基于“抗氧化-炎症-色素沉着轴”优化白芍的提取工艺与美白功效评价

doi:10.12122/j.issn.1673-4254.2026.02.22

摘要/Abstract

摘要：

目的优化白芍提取工艺，建立白芍提取物（BST）多指标含量检测方法，并初步探讨其对小鼠黑色素瘤细胞（B16F10）的酪氨酸酶（TYR）活性抑制作用和对人永生化表皮细胞（HaCaT）中ROS水平的降低作用，并对BST对体内小鼠模型治疗效果进行评估。方法以分子对接手段确定潜在TYR抑制剂成分为没食子酸、甘草苷及芍药苷；采用高效液相色谱法（HPLC）建立BST液相图谱，测定3种潜在TYR抑制剂的含量，并进行方法学验证；通过熵权法计算3种潜在TYR抑制剂的含量、TYR抑制活性及DPPH及自由基清除能力的综合评分作为响应值，采用单因素结合响应面分析法考察溶剂配比、液料比、提取时间以及粉碎目数等因素对BST提取工艺的影响，以确定最优提取工艺；通过设置黑色素-酪氨酸酶B16F10细胞实验空白组（正常培养）、模型组（α-MSH处理）、给药组（α-MSH +BST）、Hacat细胞体外抗氧化活性试验空白组［正常培养，模型组双氧水（H₂O₂）处理）、给药组（H₂O₂+BST处理）以验证作用机制。通过UVB辐射暴露系统建立小鼠黑色素瘤模型，BST（100、150、200 mg/kg）给予0.2 mL的混悬液灌胃，结合观察实验动物背部皮肤状态、Fontana-Masson银染色结果以及检测血清样本中参与调控黑色素生成、转运或代谢及抗炎相关细胞因子水平指标综合评估BST体内美白作用效果。结果分子对接结果显示BST中潜在TYR抑制剂与TYR蛋白结构的残基存在相互作用关系，建立了BST潜在TYR抑制剂的HPLC含量测定方法，方法学验证表明该方法稳定可行；单因素-响应面实验表明BST最优提取工艺为提取溶剂50%乙醇，液料比1∶15，提取时间1h，粉碎目数20目；BST对DPPH自由基以及羟自由基具有直接清除作用，能显著减少细胞中黑色素的形成并提高B16细胞内TYR蛋白的相对含量并降低HaCat细胞中ROS水平，并在体内通过抑制TYR与TNF-α （P<0.01）直接抑制黑色素合成关键酶与间接抗炎的双重美白机制，双向调节色素代谢，有效逆转UVB诱导的皮肤光老化及色素沉着。结论基于单因素响应面法优化的BST提取工艺稳定可靠、预测性良好，药理分析研究表明BST的美白作用可能是通过抑制TYR活性和调节氧化应激和炎症通路的双向调节机制而实现的。

关键词: 白芍提取物, 单因素-响应面法, B16细胞, Hacat细胞, 抗氧化, 美白

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

徐乔, 李文杰, 张馨月, 陈越, 蒋子通, 李光哲, 严铭铭. 基于“抗氧化-炎症-色素沉着轴”优化白芍的提取工艺与美白功效评价[J]. 南方医科大学学报, 2026, 46(2): 443-455.

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.

图/表 19

表1 TYR 催化反应体系

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

表2 化合物没食子酸、甘草苷及芍药苷与TYR的结合能

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

图1 化合物没食子酸(A)、芍药苷(B)、甘草苷(C)与TYR的分子对接图

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

表3 方法学考察结果

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%

图2 BST(A)及3种指标成分标准品(B~D)的HPLC图

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.

图3 各因素变化对综合评分的影响

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.

表4 因素水平表

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

表5 响应面设计方案及结果

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

表6 响应面方差分析

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

图4 等高线图和响应面图

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

图5 BST优化提取工艺TYR抑制活性比较

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

图6 优化BST提取方案的自由基清除能力比较

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.

图7 BST对B16细胞黑色素的影响

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.

图8 BST对B16细胞黑色素-酪氨酸酶荧光染色结果

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.

图9 传统水煎BST提取物与优化提取物对α-MSH刺激黑色素细胞作用的比较

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.

图10 BST对各组 HaCat 细胞中 ROS 水平的影响

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

图11 各组 C57小鼠模型背部皮肤形态学观察

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

图12 各组 C57小鼠模型背部皮肤Fontana-Masson银染色结果分析

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

图13 各组 C57小鼠模型血清中相关炎症因子含量检测

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

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