KR20140062564A - Cosmetic composition including hemolymph from silkworm larvae - Google Patents

Abstract

The present invention relates to a cosmetic composition including hemolymph derived from silkworms. The cosmetic composition of the present invention comprises hemolymph derived from silkworms, and more specifically, comprises hemolymph from silkworms obtained by putting a crude extract containing silkworm tissues remained after removing silk glands from the silkworms, treating with heat, and refining a supernatant solution obtained by continuous centrifugation. According to the present invention, the cosmetic composition comprising hemolymph derived from silkworms which is harmless to human body and has excellent anti-oxidant activity, anti-microbial activity, whitening effects, etc. is provided.

Description

COSMETIC COMPOSITION INCLUDING HEMOLYMPH FROM SILKWORM LARVAE [0002]

The present invention relates to a cosmetic composition containing silkworm derived from silkworm, and more particularly to a cosmetic composition comprising silkworm derived from silkworm which is harmless to human body and excellent in antioxidant ability, antimicrobial activity and whitening effect.

Conventionally, cosmetic compositions used in cosmetic packs are mostly composed of artificial synthetic compositions. Therefore, the effect of damaging fragile skin or regenerating the skin with healthy and rich skin is inferior.

In severe cases, it has serious disadvantages that can cause side effects such as skin rash and allergies. Accordingly, in recent years, attempts have been made to use natural products having a moisturizing effect and a whitening effect on cosmetic products. However, the natural products known to date do not show satisfactory characteristics of skin whitening effect.

Silkworm is very sensitive to the cleanliness and pesticides at the time of cultivation and is widely used as an insect for inspecting the stability.

Therefore, sericin and fibroin proteins having the effects of skin coating effect, moisturizing, antioxidant ability and skin affinity are hydrolyzed from such cocoon to be used as a main ingredient of cosmetics.

As a related prior art, Korea Patent No. 10-0494229 (registered on May 31, 2005, name: Sericin manufacturing method and mask pack using the same) and Korea Patent No. 10-0542266 (registered on Jan. 2006 2006 March 03, name: cosmetic pack composition containing sericin protein extracted from silkworm cocoon).

However, the silkworm tissue extract remaining after separating silkworm glands from the silk glands has been used as a main component of cosmetic compositions only as an additive to culture media for insect cells until now.

Such a silkworm fluid is a nutrient solution for silkworms, called a silkworm hemolymph. This hemolymph has nutritional components necessary for the growth of animal cells including insect cells. In addition, the silkworm body fluids have been approved for stability in items such as endotoxins.

Accordingly, in the present invention, as described above, utilizing the blood-lymphoma derived from silkworm, which is harmless to the human body and having utility value, as an idle resource leads to the invention as follows.

The object of the present invention is to make silkworm derived from silkworm, which has been ineffectively used though it has various efficacies, to be used as a cosmetic material and at the same time, it is harmless to the human body and has antioxidant ability, And the like.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

To achieve the above object, the cosmetic composition of the present invention is characterized by containing silkworm derived from silkworm.

The varieties of silkworms are characterized by being napped.

The blood lymph is 0.001 to 20% by weight, more preferably 0.1 to 10% by weight, based on the total weight of the cosmetic composition.

The hemolymph is characterized by removing the secretory gland from the silkworm, adding the remaining crude extract to water, heat-treating it, and then continuously centrifuging it to obtain a supernatant.

The heat treatment is performed at 40 to 80 DEG C for 10 to 120 minutes.

The cosmetic composition is characterized by having any one of a lotion, essence, lotion, cream, pack, foundation, gel, ointment or spray.

The present invention provides a cosmetic composition comprising a silkworm derived from silkworm which is harmless to human body and excellent in antioxidant ability, antibacterial activity, whitening effect and the like.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the thermal stability of a silkworm-derived hemolymph. FIG.
2 is a graph showing antioxidative activities of silkworm-derived hemolymph purified in Example 1 of the present invention.
3 is a graph showing skin cytotoxicity of silkworm-derived hemolymph purified in Example 1 of the present invention.
4 is a graph showing the melanin production inhibitory effect for confirming the whitening effect of the silkworm-derived hemolymph purified in Example 1 of the present invention.
5 is a graph showing a tyrosinase inhibitory effect for confirming whitening effect of silkworm-derived hemolymph purified in Example 1 of the present invention.

The terms, techniques, and the like described in this specification are used in the meaning commonly used in the technical field to which the present invention belongs, unless otherwise specified.

Hereinafter, a method of purifying silkworm-derived blood lymph as an active ingredient of the cosmetic composition of the present invention will be described in detail.

