WO2024051337A1 - 检测皮肤自发荧光的装置及抗糖化功效评价方法 - Google Patents

检测皮肤自发荧光的装置及抗糖化功效评价方法 Download PDF

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WO2024051337A1
WO2024051337A1 PCT/CN2023/106485 CN2023106485W WO2024051337A1 WO 2024051337 A1 WO2024051337 A1 WO 2024051337A1 CN 2023106485 W CN2023106485 W CN 2023106485W WO 2024051337 A1 WO2024051337 A1 WO 2024051337A1
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fluorescence
module
sample
skin
image sensor
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PCT/CN2023/106485
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English (en)
French (fr)
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周滢
何永红
袁登峰
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上海家化联合股份有限公司
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Publication of WO2024051337A1 publication Critical patent/WO2024051337A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0075—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by spectroscopy, i.e. measuring spectra, e.g. Raman spectroscopy, infrared absorption spectroscopy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/44—Detecting, measuring or recording for evaluating the integumentary system, e.g. skin, hair or nails
    • A61B5/441—Skin evaluation, e.g. for skin disorder diagnosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/44—Detecting, measuring or recording for evaluating the integumentary system, e.g. skin, hair or nails
    • A61B5/441—Skin evaluation, e.g. for skin disorder diagnosis
    • A61B5/443—Evaluating skin constituents, e.g. elastin, melanin, water

Definitions

  • the present invention relates to the field of efficacy detection, and specifically relates to a small non-invasive device for detecting skin AGEs content and an anti-glycation efficacy evaluation method. It can especially be used for anti-glycation of oral medicines, health care products, or external medicines, external health care products and skin external agents. effect.
  • AGEs Advanced glycation end products
  • Miillard reaction refers to the spontaneous formation of macromolecules such as proteins, lipids or nucleic acids without the participation of enzymes.
  • the stable and irreversible covalent adduct generated by the reaction with glucose or other reducing monosaccharides is the final product of the non-enzymatic glycosylation aging theory and an important indicator for studying aging.
  • the non-enzymatic glycosylation aging theory is one of the aging theories currently recognized by many scholars.
  • AGEs are brownish-yellow and have fluorescence characteristics (maximum excitation wavelength 370nm, maximum emission wavelength 440nm), irreversibility, cross-linking, structural heterogeneity, resistance to degradation, and stability to enzymes. It is present in different tissues and organs of the body, such as vascular endothelial cells, nerve cells, collagen, lens circulatory system, and tissue fibers such as kidneys, liver, lungs, blood vessels, and peritoneum, and continues to accumulate with age.
  • the sources of AGEs are divided into exogenous and endogenous sources. They mainly cause damage to the body through three mechanisms: destroying their structure and function by directly cross-linking with macromolecules such as proteins, lipids, and nucleic acids; affecting signals Transduction pathway and enzyme activity; interacts with AGEs receptors on the cell surface to trigger biological effects.
  • Glycosylation can cause protein cross-link damage and transform the normal protein structure into an old protein structure.
  • Some intracellular proteins have been proven to be glycated and inactivated in the pathology of body aging.
  • the amino acid residues mainly involved in the reaction are lysine, arginine, histidine, tyrosine, tryptophan, serine and threonine. Acid etc.
  • Glycosylation can also cause hardening of structural proteins, damage to functional enzymes, reduction of energy supply, reduction of metabolic function, and a series of aging processes such as impaired immune function, imbalance of balance function, and genetic mutations. Glycosylation also inhibits ubiquitin binding and ubiquitin-mediated degradation by occupying common protein binding sites.
  • glycosylation and oxidation are closely linked, the glycosylation process generates ROS, and AGE-protein itself is the source of ROS. Glycosylation also inhibits glycokinase transcription.
  • the loss of lysosomal regulatory functions may be partly due to the vicious cycle of AGEs promoting lipofuscin (also known as ceroid or AGE-pigmented liposomes, which is a kind of lysosome associated with caused by the formation of age-related cross-linked proteins and aggregates).
  • AGEs work by binding to specific receptors on the cell surface.
  • RAGEs members of the immunoglobulin superfamily
  • RAGEs members of the immunoglobulin superfamily
  • vascular endothelial cells smooth muscle cells, fibroblasts, lymphoid cells, glomerular mesangial cells, macrophages, neuronal cells, and tumor cells.
  • Identified species include macrophage scavenger receptor types I and II, oligosaccharyl transferase-48, 80K-H phosphoprotein, galectin-3, etc.), which can induce monocyte proliferation after binding to ligands such as AGEs.
  • Chemotactic and oxidative stress reactions and generate a large amount of oxygen free radicals, activate signaling pathways, stimulate interleukin I (IL-I), insulin-like growth factor I, tumor necrosis factor (TNF- ⁇ ), platelet-derived growth
  • IL-I interleukin I
  • TNF- ⁇ tumor necrosis factor
  • PDGF factor
  • agranulocyte-macrophage clone stimulating factor especially the activating transcription factor NF-kB, whose activation can induce the production of a variety of damage factors and produce pathogenic effects, thereby activating and damaging cells.
  • the proteins and nucleic acids in the body trigger complex biological effects, leading to abnormal cell function and structure, thus affecting the structure and function of organs.
  • AGEs accelerate human aging.
  • Sell et al. measured skin pentosin levels in eight mammal species and found that its production rate was highly correlated with the species' maximum lifespan.
  • AGEs can also cause a variety of chronic degenerative diseases, including cataracts, atherosclerosis, Alzheimer's disease, Pick's disease, Parkinson's disease, stroke, amyloidosis, kidney disease, diabetes, and neuropathy. and retinopathy, etc.
  • AGEs are the root substance and important indicator of body aging and chronic degenerative diseases of multiple organs.
  • the detection of this substance is important for the assessment of the aging stage of the body, the screening and diagnosis of chronic degenerative diseases, and the evaluation of intervention results. significance.
  • a study on non-invasive markers of vascular damage in patients with type II diabetes used a self-developed simple non-invasive device to demonstrate that skin autofluorescence is related to the level of non-fluorescent AGE in the skin; collagen-associated fluorescence is related to pentosin (fluorescent substance) and (N ⁇ The parallel relationship between )-carboxymethyl-lysine (a non-fluorescent substance) and the correlation between specific AGEs also suggests that fluorescent and non-fluorescent AGEs behave similarly. Therefore, skin autofluorescence is a marker of the entire body AGE pool and can be used to label the behavior of body AGEs. This study also suggests the feasibility of using appropriate equipment to detect AGEs content in vivo.
  • the existing AGEs measurement equipment on the market is a disease risk assessment equipment. It is mainly used for early screening and risk marking of some chronic diseases such as diabetes in medical examination institutions and hospital departments of nephrology, endocrinology, cardiology and other departments.
  • the test site can only be the forearm. Part, only the fluorescence intensity of AGEs in the 1cm*1cm area of the tested skin can be provided, and then the data is compared with the database data. Since the data of the sick population is 20% to 30% higher than that of the healthy population of the corresponding age, after comparing the range, Provide incidence rates and complication risk factors for relevant chronic diseases to prompt relevant personnel to conduct further medical examinations.
