Chrysanthemum polypeptide and application thereof

Abstract

The invention discloses a saussurea involucrata polypeptide and application thereof, and belongs to the technical field of polypeptides, the saussurea involucrata polypeptide comprises saussurea involucrata tripeptide-1, saussurea involucrata pentapeptide-4, saussurea involucrata tripeptide-5, saussurea involucrata hexapeptide-8, saussurea involucrata hexapeptide-9 or saussurea involucrata acyl snake venom tripeptide, and the saussurea involucrata polypeptide is prepared through an amide condensation method of saussurea involucrata acid. The invention has the following beneficial effects: the anti-wrinkle, firming, soothing and anti-oxidation effects of the thousand-day chrysanthemum acyl tripeptide-1, the thousand-day chrysanthemum acyl pentapeptide-4, the thousand-day chrysanthemum acyl tripeptide-5, the thousand-day chrysanthemum acyl hexapeptide-8, the thousand-day chrysanthemum acyl hexapeptide-9 or the thousand-day chrysanthemum acyl snake venom tripeptide have obvious effects on the anti-wrinkle, firming, soothing and anti-oxidation effects; particularly, the instant wrinkle removal effect is particularly outstanding.

Description

Technical Field:

[0001] The present invention belongs to the technical field of polypeptides, and more specifically relates to spilanthes polypeptides and their applications, a group of spilanthes polypeptide derivatives with excellent performance and their applications, and in particular, a group of polypeptides with high biological activity, stability, and remarkable efficacy. Background Art:

[0002] Polypeptides are core anti-aging components that have won the Nobel Prize multiple times. Their basic building blocks are amino acids, which are formed by dehydration condensation in a specific sequence and are essentially the same as proteins. Common beauty peptides are generally short peptides composed of 2-10 amino acids, and their mechanisms are clear and generally act on a certain microscopic receptor (usually called a target) on the skin, with different effects such as anti-wrinkle, whitening, moisturizing, antioxidant, and repair.

[0003] With the progress of society, the development of science and technology, and the improvement of living standards, people's awareness of pursuing natural, green, healthy, and safe has increased day by day, and green and functional cosmetics are becoming increasingly popular. With the progress of modern extraction technology and analysis technology, the active ingredients and related action mechanisms of plant extracts have gradually been discovered and confirmed.

[0004] In the beauty market, due to the green background of plant extracts, they are already loved by consumers. With the use of many synthetic chemical components being restricted, "green" will surely become the development direction of cosmetics. The development of natural plant extracts that can be applied to anti-aging cosmetics has become a trend.

[0005] Spilanthes is a plant of the Compositae, one of the nine major plant families, and its origin is South Africa. It is an annual herbaceous plant and has now appeared all over the world. Spilanthes is a traditional food ingredient, with a strong pungent and refreshing effect, is famous for its strong anesthetic effect, and is also used to treat toothache and headache. In recent years, it has gradually been applied to cosmetics.

[0006] Gattefossé SAS applied for a patent with the title: Using spilanthes extract (utilizing its botulinum-like effect) as a cosmetic ingredient for anti-wrinkle efficacy. The main purpose of the patent is to develop a product with an effect similar to botulinum toxin (inhibiting muscle contraction to reduce expression lines). Especially when in the form of spilanthes extract, it can effectively inhibit the muscle contraction activity of the skin. Spilanthol in the form of spilanthes extract has a better effect on inhibiting muscle contraction than pure Spilanthol.

[0007] Belfer Cosmetics LLC applied for a patent related to a combination of a polypeptide and spilanthes extract, which reduces skin deformation by restricting the muscle contraction of facial skin, and this plant extract can enhance the effect of the polypeptide on reducing expression lines.

[0008] At present, there is no report on the polypeptides of Centaurea cyanus L.. Summary of the Invention:

[0009] To solve the above problems, the purpose of the present invention is to provide a group of polypeptides of Centaurea cyanus L. with superior performance, which are mainly applied to the field of beauty skin care,

[0010] The specific technical solution for the present invention to solve the above technical problems is: Centaureoyl tripeptide-1, Centaureoyl pentapeptide-4, Centaureoyl tripeptide-5, Centaureoyl hexapeptide-8, Centaureoyl hexapeptide-9 or Centaureoyl snake venom-like tripeptide, which are prepared by using centaureic acid through amide condensation,

[0011] The polypeptides of Centaurea cyanus L. have the general structural formulas shown in Formula (1), Formula (2), Formula (3), Formula (4), Formula (5), and Formula (6);

[0012] Formula (1) is

[0013] Formula (2) is

[0014] Formula (3) is

[0015] Formula (4) is

[0016] Formula (5) is

[0017] Formula (6) is

[0018] The polypeptides of Centaurea cyanus L. in Formula (1), Formula (2), Formula (3), Formula (4), Formula (5), and Formula (6) have the effects of anti-wrinkle, firming, soothing, and antioxidant.

[0019] The polypeptides of Centaurea cyanus L. in Formula (1), Formula (2), Formula (3), Formula (4), Formula (5), and Formula (6) have the effect of instant wrinkle removal.

[0020] The beneficial effects of the present invention are:

[0021] Centaureoyl tripeptide-1, Centaureoyl pentapeptide-4, Centaureoyl tripeptide-5, Centaureoyl hexapeptide-8, Centaureoyl hexapeptide-9 or Centaureoyl snake venom-like tripeptide have significant effects in anti-wrinkle, firming, soothing, and antioxidant effects;

[0022] In particular: The present invention creatively discovers that Centaureoyl tripeptide-1, Centaureoyl pentapeptide-4, Centaureoyl tripeptide-5, Centaureoyl hexapeptide-8, Centaureoyl hexapeptide-9 or Centaureoyl snake venom-like tripeptide are particularly prominent in the instant wrinkle removal effect. Detailed Embodiments:

[0023] In the description of the present invention, specific details are only for a full understanding of the embodiments of the present invention. However, those skilled in the art should know that the implementation of the present invention is not limited to these details. Additionally, well-known structures and functions are not described or shown in detail to avoid obscuring the key points of the embodiments of the present invention. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0024] Specific embodiments of the present invention:

[0025] For a better understanding of the present invention, specific embodiments are used for illustration. It is emphasized that the effects of this embodiment have no substantial differences from various embodiments within the protection scope of the present invention, including the respective reagents and the content ratios of the reagents, and can all achieve the effects described in the present invention and solve the above problems. Other combinations are not elaborated here;

[0026] Example 1: Comfreyoyl Tripeptide-1

[0027] Comfreyoyl Tripeptide-1 was prepared by the method of amide condensation using comfreyic acid, and its structure is shown in formula (1):

[0028]

[0029] The specific preparation process is as follows:

[0030] Step 1: In a 500 mL three-necked flask, add 80 g of THF, 16.62 g of HOSu, and 20 g of comfreyic acid. After stirring until dissolved clearly, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (29.79 g of DCC dissolved in 20 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 100 g of water and 9.03 g of glycine, control the pH of the feed liquid to 7.0 - 7.5, and keep the temperature for reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract with 100 g of ethyl acetate once. The ethyl acetate phase is washed with acidic water with a pH of 2.0 three times, 100 g each time. The washed ethyl acetate phase is then washed with 100 g of saturated sodium chloride once, dried with 10 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 25.5 g of a white solid, with a yield of 94.9%.

[0031] Step 2: In a 500 mL three-necked flask, add 102 g of THF, 15.77 g of HOSu, and 25.5 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and slowly add a THF solution of DCC (28.28 g of DCC dissolved in 25.5 g of THF). After adding, react at an internal temperature of 25 °C for 3 h. Add 130 g of water and 17.72 g of histidine, control the pH of the feed liquid to be 7.0 - 7.5, and maintain the reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0 and extract once with 150 g of ethyl acetate. Wash the ethyl acetate phase three times with acidic water with a pH of 2.0, 100 g each time. Wash the washed ethyl acetate phase once with 100 g of saturated sodium chloride, dry with 15 g of anhydrous magnesium sulfate, and filter. Concentrate the filtrate at an external temperature of 45 °C until no more evaporation occurs to obtain 38.4 g of an off-white solid with a yield of 93.3%.

