Eel small molecule active peptide and application thereof

Abstract

The application relates to a small molecule active peptide of fish glue and application thereof, the amino acid sequence of the small molecule active peptide is Gly-Pro-Leu-Ala-Gly-Pro, the small molecule active peptide can inhibit the activity of tyrosinase, and is used in cosmetics or medicines.

Description

[0001] This application is a divisional application of application number 202111544938.6, filed on 2021-12-16, entitled "A Skin-Removing and Beautifying Composition Containing Fish Maw Oligopeptides, Its Preparation Method and Use Thereof". Technical Field

[0002] This invention belongs to the food field, specifically relating to a skin-lightening and beauty composition containing fish maw oligopeptides and its preparation method. Background Technology

[0003] The skin is the largest organ in the human body, and differences in skin color mainly depend on the amount of melanin synthesized by epidermal melanocytes. Pigmentation is usually a skin problem or disease caused by melanin deposition, such as melasma and freckles, which is a global issue. Surveys show that there are over 5 million people with melasma in the United States alone, and the number of patients in China is also increasing year by year.

[0004] The formation of skin pigmentation is related to a variety of factors, such as genetic factors, imbalance of the skin's antioxidant system, endocrine disorders (abnormal thyroid hormones, sex hormones), vascular factors, menstrual irregularities, oral contraceptives, pregnancy, nutritional abnormalities, sun damage, and mental and psychological states. Specifically, it manifests as an increase in melanocytes, an increase in the secretion of melanin granules, and the accumulation and deposition of pigment in local tissues. Research has found that the distribution of melanin to the skin requires the following process: first, melanosomes form and mature; then, melanin is synthesized; melanocytes transport melanosomes containing a large amount of melanin to adjacent keratinocytes; keratinocytes undergo some physiological and biochemical changes, leading to the deposition of melanin within the keratinocytes.

[0005] Currently, methods for removing freckles are mainly divided into three categories: oral, topical, and physical therapy. The main mechanisms are: 1. Preventing the formation of freckles by inhibiting or interfering with the formation of melanin; 2. Sun protection to reduce ultraviolet damage; 3. Removing melanin granules in the skin through laser treatment.

[0006] Oral products are mainly divided into two categories: one is various pure traditional Chinese medicine compositions, such as CN201110423325.7 A traditional Chinese medicine composition for removing freckles and beautifying the skin and its preparation method, and CN201310751085.2 A traditional Chinese medicine composition for whitening and removing freckles and its preparation method; the other is compositions with added glutathione, vitamins, etc., such as CN201710763074.4 A whitening and freckle-removing composition and its application, and CN201010292567.2 A composition for whitening and removing freckles and its preparation method. Because their effects are singular, the results are not obvious.

[0007] External treatments mainly consist of various cosmetics and ointments, such as CN201510450427.6, a whitening and freckle-removing compound preparation and its preparation method, and CN201310527260.X, a whitening and freckle-removing lotion. These usually contain ingredients such as azelaic acid, arbutin, paramethoxyphenol, tranexamic acid, or sunscreen ingredients. They can only prevent the freckles from worsening or lightening their color, and the treatment effect is poor. Moreover, azelaic acid and paramethoxyphenol mostly have certain side effects.

[0008] Physical therapy typically involves laser treatment or photofacial rejuvenation, and requires strict sun protection, usually using sunscreen in conjunction with the treatment. However, there are issues such as unstable efficacy and the risk of inflammation.

[0009] Although age spots do not affect physical health, they often appear on the face and neck, significantly impacting appearance and even causing serious social, psychological, and mental health problems. The desire for beauty is human nature, and removing age spots is a perpetual pursuit for those with them; therefore, developing a safe and effective age spot removal product has broad market prospects.

[0010] Fish maw, also known as fish glue, is one of the "Eight Treasures" alongside bird's nest and shark fin. The Compendium of Materia Medica records that fish maw can nourish the kidneys and replenish essence, nourish tendons and veins, and treat kidney deficiency, spermatorrhea, and postpartum wind spasms. The main component of fish maw is high-grade collagen, exceeding 80%, and it also contains various vitamins and trace elements such as calcium, zinc, iron, and selenium. It has been a beauty and skin-nourishing product since ancient times, possessing the effects of nourishing yin and beautifying the skin, replenishing blood and kidneys, and strengthening bodily functions. Various active peptides can be obtained from fish maw protein through enzymatic hydrolysis with different proteases. Currently, there are studies on the preparation and application of fish maw peptides, such as CN201710907667.3, a method for preparing fish maw polypeptides, and CN201810406679.2, a fish maw elastin peptide composition for postpartum conditioning and its preparation method. However, research on specific active peptide segments of fish maw peptides and their application in skin-lightening and beauty formulas has not been reported. Summary of the Invention

[0011] In view of the above-mentioned problems, current situation and development prospects, the present invention provides a skin-removing and beautifying composition containing fish maw oligopeptides, which has the effect of removing skin blemishes and beautifying the skin.

[0012] The technical solution of the present invention is as follows: a skin-removing and beauty composition containing fish maw oligopeptides, comprising 50-100 parts of fish maw oligopeptide powder, 20-40 parts of angelica powder, 30-50 parts of peach kernel powder, 10-30 parts of sea cucumber oligopeptide powder, 30-50 parts of wolfberry powder, 20-40 parts of poria cocos powder, 20-40 parts of yam powder, 20-50 parts of jujube seed powder, and 20-30 parts of rose powder.

[0013] Furthermore, the aforementioned fish maw oligopeptide powder is a small molecule active peptide powder for fish maw.

