Application of pinolenic acid methyl ester in preparation of anti-inflammatory and / or whitening products and extraction method of pinolenic acid methyl ester

Abstract

The invention provides application of pinolenic acid methyl ester in preparation of anti-inflammatory and / or whitening products and an extraction method of the pinolenic acid methyl ester, and belongs to the technical field of biological medicine. Purified Pinolenic acid methyl ester is extracted from Korean pine seed oil, and the Pinolenic acid methyl ester has excellent anti-inflammatory and whitening activity. An inflammatory factor release inhibition experiment shows that the methyl pinolenic acid ester can effectively inhibit LPS-induced RAW264.7 cell NO secretion and inflammatory mediator IL-6 and TNF-alpha production, the activity of the methyl pinolenic acid ester is superior to that of pinolenic acid, and even part of the activity of the methyl pinolenic acid ester is superior to that of potent anti-inflammatory glucocorticoid dexamethasone. Whitening activity experiments show that the methyl pinolenic acid ester also has better activity in the aspect of inhibiting the generation of melanin of mouse skin melanoma cells (B16F10). In a skin irritation experiment, the Korean pine seed oil non-saponifiable matter containing the pinolenic acid methyl ester has no irritation to the skin of the white rabbits. Therefore, the pinolenic acid methyl ester has certain potential in the aspect of being developed into whitening and anti-inflammatory products.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of methyl pinolelate in the preparation of anti-inflammatory and / or whitening products and its extraction method. Background Technology

[0002] The skin is the body's external barrier, frequently exposed to environmental stimuli, leading to a variety of skin problems. Inflammation is the root cause of many skin issues, such as acne, eczema, and dermatitis. Inflammatory responses not only directly cause skin inflammation but also activate melanocytes, promoting melanin synthesis and resulting in pigmentation. Therefore, inflammation management is a crucial perspective in skin management. By reducing inflammation, promoting skin repair, and strengthening the skin barrier function, various skin problems can be effectively improved, enhancing overall skin health.

[0003] Korean pine( Pinus koraiensis Korean pine (Sieb. et Zucc.), also known as sea pine or red pine, is an evergreen coniferous tree belonging to the Pinaceae family. It is an important economic forest tree in the Changbai Mountains, Jilin Mountains, and Lesser Khingan Mountains of China. The seeds of the Korean pine are called Korean pine nuts. The Korean pine nut oil obtained from it contains over 90% unsaturated fatty acids, including pinolenic acid (molecular formula C...). 18 H 30 O2, also known as terpineic acid (with the structural formula cis-5,9,12-octadecanoic acid), is a major Δ-5 unsaturated fatty acid unique to pine plants and their oils. It has physiological functions such as weight loss, lowering blood lipids, enhancing immunity, anti-oxidation, and enhancing insulin sensitivity. It is a new functional factor that has attracted the attention of the medical community in recent years.

[0004] Recently, under the trend of "health and wellness," functional cosmetics based on natural plants have gained popularity among consumers. Therefore, the development of cosmetics based on the active ingredients in red pine nuts has broad prospects. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide the application of methyl pinolenate in the preparation of anti-inflammatory and / or whitening products and its extraction method. The methyl pinolenate extracted and purified from red pine seed oil according to this invention exhibits excellent dual anti-inflammatory and whitening activities.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of methyl pinolenate in the preparation of anti-inflammatory and / or whitening products, wherein the methyl pinolenate has the structure shown in Formula I: Formula I.

[0007] Preferably, the product includes cosmetics or pharmaceuticals.

[0008] Preferably, the pinolenic acid methyl ester is extracted and purified from Korean pine seed oil, and the extraction and purification method comprises the following steps: The Korean pine seed oil is extracted with anhydrous methanol to obtain an anhydrous methanol part; The anhydrous methanol part is subjected to first silica gel column chromatography to obtain seven fraction segments, which are sequentially recorded as segments A, B, C, D, E, F and G in the order of component outflow; The segment E is subjected to reverse phase column chromatography to obtain eight components, which are sequentially recorded as components E1, E2, E3, E4, E5, E6, E7 and E8 in the order of component outflow; The component E3 is subjected to second silica gel column chromatography to obtain pinolenic acid methyl ester having the structure shown in Formula I.

[0009] Preferably, the volume ratio of the Korean pine seed oil to anhydrous methanol is 1:1-3; The extraction is multiple extraction, and the anhydrous methanol phases after each extraction are combined to obtain the anhydrous methanol part.

