WO2020262815A1 - Cosmetic composition comprising silkworm hemolymph-derived low-molecular material - Google Patents

Abstract

The present invention relates to a skin-whitening and antioxidant cosmetic composition comprising silkworm hemolymph-derived low-molecular material, the material having a molecular weight of less than 10 kDa. The silkworm hemolymph-derived low-molecular material utilizes material which was conventionally discarded, and thus has excellent economic efficiency, and has excellent safeness for skin and excellent skin whitening and antioxidant effects, and the high-yield production thereof is possible, and thus the material may be usefully employed for a skin-whitening and antioxidant cosmetic composition.

Description

Cosmetic composition containing low molecular weight substances derived from silkworm blood lymph

The present invention relates to a cosmetic composition comprising a low molecular weight substance derived from silkworm hemolymph having a molecular weight of less than 10 kDa.

The skin is the most basic defense organ that protects the body from external stimuli by contacting the external environment, and it is an important organ expressing external beauty. As people's external activity increases, long-term exposure to sunlight is increasing. When the skin is exposed to ultraviolet rays for a long time, skin aging proceeds, and the skin turns black due to the melanin pigment generated to protect the inner layer of the skin from ultraviolet rays. Therefore, in modern society, consumption of functional cosmetics is increasing to manage the skin, and the demand for skin that is white and clean, young and elastic like white jade is increasing. Therefore, development of ingredients with excellent whitening and anti-aging effects It is being actively conducted in the cosmetic industry.

The whitening effect of cosmetics refers to whitening the user's skin, and most whitening cosmetics have a function of inhibiting the production of melanin. Melanin is produced by melanocytes present in the basal layer of the epidermis, and the activity and distribution of melanocytes determine the color of human skin. Tyrosine, a type of amino acid, is converted into dopa (dihydroxy phenylalanine) by an enzyme called tyrosinase contained inside melanocytes, and dopaquinone and dopachrome, etc. The polymer melanin is finally produced through a series of oxidation processes. Therefore, most whitening cosmetics exhibit a whitening effect on the principle of inhibiting the production of melanin by inhibiting the activity of tyrosinase, a major enzyme in melanin production (Korean Patent Laid-Open No. 10-2019-0025329).

The anti-aging of cosmetics is to block or eliminate the causes that promote skin aging. The skin aging is caused by an imbalance between oxidation and antioxidant due to reduced antioxidant enzymes in the body and antioxidants such as vitamins E, C, glutathione and ubiquinol. As aging progresses, the reaction to produce melanin is accelerated, and damage to biological components is caused by lipid peroxidation, protein oxidation, activation of proteases, chain cleavage and abnormal crosslinking of collagen and elastin, hyaluronic acid chain cleavage, and DNA oxidation. In the end, skin aging such as reduction of skin elasticity, wrinkles and spots, and freckles occurs.

Therefore, a cosmetic composition having an antioxidant effect while inhibiting the activity of tyrosinase can prevent skin aging while effectively inducing a skin whitening effect.

As a result of studying the ingredients for inhibiting skin aging and improving the skin whitening effect, the present inventors have found that the low molecular weight substance derived from silkworm hemolymph has skin whitening and antioxidant effects. In addition, it was found that the low-molecular substance derived from silkworm hemolymph can effectively inhibit the expression of genes involved in melanin synthesis and increase antioxidant activity, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a cosmetic composition comprising a low molecular weight substance derived from silkworm hemolymph.

In order to solve the above problems, an aspect of the present invention provides a cosmetic composition comprising a low molecular weight material derived from silkworm hemolymph having a molecular weight of less than 10 kDa.

In addition, another aspect of the present invention provides a method for producing a low molecular weight substance derived from the silkworm hemolymph.

The low molecular weight substance derived from silkworm blood lymph has excellent skin safety, excellent skin whitening and antioxidant effects, and can be produced in high yield, so it can be usefully used as a cosmetic composition for skin whitening and antioxidant.

1 is a schematic diagram of a manufacturing process of a low molecular weight substance derived from silkworm blood lymph.

2 is a result of measuring the molecular weight of a low-molecular substance derived from silkworm blood lymph using a liquid chromatograph mass spectrometer.

3A and 3B are results of evaluating the cytotoxicity of silkworm hemolymph-derived small-molecular substances to dermal fibroblast and epidermal cells (Keratinocyte), respectively.

4 is a result of measuring the amount of melanin produced in melanoma cells treated with a low molecular weight substance derived from silkworm blood lymph using a microplate reader.

