TW201600118A - Application of surfactin to cosmetic - Google Patents

Abstract

This invention relates to a surfactin which is a biosurfactant generated by a microorganism- Bacillus subtilis. The surfactin is a natural cycloaliphatic peptide composed of seven amino acids to form a ring-shaped structure. Furthermore, the surfactin has functions of skin aging resistance, wrinkle resistance, skin penetration capability for enhancement of cosmetic (transdermal penetration enhancer), foaming agent, and emulsifier.

Description

Surface is applied to cosmetics

The invention relates to a method for applying a surface element, in particular to a method for applying a surface element to cosmetics.

1. Human skin structure and its aging factor

The skin is the main organ on the surface of the human body. The thickness varies depending on the location, age and gender. The main function of the skin is the first barrier of the human body, which can prevent the internal tissues of the human body from being directly damaged by external environmental factors, such as ultraviolet radiation and temperature. Changes, changes in humidity, damage from aerosols, and viruses or bacteria directly into the body, so the skin is susceptible to external factors that trigger the aging mechanism to metabolize damaged cells.

(1) Cell aging and gene defects

The aging effect gradually affects the tissues and organs from the beginning of the cells, causing the structure and function of various parts of the tissues to gradually decline. In Leonard Hayflick's 1963 study, cryopreserved human isolated cells were cultured for subsequent experiments, and it was found that the cells were deformed after a certain number of divisions; it was found that the human cells had their upper limit of the number of divisions, and the cells split about 50 times. After that, the rate of splitting and appearance will change, resulting in irregular splitting mode, resulting in abnormal cell appearance such as granular and distorted cells, causing apoptosis; this study suggests that the growth cycle of the organism is determined at the time of fertilized eggs. The life clock of an organism has been set in advance. For humans, the cell divides 50 times, about 120 years. Since the human body does not have telomerase to re-copy the fragments deleted when the cells replicate, resulting in damage to the cells during multiple cell replication and division, the cell cycle has its limitation. This limitation is also called Glass Ceiling. Aging is an irreversible and unavoidable process.

(2) UV

Skin and other organs will age over time, but the difference is that the skin will be stimulated by environmental factors in daily life, and the skin will age more intensely than other tissues and organs. Among the many environmental aging factors, ultraviolet rays (UV) are the most visible Loudness; ultraviolet wavelength range of 10~400nm, energy range from 3eV to 124eV, can be divided into three kinds of ultraviolet rays according to wavelength: (1) long-wave ultraviolet (UV-A), its wavelength is between 315~400nm, can directly penetrate the earth The atmosphere is exposed to the surface and penetrates the dermis layer of the skin, causing dark spots, aging and wrinkles on the skin. Its penetrating power is the strongest among the three wavelengths; (2) The UV-B wavelength is between 280 and 315 nm. It can cause redness, heat, pain, severe skin peeling or similar burns. When exposed to the earth, it will be absorbed by the ozone layer in the stratosphere. Only a small part will reach the surface through the ozone layer. (3) Short-wave ultraviolet light (UV- C) The wavelength is between 100 and 280 nm. It has the strongest energy and is quite dangerous. However, because its wavelength is short, it will be absorbed in the atmosphere. Only less than 0.1% of the total sunlight can reach the surface, and the sunscreen and glass barrier are generally shielded. Can effectively block. Studies have found that UV-A induces the production of matrix metalloproteinases in human skin fibroblasts. The matrix metalloproteinase family decomposes skin collagen, elastin and intercellular matrix to cause skin aging; UV-A It will force the concentration of free radicals in the cells to increase, and the excessive concentration will also cause the cells to age prematurely and even more.

(3) Free radicals

The theory of free radicals is the most widely accepted theory of aging in the scientific community. It was proposed by Denham Harmam MD of the University of Lincoln Medical School in 1954 but has not received much attention. After 20 years, the theory of free radicals has gradually been accepted. One of the mainstreams of aging theory, Denham Harmam was nominated for the Nobel Prize in Medicine in 1995. A normal atom has a pair of electrons, and a radical is an oxygen atom substance containing an unpaired electron; the electron is extremely unstable in an unpaired state, so the radical will transfer the electron of the normal atom, causing the cell to form a matrix. Change and cause cell death.

An important source of free radicals, in addition to its own metabolism or synthetic nutrients, causes aging to be environmental pollution, ultraviolet rays, radiation, smoking, pesticides and many chemicals, especially environmental pollution (automobile exhaust, factory-emitted SO) ₂ ) will increase a lot of harmful free radicals in the body.

Free radical attack can be divided into cell membrane damage and DNA damage. For human cells, oxygen-containing free radicals include superoxide anion O ₂ -, hydrogen peroxide H ₂ O ₂ , hydroxyl radical OH-, etc., all of which are collectively referred to as reactive oxygen molecules; excess free radicals are easily attacked Unsaturated fatty acids on the cell membrane. When free radicals attack unsaturated fat on the cell membrane, they form lipid peroxidase, oxidize low-density cholesterol in the blood vessel wall and inhibit prostacycline synthetase, causing arteriosclerosis, diabetes, arthritis, cataract , aging, coronary artery disease, etc. If free radicals penetrate deep into the nucleus to attack DNA, genetic information changes and lead to cancer; in addition, free radicals can induce aging genes and promote aging, and studies indicate that about 80% to 90% of aging and degenerative diseases are related to free radicals. These include cancer, Alzheimer's disease, Parkinson's disease, muscular dystrophy, skin dark spot deposition, wrinkle formation, yellow shift, degenerative heart disease, stroke, ulcer, rheumatoid arthritis and multiple sclerosis.

The defense mechanism of cells against free radicals: various antioxidant enzymes synthesized by cells, such as glutathione peroxidase (GPx), superoxide dismutase (SOD) and other enzymes and glutathione ( Glutathione, GSH), can remove superoxide anion Naturally produced substances in the body; the research report indicates that the SOD content in long-lived animals is relatively high, and humans are currently the animals with the highest SOD content in the body. Older aging, physical changes, environmental factors, etc., may cause the lack of antioxidant enzymes in the body and lead to aging.

(4) Inflammatory response

An inflammatory response is a response that occurs when a tissue is injured or infected. First, the mast cells reach the tissue and attach to the endothelial cells to release:

(1) Histamine: a derivative of amino acid, which increases the permeability of microvessels, expands local blood vessels, allows passage of substances such as plasma and phagocytic cells, and causes itching and allergic reactions. .

(2) Tumor Necrosis Factor (TNF): Cytokine kills target cells and activates the immune system, accelerates proliferation of lymphocytes and prevents the proliferation of pathogens, and attracts phagocytic cells.

(3) Prostaglandin (Prostaglandin): causes microvessels to dilate, leading to swelling and compression of nerve endings causing pain. After the inflammatory reaction, pus is formed. The pus is composed of dead cells and body fluids, and is usually digested by macrophages.

Specific cytokines activate their own cyclooxygenase (COX), which includes two major categories, COX-1 and COX-2. COX converts arachidonic acid into a large number of prostaglandins such as PGE 2 and PGF 2α. Recent research has found that Most normal tissues have not found the presence of COX-2, but it is indeed detectable in a variety of cancer patients, indicating the importance of COX-2 in cancer patients. In addition, COX-2 is an inducible enzyme whose function is to activate macrophages or other cells and to be filled with inflammatory tissues.

The inflammatory site produces a thermal sensation, mainly due to the release of vasodilators or histamine, resulting in vasodilation and increased blood flow. Some cells release IL-17 (Interleukin-1 alpha) inflammatory cytokines, or formerly known as interleukin cytokines; to activate specific white blood leukotrienes to resist allergens; Also included are chemokines that initiate cellular chemotaxis mechanisms and interferons that halt host cells for protein synthesis. Growth factors and cytotoxic molecules may also be released for tissue healing. When the above substances are secreted, the surrounding cells are also affected, resulting in loss of intercellular matrix and aging.

It can be seen from the above four aging causes that the proliferation of epidermal keratinocytes and dermal fibroblasts is slowed down during skin aging, resulting in changes in the macromolecular components and structural proteins of the extracellular matrix in the dermis of the skin. Aging phenomenon such as wrinkles of the skin, thinning of the skin, dull skin, reduced skin elasticity, and reduced moisturization. Ultraviolet light induces the formation of skin interstitial protease, which decomposes skin collagen and elastin tissue, and ultraviolet light also causes an increase in the concentration of free radicals in the cells, which in turn causes the skin to produce an inflammatory reaction and premature aging; After ultraviolet radiation, the skin will produce active oxidizing substances to destroy the cell structure, structural proteins, nucleic acids and other cellular structures, eventually leading to canceration and death of skin cells, and the loss of skin matrix caused by the accumulation of skin wrinkles.

