A compound with anti-aging and soothing effects and its application

Abstract

The present invention provides a compound with anti-aging and soothing effects and an application thereof. The compound has a structure shown in formula (I). The present invention uses bamboo leaves as a raw material, further separates and develops, and creatively separates a new compound from a bamboo leaf extract. Experimental verification shows that the compound has the effects of inhibiting the expression of collagenase, inhibiting the expression of hyaluronidase, and inhibiting the expression of three inflammatory factors IL-6, NO, and TNF-α, has excellent anti-aging, anti-inflammatory and soothing effects, and can be applied to anti-aging and soothing cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and relates to a compound with anti-aging and soothing effects and application thereof. Background Art

[0002] Lophatherum gracile Brongn. is a perennial herbaceous plant of the genus Lophatherum in the Poaceae Barnhart family. It is also known as pheasant rice, golden chicken rice, body grass, and bamboo leaf roll. It mainly grows on hillsides, woodlands or forest edges, and roadside shades below 1,200 meters above sea level.

[0003] The dried stems and leaves of the light bamboo leaf are traditional Chinese herbal medicines. They are recorded in the "Shennong's Herbal Classic" and "Compendium of Materia Medica". They belong to the category of swamp grass. Li Shizhen said: "Light bamboo leaves are everywhere in the wild. The seedlings grow in spring and are several inches high. They have thin stems and green leaves, just like the stems and leaves of thin bamboos grown by bamboo rice falling to the ground. The roots have dozens of whiskers in one nest, and the whiskers are borne on the whiskers, which are the same as Ophiopogon japonicus, but hard. It can remove irritability and heat, promote urination, and clear the heart." The "Pharmacopoeia of the People's Republic of China" since 1977 has included light bamboo leaves. It tastes sweet, light, and cold in nature. It has the effects of clearing heat and purging fire, removing irritability, and promoting urination. It is mostly used for fever, thirst, mouth sores, and painful urination.

[0004] Studies have shown that the extract of Bamboo Leaves has good eye safety and skin safety, can dose-dependently reduce the expression of IL-8 and MCP-1 in HaCaT-TRPV1-OE cells, and can significantly reduce the epidermal water loss rate and skin erythema index caused by sodium dodecyl sulfate, instantly relieve the skin tingling caused by lactic acid stimulation, and significantly reduce the skin discomfort and redness caused by capsaicin stimulation. Bamboo leaves mainly contain chemical components such as flavonoids, triterpenes, and phenolic acids. Among them, flavonoid carbon glycosides have certain antibacterial and antiviral effects, and are the main material basis for the effect of Bamboo Leaves in clearing heat and purging fire. Other studies have shown that flavonoid carbon glycosides have good whitening, anti-oxidation, radiation protection and other skin care effects.

[0005] Plant extracts are difficult to control stability between batches due to their complex composition. Isolating and purifying chemical components with clear structures and functions from the leaves of the bamboo shoots can expand ideas for the development of drugs, chemical raw materials, etc., and also provide new solutions for diseases and skin problems, which is very meaningful. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a compound with anti-aging and soothing effects and its application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a compound having anti-aging and soothing effects, the structure of the compound is shown in formula (I):

[0009] Formula (I).

[0010] The present invention uses bamboo leaves as raw materials and creatively separates a new natural compound from bamboo leaf extract. Experimental verification shows that the compound has the effects of inhibiting the expression of collagenase, inhibiting the expression of hyaluronidase and inhibiting the expression of three inflammatory factors IL-6, NO and TNF-α, and has excellent anti-aging, anti-inflammatory and soothing effects. Therefore, the compound can be used in anti-aging and soothing cosmetics. It can be foreseen that the above-mentioned compound obtained by any existing technical method in the field (isolated and purified from natural products or obtained by organic synthesis method) can obtain the same technical effect.

[0011] Exemplarily, the compound can be obtained by separation and purification from a natural product, and the compound is prepared by a method comprising the following steps:

[0012] (1) mixing the raw material of bamboo leaves with ethanol aqueous solution and then subjecting it to microwave-ultrasonic combined treatment;

[0013] (2) refluxing the treated solution to obtain an extract;

[0014] (3) concentrating the extract under reduced pressure to obtain a concentrated solution;

[0015] (4) Purifying and eluting the concentrated solution with a macroporous resin and collecting the eluate;

[0016] (5) subjecting the eluate to flocculation treatment to obtain a Bambusa chinensis leaf extract;

[0017] (6) The extract of the leaves of the bamboo shoots is separated by liquid chromatography and then dried to obtain the compound with anti-aging and soothing effects.