5A to 5D are measurement of the expression of proteins (tyrosinase, TRP-1 and TRP-2) involved in melanin synthesis in melanoma cells treated with low molecular weight substances derived from silkworm hemolymph through Western blot (FIG. 5A ), and the quantified value is shown in a graph (FIGS. 5B to 5D).

6 is a result of measuring the antioxidant activity of a low-molecular substance derived from silkworm hemolymph by DPPH analysis (diphenylpicrylhydrazyl analysis).

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention provides a cosmetic composition comprising a low molecular weight material derived from silkworm blood lymph. At this time, the molecular weight of the low molecular weight substance derived from silkworm blood lymph may be less than 10 kDa.

In the present invention, the "silkworm" is a larva of a silkworm moth belonging to the Lepidoptera silkworm moth family. In one embodiment of the present invention, the silkworm may be a silkworm, and the silkworm means a mature silkworm. The ripe silkworm is a silkworm that stops eating and moves in search of a place to build a cocoon because the growth of the last young reaches the extreme. Silkworms are mainly used for obtaining thread from cocoons, and the silkworm tissue remaining after the thread is obtained is discarded as it is or is used as an additive to a medium for insect cell culture.

In the present invention, the "hemolymph" means body fluid. Insects such as silkworms have no blood vessels, and body fluids move nutrients, discharge waste products, and maintain a balance between water and salt.

In the present invention, the "small-molecular substance derived from silkworm blood lymph" means a substance having a molecular weight of less than 10 kDa, specifically a substance having a molecular weight of less than 1 kDa, and more specifically, a molecular weight of 258 Da to 589 Da. In one embodiment of the present invention. The low molecular weight substance may be flavonoids, polysaccharides, proteins, lipids, glycoproteins, and glycolipids, and specifically, may be proteins consisting of 2 to 5 amino acids, but is not limited thereto. When the molecular weight of the low-molecular substance derived from silkworm blood lymph of the present invention is less than 10 kDa, it is easily absorbed into the skin, thereby increasing skin whitening and antioxidant effects. Specifically, the low molecular weight material derived from silkworm hemolymph may have a molecular weight pattern at the top of FIG. 2.

In one embodiment of the present invention, the low molecular weight material is prepared by lyophilizing the silkworm to prepare a dried silkworm; Removing the thread glands from the dried silkworm using a ball mill and separating the hemolymph from the dried product; Pulverizing the separated hemolymph, mixing it with distilled water of 1% to 5% based on the weight of the pulverized product, and stirring at 100 rpm to 300 rpm for 20 to 40 minutes to prepare a hemolymphatic lysate; Filtering the hemolymph lysate through a 200 µm to 1,000 µm strainer, heat treatment, and centrifuging to recover the supernatant; And the supernatant was obtained by performing the step of obtaining a low molecular weight material derived from silkworm hemolymph using an ultrafilter membrane.

When a low molecular weight material is obtained using the ultrafiltration membrane, the molecular weight of the filtered material can be easily controlled by adjusting the exclusion molecular weight of the ultrafiltration membrane. In addition, there is an advantage in that the time required in the process of obtaining can be shortened and cost can be saved. The exclusion molecular weight of the ultrafiltration membrane may be 1 kDa to 10 kDa, and in one embodiment of the present invention, an ultrafiltration membrane having an exclusion molecular weight of 10 kDa was used.

In one embodiment of the present invention, it was confirmed that the low-molecular substance derived from silkworm hemolymph of the present invention was not toxic to dermal cells and epidermal cells at all treatment concentrations (FIGS. 3A and 3B ).

In one embodiment of the present invention, when the low-molecular substance derived from silkworm blood lymph of the present invention is treated on melanoma cells, the amount of melanin production was effectively suppressed (Figure 4), and genes involved in melanin synthesis (tyrosinase, TRP- 1 and TRP-2) were effectively inhibited (FIGS. 5A to 5D ),

In addition, in one embodiment of the present invention, it was confirmed that the antioxidative activity of the low-molecular substance derived from silkworm hemolymph of the present invention is excellent (Tables 2 and 6).

Therefore, the cosmetic composition of the present invention can be used for skin whitening or antioxidant purposes.

The term "whitening" used in the present invention alleviates skin pigmentation such as blemishes, freckles, and blemishes by inhibiting the production of various biomolecules such as melanin, redox hemoglobin, carotene, and melanoids that affect skin color. , It means the effect of improving skin brightness and uniformity by relieving skin yellowness and redness. In particular, it is possible to improve skin tone by inhibiting the activity of tyrosinase, which is a melanin pigment producing enzyme.