1. Removal of silk glands from silkworms and obtaining crude extracts containing remaining silkworm tissues

First, the silkworm is freeze-dried to remove the false gland from the silkworm.

Normally silkworm blood lymph has been collected by hand. However, when the blood lym- phs are taken from the raw silkworms by hand, the amount of melanin is reduced due to the low yield and is not economical. In addition, the blood silk is destroyed by the silkworm in the extraction process (the phenomenon that the silkworm spits the digestive fluids), and the impurities are contained in a large amount.

Accordingly, the present invention first lyophilizes the silkworm to obtain a large amount of silkworm blood without impurities. In this case, it is preferable to treat the silkworm with liquid nitrogen and then freeze-dry the silkworm using a freeze-dryer to obtain a silkworm purification product.

The lyophilization is preferably performed at -40 to -70 ° C for 12 to 80 hours, and the most optimal silkworm blood cell can be obtained without impurities.

The silkworm varieties may be used regardless of which of the cultivated silkworm varieties is used, but it is preferable to use ripe silkworms in which the mulberry leaves of the digestive tissues are completely digested and the false silk is maximally developed in order to construct the silkworm cocoon.

The ripe silkworm is the 7th day of the silkworm, and when the mulberry leaf in the silkworm of the silkworm is fully digested and the silkworm is fully developed and becomes clear and transparent, the silkworm is ready to become a pupa. The beneficial silkworm stage is most economical because it can prevent the contamination of impurities due to large amount of blood lymphatic purification and digestive juice.

② After lyophilization, remove seals to obtain crude extract containing silkworm tissue.

At this time, any method may be applied as a method of removing the false gland. However, if a ball mill (diameter of 1 to 5 cm) is used, it is possible to separate the false gland, which is separated later, so that it can be used for another purpose, Collect the crude extract.

2. Obtain a purified hemolymph

The crude extract containing the separated silkworm tissue is placed in water, heat-treated, and then subjected to continuous centrifugation to obtain a supernatant to obtain a purified hemolymph.

At this time, it is preferable to add distilled water of about 10 to 20 times the weight of the crude extract containing the separated silkworm tissue.

The heat treatment is carried out at 40 to 80 ° C. for 10 to 120 minutes. When the heat treatment is performed at the same time, the crude extract containing the silkworm tissue dissolves in water, and the tyrosinase activity in the hemolymph by heat treatment Can be removed. In other words, toxic quinone protein produced by tyrosinase is terminated by heat treatment.

When the centrifugation is carried out at 12,000 to 15,000 rpm for 0.5 to 1 minute (flow rate: 0.8 L / min), the protein loss is minimized to obtain purified hemolymph.

At this time, the used centrifugal separator is a continuous centrifugal separator, which separates the supernatant while centrifuging, and is much more economical than the conventional centrifugal separation method (centrifugal separation followed by filtration to separate the supernatant), and the yield is high .

The purified hemolymph thus obtained is harmless to the human body and has excellent antioxidative, antimicrobial activity and whitening effect, and thus can be used as a main material of a cosmetic composition.

In this case, the purified hemolymph is contained in an amount of 0.001 to 20 wt%, more preferably 0.1 to 10 wt%, based on the total weight of the cosmetic composition, and the remaining balance includes a material acceptable as other cosmetic ingredients . When the purified hemolymph is contained in an amount of less than 0.001% by weight, the antioxidant activity, antimicrobial activity and whitening effect of the present invention are insufficient. When it exceeds 20% by weight, It is not suitable because it shows the effect.

The formulations of the cosmetic compositions thus prepared may be applied differently depending on the method and purpose of use.

Examples of the formulations include lotions, liquid preparations, emulsions, emulsions, suspensions, tablets, and capsules.

When used as cosmetics, it can be used as cosmetics such as foundation lotion, lipstick, mascara or makeup base such as soft lotion, milk lotion, nutrition cream, massage cream, essence, cleansing foam, cleansing water, pack or body oil And can be blended with components commonly used in external preparations for skin such as an aqueous component, an oily component, a surfactant, a moisturizing agent, a thickening agent, an antiseptic, a perfume pigment and the like.

The present invention will be described in more detail with reference to the following examples and experimental examples, but the scope of protection is not limited to the following examples and experimental examples.

<Example 1> Production of purified hemolymph from silkworm of the present invention

1kg of silkworm (5th and 7th day) was treated with liquid nitrogen, and freeze-dried at -50 ° C for 70 hours, then sericin was removed using a ball mill (diameter 1-5 cm) The crude extract was recovered.