[0018] The preparation method carries out microwave-ultrasound combined treatment on the light bamboo leaf raw material, and carries out flocculation treatment on the eluate after macroporous resin purification and elution, and combines liquid chromatography separation, on the one hand, can obtain the compound represented by formula (I) from the light bamboo leaf raw material, on the other hand, compared with other preparation processes, the preparation method also significantly improves the yield of the compound.

[0019] Preferably, the light bamboo leaf raw material in step (1) is the light bamboo leaf powder obtained by sieving through 70-90 mesh (for example, 70 mesh, 72 mesh, 74 mesh, 77 mesh, 80 mesh, 83 mesh, 86 mesh, 88 mesh, 90 mesh, etc.) after being crushed. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0020] The volume fraction of the ethanol aqueous solution in step (1) is 20%-50%, for example, 20%, 21%, 23%, 26%, 30%, 35%, 40%, 45%, 50%, etc. Other specific points within this numerical range can be selected and will not be described in detail here.

[0021] The present invention creatively finds that using an ethanol aqueous solution with a volume fraction of 20%-50% for reflux extraction can effectively increase the yield of the compound.

[0022] The mass ratio of the ethanol aqueous solution to the light bamboo leaf raw material is (5-10):1, wherein the specific point values ​​in (5-10) can be independently selected from 5, 5.2, 5.4, 5.7, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, etc., and other specific point values ​​within this numerical range can be selected, which will not be repeated here one by one.

[0023] Preferably, the temperature of the combined treatment in step (1) is 40-60°C, for example, 40°C, 41°C, 42°C, 45°C, 48°C, 50°C, 52°C, 54°C, 56°C, 60°C, etc. Other specific point values ​​within this numerical range can be selected and will not be described in detail here.

[0024] The power of the microwave treatment in the combined treatment is 50-300 W, for example, 50 W, 55 W, 60 W, 65 W, 70 W, 75 W, 80 W, 85 W, 90 W, 95 W, 100 W, 150 W, 200 W, 250 W, 300 W, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0025] The time of microwave treatment in the combined treatment is 15-25 min, for example, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0026] The power of the ultrasonic treatment in the combined treatment is 100-500 W, for example, 100 W, 110 W, 120 W, 130 W, 140 W, 150 W, 200 W, 250 W, 300 W, 350 W, 400 W, 450 W, 500 W, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0027] The time of ultrasonic treatment in the combined treatment is 5-10 min, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0028] The present invention creatively finds that when microwave-ultrasonic combined treatment is carried out, a microwave power of 50-300 W and an ultrasonic power of 100-500 W are used. Compared with other powers, the yield of the anti-aging and soothing compounds can be effectively improved. The increase in microwave power can increase the temperature inside the plant cells, accelerate the rupture of the cell wall and the release of the target compound. However, too high a microwave power will cause the heating speed to be too fast, which will lead to thermal damage or side reactions, thereby affecting the extraction effect; the shorter the time required for the collapse of the cavitation bubble with the increase in ultrasonic power, the stronger the cavitation effect generated by ultrasound per unit time, which is conducive to the improvement of the extraction rate. However, too high an ultrasonic power will produce a buffering effect, reduce the rate of energy transfer, and cause the cavitation effect to weaken, and may also cause the degradation or isomerization of the active ingredients. The present invention also finds that microwave treatment for 15-25 minutes, ultrasonic treatment for 5-10 minutes, and the temperature of the combined treatment is 40-60°C, which can further improve the yield of the anti-aging and soothing compounds.

[0029] Preferably, the number of reflux extractions in step (2) is 1-3 times, for example, 1 time, 2 times, 3 times, and the time of each extraction is independently 1-3 h, for example, 1 h, 1.2 h, 1.4 h, 1.7 h, 2.0 h, 2.4 h, 2.8 h, 3 h, etc. Other specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0030] Preferably, the purification and elution steps in step (4) include: after adsorption, standing for 2-12 hours, first eluting with water for 2-5 BV, then eluting with 5%-15% ethanol aqueous solution for 3-8 BV, and then eluting with 70%-90% ethanol aqueous solution for 5-10 BV.

[0031] The above-mentioned specific elution procedure, i.e., eluting with water first and then with ethanol-water solution, combined with a specific concentration of ethanol-water solution can effectively increase the yield of the compound with anti-aging and soothing effects.

[0032] The 2-12 h can be 2h, 2.5h, 3h, 3.5h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, etc., the 2-5 BV can be 2 BV, 3 BV, 4 BV, 5 BV, etc., the 3-8 BV can be 3 BV, 4 BV, 5 BV, 6 BV, 7 BV, 8BV, etc., the 5-10 BV can be 5 BV, 6 BV, 7 BV, 8 BV, 9 BV, 10 BV, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0033] The 5%-15% ethanol aqueous solution can be selected from ethanol aqueous solutions with concentrations of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc., and the 70%-90% ethanol aqueous solution can be selected from ethanol aqueous solutions with concentrations of 70%, 75%, 80%, 85%, 90%, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0034] Preferably, the flocculation treatment in step (5) uses a flocculant, and the amount of the flocculant used is 0.1-1% of the eluent, for example 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.8%, 0.9%, 1%, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0035] The flocculant is a combination of gelatin, ZTC1+1 and citric acid.

