Viphyllin^TM, a Standardized Black Pepper Seed Extract Exerts Antinociceptive Effects in Murine Pain Models via Activation of Cannabinoid Receptor CB2, Peroxisome Proliferator-Activated Receptor-Alpha and TRPV1 Ion Channels

Figures & data

Table 1 Grid Point Parameters Used for Molecular Docking of β-Caryophyllene with Nociceptive Receptor-Agonist Binding Sites

Receptor	Grid Centre Coordinates			Grid Size
	x	y	z	x	y	z
CB1	−43.288	−157.799	307.888	40	40	40
CB2	97.135	109.16	120.83	60	60	60
PPARα	15.295	−12.269	−32.623	46	40	54
TRPV1	24.113	−7.236	−13.071	50	50	50

Abbreviations: CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; PPARα, peroxisome proliferator-activated receptor-alpha; TRPV1, transient receptor potential cation channel subfamily V member 1.

Figure 1 Analgesic effect of Viphyllin in acetic acid–induced writhing test. Data analyzed by one-way ANOVA followed by Tukey’s test and presented as mean ± SD (n=6). *p<0.05 and ***p<0.001 vs control group; ^#p<0.05 vs reference drug (Diclofenac sodium) treatment group.

Abbreviation: BCP, β-caryophyllene.

Figure 2 Effect of pretreatment with selective antagonists of pain receptors on analgesic activity of Viphyllin during the early (A) and late phase (B) of formalin-induced paw licking test. Data analyzed by one-way ANOVA followed by Tukey’s test and presented as mean ± SD (n=6). **p<0.05 and ***p<0.001 vs control group; ^#p<0.05 vs Viphyllin treatment group.

Abbreviation: BCP, β-caryophyllene.

Figure 3 Effect of pretreatment with selective antagonists of pain receptors on analgesic activity of Viphyllin (hot plate test). Data analyzed by one-way ANOVA followed by Tukey’s test and presented as mean ± SD (n=6). ***p<0.001 vs control group; ^#p<0.05 and ^###p<0.001 vs Viphyllin treatment group.

Abbreviation: BCP, β-caryophyllene.

Figure 4 Evaluation of antinociceptive activity of Viphyllin using tail flick test. Effect of Viphyllin on the latency time of tail flick after 0, 30, 60, 90 and 120 min of treatment (A). Data analyzed by two-way ANOVA followed by Tukey’s test. Effect of pretreatment with selective antagonists of pain receptors on antinociceptive activity of Viphyllin (B). Data analyzed by one-way ANOVA followed by Tukey’s test and presented as mean ± SD (n=6). *p<0.05, **p<0.01 and ***p<0.001 vs control group; ^##p<0.01 and ^###p<0.001 vs Viphyllin treatment group.

Abbreviation: BCP, β-caryophyllene.

Table 2 Drug Likeliness of β-Caryophyllene

Lipinski’s Rule of Five
Molecular weight (<500 Da)	204.35 g/mol
MLog P (<4.15)	4.63
H-Bond donor (5)	0
H-Bond acceptor (<10)	0
Violation	1

Figure 5 Three-dimensional view of the nociceptive receptors. (A) CB1 receptor (PDB: 5XR8); (B) CB2 receptor (PDB: 6PT0); (C) PPARα receptor (PDB: 6KXY); (D) TRPV1 ion channel (PDB: 3J5R) with chain B highlighted in blue.

Table 3 Docking Score of Interaction of β-Caryophyllene with Agonist Binding Sites of Different Pain Receptors

Receptor	Binding Energy kJ/mol	Ligand Efficiency	Inhibitory Constant (µM)	Vdw_hb_Desolv_Energy	Interactions
Cannabinoid receptor 2 (CB2)	−7.83	−0.52	1.81	−7.84	Ile186, Ile110, Trp194, Phe281, Val113, Phe183, Thr114
Cannabinoid receptor 1 (CB1)	−5.16	−0.34	165.22	−5.16	Phe191, Gly194, Gly195, Ala198, Ile245, Val249
PPAR-α	−6.66	−0.44	13.05	−6.67	Ile241, Leu247, Glu251, Leu254, Arg271, Ile272, Cys275, Val332, Ala333
TRPV1	−7.58	−0.51	2.8	−7.57	Phe488, Arg491, Gly492, Glu513, Phe516, Tyr554

Figure 6 Best docking pose predictions of β-caryophyllene with the agonist binding sites of nociceptive receptors. Representative images showing the interactions of β-caryophyllene with cannabinoid receptors, CB2 (A) and CB1 (B), PPARα (C) and TRPV1 (D) binding sites. Molecular docking was performed with AutoDock 4.2 tools using Lamarckian genetic algorithm.