Application of procyanidine B2 or red rice extract in treatment of skin light injury

Abstract

The invention provides an application of procyanidine B2 or a red rice extract in treatment of skin light injury. The proanthocyanidin B2 or the red rice extract containing the proanthocyanidin B2 is combined with a key subunit NDUFA7 in NADH: ubiquinone oxidoreductase (compound I) in mitochondria to stabilize the positioning of the proanthocyanidin B2 or the red rice extract containing the proanthocyanidin B2 in the compound I and improve the activity of NADH dehydrogenase, so that the stable state of the mitochondria is maintained, the generation of ROS is reduced, the PERK-Nrf2 axis of the targeted mitochondria activates antioxidant response, and the mitochondrial activity of the mitochondria is improved. On the other hand, the excessive stress of NF-kappa B and p53-POMC axes is reduced, and the effect of repairing skin light damage is achieved by synergistically promoting cell survival and steady state maintenance through multiple channels.

Description

Technical Field

[0001] This invention relates to the use of proanthocyanidin B2 or red rice extract in the treatment of photodamage to the skin. Background Technology

[0002] The core of UV-induced photoaging is the overproduction of reactive oxygen species (ROS). When ROS levels rise, they overwhelm the skin's antioxidant defense mechanisms, triggering oxidative stress. ROS molecules, such as superoxide anions, peroxides, and singlet oxygen, damage lipids, proteins, and DNA, triggering a destructive signaling cascade closely related to photoaging mechanisms. This oxidative stress activates pro-inflammatory and pro-degradative pathways, including the NF-κB and MAPK signaling pathways. NF-κB, as a key transcription factor, regulates the expression of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, leading to chronic inflammation. Simultaneously, UV exposure activates the MAPK pathway, resulting in the upregulation of matrix metalloproteinases such as MMP-1, MMP-3, and MMP9. These enzymes degrade extracellular matrix (ECM) components, such as collagen, leading to the loss of skin structure. Nrf2 is a major regulator of cellular oxidative stress; short-term Nrf2 activation inhibits tissue damage, inflammation, and the initiation of cancer.

[0003] Mitochondria are the main organelles in skin cells that produce ROS and energy and regulate various physiological and pathological signaling pathways. UV irradiation damages mitochondrial complex I (mitochondrial NADH: ubiquinone oxidoreductase), further disrupting the homeostasis of the mitochondrial electron transport chain, leading to a further increase in ROS and apoptosis. NADUFA7 is a key subunit of complex I, and studies have shown that knockout or loss of NADUFA7 activity leads to increased mitochondrial ROS.

[0004] Chinese patent document CN103494800A discloses the application of proanthocyanidin B2 in the preparation of drugs for treating ultraviolet-induced photodamage to the skin, but it does not disclose how proanthocyanidin B2 achieves ultraviolet-induced photodamage to the skin. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides the use of proanthocyanidin B2 or red rice extract in the treatment of photodamage to the skin. The present invention discovers that proanthocyanidin B2, by binding to NDUFA7, stabilizes its localization in complex I, enhances its NADH dehydrogenase activity, thereby maintaining mitochondrial homeostasis and reducing ROS generation. Furthermore, it reduces inflammatory responses through the ROS-NF-κB / Nrf2 pathways, thereby reducing damage to cells / skin from ultraviolet and high-energy visible light.

[0006] One aspect of the present invention provides the use of proanthocyanidin B2 or red rice extract containing it in the preparation of a medicine or cosmetic for the prevention and / or treatment of photodamage to the skin, characterized in that the proanthocyanidin B2 binds to the NDUFA7 protein to achieve the prevention and / or treatment of photodamage to the skin.

[0007] In this invention, the NDUFA7 protein can be the NDUFA7 protein conventionally understood in the art. Specifically, the NDUFA7 protein referred to in this invention is the human NDUFA7 protein or its mutants. Those skilled in the art will recognize that the NDUFA7 protein is present in the mitochondria of human skin cells.