[0010] Preferably, the mesh number of the stationary phase silica gel in the first silica gel column chromatography is 200-300 mesh; The elution mode of the first silica gel column chromatography is gradient elution, and the eluent used is petroleum ether and ethyl acetate; in the gradient elution, the volume ratio of petroleum ether to ethyl acetate changes from 1:0 to 0:1.

[0011] Preferably, the fractions in the first silica gel column chromatography process are subjected to first thin layer chromatography identification, and the same components are combined; The developing agent of the first thin layer chromatography is petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to ethyl acetate is 5:1.

[0012] Preferably, the chromatographic column for the reverse phase column chromatography is an ODS medium pressure column; The elution mode of the reverse phase column chromatography is gradient elution, and the eluent used is methanol and water; in the gradient elution, the volume content of methanol changes from 70% to 100% with a change rate of 10%.

[0013] Preferably, the fractions in the reverse phase column chromatography process are subjected to second thin layer chromatography identification, and the same components are combined; The developing agent of the second thin layer chromatography is petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to ethyl acetate is 5:1.

[0014] Preferably, the mesh number of the stationary phase silica gel in the second silica gel column chromatography is 200-300 mesh; The elution mode of the second silica gel column chromatography separation is gradient elution, and the eluent used is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether and ethyl acetate in the gradient elution is 99:1 isocratic elution.

[0015] The application provides a method for extracting and purifying pinolenate methyl ester from Korean pine seed oil. The Korean pine seed oil is extracted by using anhydrous methanol to obtain an anhydrous methanol part; The anhydrous methanol part is subjected to first silica gel column chromatography separation to obtain seven fraction segments, which are sequentially recorded as segments A, B, C, D, E, F and G according to the flow order of components; The segment E is subjected to reverse phase chromatography column separation to obtain eight components, which are sequentially recorded as components E1, E2, E3, E4, E5, E6, E7 and E8 according to the flow order of components; The E3 component is subjected to second silica gel column chromatography separation to obtain pinolenate methyl ester with the structure shown in formula I.

[0016] The application provides application of pinolenate methyl ester in preparation of anti-inflammatory and / or whitening products, and the pinolenate methyl ester has the structure shown in formula I. The pinolenate methyl ester extracted and purified from Korean pine seed oil is a methyl ester of pinolenate, has better stability, solubility and permeability, and has excellent anti-inflammatory and whitening activities. Through inhibition of inflammatory factor release experiment, it is found that pinolenate methyl ester can effectively inhibit the secretion of NO and the production of inflammatory mediators IL-6 and TNF-alpha of RAW264.7 cells induced by LPS, and the activity is better than that of pinolenate, and even part of the activity is better than that of strong anti-inflammatory glucocorticoid dexamethasone. Through whitening activity experiment, it is found that the compound pinolenate methyl ester also has better activity in inhibiting the generation of melanin of mouse skin melanoma cells (B16F10). In addition, in the skin irritation experiment, the non-saponified substance of Korean pine seed oil containing pinolenate methyl ester has no irritation to the skin of white rabbits. Therefore, the compound pinolenate methyl ester has certain potential in the development of whitening and anti-inflammatory products.

[0017] The application provides a method for extracting and purifying pinolenate methyl ester from Korean pine seed oil, and the pinolenate methyl ester is finally obtained through first silica gel column chromatography separation, reverse phase chromatography column separation and second silica gel column chromatography separation after the Korean pine seed oil is extracted by using anhydrous methanol to obtain an anhydrous methanol part. The extraction and purification method is simple and easy to operate. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Preparation of the compound pinolenate methyl ester for Example 1 1 H-NMR spectrum Figure 2The compound pinolenic acid methyl ester prepared in Example 1 13 C-NMR spectrum; Figure 3 The effect of different concentrations of the compound on the survival rate of RAW264.7 cells; Figure 4 The inhibitory effect of different concentrations of the compound on the release of NO induced by LPS in RAW264.7 cells; Figure 5 The statistical diagram of the inhibition of different compounds on the production of inflammatory mediators IL-6 and TNF-α in LPS-induced RAW264.7 cells. DETAILED DESCRIPTION

[0019] The present application provides the use of pinolenic acid methyl ester in the preparation of anti-inflammatory and / or whitening products, wherein the pinolenic acid methyl ester has the structure shown in Formula I: Formula I.

