Compositions containing gamma-aminobutyric acid and natural small molecule compounds and their use in the preparation of products with anti-aging effects

Abstract

This invention relates to the field of biomedical technology, specifically disclosing a composition containing γ-aminobutyric acid (GABA) and a natural small molecule compound, and its application in the preparation of products with anti-aging effects. The natural small molecule compound has the structure shown in Formula I, Formula II, or Formula III. The composition containing GABA and the natural small molecule compound comprises GABA and any one or a mixture of more than one of the natural small molecule compounds with the structure shown in Formula I, Formula II, or Formula III. Studies have shown that the natural small molecule compound of this invention and the composition containing GABA and the natural small molecule compound have a series of effects, including anti-aging, whitening, anti-UVB exposure-induced photoaging, inhibition of tyrosinase activity, and inhibition of melanin and / or lipofuscin production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a composition containing γ-aminobutyric acid and natural small molecule compounds and application thereof in preparation of products with anti-aging effect. BACKGROUND

[0002] Melanin is a kind of dark or brown spots or patches appearing on exposed parts of the body such as face, back of hand and neck, also known as freckles, liver spots or dark spots. Ultraviolet radiation, aging and other factors can all lead to increased generation and deposition of melanin in the skin, and excessive deposition of melanin is also considered to be the main cause of the formation of skin spots such as age spots and freckles.

[0003] γ-aminobutyric acid is a neurotransmitter that can inhibit nerve transmission, regulate mood, fight anxiety and depression. Studies have shown that γ-aminobutyric acid can affect the formation and activity of lipofuscin through the signal transmission mechanism of the central nervous system, indirectly reducing melanin. In addition, γ-aminobutyric acid also has the effects of antioxidant, anti-inflammatory, promoting collagen synthesis and relaxing skin, and has excellent skin anti-aging effect. Polyphenol compound is a natural small molecule compound with multiple biological activities such as antioxidant and anti-inflammatory. Studies have shown that plant polyphenols can reduce the generation of melanin by inhibiting the activity of tyrosinase. In addition, plant polyphenols can also reduce the oxidative stress on melanocytes through antioxidant effect, and play an active role in inhibiting melanin synthesis.

[0004] At present, the treatment methods for skin age spots, freckles and the like mainly include external drugs, phototherapy and chemical exfoliation. Studies have found that although a variety of plant extracts are used in skin care products and have the activity of inhibiting or reducing melanin generation, factors such as complex composition of plant extracts and poor activity of inhibiting or reducing melanin generation greatly limit their application. Therefore, it is of great application value to develop active ingredients with the activity of inhibiting melanin generation from plant extracts. SUMMARY

[0005] In order to overcome at least one of the technical problems in the prior art, the present application provides a natural small molecule compound and a composition containing γ-aminobutyric acid and natural small molecule compounds.

[0006] The technical scheme of the present application is as follows:

[0007] The present application first provides a natural small molecule compound, which has the structure shown in formula I, formula II or formula III:

[0008]

[0009] Preferably, the composition containing γ-aminobutyric acid and natural small molecule compounds comprises γ-aminobutyric acid and any one or more than one of natural small molecule compounds with structures of Formula I, Formula II or Formula III.

[0010] Preferably, the composition containing γ-aminobutyric acid and natural small molecule compounds comprises γ-aminobutyric acid and natural small molecule compounds with structures of Formula I, Formula II and Formula III.

[0011] Further preferably, the mass ratio of γ-aminobutyric acid to natural small molecule compounds with structures of Formula I, Formula II and Formula III is 1: (1-100): (1-100): (1-100).

[0012] More preferably, the mass ratio of γ-aminobutyric acid to natural small molecule compounds with structures of Formula I, Formula II and Formula III is 1: (1-50): (1-50): (1-50).

[0013] Most preferably, the mass ratio of γ-aminobutyric acid to natural small molecule compounds with structures of Formula I, Formula II and Formula III is 1:0.5:2.4:3.9.

[0014] The application also provides use of the natural small molecule compound or the composition in preparation of a product with anti-aging and / or whitening effects.

[0015] The application also provides use of the natural small molecule compound or the composition in preparation of a product with anti-UVB exposure induced photoaging effects.

[0016] The application also provides use of the natural small molecule compound or the composition in preparation of a product with tyrosinase activity inhibiting effects.