The crude extract containing the recovered silkworm tissue was dissolved in 10 times water based on the weight of the crude extract and heat-treated at 60 ° C for 30 minutes to dissolve. Then, the resultant was centrifuged at 12,000 to 15,000 rpm in a combustion centrifuge (flow rate: Min) was used to recover the supernatant to obtain a purified hemolymph.

<Example 2> Production of cream type cosmetics of the present invention

The purified hemolymph of Example 1 was dissolved in water to prepare cosmetics having the following composition.

The purified hemolymph solution of Example 1 - 0.1 wt%

Cetanol 2.0 wt%

&Lt; tb &gt; &lt; tb &gt;

Sobutan monostearate ------- 1.0 wt%

Mineral oil -------------------- 10.0 wt%

Trioctanoate -------------- 5.0 wt%

Triethanolamine ---------------- 0.5 wt%

Carbomer ------------------------ 0.2 wt%

Glycerin 5.0 wt%

Propylene glycol - 3.0 wt%

Preservative, fragrance ---------------------

The remaining purified water

Example 3 Preparation of ointment-type cosmetics of the present invention

The purified hemolymph of Example 1 was dissolved in water to prepare cosmetics having the following composition.

The purified hemolymph solution of Example 1 - 5.0 wt%

Diethyl sebacate 8.0 wt% &lt; RTI ID = 0.0 &gt;

Sintered ----------------------------------------- 5.0 wt%

Polyoxyethylene oleyl ether phosphate - 6.0 wt%

Sodium benzoate --------------------------------

The total amount of the remaining vaseline

Example 4: Preparation of an essence type cosmetic product of the present invention

The purified hemolymph of Example 1 was dissolved in water to prepare cosmetics having the following composition.

The purified hemolymph solution of Example 1 - 10.0 wt%

Propylene glycol 10.0 wt%

Glycerin 10.0 wt%

Aqueous solution of sodium hyaluronate (1%) -------------- 5.0 wt%

Ethanol 5.0 wt%

Polyoxyethylene hydrogenated castor oil - 1.0 wt%

0.1% by weight &lt; RTI ID = 0.0 &gt;

Carbomer - 0.4 wt%

Incense ---------------------------------------

The remaining purified water

<Experimental Example 1> Analysis of components of silkworm-derived hemolymph

1. Test Method

After removing silkworm silk gland, purified silkworm blood lymphedema was lyophilized and analyzed for general components such as moisture, crude protein, crude fat, ash, and inorganic matter.

2. Experimental results

Item Example 1-5 days 7 days (% by weight) 5 days 3 days Silkworm powder (% by weight) moisture 1.09 0.75 Views min 9.51 11.76 Crude fat 8.61 4.84 Crude protein 55.74 61.13 Crude carbohydrate 25.05 21.52

As shown in Table 1, the crude protein content was 55.74% by weight, the crude carbohydrate content was 25.05% by weight, the ash content was 9.51% by weight and the crude fat was 8.61% by weight in the order of the general components of the silkworm- .

<Experimental Example 2> Thermal stability test of silkworm-derived hemolymph

1. Experimental Method

The crude extracts obtained from the silkworms obtained from the silkworms were separated before and after the heat treatment and tested for stability by heat of hemolymph. At this time, the heat treatment proceeded at 60 ° C and 121 ° C.

SDS-PAGE analysis was performed to confirm the presence of hemolymph by heat treatment.

2. Experimental results

As shown in FIG. 1, it was confirmed that the hemolymph remained in the heat treatment condition at 60 ° C. (30 minutes). On the other hand, it was confirmed that the hemolymph was completely decomposed under the heat treatment condition at 121 ° C. (20 minutes).

In other words, the toxin quinone protein produced by tyrosinase was removed under the heat treatment condition at 60 ° C (30 min) and the hemolymph remained intact. However, in the heat treatment condition at 121 ° C (20 min) It means disassembled. Therefore, in the present invention, as a purification condition suitable for separating hemolymph from silkworm, heat treatment is performed at 60 DEG C for 30 minutes.

<Experimental Example 3> Antioxidant activity analysis of purified silkworm hemolymph in the present invention

1. Experimental Method

DPPH assay was performed to measure antioxidant activity of silkworm derived hemolymph.

In other words, 40 μl of the purified hemolymph of Example 1 was added to 160 μl of 0.2 mM DPPH dissolved in methanol, blocked with light, and reacted at 30 ° C. for 30 minutes.