[0032] Step 3: In a 1000 mL three-necked flask, add 153.6 g of THF, 14.71 g of HOSu, and 38.4 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and slowly add a THF solution of DCC (26.38 g of DCC dissolved in 38.4 g of THF). After adding, react at an internal temperature of 25 °C for 3 h. Add 200 g of water and 26.24 g of Lys(Boc), control the pH of the feed liquid to be 7.0 - 7.5, and maintain the reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0 and extract once with 200 g of ethyl acetate. Wash the ethyl acetate phase three times with acidic water with a pH of 2.0, 150 g each time. Wash the washed ethyl acetate phase once with 150 g of saturated sodium chloride, dry with 20 g of anhydrous magnesium sulfate, and filter. Concentrate the filtrate at an external temperature of 45 °C until no more evaporation occurs to obtain 57.7 g of an off-white solid with a yield of 92.0%.

[0033] Step 4: In a 500 mL three-necked flask, add 120 mL of hydrochloric acid and 57.7 g of the solid from the previous step. Stir and raise the internal temperature to 90 °C and react for 3 h. Then add 300 g of water, adjust the pH of the feed liquid to 7.5 - 7.8, precipitate a white solid, stir for 2 h, and filter by suction to obtain a white solid. Vacuum dry the white solid at 45 °C to obtain a crude peptide powder. Purify it by reverse-phase C18 preparative chromatography and lyophilize to obtain chrysanthemoyl tripeptide-1, whose chemical structure is formula (1).

[0034] 1H-NMR (300 MHz, D2O), δ ppm: 13.00 (s, 1H), δ ppm: 12.66 (s, 1H), δ ppm: 8.73 (s, 1H), δ ppm: 7.66 (s, 1H), δ ppm: 8.32 - 8.38 (s, 3H), δ ppm: 6.26 (d, 1H), δ ppm: 5.47 - 6.82 (m, 5H), δ ppm: 4.92 (t, 1H), δ ppm: 4.55 (t, 1H), δ ppm: 3.85 (s, 2H), δ ppm: 2.69 - 3.17 (m, 4H), δ ppm: 1.25 - 2.00 (m, 15H); C 24 H 36 N6O5, [M + H] + = 489.27, [M - H] - = 487.27;

[0035] Example 2: Centaureoyl Pentapeptide-4

[0036] Centaureoyl Pentapeptide-4 was prepared by the method of amide condensation using centaureic acid, and its structure is shown in formula (2):

[0037]

[0038] The specific preparation process is as follows:

[0039] Step 1: In a 500 mL three-necked flask, add 80 g of THF, 16.62 g of HOSu, and 20 g of centaureic acid. After stirring to dissolve clearly, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (29.79 g of DCC dissolved in 20 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 100 g of water and 29.64 g of Lys(Boc), control the pH of the feed liquid to 7.0 - 7.5, and keep the temperature for reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract with 150 g of ethyl acetate once. The ethyl acetate phase is washed with acidic water with a pH of 2.0 three times, 100 g each time. The washed ethyl acetate phase is washed with 100 g of saturated sodium chloride once and dried with 15 g of anhydrous magnesium sulfate, and then filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 46.5 g of a white solid, with a yield of 94.1%.

[0040] Step 2: In a 1000 mL three-necked flask, add 186 g of THF, 16.28 g of HOSu, and 46.5 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (29.18 g of DCC dissolved in 46.5 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 200 g of water and 14.04 g of threonine, control the pH of the feed liquid to be 7.0 - 7.5, and carry out a heat preservation reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 150 g of ethyl acetate. The ethyl acetate phase is washed three times with acidic water with a pH of 2.0, 100 g each time. The washed ethyl acetate phase is further washed once with 100 g of saturated sodium chloride, dried with 15 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 54.3 g of a white solid, with a yield of 92.9%.

[0041] Step 3: In a 1000 mL three-necked flask, add 217.2 g of THF, 15.13 g of HOSu, and 54.3 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (27.13 g of DCC dissolved in 54.3 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 250 g of water and 13.05 g of threonine, control the pH of the feed liquid to be 7.0 - 7.5, and carry out a heat preservation reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 250 g of ethyl acetate. The ethyl acetate phase is washed three times with acidic water with a pH of 2.0, 150 g each time. The washed ethyl acetate phase is further washed once with 150 g of saturated sodium chloride, dried with 25 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 60.0 g of a white solid, with a yield of 91.7%.

[0042] Step 4: In a 1000 mL three-necked flask, add 240.0 g of THF, 13.89 g of HOSu, and 60.0 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (24.90 g of DCC dissolved in 60.0 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 300 g of water and 24.77 g of Lys(Boc), control the pH of the feed liquid to be 7.0 - 7.5, and carry out a heat preservation reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 300 g of ethyl acetate. The ethyl acetate phase is washed three times with acidic water with a pH of 2.0, 200 g each time. The washed ethyl acetate phase is further washed once with 200 g of saturated sodium chloride, dried with 30 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 75.0 g of a white solid, with a yield of 90.4%.

[0043] Step 5: In a 1000 mL three-necked flask, add 260 g of THF, 12.55 g of HOSu, and 75 g of the solid from the previous step. After stirring until clear, maintain the internal temperature at 5 - 10 °C and slowly add a THF solution of DCC (22.51 g of DCC dissolved in 75 g of THF). After addition, react at an internal temperature of 25 °C for 3 h. Add 300 g of water and 9.55 g of serine, adjust the pH of the material solution to 7.0 - 7.5, and keep the temperature for reaction for 2 h. After completion of the reaction detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0 and extract with 300 g of ethyl acetate once. Wash the ethyl acetate phase three times with acidic water at pH 2.0, 200 g each time. Wash the washed ethyl acetate phase once with 200 g of saturated sodium chloride, dry with 30 g of anhydrous magnesium sulfate, and filter. Concentrate the filtrate at an external temperature of 45 °C until no more distillate comes out, obtaining 74.8 g of an off-white solid with a yield of 90.2%.

[0044] Step 6: In a 1000 mL three-necked flask, add 225 mL of hydrochloric acid and 74.8 g of the solid from the previous step. Stir and raise the internal temperature to 90 °C for reaction for 3 h. Then add 300 g of water, adjust the pH of the material solution to 8.0 - 9.0, precipitate white solid, stir for 2 h, and filter by suction to obtain white solid. Vacuum dry the white solid at 45 °C to obtain crude peptide powder. Purify by reversed-phase C18 preparative chromatography and lyophilize to obtain chrysanthemumoyl pentapeptide-4, whose chemical structure is Formula (2).

[0045] 1 H-NMR (300 MHz, D2O), δ ppm: 12.39 (s, 1H), δ ppm: 8.32 - 8.38 (s, 5H), δ ppm: 6.62 (m, 1H), δ ppm: 5.47 - 6.26 (m, 5H), δ ppm: 5.37 (s, 2H), δ ppm: 4.94 (s, 1H), δ ppm: 4.44 - 4.62 (m, 6H), δ ppm: 3.89 - 4.06 (m, 3H), δ ppm: 2.69 (t, 4H), δ ppm: 1.25 - 2.00 (m, 16H), δ ppm: 1.58 (d, 3H), δ ppm: 1.50 (s, 4H), δ ppm: 1.16 (d, 6H);

[0046] C 33 H 57 N7O 10 , [M + H] + = 712.42, [M - H] - = 710.42;

[0047] Example 3: Chrysanthemumoyl tripeptide-5

[0048] Chrysanthemumoyl tripeptide-5 was prepared by the method of amide condensation using chrysanthemumic acid, and its structure is shown in Formula (3):

[0049]

[0050] The specific preparation process is as follows:

[0051] Step 1: In a 500 mL three-necked flask, add 80 g of THF, 16.62 g of HOSu, and 20 g of centaureic acid. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (29.79 g of DCC dissolved in 20 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 100 g of water and 29.64 g of Lys(Boc), control the pH of the feed liquid to be 7.0 - 7.5, and carry out a heat-insulated reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0 and extract once with 150 g of ethyl acetate. Wash the ethyl acetate phase three times with acidic water with a pH of 2.0, 100 g each time. Wash the washed ethyl acetate phase once with 100 g of saturated sodium chloride, dry with 15 g of anhydrous magnesium sulfate, and filter. Concentrate the filtrate at an external temperature of 45 °C until no more distillate comes out, obtaining 46.3 g of a white solid-like substance with a yield of 93.7%.