[0017] The application also provides use of the natural small molecule compound or the composition in preparation of a product with melanin and / or lipofuscin generation inhibiting effects.

[0018] Preferably, the product is a food, a skin care product or a medicine.

[0019] (1) The application provides a natural small molecule compound with a brand-new structure; researches show that the natural small molecule compound has anti-aging, whitening, anti-UVB exposure induced photoaging, tyrosinase activity inhibiting, melanin and / or lipofuscin generation inhibiting and other series of effects.

[0020] (2) The application also provides a brand new composition containing GABA and natural small molecule compounds; research shows that the composition containing GABA and natural small molecule compounds formed by combining GABA with natural small molecule compounds with structures shown in formula I, formula II and formula III has further significantly improved effects of anti-aging, whitening, anti-UVB exposure caused photoaging, inhibiting tyrosinase activity, inhibiting melanin and / or lipofuscin production, etc.; the effects of anti-aging, whitening, anti-UVB exposure caused photoaging, inhibiting tyrosinase activity, inhibiting melanin and / or lipofuscin production, etc. are significantly higher than those of the natural small molecule compounds with structures shown in formula I, formula II and formula III of the application, and also significantly higher than those of GABA; this may be the result of synergistic effect produced by combining GABA with the natural small molecule compounds with structures shown in formula I, formula II and formula III of the application.

[0021] (3) Since the natural small molecule compounds and the composition containing GABA and natural small molecule compounds both have a series of effects of anti-aging, whitening, anti-UVB exposure caused photoaging, inhibiting tyrosinase activity, inhibiting melanin and / or lipofuscin production, etc., therefore, it has important application value to use them as effective components for preparing products with corresponding effects. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is an HPLC detection spectrum of the natural small molecule compound-1 of the application.

[0023] Figure 2 It is an HR-ESI-MS spectrum of the natural small molecule compound-1 of the application.

[0024] Figure 3 It is a DEPT-135 spectrum of the natural small molecule compound-1 of the application. 1 HNMR spectrum.

[0025] Figure 4 It is a C NMR spectrum of the natural small molecule compound-1 of the application. 13

[0026] Figure 5 It is a DEPT-135 spectrum of the natural small molecule compound-1 of the application.

[0027] Figure 6 It is a DEPT-135 spectrum of the natural small molecule compound-1 of the application. 1 H- 1 H COSY spectrum.

[0028] Figure 7 It is an HMBC spectrum of the natural small molecule compound-1 of the application.

[0029] ​Figure 8 The natural small molecule compound-1 of this invention 1 H- 1 H COSY and HMBC related diagrams.

[0030] Figure 9 This is the HPLC chromatogram of the natural small molecule compound-2 of this invention.

[0031] Figure 10 This is the HR-ESI-MS spectrum of the natural small molecule compound-2 of this invention.

[0032] Figure 11 The natural small molecule compound-2 of this invention 1 HNMR spectrum.

[0033] Figure 12 The natural small molecule compound-2 of this invention 13 C NMR spectrum.

[0034] Figure 13 This is the DEPT-135 spectrum of the natural small molecule compound-2 of this invention.

[0035] Figure 14 This is the HMBC spectrum of the natural small molecule compound-2 of this invention.

[0036] Figure 15 The natural small molecule compound-2 of this invention 1 H- 1 H COSY and HMBC related diagrams.

[0037] Figure 16 This is the HPLC chromatogram of the natural small molecule compound-3 of this invention.

[0038] Figure 17 This is a high-resolution mass spectrometry of the natural small molecule compound -3 of this invention.

[0039] Figure 18 The natural small molecule compound-3 of this invention 1 HNMR spectrum.

[0040] Figure 19 The natural small molecule compound-3 of this invention 13 C NMR spectrum.

[0041] Figure 20 The natural small molecule compound-3 of this invention 1 H- 1 H COSY and HMBC related diagrams.

[0042] Figure 21This invention relates to the effects of natural small molecule compounds and compositions on the oxidative properties of nematodes damaged by UVB irradiation.