[0014] Furthermore, the fish maw oligopeptides were prepared as follows: After processing the fish maw, 15-20 times its weight volume of water was added to form a homogenate, which was then placed in an enzymatic hydrolysis tank. Then, 3-5% of the fish maw protein mass of a complex protease was added, and the mixture was enzymatically hydrolyzed at 50-55°C for 3-5 hours. The pH value of the enzyme reaction was controlled at 7.0-8.0. After the enzymatic hydrolysis was completed, the temperature was raised to 80-90°C to inactivate the enzyme for 10 minutes, resulting in a fish maw protease hydrolysate. The protease hydrolysate was centrifuged at 8000 rpm for 10 minutes to remove particulate matter. Then, membrane separation technology was used for separation, with a molecular weight cutoff of 3000 Da. The membrane solution was spray-dried to obtain fish maw oligopeptide powder. The mass ratio of the complex protease was: neutral protease: trypsin: bromelain: flavor protease = (4-6):(3-5):(1-3):(2-4).

[0015] Furthermore, over 90% of the peptides in fish maw oligopeptides have a molecular weight of less than 1000 Da.

[0016] Furthermore, the small molecule active peptides from fish maw were obtained by isolating and purifying fish maw oligopeptides: the fish maw oligopeptide powder was dissolved in water to prepare a solution with a concentration of 100 mg / mL, and then purified using Sephadex dextran gel electrophoresis. The separation and purification were performed using LH-20 column chromatography with 40% methanol as the mobile phase and a flow rate of 0.4–0.6 mL / min. The absorbance of the eluent was measured at 280 nm, and the desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC) under the following conditions: C18 column; mobile phase A: water containing 0.06%–0.08% trifluoroacetic acid (v / v); mobile phase B: acetonitrile; gradient elution conditions: 0–15 min, 3% B; 15–20 min, 3%–10% B; 20–30 min, 10% B–20% B; 30–40 min, 20% B–35% B; flow rate: 0.8 mL / min; detection wavelength: 280 nm; peaks with a retention time of 26 min were collected, concentrated, and freeze-dried to obtain small molecule active peptides from fish maw.

[0017] Furthermore, the amino acid sequence of the small molecule active peptide in fish maw is: Gly-Pro-Leu-Ala-Gly-Pro.

[0018] Furthermore, the sea cucumber oligopeptides were prepared as follows: After sea cucumber treatment, 15-20 times the mass volume of water was added to form a homogenate, which was then placed in an enzymatic hydrolysis tank. Then, 2-5% of the mass of the sea cucumber complex protease was added, and the mixture was enzymatically hydrolyzed at 40-50℃ for 4 hours. The pH value of the enzyme reaction was controlled at 8.0-9.0. After the enzymatic hydrolysis was completed, the temperature was raised to 80-90℃ to inactivate the enzyme for 10 minutes, resulting in a sea cucumber protease hydrolysate. The protease hydrolysate was centrifuged at 8000 rpm for 10 minutes to remove particulate matter. Then, membrane separation technology was used for separation, with a molecular weight cutoff of 3000 Da. The membrane solution was spray-dried to obtain sea cucumber oligopeptide powder. The mass ratio of the complex protease was: neutral protease: papain: flavor protease = (2-4):(3-5):(3-5).

[0019] Furthermore, over 90% of the peptides in sea cucumber oligopeptides have a molecular weight of less than 1000 Da.

[0020] Furthermore, the cosmetic composition is one of the following: powder, granules, tablets, capsules, pills, or oral solution.

[0021] The use of the aforementioned cosmetic composition in foods, cosmetics, or medicines for removing blemishes and enhancing beauty.

[0022] Tyrosinase (TYR) plays a crucial role in melanin synthesis, acting as a key rate-limiting enzyme. Overexpression of tyrosinase leads to increased and abnormal melanin production, resulting in pigmentation and dark spots on the skin. Melanin synthesis begins with the oxidation of L-tyrosine. TYR oxidizes L-tyrosine to dopaquinone, which then undergoes auto-oxidation to dopachrome. The products of dopachrome (5,6-dihydroxyindole carboxylic acid and 5,6-dihydroxyindole) are further oxidized to eumelanin. Typically, the expression level of tyrosinase in melanocytes of different skin tones is essentially the same; therefore, inhibiting tyrosinase activity is key to achieving effective pigmentation removal.

[0023] The tyrosinase-inhibiting active peptide (Gly-Pro-Leu-Ala-Gly-Pro) in fish maw oligopeptides can directly intervene in the melanin synthesis process. By inhibiting tyrosinase activity, it reduces the production of L-DOPA, dopaquinone, and 5,6-indolequinone, ultimately reducing melanin formation and removing age spots. Photodamage to the skin is also a contributing factor to age spots. Fish maw oligopeptides can also play an antioxidant role and delay skin aging by scavenging free radicals, increasing the activity of antioxidant enzymes, and reducing lipid peroxide content. Simultaneously, fish maw oligopeptide powder itself is a collagen peptide, providing ample nutrients to the skin.

[0024] Traditional Chinese medicine believes that the formation of age spots is related to qi and blood, as well as the internal organs, and is caused by qi stagnation and blood stasis, as well as dysfunction of the liver, spleen, and kidneys. Modern research shows that vascular factors are also an important factor in the formation of age spots. Therefore, starting with promoting blood circulation and regulating the liver, kidneys, and spleen can have a good auxiliary effect on removing age spots and beautifying the skin.

[0025] Angelica powder is made from angelica root. Angelica root is warm in nature, sweet and pungent in taste, and enters the liver, heart, and spleen meridians. It has the effects of nourishing blood, promoting blood circulation, moistening the intestines and relieving constipation. Butylphthalide in angelica root can improve blood circulation, and angelica polysaccharides can directly and / or indirectly promote hematopoiesis in the body.

[0026] Peach kernel powder is made from peach kernels. Peach kernels are neutral in nature, bitter and sweet in taste, and enter the heart, liver, and large intestine meridians. They can invigorate blood circulation, remove blood stasis, and moisten the intestines to relieve constipation. Peach kernels play a role in invigorating blood circulation and removing blood stasis by improving hemodynamics (such as increasing perfusion fluid flow and blood flow, and reducing vascular resistance).

