Compound with anti-aging and soothing effects and application thereof

Abstract

The invention provides a compound with anti-aging and soothing effects and application thereof, the compound has a structure as shown in formula (I), lophatherum gracile is used as a raw material, and a new compound is creatively separated from a lophatherum gracile extract through further separation, research and development. Experiments prove that the compound has the effects of inhibiting collagenase expression, inhibiting hyaluronidase expression and inhibiting expression of three inflammatory factors IL-6, NO and TNF-alpha, has excellent anti-aging, anti-inflammatory and soothing effects, and can be applied to cosmetics with anti-aging and soothing effects.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology and relates to a compound with anti-aging and soothing effects and its applications. Background Technology

[0002] Lophatherum gracile Brongn. is a perennial herb belonging to the genus Lophatherum in the family Poaceae (Barnhart). It is also known as mountain chicken rice, golden chicken rice, bewitching grass, and bamboo leaf heart. It mainly grows on mountain slopes, in woodlands or forest edges, and in shady places along roadsides at altitudes below 1200m.

[0003] The dried stems and leaves of *Lophatherum gracile* are a traditional Chinese herbal medicine, recorded in both the *Shennong Bencao Jing* and the *Bencao Gangmu*, belonging to the category of marshland herbs. Li Shizhen stated, "*Lophatherum gracile* grows everywhere in the fields. In spring, the seedlings grow to a height of several inches, with slender stems and green leaves, resembling the stems and leaves of slender bamboo that grows from fallen bamboo shoots. Its roots grow in clusters of dozens of fibrous roots, from which seeds form, similar to those of *Ophiopogon japonicus*, but harder. It relieves fever and irritability, promotes urination, and clears the mind." *Lophatherum gracile* has been included in all editions of the *Pharmacopoeia of the People's Republic of China* since 1977. It is sweet and bland in taste, and cold in nature, possessing the effects of clearing heat and purging fire, relieving irritability, and promoting urination. It is often used for symptoms such as feverish thirst, mouth ulcers, and painful urination.

[0004] Studies have shown that Lophatherum gracile extract has good ocular 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, immediately relieve skin stinging caused by lactic acid stimulation, and significantly reduce skin discomfort and redness caused by capsaicin stimulation. Lophatherum gracile mainly contains flavonoids, triterpenoids, phenolic acids, and other chemical components. Among them, flavonoid C-glycosides have certain antibacterial and antiviral effects, which are the main material basis for the heat-clearing and fire-purging effects of Lophatherum gracile. Other studies have shown that flavonoid C-glycosides have good skin care effects such as whitening, anti-oxidation, and anti-radiation.

[0005] Plant extracts, due to their complex composition, present challenges in controlling batch-to-batch stability. Isolating and purifying structurally and functionally defined chemical components from Lophatherum gracile leaves offers significant potential for drug and chemical raw material development, and provides new solutions for diseases and skin problems. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a compound with anti-aging and soothing effects and its applications.

[0007] To achieve this objective, the present invention adopts the following technical solution:

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

[0009] Formula (I).

[0010] This invention uses Lophatherum gracile leaves as raw material and creatively isolates a novel natural compound from its extract. Experimental verification revealed that this compound inhibits collagenase expression, hyaluronidase expression, and the expression of three inflammatory factors: IL-6, NO, and TNF-α. It exhibits excellent anti-aging, anti-inflammatory, and soothing effects, and therefore can be applied to anti-aging and soothing cosmetics. It is foreseeable that the same technical effects can be achieved by using any existing technical method in the field (either through isolation and purification from natural products or through organic synthesis).

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

[0012] (1) The raw material of light bamboo leaves was mixed with an ethanol aqueous solution and then subjected to microwave-ultrasound combined treatment;

[0013] (2) The treated solution is refluxed for extraction to obtain the extract;

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

[0015] (4) The concentrate was purified and eluted using a macroporous resin, and the eluent was collected;

[0016] (5) The eluent was subjected to flocculation treatment to obtain the Lophatherum gracile extract;

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

[0018] The above preparation method involves microwave-ultrasound combined treatment of the raw material of Lophatherum gracile, and flocculation treatment of the eluent after purification and elution with macroporous resin, combined with liquid chromatography separation. On the one hand, the compound shown in formula (I) can be obtained from the raw material of Lophatherum gracile. On the other hand, compared with other preparation processes, this preparation method also significantly improves the yield of the compound.