[0052] Step 2: In a 1000 mL three-necked flask, add 185.2 g of THF, 16.21 g of HOSu, and 46.3 g of the solid obtained in the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (29.06 g of DCC dissolved in 46.3 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 200 g of water and 13.75 g of valine, control the pH of the feed liquid to be 7.0 - 7.5, and carry out a heat-insulated reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0 and extract once with 150 g of ethyl acetate. Wash the ethyl acetate phase three times with acidic water with a pH of 2.0, 100 g each time. Wash the washed ethyl acetate phase once with 100 g of saturated sodium chloride, dry with 15 g of anhydrous magnesium sulfate, and filter. Concentrate the filtrate at an external temperature of 45 °C until no more distillate comes out, obtaining 54.1 g of a white solid-like substance with a yield of 93.4%.

[0053] Step 3: In a 1000 mL three-necked flask, add 216.4 g of THF, 15.14 g of HOSu, and 54.1 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (27.14 g of DCC dissolved in 54.1 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 300 g of water and 26.99 g of Lys(Boc), control the pH of the feed liquid to be 7.0 - 7.5, and carry out a heat-preservation reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 300 g of ethyl acetate. The ethyl acetate phase is washed three times with acidic water with a pH of 2.0, 200 g each time. The washed ethyl acetate phase is then washed once with 200 g of saturated sodium chloride, dried with 30 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more evaporation occurs, obtaining 72.1 g of a white solid-like substance with a yield of 91.1%.

[0054] Step 4: In a 500 mL three-necked flask, add 200 mL of hydrochloric acid and 72.1 g of the solid from the previous step. Stir and raise the internal temperature to 90 °C and react for 3 h; then add 300 g of water, adjust the pH of the feed liquid to 8.0 - 9.0, precipitate white solid, stir for 2 h, and carry out suction filtration to obtain white solid; vacuum-dry the white solid at 45 °C to obtain crude peptide powder; after purification by reverse-phase C18 preparative chromatography and freeze-drying, chrysanthemum acyl tripeptide-5 is obtained, and its chemical structure is the formula (3).

[0055] 1 H-NMR(300MHz,D2O),δppm:12.66(s,1H),δppm:8.32 - 8.38(s,3H),δppm:6.26(d,1H),δppm:5.47 - 6.62(m,5H),δppm:4.34 - 4.55(m,3H),δppm:2.69 - 2.73(m,5H),δppm:2.69 - 3.17(m,4H),δppm:1.25 - 2.00(m,19H),δppm:1.50(d,6H);

[0056] C 27 H 47 N5O5,[M + H] + =522.36,[M - H] - =520.36;

[0057] Example 4: Chrysanthemum acyl hexapeptide-8

[0058] Chrysanthemum acyl hexapeptide-8 is prepared by the method of amide condensation using chrysanthemum acid, and its structure is as shown in formula (4):

[0059]

[0060] The specific preparation process is as follows:

[0061] Step 1: In a 500 mL three-necked flask, add 80 g of THF, 16.62 g of HOSu, and 20 g of centauryic acid. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (29.79 g of DCC dissolved in 20 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 100 g of water and 19.39 g of L-glutamic acid 5-methyl ester, control the pH of the material liquid to 7.0 - 7.5, and maintain the reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0 and extract once with 150 g of ethyl acetate. The ethyl acetate phase is washed three times with acidic water with a pH of 2.0, 100 g each time. The washed ethyl acetate phase is then washed once with 100 g of saturated sodium chloride, dried with 15 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more evaporation occurs, obtaining 34.0 g of an off-white solid with a yield of 91.3%.

[0062] Step 2: In a 1000 mL three-necked flask, add 136 g of THF, 15.18 g of HOSu, and 34 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (27.21 g of DCC dissolved in 34 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 200 g of water and 17.71 g of L-glutamic acid 5-methyl ester, control the pH of the material liquid to 7.0 - 7.5, and maintain the reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0 and extract once with 150 g of ethyl acetate. The ethyl acetate phase is washed three times with acidic water with a pH of 2.0, 100 g each time. The washed ethyl acetate phase is then washed once with 100 g of saturated sodium chloride, dried with 15 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more evaporation occurs, obtaining 45.2 g of an off-white solid with a yield of 90.9%.

[0063] Step 3: In a 1000 mL three-necked flask, add 180.8 g of THF, 13.8 g of HOSu, and 45.2 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (24.73 g of DCC dissolved in 45.2 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 250 g of water and 14.9 g of methionine, control the pH of the material liquid to 7.0 - 7.5, and maintain the reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0 and extract once with 250 g of ethyl acetate. The ethyl acetate phase is washed three times with acidic water with a pH of 2.0, 200 g each time. The washed ethyl acetate phase is then washed once with 200 g of saturated sodium chloride, dried with 25 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more evaporation occurs, obtaining 52.5 g of an off-white solid with a yield of 90.0%.

[0064] Step 4: In a 1000 mL three-necked flask, add 210 g of THF, 12.11 g of HOSu, and 52.5 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (21.71 g of DCC dissolved in 45.2 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 250 g of water and 12.81 g of glutamine, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract with 250 g of ethyl acetate once. Wash the ethyl acetate phase with acidic water with a pH of 2.0 three times, 200 g each time. Wash the washed ethyl acetate phase with 200 g of saturated sodium chloride once, dry with 25 g of anhydrous magnesium sulfate, and filter. Concentrate the filtrate at an external temperature of 45 °C until no more distillate comes out, obtaining 57.8 g of a pale white solid with a yield of 90.3%.

[0065] Step 5: In a 1000 mL three-necked flask, add 231.2 g of THF, 11.21 g of HOSu, and 57.8 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (20.10 g of DCC dissolved in 57.8 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 300 g of water and 14.15 g of arginine, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract with 250 g of n-butanol once. Wash the n-butanol phase with acidic water with a pH of 2.0 three times, 200 g each time. Wash the washed n-butanol phase with 200 g of saturated sodium chloride once, dry with 25 g of anhydrous magnesium sulfate, and filter. Concentrate the filtrate at an external temperature of 45 °C until no more distillate comes out, obtaining 62.7 g of a pale white solid with a yield of 89.0%.

[0066] Step 6: In a 1000 mL three-necked flask, add 250.8 g of THF, 9.98 g of HOSu, and 62.7 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (17.8 g of DCC dissolved in 62.7 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 350 g of water and 12.51 g of arginine, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract with 350 g of n-butanol once. Wash the n-butanol phase with acidic water with a pH of 2.0 three times, 300 g each time. Wash the washed n-butanol phase with 300 g of saturated sodium chloride once, dry with 35 g of anhydrous magnesium sulfate, and filter. Concentrate the filtrate at an external temperature of 45 °C until no more distillate comes out, obtaining 60 g of a pale white solid with a yield of 81.2%.

[0067] Step 7: In a 500 mL three-necked flask, add 180 g of THF, 180 g of water, and 60 g of the solid from the previous step. Stir and raise the internal temperature to 45 °C, control the pH = 11.5 - 12.0, and react for 3 h; adjust the pH of the solution to 6.0 - 7.0, concentrate at 50 °C to obtain a crude peptide powder; purify by reverse-phase C18 preparative chromatography and lyophilize to obtain centaureoyl hexapeptide-8, whose chemical structure is shown in Formula (4).

[0068] 1 H-NMR (300 MHz, D2O), δ ppm: 12.01 (s, 2H), δ ppm: 8.32 - 8.38 (s, 6H), δ ppm:

[0069] 7.84 (s, 2H), δ ppm: 7.21 (s, 2H), δ ppm: 7.03 (s, 2H), δ ppm: 6.63 (s, 4H), δ ppm: 6.62 (m, 1H), δ ppm: 6.26 (d, 1H), δ ppm: 5.47 - 6.03 (m, 4H), δ ppm: 4.44 (t, 6H), δ ppm: 3.34 (t, 4H), δ ppm: 1.51 - 2.60 (m, 31H), δ ppm: 2.50 (s, 2H), δ ppm: 2.07 (s, 3H);

[0070] C 42 H 70 N 14 O 12 S, [M + H] + = 995.50, [M - H] - = 993.50;

[0071] Example 5: Centaureoyl hexapeptide-9

[0072] Centaureoyl hexapeptide-9 was prepared by the method of amide condensation using centaureic acid, and its structure is shown in Formula (5):

[0073]

[0074] The specific preparation process is as follows:

[0075] Step 1: In a 500 mL three-necked flask, add 80 g of THF, 16.62 g of HOSu, and 20 g of millennium chrysanthemum acid. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (29.79 g of DCC dissolved in 20 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 100 g of water and 9.03 g of glycine, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 150 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 100 g each time. The washed ethyl acetate phase is then washed once with 100 g of saturated sodium chloride, dried with 15 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 25.4 g of a white solid, with a yield of 94.5%.