[0027] Sour jujube seed powder is made from sour jujube seeds. Sour jujube seeds are neutral in nature, sweet and sour in taste, and enter the liver, gallbladder, and heart meridians. They have the effects of nourishing the heart and liver, calming the mind, and soothing the nerves. At the same time, the saponins and ferulic acid components in sour jujube seeds can regulate the cardiovascular system and scavenge free radicals.

[0028] Rose powder is made from rose petals. Rose petals are warm in nature, sweet and slightly bitter in taste, and enter the liver and spleen meridians. They have the effects of regulating qi and relieving depression, soothing the liver and harmonizing blood.

[0029] Sea cucumber is warm in nature and salty in taste, entering the kidney meridian. It has the effects of nourishing blood throughout the body, tonifying the kidneys and replenishing essence, and promoting bowel movement and moisturizing dryness. Sea cucumber oligopeptide powder is a small molecule active peptide prepared from sea cucumber through bio-enzymatic hydrolysis technology. It retains components such as sea cucumber saponins and sea cucumber polysaccharides, making it rich in nutrients and completely absorbed by the human body. It plays a role in nourishing the body, replenishing blood, and tonifying the kidneys. Studies have also found that sea cucumber oligopeptide powder can reduce melanin content in B16 melanoma cells, promote cell repair, and has anti-aging effects.

[0030] Goji berry powder is made from goji berries. Goji berries are neutral in nature and sweet in taste. They enter the liver and kidney meridians and can nourish the liver and kidneys, benefit essence and improve eyesight.

[0031] Poria powder is made from Poria cocos. Poria cocos is neutral in nature, sweet and bland in taste, and enters the heart, lung, spleen, and kidney meridians. It can promote diuresis and eliminate dampness, strengthen the spleen, and calm the mind. The triterpenoids in Poria cocos are very effective in treating spleen deficiency.

[0032] Yam powder is made from yam. Yam is neutral in nature and sweet in taste, and it enters the spleen, lung, and kidney meridians. It has the effects of tonifying the spleen and stomach, and nourishing the kidneys and lungs. Yam also contains a variety of amino acids, polysaccharides, trace elements, and other beneficial nutrients.

[0033] The beneficial effects of this invention are:

[0034] (1) This invention provides a novel process for the preparation, separation, and purification of tyrosinase-inhibiting active peptides derived from fish maw. These active peptides have a small molecular weight, high activity, and are easy to obtain through simple separation and purification steps. As food-derived active peptides, long-term consumption can achieve the purpose of removing blemishes and whitening the skin, and they have broad application prospects.

[0035] (2) In the early stage, the present invention identified the oligopeptides of fish maw by high performance liquid chromatography-mass spectrometry and screened their activity using online databases to obtain highly active small molecule peptides. On this basis, the active peptides were separated and purified by Sephadex LH-20 gel column chromatography and high performance liquid chromatography to verify their activity. Finally, a peptide with high tyrosinase inhibitory activity was obtained, and its amino acid sequence is: Gly-Pro-Leu-Ala-Gly-Pro. The sequence was found to be a new small molecule active peptide by searching the online databases BIOPEP and EROP-Moscow.

[0036] (3) The appearance of age spots is due to increased melanin deposition, which is caused by stagnation of qi and blood, and dysfunction of the liver, spleen, and kidneys. This invention directly reduces melanin content while improving vascular factors, regulating liver, spleen, and kidney function, and indirectly reducing the conditions for age spot formation. Through synergistic effects, it achieves the effect of removing age spots and beautifying the skin. The tyrosinase-inhibiting active peptide (Gly-Pro-Leu-Ala-Gly-Pro) in fish maw oligopeptides directly intervenes in the melanin synthesis process by inhibiting the activity of tyrosinase, thereby reducing melanin formation. Angelica sinensis powder, peach kernel powder, sea cucumber oligopeptide powder, wolfberry powder, poria cocos powder, yam powder, jujube seed powder, and rose powder are scientifically formulated to improve vascular factors, invigorate blood, soothe the liver, tonify the kidneys, and strengthen the spleen, thus restoring the body's functional balance. Attached Figure Description

[0037] Figure 1 The diagram shows the experimental results of active peptides exhibiting significant tyrosinase inhibitory activity. Detailed Implementation

[0038] Example 1

[0039] A method for isolating and purifying fish maw oligopeptides containing tyrosinase-inhibiting peptides in a skin-lightening and beauty composition includes the following steps:

[0040] (1) After processing the fish maw, add 20 times the weight volume of water to make a homogenate and place it in an enzymatic hydrolysis tank. Then add 4% of the fish maw protein mass of the complex protease and enzymatically hydrolyze at 50°C for 4 hours. The pH value of the enzyme reaction is controlled at 7.5. After the enzymatic hydrolysis is completed, the temperature is raised to 90°C to inactivate the enzyme for 10 minutes to obtain the fish maw protease hydrolysate. Centrifuge the protease hydrolysate at 8000 rpm for 10 minutes to remove particulate matter. Then, use membrane separation technology to separate the particles. The molecular weight cutoff is 3000 Da. Spray dry the membrane solution to obtain fish maw oligopeptide powder. The mass ratio of the complex protease is: neutral protease: trypsin: bromelain: flavor protease = 4:4:1:3.

[0041] (2) Dissolve the fish maw oligopeptide powder from (1) in water to prepare a solution with a concentration of 100 mg / mL, and then use Sephadex dextran gel. The separation and purification were performed using LH-20 column chromatography (3.0×100cm). The mobile phase was 40% methanol, the flow rate was 0.4mL / min, and the absorbance of the eluent was measured at 280nm. The desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC) under the following conditions: Elite C18 column (4.6mm×250mm, 5μm), mobile phase A was 0.08% trifluoroacetic acid water (v / v), mobile phase B was acetonitrile, and the gradient elution conditions were: 0–15min, 3% B; 15–20min, 3%–10% B; 20–30min, 10% B–20% B; 30–40min, 20% B–35% B; flow rate was 0.8mL / min; detection wavelength was 280nm; and the peak with a retention time of 26min was collected. After concentration, the peak was freeze-dried to obtain the small molecule active peptides of fish maw.