As used herein, the term "antioxidation" refers to inhibition of oxidation, and refers to an effect of preventing cell aging by removing free radicals generated during cell aging.

The "cosmetic composition" may be formulated as one selected from the group consisting of lotion, essence, lotion, cream, pack, foundation, gel, ointment, and spray.

In addition, the cosmetic composition may be formulated by a conventional method. Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can refer to the content disclosed in the formulation of external preparations for skin, and the International cosmetic ingredient dictionary, 6th ed (The cosmetic, Toiletry and Fragrance Association) in the formulation of cosmetic compositions. , Inc., Washington, 1995).

Specifically, the cosmetic composition may be prepared in a general emulsion formulation and solubilization formulation. For example, a lotion such as a flexible lotion or a nutritional lotion; Emulsions such as facial lotion and body lotion; Creams such as nourishing cream, moisture cream, and eye cream; essence; Cosmetic ointment; spray; Gel; pack; Sunscreen; Makeup base; It may be formulated as a liquid type, solid type, or spray type foundation, but is not limited thereto. In addition, the external preparation for skin may be formulated as an ointment, patch, gel, cream, or spray, but is not limited thereto.

In the cosmetic composition, in addition to the essential ingredients in each formulation, other ingredients may be appropriately blended within a range that does not impair the object according to the present invention depending on the type of formulation or purpose of use.

The cosmetic composition may contain a generally acceptable carrier, for example, oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately blended, but are limited thereto. no.

The acceptable carrier may vary depending on the formulation. For example, when formulated as an ointment, paste, cream or gel, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or their Mixtures can be used.

When the cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or mixtures thereof may be used as a carrier component. It may further comprise a propellant such as carbon, propane, butane or dimethyl ether.

When the cosmetic composition is formulated as a solution or emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-Butylglycol oil may be used, and in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used.

When the cosmetic composition is formulated as a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like can be used.

The cosmetic composition is a fatty substance, an organic solvent, a solubilizer, a thickener, a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, and a fragrance, which are commonly used in the industry depending on the quality or function of the final product. , Surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic activators, commonly used in cosmetics It may additionally contain adjuvants commonly used in the field of cosmetology or dermatology, such as any other ingredients.

However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

Another aspect of the present invention is to prepare a dried silkworm by lyophilizing the silkworm; Removing the thread glands from the dried silkworm using a ball mill and separating the hemolymph from the dried product; Pulverizing the separated hemolymph, mixing it with distilled water of 1% to 5% based on the weight of the pulverized product, and stirring at 100 rpm to 300 rpm for 20 to 40 minutes to prepare a hemolymphatic lysate; Filtering the hemolymph lysate through a 200 µm to 1,000 µm strainer, heat treatment, and centrifuging to recover the supernatant; And it provides a method for producing a low molecular weight material derived from silkworm hemolymph comprising the step of obtaining a low molecular weight material derived from silkworm hemolymph using the supernatant using an ultrafilter membrane.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1. Preparation of low molecular weight material derived from silkworm hemolymph

After 1 kg of soaking was treated with liquid nitrogen (-196°C, 70 hours), it was freeze-dried to prepare a dried silkworm. The silk gland was removed from the dried silkworm using a ball mill having a diameter of 1 cm to 5 cm, and the hemolymph was separated. After the separated hemolymph was pulverized, 3% of water was added to the pulverized product, and then stirred at 200 rpm for 30 minutes to prepare a hemolymphatic lysate. The hemolymphatic lysate was filtered through a 200 μm to 1,000 μm strainer and then heat-treated at 60° C. for 30 minutes in order to inactivate dopaquinone, a toxic substance. Thereafter, the supernatant was collected by centrifugation at 13,000 rpm for 20 minutes, and then the low molecular weight material derived from silkworm hemolymph was separated by filtration using a 10 kDa ultrafiltration membrane. A schematic diagram of the manufacturing process of the silkworm blood lymph-derived low-molecular material is shown in FIG. 1.

On the other hand, the unfiltered material can be used to prepare a silkworm hemolymph-derived protein (Storage Protein 2, SP2) using a Q column, and the silkworm hemolymph-derived protein is known to have skin whitening and antioxidant activity. Yes (refer to Korean Patent Registration No. 10-1501554).

