KR101738754B1 - Powder from the concentrat of silkworm oil, compositions containing the same and the process thereof - Google Patents

Abstract

The silkworm pupa of the present invention is fermented and extracted by using a culture solution of a complex strain, followed by separation and purification to obtain natural oil, which is then lyophilized to form a powder. The powder is formed into a raw material of a functional food or a drug having an effect of lowering cholesterol or a collagen biosynthesis And that it has a wrinkle-reducing effect such as inhibition of collagenase and also exhibits a skin moisturizing effect, thereby being used as a cosmetic composition for improving skin containing a powdery silkworm powder.

Description

TECHNICAL FIELD The present invention relates to silkworm powder, silkworm powder, silkworm powder, silkworm powder,

The present invention relates to a silkworm powder concentrate powder, a composition containing the silkworm powder, and a method for preparing the silkworm powder, and more particularly, to a silkworm powder concentrate powder having prevention and improvement of hyperlipidemia and a skin improving effect, .

More particularly, the present invention relates to a composition for prevention and improvement of hyperlipidemia, and more particularly, to a composition for preventing and / or treating hyperlipidemia, A composition for prevention and improvement of hyperlipidemia containing a powdery silkworm powder by confirming that triglyceride has a wrinkle-reducing effect such as lowering of arteriosclerosis index and increased collagen biosynthesis and collagenase inhibition and also showing skin moisturizing effect; To a cosmetic composition for improvement.

The silkworm pupa, which is known as a non-polluting organism, has been widely used as a functional food for treatment of adult diseases such as diabetes and hypertension and immunity function as well as a gangjeong agent using powders and extracts. Until now, the stability and clinical efficacy of silkworm as a natural product have been well known, but its activation period is still unclear.

The silkworm pupa as a natural product itself has no side effects and its efficacy as a therapeutic agent for a variety of diseases related to geriatric diseases has been continuously proved, and thus it has been widely used directly in the development of health supplements, medicines or cosmetics using the silkworm.

The silkworm pupa is known to be very effective for nourishment, blood sugar enhancement, anti-cancer, anti-aging, hangover, skin moisturizing and wrinkle removal. The silkworm extract is expected to exhibit various effects due to the high content of amino acids such as arginine and proteins such as spring bee call, hydroxyhexadecadienes and bombisin.

However, in the silkworm pupa oil, omega-3 fatty acids such as linolenic acid (ω-3 fatty acid), linoleic acid, oleic acid, Have been found to contain a large amount of polyunsaturated fatty acids.

Korean Patent No. 10-0573466 (Application No. 10-2004-076364) is an invention relating to "a method for extracting, separating and purifying oil from silkworm pupa ", wherein the silkworm powder is extracted with an organic solvent such as ethyl acetate The resulting silkworm extract was dissolved in hexane, and impurities were removed by activated charcoal. After filtration and concentration under reduced pressure, purified silkworm pupae oil was obtained , A silkworm pupa oil having an unsaturated fatty acid and a saturated fatty acid in a weight ratio of 7: 3 is introduced, Korean Patent No. 10-075117 (Application No. 10-2006-0011753) is an invention relating to "health food containing the silkworm pupa oil as an active ingredient ", wherein the silkworm pupa oil obtained in the above Patent No. 10-0573466 And the health food for the prevention of hyperlipidemia is introduced, Korean Patent No. 10-0732727 (Application No. 10-2006-0011754) is an invention related to "composition for the prevention and treatment of hyperlipidemia ", wherein said hyperpolygia prevention and prevention of hyperlipidemia comprising silkworm oil obtained in Patent No. 10-0573466 Therapeutic pharmaceutical compositions have been introduced, Korean Patent No. 10-0764928 (Application No. 10-2006-0011755) is an invention relating to "a cosmetic composition for improving skin comprising an effective amount of silkworm powder as an active ingredient ", wherein the silkworm obtained in the above Patent No. 10-0573466 A cosmetic composition for skin improvement containing a pupa oil has been introduced.