[0042] In step (1), 93.8% of the fish maw oligopeptides were peptides with a molecular weight of less than 1000 Da.

[0043] The small molecule active peptides of fish maw obtained in step (2) were detected as a single peak by liquid chromatography. The structure was determined by high performance liquid chromatography-mass spectrometry, and its amino acid sequence was: Gly-Pro-Leu-Ala-Gly-Pro.

[0044] The raw materials for fish maw oligopeptides are one or more of the following: yellow croaker (Larimichthys), cod, red-lipped croaker, grouper, yellow croaker, sturgeon, and sea bass.

[0045] Assay for tyrosinase inhibitory activity:

[0046] The freeze-dried fish maw small molecule active peptides were pre-prepared into a 5 mg / mL solution using distilled water, and then successively diluted to obtain sample solutions with concentrations of 1 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, and 0.2 mg / mL. The substrate L-tyrosine (L-Tyr) was prepared into a 0.5% L-Tyr solution using 0.1 mol / L phosphate buffer (pH = 6.8). Tyrosinase was prepared into a 100 U / mL solution using 0.1 mol / L phosphate buffer (pH = 6.8).

[0047] Experimental group: Take 1 mL of sample solution of different concentrations, add 0.5 mL of L-Tyr substrate solution first, then add 0.5 mL of tyrosinase solution, react at 37℃ for 30 min, and measure the absorbance A1 at 475 nm.

[0048] Experimental blank control group: Take 1 mL of sample solution of different concentrations, first add 0.5 mL of L-Tyr substrate solution, then add 0.5 mL of phosphate buffer solution (pH = 6.8), react at 37℃ for 30 min, and measure the absorbance A2 at 475 nm.

[0049] Negative control group: Take 1 mL of phosphate buffer solution (pH = 6.8), add 0.5 mL of L-Tyr substrate solution first, then add 0.5 mL of tyrosinase solution, react at 37 °C for 30 min, and measure the absorbance A3 at 475 nm.

[0050] Blank control group: Take 1.5 mL of phosphate buffer solution (pH = 6.8), add 0.5 mL of L-Tyr substrate solution, react at 37 °C for 30 min, and measure the absorbance A4 at 475 nm.

[0051] The tyrosinase inhibition rate of small molecule peptides at different concentrations was calculated based on absorbance values.

[0052] Inhibition rate = [(A3-A4)-(A1-A2)] / (A3-A4)×100%.

[0053] Experimental results are as follows Figure 1 As shown, the results indicate that this small molecule bioactive peptide has significant tyrosinase inhibitory activity, with an IC50 of 0.318 mg / mL.

[0054] Example 2

[0055] A skin-lightening and beauty composition containing fish maw oligopeptides, comprising 90 parts fish maw oligopeptide powder, 30 parts angelica powder, 40 parts peach kernel powder, 25 parts sea cucumber oligopeptide powder, 50 parts wolfberry powder, 30 parts poria cocos powder, 40 parts yam powder, 40 parts jujube seed powder, and 30 parts rose powder.

[0056] The preparation method is as follows:

[0057] (1) Preparation method of fish maw oligopeptides: After processing the fish maw, add 18 times the mass volume of water to make a homogenate and place it in an enzymatic hydrolysis tank. Then add 3% of the mass of fish maw protein in the complex protease and enzymatically hydrolyze at 52℃ for 5 hours. The pH value of the enzyme reaction is controlled at 7.0. After the enzymatic hydrolysis is completed, the temperature is raised to 85℃ to inactivate the enzyme for 10 minutes to obtain the fish maw protease hydrolysate. Centrifuge the protease hydrolysate at 8000 rpm for 10 minutes to remove particulate matter. Then, use membrane separation technology to separate the oligopeptides with a molecular weight cutoff of 3000 Da. Spray dry the membrane solution to obtain fish maw oligopeptide powder. The mass ratio of the complex protease is: neutral protease: trypsin: bromelain: flavor protease = 4:3:2:4.

[0058] (2) Method for separating and purifying fish maw oligopeptides: Dissolve the fish maw oligopeptide powder from (1) in water to prepare a solution with a concentration of 100 mg / mL, and then use Sephadex gel electrophoresis. The separation and purification were performed using LH-20 column chromatography (3.0×100cm). The mobile phase was 40% methanol, the flow rate was 0.5mL / min, and the absorbance of the eluent was measured at 280nm. The desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC) under the following conditions: Elite C18 column (4.6mm×250mm, 5μm), mobile phase A was 0.07% trifluoroacetic acid water (v / v), mobile phase B was acetonitrile, and the gradient elution conditions were: 0–15min, 3% B; 15–20min, 3%–10% B; 20–30min, 10% B–20% B; 30–40min, 20% B–35% B; flow rate was 0.8mL / min; detection wavelength was 280nm; and the peak with a retention time of 26min was collected. After concentration, the peak was freeze-dried to obtain the small molecule active peptides of fish maw.

[0059] (3) Preparation method of sea cucumber oligopeptide powder: After sea cucumber is processed, 20 times the weight volume of water is added to make a homogenate and placed in an enzymatic hydrolysis tank. Then, 4% of the weight of the sea cucumber complex protease is added and enzymatically hydrolyzed at 48℃ for 4 hours. The pH value of the enzyme reaction is controlled at 8.0. After the enzymatic hydrolysis is completed, the temperature is raised to 90℃ to inactivate the enzyme for 10 minutes to obtain sea cucumber protease hydrolysate. The protease hydrolysate is centrifuged at 8000 rpm for 10 minutes to remove particulate matter. Then, membrane separation technology is used for separation, and the molecular weight cutoff is 3000 Da. The membrane solution is spray-dried to obtain sea cucumber oligopeptide powder. The mass ratio of the complex protease is: neutral protease: papain: flavor protease = 3:3:4.