The yields of the silkworm hemolymph-derived protein (SP2) and the silkworm hemolymph-derived low-molecular substance were evaluated (Table 1).

As a result, referring to Table 1, the yields of the silkworm hemolymph-derived low-molecular substance and the silkworm hemolymph-derived protein (SP2) are 10% and 0.16%, respectively, and the production yield of the silkworm hemolymph-derived small-molecular substance is the silkworm hemolymph-derived protein. It was about 62 times higher than that.

Experimental Example 1. Molecular weight measurement of low molecular weight substance derived from silkworm hemolymph

Liquid chromatograph mass spectrometry was performed to confirm the molecular weight of the low molecular weight substance derived from silkworm hemolymph.

Specifically, the low molecular weight substance derived from silkworm blood lymph obtained in Example 1 was dissolved in 50% methanol at a concentration of 50 ng/ml, filtered through a 0.45 µm syringe filter, and 20 µl was injected into a liquid chromatography mass spectrometer. . In addition, an amino acid mixture of known molecular weight (Amino acid standard, #AAS18, Sigma) was injected and used as a control. The molecular weight was measured by the electron spray ionization method under the condition of 0.1% formic acid as the analysis solvent (FIG. 2).

As a result, referring to FIG. 2, the size of the low molecular weight substance derived from silkworm hemolymph was measured to be 258 Da to 589 Da.

Experimental Example 2. Evaluation of cytotoxicity of small molecule substances derived from silkworm hemolymph

In order to verify the skin stability of the silkworm hemolymph-derived small molecule substance, MTT assay was performed on human dermal fibroblasts and human keratinocytes.

Specifically, the dermal cells and epidermal cells were passaged in a DMEM (High Glucose) medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37° C., 5% CO _{2 in an} incubator. It was cultured and used in the experiment. The dermal cells and epidermal cells were dispensed into a 96 well plate at a concentration of 5 X 10 ³ cells/well, respectively, and cultured for 24 hours, and then the low molecular weight substance derived from silkworm hemolymph obtained in Example 1 was added by concentration (0, 0.1, 0.2, 0.5, 1, 2 mg/ml) and cultured for 24 hours. After incubation for 24 hours, replace with a medium containing 10% WST-1 (ez-cytox), react for 2 hours, and measure the absorbance at 450 nm using a microplate reader, and the result is measured as a control (non-treat) value. It is expressed as a ratio (Fig. 3A and Fig. 3B).

As a result, referring to FIGS. 3A and 3B, the low molecular weight substance derived from silkworm hemolymph did not show toxicity in both dermal cells and epidermal cells.

Experimental Example 3. Evaluation of skin whitening activity

Experimental Example 3.1. Evaluation of melanin production

In order to verify the melanin production inhibitory effect according to the treatment of silkworm hemolymph-derived low-molecular substances, the amount of melanin production was measured in melanoma cells (B16F10).

Specifically, the melanoma cells were aliquoted into a 6 well plate at 1×10 ⁵ cells/well and cultured for 3 days in a 37°C, 5% CO ₂ incubator. After incubation, the small-molecular substances derived from silkworm blood lymph were treated at different concentrations (0.2 mg/ml, 1 mg/ml, and 2 mg/ml), and then cultured for 3 days to recover the cells, and then 2 M NaOH was added to each experimental group. The melanin produced at 100 ℃ was dissolved. Then, the amount of dissolved melanin was measured at absorbance of 405 nm using a microplate reader (FIG. 4).

On the other hand, melanocyte stimulating hormone (α-MSH) was used as a negative control group, and arbutin and silkworm hemolymph derived protein (SP2) disclosed in Example 1 were used as a positive control and a control group, respectively. I did.

As a result, referring to FIG. 4, the experimental group treated with the low-molecular substance derived from silkworm hemolymph effectively inhibited melanin synthesis compared to the control group and the comparative group.

Experimental Example 3.2. Evaluation of protein expression related to melanin synthesis

In order to verify the inhibitory effect of melanin production according to the treatment of a low-molecular substance derived from silkworm hemolymph, the expression of protein was measured for melanin synthesis in melanoma cells (B16F10).