However, in the silkworm pupa oil of the prior art as described above, not only the fatty acid content is extremely low, but also a large amount of impurities are contained although the purification process is included, and the ratio of the unsaturated fatty acid to the saturated fatty acid is mixed to 7: 3 There has been a demand for steady development of silkworm pupa oil powder containing a large amount of fatty acids and ideally blending unsaturated fatty acids and fatty acids, pharmaceuticals containing them, and foods and cosmetic ingredient preparations containing them.

It is an object of the present invention to provide a silkworm powder concentrate powder having the effect of preventing and improving hyperlipemia and improving skin, a composition containing the same, and a process for producing the same.

In order to achieve the above object, the silkworm pupa of the present invention is fermented and extracted by using a culture solution of a complex strain, followed by separation and purification to obtain natural oil, which is lyophilized to form powder, It is confirmed that the wrinkle improvement effect such as increase of raw material or collagen biosynthesis of food or medicine, collagenase inhibition effect, and skin moisturizing effect are also confirmed. Thus, health functional auxiliary food containing skin powder of silkworm larvae as an active ingredient and skin improving And is used as a cosmetic composition for cosmetics.

The dried silkworm pupa was pulverized into powder and mixed with 10 L of a culture solution of a complex of microorganisms such as Bacillus sp. And Aspergillus sp per kilogram of silkworm pupa. The pH was adjusted to 4.5 to 6.5 and the temperature was 35 to 40 ℃ and the reaction time was 70 ~ 75 hours, and the extract was separated by centrifugation or filtration. The remaining silkworm solids were separated and extracted three times in the same manner to obtain an extract, and the extracts were combined.

10 to 30 times of hexane and an appropriate amount of activated carbon were added to the silkworm pupa oil obtained by the above method and mixed at 30 to 80 ° C. to remove impurities for 30 minutes to 2 hours. Then, the filter paper having a pore size of 5 to 50 μm , And the filtrate was concentrated under reduced pressure to completely remove the organic solvent. Thus, the oil component of the finally isolated silkworm pupa was obtained. The initial water content of the silkworm larvae was about 65 ~ 75%, and the oil content was about 10% by weight. The silkworm pupa oil obtained by this method is a liquid with almost no color and a transparent viscosity which does not give a unique smell of the silkworm pupa.

Subsequently, an excipient including Dextrose, Lecithin, Silkpeptide and the like was mixed with the obtained silkworm pupa oil. The mixture was stirred, emulsified and lyophilized to obtain silkworm powder ≪ / RTI >

[Table 1] Comparative table of general refined oil (Patent No. 10-0573466) and content ratio of major fatty acids constituting powder of silkworm powder in silkworm powder.

As can be seen from Table 1, according to the fermentation extraction method of the present invention, although the content of oleic acid and stearic acid was low in fatty acids, the content of pure alpha-linolenic acid (omega-3 fatty acid) I could increase it.

[Table 2] Structural formula of major fatty acids contained in powdered silkworm powder

The silkworm powder obtained from silkworm consisting of highly purified linoleic acid and linolenic acid according to the present invention has a cholesterol, triglyceride and atherosclerotic index lowering action, has an effect of increasing collagen biosynthesis, improving wrinkles such as collagenase inhibition, It is expected to be used as food, medicine, and cosmetics because it has effects such as efficacy.

In the present invention, the cholesterol-lowering effect of hypercholesterolemia induced by cholesterol ingestion of the powdered silkworm powder of the silkworm pupa was investigated, and the skin moisturizing condition by the test substance (silkworm unsaturated fatty acid) In order to verify the improvement efficacy.

Cholesterol and triglyceride levels in the blood biochemical test and liver were evaluated by oral administration of the test substance to the rats for 4 weeks together with induction of hyperlipidemia. The test group was set up with a normal diet (G1), a cholesterol diet negative control (G2), test substances 40 mg / kg (G3), 200 mg / kg (G4) and 1,000 mg / kg (G5) Respectively. The results of the test substance administration group were compared with the negative control group and the positive control group, and the test results are summarized as follows.

1. No abnormal symptoms associated with the administration of the test substance were observed.

2. No significant changes in body weight were observed.

3. No significant changes in feed intake were observed.

4. Blood biochemical tests showed a reproducible decrease in total cholesterol and LDL-cholesterol levels compared with negative control.