[0043] Figure 22 The present invention relates to the effects of natural small molecule compounds and compositions on oxidative indicators, tyrosinase activity, and melanin production in skin fibroblasts damaged by UVA radiation. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Example 1: Preparation of natural small molecule compounds

[0046] (1) Take cactus extract, ophiopogon japonicus extract and sophora flavescens leaf extract and mix them evenly in a weight ratio of 1:1:1 to obtain a mixed extract;

[0047] The cactus extract is prepared by a method comprising the following steps: crushing cactus and mixing it with water to obtain a cactus slurry; then adding chloroform to the cactus slurry for extraction to obtain a chloroform extract; concentrating and drying the chloroform extract to obtain the cactus extract; wherein the ratio of cactus to water and chloroform is 1kg:4L:4L.

[0048] The Ophiopogon japonicus extract is prepared by a method comprising the following steps: taking Ophiopogon japonicus and then extracting it with an organic solvent to obtain an organic solvent extract; concentrating and drying the organic solvent extract to obtain the Ophiopogon japonicus extract; wherein, the extraction is an maceration extraction; the organic solvent is ethyl acetate; the maceration extraction time is 2 days; and the ratio of Ophiopogon japonicus to ethyl acetate is 1 kg: 6 L.

[0049] The Sophora flavescens leaf extract is prepared by a method comprising the following steps: taking Sophora flavescens leaves and then extracting them with an organic solvent to obtain an organic solvent extract; concentrating and drying the organic solvent extract to obtain the Sophora flavescens leaf extract; wherein, the extraction is an maceration extraction; the organic solvent is chloroform; the maceration extraction time is 2 days; and the ratio of Sophora flavescens leaves to chloroform is 1 kg: 8 L.

[0050] (2) Load the mixed extract onto a silica gel column and elute with a mixed organic solvent consisting of chloroform and methanol in a volume ratio of 100:35 (3 column volumes). Discard the eluent obtained by eluting with the mixed organic solvent consisting of chloroform and methanol in a volume ratio of 100:35. Then elute with a mixed organic solvent consisting of chloroform and methanol in a volume ratio of 100:55 (5 column volumes). Collect the eluent obtained by eluting with the mixed organic solvent consisting of chloroform and methanol in a volume ratio of 100:55, concentrate and dry it to obtain the silica gel column elution fraction.

[0051] The silica gel in the silica gel column is 200-300 mesh silica gel, and the weight of the silica gel is 40 times the weight of the mixed extract.

[0052] (3) The eluted portion of the silica gel column is loaded onto the silica gel column again. First, it is eluted with a mixed organic solvent consisting of chloroform and methanol in a volume ratio of 100:40 (3 column volumes). The eluent obtained by eluting with the mixed organic solvent consisting of chloroform and methanol in a volume ratio of 100:40 is discarded. Then, it is eluted with a mixed organic solvent consisting of chloroform and methanol in a volume ratio of 100:45 (8 column volumes). The eluent obtained by eluting with the mixed organic solvent consisting of chloroform and methanol in a volume ratio of 100:45 is collected, concentrated and dried to obtain the active portion of the silica gel column.

[0053] The silica gel in the silica gel column is 200-300 mesh silica gel, and the weight of the silica gel is 50 times the weight of the mixed extract.

[0054] (4) The active site of the silica gel column obtained in step (3) was further prepared by preparative HPLC to obtain natural small molecule compound-1, natural small molecule compound-2 and natural small molecule compound-3, which are natural small molecule compounds with structures shown in formula I, formula II and formula III, respectively.

[0055] The preparation conditions for the preparative HPLC are as follows:

[0056] The mobile phase A was 0.1% trifluoroacetic acid-acetonitrile solution, and the mobile phase B was 0.1% trifluoroacetic acid aqueous solution. The wavelength was 220 nm, and the flow rate was 10 mL / min. The chromatographic column was an XBridge BEH C18 OBD Prep Column, 5 μm, 19 mm * 150 mm. The elution conditions were gradient elution.

[0057] The gradient elution conditions for preparing natural small molecule compound-1 are shown in Table 1. The eluent corresponding to the chromatographic peak at 9.32 min was collected, concentrated, and dried to obtain natural small molecule compound-1.

[0058] Table 1 HPLC elution time settings

[0059]

[0060] The structure of natural small molecule compound-1 is analyzed as follows:

[0061] HR-ESI-MS ( Figure 2 The peak shown is a quasi-molecular ion at m / z 373.1985 [M+Na]. + (C 20 H 30 O5Na (theoretical calculated value: 373.1985), the molecular formula is determined to be C. 20 H 30 O5 has an unsaturation degree of 7.