2. Human anti-aging protein

Studies have shown that when cells are stimulated by resveratrol, a chemical in red wine, or calorie restriction, the sirtuins family is activated to prolong the cell cycle. Sinclair and its MIT Leonard Guarente laboratory found that a special Sirtuins protein in yeast affects the aging process in two specific ways. Sirtuins can help regulate gene activity in cells and repair DNA breaks.

Philipp Oberdoerffer, Sinclair Laboratories, used the microarray platform to probe the yeast sirtuin gene in mouse cells and searched for the sirtuin gene sequence in mammals. The mouse study also confirmed that there is a sirtuin gene similar to yeast in vertebrates. . Oberdoerffer found that one of the main functions of Sirtuin in the mammalian system is to monitor the pattern of gene expression; all genes are present in all cells, but at a given time, only a few genes need to be activated, if the wrong gene is activated It can cause cell damage and cause it to enter apoptosis.

Sirtuins deacetylates genes that are inhibited from being destroyed by environmental factors, and keeps the suppressed genes in their off state, keeping the cells genetically stable; Sirtuins can help preserve chromatin, genes and groups. The protein (histone) shrinks and coats together and keeps it idle; when the DNA is damaged by ultraviolet light or free radicals, Sirtuins will assist the DNA repair mechanism in the damaged area; Sirtuins' protective function can cause permanent damage in the gene. Before sexual injury, the gene and protein are coated to achieve the protective effect. If it is not protected by Sirtuins, the histones will begin to become loose, and the genes that are preserved and suppressed will be reactivated, making the genes vulnerable to external disturbances.

When the mouse ages, the rate of DNA damage increases. This damage causes the gene expression to be out of control. The chromatin is not controlled by its control. At this time, Sirtuins will help control the loss of control of the whole genome. Many of the genes that are activated are genes that are directly related to the aging phenotype.

Other studies have found that mouse genes that are not controlled by Sirtuins are continuously present in aging mice. Oberdoerffer uses a genetically engineered mouse model of lymphoma to give mice an extra copy of the sirtuin gene, or to feed the mouse sirtuin activator, resveratrol, to find the average lifespan of the mouse. It is extended by 24% to 46%.

Leonard Guarente's research points out that through the use of new drugs, Stirtuins' redistribution will stabilize over time, and there will be new ways to protect cells from aging. According to this particular mechanism, although DNA damage can worsen aging, the cause is not DNA damage itself, but the lack of regulation of genes; the Oberdoerffer study also pointed out that this process of regulating gene activity is called epigenetics (Epigenetics, Epigenetic), unlike the substantial mutations in DNA; and by verifying this principle, it was found that aging can be reversed in a way that stimulates Sirtuins.

3. Transdermal Penetration Enhancers (TPE)

New drug research takes a lot of money and time, so the development of drug delivery systems is getting more and more attention. The most common modes of administration of drugs include oral administration, subcutaneous tissue injection, and transdermal administration. Oral administration is one of the most commonly used modes of administration. After oral administration, the drug is absorbed into the blood through the gastrointestinal mucosa to achieve a local or systemic therapeutic effect. The disadvantage of oral administration is that the absorption of the drug through the body is slow and irregular, so that the drug can not achieve the therapeutic effect; in addition, the drug must pass through the liver before reaching the blood, so that the drug effect is destroyed, and the burden on the liver is also caused; The drug cannot be taken orally because it cannot be absorbed or irritated in the intestine; the biggest disadvantage of oral administration is that the drug causes side effects in the body, and the patient may have physical discomfort such as nausea and vomiting. Another method of administration is subcutaneous tissue injection, which directly injects the drug into the skin and is transported to the whole body through subcutaneous microvascular absorption. The advantage is that the drug is not affected by gastric juice and liver, and can directly enter the blood vessel and flow through the body. The site is used for therapeutic purposes; the subcutaneous tissue injection method greatly increases the rate of administration compared with the oral method, but for patients who have long-term dependence on injection, it is necessary to bear the pain burden caused by the injection for a long time. Yet another mode of administration is the Transdermal Drug Delivery System (TDDS), in which the drug is absorbed through the skin; after administration, the drug passes through the stratum corneum of the skin within a predetermined time. After microvascular osmotic absorption, it enters the blood circulation and acts to achieve systemic therapy (Saunders et al., 1999).

The advantages of the transdermal drug delivery system include: easy production, low cost, a constant rate of entry into the body, stable concentration in the blood for a long time, reduced frequency of administration, low toxicity, and avoidance of the liver's first-pass effect, Reduce drug metabolism, reduce individual differences in medication, increase the availability of organisms and small doses to achieve therapeutic effects. In addition, transdermal administration is suitable for young, elderly or patients who are not easy to take the medicine; it is convenient to use, and can be removed immediately when the problem occurs, and the administration is stopped. Due to the above advantages of transdermal administration, this system has attracted much attention; currently research and development of transdermal drug delivery has been developed from local to systemic, targeted organs and controlled release mechanisms, and has been applied clinically (Shin et al ., 2005).

The biggest obstacle to the transdermal drug delivery system is the stratum corneum of the skin. The stratum corneum (SC) is the uppermost layer of the skin. It consists of flat long keratinocytes surrounded by stratified fat (Norlen). , 2001); The most important function of the stratum corneum is to prevent foreign substances from entering the body and prevent the water from dispersing in the body, which is the outermost barrier of the skin (Bouwstra et al., 2003). In 1973, scholar Breathnach et al. used electron microscopy to find stratum corneum cells. The gap is full of fat and begins to assume that stratum corneum fat plays an important role in skin barrier function (Breathnach et al., 1973). Studies have also shown that when the temperature of the stratum corneum rises, the fluidity of intercellular fat in the stratum corneum increases, and the percutaneous absorption permeability of the skin also increases (Golden et al., 1987); if the lipid in the stratum corneum is removed It can significantly increase the permeability of hydrophilic and amphoteric drugs to the skin, but has no significant effect on lipophilic drugs (Tsai et al., 2001).

To overcome the problem of percutaneous absorption, the following three methods can be used: The first is a physical method that gives an external energy to cause transient pores in the skin and promote drug absorption; common treatments include ultrasound, Iontophoresis, microneedle array, and thermal energy. The second is a biochemical method that uses synthetic biotransformation precursors and inhibitors with metabolic agents to increase drug absorption. The third is a chemical method in which a drug is coated with a liposome or a skin enhancer is added. The liposome is composed of a curved double-layered lipid, forming a hydrophilic outward-facing hydrophobic end. Double-sided hydrophilic interlayer can be used as a carrier for oily and aqueous substances at the same time. Oily drugs can be embedded in the lipid bilayer, while aqueous drugs can be coated in the aqueous phase of the liposome. The mechanism of action of the microlipids and cells is four. The first type is intermembrane transfer, that is, the composition of the liposome membrane is exchanged with a part of the composition of the cell membrane; the second is adsorption, and the liposome is attached to the cell membrane; The three methods are membrane fusion, the liposome is fused with the cell membrane, and the inclusion is sent into the cell; the last one is phagocytosis (endocytosis), and the liposome is swallowed by the cells. A skin penetration enhancer is a substance that accelerates the rate and content of a drug that penetrates the skin, but does not cause serious irritation and damage to the skin (Williams and Barry, 1991); the skin penetration enhancer is mainly It acts on the interstitial layer of the stratum corneum, disturbs its regular structure and improves its fluidity, and acts on keratin, relaxes the angular cell structure, increases the solubility of the drug in the stratum corneum, and increases the absorption of the drug. Purpose (Walker and Smith, 1996). The addition of skin penetration enhancers is a relatively promising method of infiltration (Saunders et al., 1999).