5. Atherogenic index was dose-correlated and reproducible.

As a result, the total cholesterol level was decreased by the silkworm powder of the silkworm pupa according to the present invention, and the atherogenic index was decreased in a reproducible manner.

The improvement of the skin moisturizing condition was evaluated by comparing the moisturizing degree of the right side of the treated hair with the moisturizing degree of the control group after the hair removal and skin dryness were induced by using the left side of the hair removal as a control group. But the moisture content increased rapidly from the second measurement to the test substance application group. In addition, in the validation of collagen biosynthesis test and collagenase inhibition test, it was found that the unsaturated fatty acid derived from the silkworm silkworm pupa increased the collagen biosynthesis to a significant level (p <0.01) as compared with the control group. , And the unsaturated fatty acid derived from the silkworm pupa was found to have an effect of improving wrinkles.

Therefore, the powdery silkworm powder according to the present invention is suitable for use as a composition for preventing and improving hyperlipidemia or as a health food, and is suitable for use as a cosmetic for improving skin. The present invention relates to a powder of silkworm powder containing a silkworm powder, A composition for prevention and improvement, a health food, and a cosmetic composition for skin improvement.

Fig. 1 is a schematic view showing a process for producing silkworm powder.
Fig. 2 is a schematic view showing the particle structure of silkworm powder.

Hereinafter, examples and experimental examples according to the present invention will be described.

Example  1: Step of fermentation extraction of silkworm pupa

The dried silkworm pupa was pulverized into powder, and 10 L of Bacillus subtilis and Aspergillus casei culture were added to the silkworm pupa at pH 4.5 to 6.5, 40 ℃, and 70 ~ 75 hours of reaction time. The extracts were collected by centrifugation or filtration. The remaining silkworm solids were separated and extracted three times in the same manner to obtain an extract, and the extracts were combined.

Example  Step 2: Separation and purification of silkworm pupa oil

The silkworm extract obtained by the method of Example 1 was filtered with a 1 占 퐉 filter and then lyophilized to obtain a silk peptide powder. In addition, the silkworm extract was mixed with 10 to 30 times of hexane and an appropriate amount of activated carbon, and the mixture was mixed at 30 to 80 ° C. for 30 minutes to 2 hours to remove impurities, and then filtered with a filter paper having a pore size of 5 to 50 μm The filtrate was concentrated under reduced pressure to completely remove the organic solvent to obtain the oil component of the finally separated silkworm pupa. The initial water content of the silkworm larvae was about 65 ~ 75%, and the oil content was about 10% by weight. The silkworm pupa oil obtained by this method is a liquid with almost no color and a transparent viscosity which does not give a unique smell of the silkworm pupa.

Example  Step 3: Concentrate the silkworm pupa oil

1) Concentration using crystallization

After adding 1.5 kg of urea to 4 L of methanol and completely dissolving at 70 캜, the silkworm oil purified by the method of Example 2 was injected by dividing it 5 to 8 times, cooled to 40 캜 and filtered. The filtrate is evaporated in a vacuum rotary evaporator and 1 L of water and a small amount of hydrochloric acid are added to the remaining solid content and stirred. Thereafter, the fatty acid layer in the upper layer is recovered. Again, add 1.5 kg of urea to 4 L of methanol and completely dissolve at 70 ° C. Add the recovered fatty acid 5 to 8 times and cool to 10 ° C. Thereafter, this liquid is filtered again to recover solid particles, and water (2 L) and hexane (2 L) are added to the solid particles, a small amount of hydrochloric acid is added and decomposed to recover the upper layer liquid. After washing with water 2-3 times, hexane is removed. To this, 700 to 1400 ml of hexane is added again and completely dissolved. The solution is cooled to -10 to -5 ° C without stirring. The resulting crystals are then filtered to remove the hexane.