  • the fluorescence intensity only includes adults over 20 years old
  • it is only used for risk assessment of early screening, it does not It provides the true content of AGEs and does not perform any calibration of standard materials. Therefore, the trace impact of the external environment or exogenous substances on skin AGEs cannot be evaluated, and it cannot be used in the field of evaluation of anti-glycation effects.
  • the volume of commercially available equipment is also limited. It is very large, larger than the size of a conventional drum washing machine, and it takes up even more space when equipped with a control computer.
  • the overall size of the detection equipment of the present invention is about 1/20 of the size of a conventional drum washing machine.
  • the equipment of the present invention is a small non-invasive detection equipment.
  • the "anti-glycation" effect of cosmetics is a type of new effect. According to the requirements of the evaluation specifications, "a scientific and reasonable analysis should be conducted based on the specific circumstances of the product efficacy claim.” However, the clinical evaluation of the anti-glycation of cosmetics is also That is, for in-vivo detection of skin AGEs content, there are no mature detection means, detection equipment and testing methods in the entire cosmetics industry.
  • the invention provides a device for detecting skin autofluorescence and its content.
  • the device includes:
  • the imaging and detection system includes a fluorescence microscopy imaging module, a fluorescence spectrum detection module and a coupling module.
  • the fluorescence microscopy imaging module is optically coupled to the coupling module
  • the fluorescence spectrum detection module is optically coupled to the coupling module.
  • Optically coupled connectivity
  • the accessory device system includes an operating module, a position fixing module and a measured part carrying module,
  • the fluorescence spectrum detection module includes a first light source and a linear array image sensor, and is optically coupled and connected to the sample to be measured in the coupling module.
  • the linear array image sensor is connected to the operating module and displays the parameters of intensity and wavelength.
  • the fluorescence microscopy imaging module includes a second light source and an area array image sensor, and is optically coupled and connected to the sample to be measured in the coupling module.
  • the area array image sensor is connected to the operation module and displays a fluorescence image of the sample.
  • the first light source is a laser light source with a wavelength of 350-390 nm
  • the second light source is a laser light source with a wavelength of 350-390 nm.
  • the fluorescent substance used for detection by the device is selected from: flavin, lipofuscin, advanced glycation end products AGEs, reduced coenzyme I NADH, carotenoids, oxidized melanin, porphyrin , collagen, amino acids, elastin, and combinations thereof.
  • the fluorescence spectrum detection module also includes a second dichroic mirror, which reflects light and converges the excitation light onto the sample to be measured through the optical path to excite the sample to produce scattered fluorescence.
  • the scattered fluorescence generated by the sample is The fluorescence is then concentrated on the linear array image sensor through the optical path.
  • the scattered fluorescence excited by the sample is transformed into parallel light through the objective lens, passes through the reflecting mirror, passes through the second dichroic mirror and filter, passes through the lens, and is converged into the spectrometer to form light.
  • the spectrum is converged onto a linear array image sensor.
  • the fluorescence microscopy imaging module further includes a first dichroic mirror, which reflects light and irradiates the excitation light onto the sample to be tested through the optical path to excite the sample to produce fluorescence, and the fluorescence excited by the sample is then The image is imaged onto the area array image sensor through the optical path.
  • the fluorescence excited by the sample passes through the objective lens, the first dichroic mirror, the fluorescence filter, and the achromatic lens, and is imaged onto the area array image sensor.
  • the operation module is used to realize real-time collection of fluorescence images and fluorescence spectra, image shooting, data analysis, reticle fitting, or a combination thereof.
  • the present invention provides a method for evaluating anti-glycation efficacy, which method includes:
  • step (e) Compare the results of step (d) with the results of step (b) to evaluate the anti-glycation efficacy.
  • step (e) includes comparing the results of step (d) and step (b) based on a standard curve of a fluorescent substance to evaluate the anti-glycation efficacy.
  • step (e) includes comparing the results of step (d) and step (b) based on a standard fluorescent color card to evaluate the anti-glycation efficacy.
  • the sample to be tested is selected from the group consisting of internal medicines, health care products, external medicines, external health care products and external skin preparations.
  • Figure 1 shows a device for detecting skin autofluorescence of the present invention.
  • Figure 2 shows the simultaneous shooting of AGEs at the tip of the finger and a standard fluorescent color card.
  • Figure 3 shows the fluorescence image of AGE-BSA standard solution (mg/mL).
  • Figure 4 shows the results of linear fitting (mg/mL) of the AGE-BSA standard curve.
  • Figure 5 shows the fluorescence image of AGE-BSA standard solution ( ⁇ g/mL).
  • Figure 6 shows the results of linear fitting of the AGE-BSA standard curve ( ⁇ g/mL).
  • this application optimizes the system band range and enhances the excitation and emission light intensity in the specific band range.
  • In-vivo image and spectral detection of fluorescent substances (for example, AGEs) on the skin surface in different parts of the body can be achieved through special small-scale non-invasive equipment.
  • the fluorescence intensity is converted into the concentration or content of fluorescent substances (for example, AGEs) through marking line fitting.
  • the detection limit can reach 1 ⁇ 10 -8 g/mL, and the impact on the body's glycation level caused by exogenous substances can be high. Sensitive identification, thereby providing a rapid, non-invasive, quantitative and highly sensitive detection method for evaluating the anti-glycation efficacy of cosmetics.
  • the invention provides a small-sized device for non-invasively detecting skin autofluorescence and its content.
  • the device includes: an imaging and detection system and an accessory device system.
  • the imaging and detection system includes a fluorescence microscopy imaging module, a fluorescence spectrum detection module and a coupling module.
  • the fluorescence microscopy imaging module is optically coupled to the coupling module, and the fluorescence spectrum detection module is optically coupled to the coupling module.
  • the accessory device system includes an operating module, a position fixing module and a measured part carrying module.
  • the fluorescence spectrum detection module includes an excitation light source 1 and a linear array image sensor, and is optically coupled and connected to the sample to be measured in the coupling module.
  • the excitation light source 1 can be a laser (1-100mW, 350-390nm) or an LED light source (3W, 350-390nm).
  • the working mode of this excitation light source can be continuous or pulsed. If the excitation light source 1 is an LED light source, the fluorescence spectrum detection module also includes a collimating lens (not shown), and the excitation light emits parallel laser light through the collimating lens.
  • the fluorescence spectrum detection module may also include a dichroic mirror 2 .
  • the transmission wavelength of the dichroic mirror 2 is 400-480 nm, and the reflection wavelength is 350-400 nm. Therefore, the dichroic mirror 2 can reflect light in the wavelength band below 400 nm and transmit the reflected excitation light above 400 nm. The reflected light converges onto the sample to be tested through a connected optical path, stimulating the sample to emit fluorescence.
  • the sample to be tested includes skin and fluorescent color cards. The emitted fluorescence is concentrated into a spectrometer and finally onto a linear array image sensor, which displays the parameters of fluorescence intensity and wavelength.
  • the two-way The reflected light from the color mirror 2 passes through the mirror, and then passes through the objective lens (for example, magnification 1x-10x) to converge the excitation light onto the sample, and the sample excites scattered fluorescence.
  • the reflection band of the mirror covers 350nm to 500nm.