[0036] The mass ratio of the gelatin, ZTC1+1 and citric acid is 1:(1-3):(2-10), wherein the specific point values ​​in (1-3) can be independently selected from 1, 1.1, 1.3, 1.5, 1.7, 2, 2.3, 2.6, 3, etc., wherein the specific point values ​​in (2-10) can be independently selected from 2, 2.2, 2.4, 2.7, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, etc., and other specific point values ​​within the above numerical range can be selected, which will not be repeated here.

[0037] The present invention also unexpectedly discovered that the yield of the anti-aging and soothing compound is significantly improved after flocculation treatment by using a flocculant composed of gelatin, ZTC1+1 and citric acid in a mass ratio of 1:(1-3):(2-10).

[0038] Preferably, the chromatographic column used for the liquid phase separation in step (6) is a C18 column.

[0039] The column temperature of the chromatographic column is 20-30°C, for example, 20°C, 22°C, 25°C, 30°C, etc. Other specific point values ​​within this numerical range can be selected and will not be described in detail here.

[0040] Preferably, the mobile phase A of the liquid chromatography in step (6) is water, the mobile phase B is acetonitrile, and the elution method is gradient elution.

[0041] The gradient elution procedure is as follows: 0-2 min, mobile phase A is maintained at 97%-99% (e.g., 97%, 98%, 99%, etc.), and mobile phase B is maintained at 1%-3% (e.g., 1%, 2%, 3%, etc.); 2-16 min, mobile phase A is uniformly reduced from 97%-99% (e.g., 97%, 98%, 99%, etc.) to 68%-72% (e.g., 68%, 69%, 70%, 71%, 72%, etc.), and mobile phase B is uniformly increased from 1%-3% (e.g., 1%, 2%, 3%, etc.) to 28%-32% (e.g., 28%, 29%, 30%, 31%, 32%, etc.); 16-17 min, mobile phase A decreases uniformly from 68%-72% (e.g. 68%, 69%, 70%, 71%, 72%, etc.) to 8%-12% (e.g. 8%, 9%, 10%, 11%, 12%, etc.), mobile phase B increases uniformly from 28%-32% (e.g. 28%, 29%, 30%, 31%, 32%, etc.) to 88%-92% (e.g. 88%, 89%, 90%, 91%, 92%, etc.); 17-18 min, mobile phase A is maintained at 8-12% (e.g. 8%, 9%, 10%, 11%, 12%, etc.), mobile phase B is maintained at 88-92% (e.g. 88%, 89%, 90%, 91%, 92%, etc.); 18-19 15-21 minutes, mobile phase A: 8%-12% (for example, 8%, 9%, 10%, 11%, 12%, etc.) uniformly increased to 97-99% (for example, 97%, 98%, 99%, etc.), mobile phase B uniformly decreased from 88%-92% (for example, 88%, 89%, 90%, 91%, 92%, etc.) to 1%-3% (for example, 1%, 2%, 3%, etc.); 19-21 minutes, mobile phase A is maintained at 97-99% (for example, 97%, 98%, 99%, etc.), mobile phase B is maintained at 1%-3% (for example, 1%, 2%, 3%, etc.); other specific point values ​​within the above numerical range can be selected, so they will not be described here one by one.

[0042] The present invention creatively discovered that the above-mentioned gradient elution process can effectively improve the yield of the compound with anti-aging and soothing effects.

[0043] Preferably, the flow rate of the gradient elution is 7-13 mL / min, for example 7 mL / min, 8 mL / min, 9 mL / min, 10 mL / min, 11 mL / min, 12 mL / min, 13 mL / min, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0044] Preferably, the detection wavelength of the liquid chromatography is 265-275 nm, for example, 265 nm, 267 nm, 270 nm, 272 nm, 275 nm, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0045] Other specific point values ​​within the above numerical range can be selected and will not be described in detail here.

[0046] In a second aspect, the present invention provides the use of the compound with anti-aging and soothing effects described in the first aspect in the preparation of a product with the effect of inhibiting the expression of collagenase, hyaluronidase or inflammatory factors.