2) Enrichment using esterification

(1) Production step of fatty acid alkyl ester

The silkworm oil purified by the method of Example 2 was added at a ratio of 1: 1 by volume to water at a temperature of 38 to 40 ° C. As the enzyme catalyst, acyl chain specificity and acyl chain specificity of triacylglycerol Lipase having 1,3-position specificity to carbon was added and the hydrolysis reaction was carried out at a stirring speed of 200 rpm for 24 hours. After 4 hours from the initiation of the reaction, alcohol for ethanol which had been heated to 40 ° C was added until the molar ratio of ethanol to ethanol reached a level of 3: 1, and ester exchange reaction was carried out to produce fatty acid ethyl ester.

(2) Preliminary distillation stage

The fatty acid ethyl ester prepared in the step (1) was continuously distilled using a molecular distillation apparatus. At this time, the temperature was maintained at 150 ° C and the degree of vacuum was maintained at 0.05 mmHg.

(3) Decompression fractionation step

In order to concentrate the distillate produced in the step (2), the column bottom temperature was 180 ° C., the column top temperature was 150 ° C. and the top degree of vacuum was 5 mmHg and the reflux ratio was 5 (reflux ratio = L / D, L = feed amount and D = output amount). The distilled water rate was 80%.

Example  4: silkworm pupa concentrated oil Powdered  step

An excipient including Dextrose (10-20%), Lecithin (10-20%) and Silkpeptide (5-10%) was added to water and mixed. The mixture was stirred for 15 minutes at 5000 r / min and 10000 r / min, respectively. The silkworm concentrate obtained in Example 3 was added to the resulting mixture and emulsified by stirring at 10000 r / min for 1 hour to prepare an emulsion. The emulsion thus prepared was frozen in liquid nitrogen and then treated with a freeze dryer To produce powdery silkworm powder (0.55 kg). Preparation of Cosmetic Composition A whitening agent such as Glabridin was added to the silkworm pupa concentrate as needed to prepare an emulsion. The emulsion was prepared by adding polyglycerylolate (Plurol Oleque), PEG-8 glyceryl caprylate At least one group selected from the group consisting of PEG-8 / Labratol, PEG-8 glyceryl linolate (Labrafil 2609), sorbitan fatty acid ester (Span), polyoxyethylene- sorbitan- fatty acid ester (0-10%) can be added.

Formulation example

1. Soft capsule (health functional food)

Prescription (㎎) in soft capsules:

Silkworm powder

Palm oil 40

Wax 15

Lecithin 15

Soybean oil 220

A total of 390 mg

Production method: A suspension is prepared by homogeneously mixing with the composition amount of the above-mentioned formulation, and filled in a gelatin soft capsule.

2. Production of lotion [Table 3]

<Manufacturing Method>

1) Raw materials of Nos. 2, 3 and 4 were weighed, and purified water of No. 1 was added thereto and dissolved by stirring at room temperature.

2) The raw materials of Nos. 5, 6, 7, 8 and 9 were weighed, dissolved by stirring, and added to 1).

3) No. 10 and silkworm pupa oil were added and dissolved, and the mixture was added to the above 1), stirred appropriately and filtered to prepare a lotion.

3. Preparation of lotion [Table 4]

<Manufacturing Method>

1) The raw materials of Nos. 1 to 12 were weighed, placed in an oil-gas heating tank, and dissolved by stirring at 75 ° C.

2) The raw materials of Nos. 13 to 18 were weighed, placed in a water tank, and dissolved by stirring at 75 캜.

3) The above 2) was transferred to the emulsification tank under vacuum decompression, then the above 1) was transferred and emulsified for 5 minutes at 3500 rpm.

4) The raw materials of No. 19 to 20 were weighed and made into a 1% solution solution. The solution was injected into an emulsification tank under a reduced vacuum, homogenized at 2500 rpm, and the pedal mixer was stirred at 25 RPM and cooled to 55 ° C .

5) The raw materials of the examples were weighed, dissolved in ethanol at room temperature, perfumed and weighed. The fractions were then homogeneously dispersed and cooled in an emulsifying tank, followed by completion of work at 30 캜 to prepare lotions.

4. Preparation of cream [Table 5]

<Manufacturing Method>

1) The raw materials of Nos. 1 to 12 were weighed, placed in an oil-gas heating tank, and dissolved by stirring at 75 ° C.