[0062] 1 HNMR (400MHz, CDCl3) spectrum Figure 3 The CCP displayed 30 proton signals. Among them, δ H 13.59 (1H, s) and 9.54 (1H, s) represent the hydroxyl hydrogen signals on the two benzene rings; δ H 5.21 (1H, t, J = 8.0 Hz) represents a single olefin proton signal; δ H 4.92 (1H, dd, J = 12.0, 4.0 Hz) represents a single hydroxymethyl hydrogen signal; in addition, seven methyl proton signals are also visible in the high-field region [δ]. H 3.73(3H,s), 3.39(3H,s), 2.68(3H,s), 1.77(3H,s), 1.70(3H,s), 0.96(3H,d,J=5.4Hz), 0.95(3H,d,J=5.4Hz)], where δ H 3.73(3H,s), 3.39(3H,s), and 2.68(3H,s) represent three hydrogen signals of the methyl group.

[0063] compound 13 C NMR (100MHz, CD3OD) spectrum Figure 4 The CCP showed 20 carbon signals, combined with the DEPT-135 spectrum ( Figure 5 It can be seen that they have 8 quaternary carbons, 3 methines, 2 methylenes, and 7 methyl groups, respectively. δ is visible in the low-field region. C 203.7 ketone carbonyl signal; δ C 131.6, 123.4 represent a pair of double bond signals; δ C 162.2, 161.3, 160.8, 114.9, 108.9, and 108.5 are carbon signals of the benzene ring.

[0064] exist 1 H- 1 H COSY spectrum ( Figure 6 ,8 In ), H-3″(δ H 1.83) and H-4″(δ H 0.95), H-5″(δ H 0.96),H-2″(δ H 1.43) is related, and it can be inferred that there are structural segments C-4″ / 5″-C-3″-C-2″; H-1″ (δ H 4.92) and H-2″(δ H 1.43) is related, suggesting the existence of the structural fragment C-1″-C-2″, thus allowing determination of the structure of the substituent on one side of the benzene ring. HMBC spectrum ( Figure 7 , 8 In ), H-1″(δ) was observed. H 4.92) and C-4′(δ C 162.2),C-5′(δ C The correlation at 108.9 indicates that the structural fragment is attached at the C-5′ position. Based on the above data, the structure of the substituent group on the benzene ring is confirmed, and the structure of the compound is deduced. Combining 1D and 2D NMR spectral information, the compound was fully assigned, as shown in Table 2.

[0065] Based on the above structural analysis, the structure of the natural small molecule compound-1 is identified as shown in Formula I. The natural small molecule compound-1 described above is the natural small molecule compound with the structure shown in Formula I of this invention.

[0066] Table 2. ¹¹D and ²¹D NMR data (CDCl₃, δinppm) of natural small molecule compounds.

[0067]

[0068]

[0069] The gradient elution conditions for preparing natural small molecule compound-2 are shown in Table 3. The eluent corresponding to the chromatographic peak at 11.51 min was collected, concentrated, and dried to obtain natural small molecule compound-2.

[0070] Table 3 HPLC elution time settings

[0071]

[0072] The structure of the natural small molecule compound-2 is determined as follows:

[0073] HR-ESI-MS ( Figure 10 The peak shown is a quasi-molecular ion at m / z 359.1825 [M+Na]. + (C 19 H 28O5Na (theoretical calculated value: 359.1828) has been determined to have the molecular formula C. 19 H 28 O5 has an unsaturation degree of 6.

[0074] compound 1 1H NMR (400MHz, CDCl3) spectrum Figure 11 The compound exhibits 27 proton signals. It contains one olefin proton signal δ. H 5.21 (1H,t,J=6.9Hz); 3 sets of methylene proton signals [δ H 3.33 (2H, d, J = 6.9 Hz), 2.59 (2H, d, J = 6.8 Hz), 1.76 (2H, overlapped)]; 6 methyl proton signals [δ H [3.69(3H,s), 2.47(3H,s), 1.83(3H,s), 1.76(3H,s), 1.30(6H,s)], of which 2 are hydroxymethyl signals [δ] H 3.69(3H,s), 2.47(3H,s)].