[0076] Step 2: In a 1000 mL three-necked flask, add 101.6 g of THF, 15.71 g of HOSu, and 25.4 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (28.17 g of DCC dissolved in 25.4 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 200 g of water and 13.1 g of L-proline, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 150 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 100 g each time. The washed ethyl acetate phase is then washed once with 100 g of saturated sodium chloride, dried with 15 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 33.2 g of a white solid, with a yield of 91.1%.

[0077] Step 3: In a 1000 mL three-necked flask, add 132.8 g of THF, 14.31 g of HOSu, and 33.2 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (25.66 g of DCC dissolved in 33.2 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 250 g of water and 15.14 g of glutamine, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After the reaction is completed as detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 250 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 200 g each time. The washed ethyl acetate phase is then washed once with 200 g of saturated sodium chloride, dried with 25 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 42.0 g of a white solid, with a yield of 90.4%.

[0078] Step 4: In a 1000 mL three-necked flask, add 168 g of THF, 12.93 g of HOSu, and 42.0 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (23.19 g of DCC dissolved in 42.0 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 250 g of water and 7.03 g of glycine, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 250 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 200 g each time. The washed ethyl acetate phase is then washed once with 200 g of saturated sodium chloride, dried with 25 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 42.6 g of a white solid-like substance, with a yield of 90.0%.

[0079] Step 5: In a 1000 mL three-necked flask, add 170.4 g of THF, 11.64 g of HOSu, and 42.6 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (20.86 g of DCC dissolved in 42.6 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 300 g of water and 9.70 g of proline, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 250 g of n-butanol. The n-butanol phase is washed 3 times with acidic water with a pH of 2.0, 200 g each time. The washed n-butanol phase is then washed once with 200 g of saturated sodium chloride, dried with 25 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 46.0 g of a white solid-like substance, with a yield of 90.5%.

[0080] Step 6: In a 1000 mL three-necked flask, add 184.0 g of THF, 10.54 g of HOSu, and 46.0 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (18.9 g of DCC dissolved in 46.0 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 350 g of water and 11.15 g of glutamine, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 350 g of n-butanol. The n-butanol phase is washed 3 times with acidic water with a pH of 2.0, 300 g each time. The washed n-butanol phase is then washed once with 300 g of saturated sodium chloride, dried with 35 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining a crude peptide powder; after purification by reverse-phase C18 preparative chromatography and freeze-drying, helianthusoyl hexapeptide-9 is obtained, and its chemical structure is the formula (5).

[0081] 1 H-NMR (300 MHz, D2O), δ ppm: 12.66 (s, 1H), δ ppm: 8.32 - 8.38 (s, 4H), δ ppm: 6.62 (m, 1H), δ ppm: 6.26 (d, 1H), δ ppm: 5.47 - 6.03 (m, 4H), δ ppm: 4.07 - 4.40 (m, 4H), δ ppm: 3.75 (s, 4H), δ ppm: 3.34 (m, 4H), δ ppm: 2.50 (s, 4H), δ ppm: 2.29 - 2.43 (m, 8H), δ ppm: 1.89 - 2.10 (m, 12H), δ ppm: 1.54 (d, 3H);

[0082] C 34 H 50 N8O 10 , [M + H] + = 731.36, [M - H] - = 729.36;

[0083] Example 6: Gomphrenoyl Viper Tripeptide

[0084] Gomphrenoyl viper tripeptide was prepared by the method of amide condensation using gomphrenic acid, and its structure is shown in formula (6):

[0085]

[0086] The specific preparation process is as follows:

[0087] Step 1: Add 80 g of THF, 16.62 g of HOSu, and 20 g of gomphrenic acid to a 500 mL three-necked flask. After stirring until clear, maintain the internal temperature at 5 - 10 °C and dropwise add a THF solution of DCC (29.79 g of DCC dissolved in 20 g of THF). After dropping, react at an internal temperature of 25 °C for 3 h. Add 100 g of water and 10.72 g of β-alanine, control the pH of the feed liquid to 7.0 - 7.5, and keep the temperature for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0 and extract with 150 g of ethyl acetate once. The ethyl acetate phase is washed three times with acidic water with a pH of 2.0, 100 g each time. The washed ethyl acetate phase is then washed once with 100 g of saturated sodium chloride and dried with 15 g of anhydrous magnesium sulfate, and then filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 26.1 g of a white solid, with a yield of 91.4%.

[0088] Step 2: In a 500 mL three-necked flask, add 108.4 g of THF, 15.19 g of HOSu, and 26.1 g of the solid from the previous step. After stirring until dissolved clearly, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (27.23 g of DCC dissolved in 26.1 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 150 g of water and 12.66 g of proline, control the pH of the feed liquid to be 7.0 - 7.5, and carry out a heat-preserving reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 150 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 100 g each time. The washed ethyl acetate phase is then washed once with 100 g of saturated sodium chloride, dried with 15 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 34.2 g of a off-white solid with a yield of 93.0%.

[0089] Step 3: In a 1000 mL three-necked flask, add 136.8 g of THF, 14.12 g of HOSu, and 34.2 g of the solid from the previous step. After stirring until dissolved clearly, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (25.32 g of DCC dissolved in 34.2 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 200 g of water and 22.32 g of Dab(Boc), control the pH of the feed liquid to be 7.0 - 7.5, and carry out a heat-preserving reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 200 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 150 g each time. The washed ethyl acetate phase is then washed once with 150 g of saturated sodium chloride, dried with 20 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 50.0 g of a off-white solid with a yield of 91.4%.

[0090] Step 4: In a 1000 mL three-necked flask, add 150.0 g of THF, 12.92 g of HOSu, and 50 g of the solid from the previous step. After stirring until dissolved clearly, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (23.16 g of DCC dissolved in 50 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 200 g of water and 10.02 g of benzylamine, control the pH of the feed liquid to be 7.0 - 7.5, and carry out a heat-preserving reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 200 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 150 g each time. The washed ethyl acetate phase is then washed once with 150 g of saturated sodium chloride, dried with 20 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 53.5 g of a off-white solid with a yield of 91.7%.

[0091] Step 5: In a 500 mL three-necked flask, add 160 mL of hydrochloric acid and 53.5 g of the solid from the previous step. Stir and raise the internal temperature to 90 °C and react for 3 h. Then add 300 g of water, adjust the pH of the solution to 8.0 - 9.0, precipitate a white solid, stir for 2 h, and filter by suction to obtain the white solid. Vacuum dry the white solid at 45 °C to obtain the crude peptide powder. Purify it by reversed-phase C18 preparative chromatography and lyophilize to obtain the centaurein-like snake venom tripeptide, whose chemical structure is formula (6).

[0092] 1 H-NMR (300 MHz, D2O), δ ppm: 8.87 (s, 1H), δ ppm: 8.32 - 8.41 (s, 2H), δ ppm: 7.23 - 7.31 (m, 5H), δ ppm: 6.62 (m, 1H), δ ppm: 6.26 (d, 1H), δ ppm: 5.47 - 5.97 (m, 4H), δ ppm: 4.40 - 4.44 (m, 4H), δ ppm: 3.44 - 3.51 (m, 4H), δ ppm: 2.63 (t, 2H), δ ppm: 1.92 - 2.44 (m, 12H), δ ppm: 1.58 (d, 3H), δ ppm: 1.50 (s, 2H).