[0047] The products include essence, lotion, emulsion, cream, mask or cleanser.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The present invention uses bamboo leaves as raw materials and creatively separates a new compound from the bamboo leaf extract. Experimental verification shows that the compound has the effects of inhibiting the expression of collagenase, inhibiting the expression of hyaluronidase and inhibiting the expression of three inflammatory factors IL-6, NO and TNF-α, and has excellent anti-aging, anti-inflammatory and soothing effects. Therefore, the compound can be used in anti-aging and soothing cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound obtained in Example 1 of the present invention;

[0051] Figure 2 is the carbon nuclear magnetic resonance spectrum of the compound obtained in Example 1 of the present invention;

[0052] Figure 3 It is the nuclear magnetic resonance DEPT 135 spectrum of the compound obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0053] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0054] In the following examples, unless otherwise specified, the reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

[0055] The gelatin involved in the following examples was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; ZTC1+1 was purchased from Beijing Zhengtiancheng Clarification Technology Co., Ltd.

[0056] Example 1

[0057] This embodiment provides a compound with the following structure that has anti-aging and soothing effects:

[0058]

[0059] The preparation method is as follows:

[0060] (1) Take 50 g of Bambusa chinensis leaves, grind them into 80 mesh fine powder, mix them with 0.5 kg of 30% ethanol aqueous solution, and then carry out microwave-ultrasonic treatment at 50°C, with microwave power of 100 W, microwave time of 20 min, and ultrasonic power of 250 W, ultrasonic time of 8 min. The Bambusa chinensis leaves powder mixture was refluxed and extracted twice, each time for 2 h, and the extracts were combined;

[0061] (2) The combined extract was concentrated to 25 g at 60°C under reduced pressure, and the concentrate was centrifuged at 3000 rpm. The supernatant was set aside. The precipitate was re-dissolved in 30 g of water and centrifuged again. The supernatant obtained by centrifugation was combined with the supernatant of the first centrifugation and set aside. 220 g of D-101 macroporous resin was loaded into the column. After elution with pure water until there was no alcohol smell, the combined supernatant of the centrifugation was taken for sample adsorption at a sample adsorption rate of 0.5 BV / h. After the sample was loaded, it was allowed to stand for 2 h. First, 2 BV was eluted with water, then 4 BV was eluted with 10% ethanol aqueous solution, and then 8 BV was eluted with 80% ethanol aqueous solution. The eluate was collected;

[0062] (3) Add 0.5% of the solution of a flocculant consisting of gelatin, ZTC1+1 and citric acid in a mass ratio of 1:1:5 to the eluent, stir at 50°C for 30 min, let stand and cool, filter, and freeze-dry at -65°C for 60 h to obtain a Bambusa chinensis leaf extract;

[0063] (4) The extract of Psoralea corylifolia was separated and purified by high performance preparative liquid chromatography. The chromatographic column used for the preparative liquid chromatography was XBridge Prep. The inner diameter of the chromatographic column was 19 mm. The length of the chromatographic column was 150 mm. The filler of the chromatographic column was OBD C18. The particle size of the filler was 5 μm. The conditions of the preparative liquid chromatography were as follows: mobile phase A was water, mobile phase B was acetonitrile, and gradient elution was performed. The gradient elution program was as follows: 0-2 min, mobile phase A: 98-98%, mobile phase B: 2-2%; 2-16 min, mobile phase A: 98-70%, mobile phase B: 2-30%; 16-17 min, mobile phase A: 70-10%, mobile phase B: 30-90%; 17-18 min, mobile phase A: 10-10%, mobile phase B: 90-90%; 18-19 min, mobile phase A: 10-10%, mobile phase B: 90-90%; min, mobile phase A: 10-98%, mobile phase B: 90-2%; 19-21min, mobile phase A: 98-98%, mobile phase B: 2-2%, elution flow rate of 10 mL / min, detection wavelength of 270 nm, preparative chromatographic column temperature of 25°C, after drying, 0.018 g of the compound with anti-aging and soothing effects was obtained, with a yield of 0.036%.

[0064] The compound obtained in step (4) was analyzed by nuclear magnetic resonance, and the obtained nuclear magnetic resonance hydrogen spectrum was as follows: Figure 1 The obtained carbon NMR spectrum is shown as Figure 2 The obtained NMR DEPT 135 spectrum is shown as Figure 3 shown.

[0065] Depend on Figure 1 and Figure 2 It can be seen that:

[0066] 1 H-NMR (500 MHz, DMSO), δ (ppm): 4.5 (1H, d, j=5.9Hz, H-1), 6.48 (1H, d, j=5.6Hz, H-5), 8.24 (1H, d, j=5.6Hz, H-6), 4.7 (1H, d, j=7.8Hz, H-1'), 3.13(1H, dt,j=2.9, 7.8Hz, H-2'), 3.2(1H, m, H-3'), 3.14(1H, m, H-4'), 3.08(1H, m, H-5'), 3.44(1H, m, H-6'), 3.66(2H, dd, j=2.6,11.6Hz, H-6').