[0075] compound 13 C10 NMR (100MHz, CDCl3) spectrum Figure 12 The CCP showed 19 carbon signals, combined with the DEPT-135 spectrum ( Figure 13 It can be seen that they have 9 quaternary carbons, 1 methine, 3 methylene, and 6 methyl groups, respectively. δ is visible in the low-field region. C A ketone carbonyl signal at 202.6; δ C 122.3 and 135.8 represent a pair of double-bonded carbon signals; δ C 155.0, 154.1, 150.8, 118.3, 111.0, and 105.3 can be inferred to be carbon signals from the benzene ring.

[0076] In HMBC spectrum ( Figure 14 , 15 In ), H-1″(δ H 3.33) and C-4′(δ C 155.0),C-5′(δ C 111.0),C-6′(δ C 154.1) is related, suggesting that the isoprene group is attached at the C-5′ position. H-1″′ (δ H 2.59) and C-2′(δ C 150.8),C-3′(δ C 105.3),C-4′(δ CThe correlation at 155.0 confirms that the 3-methyl-3-butanol fragment is attached at the C-3′ position. Based on 1D and 2D NMR spectral information, the compounds were fully assigned, as shown in Table 4.

[0077] Based on the above structural analysis, the structure of the natural small molecule compound-2 is identified as shown in Formula II. The aforementioned natural small molecule compound-2 is the natural small molecule compound with the structure shown in Formula II of this invention.

[0078] Table 4. 21D and 2DNMR data of natural small molecule compounds (CDCl3, δinppm)

[0079]

[0080]

[0081] The gradient elution conditions for preparing the natural small molecule compound-3 are shown in Table 5. The eluent corresponding to the chromatographic peak at 11.00 min was collected, concentrated, and dried to obtain the natural small molecule compound-3.

[0082] Table 5 HPLC elution time settings

[0083]

[0084] The structure of the natural small molecule compound -3 was determined as follows: HR-ESI-MS ( Figure 17 The quasi-molecular ion peak is shown at m / z 377.1603 [M+H]. + (calcd for C 20 H 25 O7,377.1595), the molecular formula of this compound was determined to be C 20 H 24 O7 has an unsaturation degree of 9.

[0085] 1 H NMR spectrum ( Figure 18 (500MHz, CDCl3) showed a signal of 3 hydroxyl protons [δ] H 18.53, 11.97, 11.02 (each 1H, s)], one unsaturated proton signal [δ H 5.97 (1H, s)], 1 characteristic methylene signal [δ H 3.49, 3.34 (each 1H, d, J = 14.9 Hz)] and 4 methyl signals [δ H 1.54, 1.47 (each 3H, s), 1.17 (3H, t, J = 7.2Hz), 0.96 (3H, t, J = 7.5Hz)]. 13 C NMR spectrum (Figure 19 (125MHz, CDCl3) showed 20 carbon atom signals, which, combined with its DEPT-135 analysis, can be assigned to 11 quaternary carbons, 1 methine, 4 methylene and 4 methyl atoms.

[0086] Further analysis of the 2D NMR (Table 6) signals of this compound revealed methylene (δ) C 34.8) proton signal H-9 (δ H 3.19) and methyl (δ) C 8.6) Proton H-10 (δ H 1.16) Yes 1 H- 1 H COSY( Figure 20 It is related to H-9 and the carbonyl carbon C-8 (δ) C 207.3) HMBC long-range correlation was found; combined with 1D and 2D NMR spectral information, the compounds were fully assigned, as shown in Table 6.

[0087] Based on the above structural analysis, the structure of the natural small molecule compound-3 is identified as shown in Formula III. The aforementioned natural small molecule compound-3 is the natural small molecule compound with the structure shown in Formula III of this invention.

[0088] Table 6. 31D and 2DNMR data of natural small molecule compounds (CDCl3, δinppm, JinHz)

[0089]

[0090]

[0091] Example 2: Preparation method of a composition containing γ-aminobutyric acid and natural small molecule compounds

[0092] γ-aminobutyric acid is mixed with natural small molecule compound-1, natural small molecule compound-2, and natural small molecule compound-3 in a mass ratio of 1:0.5:2.4:3.9 to obtain the γ-aminobutyric acid and natural small molecule composition, that is, the composition containing γ-aminobutyric acid and natural small molecule compounds of the present invention.

[0093] The natural small molecule compound-1, natural small molecule compound-2, and natural small molecule compound-3 are natural small molecule compounds with structures shown in Formula I, Formula II, and Formula III of this invention, respectively.