  • the emitted scattered fluorescence first passes through the objective lens, then passes through the reflecting mirror, passes through the dichroic mirror 2 and the filter, and passes through the lens (focal length f 30-100 mm), so that the scattered fluorescence light is converged into the spectrometer.
  • the bandpass of the filter is 400-480nm, which can filter out light in the 400nm and below band.
  • the spectrum is dispersed through a spectrometer, and the resulting spectrum is converged onto a linear array image sensor (including CCD and CMOS).
  • the image sensor is connected to the operating module and displays the parameters of intensity and wavelength.
  • the fluorescence microscopy imaging module includes an excitation light source 2 and an area array image sensor, and is optically coupled and connected to the sample to be measured in the coupling module.
  • the excitation light source 2 can be a laser (1-100mW, 350-390nm) or an LED light source (3W, 350-390nm).
  • the working mode of this excitation light source can be continuous or pulsed.
  • the excitation light source 2 is an LED light source
  • the fluorescence spectrum detection module also includes a collimation lens 1, through which the excitation light emits parallel laser light.
  • the collimating lens 1 includes but is not limited to spherical lenses and aspherical lenses.
  • collimating lens 1 has Focal length f 20-50mm.
  • the fluorescence microscopy imaging module may also include a dichroic mirror 1 .
  • the transmission wavelength of the dichroic mirror 1 is 400-480 nm, and the reflection wavelength is 350-400 nm.
  • the excitation light is focused at the front focal length of the objective lens (for example, magnification 1x-10x), and the excitation light is emitted parallel to the objective lens, so that the excitation light is uniformly illuminated on the sample, and the sample to be measured is excited to produce fluorescence.
  • samples to be tested include skin and fluorescent color charts.
  • the generated fluorescence passes through the objective lens, the dichroic mirror 1, the fluorescence filter, and the achromatic lens 1, and is imaged into an area array image sensor (including CCD or CMOS, black and white or color).
  • an area array image sensor including CCD or CMOS, black and white or color.
  • the bandpass of a fluorescence filter is 400-480nm, which can filter out light in the 400nm and below band.
  • the area array image sensor is connected to the operation module, and the fluorescence image of the sample is displayed on the operation module.
  • the present application provides a small non-invasive device for detecting skin AGEs content.
  • the device includes: an imaging and detection system, and an accessory device system.
  • the imaging and detection system includes a fluorescence microscopy imaging module, a fluorescence spectrum detection module and a coupling module;
  • the accessory device system includes an operation module, a position fixing module and a measured part carrying module.
  • the sample to be tested including skin and fluorescent color cards
  • the dichroic mirror 1 After stimulating the AGEs of the sample to be tested (including skin and fluorescent color cards) to generate fluorescence, it passes through the objective lens, the dichroic mirror 1, the fluorescence filter (bandpass 400-480nm), and the achromatic lens 1 to form an area array image.
  • the fluorescence image of the sample is displayed on the operating module.
  • laser light source 1 laser (1-100mW, 350-390nm), working mode is continuous
  • laser light source 2 emits parallel laser light, which passes through the dichroic mirror 2 (transmission wavelength 400-480nm, reflection wavelength 350-400nm) , reflects light in the band below 400nm, passes through the mirror (the reflection band covers 350nm to 500nm), and finally, passes through the objective lens to converge the excitation light on the sample.
  • the sample excites scattered fluorescence.
  • the fluorescence first passes through the objective lens, causing the scattered fluorescence light to become Parallel light passes through the reflecting mirror, passes through the dichroic mirror 2 and the filter (bandpass 400-480nm), filters out the light in the 400nm and below band, passes through the lens (focal length f30-100mm), and concentrates the fluorescent scattered light into the spectrometer. , the spectrum is dispersed through the spectrometer, and the formed spectrum is converged on the linear array image sensor (CCD).
  • the image sensor is connected to the operating module and displays the parameters of intensity and wavelength.
  • the operation module is used to realize functions such as real-time collection of fluorescence images and spectra, image shooting, data analysis, and line fitting. It is a computing system equipped with control software. It can select fluorescent color cards and different areas of skin samples for differential analysis, and process them to obtain the sense. Integration of the intensity of the spectrum of interest, including but not limited to computers, tablets, embedded systems, etc.; standards required for line fitting include, but are not limited to, standard fluorescent color cards, standard solutions (including but not limited to quinine sulfate standard products, AGEs-BSA standard products, etc.).
  • the position fixing module is used to adjust the height, steering, inclination and other spatial positions of the imaging and detection system according to the difference between the parts being tested, to ensure that the test window is perpendicular to the surface of the sample/skin being tested during testing, including but not limited to lifting platforms, multi-directional machinery Arm etc.
  • the tested part carrying module is used to ensure that the sample/subject tested part is in the correct position during each test. Constant, including but not limited to sample holders, head drags, arm drags, special templates, etc.
  • the present invention provides a method for testing the autofluorescence and content of fluorescent substances (eg, AGEs) in human skin.
  • fluorescent substances eg, AGEs
  • the test method includes the following process: turn on the equipment, adjust the exposure time and gain multiple according to the pre-test intensity and content range, fit the standard curve with a standard with an appropriate concentration gradient, and select the fitting method. Calculate the fitting data to obtain the correlation coefficient R 2 corresponding to the fitting function, ensuring that it is greater than 0.95, or directly use a standard fluorescent color card with an appropriate concentration gradient to calibrate the fluorescence intensity. Volunteers visit, sign informed consent forms, and are screened according to the inclusion and exclusion criteria. Volunteers who meet the criteria clean the test site according to the test requirements, pat dry with paper towels, and enter the constant temperature and humidity room (for example, the test temperature is 21°C ⁇ 1 °C, test humidity 50% ⁇ 10%), sit and rest for 30 minutes.
  • the volunteer remains stationary in a sitting/supine/lying position, and marks the location of the test area on the site to be tested (for example, the junction of the nose baseline and the perpendicular line passing the lateral canthus of the eye, a 2cm*4cm rectangular area, etc.).
  • the location of the test area on the site to be tested for example, the junction of the nose baseline and the perpendicular line passing the lateral canthus of the eye, a 2cm*4cm rectangular area, etc.
  • the software displays the average fluorescence intensity and intensity distribution diagram in the area, and calculates the content of fluorescent substances (for example, AGEs) in the area through the aforementioned standard curve.
  • the present invention provides a method for non-invasively evaluating anti-glycation efficacy in vivo.
  • the method for non-invasively evaluating the anti-glycation efficacy in vivo includes the following process: turning on the equipment, adjusting the exposure duration and gain multiple according to the pre-test intensity and content range, and modeling the standard curve with a suitable concentration gradient standard. Combined, through the selection of the fitting method and the calculation of the fitting data, the correlation coefficient R 2 corresponding to the fitting function is obtained, ensuring that it is greater than 0.95, or directly calibrating the fluorescence intensity using a standard fluorescent color card with an appropriate concentration gradient. Volunteers visit, sign informed consent forms, and are screened according to the inclusion and exclusion criteria.
  • the volunteer remains stationary in a sitting/supine/lying position, and marks the location of the test area on the site to be tested (for example, the junction of the nose baseline and the perpendicular line passing the lateral canthus of the eye, a 2cm*4cm rectangular area, etc.).