[0067] 13C-NMR (125 MHz, DMSO), δ (ppm): 56.29 (C-1), 175.11 (C-2), 162.40 (C-3), 141.12 (C-4), 116.92 (C-5), 156. 58(C-6), 103.33(C-1'), 77.45(C-2'), 76.69(C-3'), 74.22(C-4'), 70.26(C-5'), 61.45(C-6').

[0068] Example 2

[0069] This embodiment provides a compound with anti-aging and soothing effects, and its preparation method is as follows:

[0070] (1) Take 50 g of Bambusa chinensis leaves, grind them into 70 mesh fine powder, mix them with 0.25 kg of 40% ethanol aqueous solution, and then carry out microwave-ultrasonic treatment at 60°C, with microwave power of 280 W, microwave time of 18 min, and ultrasonic power of 150 W, ultrasonic time of 10 min. The treated Bambusa chinensis leaves powder mixture was refluxed and extracted 3 times, each time for 1 h, and the extracts were combined;

[0071] (2) The combined extract was concentrated to 25 g at 60°C under reduced pressure, and the concentrate was centrifuged at 3000 rpm. The supernatant was set aside. The precipitate was re-dissolved in 30 g of water and centrifuged again. The supernatant obtained by centrifugation was combined with the supernatant of the first centrifugation and set aside. 220 g of AB-8 macroporous resin was loaded into the column. After elution with pure water until there was no alcohol smell, the combined supernatant of the centrifugation was taken for sample adsorption at a sample adsorption rate of 0.5 BV / h. After the sample was loaded, it was allowed to stand for 10 h. First, 4 BV was eluted with water, then 6 BV was eluted with 8% ethanol aqueous solution, and then 8 BV was eluted with 85% ethanol aqueous solution. The eluate was collected;

[0072] (3) Add 0.1% of the solution of a flocculant consisting of gelatin, ZTC1+1 and citric acid in a mass ratio of 1:2:8 to the eluent, stir at 50°C for 30 min, let stand and cool, and then filter to obtain a light bamboo leaf extract;

[0073] (4) The extract of Psoralea corylifolia was separated and purified by high performance preparative liquid chromatography. The chromatographic column used for the preparative liquid chromatography was XBridge Prep. The inner diameter of the chromatographic column was 19 mm. The length of the chromatographic column was 150 mm. The filler of the chromatographic column was OBD C18. The particle size of the filler was 5 μm. The conditions of the preparative liquid chromatography were as follows: mobile phase A was water; mobile phase B was acetonitrile. Gradient elution was performed. The gradient elution program was as follows: 0-2 min, mobile phase A: 97-97%, mobile phase B: 3-3%; 2-16 min, mobile phase A: 97-68%, mobile phase B: 3-32%; 16-17 min, mobile phase A: 68-8%, mobile phase B: 32-92%; 17-18 min, mobile phase A: 8-8%, mobile phase B: 92-92%; 18-19 min, mobile phase A: 8-8%, mobile phase B: 92-92%; min, mobile phase A: 8-97%, mobile phase B: 92-3%; 19-21min, mobile phase A: 97-97%, mobile phase B: 3-3%, elution flow rate of 7 mL / min, detection wavelength of 270 nm, preparative chromatographic column temperature of 30°C, after drying, 0.0153 g of the compound with anti-aging and soothing effects was obtained, with a yield of 0.0306%.

[0074] The obtained compound was confirmed by nuclear magnetic resonance to be the compound represented by formula (I).

[0075] Example 3

[0076] This embodiment provides a compound with anti-aging and soothing effects, and its preparation method is as follows:

[0077] (1) Take 50 g of Bambusa chinensis leaves, grind them into 90 mesh fine powder, mix them with 0.4 kg of 25% ethanol aqueous solution, and carry out microwave-ultrasonic combined treatment at 55 °C, with microwave power of 100 W, microwave time of 25 min, and ultrasonic power of 400 W, ultrasonic time of 5 min. The treated Bambusa chinensis leaves powder mixture was refluxed and extracted once, each time for 3 h, to obtain an extract;

[0078] (2) The extract was concentrated to 25 g at 60 °C under reduced pressure, and the concentrate was centrifuged at 3000 rpm. The supernatant was set aside. The precipitate was re-dissolved in 30 g of water and centrifuged again. The supernatant obtained by centrifugation was combined with the supernatant of the first centrifugation and set aside. 220 g of D-101 macroporous resin was loaded into the column. After elution with pure water until there was no alcohol smell, the combined centrifuged supernatant was taken for sample adsorption at a sample adsorption rate of 0.5 BV / h. After the sample was loaded, it was allowed to stand for 10 h. First, 5 BV was eluted with water, then 3 BV was eluted with 12% ethanol aqueous solution, and then 10 BV was eluted with 75% ethanol aqueous solution. The eluate was collected;