[0019] Preferably, the light bamboo leaf raw material mentioned in step (1) is light bamboo leaf powder obtained by crushing and passing it through a 70-90 mesh sieve (e.g., 70 mesh, 72 mesh, 74 mesh, 77 mesh, 80 mesh, 83 mesh, 86 mesh, 88 mesh, 90 mesh, etc.). Other specific point values ​​within this range can be selected, and will not be described in detail here.

[0020] The volume fraction of the ethanol aqueous solution in step (1) is 20%-50%, such as 20%, 21%, 23%, 26%, 30%, 35%, 40%, 45%, 50%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0021] This invention creatively discovers that using a reflux extraction with an ethanol aqueous solution of 20%-50% by volume can effectively improve the yield of the compound.

[0022] The mass ratio of the ethanol aqueous solution to the raw material of light bamboo leaves is (5-10):1. The specific point values ​​in (5-10) can be independently selected as 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. Other specific point values ​​within this range can also be selected, and will not be elaborated here.

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

[0024] The power of microwave processing in the combined processing is 50-300 W, such as 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 values ​​within this range can be selected, and will not be elaborated here.

[0025] The microwave processing time in the combined processing is 15-25 min, such as 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 range can be selected, which will not be elaborated here.

[0026] The power of the ultrasonic treatment in the combined processing is 100-500 W, such as 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 values ​​within this range can be selected, and will not be elaborated here.

[0027] The duration of ultrasonic treatment in the combined process is 5-10 minutes, such as 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, etc. Other specific values ​​within this range can be selected, which will not be elaborated here.

[0028] This invention creatively discovers that, in microwave-ultrasound combined treatment, using microwave power of 50-300 W and ultrasonic power of 100-500 W, compared to other power levels, can effectively improve the yield of the compound with anti-aging and soothing effects. Increased microwave power can raise the internal temperature of plant cells, accelerating cell wall rupture and the release of the target compound. However, excessively high microwave power can lead to excessively rapid heating, resulting in thermal damage or side reactions, thus affecting the extraction effect. Increased ultrasonic power shortens the time required for cavitation bubble collapse, enhancing the cavitation effect generated by ultrasound per unit time, which is beneficial for improving the extraction rate. However, excessively high ultrasonic power can produce a buffering effect, reducing the energy transfer rate and weakening the cavitation effect, and may also cause degradation or isomerization of the active ingredients. This invention also finds that microwave treatment for 15-25 min, ultrasonic treatment for 5-10 min, and a combined treatment temperature of 40-60℃ can further improve the yield of the compound with anti-aging and soothing effects.

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

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

[0031] Using the specific elution procedure described above, namely elution with water followed by elution with an aqueous ethanol solution, and combining it with an aqueous ethanol solution of a specific concentration, can effectively improve 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, 8 BV, 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 will not be elaborated 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 range can be selected, and will not be elaborated here.

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

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

[0036] The mass ratio of gelatin, ZTC1+1, and citric acid is 1:(1-3):(2-10). Specific 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. Specific 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. Other specific values ​​within the above ranges can be selected, and will not be elaborated further here.

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

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

[0039] The column temperature of the chromatographic column is 20-30℃, such as 20℃, 22℃, 25℃, 30℃, etc. Other specific point values ​​within this range can be selected, which will not be elaborated here.

[0040] Preferably, in step (6), the mobile phase A of the liquid chromatography 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... At 17-18 minutes, 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.), while 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.); at 18-19 minutes, mobile phase A remains at 8-12% (e.g., 8%, 9%, 10%, 11%, 12%, etc.), and mobile phase B remains at 88-92% (e.g., 88%, 89%, 90%, 91%, 92%, etc.); at 18-19 minutes... For 1 minute, mobile phase A is increased uniformly from 8%-12% (e.g., 8%, 9%, 10%, 11%, 12%) to 97-99% (e.g., 97%, 98%, 99%), while mobile phase B is decreased uniformly from 88%-92% (e.g., 88%, 89%, 90%, 91%, 92%) to 1%-3% (e.g., 1%, 2%, 3%). For 19-21 minutes, mobile phase A is maintained at 97-99% (e.g., 97%, 98%, 99%), and mobile phase B is maintained at 1%-3% (e.g., 1%, 2%, 3%). Other specific point values ​​within the above range can be selected, and will not be elaborated here.