[0060] (4) Take 90 parts of fish maw oligopeptide powder, 30 parts of angelica powder, 40 parts of peach kernel powder, 25 parts of sea cucumber oligopeptide powder, 50 parts of wolfberry powder, 30 parts of poria powder, 40 parts of yam powder, 40 parts of jujube kernel powder, and 30 parts of rose powder, mix them in a three-dimensional motion mixer for 15 minutes to fully mix them and obtain the powder.

[0061] Example 3

[0062] A skin-lightening and beauty composition containing fish maw oligopeptides, comprising 70 parts fish maw oligopeptide powder, 28 parts angelica powder, 35 parts peach kernel powder, 20 parts sea cucumber oligopeptide powder, 40 parts wolfberry powder, 25 parts poria cocos powder, 30 parts yam powder, 35 parts jujube kernel powder, and 20 parts rose powder.

[0063] The preparation method is as follows:

[0064] (1) Preparation method of fish maw oligopeptide powder: After processing the fish maw, add 16 times the mass volume of water to make a homogenate and place it in an enzymatic hydrolysis tank. Then add 5% of the mass of fish maw protein complex protease and enzymatically hydrolyze at 55℃ for 3 hours. The pH value of the enzyme reaction is controlled at 8.0. After the enzymatic hydrolysis is completed, the temperature is raised to 80℃ to inactivate the enzyme for 10 minutes to obtain fish maw protease hydrolysate. Centrifuge the protease hydrolysate at 8000 rpm for 10 minutes to remove particulate matter. Then, use membrane separation technology to separate the particles. The molecular weight cutoff is 3000 Da. Spray dry the membrane solution to obtain fish maw oligopeptide powder. The mass ratio of the complex protease is: neutral protease: trypsin: bromelain: flavor protease = 6:3:3:3.

[0065] (2) Method for separating and purifying fish maw oligopeptides: Dissolve the fish maw oligopeptide powder from (1) in water to prepare a solution with a concentration of 100 mg / mL, and then use Sephadex gel electrophoresis. The separation and purification were performed using LH-20 column chromatography (3.0×100cm). The mobile phase was 40% methanol, the flow rate was 0.6mL / min, and the absorbance of the eluent was measured at 280nm. The desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC) under the following conditions: Elite C18 column (4.6mm×250mm, 5μm), mobile phase A was 0.07% trifluoroacetic acid in water (v / v), mobile phase B was acetonitrile, and the gradient elution conditions were: 0–15min, 3% B; 15–20min, 3%–10% B; 20–30min, 10% B–20% B; 30–40min, 20% B–35% B; flow rate was 0.8mL / min; detection wavelength was 280nm; and the peak with a retention time of 26 minutes was collected. After concentration, the peak was freeze-dried to obtain the small molecule active peptides of fish maw.

[0066] (3) Preparation method of sea cucumber oligopeptide powder: After sea cucumber is processed, 15 times the weight volume of water is added to make a homogenate and placed in an enzymatic hydrolysis tank. Then, 5% of the weight of the sea cucumber complex protease is added and enzymatically hydrolyzed at 50°C for 4 hours. The pH value of the enzyme reaction is controlled at 8.5. After the enzymatic hydrolysis is completed, the temperature is raised to 85°C to inactivate the enzyme for 10 minutes to obtain sea cucumber protease hydrolysate. The protease hydrolysate is centrifuged at 8000 rpm for 10 minutes to remove particulate matter. Then, membrane separation technology is used for separation, and the molecular weight cutoff is 3000 Da. The membrane solution is spray-dried to obtain sea cucumber oligopeptide powder. The mass ratio of the complex protease is: neutral protease: papain: flavor protease = 2:5:4.

[0067] (4) Take 70 parts of fish maw oligopeptide powder, 28 parts of angelica powder, 35 parts of peach kernel powder, 20 parts of sea cucumber oligopeptide powder, 40 parts of wolfberry powder, 25 parts of poria powder, 30 parts of yam powder, 35 parts of jujube kernel powder, and 20 parts of rose powder, mix them in a three-dimensional motion mixer for 20 minutes, mix them thoroughly, and obtain the powder.

[0068] Example 4

[0069] A skin-lightening and beauty composition containing fish maw oligopeptides, comprising 80 parts fish maw oligopeptide powder, 32 parts angelica powder, 40 parts peach kernel powder, 24 parts sea cucumber oligopeptide powder, 40 parts wolfberry powder, 32 parts poria cocos powder, 32 parts yam powder, 40 parts jujube seed powder, and 24 parts rose powder.

[0070] The preparation method is as follows:

[0071] (1) Preparation method of fish maw oligopeptide powder: After processing the fish maw, add 18 times the mass volume of water to make a homogenate and place it in an enzymatic hydrolysis tank. Then add 5% of the mass of fish maw protein complex protease and enzymatically hydrolyze at 50℃ for 4 hours. The pH value of the enzyme reaction is controlled at 8.0. After the enzymatic hydrolysis is completed, the temperature is raised to 80℃ to inactivate the enzyme for 10 minutes to obtain fish maw protease hydrolysate. Centrifuge the protease hydrolysate at 8000 rpm for 10 minutes to remove particulate matter. Then, use membrane separation technology to separate the particles. The molecular weight cutoff is 3000 Da. Spray dry the membrane solution to obtain fish maw oligopeptide powder. The mass ratio of the complex protease is: neutral protease: trypsin: bromelain: flavor protease = 5:5:3:3.

