Optimized extraction of active components of Paeonia lactiflora and their antioxidant, anti-inflammatory and pigmentation-reducing effects for skin whitening

doi:10.12122/j.issn.1673-4254.2026.02.22

Abstract

Abstract:

Objective To optimize the extraction process of the active components of Radix Paeoniae Alba and assess the antioxidant, anti-inflammatory and pigmentation-reducing effects of Radix Paeoniae Alba extract (BST). Methods Molecular docking identified gallic acid, liquiritin, and paeoniflorin as potential tyrosinase inhibitors, and their contents in BST were determined with high-performance liquid chromatography (HPLC). Based on the contents of the 3 inhibitors, their tyrosinase-inhibiting activity, and DPPH radical scavenging capacity calculated using entropy weight method, the extraction conditions were optimized for solvent ratio, liquid-to-solid ratio, extraction time, and particle size. The inhibitory effects of BST on tyrosinase activity and melanin were assessed in α‑MSH-treated B16F10 cells, and its effect on reactive oxygen species (ROS) production was evaluated in H₂O₂-treated HaCaT cells. In a mouse model of melanogenesis induced by UVB radiation, the effect of BST gavage (100, 150, and 200 mg/kg) were evaluated by assessing skin conditions of the mice, Fontana-Masson staining, and detecting serum levels of cytokines related to melanogenesis, melanosome transport, metabolism, and inflammation. Results The optimized parameters for BST extraction included 50% ethanol, liquid-to-solid ratio of 15:1, extraction time of 1 h, and 20-mesh particle size. BST exhibited strong free radical-scavenging activity and significantly reduced melanogenesis and increased tyrosinase protein in B16 cells, and lowered ROS production in HaCaT cells. In the mouse models of melanogenesis, BST significantly suppressed the melanogenic enzymes and inflammatory responses by reducing the levels of tyrosinase and TNF-α, thereby effectively reversed UVB-induced photoaging and hyperpigmentation. Conclusion The optimized BST extraction process is stable and predictable. BST shows good skin-whitening effect, which is mediated possibly by inhibiting tyrosinase activity and modulating oxidative stress and inflammatory pathways.

Key words: white peony extract, univariate-response surface method, B16 cells, Hacat cells, antioxidant effect, skin whitening

Qiao XU, Wenjie LI, Xinyue ZHANG, Yue CHEN, Zitong JIANG, Guangzhe LI, Mingming YAN. Optimized extraction of active components of Paeonia lactiflora and their antioxidant, anti-inflammatory and pigmentation-reducing effects for skin whitening[J]. Journal of Southern Medical University, 2026, 46(2): 443-455.

Figures/Tables 19

Tab.1 Tyrosinase （TYR） catalytic reaction system

Reagent	C₀	C	T₀	T
PBS	2.5	2	2	1.5
TYR	0.5	0.5	0.5	0.5
L-tyrosine	-	0.5	-	0.5
Sample	-	-	0.5	0.5

Tab.2 Binding energy of gallic acid, paeoniflorin, glycyrrhizic acid, ferulic acid, resveratrol, and quercetin with tyrosinase

Serial number	Compound	Molecular weight (Da)	Binding site on TYR	Binding energy (kcal/mol)
1	Glycyrrhizin	418.4	ALA-127,MET-129,SER-77, GLN-78	-8.5
2	Paeoniflorin	480.45	LYS-35,PHE-93,ARG-90,PRO-91	-6.6
3	Gallic acid	170.12	GLU-97,GLY-126,SER-77	-4.8

Fig.1 Molecular docking diagrams showing binding of gallic acid (A), paeoniflorin (B), and glycyrrhizin (C) with TYR.

Tab.3 Results of methodological evaluation

Result	Gallic acid	Glycyrrhizin	Paeoniflorin
Linear regression equation	y=12794x+31480	y=19188x-22744	y=8751.8x+15812
Coefficient of linear correlation (R²)	0.9987	0.9957	0.9959
Precision RSD value (%)	1.17	0.74	0.69%
Stability RSD value (%)	0.99	0.24	1.05%
Repeatability RSD value (%)	0.77	1.33	1.40%
Average sample recovery rate (%)	101.6	102.4	100.9%
RSD value for sample recovery (%)	0.67%	1.20	0.23%

Fig.2 HPLC chromatograms of Paeonia lactiflora extract (A) and the standard samples of the 3 indicator components (B, C, D). S1: Extract of Paeonia lactiflora extract; S2: Gallic acid; S3: Paeoniflorin; S4: Liquiritin.