[0094] Example 1: Evaluation of anti-photoaging activity and activity in inhibiting UV irradiation-induced lipofuscin production in *C. elegans*

[0095] Methods: N2 wild-type nematodes were cultured at 20℃ and randomly divided into 9 groups (each group containing no fewer than 60 nematodes), with 3 parallel control groups per group. The normal control group received phosphate-buffered saline solution instead of the active ingredient. Except for the normal control group, all other groups of L4 stage nematodes were cultured normally on NGM / OP50 plates for 2 days, then exposed to a UV lamp equipped with a 254nm UV bulb (20 watts) and a UV intensity of 1000 J / m². 2 After irradiating with the specified intensity for 3 hours, the nematodes were transferred to NGM / OP50 plates containing 50 μg / mL of active ingredient and cultured for another 3 days. Then, live nematodes were collected, and the contents of ROS, MDA, lipofuscin, and the activities of SOD and CAT in the same number (30 nematodes) were measured. The experimental groups are shown in Table 7.

[0096] Table 7 Experimental Groups

[0097]

[0098]

[0099] The results are as follows Figure 21As shown in a, 21b, 21c, 21d, and 21e, after UVB irradiation treatment, compared with the blank control group, the contents of ROS, MDA, and lipofuscin in the nematodes of the UVB irradiation group were significantly increased, while the activities of antioxidant enzymes SOD and CAT in the nematodes were significantly decreased. This indicates that UVB irradiation significantly induced oxidative stress in nematodes, causing severe oxidative damage and accelerating senescence. Analysis of the results of this experiment revealed that, compared with the blank control group, the contents of ROS, MDA, and lipofuscin in the nematodes of the UVB irradiation group increased by 265.6% (p<0.01), 178.3% (p<0.01), and 334.1% (p<0.01), respectively; while the activities of antioxidant enzymes SOD and CAT in the nematodes decreased by 44.1% (p<0.01) and 34.7% (p<0.01), respectively. Unlike the UVB irradiation group, the SOD and CAT activities in nematodes pretreated with γ-aminobutyric acid (GABA), the natural small molecule compound of this invention, and the GABA and natural small molecule composition were effectively increased. Furthermore, the ROS, MDA, and lipofuscin contents in nematodes treated with these compounds were lower than those in the UVB irradiation group. Specifically, compared to the UVB irradiation group, the ROS, MDA, and lipofuscin contents in nematodes treated with the GABA and natural small molecule composition group decreased by 60.6% (p<0.01), 41.1% (p<0.01), and 59.7% (p<0.01), respectively. This indicates that the natural small molecule compound of this invention and the GABA and natural small molecule composition of this invention can effectively inhibit oxidative damage in nematodes caused by UV irradiation, exerting their antioxidant activity. Simultaneously, they can inhibit lipofuscin formation caused by UV irradiation, demonstrating that the GABA and natural small molecule combination possesses excellent anti-aging activity.

[0100] Further analysis of the experimental results revealed that the natural small molecule compounds of the present invention, as well as the γ-aminobutyric acid and natural small molecule composition of the present invention, can effectively enhance the activity of antioxidant enzymes SOD and CAT in nematodes. Among them, compared with the UVB irradiation group, the activities of antioxidant enzymes SOD and CAT in nematodes in the γ-aminobutyric acid and natural small molecule composition groups increased by 69.7% (p<0.01) and 40.2% (p<0.01), respectively.

[0101] The experimental results in this section show that γ-aminobutyric acid (GABA) and the natural small molecule composition have stronger effects on improving the activity of nematodes in clearing ROS and MDA and inhibiting the formation of lipofuscin than GABA, resveratrol, quercetin, or natural small molecule compounds-1, 2, and 3 at the same concentration. We speculate that GABA and the natural small molecules in the GABA and natural small molecule composition of this invention can synergistically enhance the activity of antioxidant enzymes SOD and CAT in nematodes and improve the activity of nematodes in clearing ROS and MDA and inhibiting the formation of lipofuscin, thereby better resisting photoaging caused by UVB exposure.

[0102] Example 2: Evaluation of effects on skin cell vitality, inhibition of UV irradiation-induced increase in tyrosinase activity, and inhibition of melanin production.