[0008] One aspect of the present invention also provides the use of proanthocyanidin B2 or a red rice extract containing it in the preparation of a medicament or cosmetic for the prevention and / or treatment of photodamage to the skin, wherein proanthocyanidin B2 binds to NDUFA7 protein to achieve the prevention and / or treatment of photodamage to the skin; the amino acid sequence of the NDUFA7 protein is as shown in SEQ ID No: 1; or an amino acid sequence having at least 90% or more homology with the amino acid shown in SEQ ID No: 1.

[0009] In this invention, the amino acid sequence shown in SEQ ID No: 1 is: MASATRLIQRLRNWASGHDLQGKLQLRYQEISKRTQPPPKLPVGPSHKLSNNYYCTRDGRRESVPPSIIMSSQKALVSGKPAESSAVAATEKKAVTPAPPIKRWELSSDQPYL.

[0010] In a specific embodiment of the present invention, the Uniprot ID of the amino acid sequence of the NDUFA7 protein is O95182.

[0011] UV irradiation of the skin leads to an increase in reactive oxygen species (ROS), affecting both the ROS-NF-κB and Nrf2 pathways, thereby causing cell or skin damage. This invention discovers that proanthocyanidins bind to NDUFA7, stabilizing the activity of NADH:ubiquinone oxidoreductase in mitochondria. The presence of this enzyme reduces ROS production, thus targeting both the ROS-NF-κB and Nrf2 pathways and reducing cell / skin damage. In this invention, the red rice extract also achieves binding to NDUFA7 through its proanthocyanidin B2 content.

[0012] In this invention, the proanthocyanidin B2 and the NDUFA7 protein are preferably bound by hydrogen bonds.

[0013] In this invention, the 102nd amino acid residue of the NDUFA7 protein is preferably a lysine residue. In a specific embodiment of this invention, the proanthocyanidin B2 or the proanthocyanidin B2 in the red rice extract binds to the lysine residue at the 102nd position of the amino acid sequence shown in SEQ ID No: 1.

[0014] In this invention, the red rice extract preferably contains at least 5 μM of proanthocyanidin B2.

[0015] In this invention, the red rice extract preferably further includes at least other polyphenolic compounds, phenolic acid compounds, flavonoids, flavonols, flavonealkyls, and coumarin compounds; optionally, the phenolic acid compounds include caffeic acid, ferulic acid, and p-coumaric acid; optionally, the flavonoid compounds include apigenin; optionally, the flavonols include quercetin; optionally, the flavonealkyls include naringenin.

[0016] In the red rice extract, polyphenolic compounds preferably account for more than 6% of the total mass of the red rice extract, such as 10%, 20%, 25%, 30%, 40%, or 50%, and more preferably 20%-30% of the total mass of the red rice extract. The polyphenolic compounds include proanthocyanidins B2.

[0017] In this invention, the preferred method for preparing the red rice extract includes the following steps: red rice is extracted with an aqueous solution of alcohol, and the supernatant is collected by solid-liquid separation; wherein, the substance remaining after removing the solvent from the supernatant is the red rice extract; the alcohol may include one or more of monohydric alcohols, dihydric alcohols, and trihydric alcohols. It is expected by those skilled in the art that the substance remaining after removing the solvent from the supernatant is typically a solid.

[0018] The red rice is preferably red rice bran. The D50 particle size of the red rice bran is preferably 20-100 micrometers.

[0019] The monohydric alcohol may be an alkyl monohydric alcohol, preferably ethanol.

[0020] The diol may be an alkyl diol, preferably propylene glycol and / or butanediol; wherein the propylene glycol is preferably 1,2-propanediol.

[0021] The preferred mass ratio of the red rice to the aqueous solution of the alcohol is 1:(4-19), for example, 1:5 or 1:6.

[0022] The alcohol concentration in the aqueous solution is preferably 30-80%, for example 40%, 50%, 60% or 70%.

[0023] The extraction temperature is preferably 35-55℃.

[0024] The extraction time is preferably 8-13.0 hours.