  • a blank control can be set according to the test requirements, and the basic values of each parameter can be tested before the sample is applied. Test the fluorescence intensity of AGEs in the labeled area and content and/or other parameters to be measured.
  • Other parameters to be measured include but are not limited to skin yellowness (instrument Konica Minolta CM-700d), elasticity and firmness (instrument Cutometer dual MPA580), and skin texture parameters (instrument Visioscan VC98).
  • the stepwise dilution method prepare the AGE-BSA standard solution with concentration gradients of 0.0mg/mL, 0.5mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL, 2.5mg/mL and 10 ⁇ g/mL, Pipette 10 ⁇ L of AGE-BSA standard solutions of 20 ⁇ g/mL, 30 ⁇ g/mL, 40 ⁇ g/mL, 50 ⁇ g/mL, and 60 ⁇ g/mL into the slide channel of 1 cm ⁇ 1 cm ⁇ 0.1 mm, and photograph the corresponding fluorescence images in sequence. (Figure 3, Figure 5), perform first-order linear fitting to obtain the fitting function and correlation coefficient R 2 . The corresponding average fluorescence intensity of the standard solution and the fitting function results are shown in Table 1, Table 2 and Figure 4 and Figure 6.
  • the equipment continues to run for 24 hours.
  • a total of 4 groups were taken 10 times to obtain the average fluorescence intensity of the corresponding fluorescence images.
  • the average value, standard deviation, standard deviation deviation of each group of values and the overall accuracy of the 40 data were calculated, as shown in Table 3.
  • the anti-glycation efficacy evaluation test of cosmetics was conducted on 10 healthy female volunteers (average age 43.8 ⁇ 2.0 years old) with no visible blood vessels, scars, lichenification or other skin abnormalities on the volar side of the forearm between 40 and 45 years old.
  • the test samples were tested It was a 5 wt% ergothioneine aqueous solution purchased from the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences.
  • the sample dosage is 2 mg/cm 2 and massage it fully until absorbed; the interval is at least 4H.
  • the second sample application use the test sample in the sample area according to the random table.
  • the dosage of the sample is 2 mg/cm 2 and massage thoroughly until absorbed; the sample application is repeated for 1 week.
  • the subjects are required to return as agreed, and the experimenter applies the sample in the same way; after the sample is used for 3 days and 1 After several weeks, repeat the skin test procedure.
  • the present invention is applied to the in vivo AGEs autofluorescence test to evaluate the "anti-glycation" effect of exogenous substances. It does not require pretreatment, real-time testing, high detection limit, good parallel test repeatability, and good short-term test reproducibility in the same area. , High test sensitivity.
  • the present invention can keenly detect the improvement of skin glycation level by exogenous substances, and can be used to evaluate the "anti-glycation" effect of exogenous substances, and the time point is significantly earlier than other indicators related to skin aging indicators, confirming that it can indeed As a target indicator for the "anti-aging” claim, it has guiding significance for the evaluation of "anti-aging” related claims.