[0072] (2) Method for separating and purifying fish maw oligopeptides: Dissolve the fish maw oligopeptide powder from (1) in water to prepare a solution with a concentration of 100 mg / mL, and then use Sephadex gel electrophoresis. The separation and purification were performed using LH-20 column chromatography (3.0×100cm). The mobile phase was 40% methanol, the flow rate was 0.5mL / min, and the absorbance of the eluent was measured at 280nm. The desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC) under the following conditions: Elite C18 column (4.6mm×250mm, 5μm), mobile phase A was 0.06% trifluoroacetic acid water (v / v), mobile phase B was acetonitrile, and the gradient elution conditions were: 0–15min, 3% B; 15–20min, 3%–10% B; 20–30min, 10% B–20% B; 30–40min, 20% B–35% B; flow rate was 0.8mL / min; detection wavelength was 280nm; and the peak with a retention time of 26min was collected. After concentration, the peak was freeze-dried to obtain the small molecule active peptides of fish maw.

[0073] (3) Preparation method of sea cucumber oligopeptide powder: After sea cucumber is processed, 17 times the mass volume of water is added to make a homogenate and placed in an enzymatic hydrolysis tank. Then, 3% of the mass of the sea cucumber complex protease is added and enzymatically hydrolyzed at 45℃ for 4 hours. The pH value of the enzyme reaction is controlled at 9.0. After the enzymatic hydrolysis is completed, the temperature is raised to 80℃ to inactivate the enzyme for 10 minutes to obtain sea cucumber protease hydrolysate. The protease hydrolysate is centrifuged at 8000 rpm for 10 minutes to remove particulate matter. Then, membrane separation technology is used for separation, and the molecular weight cutoff is 3000 Da. The membrane solution is spray-dried to obtain sea cucumber oligopeptide powder. The mass ratio of the complex protease is: neutral protease: papain: flavor protease = 2:4:4.

[0074] (4) Take 80 parts of fish maw oligopeptide powder, 32 parts of angelica powder, 40 parts of peach kernel powder, 24 parts of sea cucumber oligopeptide powder, 40 parts of wolfberry powder, 32 parts of poria powder, 32 parts of yam powder, 40 parts of jujube kernel powder, and 24 parts of rose powder, mix them in a three-dimensional motion mixer for 18 minutes to fully mix them and obtain the powder.

[0075] Example 5

[0076] A skin-lightening and beauty composition containing fish maw oligopeptides, comprising fish maw oligopeptide powder, angelica powder, peach kernel powder, sea cucumber oligopeptide powder, wolfberry powder, poria cocos powder, yam powder, jujube seed powder, and rose powder.

[0077] A skin-lightening and beauty composition containing fish maw oligopeptides, comprising: 50-100 parts fish maw oligopeptide powder, 20-40 parts angelica powder, 30-50 parts peach kernel powder, 10-30 parts sea cucumber oligopeptide powder, 30-50 parts wolfberry powder, 20-40 parts poria cocos powder, 20-40 parts yam powder, 20-50 parts jujube seed powder, and 20-30 parts rose powder.

[0078] Furthermore, the components of this formula are: 65-80 parts of fish maw oligopeptide powder, 26-32 parts of angelica powder, 30-40 parts of peach kernel powder, 15-24 parts of sea cucumber oligopeptide powder, 34-40 parts of wolfberry powder, 25-32 parts of poria powder, 20-32 parts of yam powder, 25-40 parts of jujube seed powder, and 20-24 parts of rose pollen.

[0079] The formula may also include: 55-90 parts of fish maw oligopeptide powder, 22-36 parts of angelica powder, 33-45 parts of peach kernel powder, 11-27 parts of sea cucumber oligopeptide powder, 33-45 parts of wolfberry powder, 22-36 parts of poria cocos powder, 22-36 parts of yam powder, 22-45 parts of jujube seed powder, and 22-27 parts of rose pollen.

[0080] This formulation can be prepared in the form of powder, granules, tablets, capsules, pills, oral solutions, etc., with powder and granules being the preferred forms.

[0081] This invention also relates to the preparation, separation, and purification of fish maw oligopeptides in this formulation, as well as a method for preparing sea cucumber oligopeptides:

[0082] (1) Preparation method of fish maw oligopeptides: After processing the fish maw, add 15-20 times the weight volume of water to make a homogenate and place it in an enzymatic hydrolysis tank. Then add 3-5% of the fish maw protein mass of a complex protease and hydrolyze at 50-55℃ for 3-5 hours. The pH value of the enzyme reaction is controlled at 7.0-8.0. After the enzymatic hydrolysis is completed, heat to 80-90℃ to inactivate the enzyme for 10 minutes to obtain the fish maw protease hydrolysate. Centrifuge the protease hydrolysate at 8000 rpm for 10 minutes to remove particulate matter. Then, use membrane separation technology to separate the oligopeptides with a molecular weight cutoff of 3000 Da. Spray dry the membrane solution to obtain fish maw oligopeptide powder. The mass ratio of the complex protease is: neutral protease: trypsin: bromelain: flavor protease = (4-6):(3-5):(1-3):(2-4).

[0083] (2) Method for separating and purifying fish maw oligopeptides: Dissolve the fish maw oligopeptide powder from (1) in water to prepare a solution with a concentration of 100 mg / mL, and then use Sephadex gel electrophoresis. The separation and purification were performed using LH-20 column chromatography (3.0×100cm). The mobile phase was 40% methanol, and the flow rate was 0.4–0.6 mL / min. The absorbance of the eluent was measured at 280 nm, and the desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC) under the following conditions: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase A was water containing 0.06%–0.08% trifluoroacetic acid (v / v); mobile phase B was acetonitrile; gradient elution conditions were: 0–15 min, 3% B; 15–20 min, 3%–10% B; 20–30 min, 10% B–20% B; 30–40 min, 20% B–35% B; flow rate was 0.8 mL / min; detection wavelength was 280 nm; and the peak with a retention time of 26 min was collected. After concentration, the peak was freeze-dried to obtain the small molecule active peptides of fish maw.