[0093] C 29 H 41 N5O4, [M + H] + = 524.32;

[0094] Detect the anti-wrinkle, firming, soothing, and antioxidant effects of centaurein pentapeptide-4, centaurein tripeptide-5, centaurein tripeptide-1, centaurein hexapeptide-9, centaurein hexapeptide-8, and centaurein-like snake venom tripeptide in the above Examples 1 - 6. The detection data are as follows:

[0095] Table 1: Experimental data of the anti-wrinkle effect of the examples of the present invention

[0096]

[0097] It can be seen from the data analysis in Table 1 that:

[0098] The centaurein pentapeptide-4, centaurein tripeptide-5, centaurein tripeptide-1, centaurein hexapeptide-9, centaurein hexapeptide-8, and centaurein-like snake venom tripeptide prepared by the present invention have significant effects in anti-wrinkle compared with the commercially available polypeptide tetrapeptide-9 and hexapeptide-8.

[0099] Table 2: Experimental data of the firming effect of the examples of the present invention

[0100]

[0101] It can be seen from the data analysis in Table 2 that:

[0102] The synthesized Centaureoyl Pentapeptide-4, Centaureoyl Tripeptide-5, Centaureoyl Tripeptide-1, Centaureoyl Hexapeptide-9, Centaureoyl Hexapeptide-8 and Centaureoyl Viperin Tripeptide of the present invention have significant effects on firming efficacy as compared with the commercially available polypeptides Tetrapeptide-9 and Hexapeptide-8.

[0103] Table 3: Experimental data of the soothing efficacy of the examples of the present invention

[0104]

[0105] It can be seen from the data analysis in Table 3 that:

[0106] The synthesized Centaureoyl Pentapeptide-4, Centaureoyl Tripeptide-5, Centaureoyl Tripeptide-1, Centaureoyl Hexapeptide-9, Centaureoyl Hexapeptide-8 and Centaureoyl Viperin Tripeptide of the present invention have significant effects on soothing efficacy as compared with the commercially available polypeptides Tetrapeptide-9 and Hexapeptide-8.

[0107] Table 4: Experimental data of the antioxidant efficacy of the examples of the present invention

[0108]

[0109]

[0110] It can be seen from the data analysis in Table 4 that:

[0111] The synthesized Centaureoyl Pentapeptide-4, Centaureoyl Tripeptide-5, Centaureoyl Tripeptide-1, Centaureoyl Hexapeptide-9, Centaureoyl Hexapeptide-8 and Centaureoyl Viperin Tripeptide of the present invention have significant effects on antioxidant efficacy as compared with the commercially available polypeptides Tetrapeptide-9 and Hexapeptide-8.

[0112] In order to more intuitively demonstrate the process advantages of the present invention, a comparison is made by using the replacement method of the present invention.

[0113] Comparative Example 1-1:

[0114] Decanoyl Tripeptide-1, i.e., formula (7), is synthesized in the same manner as Centaureoyl Tripeptide-1, except that centaureic acid is replaced with capric acid.

[0115]

[0116] 1H-NMR (300 MHz, D2O), δ ppm: 13.00 (s, 1H), δ ppm: 12.66 (s, 1H), δ ppm: 9.04 (s, 1H), δ ppm: 8.73 (s, 1H), δ ppm: 8.32 (s, 2H), δ ppm: 7.66 (s, 1H), δ ppm: 4.92 (t, 1H), δ ppm: 4.55 (t, 1H), δ ppm: 4.09 (s, 2H), δ ppm: 2.69 - 3.17 (m, 4H), δ ppm: 2.13 (t, 2H), δ ppm: 1.53 - 1.76 (m, 6H), δ ppm: 1.50 (s, 2H), δ ppm: 1.25 - 1.30 (m, 14H), δ ppm: 0.88 (t, 3H);

[0117] C 24 H 42 N6O5, [M + H] + = 495.32, [M - H] - = 493.32;

[0118] Comparative Example 1 - 2:

[0119] Octanoyl tripeptide - 1, namely formula (8), was synthesized in the same way as Tagetesoyl tripeptide - 1, except that Tagetes acid was replaced with octanoic acid.

[0120]

[0121] 1 H-NMR (300 MHz, D2O), δ ppm: 13.00 (s, 1H), δ ppm: 12.66 (s, 1H), δ ppm: 9.04 (s, 1H), δ ppm: 8.73 (s, 1H), δ ppm: 8.32 (s, 2H), δ ppm: 7.66 (s, 1H), δ ppm: 4.92 (t, 1H), δ ppm: 4.55 (t, 1H), δ ppm: 4.09 (s, 2H), δ ppm: 2.69 - 3.17 (m, 4H), δ ppm: 2.13 (t, 2H), δ ppm: 1.53 - 1.76 (m, 6H), δ ppm: 1.50 (s, 2H), δ ppm: 1.25 - 1.30 (m, 10H), δ ppm: 0.88 (t, 3H);

[0122] C 22 H 38 N6O5, [M + H] + = 467.29, [M - H] - = 465.29;

[0123] Comparative Example 2 - 1:

[0124] Decanoyl pentapeptide-4, namely formula (9), is synthesized in the same way as Compositae acyl pentapeptide-4, except that the Compositae acid is replaced by decanoic acid.

[0125]

[0126] 1 H-NMR (300 MHz, D2O), δ ppm: 12.39 (s, 1H), δ ppm: 8.32 (s, 5H), δ ppm: 5.37 (s, 2H), δ ppm: 4.94 (s, 1H), δ ppm: 3.89 - 4.62 (m, 9H), δ ppm: 2.69 (t, 4H), δ ppm: 1.53 - 2.05 (m, 12H), δ ppm: 1.50 (s, 4H), δ ppm: 1.16 - 1.30 (m, 22H), δ ppm: 0.88 (t, 3H);

[0127] C 33 H 63 N7O 10 , [M + H] + = 718.46, [M - H] - = 716.46;

[0128] Comparative Example 2-2:

[0129] Octanoyl pentapeptide-4, namely formula (10), is synthesized in the same way as Compositae acyl pentapeptide-4, except that the Compositae acid is replaced by octanoic acid.

[0130]

[0131] 1 H-NMR (300 MHz, D2O), δ ppm: 12.39 (s, 1H), δ ppm: 8.32 (s, 5H), δ ppm: 5.37 (s, 2H), δ ppm: 4.94 (s, 1H), δ ppm: 3.89 - 4.62 (m, 9H), δ ppm: 2.69 (t, 4H), δ ppm: 1.53 - 2.05 (m, 12H), δ ppm: 1.50 (s, 4H), δ ppm: 1.16 - 1.30 (m, 18H), δ ppm: 0.88 (t, 3H);

[0132] C 31 H 59 N7O 10 , [M + H] + = 690.43, [M - H] - = 688.43;

[0133] Comparative Example 3-1:

[0134] Decanoyl tripeptide-5, namely formula (11), was synthesized in the same way as gomphrenoyl tripeptide-5, except that gomphrenic acid was replaced by decanoic acid.

[0135]

[0136] 1 H-NMR (300 MHz, D2O), δ ppm: 12.66 (s, 1H), δ ppm: 8.32 (s, 3H), δ ppm: 4.34 (d, 1H), δ ppm: 4.44 (t, 1H), δ ppm: 4.55 (t, 1H), δ ppm: 2.73 (m, 1H), δ ppm: 2.69 (t, 4H), δ ppm: 1.53 - 2.05 (m, 12H), δ ppm: 1.50 (s, 4H), δ ppm: 1.25 - 1.30 (m, 16H), δ ppm: 0.96 (d, 6H) δ ppm: 0.88 (t, 3H);

[0137] C 27 H 53 N5O5, [M + H] + = 528.40, [M - H] - = 526.40;

[0138] Comparative Example 3-2:

[0139] Octanoyl tripeptide-5, namely formula (12), was synthesized in the same way as gomphrenoyl tripeptide-5, except that gomphrenic acid was replaced by octanoic acid.

[0140]

[0141] 1 H-NMR (300 MHz, D2O), δ ppm: 12.66 (s, 1H), δ ppm: 8.32 (s, 3H), δ ppm: 4.34 (d, 1H), δ ppm: 4.44 (t, 1H), δ ppm: 4.55 (t, 1H), δ ppm: 2.73 (m, 1H), δ ppm: 2.69 (t, 4H), δ ppm: 1.53 - 2.05 (m, 12H), δ ppm: 1.50 (s, 4H), δ ppm: 1.25 - 1.30 (m, 12H), δ ppm: 0.96 (d, 6H) δ ppm: 0.88 (t, 3H);

[0142] C 25 H 49 N5O5, [M + H] + = 500.37, [M - H] - = 498.37;

[0143] Comparative Example 4-1:

[0144] Decanoyl hexapeptide-8, namely formula (13), was synthesized in the same way as Centaureoyl hexapeptide-8, except that centaureic acid was replaced with capric acid.