[0042] The present invention creatively discovers that by employing the above-described gradient elution process, the yield of the compound with anti-aging and soothing effects can be effectively improved.

[0043] Preferably, the gradient elution flow rate is 7-13 mL / min, such as 7 mL / min, 8 mL / min, 9 mL / min, 10 mL / min, 11 mL / min, 12 mL / min, 13 mL / min, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

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

[0045] Other specific point values ​​within the above range can be selected, and will not be elaborated on here.

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

[0047] The products include serums, toners, lotions, creams, masks, or facial cleansers.

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

[0049] This invention uses Lophatherum gracile as raw material and creatively isolates a new compound from Lophatherum gracile extract. Experimental verification has shown that this compound has the effects of inhibiting collagenase expression, inhibiting hyaluronidase expression, and inhibiting the expression of three inflammatory factors IL-6, NO, and TNF-α. It has excellent anti-aging, anti-inflammatory and soothing effects. Therefore, this compound can be applied to anti-aging and soothing cosmetics. Attached Figure Description

[0050] Figure 1 This is the 1H NMR spectrum of the compound obtained in Example 1 of this invention;

[0051] Figure 2 This is the carbon NMR spectrum of the compound obtained in Example 1 of this invention;

[0052] Figure 3 This is the DEPT 135 nuclear magnetic resonance spectrum of the compound obtained in Example 1 of this invention. Detailed Implementation

[0053] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0054] Unless otherwise specified, the reagents and consumables used in the following embodiments were purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and techniques used were conventional methods and techniques in the art.

[0055] The gelatin used in the following examples was purchased from Shanghai McLean 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] Its preparation method is as follows:

[0060] (1) Take 50 g of light bamboo leaves, grind them into 80 mesh fine powder, mix them with 0.5 kg of 30% ethanol aqueous solution, and then perform microwave-ultrasound combined treatment at 50℃. The microwave power is 100 W and the microwave time is 20 min. The ultrasonic power is 250 W and the ultrasonic time is 8 min. The treated light bamboo leaf powder mixture is refluxed and extracted twice, each time for 2 h. The extracts are then combined.

[0061] (2) The combined extract was concentrated to 25 g under reduced pressure at 60℃. The concentrate was centrifuged at 3000 rpm. The supernatant was kept for later use. The precipitate was reconstituted with 30 g of water and centrifuged again. The supernatant obtained by centrifugation was combined with the supernatant from the first centrifugation and kept for later use. 220 g of D-101 macroporous resin was packed into a column. After elution with pure water until there was no alcohol smell, the combined centrifugation supernatant was used for adsorption. The adsorption rate was 0.5 BV / h. After the adsorption was completed, the column 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 eluent was collected.

[0062] (3) Add 0.5% of a flocculant composed 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 light bamboo leaf extract.

[0063] (4) The extract of *Lophatherum gracile* was separated and purified using high-performance liquid chromatography (HPLC). The HPLC column used was an XBridge Prep with an inner diameter of 19 mm and a length of 150 mm. The packing material was OBD C18 with a particle size of 5 μm. The HPLC conditions 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 B: 90-90%; 18-19 min, mobile phase B: 90-90%; 19-10 ... The elution flow rate was 10 mL / min, the detection wavelength was 270 nm, the preparative chromatographic column temperature was 25 °C, and 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 using nuclear magnetic resonance (NMR), and the resulting proton NMR spectrum is shown below. Figure 1 As shown, the obtained carbon NMR spectrum is as follows: Figure 2 As shown, the obtained NMR DEPT 135 spectrum is as follows. Figure 3 As shown.

[0065] Depend on Figure 1 and Figure 2 It can be known 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 light bamboo leaves, grind them into 70 mesh fine powder, mix them with 0.25 kg of 40% ethanol aqueous solution, and then perform microwave-ultrasound combined treatment at 60℃. The microwave power is 280 W and the microwave time is 18 min. The ultrasonic power is 150 W and the ultrasonic time is 10 min. The treated light bamboo leaf powder mixture is refluxed and extracted 3 times, 1 h each time. The extracts are combined.