Absorbance was measured at 517 nm using a microplate reader for the sample which had been completed.

Vitamin C was used as a control.

The antioxidant capacity of the measured hemolymph was shown in Table 1 and FIG.

2. Experimental results

sample IC ₅₀ ([mu] g / ml) Vitamin C 0.1 ± 0.06 Silkworm-derived hemolymph (Example 1) 15.3 + 4.8

As shown in Table 1 and FIG. 2, the antioxidant power of Example 1 of the present invention was 15.3 ± 4.8 g / ml, while the antioxidative power of 15% vitamin C, which is commonly used as a cosmetic material exhibiting antioxidant activity, was 0.1 ± 0.06 Mu] g / ml. That is, the antioxidative effect of the purified hemolymph of Example 1 of the present invention was found to be similar to that of 15% vitamin C.

<Experimental Example 4> Antioxidant activity analysis by silkworm variety

The antioxidative effect was measured in the same manner as in Experimental Example 1 above.

The antioxidant effect was measured by using a blood lymphedema (Example 1), sericin hemolymph, and sericin (a commercially available reference drug).

sample IC ₅₀ ([mu] g / ml) (Example 1) 6.4 ± 0.3 Sericin sleep 36.2 ± 3.2 Sericin 202.9 ± 33.1 Vitamin C 0.57 + 0.02

As a result, as shown in Table 2, it was found that the stool-derived hemolymph of the general incentive silkworm varieties was superior to sericin hypoxia and sericin purified from commercially available silkworm cocin.

<Experimental Example 5> The skin cytotoxicity of purified silkworm hemolymph from the present invention

1. Experimental Method

Human epidermal keratinocyteeonatal (HEKn) used in this study was Epilife medium containing 1% HKGs (human keratinocyte growth supplement) and 1% penicillin / streptomycin at 37 ℃ in a 5% CO ² incubator And cultured.

MTT assay experiments were conducted to determine the toxicity of HEKn to the silkworm blood limb of Example 1 used in this experiment.

The cells were seeded at a density of 1 × 10 ⁴ cells / well in a 96-well plate and cultured for 24 hours. Then, the silkworms were seeded at a concentration of 0, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000 μg / Blood lymph was treated and cultured for 24, 48, and 72 hours.

MTT (5 mg / ㎖) solution was added to each well and allowed to react for 3 hours. The MTT solution was removed, DMSO was added to each well, and formazan was dissolved. The absorbance at 570 nm was measured and the result was expressed as a ratio to the control value.

2. Experimental results

As a result, as shown in FIG. 3, in the present invention, the silkworm-derived hemolymph decreased by 3.43% at 1000 ㎍ / ㎖ after 24 hours and 5.71% at 500 ㎍ / Respectively. This indicates that the cell survival rate is decreased depending on time and concentration, meaning that silkworm-derived hemolymph has no strong toxicity to skin cells.

<Experimental Example 6> Antimicrobial activity of the purified silkworm hemolymph from the present invention

1. Experimental Method

The antibacterial activity of the purified silkworm hemolymph was confirmed in Example 1 of the present invention.

Escherichia coli KCTC 1039, Staphylococcus aureus KCTC 1621, Staphylococcus epidermidis KCTC 1917 and Candida albicans KCTC 7965 were prepared as the strains used .

All four of the above strains can be antimicrobial to other similar strains by observing the antimicrobial activity of these three strains as gram-negative, gram-positive, yeast (fungi) have.

First, the four strains were diluted to measure the viable cell count of each microorganism present on the diluted solution. In the following description, a small amount of each strain cultured in a pre-cultured plate medium was inoculated into 10 mL of a liquid medium suitable for each strain, and subcultured for 18 to 24 hours. 0.1 ml of the culture solution was inoculated therein and cultured for 12 to 24 hours.

After the cultivation, each strain was diluted with physiological saline to 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , and 10 ^-5 .

The diluted solution was applied to the plate medium three times each time, and the number of bacteria was measured after 24 hours.

E. coli , which is a coliform bacterium, showed a rapid growth rate as gram negative bacteria and C. albicans showed a low growth rate because it was a fungus as a fungus. In other words, in the case of E.coli ^10-4, S. aureus, C. albicans is 10 ^-3, S. epidermidis was applied to the experiment to ^10-4.

The antimicrobial effect of each of the strains prepared as described above was measured.

