In vitro anti-inflammatory efficacy testing of cosmetics

In vitro anti-inflammatory efficacy testing of cosmetics

Detection principle and experimental model construction

The in vitro anti-inflammatory efficacy testing of cosmetics is based on the lipopolysaccharide (LPS) - induced macrophage inflammation model, which simulates the pathophysiological process of skin inflammation response to evaluate the inhibitory effect of the test substance on the release of inflammatory factors. The experiment selected mouse monocyte macrophage RAW 264.7 as the model cell, which can activate the NF - κ B signaling pathway under LPS stimulation and secrete a large amount of inflammatory mediators such as nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor - α (TNF - α). The release level of these mediators is positively correlated with the degree of inflammation.

Key parameters for model construction:

Cell seeding density: 5 × 10 ⁴ cells/well (96 well plate)

LPS stimulation concentration: 1 μ g/mL (Sigma Aldrich L2630)

Test substance processing time: pre-treatment for 2 hours followed by LPS stimulation for 24 hours

Positive control: Dexamethasone (1 μ mol/L, inhibition rate ≥ 80%)

The experiment requires setting up 5 concentration gradients (0.01%~1%) and a blank control group. The MTT method is used to verify that the cell survival rate is>80%, ensuring that the detection results are not affected by cytotoxicity interference.

Core detection indicators and methodological validation

Determination of nitric oxide (NO) release

The Griess reagent colorimetric method (Sigma Aldrich G4410) was used to quantitatively detect the content of nitrite, a metabolite of NO, in the cell supernatant. The testing process includes:

Sample pretreatment: Mix 50 μ L of cell supernatant with an equal volume of Griess reagent

Incubation conditions: react at 37 ℃ in the dark for 15 minutes

Detection instrument: Enzyme linked immunosorbent assay (Thermo Multiskan FC) 540nm wavelength for measuring absorbance

Calculation method: Calculate the concentration of NO using the standard curve of sodium hyaluronate (0-100 μ mol/L), and the inflammation inhibition rate is calculated as (model group mean - test group mean)/(model group mean - blank group mean) × 100%

Methodological validation requirements: Standard curve R ²>0.99. Within day precision RSD<5%, recovery rate 90%~110%.

Detection of inflammatory factors (IL-6/TNF - α) levels

Adopting the double antibody sandwich ELISA method (R&D Systems kit), the specific parameters are as follows:

IL-6 detection range: 7.8~500pg/mL, with a maximum detection limit of 3.9pg/mL

The detection range of TNF - α is 15.6-1000pg/mL, with a maximum detection limit of 7.8pg/mL

Operation steps: Sample dilution → Sample incubation → Plate washing → Enzyme labeled secondary antibody binding → Color development (TMB substrate) → Termination of reaction (2mol/L H ₂ SO ₄) → 450nm reading

Data requirements: The IL-6/TNF - α levels in the test group were reduced by ≥ 30% compared to the model group, and the difference was statistically significant (independent sample t-test, p<0.05)

Standard basis and quality control system

The entire testing process strictly follows the OECD TG 442E (2018) "In vitro testing methods for skin irritations/corrosives" and GB/T 35892-2018 "In vitro 3D skin model irritations testing methods for cosmetics". Key quality control measures include:

Cell line authentication: RAW 264.7 cells (ATCC TIB-71) identified by STR were used, and the number of passages was controlled within 20

Reagent traceability: LPS endotoxin activity ≥ 1 × 10 ⁶ EU/mg, ELISA kit within batch coefficient of variation<10%

Environmental control: CO ₂ incubator (37 ℃± 0.5 ℃, 5% CO ₂), ultra clean workbench (ISO level 5)

Data validity: Three replicates were set for each concentration, and the experiment was repeated three times. The intra group coefficient of variation was less than 15%

Testing process and criteria for determining results

Complete experimental process

Cell preparation: Resuscitate RAW 264.7 cells and subculture them in DMEM medium containing 10% fetal bovine serum

Test substance treatment: Dilute the cosmetic sample with culture medium to the set concentration and sterilize it through a 0.22 μ m filter membrane

Inflammation induction: LPS (final concentration 1 μ g/mL) was added to stimulate for 24 hours, and cell supernatant was collected

Indicator detection: Simultaneous determination of NO (Griess method) and IL-6/TNF - α (ELISA method)

Data analysis: GraphPad Prism 9.0 was used for statistical analysis to calculate the IC ₅₀ value

Result judgment criteria

Note: To determine potency, it is necessary to simultaneously meet the requirements of NO inhibition rate ≥ 50% and IL-6/TNF - α inhibition rate ≥ 40%

Technical capability of testing institutions

The Zhongke Testing Laboratory has CNAS accredited qualifications (certificate number CNAS L22006) and a comprehensive in vitro anti-inflammatory testing technology platform, including:

Multi factor detection system: Luminex xMAP 200 (can simultaneously detect 10 inflammatory factors)

High connotation imaging instrument: PerkinElmer Operatta CLS (Cell Morphology Analysis)

Automated Workstation: Beckman Biomek 4000 (Sample Preprocessing)

In the past three years, the laboratory has completed more than 500 batches of cosmetics anti-inflammatory testing projects, covering face cream, essence, facial mask and other dosage forms, with a data passing rate of 92.3%. Typical cases show that a soothing essence containing purslane extract has an inhibition rate of 62.7% for NO and 58.3% for IL-6 at a concentration of 0.5%, both of which are better than the industry average (41.2% for NO).

Application scenarios and industry value

In vitro anti-inflammatory testing is mainly used for the efficacy verification of sensitive muscle repair, anti acne, and post sun repair cosmetics, and can provide the following technical support for enterprises:

Formula optimization: Select the optimal amount of anti-inflammatory active ingredients (such as centella asiatica glycoside and bisabolol) to be added

Declaration of compliance: meets the testing requirements for the "soothing" effect in the "Cosmetic Efficacy Declaration Evaluation Specification"

Market competition: Enhancing product competitiveness through third-party testing data, such as an international brand increasing product premium by 20% through this testing

With the increasing demand of consumers for the safety of cosmetics, in vitro anti-inflammatory testing has become a core item in efficacy evaluation. The laboratory suggests that companies introduce this testing during the product development stage, combined with human patch testing to form a complete evidence chain, which not only ensures product compliance but also enhances market trust.