[0020] In the present application, the product preferably includes cosmetics or drugs.

[0021] In the present application, the pinolenic acid methyl ester is preferably extracted and purified from Korean pine seed oil, and the method for extracting and purifying pinolenic acid methyl ester from Korean pine seed oil in the present application comprises the following steps: The Korean pine seed oil is extracted with anhydrous methanol to obtain an anhydrous methanol part; The anhydrous methanol part is subjected to first silica gel column chromatography to obtain 7 fractions, which are sequentially recorded as A, B, C, D, E, F, and G fractions in the order of component outflow; The E fraction is subjected to reverse phase chromatography column separation to obtain 8 components, which are sequentially recorded as E1, E2, E3, E4, E5, E6, E7, and E8 components in the order of component outflow; The E3 component is subjected to second silica gel column chromatography to obtain pinolenic acid methyl ester having the structure shown in Formula I.

[0022] In the present application, the Korean pine seed oil is extracted with anhydrous methanol to obtain an anhydrous methanol part. In the present application, the volume ratio of the Korean pine seed oil to anhydrous methanol is preferably 1:1~3, and more preferably 1:1~2. In the present application, the extraction is multiple extractions, and the number of extractions is preferably 5~13, and more preferably 8~10. In the present application, the temperature of single extraction is preferably 20~25℃, and the time is preferably 30~60min, and more preferably 40~50min. In the present application, the anhydrous methanol phase is combined after each extraction to obtain the anhydrous methanol part. In the present application, the non-saponified substance of Korean pine seed oil can be extracted by anhydrous methanol extraction.

[0023] After obtaining the anhydrous methanol part, the present application carries out first silica gel column chromatography on the anhydrous methanol part to obtain 7 fraction segments, which are sequentially recorded as segments A, B, C, D, E, F and G in the order of component outflow. In the present application, the mesh number of the stationary phase silica gel in the first silica gel column chromatography is preferably 200-300 mesh, the column length is 1.2 m, and the inner diameter is 7.5 cm. In the present application, the sample loading mode of the first silica gel column chromatography is preferably as follows: the anhydrous methanol part is redissolved with anhydrous methanol, then 1.5 times the sample amount of silica gel is mixed with the sample, and then silica gel column chromatography is carried out.

[0024] In the present application, the elution mode of the first silica gel column chromatography is preferably gradient elution, and the eluent used is preferably petroleum ether and ethyl acetate; the volume ratio of petroleum ether and ethyl acetate in the gradient elution is preferably changed from 1:0 to 0:1, and is more preferably 1:0, 120:1, 100:1, 50:1, 20:1, 10:1, 5:1 and 0:1, and the flow rate is preferably 10-15 mL / min.

[0025] In the present application, the first thin layer chromatography is preferably used to identify the fractions during the first silica gel column chromatography, and the same components are combined; the developing agent of the first thin layer chromatography is preferably petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether and ethyl acetate is preferably 5:1.

[0026] After obtaining the E segment fraction segment, the present application carries out reverse phase chromatography column separation on the E segment to obtain 8 components, which are sequentially recorded as E1, E2, E3, E4, E5, E6, E7 and E8 in the order of component outflow. In the present application, the chromatography column for the reverse phase chromatography column separation is an ODS medium pressure column, the column length is 50 cm, and the inner diameter is 4 cm. In the present application, the sample loading mode of the reverse phase chromatography separation is preferably as follows: the E segment is redissolved with anhydrous methanol, then 1.5-2.0 times the sample amount of reverse phase silica gel is mixed with the sample, and then reverse phase silica gel column chromatography is carried out.

[0027] In the present application, the elution mode of the reverse phase chromatography column separation is preferably gradient elution, and the eluent used is preferably methanol and water; the volume ratio of methanol and water in the gradient elution is preferably changed from 70% to 100% with a change rate of 10%, and is more preferably 70%, 80%, 90% and 100%.

[0028] In the present application, the second thin layer chromatography is preferably used to identify the fractions during the reverse phase chromatography column separation, and the same components are combined; the developing agent of the second thin layer chromatography is preferably petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether and ethyl acetate is preferably 5:1.

[0029] After obtaining the E3 component, the present application performs second silica gel column chromatography on the E3 component to obtain pinolenic acid methyl ester with the structure shown in Formula I. In the present application, the mesh number of the stationary phase silica gel in the second silica gel column chromatography is preferably 200-300 mesh, the column length is preferably 45 cm, and the inner diameter is preferably 1.5 cm.