[0079] (3) Add 1% of the solution of a flocculant consisting of gelatin, ZTC1+1 and citric acid in a mass ratio of 1:3:7 to the eluent, stir at 50°C for 30 min, let stand and cool, and then filter to obtain a light bamboo leaf extract;

[0080] (4) The extract of Psoralea corylifolia was separated and purified by high performance preparative liquid chromatography. The chromatographic column used for the preparative liquid chromatography was XBridge Prep. The inner diameter of the chromatographic column was 19 mm. The length of the chromatographic column was 150 mm. The filler of the chromatographic column was OBD C18. The particle size of the filler was 5 μm. The conditions of the preparative liquid chromatography were as follows: mobile phase A was water; mobile phase B was acetonitrile. Gradient elution was performed. The gradient elution program was as follows: 0-2 min, mobile phase A: 99-99%, mobile phase B: 1-1%; 2-16 min, mobile phase A: 99-72%, mobile phase B: 1-28%; 16-17 min, mobile phase A: 72-12%, mobile phase B: 28-88%; 17-18 min, mobile phase A: 12-12%, mobile phase B: 88-88%; 18-19 min, mobile phase A: 12-99%, mobile phase B: 88-1%; 19-21min, mobile phase A: 99-99%, mobile phase B: 1-1%, elution flow rate of 13 mL / min, detection wavelength of 270 nm, preparative chromatographic column temperature of 35°C, after drying, 0.0147 g of the compound with anti-aging and soothing effects was obtained, with a yield of 0.0294%.

[0081] The obtained compound was confirmed by nuclear magnetic resonance to be the compound represented by formula (I).

[0082] Example 4

[0083] This embodiment provides a compound with anti-aging and soothing effects. The preparation method thereof is different from that of Embodiment 1 only in that the mass fraction of the ethanol aqueous solution in step (1) is 15%, and other preparation conditions remain unchanged. Finally, 0.0093 g of the compound is obtained, and the yield is 0.0186%.

[0084] The obtained compound was confirmed by nuclear magnetic resonance to be the compound represented by formula (I).

[0085] Example 5

[0086] This embodiment provides a compound with anti-aging and soothing effects. The preparation method thereof is different from that of Example 1 only in that the mass fraction of the ethanol aqueous solution in step (1) is 60%, and other preparation conditions remain unchanged. Finally, 0.0106 g of the compound is obtained, and the yield is 0.0212%.

[0087] The obtained compound was confirmed by nuclear magnetic resonance to be the compound represented by formula (I).

[0088] Example 6

[0089] This example provides a compound with anti-aging and soothing effects. The preparation method thereof is different from that of Example 1 only in that after the sample is loaded in step (2), after standing for 2 h, 2 BV of the compound is first eluted with water, then 4 BV of the compound is eluted with a 20% ethanol aqueous solution, and then 8 BV of the compound is eluted with a 60% ethanol aqueous solution. The eluate is collected, and other preparation conditions remain unchanged. Finally, 0.0125 g of the compound is obtained, and the yield is 0.025%.

[0090] The obtained compound was confirmed by nuclear magnetic resonance to be the compound represented by formula (I).

[0091] Example 7

[0092] This example provides a compound with anti-aging and soothing effects. The preparation method thereof is different from that of Example 1 only in that after the sample is loaded in step (2), after standing for 2 h, 2 BV of the compound is first eluted with water, then 4 BV of the compound is eluted with a 50% ethanol aqueous solution, and then 8 BV of the compound is eluted with an 80% ethanol aqueous solution. The eluate is collected, and other preparation conditions remain unchanged. Finally, 0.0131 g of the compound is obtained, and the yield is 0.0262%.

[0093] The obtained compound was confirmed by nuclear magnetic resonance to be the compound represented by formula (I).

[0094] Example 8

[0095] This example provides a compound with anti-aging and soothing effects. The preparation method thereof is different from that of Example 1 only in that isocratic elution is used in step (4), with mobile phase A: 70%, mobile phase B: 30%, and elution time 21 min. Other preparation conditions remain unchanged. Finally, 0.0113 g of the compound is obtained, with a yield of 0.0226%.

[0096] The obtained compound was confirmed by nuclear magnetic resonance to be the compound represented by formula (I).