[0145]

[0146] 1 H-NMR (300 MHz, D2O), δ ppm: 12.01 (s, 2H), δ ppm: 8.32 (s, 6H), δ ppm: 7.84 (s, 2H), δ ppm: 7.21 (s, 2H), δ ppm: 7.03 (s, 2H), δ ppm: 6.63 (s, 4H), δ ppm: 4.44 (t, 6H), δ ppm: 3.34 (t, 4H), δ ppm: 1.51 - 2.60 (m, 31H), δ ppm: 2.50 (s, 2H), δ ppm: 1.26 - 1.30 (m, 12H), δ ppm: 0.88 (t, 3H);

[0147] C 42 H 76 N 14 O 12 S, [M + H] + = 1001.55, [M - H] - = 999.55;

[0148] Comparative Example 4-2:

[0149] Octanoyl hexapeptide-8, namely formula (14), was synthesized in the same way as Centaureoyl hexapeptide-8, except that centaureic acid was replaced with octanoic acid.

[0150]

[0151] 1 H-NMR (300 MHz, D2O), δ ppm: 12.01 (s, 2H), δ ppm: 8.32 (s, 6H), δ ppm: 7.84 (s, 2H), δ ppm: 7.21 (s, 2H), δ ppm: 7.03 (s, 2H), δ ppm: 6.63 (s, 4H), δ ppm: 4.44 (t, 6H), δ ppm: 3.34 (t, 4H), δ ppm: 1.51 - 2.60 (m, 31H), δ ppm: 2.50 (s, 2H), δ ppm: 1.26 - 1.30 (m, 8H), δ ppm: 0.88 (t, 3H);

[0152] C 40 H 72 N 14 O 12 S, [M + H]+ = 973.52, [M-H] - = 971.52;

[0153] Comparative Example 5-1:

[0154] Decanoyl hexapeptide-9, i.e., formula (15), was synthesized in the same manner as centaureoyl hexapeptide-9, except that centaureic acid was replaced with decanoic acid.

[0155]

[0156] 1 H-NMR(300MHz, D2O), δ ppm: 12.01(s, 2H), δ ppm: 8.32(s, 4H), δδ ppm: 4.07 - 4.40(t, 4H), δ ppm: 3.75(s, 4H), δ ppm: 3.40 - 3.58(m, 4H), δ ppm: 1.89 - 2.34(m, 16H), δ ppm: 2.50(s, 4H), δ ppm: 1.53(m, 2H), δ ppm: 1.23 - 1.28(m, 12H), δ ppm: 0.88(t, 3H);

[0157] C 34 H 56 N8O 10 , [M+H] + = 737.41, [M-H] - = 735.41;

[0158] Comparative Example 5-2:

[0159] Octanoyl hexapeptide-9, i.e., formula (16), was synthesized in the same manner as centaureoyl hexapeptide-9, except that centaureic acid was replaced with octanoic acid.

[0160]

[0161] 1 H-NMR(300MHz, D2O), δ ppm: 12.01(s, 2H), δ ppm: 8.32(s, 4H), δδ ppm: 4.07 - 4.40(t, 4H), δ ppm: 3.75(s, 4H), δ ppm: 3.40 - 3.58(m, 4H), δ ppm: 1.89 - 2.34(m, 16H), δ ppm: 2.50(s, 4H), δ ppm: 1.53(m, 2H), δ ppm: 1.23 - 1.28(m, 8H), δ ppm: 0.88(t, 3H);

[0162] C 32 H 52 N8O 10, [M+H] + = 709.38, [M-H] - = 707.38;

[0163] Comparative Example 6-1:

[0164] Decanoyl venom tripeptide, namely formula (17), was synthesized in the same way as the tagetesoyl venom tripeptide, except that tagetesic acid was replaced by decanoic acid.

[0165]

[0166] 1 H-NMR (300 MHz, D2O), δ ppm: 8.87 (s, 1H), δ ppm: 8.32 (s, 1H), δ ppm: 8.01 (s, 1H), δ ppm: 7.23 - 7.31 (m, 5H), δ ppm: 4.40 - 4.44 (m, 4H), δ ppm: 3.68 (t, 2H), δ ppm: 3.41 - 3.51 (t, 2H), δ ppm: 1.92 - 2.63 (m, 12H), δ ppm: 1.53 (m, 2H), δ ppm: 1.50 (s, 2H), δ ppm: 1.26 - 1.30 (m, 12H), δ ppm: 0.88 (t, 3H);

[0167] C 29 H 47 N5O4, [M+H] + = 530.36;

[0168] Comparative Example 6-2:

[0169] Octanoyl venom tripeptide, namely formula (18), was synthesized in the same way as the tagetesoyl venom tripeptide, except that tagetesic acid was replaced by octanoic acid.

[0170]

[0171] 1 H-NMR (300 MHz, D2O), δ ppm: 8.87 (s, 1H), δ ppm: 8.32 (s, 1H), δ ppm: 8.01 (s, 1H), δ ppm: 7.23 - 7.31 (m, 5H), δ ppm: 4.40 - 4.44 (m, 4H), δ ppm: 3.68 (t, 2H), δ ppm: 3.41 - 3.51 (t, 2H), δ ppm: 1.92 - 2.63 (m, 12H), δ ppm: 1.53 (m, 2H), δ ppm: 1.50 (s, 2H), δ ppm: 1.26 - 1.30 (m, 8H), δ ppm: 0.88 (t, 3H);

[0172] C27 H 43 N5O4, [M+H] + = 502.33;

[0173] Comparative Example 7: Zinniaoyl Tripeptide-17

[0174] Zinniaoyl Tripeptide-17 was prepared by the method of amide condensation, and its structure is shown in Formula (19):

[0175]

[0176] The specific preparation process is as follows:

[0177] Step 1: In a 500 mL three-necked flask, add 80 g of THF, 16.62 g of HOSu, and 20 g of zinnic acid. After stirring until dissolved clearly, keep the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (29.79 g of DCC dissolved in 20 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 100 g of water and 9.03 g of glycine, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract with 100 g of ethyl acetate once. The ethyl acetate phase is washed with acidic water with a pH of 2.0 three times, 100 g each time. The washed ethyl acetate phase is then washed with 100 g of saturated sodium chloride once, dried with 10 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 25.5 g of a white solid-like product with a yield of 94.9%.

[0178] Step 2: In a 500 mL three-necked flask, add 102 g of THF, 15.77 g of HOSu, and 25.5 g of the solid obtained in the previous step. After stirring until dissolved clearly, keep the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (28.28 g of DCC dissolved in 25.5 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 130 g of water and 13.15 g of proline, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract with 150 g of ethyl acetate once. The ethyl acetate phase is washed with acidic water with a pH of 2.0 three times, 100 g each time. The washed ethyl acetate phase is then washed with 100 g of saturated sodium chloride once, dried with 15 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 34.0 g of a white solid-like product with a yield of 92.9%.

[0179] Step 3: In a 1000 mL three-necked flask, add 216.4 g of THF, 14.66 g of HOSu, and 34.0 g of the solid from the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (26.28 g of DCC dissolved in 54.1 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 300 g of water and 15.61 g of glutamic acid, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After completion of the reaction detected by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 300 g of ethyl acetate. The ethyl acetate phase is washed three times with acidic water with a pH of 2.0, 200 g each time. The washed ethyl acetate phase is then washed once with 200 g of saturated sodium chloride, dried with 30 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out to obtain a crude peptide powder; after purification by reverse-phase C18 preparative chromatography and freeze-drying, sesquiterpene tripeptide-17 is obtained, and its chemical structure is the formula (19).