A small amount of each strain cultured on a plate culture medium was cultured for 18-24 hours in 10 mL of a liquid medium for each strain. The cultured broth was inoculated at 0.1 mL and cultured for 12 to 24 hours. Thereafter, 1 g each of 1% (sample 1), 2% (sample 2) and 3% (sample 3) of the silkworm derived from the silkworm of Example 1 of the present invention was added to 100 mL of sterilized physiological saline, , And the strain was inoculated with the strain at an optimal concentration. That is, the physiological saline inoculated with the sample and the strain was incubated in a 35 ° C incubator for 1 hour. As a blank, physiological saline solution treated only with a homogenate was used.

Then, each of physiological saline was diluted to final concentrations of 10 ^-6 , 10 ^-7 , and 10 ^-8 , and the cells were plated on a plate medium three times to determine the number of viable cells.

2. Experimental results

The results of the experiment are shown in Table 3 below.

Flour blank
(CFU / ml) sample1
(CFU / ml) sample2
(CFU / ml) sample3
(CFU / ml) E. coli
(Using 10 ^-4 dilution) Approximately 4000 Approximately 4000 0.0 2.33 S. aureus
(Using 10 ^-5 dilution) 51.0 78.7 0.0 0.3 S. epidermidis
(Using 10 ^-6 dilution) 12.3 5.0 0.0 0.0 C. albicans
(Using 10 ^-4 dilution) 435.0 324.0 314.0 398.0

As shown in Table 3, complete killing of the bacteria was confirmed in E. coli, S. aureus, and S. epidermidis . Thus, the silkworm-derived hemolymph of Example 1 of the present invention showed excellent antimicrobial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus aureus and Candida bacteria.

<Experimental Example 7> Whitening effect of purified silkworm-derived hemolymph in the present invention

1. Experimental Method

Melanogenesis inhibitory effect was measured by cell culture. Arbutin and morphine were used as comparative materials. It is one of the materials widely used in existing cosmetics because it is excellent in whitening effect with mulberry root shell. Cells were tested using BL6 melanocytes. BL6 melanocytes were dispensed into a 96 well microplate at 5,000 / well. After 24 hours, the samples were treated at different concentrations. After 72 hours, the amount of melanin produced was measured at 475 nm. As a control, Cells were excluded from the sample and the medium was measured at the same time.

The results were compared with those of arbutin by measuring the cytotoxicity of the samples and considering the concentration of cytotoxicity and considering the number of cells. The cytotoxicity and proliferation experiments were performed as follows. 3T3 cells were seeded at 5,000 / well in a 96-well microplate, and after 24 hours, the samples were treated by concentration. Thiazoline blue was administered 44 hours later and the absorbance was measured at 570 nm after 4 hours (using 10% Fetal Bovine Serum (FBS) medium in the test).

2. Experimental results

The results of the experiment are shown in Table 4 and FIG.

Item Experimental concentration (%, v / v) One 5 10 The hemolymph of Example 1 21.30 30.88 50.23 Mallow 12.15 22.41 25.11 Arbutin 9.74 14.9 20.68

As shown in Table 4 and FIG. 4, it was confirmed that the melanin formation inhibitory effect of the hemolymph of Example 1 was superior to that of the control monobasic and arbutin. As shown in Fig. 5, the silkworm-derived hemymph from Example 1 exhibited superior effects to the bark of alfalfa sprouts.

By confirming whether or not the silkworm derived from the silkworm, which has various abilities and has been discarded and has not been properly used, can be used as a cosmetic material, the cosmetic composition having excellent antioxidant ability, antibacterial activity and whitening effect . &Lt; / RTI &gt;

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. You can implement the examples. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

Claims (7)

Wherein the composition comprises silkworm-derived hemolymph.
The method according to claim 1,
Wherein said silkworm is a silkworm of 5 days 7 days,
Cosmetic composition.
The method according to claim 1,
Wherein the hemolymph is contained in an amount of 0.001 to 20% by weight based on the total weight of the cosmetic composition,
Cosmetic composition.
The method according to claim 3,
Wherein the hemolymph is contained in an amount of 0.1 to 10% by weight based on the total weight of the cosmetic composition,
Cosmetic composition.
The method according to claim 1,
Wherein the hemolymph is purified by removing the secretory gland from the silkworm and removing the residual crude extract containing the silkworm tissue into water and heat-treating it, followed by continuous centrifugation to obtain a supernatant.
Cosmetic composition.
The method of claim 5,
Wherein the heat treatment is performed at 40 to 80 DEG C for 10 to 120 minutes,
Cosmetic composition.
The cosmetic composition according to any one of claims 1 to 6, which has a formulation of any one of lotion, essence, lotion, cream, pack, foundation, gel, ointment or spray,
Cosmetic composition.

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