[0097] Example 9

[0098] This embodiment provides a compound with anti-aging and soothing effects. The preparation method thereof is different from that of Example 1 only in that the elution procedure in step (4) is: 0-2 min, mobile phase A: 98-98%, mobile phase B: 2-2%; 2-17 min, mobile phase A: 98-10%, mobile phase B: 2-90%; 17-18 min, mobile phase A: 10-10%, mobile phase B: 90-90%; 18-19 min, mobile phase A: 10-98%, mobile phase B: 90-2%; 19-21 min, mobile phase A: 98-98%, mobile phase B: 2-2%. The other preparation conditions remain unchanged. Finally, 0.0098 g of the compound was obtained with a yield of 0.0196%.

[0099] The obtained compound was confirmed by nuclear magnetic resonance to be the compound represented by formula (I).

[0100] Comparative Example 1

[0101] This comparative example provides a compound with anti-aging and soothing effects. The preparation method thereof is different from that of Example 1 only in that in step (1), microwave-ultrasonic combined treatment is not performed, and after the light bamboo leaf powder is mixed with 0.5 kg of 30% ethanol aqueous solution, it is directly refluxed and extracted twice, each time for 2 h, and other preparation conditions remain unchanged. Finally, 0.0051 g of the compound is obtained, and the yield is 0.0102%.

[0102] The obtained compound was confirmed by nuclear magnetic resonance to be the compound represented by formula (I).

[0103] Comparative Example 2

[0104] This comparative example provides a compound with anti-aging and soothing effects. The preparation method thereof is different from that of Example 1 only in that the flocculation treatment in step (3) is not performed, and the eluate obtained in step (2) is directly subjected to the high-efficiency preparative liquid phase separation in step (4). Other preparation conditions remain unchanged, and 0.0078 g of the compound is finally obtained, with a yield of 0.0156%.

[0105] The obtained compound was confirmed by nuclear magnetic resonance to be the compound represented by formula (I).

[0106] Test Example 1

[0107] In this test example, tetracycline was used as a positive control to measure the collagenase inhibitory activity of the compound prepared in Example 1:

[0108] To each well of a 96-well plate, 20 μL of enzyme solution with different concentrations (10, 5, 2.5, 1.25, 0.625, 0.3125 U / mL), 120 μL of buffer salt, and 60 μL of sample solvent were added. After incubation at 37°C for 20 min, 40 μL of substrate solution was added. The total reaction volume was 240 μL. Two parallel groups were set up. The total enzyme kinetic cycle time was 40 mins. The absorbance at 330 nm was measured every 1 min, the average value was calculated, and the curve was drawn to find the most suitable enzyme concentration.

[0109] 40 μL of collagenase solution and 20 μL of 0.5 mg / mL of the compound prepared in Example 1, as well as 40 μL of collagenase solution and 20 μL of 0.5 mg / mL tetracycline were added to a 96-well plate, respectively. After incubation at 37° C. for 20 min, 40 μL of substrate solution FALGPA (N-(3-[2-furanyl]acryloyl)-Leu-Gly-Pro-Ala) was added, and the absorbance change value at 330 nm from 0 to 20 minutes was measured.

[0110] The inhibition rate of collagenase in the positive control group was 100%. When the concentration of the compound prepared in Example 1 was 0.5 mg / mL, the inhibition rate of collagenase was 38.69%. It can be seen that the compound prepared in Example 1 can effectively inhibit the activity of collagenase and achieve anti-aging effects.

[0111] Test Example 2

[0112] In this test example, tannic acid was used as a positive control to determine the hyaluronidase inhibitory activity of the compound prepared in Example 1:

[0113] Add 50 μL of hyaluronidase solution and 50 μL of 0.5 mg / mL of the compound prepared in Example 1 (sample group) to the high-throughput tube, set up a positive control group (replacing the compound in the sample group with tannic acid), set up a control group (replacing the hyaluronidase solution in the sample group or the positive control group with acetate buffer), set up a blank control group (adding 100 μL of acetate buffer), incubate at 37°C for 20 min, then add 100 μL of calcium chloride solution to each tube, incubate at 37°C for 20 min, then add 50 μL of sodium hyaluronate, wherein the control group replaces the sodium hyaluronate solution with acetate buffer, and incubate at 37°C for 40 min. After the incubation is completed, add 100 μL of sodium hydroxide solution and potassium borate solution to each tube, respectively, incubate at 85°C for 5 min, ice bath for 2 min, and place at room temperature for 5 min, take 100 μL of the reaction solution in a 96-well plate, and add 100 μL of color development solution to each well. The 96-well plate was quickly placed in an ELISA reader and shaken at 37°C for 1 min. The absorbance was measured at 585 nm 10 min after the color developing solution was added.

[0114] The inhibition rate of hyaluronidase in the positive control group was 100%. When the concentration of the compound prepared in Example 1 was 0.5 mg / mL, the inhibition rate of hyaluronidase was 26.59%. It can be seen that the compound prepared in Example 1 can effectively inhibit the activity of collagenase and achieve a soothing effect.