[0071] (2) The combined extract was concentrated to 25 g under reduced pressure at 60℃. The concentrate was centrifuged at 3000 rpm. The supernatant was kept for later use. The precipitate was reconstituted with 30 g of water and centrifuged again. The supernatant obtained by centrifugation was combined with the supernatant from the first centrifugation and kept for later use. 220 g of AB-8 macroporous resin was packed into a column. After elution with pure water until there was no alcohol odor, the combined centrifugation supernatant was used for adsorption. The adsorption rate was 0.5 BV / h. After the adsorption was completed, the column 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 eluent was collected.

[0072] (3) Add 0.1% 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 the Lophatherum gracile extract.

[0073] (4) The extract of *Lophatherum gracile* was separated and purified using high-performance liquid chromatography (HPLC). The HPLC column used was an XBridge Prep with an inner diameter of 19 mm and a length of 150 mm. The packing material was OBD C18 with a particle size of 5 μm. The HPLC conditions 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: 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 B: 92-92%; 18-19 min, mobile phase B: 92-92%; 19-10 min, mobile phase B: 92-92%; 19-10 min, mobile phase B: 92-92%; 19-10 min, mobile phase B: 97-68%, ...7-68%, mobile phase B: 97-6 The elution flow rate was 7 mL / min, the detection wavelength was 270 nm, the preparative chromatographic column temperature was 30 °C, and the solution was dried to obtain 0.0153 g of the compound with anti-aging and soothing effects, with a yield of 0.0306%.

[0074] After verification by nuclear magnetic resonance, the obtained compound is the compound shown in 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 light bamboo leaves, grind them into 90 mesh fine powder, mix them with 0.4 kg of 25% ethanol aqueous solution, and then perform microwave-ultrasound combined treatment at 55℃. The microwave power is 100 W and the microwave time is 25 min. The ultrasonic power is 400 W and the ultrasonic time is 5 min. The treated light bamboo leaf powder mixture is refluxed and extracted once, each time for 3 h, to obtain the extract.

[0078] (2) The extract was concentrated to 25 g under reduced pressure at 60℃. The concentrate was centrifuged at 3000 rpm. The supernatant was kept for later use. The precipitate was reconstituted with 30 g of water and centrifuged again. The supernatant obtained by centrifugation was combined with the supernatant obtained by the first centrifugation and kept for later use. 220 g of D-101 macroporous resin was packed into the column. After elution with pure water until there was no alcohol smell, the combined supernatant was used for adsorption. The adsorption rate was 0.5 BV / h. After the adsorption was completed, the column 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 eluent was collected.

[0079] (3) Add 1% 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 the Lophatherum gracile extract.

[0080] (4) The extract of *Lophatherum gracile* was separated and purified using high-performance liquid chromatography (HPLC). The HPLC column used was an XBridge Prep with an inner diameter of 19 mm and a length of 150 mm. The packing material was OBD C18 with a particle size of 5 μm. The HPLC conditions 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: 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: 12-12%, mobile phase B: 88-88%; 18-19 min, mobile phase B ... The elution flow rate was 13 mL / min, the detection wavelength was 270 nm, the preparative chromatographic column temperature was 35 °C, and after drying, 0.0147 g of the compound with anti-aging and soothing effects was obtained, with a yield of 0.0294%.

[0081] After verification by nuclear magnetic resonance, the obtained compound is the compound shown in formula (I).

[0082] Example 4

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

[0084] After verification by nuclear magnetic resonance, the obtained compound is the compound shown in formula (I).

[0085] Example 5

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

[0087] After verification by nuclear magnetic resonance, the obtained compound is the compound shown in formula (I).

[0088] Example 6

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

[0090] After verification by nuclear magnetic resonance, the obtained compound is the compound shown in formula (I).

[0091] Example 7

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

[0093] After verification by nuclear magnetic resonance, the obtained compound is the compound shown in formula (I).

[0094] Example 8

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

[0096] After verification by nuclear magnetic resonance, the obtained compound is the compound shown in formula (I).