[0084] (3) Preparation method of sea cucumber oligopeptides: After sea cucumber is processed, 15-20 times the weight volume of water is added to make a homogenate and placed in an enzymatic hydrolysis tank. Then, 2-5% of the weight of the sea cucumber complex protease is added and enzymatically hydrolyzed at 40-50℃ for 4 hours. The pH value of the enzyme reaction is controlled at 8.0-9.0. After the enzymatic hydrolysis is completed, the temperature is raised to 80-90℃ to inactivate the enzyme for 10 minutes to obtain sea cucumber protease hydrolysate. The protease hydrolysate is centrifuged at 8000 rpm for 10 minutes to remove particulate matter. Then, membrane separation technology is used for separation, and the molecular weight cutoff is 3000 Da. The membrane solution is spray-dried to obtain sea cucumber oligopeptide powder. The mass ratio of the complex protease is: neutral protease: papain: flavor protease = (2-4):(3-5):(3-5).

[0085] In step (1), more than 90% of the fish maw oligopeptides have a molecular weight of less than 1000 Da.

[0086] The small molecule active peptide obtained in step (2) was detected as a single peak by liquid chromatography. Its structure was determined by high-performance liquid chromatography-mass spectrometry, and its amino acid sequence was: Gly-Pro-Leu-Ala-Gly-Pro. Tyrosinase inhibitory activity was measured, and the results showed that the small molecule peptide has strong tyrosinase inhibitory activity.

[0087] In step (3), more than 90% of the sea cucumber oligopeptides have a molecular weight of less than 1000 Da.

[0088] Comparative Example 1

[0089] A skin-lightening and beauty composition containing fish maw, comprising 90 parts fish maw powder, 30 parts angelica powder, 40 parts peach kernel powder, 25 parts sea cucumber oligopeptide powder, 50 parts wolfberry powder, 35 parts poria cocos powder, 40 parts yam powder, 40 parts jujube seed powder, and 25 parts rose powder.

[0090] The preparation method is as follows:

[0091] (1) Preparation method of fish maw powder: crush the fish maw with a pulverizer for 5 minutes and pass it through an 80-mesh sieve to obtain fish maw powder.

[0092] (2) Preparation method of sea cucumber oligopeptide powder: After sea cucumber is processed, 17 times the weight volume of water is added to make a homogenate and placed in an enzymatic hydrolysis tank. Then, 3% of the weight of the sea cucumber complex protease is added and enzymatically hydrolyzed at 45℃ for 4 hours. The pH value of the enzyme reaction is controlled at 9.0. After the enzymatic hydrolysis is completed, the temperature is raised to 80℃ to inactivate the enzyme for 10 minutes to obtain sea cucumber protease hydrolysate. The protease hydrolysate is centrifuged at 8000 rpm for 10 minutes to remove particulate matter. Then, membrane separation technology is used for separation, and the molecular weight cutoff is 3000 Da. The membrane solution is spray-dried to obtain sea cucumber oligopeptide powder. The mass ratio of the complex protease is: neutral protease: papain: flavor protease = 2:4:4.

[0093] (4) Take 90 parts of fish maw powder, 30 parts of angelica powder, 40 parts of peach kernel powder, 25 parts of sea cucumber oligopeptide powder, 50 parts of wolfberry powder, 35 parts of poria powder, 40 parts of yam powder, 40 parts of jujube kernel powder, and 25 parts of rose powder, mix them in a three-dimensional motion mixer for 18 minutes to fully mix them and obtain the powder.

[0094] Comparative Example 2

[0095] A skin-lightening and beauty composition containing fish maw oligopeptides, comprising 85 parts fish maw oligopeptide powder, 40 parts peach kernel powder, 40 parts wolfberry powder, 35 parts poria cocos powder, 30 parts yam powder, 38 parts jujube seed powder, and 20 parts rose powder.

[0096] The preparation method is as follows:

[0097] (1) Preparation method of fish maw oligopeptide powder: After processing the fish maw, add 17 times the mass volume of water to make a homogenate and place it in an enzymatic hydrolysis tank. Then add 3.5% of the mass of fish maw protein in the complex protease and enzymatically hydrolyze at 50℃ for 4 hours. The pH value of the enzyme reaction is controlled at 7.6. After the enzymatic hydrolysis is completed, the temperature is raised to 85℃ to inactivate the enzyme for 10 minutes to obtain the fish maw protease hydrolysate. Centrifuge the protease hydrolysate at 8000 rpm for 10 minutes to remove particulate matter. Then, use membrane separation technology to separate the particles. The molecular weight cutoff is 3000 Da. Spray dry the membrane solution to obtain fish maw oligopeptide powder. The mass ratio of the complex protease is: neutral protease: trypsin: bromelain: flavor protease = 4:5:2:4.

[0098] (2) Method for separating and purifying fish maw oligopeptides: Dissolve the fish maw oligopeptide powder from (1) in water to prepare a solution with a concentration of 100 mg / mL, and then use Sephadex gel electrophoresis. The separation and purification were performed using LH-20 column chromatography (3.0×100cm). The mobile phase was 40% methanol, the flow rate was 0.4mL / min, and the absorbance of the eluent was measured at 280nm. The desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC) under the following conditions: Elite C18 column (4.6mm×250mm, 5μm), mobile phase A was 0.07% trifluoroacetic acid in water (v / v), mobile phase B was acetonitrile, and the gradient elution conditions were: 0–15min, 3% B; 15–20min, 3%–10% B; 20–30min, 10% B–20% B; 30–40min, 20% B–35% B; flow rate was 0.8mL / min; detection wavelength was 280nm, and the retention time was collected for the small molecule active peptides from fish maw.

[0099] (3) Take 85 parts of fish maw oligopeptide powder, 40 parts of peach kernel powder, 40 parts of wolfberry powder, 35 parts of poria powder, 30 parts of yam powder, 38 parts of jujube kernel powder, and 20 parts of rose powder, mix them in a three-dimensional motion mixer for 18 minutes, and mix them thoroughly to obtain the powder.