Surfactant is a good skin penetration enhancer that aids in the penetration of biofilms and skin (Lopez et al., 2000) and has been widely used in drug penetration in recent years (Nokhodchi et al., 2003). Shokri et al., 2001). In 2001, Nokhodchi et al. pointed out that the surfactant lasulfate sulfate (SLS), cetyl bromide Cetyltrimethylammonium bromide (CTAB) and Benzalkonium Chloride promote the absorption of the skin of the mouse against the depressive drug Diazepam (Shokri et al., 2001). Nokhodchi et al. pointed out another study on antidepressant drugs, such as sodium lauryl sulfate (SLS), cetyltrimethylammonium bromide (CTAB) and chlorinated phthalate. Benzalkonium Chloride promotes the absorption of the mouse skin against the depressive drug lorazepam (Nokhodchi et al., 2003). Surface elements have a fairly good affinity for synthetic cell membranes, cell membranes of protists, and cell membranes of eukaryotes (Maget-Dana and Ptak, 1995; Sheppard et al., 1991; Tsukagoshi et al., 1970b); The binding of the element to the cell membrane is highly selective because the affinity of the surface element for cholesterol (cholesterol) and phospholipid is high, and the structure of the cell membrane is dominated by these two substances (Hosono and Suzuki). , 1985). If it is milder than chemically synthesized surfactants, it will not cause damage to the skin.

In the conventional method, the drug or nutrient active substance can only penetrate into the barrier layer of the epidermis (ie, the stratum corneum), and the effect is not significant (only about 0.3% of the effect); to solve this problem, there are many The skin absorption technology has been developed with the aim of effectively improving the penetration of nutrients and allowing nutrients to pass through the epidermal and dermal cells of the skin. Therefore, the nutrient delivery method has become one of the research priorities of skin care technology.

Although the stratum corneum of human skin is very thin, its thickness is only 10-25 microns. The thinnest stratum corneum is only 6 microns thick in the upper eyelid, but it is quite "tough" and is the most important protective layer of skin. The general cosmetic raw materials percutaneously infiltrated mainly through three ways: 1. through the sweat duct; 2. directly through the stratum corneum (stratum corneum); and 3. through the hair follicle (hair follicle).

Cosmetics raw materials take gold as an example. Modern gold beauty research has confirmed that gold has the functions of detoxification, lock, cleansing and wrinkle removal. After use, it can reconstitute cytokines of skin, promote physiological functions, metabolism, balance oil secretion and maintain natural moisture. It can also resist allergies caused by the outside world. Nano-gold, which is commonly used in cosmetic raw materials, is about one-twentieth the size of human hair pores. Therefore, when using nano-pure skin care products applied to the skin, it is hoped that nano-gold energy will penetrate into the dermis cells instantaneously.

When nanogold enters the dermis, nanogold regulates dermal cell function at the gene level, including the promotion of dermal cells to produce a range of active substances, including SOD, metallothionein, and EGF. Since SOD can scavenge hydroxyl radicals, and metallothionein can resist the damage of cortical cells by ultraviolet rays, nanogold has an anti-aging effect on dermal cells. Other studies have shown that nanogold can stimulate fiber cells, secrete new synthetic extracellular matrix (ECM), stimulate fibroblasts to secrete collagen, and stimulate fiber cells to present and secrete epidermal growth factor (EGF) to differentiate targets. Cell-specific keratinocyte growth factor (KGF); at the same time, it strengthens the skin's firmness, leaving the skin smooth and elastic, giving off a sparkling glow.

4. Bioemulsifier

In addition to being used as an antimicrobial peptide, surface keratin plays another important role as a bioemulsifier. In 1999, Deleu et al found that in the test for inhibiting lactation and flocculation, the effect of italin was better than that of surface. In the test of the emulsifying effect on alkanes, the emulsifying effect of surface elements on alkanes was much greater than that. The most emulsified effect of the herbicidal and abundance is the chemically synthesized anionic surfactant Sodium dodecyl sulfate (SDS). Other studies have shown that the addition of 20mg / l of surface hormone can increase the biodegradability of diesel, and further pointed out that the pH value will affect the emulsifying efficiency of surface oil to diesel, the biodegradability of surface oil to diesel when the pH is adjusted to 7.4. optimal.

5. Foaming agent

In addition to the emulsifying ability of general surfactants, surface elements also have foaming ability (Razafindralambo et al., 1998). The so-called foaming phenomenon means that surface elements are present in the interface between the gas phase and the liquid phase, and the shaking is vigorously caused to cause the surfactant to catch the air to form a film containing air (Halling, 1981). Razafindralambo et al. also pointed out that surface priming has superior foaming effect compared with italin, presumably related to the structure between the two, surface element is an anionic surfactant, and its fatty acid carbon chain is shorter. While imipenem is a nonionic surfactant with a long fatty acid carbon chain (Razafindralambo, et al., 1998).

It is an object of the present invention to provide a method of preparing an anti-aging (or anti-wrinkle) cosmetic composition using surfactin, wherein the composition comprises a surface element and a pharmaceutically acceptable carrier, excipient, diluent , an adjuvant, etc.; wherein the surface element is a cyclolipopeptide molecule, A cyclic heptapeptide structure consisting of 7 amino acids (L-Aspartic acid, L-leucine, glutamic acid, L-leucine, L-valine and two D-leucines). The cyclized 7 peptide is linked to a β-hydroxy fatty acid, which is dominated by an iso14 (isoC14) hydroxyl group (17% to 35%); wherein the fatty acid distribution of the surface element at the fatty acid terminal is as follows: (1) the iso C13 is greater than 3%; (2) positive C13 is greater than 0.65%; (3) iso-C14 is greater than 17%; (4) positive C14 is less than 41%; and (5) iso-C15 is less than 11%; wherein the fatty acid terminal fatty acid distribution is below The column distribution is preferably: (1) different C13 is greater than 10%; (2) positive C13 is greater than 25%; (3) iso C14 is greater than 35%; (4) positive C14 is less than 25%; and (5) less than C15 is less 3%; wherein the fatty acid distribution at the terminal of the fatty acid is optimal in the following distributions: (1) 11% for the iso C 13; (2) 26% for the positive C13; (3) 37% for the different C 14; (4) Positive C14 is 24%; and (5) iso C15 is 2%; wherein the surface element has a molecular weight of 1022 or 1036 Da; wherein the surface element comprises its chemical isomer. (See Republic of China Patent Application No. 097137532).

Wherein the anti-aging (or anti-wrinkle) cosmetic composition may further comprise at least one of the following: an alcohol, an ester, a complex polysaccharide, a nut oil, and a vitamin; wherein the alcohol comprises at least one of the following: C16-18 alcohol Butanediol, pentanediol, octanediol, glycerol, 16 alcohol, 18 alcohol, 22 alcohol and propylene glycol; wherein the ester comprises at least one of the following: olive oil cetyl alcohol ester (OLIVEM 1000), Glyceryl monostearate (GSM), isopropyl myristate (IPM), isopropyl palmitate (IPP) and triglyceride; wherein the composite polysaccharide system comprises at least one of the following: xanthan gum, white fungus a polysaccharide body, a glucoside polysaccharide, a senna seed polysaccharide; wherein the nut oil comprises at least one of the following: argan oil, Hawaiian stone oil, avocado oil, wheat germ oil, olive oil; wherein the vitamin system comprises At least one of the following: vitamin A, vitamin B, vitamin C, vitamin E, vitamin F, vitamin K.

The anti-aging (or anti-wrinkle) cosmetic composition is used for promoting the proliferation of fibroblasts, anti-UV aging, anti-oxidation, promoting the expression of sertuin 1 gene, promoting collagen proliferation and inhibiting matrix metalloproteinase (Matrix metallopeptidase). Wherein the matrix metalloproteinase is Matrix metallopeptidase 9 (Matrix metallopeptidase 9).