[0030] In the present application, the elution mode of the second silica gel column chromatography is preferably gradient elution, and the eluent used is preferably petroleum ether and ethyl acetate; in the gradient elution, the volume of petroleum ether and ethyl acetate is preferably 99:1 isocratic elution.

[0031] The present application provides a method for extracting and purifying pinolenic acid methyl ester from Korean pine seed oil, comprising the following steps: The Korean pine seed oil is extracted with anhydrous methanol to obtain an anhydrous methanol part; The anhydrous methanol part is subjected to first silica gel column chromatography to obtain 7 fractions, which are sequentially recorded as A, B, C, D, E, F, and G fractions according to the order of component outflow; The E segment is subjected to reverse phase chromatography column separation to obtain 8 components, which are sequentially recorded as E1, E2, E3, E4, E5, E6, E7, and E8 components according to the order of component outflow; The E3 component is subjected to second silica gel column chromatography to obtain pinolenic acid methyl ester with the structure shown in Formula I.

[0032] In the present application, the specific method for extracting and purifying pinolenic acid methyl ester from Korean pine seed oil is the same as above, and will not be repeated here.

[0033] The application of pinolenic acid methyl ester in preparing anti-inflammatory and / or whitening products and its extraction method will be described in detail below with examples, but they should not be understood as limiting the scope of protection of the present application.

[0034] Example 1 The method for extracting and purifying pinolenic acid methyl ester from Korean pine seed oil adopts the following steps: (1) Take Korean pine seed oil and extract it with anhydrous methanol, the volume ratio of Korean pine seed oil to anhydrous methanol is 1:1, the extraction temperature is 24℃, and the time is 1h; repeat 13 times, combine the anhydrous methanol phase to obtain an anhydrous methanol part.

[0035] (2) Take 218.0 g of anhydrous methanol fraction and redissolve it in anhydrous methanol. Mix the sample with 200-300 mesh silica gel (1.5 times the sample amount). After mixing, perform the first silica gel column chromatography separation. The column length is 1.2 m and the inner diameter is 7.5 cm. The eluent is petroleum ether and ethyl acetate. The volume ratio of petroleum ether to ethyl acetate varies from 1:0 to 0:1, specifically 1:0, 120:1, 100:1, 50:1, 20:1, 10:1, 5:1 and 0:1, which are eight gradients. Thin-layer chromatography (TLC) is used for identification. The developing solvent is petroleum ether and ethyl acetate. The volume ratio of petroleum ether to ethyl acetate is 5:1. Seven fractions are obtained. According to the elution order of the components, they are named A, B, C, D, E, F and G.

[0036] (3) Take 8.0 g of segment E and pass it through an ODS medium-pressure column (column length 50 cm, inner diameter 4 cm) for reversed-phase chromatography separation. The eluent is methanol and water, with the volume ratio of methanol to water ranging from 70% to 100%, a change rate of 10%, specifically 70%, 80%, 90%, and 100%, ultimately yielding eight components: E1, E2, E3, E4, E5, E6, E7, and E8. During the reversed-phase chromatography separation process, the fractions are identified by a second thin-layer chromatography method, and identical components are combined. The developing solvent for the second thin-layer chromatography is petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 5:1.

[0037] (4) The E3 component was separated and purified by silica gel column chromatography for the second time. The silica gel particle size was 200-300 mesh, the column length was 45 cm, the inner diameter was 1.5 cm, and the eluent was petroleum ether and ethyl acetate. The volume ratio of petroleum ether and ethyl acetate was 99:1 for isocratic elution to obtain methyl pinolenic acid with the structure shown in Formula I.

[0038] The obtained methyl pinolenate 1 H-NMR spectrum as follows Figure 1 As shown, 13 C-NMR spectrum as follows Figure 2 As shown.

[0039] Test Example 1: In vitro anti-inflammatory activity experiment In this experiment, the Griess method was used to detect the inhibitory effect of the target compound on the production of the inflammatory mediator NO in a mouse monocyte / macrophage RAW264.7 inflammation model induced by LPS lipopolysaccharide, and to evaluate the anti-inflammatory effect of the compound. The test compounds were methyl pinolenate and pinolenic acid prepared in Example 1.