Specifically, the melanoma cells were aliquoted into a 6 well plate at 1×10 ⁵ cells/well and cultured for 3 days in a 37°C, 5% CO ₂ incubator. After incubation, the cells were recovered by treating the silkworm hemolymph-derived low-molecular substances at different concentrations (0.2 mg/ml, 1 mg/ml, and 2 mg/ml) and cultured for 3 days to recover the cells. , PPI1015, Quartett) to extract proteins by processing RIPA buffer containing tyrosinase, tyrosinase-related protein 1 (TRP1) and Thai through western blot The expression of tyrosinase related protein 2 (TRP2) was analyzed. In addition, α-MSH was used as a negative control and arbutin was used as a positive control (FIGS. 5A to 5D).

As a result, referring to FIGS. 5A to 5D, the experimental group treated with the small molecule derived from silkworm hemolymph significantly inhibited the expression of tyrosinase, TRP1 and TRP2 compared to the control group. In particular, in the case of the experimental group treated with a small molecule derived from silkworm hemolymph at 2 mg/ml, even compared with the positive control group, the inhibitory activity of tyrosinase, TRP1 and TRP2 expression was equal or superior.

Experimental Example 4. Evaluation of antioxidant activity

DPPH analysis (diphenylpicrylhydrazyl analysis) was performed to verify the antioxidant activity according to the treatment of low molecular weight substances derived from silkworm hemolymph.

Specifically, the low-molecular substances derived from silkworm hemolymph prepared in Example 1 were analyzed by concentration (0.16 µg/ml, 0.31 µg/ml, 0.62 µg/ml, 1.25 µg/ml, 2.5 µg/ml, 5 µg/ml, 10 µg/ml and 20 µg/ml) were prepared at 40 µl each, and then 160 µl of 0.2 mM DPPH reagent dissolved in methanol was added and reacted for 30 minutes after blocking the light. Then, the absorbance was measured at 517 nm using a microplate reader.

As a comparative group, silkworm hemolymph-derived protein (SP2), arbutin and vitamin C (ascorbic acid) disclosed in Example 1, which are known to have excellent antioxidant activity, were used (Tables 2 and 6).

As a result, referring to Table 2 and FIG. 6, the low-molecular substance derived from silkworm hemolymph of the present invention exhibited equivalent or superior antioxidant activity compared to the comparative group.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof, are included in the scope of the present invention.

Claims (8)

1. Contains low molecular weight substances derived from silkworm blood lymph,

   The molecular weight of the low molecular weight substance derived from silkworm blood lymph is less than 10 kDa cosmetic composition.
2. The method of claim 1,

   The molecular weight of the low molecular weight material is less than 1 kDa, cosmetic composition.
3. The method of claim 1,

   The molecular weight of the low molecular weight material is 258 Da to 589 Da, cosmetic composition.
4. The method of claim 1,

   The low molecular weight material, freeze-drying the silkworm to prepare a dried silkworm;

   Removing the thread glands from the dried silkworm using a ball mill and separating the hemolymph from the dried product;

   Pulverizing the separated hemolymph, mixing it with distilled water of 1% to 5% based on the weight of the pulverized product, and stirring at 100 rpm to 300 rpm for 20 to 40 minutes to prepare a hemolymphatic lysate;

   Filtering the hemolymph lysate through a 200 µm to 1,000 µm strainer, heat treatment, and centrifuging to recover the supernatant; And

   A cosmetic composition obtained by performing the step of obtaining a low molecular weight substance derived from silkworm blood lymph using the supernatant liquid using an ultrafilter membrane.
5. The method of claim 4,

   The ultrafiltration membrane has an exclusion molecular weight of 1 kDa to 10 kDa, a cosmetic composition.
6. The method according to any one of claims 1 to 5,

   The cosmetic composition is for antioxidant or skin whitening, cosmetic composition.
7. The method according to any one of claims 1 to 5,

   The cosmetic composition has any one formulation selected from the group consisting of lotion, essence, lotion, cream, pack, foundation, gel, ointment and spray, cosmetic composition.
8. Lyophilizing the silkworm to prepare a dried silkworm;

   Removing the thread glands from the dried silkworm using a ball mill and separating the hemolymph from the remaining dried material;

   Pulverizing the separated hemolymph, mixing it with distilled water of 1% to 5% based on the weight of the pulverized product, and then stirring at 100 rpm to 300 rpm for 20 to 40 minutes to prepare a hemolymphatic lysate;

   Filtering the hemolymph lysate through a 200 µm to 1,000 µm filter, heat treatment, and centrifuging to recover the supernatant; And

   A method for producing a low molecular weight material derived from silkworm hemolymph comprising the step of obtaining a low molecular weight material derived from silkworm hemolymph using the supernatant using an ultrafilter membrane.

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