The present invention further provides a method of preparing a composition for increasing skin penetration by surfactin, wherein the composition comprises a surface element and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, etc. Wherein the surface element is a cyclolipin peptide, the surface element Including a seven-peptide sequence (L) Glu-(L)Leu-(D)Leu-(L)Val-(L)Asp-(D)Leu-(L)Leu linked to a beta hydroxy fatty acid, the surface element is The fatty acid distribution at the fatty acid terminal is as follows: (1) the iso C13 is greater than 3%; (2) the positive C13 is greater than 0.65%; (3) the iso C14 is greater than 17%; (4) the positive C14 is less than 41%; and (5) the iso C15 Less than 11%; wherein the fatty acid distribution at the end of the fatty acid is preferably the following distribution: (1) different C13 is greater than 10%; (2) positive C13 is greater than 25%; (3) iso-C14 is greater than 35%; (4) positive C14 is less than 25%; and (5) iso C15 is less than 3%; wherein the fatty acid distribution at the terminal of the fatty acid is optimal in the following distributions: (1) 11% of iso C 13; (2) 26% of positive C13; (3) the specific C 14 is 37%; (4) the positive C14 is 24%; and (5) the iso C15 is 2%; wherein the surface element has a molecular weight of 1022 or 1036 Da; wherein the surface element comprises its chemical isomer The composition for increasing skin penetration may further comprise at least one of the following: an alcohol, an ester, a complex polysaccharide, a nut oil, and a vitamin; wherein the alcohol comprises at least one of the following: C16-18 alcohol, Butanediol, pentanediol, octanediol, glycerol, 16 alcohol, 18 alcohol, 22 alcohol and propylene glycol; wherein the ester is packaged Including at least one of the following: olive oil cetyl alcohol ester (OLIVEM 1000), glyceryl monostearate (GSM), isopropyl myristate (IPM), isopropyl palmitate (IPP) and triglyceride The complex polysaccharide system comprises at least one of the following: xanthan gum, tremella polysaccharide, glucoside polysaccharide, senna seed polysaccharide; wherein the nut oil comprises at least one of the following: argan oil, Hawaiian stone oil , avocado oil, wheat germ oil, olive oil; wherein the vitamin includes at least one of the following: vitamin A, vitamin B, vitamin C, vitamin E, vitamin F, vitamin K. .

The composition for increasing skin penetration is used to promote the penetration of cosmetic raw materials into the skin; wherein the cosmetic raw materials are dexamethasone, hyaluronic acid, gamma-polyglutamic acid and nanometer. Gold-nanoparticles.

The present invention further provides a method for preparing an emulsifier composition by surfactant, wherein the emulsifier composition comprises a surface element and a pharmaceutically acceptable carrier, an excipient, a diluent, an adjuvant, etc.; The surface element is a cyclolipin peptide comprising a seven-peptide sequence (L) Glu-(L)Leu-(D)Leu-(L)Val-(L)Asp- linked to a beta hydroxy fatty acid. D) Leu-(L)Leu, the fatty acid distribution of the surface element at the fatty acid end is as follows: (1) the iso-C13 is greater than 3%; (2) the positive C13 is greater than 0.65%; (3) the iso-C14 is greater than 17%; (4) Positive C14 is less than 41%; and (5) iso C15 is less than 11%; wherein the fatty acid distribution at the terminal of the fatty acid is preferably the following distribution: (1) different C13 is greater than 10%; (2) positive C13 is greater than 25%; (3) iso-C14 is greater than 35%; (4) positive C14 is less than 25%; and (5) iso-C15 is less than 3%; wherein the fatty acid terminal fatty acid The distribution is optimal with the following distributions: (1) iso C 13 is 11%; (2) positive C13 is 26%; (3) iso C 14 is 37%; (4) positive C14 is 24%; and (5) The iso-C15 is 2%; wherein the surface element has a molecular weight of 1022 or 1036 Da; wherein the surface element comprises a chemical isomer thereof; wherein the emulsifier composition further comprises at least one of the following: fatty acid glyceride, fatty acid sorbitan Ester, fatty acid sucrose ester, fatty acid propylene glycol ester, lecithin.

Wherein the emulsifier composition is used to increase foaming power, increase emulsification, and increase smoothness; wherein the emulsifier composition is used to increase the foaming power of the cleaning article, which is shampoo and facial cleansing. Milk, hand lotion, bath essence, etc.; wherein the emulsifier composition is used to increase the emulsifying power of the cosmetic, the body is a body lotion, a cream, an essence, a cream, etc.; wherein the emulsifier composition is used to increase The smoothness of the cleaning products, such as shampoo, facial cleanser, hand lotion and bath essence.

Figure 1 Effect of different concentrations of surface hormone on the expression of longevity gene ( sertuin 1 gene) in mouse embryonic fibroblasts (BALB/3T3 clone A31 Mus musculu s embryo fibroblast); culture of mouse embryonic fibroblasts at different concentrations Surface elements (0, 25, 50, 75, 100 μM), culture time was 6, 12, 24, 36, 48 hours.

Figure 2 Effect of Resveratrol, Palmitoyl pentapeptide-3 and surface cytokines on cell proliferation; Mouse embryonic fibroblasts were cultured at different concentrations of surface proteins (0, 25, 50, 75, 100 μM), resveratrol (0, 5, 10, 20, 30 μM) and pentapeptide (0, 3, 5, 8, 10 μM), culture time was 48 hours.

Fig. 3 The effect of surface keratin on photoaging; the effect of different concentrations of surface elements on the survival rate of mouse embryonic fibroblasts after exposure to ultraviolet light; cells with UV of 5 J/cm2, 10 J/cm2 and 15 J/cm2 After light irradiation, surface concentrations of 25, 50, 75, 100, and 125 μM were added, and after 24 hours of culture, the cell survival rate was measured by the MTT method.

Figure 4 The protective effect of surface elements on the anti-oxidative stress of mouse embryonic fibroblasts; After treatment with hydrogen peroxide at concentrations of 100, 150, 200 and 250 μM, surface concentrations of 0, 25, 50, 75 and 100 μM were added and cultured for 24 hours, and then cell viability was tested by MTT assay.

Figure 5: Effect of surface and pentapeptide on the collagen dose of mouse embryonic fibroblasts; collagen content is measured by Sircol collagen assay; mouse embryonic fibroblasts are at different concentrations Surface elements (0, 25, 50, 75, 100 μM) and pentapeptide (0, 3, 5, 8, 10 μM) were cultured for 24 hours; PBS: phosphate buffer solution; a, b, ab, c, d represents There is no statistical difference in the same symbol.

Figure 6: Surface metalloproteinase (SF) and pentapeptide (PPP-3) inhibit matrix metalloproteinase (Matrix metallopeptidase 9); matrix metalloproteinase is measured by enzyme immunoassay (ELISA); mouse embryo Fibroblasts were cultured in different concentrations of five peptides (0, 3, 5, 8 and 10 μM) and surface proteins (0, 25, 50, 75 and 100 μM); a, b, c represent no statistics if the symbols are the same The difference.

Figure 7 Surface surface promotes skin absorption of corticosteroids; the experiment is divided into two groups. The control group is PBS (pH=7.4) 100 μl mixed with fluorescein isothiocyanate (FITC) labeled corticosteroid (dexamethasone), added to 1 cm. ² Sterile absorbent cotton and fixed on the back of the mouse with a breathable patch; the experimental group was treated with isothiocyanate-labeled corticosteroids with 0, 0.2, 0.5, 1, 2, 5% surface elements; the experimental results were conjugated. The focal microscope was used to detect the fluorescence brightness; (A)~(F) was sequentially added to the corticosteroids with 0, 0.2, 0.5, 1, 2, 5% surface elements; SF: surface protein.

Figure 8 shows the test of the skin's absorption of the moisturizing factor hyaluronic acid (HA). The experiment was divided into two groups. The control group was PBS (pH=7.4) 100 μl mixed with fluorescein isothiocyanate (FITC)-labeled hyaluronic acid, added to 1 cm ² sterile cotton wool, and fixed with a breathable patch. The back of the mouse. The experimental group was treated with isothiocyanate-labeled hyaluronic acid with 0, 0.2, 0.5, 1, 2, and 5% of surface keratin for one hour; the experimental results were observed by conjugated focal microscope to detect the fluorescence brightness. (A) ~ (F) map sequentially added 0, 0.2, 0.5, 1, 2, 5% of surface acid for hyaluronic acid; SF: surface element.

Figure 9 Surface surface promotes skin absorption moisturizing factor (γ-GPA) test; the experiment is divided into two groups, the control group is PBS (pH = 7.4) 100μl mixed with fluorescein isothiocyanate (FITC) labeled γ-GPA, Add to 1 cm ² sterile cotton wool and fix it on the back of the mouse with a breathable patch; the experimental group added 0, 1%, 2%, 5%, 10%, 15%, 20% with isothiocyanate-labeled γ-GPA. The surface element was treated for one hour, and the experimental results were detected by a conjugated focal microscope to detect the brightness of the fluorescence; (A)~(G) were sequentially added to the γ-GPA by 0, 1%, 2%, 5%, and 10%. , 15%, 20% of surface elements.