[0040] The experimental steps are as follows: Cell culture: RAW264.7 cells in the logarithmic growth phase were scraped off using a cell scraper and suspended in high-glucose DMEM complete culture medium containing 10% fetal bovine serum to form a single-cell suspension. The suspension was cultured at 1.0 × 10⁻⁶ cells / cells. 5Seeds were inoculated into 96-well plates at a density of 100 μL per well and cultured overnight.

[0041] Add the test compound: Induce stimulation with 1 μg / mL LPS, and simultaneously add the test compound (concentrations set at 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM). Set up a drug-free group (and model group), a normal group (without LPS), and a positive control group (dexamethasone, 10 μM). Each treatment was performed in triplicate and incubated at 37°C with 5% CO2 for 24 hours.

[0042] Detection of inflammatory mediators: Take 50 μL of cell culture supernatant and use the Griess kit to detect NO production, and read the absorbance at 540 nm.

[0043] Cell viability assay: Discard the culture medium in each well, add a 4:1 mixture of culture medium and MTS (100 μL per well), and set up 3 blank replicates. Incubate for 2–4 hours and read the absorbance at 490 nm. Calculate the cell viability using the following formula.

[0044] Survival rate % = (OD value of experimental group - OD value of blank group) / (OD value of negative group - OD value of blank group) × 100%.

[0045] The effects of different concentrations of compounds on the survival rate of RAW264.7 cells, such as Figure 3 As shown.

[0046] The inhibitory effects of different concentrations of compounds on LPS-induced NO release in RAW264.7 cells were as follows: Figure 4 As shown.

[0047] Different compounds inhibit LPS-induced NO release from RAW264.7 cells in ECMO cells 50 The values ​​are shown in Table 1.

[0048] Table 1. Different compounds inhibiting LPS-induced NO release from RAW264.7 cells in ECMO cells 50 value

[0049] Experimental results showed that, within the concentration range of 1–100 μM, methyl pinolenate had no significant cytotoxicity to RAW264.7 cells and could inhibit LPS-induced NO production in a dose-dependent manner, with better inhibitory effects than pinolenate and comparable to the positive control (dexamethasone).

[0050] The effects of the compound on the release of inflammatory factors were further investigated. Using dexamethasone as a positive control (10 μM), the effects of the compound (20 μM) on the levels of the inflammatory factors IL-6 and TNF-α in LPS-induced RAW 264.7 cells were measured. (Table 2 and...) Figure 5 Results of different compounds inhibiting LPS-induced production of inflammatory mediators IL-6 and TNF-α in RAW264.7 cells.

[0051] Table 2. Different compounds inhibit LPS-induced inflammatory mediators IL-6 and TNF-α in RAW264.7 cells. α The results (compared to the control group) ### p<0.001; compared with the LPS group, p<0.01, *p<0.001)

[0052] Experimental results show that, compared with the LPS model group, the methyl pinolenic acid extracted from red pine nut oil in this invention can effectively inhibit the release of LPS-induced IL-6 and TNF-α, which is superior to pinolenic acid. In terms of inhibiting the release of LPS-induced IL-6, it is more effective than dexamethasone (10 μM).

[0053] Test Example 2: Whitening Activity Experiment The cells used in this test case were B16F10 mouse skin melanoma cells, and the compounds to be tested were methyl pinolenic acid prepared in Example 1 and the compound pinolenic acid.

[0054] The experimental equipment and main reagents used in this embodiment are shown in Table 3.

[0055] Table 3 Experimental Equipment and Reagents

[0056] The experimental steps are as follows: Cell culture: Mouse skin melanoma B16F10 cells were cultured in 1640 medium containing 10% FBS and 100 U / mL antibiotic solution, and placed in a 37°C, 5% CO2 cell culture incubator. When the cells reached 90% confluence, they were digested with trypsin containing 0.25% EDTA and passaged.