2) The raw materials of Nos. 13 to 18 were weighed, placed in a water tank, and dissolved by stirring at 75 캜.

3) The above 2) was transferred to the emulsification tank under vacuum decompression, then the above 1) was transferred and emulsified for 5 minutes at 3500 rpm.

4) The raw materials of No. 19 to 20 were weighed and made into a 1% solution solution, injected into an emulsification tank under vacuum and reduced pressure, homogeneously mixed at 2500 rpm, and the pedal mixer was stirred at 25 RPM, And cooled.

5) The raw materials of the examples were weighed, dissolved in ethanol at room temperature, perfumed and weighed in the emulsified tanks, and homogenously dispersed and cooled, followed by work-up at 30 ° C to prepare a cream.

5. Preparation of mask pack [Table 6]

<Manufacturing Method>

1) The raw materials No. 1 to No. 3 were weighed and dissolved by stirring at 75 ° C for 30 minutes.

2) The raw materials of Nos. 4 to 11 were weighed and dissolved by stirring at 75 캜.

3) The raw materials of No. 12 to 19 were weighed and dissolved with stirring with purified water 1.

4) The raw materials of numbers 20 to 25 were weighed and dissolved with stirring with purified water 1.

5) Mixtures 1) to 4) were mixed to prepare a mixed solution, followed by vacuum and defoaming to prepare a mask pack composition.

Experimental Example  1: Cholesterol lowering effect test

1. Dose and composition of the test group

The dose, the composition of the test group, the dose and the dose are shown in Table 7 below.

[Table 7]

2. Group Separation and Animal Identification

A) The day before the administration, the body weight was measured and ranked, and the SPF SD rats were divided into 6 groups of 6 mice per group.

B) Individual identification is indicated on the back of the animal using the identification label and picric acid attached to the front of the box.

3. Induction of hyperlipemia and administration of test substance

A) inducing hyperlipidemia

AIN-76 was supplemented with cholesterol diet supplemented with 1% cholesterol, and was supplemented with 0.5% ethanol-containing water to induce hyperlipemia.

B) Method of preparing test substance

The highest dose of the test substance was set at 1,000 mg / kg and the two dose groups were added below with an air ratio of 5. The test substance was weighed and dissolved in an excipient, corn oil, to prepare a test substance to be administered in a maximum dose of 1,000 mg / kg. The test substance to be administered in the following dose group was prepared by diluting the test substance to be administered in the highest dose group stepwise with the same excipient. Preparation of the test substance was performed before the daily administration.

Animals were anesthetized with a subcutaneous skin fixation method and the test substance was orally administered once daily for 4 weeks from the day of feeding of diets for inducing hyperlipemia (Day 0).

4. Test items

A) General symptom observation

All animals were observed at least once a day during the experimental period, and no abnormal abnormalities were observed.

B) Weight measurement

All animals were weighed at the time of collection, group separation, starting of test substance administration, once a week during the test period, and on the day of the autopsy.

[Table 8] Weight change (unit: g)

As shown in Table 8, no significant change in body weight was observed.

C) Feed intake

Once a week.

[Table 9] Dietary intake (unit: g)

As shown in Table 9, no significant change in feed intake was observed.

D) Blood biochemical examination

Blood biochemical tests were performed twice. The first test was performed at 2 weeks after the test substance administration, and the second test at the autopsy. At the first test, the animals were fasted overnight, and blood was collected from the venous vein of the animals. At the second test, the animals were fasted overnight and lavaged under anesthesia to collect blood from the posterior vena cava. The collected blood was centrifuged at 3,000 rpm for 10 minutes, and serum was measured for the following items. (See Table 10)

[Table 10]

Atherogenic index (AI) was calculated by dividing the total cholesterol (HDL - cholesterol) by the HDL - cholesterol level.

① 1st test (2nd week of test substance administration)

[Table 11] Serum Lipid Content and Arteriosclerosis Index (AI)

The total cholesterol and LDL-cholesterol of the test substance-administered group tended to decrease in a dose-related manner as compared with the negative control group (G2). The atherogenic index tended to decrease to 25% (G3), 44% (G4) and 64% (G5), respectively, compared with the negative control group (G2). And it was confirmed that it was better than 40% when compared with the positive control group (G6).