[0025] The extraction method is stirring extraction. The stirring speed is preferably 150-200 rpm.

[0026] The solid-liquid separation is preferably performed by centrifugation.

[0027] The supernatant is preferably the supernatant after membrane filtration. The membrane filtration is to fully remove residual particulate matter. The filter membrane size can be conventional in the art, generally 0.22-8.00 μm, preferably 0.50 μm.

[0028] In the supernatant, the mass content of the red rice extract is preferably 0.5% or more, more preferably 0.5%-1%, for example 0.7%. Those skilled in the art will understand that the mass content of the red rice extract can be expressed as the solid content in the supernatant.

[0029] In this invention, the concentration of proanthocyanidin B2 in the drug or cosmetic is preferably 5-100 μM, more preferably 60 μM.

[0030] In this invention, the skin photodamage is preferably caused by ultraviolet and high-energy visible light irradiating the skin, more preferably by medium-wave ultraviolet light. The medium-wave ultraviolet light refers to ultraviolet light in the 280-320 nm wavelength range.

[0031] Positive and progressive effects: This invention discovers that proanthocyanidin B2 and its contained red rice extract can bind with high affinity to NDUFA7, thereby stabilizing the structure of NDUFA7 to regulate the activity of NADH: ubiquinone oxidoreductase (complex I) and further reducing ROS production, ultimately achieving the effect of repairing photodamage to the skin. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the docking of proanthocyanidin B2 with the NDUFA7 target molecule.

[0033] Figure 2 The image shows the results of LC-MS detection of proanthocyanidin B2 in red rice extract.

[0034] Figure 3 This study validated the targeted binding of red rice extract or proanthocyanidin B2 to NDUFA7 protein using cellular thermal displacement analysis-mass spectrometry.

[0035] Figure 4 Figure showing the detection results of different concentrations of proanthocyanidin B2 in alleviating UVB-induced HaCaT cell damage.

[0036] Figure 5 Figure showing the detection results of different concentrations of proanthocyanidin B2 in reducing UV-induced ROS in HaCaT cells.

[0037] Figure 6 Figure showing the detection results of red rice extract reducing UV-induced ROS in HaCaT cells. Detailed Implementation

[0038] To make the technical means, inventive features, objectives, and effects of the invention readily understandable, the invention is further described below with reference to specific illustrations and embodiments. However, the invention is not limited to the embodiments described below.

[0039] Example 1: Red Rice Extract Take a clean beaker and weigh 10g of the above-mentioned red rice bran plant powder. Grind the red rice bran plant powder to a D50 particle size of 20-100μm. Place the ground red rice bran plant powder in the beaker, add 50g of a 70% aqueous solution of butanediol, seal the beaker, and extract by stirring at 35-55℃ for 8-13 hours at a stirring speed of 150-200rpm. Centrifuge to separate the solid and liquid. Filter the supernatant through a 0.50μm filter membrane to obtain the plant extract, then concentrate under reduced pressure to one-third of its volume, filter through a 0.5μm filter membrane, and prepare a red rice extract solution with water (total solvent amount remains 50g). In this example, the alcohol in the 50g 70% aqueous solution of butanediol can be replaced with alkyl monohydric alcohols and / or other alkyl dihydric alcohol solvents.

[0040] The solid content of the plant extract was found to be 0.7% (w / w), meaning that 1 mL of the plant extract contained 7 mg of red rice extract, and the density of the plant extract was approximately 1 g / mL.

[0041] Example 1: Component confirmation of red rice extract The components in the red rice extract obtained in Example 1 were detected using LC-MS / MS. LC-MS data were processed using MS-DIAL (version 4.70) for feature extraction and peak area quantification. Metabolite identification was performed by using retention time, mass-to-charge ratio (m / z), isotopic mode, and MS / MS spectral similarity matching reference databases. The identification of proanthocyanidin B2 was taken as an example. Figure 2 The extractive ion chromatogram (EIC) showed a distinct characteristic peak at a retention time of 6.616 min. Figure 2 A). By mirroring the secondary mass spectra acquired in the experiment with reference spectra in the database (detection value vs. reference value), Figure 2 B), the main characteristic fragment ions showed a high degree of agreement. Among them, the molecular ion peak m / zThe concentrations 577.1394 and the characteristic monomeric fragment ion m / z 289.0718 were consistent with the standard reference spectrum (m / z 577.1335, 289.0710), confirming that the component was proanthocyanidin B2. Further analysis identified 210 components, and Table 1 lists the main active ingredients detected in the red rice extract.