  • the present invention illustrates the detailed methods of the present invention through the above embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must rely on the above detailed methods to be implemented.
  • Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

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Abstract

本发明提供了一种检测皮肤自发荧光及其含量的装置，包括：成像及检测系统和附属器件系统，成像及检测系统包括荧光显微成像模块、荧光光谱检测模块及耦合模块，荧光显微成像模块与耦合模块光学耦合连通，荧光光谱检测模块与耦合模块光学耦合连通，附属器件系统包括操作模块、位置固定模块和被测部位承载模块。本发明还涉及采用所述小型无创装置评价抗糖化功效的方法。

Description

检测皮肤自发荧光的装置及抗糖化功效评价方法 技术领域
本发明涉及功效检测领域，具体涉及一种小型无创检测皮肤AGEs含量的装置及抗糖化功效评价方法，尤其可以用于内服药品、保健品，或外用药品、外用保健品及皮肤外用剂的抗糖化功效。
背景技术
晚期糖基化终末产物(advanced glycation end products AGEs)是非酶糖基化反应(Maillard反应)的终末产物，是指蛋白质、脂质或核酸等大分子在没有酶参与的条件下，自发的与葡萄糖或其他还原单糖反应所生成的稳定且不可逆的共价加成物，是非酶糖基化衰老理论的最终产物，是研究衰老的一项重要指标。非酶糖基化衰老理论是目前已被众多学者公认的衰老理论之一。
1912年，法国科学家Maillard最早报道了AGEs。AGEs呈棕黄色，具有荧光特性(最大激发波长370nm，最大发射波长440nm)、不可逆性、交联性、结构异质性、不易被降解、对酶稳定等特性。在肌体的不同组织器官中，如血管内皮细胞、神经细胞、胶原、晶状体循环系统以及肾脏、肝脏、肺脏、血管、腹膜等组织纤维中，都有存在，且随着年龄的增长不断积聚。截至目前尚未发现能够使AGEs生成逆转的细胞系，且尚未发现任一蛋白水解途径可以清除细胞内的AGEs，尤其是涉及到蛋白质的交联时；体内AGEs的清除主要是通过单核巨噬细胞的吞噬作用经肾脏清除。
AGEs的来源分为外源性和内源性两种，其主要通过三种机制对肌体造成损害：通过与蛋白质、脂质、核酸等大分子物质直接交联结合破坏其结构和功能；影响信号传导途径和酶活性；与细胞表面的AGEs受体相互作用，引发生物学效应。
糖基化会造成蛋白质交联损伤，使正常的蛋白结构转变成老年蛋白结构， 一些胞内蛋白已被证明在肌体老化的病理中被糖化和失活，主要参与反应的氨基酸残基有赖氨酸、精氨酸、组氨酸、酪氨酸、色氨酸、丝氨酸以及苏氨酸等。糖基化还会造成结构蛋白的硬化，功能酶的损伤，能量供应的减少，代谢功能的降低，并引起免疫功能受损，平衡机能失调，基因突变等一系列老化过程。由于占据共同的蛋白结合位点，糖基化还可抑制泛素结合和泛素介导的降解。此外，糖基化和氧化联系紧密，糖基化过程产生ROS，且AGE-蛋白自身就是ROS的来源。糖基化还抑制糖激酶的转录。溶酶体调节功能(大自噬、蛋白酶表达)的缺失可能部分是由于AGEs的恶性循环促进脂褐素(亦称蜡样质或AGE-色素样脂质体，是溶酶体中一种与年龄相关的交联蛋白和聚集物质)的形成而造成的。
众多研究表明AGEs通过与细胞表面的特异性受体结合起作用。细胞表面的RAGEs(免疫球蛋白超家族成员，可表达于血管内皮细胞、平滑肌细胞、成纤维细胞、淋巴样细胞、肾小球系膜细胞、巨噬细胞、神经元细胞以及肿瘤细胞等，目前已识别的种类包括巨噬细胞清道夫受体I型和II型、寡糖转移酶-48、80K-H磷蛋白、galectin-3等)与AGEs等配体结合后，可诱导单核细胞的趋化和氧化应激反应，并生成大量的氧自由基，激活信号传导通路，刺激白细胞介素I(IL-I)、胰岛素样生长因子I、肿瘤坏死因子(TNF-α)、血小板来源生长因子(PDGF)、无粒白细胞-巨噬细胞克隆刺激因子的释放和表达，尤其是激活转录因子NF-kB，它的激活可诱导产生多种损伤因子，产生致病效应，从而激活和损害细胞内的蛋白质、核酸，引发复杂的生物学效应，导致细胞的功能和结构异常，从而影响器官的结构和功能。
研究表明，AGEs会加速人体衰老。Sell等人检测八种哺乳动物的皮肤戊糖素水平，发现其生成率与物种的最大寿限高度相关。此外，AGEs还会导致多种慢性退化型疾病的发生，包括白内障、动脉粥样硬化、阿尔兹海默病、皮克氏病、帕金森病、中风、淀粉样变性、肾病、糖尿病、神经病变和视网膜病变等。
就皮肤而言，AGEs与胶原蛋白交联结合，造成其断裂，致使皮肤弹性下降，出现萎缩，进而产生皱纹。不仅如此，过多的AGEs附着在肌肤上，还会使其逐渐变黄，黯淡无光。早期的一项研究证实蛋白代谢是不同类型胶原蛋白 AGEs积累的主要决定因素，并估算出皮肤胶原蛋白的半衰期为15年，软骨胶原蛋白的半衰期为117年。如此长的半衰期，如不加以干预，很容易蓄积AGEs，造成皮肤老态。
因此，AGEs是肌体衰老以及多脏器慢性退化型疾病的根源物质及重要指征，该物质的检测对肌体衰老阶段的评估、慢性退化型疾病的筛查、诊断以及干预结果的评估均具有重要意义。
90年代早期，多采用胶原关联荧光(collagen-linked fluorescence CLF)或特定AGE测量法(戊糖素、羧甲基赖氨酸)检测活检组织匀浆提取物中的AGEs，取样时会造成生物体的创伤甚至死亡，且前处理过程复杂，通常需要1～2天完成，该过程还会造成被测物质的损耗，造成痕量物质或含量变化不明显的物质测试的精确度和准确性无法保证的问题。近年，越来越多的人使用高效液相色谱法、酶联免疫吸附法、质谱联用或荧光法在血清或血浆中测定AGEs。与需要活检组织相比，血浆更适合反复测量，但血浆AGE测定法的可重复性较差，而且与AGEs的组织含量相关性较差，血液和尿液样本中的AGE未必能反映组织AGE的水平。复杂性、成本、缺乏可重复性、缺乏标准化限制了这些检测方法的广泛应用。
一项关于II型糖尿病患者血管损伤非侵入性标记物的研究利用自研的简易无创装置证明皮肤自身荧光与皮肤非荧光AGE的水平相关；胶原关联荧光与戊糖素(荧光物质)和(Nε)-羧甲基-赖氨酸(非荧光物质)的平行关系以及特定AGE间的相互关系也表明荧光和非荧光AGE表现相似。因此，皮肤自身荧光是整个肌体AGE池的一个标志，可用于标记肌体AGE的行为。该研究也提示采用适当设备进行在体AGEs含量检测的可行性。