Figure 10 Surface surface promotes the absorption of nanogold through the skin; the amount of nanogold is measured by a fluorescence microscope; the green fluorescence represents the presence of nanogold, and the nanogold mixed with surface elements can More nanogold was found in the dermis of mice (BALB/c); green highlights were hair follicles autofluorescence; SC surface: stratum corneum surface.

Figure 11 Emulsion force analysis of surface factors; dissolve different concentrations of surface pigment in a buffer solution of pH 6.4, 7.4 and 8.4, take 2ml and add 3ml of diesel oil in a test tube, shake (vortex) for 2 minutes, in the room After standing for 24 hours under temperature, the emulsification index (E ₂₄ ) was measured.

Figure 12: Foaming force analysis of surface pigments; different concentrations of crude purified surface pigment were dissolved in a buffer solution of pH 7.4, and after vortexing for two minutes, after standing for 1 hour, the foam height was measured and calculated. Maximum foam density (MD).

Figure 13 shows the relationship between surface element concentration and foaming height.

Figure 14 shows the relationship between surface element concentration and turbidity (Turbidity %).

Figure 15 After dilution of the polymer-surfactant complex, surface-containing shampoos will precipitate.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples. The following examples are carried out by a surface element having the following characteristics: the surface element is a cyclolipin peptide, and the surface element comprises a beta-hydroxy fatty acid (linked to 13 to 15 hydrocarbons). Chain) of the seven-peptide sequence (L) Glu-(L)Leu-(D)Leu-(L)Val-(L)Asp-(D)Leu-(L)Leu; wherein the fatty acid has a different C14 37%.

Example 1 Surface anti-aging of human skin and mouse embryonic fibroblasts Chemical test

Experimental materials and methods

Experimental cell strain

Human skin fibroblast (CCD-966SK) was purchased from the BCRC Bioresource Conservation and Research Center - ID: 60153.

2. Mouse BALB/3T3 embryo (BALB/3T3 Clone A31) was purchased from the BCRC Bioresource Conservation and Research Center - ID: 60009.

3. Experimental animals

The BALB/cByJNarl strain of single sex was purchased from the National Laboratory Animal Center and weighed about 200-250 grams. The breeding conditions were artificial light 12 hours a day, humidity 75% and temperature 25 ° C. It was raised in the ocean with air-conditioning equipment. The University Life Science Animal Room is cared for by a dedicated person to give enough feed and water.

4. Anti-aging gene performance test

(1) Human and mouse gene cDNA sequence selection

This experiment used TRIzol reagent (Invitrogen, USA) to extract Total RNA from human skin fibroblasts and mouse embryonic fibroblasts, and analyzed by RNA electrophoresis; human skin fibrils were performed by reverse transcription polymerase chain reaction (RT-PCR). The sirtuin 1, sirtuin 3 and mouse embryonic fibroblast sirtuin 1 gene sequence was selected; and the known sirtuins gene sequence was searched by gene bank (GeneBank), and the similar sequence was searched by GCG program to design a pair of primers. (Primer), performing a polymerase chain reaction (PCR); a small portion of the reaction product is analyzed by 1.2% colloidal electrophoresis. When a product corresponding to the size of the expected gene fragment appears, the remaining product is passed through After electrophoresis of the low melting point agar colloid, the product of the desired colloid is excised from the colloid and subjected to colloidal extraction to obtain a purified DNA fragment of a specific size. The purified fragment and the selection vector pGEM-T-easy vector (pGEM-T-easy The cloning kit, Promega, USA) is ligated into a competent cell; finally, after a small amount of culture, the selection vector is selected for sequencing. Sequence information using the BLAST program American Center for Biological Information (NCBI) website for comparison. via After the above steps, the correct part of human skin fibroblast sirtuin 1, sirtuin 3 and mouse embryonic fibroblast sirtuin 1 cDNA sequence were selected, and the selected strain was transformed into competent cells for replication and high extraction. Purity plastids are preserved for subsequent experiments.

(2) Extraction of cellular Total RNA

RNA is easily decomposed by RNase in the natural environment, so the equipment used must be sterilized at high temperature and dried in an oven before the experiment. Wear gloves and a mask during operation. For the cell experiment group to be tested, 800 μL of TRIzolTM reagent was quickly added to the culture dish, and the washed cell liquid was placed in a sterilized Tube, and chloroform (Chloroform) of 1/5 of the total volume of TRIzolTM reagent was added, and the mixture was vigorously shaken. After 30 seconds, it was allowed to stand at room temperature for 15 minutes, and then centrifuged at 13200 rpm for 15 minutes at 4 ° C, and the supernatant was withdrawn into a new 1.5 mL microcentrifuge tube. At this point, the RNA will be dissolved in the aqueous layer of the supernatant, and the supernatant will be inhaled into another clean 1.5 mL microcentrifuge tube. Isopropanol and high salt solution of 1/2 volume of TRIzolTM reagent will be added. (1.2M Sodium Chloride and 0.8M Sodium citrate), shake evenly at -20 °C for 30 minutes, then centrifuge at 13200 rpm for 15 minutes at 4 °C to precipitate RNA; carefully remove the supernatant, first add 800 μL After 100% absolute alcohol shaking and rinsing, after centrifugation at 13200 rpm for 10 minutes at 4 ° C, 100% alcohol was removed. The supernatant was carefully aspirated and removed, and the RNA pellet was dried at 55 ° C for 5 minutes. The precipitate was dissolved in 15 μL of DEPC water. The RNA was dissolved at 55 ° C for 10 minutes and finally stored in a -80 ° C freezer for later use.

(3) Measurement of RNA concentration and purity

The OD260 was measured by a spectrophotometer, and the standard for calculating the RNA concentration was 1 OD260 = 40 μg of RNA/mL, and the formula was 40 μg × dilute factor × A260 = μg / mL. RNA purity is based on the ratio of OD260/OD280, and the ratio is between 1.9 and 2.0 for higher purity RNA.

(4) Reverse Transcription (RT)

Take 5 μg of Total RNA in a 200 μL microcentrifuge tube and use DEPC water. The total volume was adjusted to 13 μL, and 1 μL of oligo(dT)18, 10 mM dNTPs was added and placed on ice. Further, 5 μM of 5×AMV buffer and 1 μl of AMV (40 units/μL) were added, uniformly mixed, and reacted at 42° C. for 60 minutes to synthesize cDNA, and then reacted at 70° C. for 10 minutes to terminate the reaction.

(5) Polymerase chain reaction (PCR)

A cDNA template (RT) prepared by RT was subjected to polymerase chain reaction (PCR) amplification analysis using Taq polymerase. 1 μL of cDNA was added to 0.5 μL of 10 mM dNTPs, 2.5 μl of 10× PCR buffer, Gene specific primer (GSP) of 0.5 μL and 0.25 μL of Taq polymerase, and the total volume was adjusted to 25 μL by adding secondary sterilized water. The reaction tube was placed in a PCR reactor (DNA thermal cycler; Applied Biosystems 2720 Thermal Cycler), and the following conditions were set: dissociation at 94 ° C for 2 minutes; dissociation at 94 ° C for 30 seconds; bonding at 64 ° C for 30 seconds; synthesis at 72 ° C for 1 minute; The dissociation, bonding, and synthesis steps were repeated for 30 cycles and stored at 4 °C. 5~15μL of the PCR product after reaction was analyzed by electrophoresis in 1.5% agar colloid. After staining with ethidium bromide (EtBr) for 10 minutes, it was deproteinized with water for about 10 minutes and placed in the camera system Digital Gel Image System. Image and quantitative analysis by UN-SCAN-IT gel-Gel Analysis Software Version 6.1.

(6) Real-time quantitative PCR

The fluorescent dye SYBR green I can be embedded in the double groove of the DNA, and the fluorescent light is excited by the halogen lamp to detect the amount of fluorescence. When SYBR green I is not embedded in double-stranded DNA, the fluorescent background value is very low; when SYBR green I is first embedded in the PCR-amplified target gene fragment, the fluorescence value signal is also relatively increased. If there is no interference of non-specific primers or interference of Genomic DNA, the state of the PCR-synthesized target gene can be distinguished by a two-fold multiplication of geometric phase and insufficient reactants. Gene synthesis is not a two-fold multiplication linear phase; and finally the reactants are depleted, failed, and reach the Plateau phase of the end of the reaction. Therefore, to detect the amount of expression of the target gene in tissues and organs, It must be quantified to quantify the geometric progression of the PCR reaction. The principle of Real-time quantitative PCR is to use a different concentration of template under the same PCR reaction conditions, the reaction containing high concentration of template will reach the geometric multiplication period faster, and the relatively low concentration template will be reached slowly, and the definition will reach the geometry. The number of critical PCR cycles at the midpoint of the series multiplication period is determined as CT (Threshold Cycle), which means that the CT value increases as the template concentration decreases. Using the CT values of the simultaneous quantitative PCR reactions of different concentration standards, the standard curve and the regression formula were calculated by software, and the absolute expression of the target gene contained in the sample was calculated by interpolation. At the same time, if you want to get Melting Curve, you can perform continuous fluorescence detection at 40 °C~99 °C after the target gene amplification cycle. The results of melting curve can be used to understand whether the primers have complementary phenomena or the specificity of the primers. Sex, to better understand the correctness of experimental quantification.