[0042] Table 1 Example 2: Model Prediction of Target Points and Validation Method SCOPE-DTI prediction is a practical application of deep learning-based drug-target interaction (DTI prediction). By constructing the largest semi-inductive human DTI dataset to date, it is possible to rapidly predict the protein targets of small molecules by inputting their structures (Yigang Chen et al., Nat Commun, 2025).

[0043] For the 210 ingredients, SCOPE-DTI was used for prediction. Since the target of red rice extract and proanthocyanidin B2 overlaps, proanthocyanidin B2 was ultimately selected. Its target is the NDUFA7 protein.

[0044] The amino acid sequence of the NDUFA7 protein used in this efficacy example is the amino acid sequence shown in SEQ ID No: 1: MASATRLIQRLRNWASGHDLQGKLQLRYQEISKRTQPPPKLPVGPSHKLSNNYYCTRDGRRESVPPSIIMSSQKALVSGKPAESSAVAATEKKAVTPAPPIKRWELSSDQPYL.

[0045] CETSA-MS verification: Experimental methods: A mixed cell system of human keratinocytes (HaCaT), human skin fibroblasts (HSF), and human melanoma cells (MNT-1) was cultured. The pellet was collected and resuspended in PBS containing 1% protease inhibitor. The cells were lysed by three rapid freeze-thaw cycles in liquid nitrogen, centrifuged at 20,000g for 20 minutes at 4°C, and the supernatant was collected. The concentration was measured by the BCA method and diluted to 1 mg / mL.

[0046] The cell lysates were treated with 10 µmol / L proanthocyanidin B2, red rice extract solution (concentration 0.01% (w / v)) or DMSO for 10 minutes, respectively. Sera-Mag carboxyl-modified magnetic particles were added at a mass ratio of 5:1, and the mixture was incubated at 52°C for 3 minutes and then cooled. The magnetic beads were separated and washed, and then alkylated and digested with trypsin.

[0047] Peptide samples were analyzed using label-free quantitative proteomics by SWATH-MS on an Ekspert nanoLC 400 and ABSciex TripleTOF 6600 plus system. After gradient elution on a C18 column for 90 minutes, LC-MS / MS analysis was performed. Raw data were processed using DIA-NN 1.9.1 software in conjunction with a human protein library, and differential expression analysis was performed using Limma software.

[0048] CETSA-MS experimental results: Based on the predicted target of proanthocyanidin B2 in the red rice extract of Example 1, which is NDUFA7, experiments were conducted to verify whether they could bind. CETSA-MS confirmed that both the red rice extract and proanthocyanidin B2 could bind to the NDUFA7 target. The verification results are as follows: Figure 3 As shown.

[0049] Figure 3 Results of thermostability experiments for NDUFA7 protein: Figure 3 Results A showed that the control group ( Figure 3 The melting temperature (Tm) of NDUFA7 protein in the control group (A) was 48.5℃, while that obtained from red rice extract ( Figure 3 After treatment with the TPFD group in group A, the Tm was significantly increased to 56.8℃ (ΔTm=8.3℃), and the difference was statistically significant. This result indicates that the active ingredient in red rice extract can bind to NDUFA7 and increase Tm by stabilizing its protein structure. Figure 3 Results B showed that the Tm of NDUFA7 protein in the control group was 48.5℃, while that of NDUFA7 protein after proanthocyanidin B2 (… Figure 3 After treatment with group B2 (of B), the Tm of NDUFA7 protein was significantly increased to 61.0℃ (ΔTm=12.5℃), and the difference was statistically significant. This result indicates that proanthocyanidin B2 can bind to NDUFA7 and increase its Tm by stabilizing its protein structure. The increase in Tm of NDUFA7 protein by both red rice extract and proanthocyanidin B2 suggests that the binding of proanthocyanidin B2 to NDUFA7 is more conducive to the stability of the NDUFA7 protein.