[0115] Test Example 3

[0116] This test example measured the inhibitory effects of the compound prepared in Example 1 on three inflammatory factors: IL-6, NO, and TNF-α:

[0117] Mouse macrophage RAW264.7 cells were selected and plated in 96-well plates. The initial cell number was 10 4 -10 5 After the cell coverage rate reached more than 90%, 120 μg / mL of the compound prepared in Example 1 was used for intervention for 1 h, and the control group was intervened with cell culture medium for 1 h. After intervention, LPS lipopolysaccharide was added and cultured for 24 h. The supernatant was collected, and the inflammatory factor generation ability of the sample was detected by the kit, and the absorbance was detected at 540 nm to test the relative expression of each inflammatory factor, and the anti-inflammatory effect of the compound was evaluated.

[0118] The compound obtained in Example 1 has an inhibition rate of 60% on IL-6, 30% on NO, and 40% on TNF-α at 120 μg / mL. It can be seen that the compound prepared in Example 1 can effectively inhibit IL-6, NO, and TNF-α inflammatory factors, and achieve anti-inflammatory and soothing effects.

[0119] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0120] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0121] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A compound with anti-aging and soothing effects, characterized in that: The structure of the compound is shown in formula (I): Formula (I).

2. A method for preparing the compound according to claim 1, characterized in that: The preparation method comprises the following steps: (1) mixing the raw material of bamboo leaves with ethanol aqueous solution and then subjecting it to microwave-ultrasonic combined treatment; (2) refluxing the treated solution to obtain an extract; (3) concentrating the extract under reduced pressure to obtain a concentrated solution; (4) Purifying and eluting the concentrated solution with a macroporous resin and collecting the eluate; (5) subjecting the eluate to flocculation treatment to obtain a Bambusa chinensis leaf extract; (6) The extract of the leaves of the bamboo shoots is separated by liquid chromatography and then dried to obtain the compound with anti-aging and soothing effects.

3. The preparation method according to claim 2, characterized in that: The volume fraction of the ethanol aqueous solution in step (1) is 20%-50%; The mass ratio of the ethanol aqueous solution to the light bamboo leaf raw material is (5-10):

1.

4. The preparation method according to claim 2, characterized in that: The temperature of the combined treatment in step (1) is 40-60°C; The power of the microwave treatment is 50-300 W, and the time of the microwave treatment is 15-25 min; The power of the ultrasonic treatment is 100-500 W, and the time of the ultrasonic treatment is 5-10 min.

5. The preparation method according to claim 2, characterized in that: The number of reflux extractions in step (2) is 1-3 times, and the time for each extraction is independently 1-3 h.

6. The preparation method according to claim 2, characterized in that: The purification and elution steps in step (4) include: after adsorption, standing for 2-12 hours, first eluting with water for 2-5 BV, then eluting with 5%-15% ethanol aqueous solution for 3-8 BV, and then eluting with 70%-90% ethanol aqueous solution for 5-10 BV.

7. The preparation method according to claim 2, characterized in that: The flocculation treatment in step (5) uses a flocculant, and the amount of the flocculant used is 0.1%-1% of the eluent; The flocculant consists of gelatin, ZTC1+1 and citric acid in a mass ratio of 1:(1-3):(2-10).

8. The preparation method according to claim 2, characterized in that: The chromatographic column used for the liquid chromatography separation in step (6) is a C18 column, and the column temperature of the chromatographic column is 25-35°C.

9. The preparation method according to claim 2, characterized in that: The mobile phase A of the liquid chromatography in step (6) is water, the mobile phase B is acetonitrile, and the elution method is gradient elution; The gradient elution procedure is as follows: 0-2 min, mobile phase A is maintained at 97%-99%, and mobile phase B is maintained at 1%-3%; 2-16 min, mobile phase A is uniformly reduced from 97%-99% to 68%-72%, and mobile phase B is uniformly increased from 1%-3% to 28%-32%; 16-17 min, mobile phase A is uniformly reduced from 68%-72% to 8%-12%, and mobile phase B is uniformly increased from 28%-32% to 88%-92%; 17-18 min, mobile phase A is maintained at 8-12%, and mobile phase B is maintained at 88-92%; 18-19 min, mobile phase A: 8%-12% is uniformly increased to 97-99%, and mobile phase B is uniformly reduced from 88%-92% to 1%-3%; 19-21 min, mobile phase A is maintained at 97-99%, and mobile phase B is maintained at 1%-3%.

10. Use of the compound with anti-aging and soothing effects according to claim 1 in the preparation of a product with the effect of inhibiting the expression of collagenase, hyaluronidase or inflammatory factors; The product is essence, lotion, emulsion, cream, facial mask or facial cleanser.

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