[0100] Experimental verification

[0101] The products obtained in Examples 2, 3, and 4 of this invention and Comparative Examples 1 and 2 were used as samples, and their skin-removing and beautifying effects were verified through animal experiments.

[0102] Seventy female mice weighing 18–22g and aged 7 weeks were randomly divided into 7 groups (normal control group, model group, and experimental groups 1, 2, 3, 4, and 5), with 10 mice in each group.

[0103] Treatment of model group and experimental group mice: The backs of mice were shaved to fully expose the skin, once a week, with a shaved area of ​​4cm × 3cm. The skin of the mice was locally irradiated with medium-wave ultraviolet light at a light source of 20cm, once a day for 30 minutes each time. Progesterone injection solution (20mg·kg) was injected intramuscularly into the hind limbs. -1 The mice were injected alternately into both hind limbs once daily. Simultaneously, the mice were restrained in a mouse restraint device for one hour daily. This continued for 30 days, resulting in a mouse model of chloasma caused by liver qi stagnation.

[0104] Treatment of normal control mice: Mice were locally irradiated with medium-wave ultraviolet light at a distance of 20 cm from the light source, once daily for 30 minutes each time. Physiological saline (20 mg / kg) was injected intramuscularly into the hind limbs. -1 ), administer injections alternately to both hind limbs, once daily. Continue for 30 days.

[0105] According to the requirements in Table 1, mice in each group were administered the gavage test once daily for 30 consecutive days. All mice were euthanized at the end of the experiment.

[0106] Table 1. Gavage experimental protocols for each group of mice

[0107] Group Mouse categories Gavage sample Daily dose normal control group normal control mice physiological saline 0.2ml / 10g Model group Mice with liver qi stagnation type of melasma physiological saline 0.2ml / 10g Experimental group 1 Mice with liver qi stagnation type of melasma Example 2 Product 500mg / kg Experimental group 2 Mice with liver qi stagnation type of melasma Example 3 Product 500mg / kg Experimental group 3 Mice with liver qi stagnation type of melasma Example 4 Product 500mg / kg Experimental group 4 Mice with liver qi stagnation type of melasma Comparative Example 1 Product 500mg / kg Experimental group 5 Mice with liver qi stagnation type of melasma Comparative Example 2 Product 500mg / kg

[0108] Mouse skin tissue analysis: 1. Take an appropriate amount of mouse skin tissue irradiated with ultraviolet light, add 10 times the volume of ice water, homogenize, centrifuge, and detect the content of tyrosine, lipofuscin, superoxide dismutase (SOD), and malondialdehyde (MDA) in the supernatant. 2. Take a 1cm × 1cm piece of mouse skin tissue irradiated with ultraviolet light to observe the distribution of melanocytes.

[0109] Mouse liver tissue analysis: Take an appropriate amount of mouse liver tissue, add 10 times the volume of ice water, homogenize and centrifuge, and detect the content of tyrosine, superoxide dismutase (SOD) and malondialdehyde (MDA) in the supernatant.

[0110] The assay employed high-performance liquid chromatography (HPLC) to determine tyrosine, fluorescence colorimetry to determine lipofuscin, xanthine oxidase (SOD) to determine superoxide dismutase (SOD), and thiobarbituric acid (MDA) to determine MDA. Mouse skin was fixed in 10% formalin, then routinely embedded in paraffin and sectioned. After dewaxing to water and antigen retrieval, HMB45 (melanoma) was used as the primary antibody to visualize melanin granules in the epidermis using immunohistochemistry, thus observing the distribution of melanocytes.

[0111] The experimental results of the mouse skin tissue in each group are shown in Table 2.

[0112] The experimental results of liver tissue from each group of mice are shown in Table 3.

[0113] Firstly, compared with the normal control group, the number of melanocytes, tyrosine, lipofuscin, and MDA content in the skin of mice in the model group were significantly increased, while the SOD content was decreased. The tyrosine and MDA content in the liver were also significantly increased, indicating that the model was successfully established.

[0114] Experimental results on skin tissue showed that experimental groups 1, 2, and 3 could significantly reduce the number of melanocytes in mouse skin, while also reducing the content of tyrosine, lipofuscin, and MDA in the skin, and significantly increasing the activity of SOD in the skin. Experimental groups 4 and 5 could also reduce the number of melanocytes, the content of tyrosine, lipofuscin, and MDA in mouse skin, and increase the activity of SOD, but the therapeutic effect was not as good as that of experimental groups 1, 2, and 3.

[0115] Experimental results on liver tissue showed that experimental groups 1, 2, and 3 could significantly reduce the content of tyrosine and MDA in mouse liver and increase the activity of SOD in skin; experimental groups 4 and 5 could also reduce the content of tyrosine and MDA in mouse liver and increase SOD activity to a certain extent, but the therapeutic effect was worse than that of experimental groups 1, 2, and 3.

[0116] Conclusion: The experimental results show that the formulation of the present invention has good skin-removing and beautifying effects. Moreover, the formulations of Examples 2, 3, and 4 are more effective than those of Comparative Examples 1 and 2, which proves that the raw materials of the formulation of the present invention have a synergistic and promoting effect.

[0117] Although the invention has been described through specific embodiments, those skilled in the art will understand that the invention may also be covered by other embodiments within the scope of the invention as stated herein.

[0118] Table 2. Number of melanocytes, tyrosine, lipofuscin, MDA content, and SOD activity in the skin tissue of mice in each group.

[0119]

[0120] Table 3. Tyrosine, MDA content, and SOD activity in liver tissue of mice in each group.

[0121]

[0122]

[0123] Although the invention has been described through specific embodiments, those skilled in the art will understand that the invention may also encompass other embodiments within the scope of the invention as described herein.

Claims

1. A small molecule active peptide from fish maw, characterized in that, Its amino acid sequence is Gly-Pro-Leu-Ala-Gly-Pro.

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