[0103] Methods: Human skin fibroblasts were cultured normally. When the cells reached 90% confluence, they were digested with 0.25% trypsin and seeded into 24-well plates. When the cells reached 80% confluence, ultraviolet (UV) irradiation was performed. A 360 nm UVA lamp was used. The UV lamp was first sterilized and placed in a clean bench. A constant-temperature water bath was then added and placed in the clean bench, where the UV lamp was sterilized for 30 minutes. Before irradiation, the long-wave UV lamp was placed on the constant-temperature water bath, and the distance between the lamp and the irradiation plane of the 24-well plate was measured to be 15 cm. The long-wave UV lamp was preheated for 30 minutes before measurement. After the irradiation power stabilized, it was measured to be 20.0 mJ / s. During irradiation, the culture medium was replaced with PBS, and the cells were exposed to the UVA irradiation device in the constant-temperature water bath at a distance of 15 cm. Two hours before irradiation, PBS solution was added, and the culture plate was placed in a 37°C constant temperature water bath. After irradiation, the plate was replaced with DMEM containing 10% fetal bovine serum and active ingredients (final concentration 30 μg / mL) and placed in the incubator for continued culture. After 24 hours, cells and culture supernatant were collected, and cell viability was detected using the CCK 8 kit. The contents of ROS, SOD, MDA, CAT, tyrosinase activity, and melanin content were detected according to the instructions of the ELISA kits. The experimental groups are shown in Table 8.

[0104] Table 8 Experimental Groups

[0105]

[0106] Analysis of the experimental results of this effect embodiment ( Figure 22a) The results showed that UVA irradiation reduced cell viability by 27.5% compared to the blank control group (p<0.01). However, analysis of the experimental results revealed that cells treated with the natural small molecules of this invention, especially γ-aminobutyric acid (GABA) and the natural small molecule composition, exhibited significantly higher cell viability than the UVA irradiation group. Specifically, compared to the UVA irradiation group, the GABA and natural small molecule composition group showed a 33.8% increase in cell viability (p<0.01). Compared to the same concentrations of GABA, resveratrol, natural small molecule compound-1, natural small molecule compound-2, natural small molecule compound-3, or quercetin, the GABA and natural small molecule combination significantly improved cell viability under UVA irradiation conditions, indicating that the GABA and natural small molecule composition can better enhance the anti-UVA irradiation damage activity of cells.

[0107] Further analysis of the experimental results of this efficacy example revealed that, compared with the groups containing γ-aminobutyric acid (GABA), resveratrol, natural small molecule compound-1, natural small molecule compound-2, natural small molecule compound-3, or quercetin, the group containing GABA and the natural small molecule composition showed a more significant increase in cell viability. This indicates that the activity of GABA and the natural small molecule composition in enhancing cell viability is stronger than that of GABA, resveratrol, natural small molecule compound-1, natural small molecule compound-2, natural small molecule compound-3, or quercetin. We speculate that GABA and the natural small molecule compounds in the GABA and natural small molecule composition of this invention may enhance cell viability and improve cell resistance to UVA damage through synergistic effects.

[0108] Further analysis of the experimental results of this effect embodiment ( Figure 22 b) and 22c) show that, compared with the blank control group, UVA irradiation increased the ROS and MDA content of cells by 148.3% (p<0.01) and 191.5% (p<0.01), respectively. However, analysis of the experimental results of this effective example revealed that the intracellular ROS and MDA content of cells treated with the natural small molecule compounds of this invention, especially the γ-aminobutyric acid and natural small molecule composition of this invention, was significantly lower than that of the UVA irradiation group. Specifically, compared with the UVA irradiation group, the ROS and MDA content of cells in the γ-aminobutyric acid and natural small molecule composition group decreased by 37.5% (p<0.01) and 51.2% (p<0.01), respectively. Further analysis of the experimental results revealed that, compared with the same concentrations of γ-aminobutyric acid (GABA), resveratrol, natural small molecule compound-1, natural small molecule compound-2, natural small molecule compound-3, or quercetin, GABA and the natural small molecule combination could better enhance the activity of cells in clearing ROS and MDA under UVA irradiation conditions, indicating that GABA and the natural small molecule combination have better activity in enhancing the activity of cells in resisting UVA irradiation damage.