The 96 well PCR plate was placed on a pre-cooled 96 well PCR plate ice box at 4 °C, and 10 μL of SYBR green and 4 μL of Real-time PCR specific primers were added (Table 1). Finally, 2 μL of RT product was added as PCR. The template of the reaction was attached to a 96 well PCR plate membrane and placed in a 4 ° C centrifuge for 3 minutes at 1500 rpm, and the sample was centrifuged to the bottom of the PCR plate. The PCR plate was placed in a real-time quantitative PCR reactor (Roche lightcycler 480 Real time PCR), and the following reaction conditions were set: hot start reaction was carried out at 95 ° C for 5 minutes; amplification reaction (Amplification) was dissociated at 95 ° C 40 seconds, 60 ° C bonding for 30 seconds, 40 cycles; and continuously detect the fluorescence value to determine whether the amplified products are the same dissociation temperature. After completion of the reaction, the reaction data was analyzed in Roche software. The data obtained from the experiment were analyzed by SPSS software using One-way analysis of variance, and the degree of significant difference between the factors was compared according to Duncan's Multiple Range Test (p<0.05). The obtained data were average±standard deviation (Mean). ±SD) representation.

Table 1 Real-time PCR specific primer

Results Surface element promotes the expression and proliferation of longevity gene (sertuin 1 gene) in mouse embryonic fibroblasts

As can be seen from Fig. 1, surface stimulants can promote the expression of the longevity gene ( sertuin 1 gene) of mouse embryonic fibroblasts; when different concentrations of surface elements are administered to mouse embryonic fibroblasts, different concentrations of surface elements for sertuin 1 Gene expression has different effects; when administered to mouse embryonic fibroblast surface hormone for 6 hours, 50μM, 75μM surface element has the effect of promoting sertuin 1 gene expression; when mouse embryonic fibroblast surface factor is administered for 12 hours After that, 25 μM of surface element has the effect of promoting the expression of sertuin 1 gene; when the mouse embryonic fibroblast surface element 36 is administered, 50 μM, 100 μM of surface element has the effect of promoting the expression of sertuin 1 gene; when the mouse embryonic fiber is administered After the parent cell surface 48, 50 μM of surface element has the effect of promoting the expression of the sertuin 1 gene. Among them, 50 μM of surface hormone has the best effect on promoting the performance of sertuin 1 gene.

Figure 2 shows the effects of Resveratrol, Palmitoyl pentapeptide-3 and surface hormone on the proliferation of mouse embryonic fibroblasts; Figure 2 shows that surface proteins promote mouse embryos. The effect of fibroblast proliferation, and promotes cell proliferation with 75 μM surface protein The best effect is that the 50 μM surface element promotes cell proliferation with the second best effect. In addition, the effect of surface stimulating cells on cell proliferation is better than that of resveratrol and pentapeptide.

Example 2 Surface elements have anti-photoaging and anti-oxidation effects

1. Surface element has anti-photoaging repair function

After the cells were plated, cultured for 4 hours and fixed in a culture dish, the UV-A wavelength ultraviolet light tube was used as a light source, and the experimental cells were irradiated with an energy concentration of 0 J/cm ² in an ultraviolet light hybridization box. 10 J/cm ² and 15 J/cm ² were added, and surface concentrations of 25, 50, 75, 100, and 125 μM were added after the irradiation, and after 24 hours of culture, the cell survival rate was measured by the MTT method.

It can be seen from Fig. 3 that the surface element has anti-photoaging repair function; when the mouse embryonic fibroblast is irradiated with ultraviolet light of 10 J/cm ² , the surface elements at 25, 50, 75, 100 and 125 μM concentration are increased. The effect of cell survival rate was the best at 75 μM concentration; when the mouse embryonic fibroblasts were irradiated with 15 J/cm ² of ultraviolet light, the surface elements at 25, 50, 75, and 100 μM concentrations increased. The effect of cell viability was the best, and the surface effect at a concentration of 50 μM was the best.

2. Surface element has anti-oxidation repair function

The mouse embryonic fibroblasts were plated, cultured for 4 hours to be fixed in a culture dish, and hydrogen peroxide was used as an urgent source of oxidation; the cells were treated with hydrogen peroxide at concentrations of 100, 150, 200, and 250 μM, and then added. Surface proteins at concentrations of 25, 50, 75 and 100 μM were cultured for 24 hours and then tested for cell viability by MTT assay.

As can be seen from Fig. 4, the surface element can increase the survival rate of mouse embryonic fibroblasts treated with hydrogen peroxide. Among them, 25, 50, 75 μM concentration of surface elements have a good effect, while the 75 μM concentration of surface elements has the most significant effect.

Example 3 Effect of surface element on collagen growth of mouse embryonic fibroblasts

1. Collagen concentration analysis

The collagen concentration analysis kit (Sircol ^TM soluble collagen assay kit) were analyzed for total collagen concentration in the medium. The analysis method is briefly described as follows:

The sample to be analyzed and 0.1 mL of different concentrations of the standard were placed in a 1.5 mL centrifuge tube. Add 1 mL of dye reagent and shake with a vibrating mixer for 35 min. Centrifuge at 12000 rpm for 10 min and pour off the supernatant. Carefully remove the residual liquid from the edge of the tube with absorbent paper and do not touch the sediment. The precipitate was dissolved by adding 1 mL of Alkali reagent, and after color stabilization, 0.2 mL was taken out and placed in a 96-well plate, and the absorbance at 570 nm was read by an ELISA reader.

2. Surface elements have the effect of increasing the collagen of mouse embryonic fibroblasts

As can be seen from Fig. 5, surface keratin can increase the proliferation of collagen in mouse embryonic fibroblasts; compared with the control group cultured with Phosphate buffered saline (PBS), 25, 50, 75 and 100 μM Surface elements have a good effect on increasing intracellular collagen, and 100 μM is the best. The Panmitoyl pentapeptide-3 (PPP-3) can also increase the collagen content in the cells, but the effect of the surface elements seems to be better than that of the surface substances.

Example 4 Surface elements have the effect of inhibiting matrix metallopeptide 9

1. Matrix metalloproteinase (Matrix metallopeptidase 9) inhibition effect determination

After the mouse embryonic fibroblasts were cultured, the commercial active anti-wrinkle substances 3, 5, 8 and 10 μM Palmitoyl pentapeptide-3 were added and compared with the addition of 25, 50, 75 and 100 μM surface elements by Abnova MMP- 9 (Mouse) ELISA Kit and ELISA Reader were used to analyze the concentration of matrix metalloproteinase 9 in cells.

2. Surface element has the effect of inhibiting matrix metalloproteinase

As can be seen from Fig. 6, surface molecules of 50, 75 and 100 μM have an effect of inhibiting matrix metalloproteinase; and the surface inhibitory effect at 50 μM is optimal.

Example 5 Surface element promotes skin penetration of cosmetic raw materials

Experimental method

Before the experiment, the back shaving action was performed. The 6-8 week mice were anesthetized by intraperitoneal injection with an anesthetic Zoletil. After the animals were laid flat, the back hair was gently shaved with an electric razor, and the remaining body hair was removed with a depilatory cream. The animals are placed in a cage with a warm light and given care of the drinking water. Prior to the experiment, the mice were anesthetized with Zoletil. The experiment was divided into two groups. The control group was PBS (pH=7.4) 100 μl mixed with fluorescein isothiocyanate (FITC)-labeled hyaluronic acid or dexamethasone, added to 1 cm ² sterile cotton wool and ventilated. The cloth was fixed to the back of the mouse. The experimental group was treated with hyaluronic acid or corticosteroids with 0.2, 0.5, 1, 2, and 5% surfactin.