② 2nd test (4th week of test substance administration)

[Table 12] Serum Lipid Content and Arteriosclerosis Index (AI)

Total cholesterol, triglyceride and LDL-cholesterol tended to decrease in the test substance 1,000 mg / kg group compared to the negative control group (G2). The atherogenic index tended to decrease to 22% (G3), 42% (G4) and 60% (G5), respectively, compared with the negative control (G2). And it was confirmed that the efficiency was better than 24% when compared with the positive control group (G6).

Experimental Example  2: Acute Toxicity Experiment

In order to examine the acute toxicity of the powdered silkworm powder of the present invention, acute toxicity test was carried out as follows.

Six - week old SPF SD rats were used as experimental animals, and 5 rats were divided into groups. The powdery silkworm powder obtained in the above example was suspended in a 0.5% methylcellulose solution and administered once orally at a dose of 0.1 g / kg.

After the administration of the test substance, the mortality, clinical symptoms, and weight changes of the animals were observed, and hematological tests and blood biochemical tests were carried out, and autopsy was performed to observe the abnormalities of the abdominal organs and thoracic organs.

As a result of the test, there was no clinically symptomatic symptom in all the animals to which the test substance was administered, no mortality was observed, and no toxic change was observed in weight change, blood test, blood biochemical test or autopsy.

As a result, the silkworm larvae concentrate did not show any toxic change up to 0.1 g / kg in rats, and the minimum LD ₅₀ (oral dose) was at least 0.1 g / kg or more.

Experimental Example  3: skin moisturizing effect test

1. Dose and composition of the test group

The dose, composition and dose of the test group are shown in Table 13 below.

[Table 13]

2. Administration of powdered silkworm powder

The powdery silkworm powder of the silkworm larvae was weighed and dissolved in corn oil, which is an excipient. The amount of application was weighed using a pipette, and then applied twice a day at 6 hour intervals evenly on the epilating site.

3. Test items

(1) Prior to application of test substance, hair removal and skin dry induction method

A) Hair removal

The flank of the guinea pig was firstly epilated with a clipper and secondly epilated with an electric shaver (hair removal area 3.0 cm x 3.0 cm).

B) After epilating, the face pad wetted with 2% SLS (Sodium Lauryl Sulfate) was contacted to the right flank for 5 minutes. After 1 and 2 days, SLS was applied in the same way to induce skin dryness.

C) 2% SLS was applied for 3 consecutive times and left for 2 days.

(2) Application of test substance and measurement of moisturizing degree

A) Day 1 (2 days after application of 2% SLS 3 times, first degree of moisturizing)

Both sides of the flank were shaved with an electric shaver and washed with running water. After washing, the animals were allowed to stand in the breeding box containing the animal for 2 hours. Moisture resistance (capacitance) of both flanks was measured with a skin moisture meter (Corneometer, CM 825, Germany). Moisture was measured at one site more than 3 times and the average value was recorded. After moisturizing, the test substance was applied to the area treated with 2% SLS (repeated 6 hours later).

B) Day 2

The test material was applied in the same manner as the first day.

C) Day 3 (Secondary Moisture Level Measurement)

The hair was removed with an electric shaver and the degree of moisturization was measured with a skin moisturizer (corneometer) after 1 hour and 30 minutes. The test material was applied after the measurement. The left side was the control in the measurement and the right side was the treatment of the test material and the difference in moisture between the two sides was measured. The moisture content was measured and the test material was applied twice at intervals of 6 hours.

D) Day 4

The test material was applied in the same manner as the first day.

E) Day 5 (third degree of moisture measurement)

The degree of moisturization after epilating was measured in the same manner as in the third day, and the test material was applied.

F) Day 6

The test material was applied in the same manner as the first day.

G) Day 7

The test material was left without application.

A) Day 8 (measure the fourth degree of moisturizing)

The degree of moisturizing was measured after depilating.

(3) Observation and examination items

(1) The measured moisture level was measured by the difference between the left untreated pupa oil and the right skin induced dry skin test data.