[0180] 1 H-NMR (300 MHz, D2O), δ ppm: 12.66 (s, 1H), δ ppm: 8.32 - 8.38 (s, 2H), δ ppm: 7.03 (s, 2H), δ ppm: 6.26 (d, 1H), δ ppm: 5.47 - 6.62 (m, 5H), δ ppm: 4.44 - 4.55 (t, 2H), δ ppm: 3.85 (s, 2H), δ ppm: 3.41 - 3.51 (m, 2H), δ ppm: 1.92 - 2.33 (m, 12H), δ ppm: 1.58 (d, 3H);

[0181] C 22 H 32 N4O6, [M + H] + = 449.23, [M - H] - = 447.23;

[0182] Comparative Example 8: Sesquiterpene pentapeptide-1

[0183] The preparation of sesquiterpene pentapeptide-1 is obtained by the method of amide condensation, and its structure is as shown in formula (20):

[0184]

[0185] The specific preparation process is as follows:

[0186] Step 1: In a 500 mL three-necked flask, add 80 g of THF, 16.62 g of HOSu, and 20 g of centaureic acid. After stirring until dissolved clearly, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (29.79 g of DCC dissolved in 20 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 100 g of water and 20.98 g of arginine, control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 150 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 100 g each time. The washed ethyl acetate phase is further washed once with 100 g of saturated sodium chloride, dried with 15 g of anhydrous magnesium sulfate, and filtered. Concentrate the filtrate at an external temperature of 45 °C until no more distillate comes out, obtaining 36.3 g of a white solid, with a yield of 93.6%.

[0187] Step 2: In a 1000 mL three-necked flask, add 145.2 g of THF, 15.55 g of HOSu, and 36.3 g of the solid from the previous step. After stirring until dissolved clearly, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (27.88 g of DCC dissolved in 36.3 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 200 g of water and 27.73 g of Lys(Boc), control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 150 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 100 g each time. The washed ethyl acetate phase is further washed once with 100 g of saturated sodium chloride, dried with 15 g of anhydrous magnesium sulfate, and filtered. Concentrate the filtrate at an external temperature of 45 °C until no more distillate comes out, obtaining 57.6 g of a white solid, with a yield of 92.9%.

[0188] Step 3: In a 1000 mL three-necked flask, add 230.4 g of THF, 14.45 g of HOSu, and 57.6 g of the solid from the previous step. After stirring until dissolved clearly, maintain the internal temperature at 5 - 10 °C, and dropwise add a THF solution of DCC (25.90 g of DCC dissolved in 57.6 g of THF); after dropping, react at an internal temperature of 25 °C for 3 h; add 250 g of water and 19.79 g of Asp(OtBu), control the pH of the feed liquid to be 7.0 - 7.5, and keep the temperature for reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 250 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 150 g each time. The washed ethyl acetate phase is further washed once with 150 g of saturated sodium chloride, dried with 25 g of anhydrous magnesium sulfate, and filtered. Concentrate the filtrate at an external temperature of 45 °C until no more distillate comes out, obtaining 68.0 g of a white solid, with a yield of 90.0%.

[0189] Step 4: In a 1000 mL three-necked flask, add 272.0 g of THF, 13.01 g of HOSu, and 68.0 g of the solid obtained in the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and slowly add dropwise a THF solution of DCC (23.32 g of DCC dissolved in 68.0 g of THF); after adding dropwise, react at an internal temperature of 25 °C for 3 h; add 300 g of water and 11.03 g of valine, control the pH of the feed liquid to be 7.0 - 7.5, and carry out a heat-preservation reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 300 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 200 g each time. The washed ethyl acetate phase is then washed once with 200 g of saturated sodium chloride, dried with 30 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 70.2 g of an off-white solid with a yield of 90.5%.

[0190] Step 5: In a 1000 mL three-necked flask, add 280.8 g of THF, 11.78 g of HOSu, and 70.2 g of the solid obtained in the previous step. After stirring until dissolved and clear, maintain the internal temperature at 5 - 10 °C, and slowly add dropwise a THF solution of DCC (21.12 g of DCC dissolved in 70.2 g of THF); after adding dropwise, react at an internal temperature of 25 °C for 3 h; add 300 g of water and 15.45 g of tyrosine, control the pH of the feed liquid to be 7.0 - 7.5, and carry out a heat-preservation reaction for 2 h. After detecting the completion of the reaction by HPLC, filter, and concentrate the filtrate at an external temperature of 45 °C. Adjust the pH to 2.0, and extract once with 300 g of ethyl acetate. The ethyl acetate phase is washed 3 times with acidic water with a pH of 2.0, 200 g each time. The washed ethyl acetate phase is then washed once with 200 g of saturated sodium chloride, dried with 30 g of anhydrous magnesium sulfate, and filtered. The filtrate is concentrated at an external temperature of 45 °C until no more distillate comes out, obtaining 75.2 g of an off-white solid with a yield of 89.6%.

[0191] Step 6: In a 1000 mL three-necked flask, add 225 mL of hydrochloric acid and 75.2 g of the solid obtained in the previous step. Stir and raise the internal temperature to 90 °C and react for 3 h; then add 300 g of water, adjust the pH of the feed liquid to 6.0 - 7.0, precipitate a white solid, stir for 2 h, and carry out suction filtration to obtain a white solid; vacuum-dry the white solid at 45 °C to obtain a crude peptide powder; after purification by reverse-phase C18 preparative chromatography and freeze-drying, chrysanthemoyl pentapeptide-1 is obtained, and its chemical structure is the formula (20).

[0192] 1H-NMR (300 MHz, D2O), δ ppm: 12.89 (s, 1H), δ ppm: 12.39 (s, 1H), δ ppm: 9.06 (s, 1H), δ ppm: 8.32 - 8.38 (s, 5H), δ ppm: 7.84 (s, 1H), δ ppm: 6.68 - 6.96 (d, 4H), δ ppm: 6.63 (s, 2H), δ ppm: 6.62 (m, 1H), δ ppm: 6.26 (d, 1H), δ ppm: 5.47 - 6.03 (m, 4H), δ ppm: 4.44 - 4.86 (m, 4H), δ ppm: 4.34 (d, 1H), δ ppm: 2.67 - 3.34 (m, 9H), δ ppm: 2.50 (s, 1H), δ ppm: 1.25 - 2.00 (m, 14H), δ ppm: 1.58 (d, 3H), δ ppm: 1.50 (s, 2H), δ ppm: 0.96 (d, 6H);

[0193] C 40 H 61 N9O 10 , [M + H] + = 828.45, [M - H] - = 826.45;

[0194] Comparative Example 9: Centaureic acid

[0195] The centaureic acid used in this group of compounds was synthesized by ourselves, and its structure is shown in formula (21):

[0196]

[0197] The specific preparation process is as follows:

[0198] Step 1: In a 500 mL sealed container, add 100 g of water, 100 g of 4-bromobutanal, and 104.99 g of propanediol cyclo(iso)propylidene carbonate. Keep the internal temperature at 5 - 75 °C and stir the reaction for 2 h in a sealed manner; then naturally cool to 25 °C; add 300 g of saturated sodium carbonate solution and stir for 30 min. Then add 500 ml of diethyl ether for extraction once; add 50 g of anhydrous magnesium sulfate to the organic phase for drying for 30 min, filter by suction, and concentrate the filtrate to dryness at 45 °C to obtain 132 g of a yellow solid with a yield of 71.9%.

[0199] Step 2: Add 132 g of the above solid, 137.43 g of triphenylphosphine, and 528 g of acetonitrile to a 2 L reaction flask, fill with a nitrogen balloon, replace with vacuum three times, and reflux the reaction in an oil bath at 90 °C for 15 h; concentrate the feed liquid under reduced pressure at 45 °C until no more distillate comes out, add 528 g of toluene, stir at room temperature for 2 h, filter by suction, and dry the filter cake under vacuum at 65 °C for 1 h to obtain 210.7 g of a white solid with a yield of 85%.

[0200] Step 3: Add 210.7 g of the solid from the previous step, 215.94 g of potassium carbonate, and 800 g of toluene into a 2 L reaction flask. Then add 245.2 g of crotonaldehyde, and react at 65 °C in an oil bath for 16 h. After returning to room temperature, add 800 g of water to the liquid material, stir for 10 min, and then separate the liquid. Concentrate the organic phase under reduced pressure at 65 °C until no more distillate comes out. Add 800 g of n-hexane, stir at room temperature for 1 h, and a solid gradually precipitates. Filter by suction, wash the filtrate twice with 800 ml of water. Dry the organic phase with 30 g of anhydrous magnesium sulfate for 30 min, filter by suction, and concentrate the filtrate under reduced pressure at 45 °C until no more distillate comes out to obtain 95 g of a yellow liquid product with a yield of 97.2%.