One hour after the treatment, the mice were sacrificed by CO ₂ asphyxiation, the residual drug on the skin was cleaned with alcohol, and the back skin of the mouse was cut out and embedded in Optimum cutting temperature compound (OCT) (SAKURA®, Japen). Frozen sections (CM-2000, Leica, Germany) were performed.

2. Surface substances can promote the penetration of corticosteroids into the skin

As can be seen from Figure 7, surface keratin can promote the penetration of corticosteroids into the skin; by observing the intensity of the fluorescence, the amount of corticosteroid penetration into the skin can be known in each experimental group; the stronger the fluorescence intensity, the penetration of the skin. The greater the amount of corticosteroids; (B), (C), (D), (E) in Figure 7 can be observed more fluorescence than the control group (A); that is, 0.2, 0.5, 1, 2% of surface substances can increase the amount of corticosteroids penetrating the skin.

3. Surface pigment promotes skin absorption and moisturizing factor hyaluronic acid (HA)

As can be seen from Fig. 8, the surface element can promote the absorption of hyaluronic acid in the skin; the stronger the fluorescence intensity, the more the amount of hyaluronic acid that penetrates the skin; the fluorescence intensity observed by (A) in Fig. 8 is the strongest. That means that 1% of the surface pigment promotes the skin's absorption of the moisturizing factor hyaluronic acid.

4. Surface pigment promotes skin absorption and absorption of glutamic acid (Gamma-polyglutamic acid, γ-GPA)

It can be seen from Fig. 9 that surface stimulating substances can promote skin absorption and moisturizing factor (γ-GPA); the stronger the fluorescence intensity, the more the amount of polyglutamic acid absorbed by the skin; (B) in Fig. 9 ( More fluorescence can be observed in C), (D), (E), (F), (G) than in the control group (A); that is, 1, 2, 5, 10, 10, 15, 20 % of the surface pigments can increase the amount of polyglutaric acid absorbed by the skin.

5. Surface element promotes the absorption of gold-nanoparticles (2nm) through the skin.

When we use nano gold and surface pigment to smear on the mouse epidermis, after a period of time, the skin is taken for cryosection. Since nano gold can self-illuminate, it can directly observe the nano-gold in the skin epidermis with an optical microscope. Fluorescence content of the dermis layer. The results show that the skin cream obtained by the emulsification of the conventional process has an internal structure of micelle or micelle, which is disadvantageous for the skin to absorb nano gold, and nano gold is only found in the epidermis. The addition of 12.5 ppm of surface pigment significantly promoted the absorption of nanogold through the skin (Fig. 10).

It can be seen from the above research results that the surface element can replace the chemically synthesized surfactant and is widely used in the transdermal drug delivery system.

Example 6 Surface emulsification analysis

The emulsification index is one of the important indexes of the surfactant. The emulsification index of the surfactant to diesel oil is measured by the method used by Cooper et al. Different concentrations of crude purified surface pigment were dissolved in a buffer solution of pH 6.4, 7.4 and 8.4, 2 ml was taken out and 3 ml of diesel oil was added to the test tube, vortexed for 2 minutes, and allowed to stand at room temperature for 24 hours, and the emulsion was measured. The ratio of the height of the layer to the total height of the solution multiplied by 100% is the emulsification index of the liquid to be tested. The emulsification capacity of the oil is expressed by the emulsification index (E ₂₄ ), and the formula of E ₂₄ is as follows:

It can be seen from Fig. 11 that the surface element has a better emulsifying power at pH=7.4, and when the surface is The optimum emulsifying power is obtained at pH=7.4 and 160 μg/ml.

Example 7 Surface element can be used as a foaming agent

Foaming force analysis

The surfactant has the property of low foaming property, and the present invention is to test whether the crude purified surface element has foaming property. Referring to the method used by Razafindralambo et al., different concentrations of crude purified surface flavonoids were dissolved in a buffer solution having a pH of 7.4, and allowed to stand for 1 hour after shaking for 2 minutes, and the foam height was measured. The foaming force is expressed by the maximum foam density (MD), and the maximum foaming density is obtained by dividing the total liquid height by the maximum foaming height.

2. Surface element can be used as a foaming agent

Surface elements have many biological properties, including the ability to foam and emulsify common surfactants (Razafindralambo et al., 1998). The so-called foaming phenomenon means that surface elements are present in the interface between the gas phase and the liquid phase, and the shaking is vigorously caused to cause the surfactant to catch the air to form a film containing air (Halling, 1981). The present invention tests whether the surface element produced by Bacillus subtilis TH has foaming activity, and the results show that the addition of more than 150 μg of surface element in the buffer has no significant decrease in MD value, and the lowest MD value is 1.23 (see Fig. 12); The concentration of the surface element added to the deionized water has reached the critical micelle concentration (CMC), and the surface element forms a microcell, leaving only the monomer to act in the solution. Figure 13 shows the relationship between surface element concentration and foaming height. Razafindralambo et al. pointed out that the lowest MD value of surface element can reach 0.10. Compared with iturin, surface element has excellent foaming effect, which is presumed to be related to the structure between the two. Surface element belongs to anionic interface activity. And its fatty acid carbon chain is shorter, while itrathecin is a nonionic surfactant with a long fatty acid carbon chain (Razafindralambo et al., 1998). The surface pigments produced by Bacillus subtilis TH have foaming ability and can be used as personal care products such as detergents, shampoos and hand lotions.

Example 8 Surface element as a smoothing agent

The surface is excellent in Flushing will show good sense of smooth, and at low concentrations, it will produce a corecervate phenomenon.

1. Surface element can reduce turbidity

(1) Adjust the relative concentration of shampoo to 0.01-1.0 relative concentration = shampoo stock solution (g) / (shampoo stock solution (g) + water (g))

(2) Measurement (1) Transmittance (%, temperature: 40 ° C) in 420 ml of the solution, simply indicating its turbidity (%) = 100 - transmittance (%)

It can be seen from Fig. 14 that the surface element of the present invention is more capable of reducing the turbidity of the shampoo, i.e., increasing the shampoo, compared with the sodium laurate sulfate, which is commonly used in cleaning products. Fine smoothness.

2. Surface molecules produce droplets (corecervate)

Surfaces exhibit excellent sense of smoothness when rinsed, and at low concentrations, corecervate occurs.

It can be seen from Fig. 15 that after dilution of the polymer-surfactant complex - meaning the corecervate phenomenon, the surface-containing shampoo will have a precipitate; therefore Reduced friction sensitivity and increased sense of smoothness.

The above-mentioned embodiments and the drawings are only the preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, that is, the equivalent variations and modifications of the scope of the present invention should be covered by the present invention. Within the scope.

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Claims (43)