(2) Statistical analysis of the measured moisture level was carried out by using one-way ANOVA using SPSS 10.1, a commercial statistical program, and post-test was conducted when there was significance between groups. The P value of each test substance application group and the control group Were compared. Or independent sample T test was performed to compare the P values of the control group with the silkworm pupa oil treatment group.

4. Results

2% SLS was used to induce dry skin on the right skin, and the moisturizing degree of the right skin applied on the left side and the silkworm powder of the silkworm powder was shown in Table 9 below. The less the difference in moisturizing degree, the more moisturizing the dry skin inducing part.

In the first measurement, moisturizing degree of silkworm powder concentrate oil powder group tended to increase slightly compared with control group. In the second measurement, the moisturizing degree of the silkworm powder powdered group was significantly increased to 179% (G2), 167% (G3) and 219% (G4) in the control group (p <0.05). It has more than 115% efficiency than ordinary refined oil. In the third measurement, the moisturizing degree of the silkworm powdery powder coating group was increased to 129% (G2), 137% (G3) and 188% (G4) in the control group as compared with the control group. It has an efficiency of more than 117% than general refined oil. In the fourth measurement, the moisturizing degree of the silkworm powdery powder coating group increased to 112% (G2), 134% (G3) and 158% (G4) of the control group. It has an efficiency of 129% or more than that of general refined oil.

[Table 14] Measurement of skin moisturizing degree

The moisturizing degree of the control group was gradually increased with the lapse of time, whereas the silkworm powdery powder application group showed a rapid increase in moisture retention from the second measurement, and the state was maintained. As a result, the application of powdery silkworm powder of the silkworm pupa was found to increase the moisturizing degree of the skin under the test conditions which caused the skin dryness, so that the moisturizing condition of the skin was improved.

Experimental Example  4: Confirm collagen biosynthesis

Collagen (types I, II, III, IV and V) are synthesized in the form of precursors called procollagen. Procollagen comprises a peptide base sequence called a propeptide at the amino terminus and at the carboxy terminus. The function of the propeptide is known to aid folding of procollagen molecules in the endoplasmic reticulum and to cleave and separate from collagen molecules when collagen polymerization occurs. Thus, by measuring the amount of the separated propeptide, the degree of collagen biosynthesis in the cell can be grasped.

* Test Methods

Human normal fibroblast cells were seeded in a 6-well dish and cultured for a day.

② Treat each sample with each concentration containing 0.05% FBS, and then re-cultivate for 48 hours.

After 48 hours, the cell culture medium was collected.

④ 100 μl of each sample collected was placed in each well of a microplate and incubated at 37 ° C for 2 hours.

⑤ Each well was washed three times with 400 μl of PBS.

⑥ 100 ㎕ of solution I (Antibody-POD conjugate solution) was added to each well, followed by incubation at 37 캜 for 1 hour.

⑦ Each well was washed three times with 400 ㎕ of PBS.

⑧ 100 μl of substrate solution (TMBZ) was added to each well, followed by incubation at room temperature (20 to 30 ° C) for 15 minutes.

⑨ 100 ㎕ of 1N H2SO4 was added to each well and mixed well.

⑩ Analyzed with an ELISA reader at 450 nm.

* Statistical processing

The measured values were expressed as mean ± standard deviation and the significance was tested by t-test using SPSS / PC + program.

Table 15 Effect of Powdered Silkworm Powder on Collagen Production

[Table 16] Collagen production rate of experimental group for control group

As a result of observing the effect of collagen synthesis on the silkworm powder, silkworm powder was found to increase the collagen biosynthesis in 0.01% and 0.05% concentration-dependently (p <0.01) I could confirm a good thing. Therefore, it was confirmed that the silkworm larvae concentrated oil used as the sample of this experiment had excellent collagen production in the cell.

Experimental Example  5: Collagenase  Inhibition test

An antibody against collagenase was used as a method for measuring the activity of collagenase, an enzyme that degrades collagen.

In this test, type I collagenase assay kit (Amersham) was used and absorbance was measured by ELISA.