[0057] Cellular melanin content detection: B16F10 cells were processed at a ratio of 5 × 10⁻⁶. 4Cells were seeded at a density of 2 mL / mL in 6-well cells culture plates. After 24 hours, once the cells had adhered, they were divided into a blank control group, an α-MSH model group, an α-MSH positive control group (arbutin), and an α-MSH administration group, with three replicates per group. The blank control group was treated with DMEM medium containing 10% FBS, the model group with medium containing 100 nmol / L α-MSH (M=1664.9), the positive control group with 100 nmol / L α-MSH and 500 μg / mL arbutin, and the model + administration group with medium containing 100 nmol / L α-MSH and a final concentration of 50 μM, 2 mL per well. The cells were then incubated in a cell culture incubator for 72 hours. Collect the supernatant and transfer it to a 96-well plate. Measure the absorbance at 405 nm to compare the melanin secretion of the cells. Wash the cells twice with PBS, digest them with trypsin, and centrifuge at 900 rpm for 4 min, then collect them in centrifuge tubes. Add 400 μL of 1 mol / L NaOH solution containing 10% dimethyl sulfoxide to each sample to lyse the cells. Incubate at 80℃ for 1 h, then transfer 100 μL to each well of a 96-well plate. Measure the absorbance at 405 nm. Use the blank group as a control to compare the relative melanin content in the cells of each group.

[0058] Cellular melanin inhibition rate IC 50 Measurement: The IC50 inhibition rate of melanin in the drug-treated groups with similar results to the positive control group was measured. 50 Five drug concentrations were set for each sample: 2.5 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, and 40 μmol / L. The experimental procedure was the same as that for the detection of cell melanin content.

[0059] Data processing and analysis: Each sample was measured in triplicate, and the result was the average of the three parallel measurements. GraphPad Prism 9 statistical software was used for data analysis. Values ​​are expressed as mean ± standard deviation (±s). One-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0060] Table 4 shows the relative melanin content in the cell supernatant; Table 5 shows the relative melanin content in the precipitated cells; Table 6 shows the melanin inhibition rate (IC50). 50 value.

[0061] Table 4. Relative melanin content values ​​in cell supernatant

[0062] Table 5. Relative content of melanin deposited in cells

[0063] Table 6 Melanin Inhibition Rate IC 50 value

[0064] Experimental results show that the melanin content in cells of methyl pinolenate in the red pine seed oil provided by this invention is lower than that in the model group, and methyl pinolenate is comparable to the positive control arbutin, indicating that methyl pinolenate can inhibit the production of melanin in B16F10 cells.

[0065] Test Example 3: Skin Irritation Test The experimental animal used in this test case was the Japanese white rabbit, and the compound to be tested was the unsaponifiables of red pine nut oil (the anhydrous methanol fraction obtained by extracting red pine nut oil with anhydrous methanol in Example 1, which contains methyl pinolenic acid prepared in Example 1).

[0066] The experimental steps are as follows: Three Japanese white rabbits were used. Twenty-four hours before drug administration, all rabbits underwent hair removal (both sides of the spine) and disinfection. The hair removal area was 5 cm × 5 cm. For the damaged skin group, a crisscross pattern was made on the bald area using a sterile syringe needle, 1 mm deep, until slight bleeding occurred in the epidermis. Each incision was approximately 2 cm long and considered as damaged skin. The experiment used a self-comparison method between the left and right sides of the same animal. Two 5 cm × 5 cm areas were taken from each rabbit on both sides of the spine, resulting in four skin test areas per rabbit. The three Japanese white rabbits had a total of 12 skin test areas: 6 for intact skin and 6 for damaged skin. The intact skin areas were encapsulated with transparent film, covered with non-irritating gauze, and secured with tape. The test drug was removed with physiological saline 24 hours after drug administration. Local skin reactions such as erythema and edema, and their resolution, were observed and recorded at 1 h, 24 h, 48 h, and 72 h after drug removal.

[0067] Experimental results showed that after 72 hours of application of the unsaponifiables of red pine nut oil, the skin gloss L-value was measured using a WR colorimeter. The gloss L-values ​​of intact and damaged skin samples are shown in Table 7. It can be seen that the gloss L-values ​​(fair and smooth) of both intact and damaged skin samples were higher than those of the control group. Compared with the control group, there was no erythema or edema, indicating that it was non-irritating to the skin of rabbits. This demonstrates that the methyl pinoleate in the unsaponifiables of red pine nut oil provided in this invention is non-irritating to the skin of rabbits.

[0068] Table 7 Skin Gloss L-value

[0069] Application Example 1: Preparation of Whitening and Soothing Essence This application example provides an essence containing methyl pinolenate of the present invention or an oil or unsaponifiable matter containing methyl pinolenate. The specific formula of the essence is shown below, with the total mass fraction being 100%.

[0070] Essence Formula 1: Pinolenic acid methyl ester 0.1%, trehalose 1%, glycerin 3%, PEG-100 glyceryl stearate 0.5%, p-hydroxyacetophenone 0.5%, xanthan gum 0.1%, disodium EDTA 0.05%, balance deionized water.