[0050] Molecular docking: Molecular docking experiments were conducted using AutoDock Vina. The results are as follows: Figure 1As shown, the docking cascade area is marked in orange. Proanthocyanidin B2 can bind to NDUFA7 protein with high affinity, with a binding energy of −9.0 kcal / mol, indicating that proanthocyanidin B2 and NDUFA7 can spontaneously form a stable complex in the in vitro molecular simulation system, and the binding conformation is highly plausible. LigPlot analysis shows that this binding stability is partly due to the hydrogen bond formed between proanthocyanidin B2 and the lysine residue at position 102 of the NDUFA7 subunit.

[0051] Example 3: Experiment on the reduction of UVB-induced HaCaT cell damage by proanthocyanidin B2 Experimental method: HaCaT cells in the logarithmic growth phase were injected at a dose of 1×10⁻⁶. 5 / wells were inoculated into 96-well plates and cultured. When the plate was approximately 80% covered, the culture medium was washed off, and different concentrations of proanthocyanidin B2 / VC were added and incubated for 24 h (as shown in Table 2 below). After removing the proanthocyanidin B2 / VC, 1×PBS was added, and 15 mJ / cm² was used as the incubator. 2 Cells were irradiated with UVB and cultured for 24 hours after irradiation. Cell viability was then assessed using the CCK8 assay after 24 hours.

[0052] Relative cell viability (%) = A 实验组 / A 对照组 *100%, A refers to the absorption intensity at 450 nm.

[0053] Perform a t-test analysis. P>0.05 is considered as no significant difference, P<0.05 is marked as "*", and P<0.01 is marked as "**". Significant and highly significant differences are statistically significant.

[0054] Table 2 HaCaT cells were pretreated with different concentrations of proanthocyanidin B2 to assess whether proanthocyanidin B2 (PB2) has a protective effect against UVB-induced photodamage. Figure 4 As shown, the results revealed that the survival rate exhibited a gradual increasing trend (from 38%→60%→69%), reaching its peak at 60 μM, and then stabilizing at 100 μM. This indicates that proanthocyanidin B2 begins to exert a protective effect and exhibits a dose-response relationship; furthermore, in the UVB irradiation cell model, the cell survival rate reached ~69% at 60 μM proanthocyanidin B2. This demonstrates that proanthocyanidin B2 is an effective UVB protectant, exhibiting photoprotective effects on HaCaT cells within the tested range of 5-100 μM, with the best effect observed at 60 μM.

[0055] Example 4: Experiment on the reduction of UVB-induced ROS in HaCaT cells by proanthocyanidin B2 Experimental methods: HaCaT cells were seeded in 12-well plates and cultured until the plate formation rate reached 40%. Different concentrations of proanthocyanidin B2 and positive control vitamin C were added for 30 min. The culture medium was then removed, and 1×PBS was added. The solution was applied at 15 mJ / cm². 2 After UVB irradiation, DCFHDA was added and the mixture was incubated at 37°C for 30 min. The mixture was then washed with 1×PBS, and 1×PBS was added to each well. The images were taken at 488 nm using an inverted fluorescence microscope, and the mean fluorescence intensity (MFI) was analyzed using ImageJ, as shown in Table 3.

[0056] The inhibition rate is calculated using the following formula: Inhibition rate = 100% * (model group - sample group) / (model group - control group).

[0057] Table 3 like Figure 5 As shown, proanthocyanidin B2 at concentrations of 20, 60, and 100 μM can alleviate UV-induced ROS production in HaCaT cells. Calculations showed that the inhibition rates of UVB-induced ROS production in HaCaT cells at 20, 60, and 100 μM proanthocyanidin B2 were 152.26%, 175.01%, and 68.57%, respectively. Proanthocyanidin B2 exhibited inhibitory effects on ROS in HaCaT cells within the tested range of 20-100 μM, with the best effect observed at 60 μM.