市面上现有的AGEs测量设备为疾病风险评估设备，主要用于体检机构及医院肾内科、内分泌科、心内科等科室糖尿病等部分慢性疾病的早期筛查、风险标记，测试部位仅能选择前臂部位，仅可提供被测皮肤1cm*1cm区域的AGEs荧光强度，再将该数据与数据库数据比对，由于患病人群的数据较对应年龄健康人群高20％～30％，比对范围后，提供相关慢性病的发病几率及并发症风险因子，以提示相关人员进行进一步医疗检查。由于该设备数据库范围有限(荧光强度只包含20岁以上的成年人的情况)，且仅作前期筛查的风险评估，不 提供真实的AGEs的含量，也未进行任何标准物质的校准，故对外界环境或外源物质对皮肤AGEs的微量影响无法评估，不能用于抗糖化效果的评价领域；此外，市售设备体积也十分庞大，超过常规滚筒洗衣机的大小，配置操控电脑后，更为占据空间。占地大、测试面积小、测试部位局限(仅为前臂部位)、非定量、测试数据仅供风险提示、无法识别抗糖化短期效应等，均限制其在相关领域的应用。本发明所述检测设备整体大小为常规滚筒洗衣机大小的约1/20，占地面积小、可操作性空间大、测试面积大、可测试多处部位(包括但不限于：面部、躯干部、手足部、毛发等)、可定性定量提供准确荧光强度和对应物质含量，检出限低至10-8量级，且无需对被测样品进行预处理，故可对外源物质(待测样品)的痕量影响进行无创、高敏感、高精度探测，故本发明所述设备为小型无创检测设备。
2021年1月1日起《化妆品监督管理条例》施行，对化妆品进行全过程管理。为规范化妆品生产经营活动、化妆品功效宣称评价工作，保障化妆品的质量安全，保证功效宣称评价结果的科学性、准确性和可靠性，维护消费者合法权益，推动社会共治和化妆品行业健康发展，根据《化妆品监督管理条例》等有关法律法规要求，《化妆品分类规则和分类目录》、《化妆品功效宣称评价规范》制定实施。分类目录将化妆品功效分为26类+新功效，新功效即不符合26类规则的功效宣称。
基于上述法规，化妆品“抗糖化”功效属于新功效的一种，按照评价规范要求，“应当根据产品功效宣称的具体情况，进行科学合理的分析”，但化妆品抗糖基化的临床评估，亦即皮肤AGEs含量的在体检测，在整个化妆品行业未有成熟检测手段、检测设备和测试方法。
目前市场上的相关产品由于缺乏足够的临床数据支持，大多放弃了“抗糖基化”的宣称点，或在新法规的“窗口期”(2023年5月1日)前，意图混淆“抗糖基化”和“抗氧化”的概念，或即便有数据支持，也是来自原料的体外实验，无法迁移推演至在体应用效果，不符合功效宣称评价规范对新功效评价方法的要求。
此外，化妆品“抗初老”宣称目前仅将靶向指标设定为胶原蛋白或是皮肤表征(肤色、弹性、细纹、皱纹等)，事实上这些指标的出现已远远晚于皮肤 AGEs水平的改变，作为“抗初老”宣称的靶向指标并不合适。皮肤AGEs在体检测设备及方法可通过获得初老～AGEs～生理指标的关系，建立行业内的肌肤早衰新标准，重新定义初老指数。
发明内容
本发明提供了一种检测皮肤自发荧光及其含量的装置，所述装置包括：
(i)成像及检测系统；和
(ii)附属器件系统，
其中，所述成像及检测系统包括荧光显微成像模块、荧光光谱检测模块及耦合模块，所述荧光显微成像模块与所述耦合模块光学耦合连通，所述荧光光谱检测模块与所述耦合模块光学耦合连通，
其中，所述附属器件系统包括操作模块、位置固定模块和被测部位承载模块，
其中，所述荧光光谱检测模块包括第一光源和线阵图像传感器，并且与耦合模块中的待测样品光学耦合连通，所述线阵图像传感器连接操作模块，显示出强度和波长的参数，
其中，所述荧光显微成像模块包括第二光源和面阵图像传感器，并且与耦合模块中的待测样品光学耦合连通，所述面阵图像传感器连接操作模块，显示出样品荧光图像。
在优选的实施方式中，所述第一光源是波长350-390nm的激光光源，所述第二光源是波长350-390nm的激光光源。
在优选的实施方式中，所述装置用于检测的荧光物质选自：黄素、脂褐素、晚期糖基化终末产物AGEs、还原型辅酶I NADH、类胡萝卜素、氧化黑色素、卟啉、胶原蛋白、氨基酸、弹性蛋白以及它们的组合。
在优选的实施方式中，所述荧光光谱检测模块还包括第二二向色镜，其反射光并通过光路使激发光汇聚到待测样品上以激发样品产生散射的荧光，样品激发出的散射的荧光再通过光路汇聚到线阵图像传感器上。在更优选的实施方式中，样品激发出的散射的荧光通过物镜变为平行光，经反射镜，透过第二二向色镜和滤波片，经过透镜，汇聚到光谱仪中，形成光 谱汇聚到线阵图像传感器上。
在优选的实施方式中，所述荧光显微成像模块还包括第一二向色镜，其反射光并通过光路使激发光照射到待测样品上以激发样品产生荧光，样品激发出的荧光再通过光路成像到面阵图像传感器上。在更优选的实施方式中，样品激发出的荧光通过物镜，透过第一二向色镜，荧光滤光片，消色差透镜，成像到面阵图像传感器上。
在优选的实施方式中，所述操作模块用于实现荧光图像及荧光光谱的实时采集、图像拍摄、数据分析、标线拟合或其组合。
另一方面，本发明提供了一种抗糖化功效的评价方法，所述方法包括：
(a)标记皮肤待测区域；
(b)采用检测皮肤自发荧光及其含量的装置测定标记区域的皮肤荧光强度及含量的基础值；
(c)使用待测样品；
(d)采用检测皮肤自发荧光及其含量的装置测定使用样品后标记区域的皮肤荧光强度及含量；
(e)将步骤(d)的结果与步骤(b)的结果进行比较，以评价抗糖化功效。
在优选的实施方式中，所述步骤(e)包括基于荧光物质的标准曲线比较步骤(d)和步骤(b)的结果，以评价抗糖化功效。
在优选的实施方式中，所述步骤(e)包括基于标准荧光色卡比较步骤(d)和步骤(b)的结果，以评价抗糖化功效。
在优选的实施方式中，所述待测样品选自：内服药品、保健品、外用药品、外用保健品和皮肤外用剂。
附图说明
下面结合附图进一步说明本发明。
图1显示了本发明的检测皮肤自发荧光的装置。
图2显示了同时拍摄手指前端AGEs及标准荧光色卡。
图3显示了AGE-BSA标准溶液荧光图像(mg/mL)。
图4显示了AGE-BSA标准曲线线性拟合(mg/mL)的结果。
图5显示了AGE-BSA标准溶液荧光图像(μg/mL)。
图6显示了AGE-BSA标准曲线线性拟合(μg/mL)的结果。
具体实施方式
本申请针对某些荧光物质(例如，AGEs)激发光谱和发射光谱的特异性，对系统波段范围进行优化，增强特定波段范围激发和发射光强。通过特制小型无创设备实现不同部位皮肤表面荧光物质(例如，AGEs)的在体图像及光谱检测。此外，通过标线拟合将荧光强度转换为荧光物质(例如，AGEs)的浓度或含量，检出限可达1×10-8g/mL，对外源物质造成的肌体糖化水平的影响可高敏感识别，从而提供一种化妆品抗糖化功效评估的快速、无创、定量、高灵敏检测手段。
本发明提供了一种小型无创地检测皮肤自发荧光及其含量的装置，所述装置包括：成像及检测系统和附属器件系统。成像及检测系统包括荧光显微成像模块、荧光光谱检测模块及耦合模块，所述荧光显微成像模块与所述耦合模块光学耦合连通，所述荧光光谱检测模块与所述耦合模块光学耦合连通。附属器件系统包括操作模块、位置固定模块和被测部位承载模块。
参照图1所示，荧光光谱检测模块包括激发光源1和线阵图像传感器，并且与耦合模块中的待测样品光学耦合连通。激发光源1可以是激光器(1-100mW，350-390nm)或LED光源(3W，350-390nm)。这种激发光源的工作方式可为连续式或者脉冲式。如果激发光源1是LED光源，荧光光谱检测模块还包括准直透镜(未显示)，激发光通过准直透镜发出平行激光。荧光光谱检测模块还可以包括二向色镜2。在一些实施方式中，二向色镜2的透射波长为400-480nm，反射波长为350-400nm。因此，二向色镜2能够反射400nm以下波段的光，透过400nm以上的反射激发光。反射光经过连通的光路汇聚到待测样品上，激发样品发出荧光，例如，待测样品包括皮肤和荧光色卡。发出的荧光则汇聚到光谱仪中，并最终汇聚到线阵图像传感器上，从而显示荧光强度和波长的参数。在一些实施方式中，二向 色镜2的反射光经过反射镜，再经过物镜(例如，放大倍数1x-10x)使激发光汇聚到样品上，样品激发出散射的荧光。例如，反射镜的反射波段覆盖350nm到500nm。在一些实施方式中，发出的散射的荧光先通过物镜，再经反射镜，通过二向色镜2和滤波片，经过透镜(焦距f 30-100mm)，使荧光散射光汇聚到光谱仪当中。例如，滤波片的带通为400-480nm，能够滤掉400nm及以下波段的光。在一些实施方式中，经过光谱仪进行光谱的色散，形成的光谱汇聚到线阵图像传感器(包含CCD和CMOS)上。图像传感器连接操作模块，显示出强度和波长的参数。
