± 0.816	0	95.6 × 10^3 ± 0.816	0
EMB medium	98 × 10^6 ± 3.949	9,994 × 10^6 ± 8.256	9.1 × 10^6 ± 0.983	9,994 × 10^6 ± 5.683
Chapman medium	0	0	993 ± 3.911	0

Figure 2. Histopathological effect of the extract on the intestine of S.littoralislarvae (a): Normal appearance. (b): Histopathological aspect of the intestine of S.littoralistreated larvae. V:vesicles formation L:Lysis of epithelial cells. G:Large vacuolation of epithelial cells. Lu:lumen. Ap:apical membrane. Bm:basal membrane. (Magnification × 40).

Table 5. Docking binding energies, conventional hydrogen bonding, and the number of closest residues to the docked compounds into the active sites of S. aureus Gyrase (pdb 2 × CT), S. aureus tyrosyl-tRNA synthetase (pdb 1JIJ), human peroxiredoxin 5 (pdb 1HD2), cyclooxygenase-2 (pdb 1C × 2), and trypanothione reductase (pdb 2JK6).

Name of compound	Binding energy (kcal/mol)	H-bonds (HBs)	Number of closest residues to the docked ligand in the active site
2 × CT
Gallic acid	1.22	4	2
Juglone	−2.93	1	2
Catechin	−2.29	2	2
Vanillic acid	−2.15	2	2
Coumarin	−3.32	2	2
Epicatechin	−2.23	3	3
1JIJ
Gallic acid	−3.74	8	6
Juglone	−5.65	2	3
Catechin	−7.58	6	7
Vanillic acid	−3.55	4	5
Coumarin	−5.57	0	3
Epicatechin	−7.74	6	7
1HD2
Gallic acid	−4.34	5	3
Juglone	−5.23	4	4
Catechin	−4.66	5	7
Vanillic acid	−4.23	3	4
Coumarin	−4.59	3	5
Epicatechin	−4.66	4	7
1C×2
Gallic acid	−4.60	3	5
Juglone	−6.20	2	6
Catechin	−7.84	5	8
Vanillic acid	−4.80	3	7
Coumarin	−6.08	1	6
Epicatechin	−7.97	6	6
2JK6
Gallic acid	−5.38	5	6
Juglone	−6.39	3	6
Catechin	−8.14	5	8
Vanillic acid	−5.47	6	8
Coumarin	−5.87	3	6
Epicatechin	−8.43	8	9

Figure 3. 3D (right) and 2D (left) binding interactions of the stable Coumarin- S. aureus Gyrase, Epicatechin- S. aureus tyrosyl-tRNA synthetase, Juglone- human peroxiredoxin 5, Epicatechin- cyclooxygenase-2, and Epicatechin-trypanothione reductase complexes.

Table 6. ADME properties of the identified compounds.

	Gallic acid	Juglone	Catechin	Vanillic acid	Coumarin
Physicochemical properties/lipophilicity/druglikeness
Molecular weight	170.12	174.15	168.15	146.14	290.27
Num. heavy atoms	12	13	12	11	21
Num. arom. heavy atoms	6	6	6	10	12
Fraction Csp3	0.00	0.00	0.12	0.00	0.20
Num. rotatable bonds	1	0	2	0	1
Num. H-bond acceptors	5	3	4	2	6
Num. H-bond donors	4	1	2	0	5
Molar refractivity	39.47	46.27	41.92	42.48	74.33
TPSA	97.99	54.37	66.76	30.21	110.38
Consensus Log Po/w	0.21	1.36	1.08	1.82	0.85
Lipinskiˈs rule	Yes	Yes	Yes	Yes	Yes
Bioavailability score	0.56	0.55	0.85	0.55	0.55
Pharmacokinetics
GI absorption	High	High	High	High	High
BBB permeant	No	Yes	No	Yes	No
P-gp substrate	No	No	No	No	Yes
CYP1A2 inhibitor	No	No	No	Yes	No
CYP2C19 inhibitor	No	No	No	No	No
CYP2C9 inhibitor	No	No	No	No	No
CYP2D6 inhibitor	No	No	No	No	No
CYP3A4 inhibitor	Yes	No	No	No	No
Log Kp (cm/s)	−6.84	−6.00	−6.31	−6.20	−7.82

Figure 4. (A) Bioavailability radar of identified compounds based on physicochemical indices ideal for oral bioavailability. LIPO, Lipophilicity: −0.7 < XLOGP3 < þ 5; SIZE, Molecular size: 150 g/mol < mol. wt.<500 g/mol; POLAR, Polarity: 20 Å2 < TPSA<130 Å2; INSOLU, Insolubility: 0 < Log S (ESOL) < 6; INSATU, Insaturation: 0.25 < Fraction Csp3 < 1; FLEX, Flexibility: 0 < Number of rotatable bonds<9. The colored zone is the suitable physicochemical space for oral bioavailability. (B) Boiled-egg (B) model of compounds.

Figure 5. Top-50% of target prediction for identified compounds.