[0058] Example 5: Experiment on the reduction of UV-induced ROS in HaCaT cells by red rice extract. Experimental methods: HaCaT cells were seeded in 12-well plates and cultured until the plate formation rate reached 40%. Different concentrations of red rice extract from Example 1 and positive control VC were added for 2 hours. The culture medium was removed, 1×PBS was added, and the plates were irradiated with ultraviolet light. DCFHDA was added and the plates were incubated at 37°C for 30 min. The plates were washed with 1×PBS, and 1×PBS was added to each well. The plates were photographed at 488 nm using an inverted fluorescence microscope, and the mean fluorescence intensity (MFI) was analyzed using ImageJ.

[0059] The inhibition rate is calculated using the following formula: Inhibition rate = 100% * (model group - sample group) / (model group - control group).

[0060] Experimental results are as follows Figure 6 As shown, the red rice extract in Example 1 can reduce ROS produced by UV-induced HaCaT cells. Figure 6 The inhibition rates of red rice extract at concentrations of 10, 100, and 1000 ppm (solid content) were 78.74%, 76.83%, and 87.54%, respectively.

[0061] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. Use of procyanidin B2 or a red rice extract containing the same in the preparation of a medicine or a cosmetic for preventing and / or treating skin photo-damage, characterized in that, The procyanidin B2 is combined with the NDUFA7 protein to achieve the prevention and / or treatment of the skin photo-damage.

2. Use of procyanidin B2 or a red rice extract containing the same in the preparation of a medicine or a cosmetic for preventing and / or treating skin photo-damage, characterized in that, The procyanidin B2 is combined with the NDUFA7 protein to achieve the prevention and / or treatment of the skin photo-damage. The amino acid sequence of the NDUFA7 protein is as shown in SEQ ID No: 1 or an amino acid sequence homologous to the amino acid sequence shown in SEQ ID No: 1 by at least 90% or above.

3. Use according to claim 1 or 2, characterized in that, The amino acid sequence of the NDUFA7 protein is as shown in SEQ ID No: 1 or an amino acid sequence homologous to the amino acid sequence shown in SEQ ID No: 1 by at least 90% or above.

4. The use according to claim 2, characterized in that, The procyanidin B2 is combined with the lysine residue at the 102nd position of the amino acid sequence shown in SEQ ID No: 1, or the procyanidin B2 in the red rice extract is combined with the lysine residue at the 102nd position of the amino acid sequence shown in SEQ ID No:

1.

5. Use according to claim 1 or 2, characterized in that, The red rice extract contains at least 5 μM of procyanidin B2.

6. Use according to claim 1 or 2, characterized in that, The red rice extract further contains at least other polyphenol compounds, phenolic acid compounds, flavonoid compounds, flavonol compounds, flavan compounds and coumarin compounds. Optionally, the phenolic acid compounds include caffeic acid, ferulic acid and p-coumaric acid. Optionally, the flavonoid compounds include apigenin; optionally, the flavonol compounds include quercetin; and optionally, the flavan compounds include naringenin.

7. Use according to claim 1 or 2, characterized in that, The preparation method of the red rice extract comprises the following steps: after the red rice is extracted by an aqueous alcohol solution, the supernatant is collected by solid-liquid separation; and the substance obtained by removing the solvent from the supernatant is the red rice extract.

8. Use according to claim 1 or 2, characterized in that, The concentration of the procyanidin B2 in the medicine or the cosmetic is 5-100 μM, preferably 60 μM.

9. Use according to claim 1 or 2, characterized in that, The skin photo-damage is caused by the irradiation of the skin by ultraviolet rays and high-energy visible light.

10. Use according to claim 1 or 2, characterized in that, The skin photo-damage is caused by the irradiation of the skin by medium-wave ultraviolet rays and high-energy visible light.

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