[0109] Further analysis of the experimental results of this effect embodiment ( Figure 22 As shown in d and 22e), compared with the blank control group, UVA irradiation reduced the activities of SOD and CAT in cells by 22.9% (p<0.01) and 24.5% (p<0.01), respectively. However, analysis of the experimental results of this effective example revealed that the activities of SOD and CAT in cells treated with the natural small molecule compounds of this invention, especially γ-aminobutyric acid and the natural small molecule composition of this invention, were significantly higher than those in the UVA irradiation group. Specifically, compared with the UVA irradiation group, the activities of SOD and CAT in cells treated with γ-aminobutyric acid and the natural small molecule composition group increased by 23.4% (p<0.01) and 40.2% (p<0.01), respectively. Further analysis of the experimental results revealed that, compared with the same concentrations of γ-aminobutyric acid, resveratrol, natural small molecule compound-1, natural small molecule compound-2, natural small molecule compound-3, or quercetin, the γ-aminobutyric acid and natural small molecule composition of the present invention can better enhance the activity of SOD and CAT in cells under UVA irradiation conditions, indicating that the γ-aminobutyric acid and natural small molecule composition of the present invention has better activity in enhancing the resistance of cells to UVA irradiation damage.

[0110] Analysis of the experimental results of this example showed that, compared with the blank control group, the UVA irradiation group ( Figure 22 The activity of f) increased significantly, and the melanin content also increased significantly. Figure 22 g) This indicates that UVA irradiation can damage skin fibroblasts, activate tyrosinase, or promote its expression or secretion, thereby promoting melanin expression and secretion. Compared with the blank control group, the UVA irradiation group showed a 266.1% increase in tyrosinase activity (p<0.01). However, analysis of the experimental results of this effective example revealed that the tyrosinase activity of the natural small molecule compounds of this invention, especially the γ-aminobutyric acid and natural small molecule composition groups, was significantly lower than that of the UV irradiation group. Specifically, compared with the UVA irradiation group, the tyrosinase activity of the γ-aminobutyric acid and natural small molecule composition groups decreased by 56.8% (p<0.01). Compared with the blank control group, the UVA irradiation group showed a 156.3% increase in melanin content (p<0.01). However, analysis of the experimental results of this effective example revealed that the melanin content of the natural small molecule compound groups, especially the γ-aminobutyric acid and natural small molecule composition groups, was significantly lower than that of the UVA irradiation group. Compared with the UVA irradiation group, the melanin content of the γ-aminobutyric acid and natural small molecule composition of the present invention was reduced by 46.1% (p<0.01).

[0111] Further analysis of the experimental results of this effective embodiment revealed that, under the same concentration conditions, the tyrosinase activity and melanin content of the γ-aminobutyric acid (GABA) and natural small molecule composition group of this invention were lower than those of the GABA, resveratrol, natural small molecule compound-1, natural small molecule compound-2, natural small molecule compound-3, or quercetin group. We speculate that the GABA and natural small molecule compounds in the GABA and natural small molecule composition of this invention may synergistically inhibit the increase in tyrosinase activity induced by UVA irradiation in human skin fibroblasts, or inhibit its expression or secretion, thereby inhibiting the expression or secretion of melanin in human skin fibroblasts.

[0112] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A composition comprising γ-aminobutyric acid and a natural small molecule compound, characterized in that, A natural small molecule compound comprising γ-aminobutyric acid and a structure represented by Formula I, Formula II and Formula III; 2. The composition containing γ-aminobutyric acid and natural small molecule compounds according to claim 1, characterized by, The mass ratio of γ-aminobutyric acid to the natural small molecule compound of the structure represented by Formula I, Formula II and Formula III is 1: (1-100): (1-100): (1-100).

3. The composition containing γ-aminobutyric acid and natural small molecule compounds according to claim 2, characterized by, The mass ratio of γ-aminobutyric acid to the natural small molecule compound of the structure represented by Formula I, Formula II and Formula III is 1: (1-50): (1-50): (1-50).

4. The composition containing γ-aminobutyric acid and natural small molecule compounds according to claim 1, characterized by, The mass ratio of γ-aminobutyric acid to the natural small molecule compound of the structure represented by Formula I, Formula II and Formula III is 1:0.5:2.4:3.

9.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of a product having an anti-aging effect and / or a whitening effect.

6. Use of the composition according to any one of claims 1 to 4 in the preparation of a product having an anti-photoaging effect caused by UVB exposure.

7. Use of the composition according to any one of claims 1 to 4 in the preparation of a product having an inhibitory effect on the activity of tyrosinase.

8. Use of the composition according to any one of claims 1 to 4 in the preparation of a product having an inhibitory effect on the production of melanin and / or lipofuscin.

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