[0071] Essence Formula 2: 0.2% unsaponifiables of red pine seed oil, 1% trehalose, 3% glycerin, 0.5% PEG-100 glyceryl stearate, 0.5% p-hydroxyacetophenone, 0.1% xanthan gum, 0.05% disodium EDTA, and the balance being deionized water.

[0072] Essence Formula 3: 1% red pine seed oil, 1% trehalose, 3% glycerin, 0.5% PEG-100 glyceryl stearate, 0.5% p-hydroxyacetophenone, 0.1% xanthan gum, 0.05% disodium EDTA, and the remainder is deionized water.

[0073] Nine volunteers of different ages were selected and divided into three groups (Group 1, Group 2, and Group 3), with three participants in each group. After cleansing their faces, the serums were applied to the facial skin once daily for three consecutive days. No skin irritation or allergic reactions were observed in any of the three groups, and all participants reported that the serums had good soothing and whitening effects.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of methyl pinolenate in the preparation of anti-inflammatory and / or whitening products, characterized in that, The methyl pinolenic acid has the structure shown in Formula I: Formula I.

2. The application according to claim 1, characterized in that, The products mentioned include cosmetics or pharmaceuticals.

3. The application according to claim 1, characterized in that, The methyl pinolenic acid is obtained by extraction and purification from red pine seed oil. The extraction and purification method includes the following steps: Red pine seed oil was extracted with anhydrous methanol to obtain anhydrous methanol fraction; The anhydrous methanol fraction was subjected to a first silica gel column chromatography separation to obtain 7 fractions, which were named A, B, C, D, E, F and G in the order of elution of the components. The E segment was separated by reversed-phase chromatography to obtain 8 components, which were named E1, E2, E3, E4, E5, E6, E7 and E8 in the order of elution. The E3 component was subjected to a second silica gel column chromatography separation to obtain methyl pinolenic acid with the structure shown in Formula I.

4. The application according to claim 3, characterized in that, The volume ratio of the red pine seed oil to anhydrous methanol is 1:1~3; The extraction process involves multiple extractions, with the anhydrous methanol phases combined after each extraction to obtain the anhydrous methanol fraction.

5. The application according to claim 3, characterized in that, The silica gel used as the stationary phase in the first silica gel column chromatography separation has a mesh size of 200-300 mesh. The first silica gel column chromatography separation was performed using gradient elution with petroleum ether and ethyl acetate as eluents; the volume ratio of petroleum ether to ethyl acetate varied from 1:0 to 0:1 during the gradient elution.

6. The application according to claim 3 or 5, characterized in that, It also includes performing a first thin-layer chromatography method to identify the fractions in the first silica gel column chromatography separation process and combining the same components; The developing solvent for the first thin-layer chromatography is petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate is 5:

1.

7. The application according to claim 3, characterized in that, The reversed-phase chromatography column used for separation is an ODS medium-pressure column; The elution method of the reversed-phase chromatographic column is gradient elution, and the eluent used is methanol and water; the volume content of methanol in the gradient elution changes from 70% to 100%, with a change rate of 10%.

8. The application according to claim 3 or 7, characterized in that, It also includes performing a second thin-layer chromatography method to identify the fractions in the separation process of the reversed-phase chromatographic column and to combine the same components; The developing solvent for the second thin-layer chromatography is petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate is 5:

1.

9. The application according to claim 3, characterized in that, The silica gel used as the stationary phase in the second silica gel column chromatography separation has a mesh size of 200-300 mesh. The second silica gel column chromatography separation uses gradient elution with petroleum ether and ethyl acetate as eluents, and the volume ratio of petroleum ether to ethyl acetate in the gradient elution is 99:1 isocratic elution.

10. A method for extracting and purifying methyl pinolenate from red pine nut oil, characterized in that, Includes the following steps: Red pine seed oil was extracted with anhydrous methanol to obtain anhydrous methanol fraction; The anhydrous methanol fraction was subjected to a first silica gel column chromatography separation to obtain 7 fractions, which were named A, B, C, D, E, F and G in the order of elution of the components. The E segment was separated by reversed-phase chromatography to obtain 8 components, which were named E1, E2, E3, E4, E5, E6, E7 and E8 in the order of elution. The E3 component was subjected to a second silica gel column chromatography separation to obtain methyl pinolenic acid with the structure shown in Formula I.

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