[0201] Step 4: Add 95 g of the product from the previous step, 0.5 g of ferric chloride hexahydrate, and 300 g of nitromethane into a 1000 mL three-necked flask, and reflux at an internal temperature of 110 °C for 24 h. After returning to room temperature, add 400 ml of saturated sodium bicarbonate solution, and extract twice with 100 ml of dichloromethane. Adjust the pH of the aqueous phase to 2.0, then add 200 ml of dichloromethane dropwise and extract twice. Dry the dichloromethane phase with 30 g of anhydrous magnesium sulfate, filter, and concentrate the filtrate at an external temperature of 45 °C until no more distillate comes out. Purify by reverse-phase C18 preparative chromatography and lyophilize to obtain acmella oleracea acid, whose chemical structure is formula (21);

[0202] 1 1H-NMR (300 MHz, CDCl3), δ ppm: 12.05 (s, 1H), δ ppm: 7.11 (m, 1H), δ ppm: 5.47 - 6.03 (m, 5H), δ ppm: 2.00 (m, 4H), δ ppm: 1.58 (d, 3H);

[0203] C 10 H 14 O2, [M - H] - = 165.10;

[0204] Test on the immediate anti-wrinkle effect of polypeptide on human body

[0205] Immediate anti-wrinkle treatment instantaneously fades wrinkles by blocking the electrical conduction between nerves and muscles, rejuvenating the skin to make it look younger, that is, by reducing the release amount of acetylcholine. The test is based on neuron cells, and the immediate anti-wrinkle effect of the sample to be tested is evaluated by detecting the change and change rate of acetylcholine content.

[0206] Test method

[0207] 1) Cell seeding: After resuscitating the cells, when the plating rate reaches about 60%, seed the cells into a 6-well plate and incubate overnight in a CO2 incubator (37 °C, 5% CO2).

[0208] 2) Solution preparation: Prepare the working solution of the test substance according to the test grouping (Table 1).

[0209] 3) Administration: According to the test grouping, when the cell seeding rate in the 96-well plate reaches 40% - 60%, group administration is carried out. The dosage per well is 2 mL, and 3 replicate wells are set for each group. Incubate in a CO2 incubator (37 °C, 5% CO2) for 30 min.

[0210] 4) Detection: Collect the cell culture supernatant and perform detection according to the instructions of the detection kit.

[0211] 5) Inhibition rate calculation: Inhibition rate (%) = (blank control group - sample group) / blank control group × 100%

[0212] 6) Result statistical analysis: Use GraphPad Prism to plot graphs, and the results are expressed as Mean ± SD. The t-test statistical analysis is used for comparison between groups. All statistical analyses are two-tailed. P < 0.05 is considered to have a significant difference, and P < 0.01 is considered to have a highly significant difference.

[0213] Test results: The test data are shown in Table 5:

[0214] Table 5 Test results of the immediate anti-wrinkle effect on human body in different examples and comparative examples

[0215]

[0216]

[0217] As can be seen from Table 5,

[0218] (1) From the comparison between Examples 1 - 6 and the comparative examples, it can be seen that:

[0219] The test is based on neuron cells. By detecting the change and change rate of acetylcholine content, the immediate anti-wrinkle effect of the sample to be tested is evaluated. The clearance rates are all greater than 18%. In particular, the clearance rates of chrysanthemumoyl hexapeptide - 8 and chrysanthemumoyl-like snake venom tripeptide are as high as 25.19% and 26.21%,

[0220] Compared with the BC group, the comparative examples of each example are much lower than the clearance rates of Examples 1 - 6. The immediate anti-wrinkle effect of the compounds in Examples 1 - 6 is significant, while the effect of the compounds conjugated with capric acid and caprylic acid is relatively poor;

[0221] It can be seen from this that although replacing chrysanthemumic acid with capric acid or caprylic acid can also prepare similar polypeptides, the immediate anti-wrinkle effect is much lower than that of the polypeptides prepared by the amide condensation method using chrysanthemumic acid;

[0222] (2) From Comparative Example 9, it can be seen that:

[0223] To further investigate the immediate anti-wrinkle effect of centaureic acid, Comparative Example 9 separately studied the immediate anti-wrinkle effect of centaureic acid, and the clearance rate was only 11.21%. Thus, although centaureic acid has an immediate anti-wrinkle effect, its effect is far lower than that of the polypeptides in Examples 1-6;

[0224] (3) As can be seen from Comparative Examples 7-8:

[0225] To further investigate the polypeptides prepared by the amide condensation of different amino acids and centaureic acid, centaureic tripeptide-17 in Comparative Example 7 and centaureic pentapeptide-1 in Comparative Example 8, although also prepared by the amide condensation method using centaureic acid, have extremely low immediate anti-wrinkle effects. Centaureic pentapeptide-1 and centaureic tripeptide-17 have almost no effect. It can be seen that not all polypeptides conjugated with centaureic acid can increase the immediate anti-wrinkle effect;

[0226] (4) As can be seen from Control Groups 1-5:

[0227] Among them, tripeptide-1, tripeptide-5, pentapeptide-4, and hexapeptide-9 have no immediate anti-wrinkle effect, and their effects are enhanced after being conjugated with centaureic acid compared to centaureic acid alone;

[0228] However, not all polypeptides conjugated with centaureic acid have an immediate anti-wrinkle effect. For example, pentapeptide-1 and tripeptide-17 have worse effects after being conjugated with centaureic acid than centaureic acid alone;

[0229] Hexapeptide-8 and snake venom-like tripeptide itself have an immediate anti-wrinkle effect. After being conjugated with centaureic acid, their effects are significantly enhanced. However, when conjugated with capric acid and caprylic acid, there are still effects, but compared with the control conjugated with centaureic acid, the effect is significantly enhanced after being conjugated with centaureic acid.

[0230] In summary:

[0231] (1) Centaureic pentapeptide-4, centaureic tripeptide-5, centaureic tripeptide-1, centaureic hexapeptide-9, centaureic hexapeptide-8, and centaureic snake venom-like tripeptide have relatively excellent anti-wrinkle, firming, soothing, and antioxidant effects;

[0232] (2) Centaureic acid can react with tripeptide-1, tripeptide-5, pentapeptide-4, and hexapeptide-9, which have no immediate anti-wrinkle effect, through amide condensation to obtain polypeptides with an immediate anti-wrinkle effect higher than that of centaureic acid itself;

[0233] (3) Centaureic acid can also greatly enhance the immediate anti-wrinkle effect of polypeptides by reacting with hexapeptide-8 and snake venom-like tripeptide, which have an immediate anti-wrinkle effect, through amide condensation;

[0234] (4) The instant wrinkle-removing effect of chrysanthemumoyl tripeptide-1, chrysanthemumoyl pentapeptide-4, chrysanthemumoyl tripeptide-5, chrysanthemumoyl hexapeptide-8, chrysanthemumoyl hexapeptide-9 or chrysanthemumoyl-like snake venom tripeptide prepared by amide condensation of chrysanthemumic acid has a significant effect.

Claims

1. A Tagetes erecta L. polypeptide, characterized in that The Centaurea cyanus polypeptide is Centaureoyl tripeptide-1, Centaureoyl pentapeptide-4, Centaureoyl tripeptide-5, Centaureoyl hexapeptide-8, Centaureoyl hexapeptide-9 or Centaureoyl-snake venom-like tripeptide.

2. The chrysanthemum polypeptide according to claim 1, wherein The Centaurea cyanus polypeptide has a general structural formula as shown in Formula (1), Formula (2), Formula (3), Formula (4), Formula (5), and Formula (6); Equation (1) is as follows; Equation (2) is Equation (3) is Equation (4) is Equation (5) is Equation (6) is 3. The chrysanthemum morifolium Ramat polypeptide according to claim 1 or 2, characterized in that The Centaurea cyanus polypeptide is prepared by an amide condensation method using Centaurea cyanus acid.

4. The chrysanthemum morifolium polypeptide according to claim 1 or 2, characterized in that The Centaurea cyanus polypeptide has the effects of anti-wrinkle, firming, soothing, and antioxidant.

5. The chrysanthemum morifolium polypeptide according to claim 1 or 2, characterized in that The Centaurea cyanus polypeptide has an immediate anti-wrinkle effect.

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