同样参照图1所示，荧光显微成像模块包括激发光源2和面阵图像传感器，并且与耦合模块中的待测样品光学耦合连通。激发光源2可以是激光器(1-100mW，350-390nm)或LED光源(3W，350-390nm)。这种激发光源的工作方式可为连续式或者脉冲式。如果激发光源2是LED光源，荧光光谱检测模块还包括准直透镜1，激发光通过准直透镜1发出平行激光。例如，准直透镜1包括但不限于球面镜和非球面镜。例如，准直透镜1具有焦距f 20-50mm。荧光显微成像模块还可以包括二向色镜1。在一些实施方式中，二向色镜1的透射波长为400-480nm，反射波长为350-400nm。经过二向色镜1反射，激发光在物镜(例如，放大倍数1x-10x)的前焦距聚焦，在物镜平行射出激发光，使激发光均匀照射在样品之上，激发待测样品产生荧光。例如，待测样品包括皮肤和荧光色卡。在一些实施方式中，产生的荧光通过物镜，透过二向色镜1，荧光滤光片，消色差透镜1，成像到面阵图像传感器(包含CCD或CMOS，黑白或彩色)中。例如，荧光滤光片的带通为400-480nm，能够滤掉400nm及以下波段的光。面阵图像传感器连接操作模块，在操作模块上显示样品荧光图像。
在一个具体实施方式中，本申请提供了一种小型无创检测皮肤AGEs含量的设备，该设备包括：成像及检测系统、附属器件系统。其中成像及检测系统包括荧光显微成像模块、荧光光谱检测模块及耦合模块；附属器件系统包括操作模块、位置固定模块和被测部位承载模块。
荧光显微成像模块中，激发光从激发光源2(LED光源(3W，350-390nm)，工作方式为连续式)发出，通过调节准直透镜1(球面镜， 焦距f＝25mm)，经过二向色镜1(透射波长400-480nm，反射波长350-400nm)反射，使LED激发光在物镜(放大倍数1x-10x)的前焦距聚焦，在物镜平行射出激发光，使激发光均匀照射在样品之上。激发被测样品(包含皮肤和荧光色卡)AGEs产生荧光后，通过物镜，透过二向色镜1，荧光滤光片(带通400-480nm)，消色差透镜1，成像到面阵图像传感器(CCD，彩色)中，在操作模块上显示样品荧光图像。
荧光光谱检测模块中，激光光源1(激光器(1-100mW，350-390nm)，工作方式为连续式)发出平行激光，经过二向色镜2(透射波长400-480nm，反射波长350-400nm)，反射400nm以下波段的光，经过反射镜(反射波段覆盖350nm到500nm)，最后，经过物镜使激发光汇聚到样品上，样品激发出散射的荧光，荧光先通过物镜，使荧光散射光变为平行光，经反射镜，通过二向色镜2和滤波片(带通400-480nm)，滤掉400nm及以下波段的光，经过透镜(焦距f30-100mm)，使荧光散射光汇聚到光谱仪当中，经过光谱仪进行光谱的色散，形成的光谱汇聚到线阵图像传感器(CCD)上。图像传感器连接操作模块，显示出强度和波长的参数。
操作模块用于实现荧光图像及光谱实时采集、图像拍摄、数据分析、标线拟合等功能，为搭载控制软件的计算系统，可选取荧光色卡和皮肤样品不同区域做差分分析，处理得到感兴趣光谱的强度积分，包括但不限于计算机、平板电脑、嵌入式系统等；标线拟合所需标准品包括但不限于标准品荧光色卡、标准品溶液(包括但不限于硫酸奎宁标准品、AGEs-BSA标准品等)。
此外，除了使用常规标准品溶液外，还开发了适用于不同浓度测试场景的多套标准荧光色卡进行定标，样品拍摄时将对应浓度梯度的标准荧光色卡置于样品一侧，实现标准色卡和待测样品的同时拍摄及测量，可极大地减少外部环境、人员操作的改变带来的系统误差(如图2所示)。
位置固定模块用于根据被测部位的区别调整成像及检测系统的高低、转向、倾角等空间位置，确保测试时测试窗垂直于被测样品/皮肤表面，包括但不限于升降平台、多向机械臂等。
被测部位承载模块用于确保样品/受试者被测部位在每次测试时位置 恒定，包括但不限于样品支架、头拖、臂拖、特制模板等。
另一方面，本发明提供了人体皮肤荧光物质(例如，AGEs)自发荧光及含量测试方法。
在一个实施方式中，测试方法包括以下过程：打开设备，根据预测试强度及含量范围调节曝光时长和增益倍数，用合适浓度梯度的标准品对标准曲线进行拟合，通过拟合方式的选择、拟合数据的计算，得到拟合函数对应的相关系数R2，确保其大于0.95，或直接采用合适浓度梯度的标准荧光色卡对荧光强度进行校准。志愿者到访，签署知情同意书后，按照入选和排除标准进行筛选，符合标准的志愿者根据测试需求清洁受试部位，用纸巾拍干，进入恒温恒湿室(例如，测试温度21℃±1℃、测试湿度50％±10％)，静坐休息30分钟。志愿者保持坐位/卧位/躺位不动，于待测部位标记测试区域位置(例如，鼻基准线和过眼外眦垂线交界处，2cm*4cm矩形区域等)。于标记区域拍摄荧光物质(例如，AGEs)荧光图像，勾选合适测量区域，软件显示该区域平均荧光强度、强度分布图，并通过前述标准曲线计算得到该区域荧光物质(例如，AGEs)含量。
又一方面，本发明提供了一种在体、无创地评价抗糖化功效的方法。
在一个实施方式中，在体、无创地评价抗糖化功效的方法包括以下过程：打开设备，根据预测试强度及含量范围调节曝光时长和增益倍数，用合适浓度梯度的标准品对标准曲线进行拟合，通过拟合方式的选择、拟合数据的计算，得到拟合函数对应的相关系数R2，确保其大于0.95，或直接采用合适浓度梯度的标准荧光色卡对荧光强度进行校准。志愿者到访，签署知情同意书后，按照入选和排除标准进行筛选，符合标准的志愿者根据测试需求清洁受试部位，用纸巾拍干，进入恒温恒湿室(例如，测试温度21℃±1℃、测试湿度50％±10％)，静坐休息30分钟。志愿者保持坐位/卧位/躺位不动，于待测部位标记测试区域位置(例如，鼻基准线和过眼外眦垂线交界处，2cm*4cm矩形区域等)。可根据测试需求设置空白对照，在未涂抹样品前，进行各参数基础值的测试。于标记区域测试AGEs荧光强度 及含量和/或其他待测参数，其他待测参数包括但不限于皮肤黄度(仪器Konica Minolta CM-700d)、弹性和紧致度(仪器Cutometer dual MPA580)、皮肤纹理参数(仪器Visioscan VC98)的基础值和涂抹样品后不同时间点的测量值。测试完成后进行空白对照比较/使用前后比较判断是否具有统计学意义，从而评价抗糖化功效。
下面结合具体的实施例进一步阐述本发明。但是，应该明白，这些实施例仅用于说明本发明而不构成对本发明范围的限制。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另有说明，所有的百分比和份数按重量计。
实施例1：AGE-BSA(ab51995)标准曲线线性拟合
按逐级稀释法配置浓度梯度依次为0.0mg/mL、0.5mg/mL、1.0mg/mL、1.5mg/mL、2.0mg/mL、2.5mg/mL的AGE-BSA标准溶液和10μg/mL、20μg/mL、30μg/mL、40μg/mL、50μg/mL、60μg/mL的AGE-BSA标准溶液，分别移取10μL至1cm×1cm×0.1mm的载玻片流道中，依次拍摄得到对应荧光图像(图3、图5)的平均荧光强度，进行一阶线性拟合，得到拟合函数和相关系数R2。标准溶液对应平均荧光强度、拟合函数结果如表1、表2和图4、图6所示。
