Potent inhibition of human tyrosinase inhibitor by verproside from the whole plant of Pseudolysimachionrotundum var. subintegrum

Figures & data

Figure 1. (A) UPLC-PDA and (B) UPLC-QTOF-MS data of the Pseudolysimachion rotundum var. subintegrum extract after the binding experiment. Black: not exposed to enzyme; red, blue, and green; after incubation with tyrosinase for 15 min. Peaks numbered 1 − 9 represent the compounds isolated from P. rotundum var. subintegrum in our study.

Table 1. UPLC chromatographic, UV–Vis, and mass spectral data of the compounds identified from P. rotundum var. subintegrum.

Peak	tR (min)	λmax (nm)	Detected ion [M − H]^−	Calculated ion [M − H]^−	ppm	Molecular Formula	Identification	Control (area)	after tyrosinase (area)
1	3.75	296, 263, 220	497.1292	497.1295	−0.6	C22H26O13	Verproside	112306	61907
2	4.84	296, 263, 220	533.1068	533.1062	1.1	C22H27ClO13	Longifolioside A	10677	3852
3	5.39	258	481.1327	481.1346	−3.9	C22H26O12	Catalposide	26121	21250
4	5.88	328, 218	523.1436	523.1452	−3.1	C24H28O13	Verminoside	16481	2886
5	6.01	294, 263, 220	511.1451	511.1452	−0.2	C23H28O13	Picroside II	17798	12194
6	6.10	296, 263, 220	533.1064	533.1062	0.4	C22H27ClO13	Piscroside C	44999	22171
7	6.42	296, 263, 220	511.1447	511.1452	−0.1	C23H28O13	Isovanillyl catapol	12844	8093
8	9.04	327, 220	537.1647	537.1608	7.3	C25H30O13	Minecoside	976	465
9	9.17	328, 220	525.1627	525.1613	2.5	C24H30O13	6-O-Veratroyl catalpol	15756	11932

Figure 2. Chemical structures of the major compounds isolated from the extract of the whole plant of Pseudolysimachion rotundum var. subintegrum.

Figure 3. (A) Effects of compounds 1, 2, and 8 on the activity of tyrosinase to catalyse L-tyrosine. (B) Lineweaver-Burk plots to investigate the ability of compound 1 to inhibit the monophenolase activity of tyrosinase. (C) Dixon plots to investigate the ability of compound 1 to inhibit the monophenolase activity of tyrosinase. (D) Time-dependent chromatograms of tyrosinase in the presence of compound 1 using UPLC-PDA. (E) Inhibition as a function of preincubation time for the most active compound 1 (5, 10, and 20 mM). (F) Plot of k_obs as a function of the concentration of the slow-binding inhibitor 1 fitted by Equation (4)(4) $$k_{\text{obs}}=k_6+\left[(k_5\hspace{0.17em}\times \hspace{0.17em}\right[\mathrm{I}\left])/(K_{\mathrm{i}}{}^{\text{app}}\hspace{0.17em}+\right[\mathrm{I}])]$$(4) .

Table 2. Inhibitory effects of isolated iridoids 1 − 9 on tyrosinase activity.

Compounds	Mushroom Tyrosinase L-Tyrosine			Mushroom Tyrosinase L-DOPA	Human Tyrosinase L-Tyrosine	Human Tyrosinase L-DOPA
	IC50 (µM)^a	100 µM inhibition (%)	Type of inhibition (Ki, µM)^b	IC50 (µM)^a	IC50 (µM)^a	IC50 (µM)^a
Verproside (1)	31.2 ± 2.1	98.4	Competitive (5.1 ± 0.7)	177.2 ± 33.1	197.3	842.7
Longifolioside A (2)	192.9 ± 10.5	36.0	NT	> 200	NT	NT
Catalposide (3)	>200	9.9	NT	> 200	NT	NT
Verminoside (4)	>200	8.9	NT	> 200	NT	NT
Picroside II (5)	>200	5.3	NT	> 200	NT	NT
Piscroside C (6)	>200	6.7	NT	> 200	NT	NT
Isovanily catapol (7)	>200	10.1	NT	> 200	NT	NT
Minecoside (8)	131.8 ± 8.3	41.7	NT	> 200	NT	NT
6-O-Veratroyl catalpol (9)	>200	6.1	NT	> 200	NT	NT
Kojic acid^d	14.8 ± 0.6	NT	NT	37.1 ± 1.3	1109.3	1819.7
					(117.0)^e	(185.0)^e

All compounds were examined in a set of experiments repeated three times

^aIC₅₀ values of compounds represent the concentration that caused 50% enzyme activity loss

^bValues of inhibition constant.