Fig.3 Impact of changes in various factors on the comprehensive score. A: Solvent ratio; B: Solid-liquid ratio; C: Extraction time; D: Crushing mesh size.

Tab.4 Factor level table

Level	Factor
Level	Solvent ratio (A)	Liquid-to-solid ratio (B)	Extraction time (C)	Crushing mesh size (D)
-1	40%	10	0.5	The entire film
0	50%	15	1	20 mesh
1	60%	20	1.5	40 mesh

Tab.5 Response surface design scheme and results

Number unit	A	B	C	D	Overall rating
1	-1	-1	0	0	79.4
2	1	-1	0	0	69.6
3	-1	1	0	0	56.7
4	1	1	0	0	49.7
5	0	0	-1	-1	49.0
6	0	0	1	-1	9.0
7	0	0	-1	1	52.0
8	0	0	1	1	9.6
9	-1	0	0	-1	54.4
10	1	0	0	-1	36.8
11	-1	0	0	1	57.9
12	1	0	0	1	50
13	0	-1	-1	0	81.5
14	0	1	-1	0	58.6
15	0	-1	1	0	15.3
16	0	1	1	0	6.0
17	-1	0	-1	0	77.1
18	1	0	-1	0	66.6
19	-1	0	1	0	14.3
20	1	0	1	0	12.3
21	0	-1	0	-1	54.4
22	0	1	0	-1	27.4
23	0	-1	0	1	58.7
24	0	1	0	1	41.9
25	0	0	0	0	100.0
26	0	0	0	0	100.0
27	0	0	0	0	100.0

Tab.6 Response surface analysis of variance

Sources of variance	Sum of squares	Degree of freedom	Mean square	F	P
Model	20577.88	14	1469.85	55.26	<0.0001
A	250.25	1	250.25	9.41	0.0098
B	1172.16	1	1172.16	44.06	<0.0001
C	8442.91	1	8442.91	317.39	<0.0001
D	127.4	1	127.4	4.79	0.0491
AB	1.96	1	1.96	0.0737	0.7907
AC	18.06	1	18.06	0.679	0.426
AD	23.52	1	23.52	0.8843	0.3656
BC	46.24	1	46.24	1.74	0.212
BD	26.01	1	26.01	0.9778	0.3423
CD	1.44	1	1.44	0.0541	0.8199
A²	1585.47	1	1585.47	59.6	<0.0001
B²	2228.6	1	2228.6	83.78	<0.0001
C²	8082.56	1	8082.56	303.85	<0.0001
D²	5704.33	1	5704.33	214.44	<0.0001
Residual	319.21	12	26.6
Missing item	319.21	10	31.92
Pure error	0	2	0
Total deviation	20897.09	26

Fig.4 Contour maps and response surface maps. A: Solvent ratio; B: Solid-liquid ratio; C: Extraction time; D: Crushing mesh size.

Fig.5 Comparison of TYR inhibitory activity of Paeonia lactiflora extract (BST) obtained using optimized extraction process.

Fig.6 Free radical scavenging capacity of BST obtained using optimized extraction protocol. (A) Scavenging capacity for DPPH free radicals. (B) Scavenging capacity for hydroxyl free radicals.

Fig.7 Effect of BST on melanin quantity in B16 cells (Scale bar=200 μm). A: Blank group cells; B: Model group cells; C: Cells in 50 μg/mL BST.

Fig.8 Fluorescent staining rfor melanin-tyrosinase in B16 cells (Scale bar=200 μm). A: Blank group cells; B: Model group cells; C: Cells in 50 μg/mL BST.

Fig.9 Comparison of the effects of traditional water-decocted BST extract and the optimized extract on α‑MSH-stimulated melanocytes. A: Inhibition rate of TYR. B: Cell viability. **P<0.01.

Fig.10 Effect of BST on ROS levels in HaCat cells in different groups (Scale bar=400 μm).

Fig.11 Morphological observation of dorsal skin of C57 mouse models in different groups.

Fig.12 Fontana-Masson silver staining of dorsal skin from C57 mouse models in each group (Scale bar=500 μm).

Fig.13 Serum levels of TRY and TNF-α in C57 mouse models in different groups. **P<0.01.

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