A method of preparing an anti-aging cosmetic composition by surfactin, wherein the composition comprises a surface element and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, and the like. A method for preparing an anti-aging cosmetic composition with a surface element as claimed in claim 1, wherein the surface element is a cyclolipin peptide comprising a seven-peptide sequence (L) Glu-linked to a β-hydroxy fatty acid. (L) Leu-(D)Leu-(L)Val-(L)Asp-(D)Leu-(L)Leu, the fatty acid distribution of the surface element at the fatty acid terminal is as follows: (1) the iso C13 is greater than 3%; (2) positive C13 is greater than 0.65%; (3) iso-C14 is greater than 17%; (4) positive C14 is less than 41%; and (5) iso-C15 is less than 11%. A method for preparing an anti-aging cosmetic composition by surface element as claimed in claim 2, wherein the fatty acid at the terminal of the fatty acid is distributed as follows: (1) an iso C13 is greater than 10%; (2) a positive C13 is greater than 25%; (3) The iso-C14 is greater than 35%; (4) the positive C14 is less than 25%; and (5) the iso-C15 is less than 3%. A method for preparing an anti-aging cosmetic composition with a surface element according to the third aspect of the patent application, wherein the fatty acid at the terminal of the fatty acid is distributed as follows: (1) an iso C 13 is 11%; (2) a positive C13 is 26%; (3) The iso C 14 is 37%; (4) the positive C14 is 24%; and (5) the iso C15 is 2%. A method of preparing an anti-aging cosmetic composition with a surface element as claimed in claim 1, wherein the surface element has a molecular weight of 1022 or 1036 Da. A method of preparing an anti-aging cosmetic composition with a surface element according to the first aspect of the patent application, wherein the surface element comprises a chemical isomer thereof. The method of preparing an anti-aging cosmetic composition by surface factor as claimed in claim 1, wherein the anti-aging cosmetic composition further comprises at least one of the following: an alcohol, an ester, a complex polysaccharide, a nut oil, and a vitamin. A method for preparing an anti-aging cosmetic composition by surface factor as claimed in claim 7 wherein the alcohol comprises at least one of the following: C16-18 alcohol, butanediol, pentanediol, octanediol, and C3. Alcohol, 16 alcohol, 18 alcohol, 22 alcohol and propylene glycol. A method of preparing an anti-aging cosmetic composition with a surface element as claimed in claim 7 wherein the ester comprises at least one of the following: olive oil cetyl alcohol ester (OLIVEM 1000), glyceryl monostearate (GSM) ), isopropyl myristate (IPM), isopropyl palmitate (IPP) and triglyceride. The method for preparing an anti-aging cosmetic composition by surface element according to the seventh aspect of the patent application, wherein the composite polysaccharide system comprises at least one of the following: xanthan gum, tremella polysaccharide, glucoside polysaccharide, senna seed polysaccharide . A method of preparing an anti-aging cosmetic composition with a surface element according to the seventh aspect of the patent application, wherein the nut oil comprises at least one of the following: arganic acid, Hawaiian nuclear oil, avocado oil, wheat germ oil, olive oil. A method for preparing an anti-aging cosmetic composition with a surface element according to the seventh aspect of the patent application, wherein the vitamin system comprises at least one of the following: vitamin A, vitamin B, vitamin C, Vitamin E, vitamin F, vitamin K. A method for preparing an anti-aging cosmetic composition by surface factor as claimed in claim 1, wherein the anti-aging cosmetic composition is for promoting proliferation of fibroblasts, proliferation of collagen, or expression of a sertuin 1 gene. A method of preparing an anti-aging cosmetic composition with a surface element according to the first aspect of the patent application, wherein the anti-aging cosmetic composition is used for anti-UV aging or anti-oxidation. A method of preparing an anti-aging cosmetic composition with a surface element as claimed in claim 1, wherein the anti-aging cosmetic composition is for inhibiting matrix metallopeptidase. A method for preparing an anti-aging cosmetic composition with a surface element according to the fifteenth aspect of the patent application, wherein the matrix metalloproteinase is Matrix metallopeptidase 9 (Matrix metallopeptidase 9). A method of preparing a composition for increasing skin penetration by surfactin, wherein the composition comprises a surface element and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, and the like. A method for preparing a composition for increasing skin penetration by a surface element according to claim 17, wherein the surface element is a cyclolipopeptide comprising a seven-peptide sequence linked to a beta hydroxy fatty acid (L) Glu-(L)Leu-(D)Leu-(L)Val-(L)Asp-(D)Leu-(L)Leu, the fatty acid distribution of the surface element at the fatty acid terminal is as follows: (1) the hetero-C13 is greater than 3%; (2) positive C13 is greater than 0.65%; (6) iso C14 is greater than 17%; (7) positive C14 is less than 41%; (8) The difference of C15 is less than 11%. A method for preparing a composition for increasing skin penetration by a surface element according to claim 18, wherein the fatty acid at the terminal of the fatty acid is distributed as follows: (1) an iso C13 is greater than 10%; and (2) a positive C13 is greater than 25%; (3) The iso-C14 is greater than 35%; (4) the positive C14 is less than 25%; and (5) the iso-C15 is less than 3%. A method for preparing a composition for increasing skin penetration by a surface element according to claim 19, wherein the fatty acid at the terminal of the fatty acid is distributed as follows: (1) an iso C 13 is 11%; and (2) a positive C13 is 26%. (3) Iso C 14 is 37%; (4) C14 is 24%; and (5) Iso C15 is 2%. A method of preparing a composition for increasing skin penetration by a surface element as claimed in claim 17, wherein the surface element has a molecular weight of 1022 or 1036 Da. A method of preparing a composition for increasing skin penetration by a surface element, as in claim 17, wherein the surface element comprises a chemical isomer thereof. A method for preparing a composition for increasing skin penetration by a surface element according to claim 17, wherein the composition for increasing skin penetration further comprises at least one of the following: an alcohol, an ester, a complex polysaccharide. , nut oil and vitamins. A method for preparing a composition for increasing skin penetration by a surface element according to the scope of claim 23, wherein the alcohol comprises at least one of the following: C16-18 alcohol, butanediol, pentanediol, octanediol , glycerol, 16 alcohol, 18 alcohol, 22 alcohol and propylene glycol. A method of preparing a composition for increasing skin penetration by a surface element according to claim 23, wherein the ester comprises at least one of the following: olive oil cetyl alcohol ester (OLIVEM 1000), glyceryl monostearate Ester (GSM), isopropyl myristate (IPM), isopropyl palmitate (IPP) and triglycerides. A method for preparing a composition for increasing skin penetration by a surface element according to claim 23, wherein the composite polysaccharide system comprises at least one of the following: xanthan gum, tremella polysaccharide, glucoside polysaccharide, senna Seed polysaccharide. A method for preparing a composition for increasing skin penetration by a surface element according to the scope of claim 23, wherein the nut oil comprises at least one of the following: argan oil, Hawaiian stone oil, avocado oil, wheat germ oil, olive oil. A method for preparing a composition for increasing skin penetration by a surface element according to claim 23, wherein the vitamin comprises at least one of the following: vitamin A, vitamin B, vitamin C, vitamin E, vitamin F, vitamin K . A method of preparing a composition for increasing skin penetration by a surface element, as in claim 17, wherein the composition is for promoting penetration of a cosmetic material into the skin. A method of preparing a composition for increasing skin penetration by a surface element according to claim 29, wherein the cosmetic material is dexamethasone. A method of preparing a composition for increasing skin penetration by a surface element according to claim 29, wherein the cosmetic material is hyaluronic acid. A method of preparing a composition for increasing skin penetration by a surface element according to claim 29, wherein the cosmetic material is gamma-polyglutamic acid. A method for preparing a composition for increasing skin penetration by using a surface element as claimed in claim 29 The method wherein the cosmetic material is gold-nanoparticles. A method of preparing an emulsifier composition by surfactin, wherein the composition comprises a surface element and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, and the like. A method for preparing an emulsifier composition by a surface element according to claim 34, wherein the surface element is a cyclolipin peptide comprising a seven-peptide sequence (L) Glu- (linked to a β-hydroxy fatty acid). L) Leu-(D)Leu-(L)Val-(L)Asp-(D)Leu-(L)Leu, the fatty acid distribution of the surface element at the fatty acid terminal is as follows: (1) the iso-C13 is greater than 3%; 2) positive C13 is greater than 0.65%; (9) iso-C14 is greater than 17%; (10) positive C14 is less than 41%; and (11) iso-C15 is less than 11%. A method for preparing an emulsifier composition by a surface element according to claim 35, wherein the fatty acid at the terminal of the fatty acid is distributed as follows: (1) an iso C13 is greater than 10%; (2) a positive C13 is greater than 25%; (3) C14 is greater than 35%; (4) positive C14 is less than 25%; and (5) iso-C15 is less than 3%. A method for preparing an emulsifier composition by a surface element according to claim 36, wherein the fatty acid at the terminal of the fatty acid is distributed as follows: (1) 11% of iso C 13; (2) 26% of positive C13; (3) The iso C 14 is 37%; (4) the positive C14 is 24%; and (5) the iso C15 is 2%. A method of preparing an emulsifier composition by a surface element according to claim 34, wherein the surface element has a molecular weight of 1022 or 1036 Da. A method of preparing an emulsifier composition by a surface element as claimed in claim 34, wherein the surface element comprises a chemical isomer thereof. A method for preparing an emulsifier composition by a surface element according to claim 34, wherein the emulsifier composition further comprises at least one of the following: a fatty acid glyceride, a fatty acid sorbitan ester, a fatty acid sucrose ester, a fatty acid propylene glycol ester. , lecithin. A method of preparing an emulsifier composition by a surface element as claimed in claim 34, wherein the emulsifier composition is for increasing the foaming power. A method of preparing an emulsifier composition by a surface element as claimed in claim 34, wherein the emulsifier composition is used to increase emulsification. A method of preparing an emulsifier composition by a surface element as claimed in claim 34, wherein the emulsifier composition is used to increase slipperiness.