^cNT is not test.

^dPositive control.

^eMonophenolase activity 117 µM and diphenol oxidase activity 185 µM IC₅₀ value calculated from Dolinska et al., 2014.³⁶

Table 3. Docking simulation results of isolated iridoids 1 − 9 on tyrosinase activity.

Compounds	Lowest energy (kcal/mol)	Number in group^a	Average energy in group (kcal/mol)	Binding location^b
Verproside (1)	−6.9	43	−3.72	Active
Longifolioside A (2)	−6.6	30	−5.04	Other
Catalposide (3)	−7.2	44	11.36	Active
Verminoside (4)	−6.6	24	−5.07	Other
Picroside II (5)	−6.7	29	−1.91	Other
Piscroside C (6)	−7.2	29	−4.64	Active
Isovanillyl catapol (7)	−6.3	48	4.82	Other
Minecoside (8)	−6.2	31	6.63	Other
6-O-Veratroyl catalpol (9)	−6.2	24	−4.60	Other

^aThe number of compounds existing in the same pocket.

^bActive: a compound docks on the tyrosinase active site.

Other: a compound docks on the other side which is not related on the enzyme reaction.

Figure 4. Molecular docking views of two compounds, verproside and kojic acid, on two tyrosinases: (A) verproside-mTyr, (B) verproside-hTyr, (C) kojic acid-mTyr, and (D) kojic acid-hTyr. The tyrosinases are represented by a white surface protein model, and the two Copper ions present in the tyrosinases are represented by a yellow space-filling model. The active site is highlighted by a red transparent box. The compounds are represented by ball and stick models with atom colours: red for Oxygen, cyan for Carbon, and white for Hydrogen.

Figure 5. Hydrogen bonding patterns of two compounds, verproside and kojic acid, on two tyrosinases, mTyr and hTyr: (A) verproside-mTyr, (B) verproside-hTyr, (C) kojic acid-mTyr, and (D) kojic acid-hTyr. The active site, highlighted by the red transparent box in Figure 4, is magnified to show the detailed hydrogen bonding pattern. The compounds are drawn using the ball and stick model and coloured by atom: red for Oxygen, cyan for Carbon, and white for Hydrogen. The hydrogen bonding interactions are indicated by dotted lines with the interaction numbers. The detailed hydrogen bonding patterns are provided in Supplementary material Table S2.

Table 4. Docking simulation comparisons of verproside and kojic acid on mushroom (mTyr) and human tyrosinase (hTyr).

	IC50 (μM)		Docking energy^a (rank in group)		Number of Hydrogen bond^b
	mTyr	hTyr	mTyr	hTyr	mTyr	hTyr
Verproside	31.2	197.3	−6.9 (1)	−7.8 (2)	7	4
Kojic acid	14.8	1109.3	−5.9 (1)	−5.6 (3)	2	1

^aEnergy unit: kcal/mol

^bThe detail hydrogen bond patterns are shown on Table S2.

Figure 6. Molecular dynamics simulation results. (A) The final structures of mTyr-verproside complex. The tyrosinases are depicted using a grey surface model, while the active site revealed by docking simulation is coloured yellow. The verproside is represented by a blue stick model. For clarity, the explicit water models and counter ions are not included in the figure. (B) Verproside grouping. The centre of geometry (COG) of the atoms comprising the rings was utilised to measure the interacting distance. The measurement was conducted between the COG and the alpha Carbon atom of the interacting residues. (C) The interaction distance between verproside and mTyr. It consists of 33 sub-panels, each displaying the distance-time profile (X-axis: time from 0 to 100 ns; Y-axis: distance from 0 to 20 Å). The Y panel is divided into three groups, namely G1, G2, and G3, as mentioned in Figure 6B. The red dotted line represents the distance of 10 Å, which serves as the lower limit distance.

Gębalski J, Graczyk F, Załuski D. Paving the way towards effective plant-based inhibitors of hyaluronidase and tyrosinase: a critical review on a structure–activity relationship. J Enzyme Inhib Med Chem. 2022;37(1):1120–1195.

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