[0097] Example 9

[0098] This embodiment provides a compound with anti-aging and soothing effects. The preparation method differs from that of Example 1 only in the elution procedure in step (4): 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%. Other preparation conditions remain unchanged, and finally 0.0098 g of the compound is obtained, with a yield of 0.0196%.

[0099] After verification by nuclear magnetic resonance, the obtained compound is the compound shown in formula (I).

[0100] Comparative Example 1

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

[0102] After verification by nuclear magnetic resonance, the obtained compound is the compound shown in formula (I).

[0103] Comparative Example 2

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

[0105] After verification by nuclear magnetic resonance, the obtained compound is the compound shown in formula (I).

[0106] Test Example 1

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

[0108] Add 20 μL of enzyme solution, 120 μL of buffer salt, and 60 μL of sample solvent at different concentrations (10, 5, 2.5, 1.25, 0.625, 0.3125 U / mL) to each well of a 96-well plate. After incubation at 37℃ for 20 min, add 40 μL of substrate solution, for a total reaction volume of 240 μL. Set up two parallel groups. The total enzyme kinetic cycle time is 40 mins. Measure the absorbance at 330 nm every 1 min, calculate the average value, plot the curve, and find the optimal enzyme concentration.

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

[0110] The positive control group showed a 100% inhibition rate of collagenase. In Example 1, the compound prepared at a concentration of 0.5 mg / mL showed a 38.69% inhibition rate of collagenase. This indicates that the compound prepared in Example 1 can effectively inhibit the activity of collagenase and achieve anti-aging effects.

[0111] Test Example 2

[0112] This test example uses tannic acid 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 the compound prepared in Example 1 (0.5 mg / mL) to high-throughput tubes (sample group). Set up a positive control group (replacing the compound in the sample group with tannic acid), a control group (replacing the hyaluronidase solution in the sample group or positive control group with acetate buffer), and 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 and incubate at 37°C for 20 min. Next, add 50 μL of sodium hyaluronate, with the control group using acetate buffer instead of sodium hyaluronate solution. Incubate at 37°C for 40 min. After incubation, add 100 μL of sodium hydroxide solution and potassium borate solution to each tube, incubate at 85°C for 5 min, on ice for 2 min, and at room temperature for 5 min. Transfer 100 μL of the reaction solution to a 96-well plate and add 100 μL of colorimetric reagent to each well. The 96-well plate was quickly placed in an ELISA reader and shaken at 37°C for 1 min. After adding the chromogenic solution and 10 min later, the absorbance was measured at 585 nm.

[0114] The positive control group showed a 100% inhibition rate against hyaluronidase. In Example 1, the compound prepared at a concentration of 0.5 mg / mL showed a 26.59% inhibition rate against hyaluronidase. This indicates that the compound prepared in Example 1 can effectively inhibit collagenase activity and achieve a soothing effect.

[0115] Test Example 3

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

[0117] Mouse macrophages RAW264.7 were selected and seeded in 96-well plates with an initial cell number of 10. 4 -10 5 After the cell coverage reached over 90%, the cells were treated with the compound prepared in Example 1 at a concentration of 120 μg / mL for 1 h, while the control group was treated with cell culture medium for 1 h. LPS lipopolysaccharide was added after the intervention, and the cells were cultured for another 24 h. The supernatant was collected, and the ability of the samples to generate inflammatory factors was detected using a kit. Absorbance was measured at 540 nm to determine the relative expression levels of each inflammatory factor and evaluate the anti-inflammatory efficacy of the compound.

[0118] The compound obtained in Example 1 showed an inhibition rate of 60% against IL-6, 30% against NO, and 40% against TNF-α at a concentration of 120 μg / mL. This indicates that the compound prepared in Example 1 can effectively inhibit IL-6, NO, and TNF-α inflammatory factors, achieving anti-inflammatory and soothing effects.

[0119] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0120] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within 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 describe the various possible combinations separately.

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. The compound according to claim 1, characterized in that The compound is prepared by a method comprising 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 compound 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 compound 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 compound 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 compound 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 compound 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 compound 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 compound 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 any one of claims 1 to 9 in the preparation of a product with the effect of inhibiting the expression of collagenase, hyaluronidase or inflammatory factors; The products include essence, lotion, emulsion, cream, mask or cleanser.

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