表1：AGE-BSA标准溶液荧光强度(mg/mL)
表2：AGE-BSA标准溶液荧光强度(μg/mL)
通过表1、表2和图4、图6的结果可以看出，本发明可识别并区分极低浓度(低至10-8量级)且浓度间隔较小的AGEs对应的自发荧光强度，且均表现出很好的线性度。
实施例2：设备精确度和稳定性测试
设备持续运行24小时，配置1.0mg/mL的AGE-BSA标准溶液，移取10μL至1cm×1cm×0.1mm的载玻片流道中，每隔1小时进行一组荧光图像的拍摄，每组拍摄10次，共拍摄4组，得到对应荧光图像的平均荧光强度，计算每组数值的平均值、标准差、标准差偏离以及40个数据的整体精度情况，如表3所示。
表3：设备重复度、精度及稳定性测试结果
通过表3可以看出，即便设备在长时间运行(超过24h)后，对低浓度的标准品，同一时段多次测试之间重复性依旧很好(组内偏差＜0.4％)，且不同时段的测试值相当，体现出很好的精确度和稳定性。
实施例3：化妆品抗糖化功效评价测试
打开设备，根据预测试强度及含量范围调节曝光时长为10ms、增益倍数为16，用标准荧光色卡对荧光强度进行校准。以10名40～45岁的前臂掌侧没有可见血管、疤痕、苔藓样变或其他皮肤异常情况的健康女性志愿者(平均年龄43.8±2.0岁)为对象进行化妆品抗糖化功效评价测试，测试样品为5重量％麦角硫因水溶液，购于中国科学院上海有机化学研究所。
志愿者到访，签署知情同意书后，按照入选和排除标准进行筛选，符合标准的志愿者用温水清洁双侧前臂内侧肌肤，用纸巾拍干，进入恒温恒湿室(测试温度21℃±1℃、测试湿度50％±5％)，静坐休息30分钟。志愿者保持坐位，于双侧肘部褶皱下15cm的前臂掌侧皮肤标记3cm*3cm测试区域位置，左右各标记一处，一处为样品区域，一处为空白区域，在未涂抹样品前，进行各参数基础值的测试。
于双侧前臂内侧标记区域测试AGEs荧光强度、皮肤黄度(仪器Konica Minolta CM-700d)、弹性和紧致度(仪器Cutometer dual MPA580)的基础值，每个区域平行测试三次，计算平均值。
第一次样品涂抹，按随机表在样品区域使用测试样品，样品使用量为2mg/cm2，充分按摩至吸收；间隔至少4H，第二次样品涂抹，按随机表在样品区域使用测试样品，样品使用量为2mg/cm2，充分按摩至吸收；样品涂抹重复1周，在测试周期内，要求受试者按约定回访，由实验人员按同样方法涂抹样品；分别在样品使用3天、1周后，重复皮肤测试操作。
数据使用SPSS 23.0软件进行统计，检验水准为a＝0.05。使用前后对照：如数据呈现正态分布，则采用参数检验(配对t检验)，如数据不符合正态分布，则采用非参数检验(Wilcoxon符号秩检验)；空白对照比较：如数据呈现正态分布，则采用参数检验(独立样本t检验)，如数据不符合正态分布，则采用非参数检验(Mann-Whinty U检验)。
1)AGEs荧光强度
表4
2)皮肤黄度
表5

3)紧致度R0
表6

4)弹性R2
表7
将本发明应用于在体AGEs自发荧光测试，以评估外源物质的“抗糖化”功效，无需预处理、实时测试、检出限高、平行测试重复性好、同区域短期测试复现性好、测试灵敏度高。
空白区域在3天、1周后，AGEs自发荧光强度均无显著性变化 (p＝0.586，p＝0.328)；而在抗糖原料水溶液使用3天后，AGEs自发荧光强度有下降趋势(p＝0.059)，使用1周后，AGEs自发荧光强度显著下降，改善率为13.89％(p＝0.003)，与空白区域具有显著性差异(p＝0.007)，且此时皮肤黄度、紧致度和弹性指标还未有明显变化。说明本发明可敏锐检测到外源物质对皮肤糖化水平的改善情况，即可用于评价外源物质的“抗糖化”功效，且时间节点明显早于其他皮肤衰老指征相关指标，证实其确实可作为“抗初老”宣称的靶向指标，对“抗初老”相关宣称的评价具有指导意义。
本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (12)

  1. 一种检测皮肤自发荧光及其含量的装置，所述装置包括：
    (i)成像及检测系统；和
    (ii)附属器件系统，
    其中，所述成像及检测系统包括荧光显微成像模块、荧光光谱检测模块及耦合模块，所述荧光显微成像模块与所述耦合模块光学耦合连通，所述荧光光谱检测模块与所述耦合模块光学耦合连通，
    其中，所述附属器件系统包括操作模块、位置固定模块和被测部位承载模块，
    其中，所述荧光光谱检测模块包括第一光源和线阵图像传感器，并且与耦合模块中的待测样品光学耦合连通，所述线阵图像传感器连接操作模块，显示出强度和波长的参数，
    其中，所述荧光显微成像模块包括第二光源和面阵图像传感器，并且与耦合模块中的待测样品光学耦合连通，所述面阵图像传感器连接操作模块，显示出样品荧光图像。
  2. 如权利要求1所述的装置，其中，所述第一光源是波长350-390nm的激光光源，所述第二光源是波长350-390nm的激光光源。
  3. 如权利要求1所述的装置，其中，所述装置用于检测的荧光物质选自：黄素、脂褐素、晚期糖基化终末产物AGEs、还原型辅酶I NADH、类胡萝卜素、氧化黑色素、卟啉、胶原蛋白、氨基酸、弹性蛋白以及它们的组合。
  4. 如权利要求1所述的装置，其中，所述荧光光谱检测模块还包括第二二向色镜，其反射光并通过光路使激发光汇聚到待测样品上以激发样品产生散射的荧光，样品激发出的散射的荧光再通过光路汇聚到线阵图像传感器上。
  5. 如权利要求4所述的装置，其中，样品激发出的散射的荧光通过物镜变为平行光，经反射镜，透过第二二向色镜和滤波片，经过透镜，汇聚到光谱仪中，形成光谱汇聚到线阵图像传感器上。
  6. 如权利要求1所述的装置，其中，所述荧光显微成像模块还包括第一二向色镜，其反射光并通过光路使激发光照射到待测样品上以激发样品产生荧光，样品激发出的荧光再通过光路成像到面阵图像传感器上。
  7. 如权利要求6所述的装置，其中，样品激发出的荧光通过物镜，透过第一二向色镜，荧光滤光片，消色差透镜，成像到面阵图像传感器上。
  8. 如权利要求1所述的装置，其中，所述操作模块用于实现荧光图像及荧光光谱的实时采集、图像拍摄、数据分析、标线拟合或其组合。
  9. 一种抗糖化功效的评价方法，所述方法包括：
    (a)标记皮肤待测区域；
    (b)采用如权利要求1-8中任一项所述的装置测定标记区域的皮肤荧光强度及含量的基础值；
    (c)使用待测样品；
    (d)采用如权利要求1-8任一项所述的装置测定使用样品后标记区域的皮肤荧光强度及含量；
    (e)将步骤(d)的结果与步骤(b)的结果进行比较，以评价抗糖化功效。
  10. 如权利要求9所述的方法，其中，所述步骤(e)包括基于荧光物质的标准曲线比较步骤(d)和步骤(b)的结果，以评价抗糖化功效。
  11. 如权利要求9所述的方法，其中，所述步骤(e)包括基于标准荧光色卡比较步骤(d)和步骤(b)的结果，以评价抗糖化功效。
  12. 如权利要求9所述的方法，其中，所述待测样品选自：内服药品、保健品、外用药品、外用保健品和皮肤外用剂。
PCT/CN2023/106485 2022-09-09 2023-07-10 检测皮肤自发荧光的装置及抗糖化功效评价方法 WO2024051337A1 (zh)

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