* Test Methods

Human normal fibroblasts were cultured and maintained in a suitable medium at 37 ° C in a 5% CO ₂ incubator.

For the test, a certain number of cells were dispensed into appropriate culture vessels and incubated for a certain period of time. Cell adhesion was confirmed, and samples (0.01%, 0.05%, 0.1%) and TNF-alpha were added to the cell culture medium for 24 hours Lt; / RTI &gt; At this time, TNF-alpha was used as a substance activating MMP-1.

After culturing for 24 hours, the cell culture medium was collected, and 100 μl thereof was added to assay wells and incubated overnight at 2-8 ° C.

④ Each assay well was washed 4 times with wash buffer solution.

⑤ Add 50 μl of APMA, 50 μl of assay buffer and detection enzyme + substrate to each well, and mix well.

(6) Incubation at 37 ° C for 90 minutes, followed by measurement at 450 nm using an ELISA reader.

* Statistical processing

The measured values were expressed as mean ± SD and the significance was tested by t-test using SPSS / PC + program.

Table 17 Effect of Powdered Silkworm Powder from Silkworm Pupae on Collagenase Activity

Human normal fibroblasts were divided into appropriate culture vessels and incubated for a certain period of time to confirm cell adhesion. Cell culture medium was added to the cell culture medium for a certain period of time, and all the experimental values were found to be significant for the control group (p < 0.01).

[Table 18] Inhibition rate of collagenase activity by powdery silkworm powder of silkworm powder

Inhibition rate of collagenase activity by the powdery silkworm powder of the silkworm pupa was set at 100% of the control value, and the measured values of the samples were expressed in relative proportions.

Human normal fibroblasts were cultured and subjected to a collagenase activity test using a kit in cells that had been subjected to a sample treatment (0.01%, 0.05%, 0.1%) by concentration. As a result, there was a significant concentration-dependent inhibition of collagenase activity (p < 0.01). However, the inhibition of collagenase activity was not observed in the case of 0.1% concentration of general tablets oil.

Experimental Example  6: Skin safety test

First skin irritation test was conducted using New Zealand white rabbits. The left and right sides of the midline of the back part of the rabbit were respectively removed in a forward direction of about 10 cm, and the four test sites were set by dividing the front and back of each part. Two sections of left and right diagonal were made with # gauge frame so as not to bleed with 18 gauge injection needle and the remaining 2 parts were placed on the upper part of the gun. The powdery silkworm powder was dissolved in butylene glycol to prepare a 20% solution. 0.5 ml of this solution was impregnated in two 2.5 x 2.5 cm Lint pieces and then obliterated at the right two sites. The left two sites were filled with butylene The glycols were impregnated in two 2.5 × 2.5 cm Lint, and then obturator was attached to the left two sites, and the skin was observed for 24, 48 and 72 hours.

As a result of the test, skin adverse reactions such as redness and swelling did not occur in the dry and damaged areas where silkworm powder was applied.

Experimental Example  7: Confirmation of tyrosinase inhibitory effect

The method of colorimetrically measuring the DOPA chrome produced as a result of the tyrosinase action was modified to include a silkworm powdery concentrate oil added with a whitening agent such as glabridin and another kind of whitening functional drug symwhite 377 Tyrosinase activity inhibitory activity was measured.

10 μl of silkworm concentrate powder or Symwhite 377 supplemented with 40 μl of 1.5 mM L-tyrosine solution and a whitening agent were added to 110 μl of 0.1 M phosphate buffer (pH 6.5) in a 96-well plate and then mushroom tyrosinase (2000 units / ml) was added, and the reaction was carried out at 37 ° C for 10 minutes. Dopachrome generated in the reaction solution was measured at 490 nm using a microplate counter.

Arbutin was used as a positive control for comparison of experimental results.

[Table 19] Measurement of inhibition of tyrosinase activity by powdery mildew powder of silkworm powder

Experimental Example  8: Confirmation of cytotoxicity and inhibition of melanin formation

The cytotoxicity and melanin production inhibitory effect on the pigment cells were measured.

MB-16 melanoma cells were inoculated into DMEM medium containing 10% serum at 5 × 10 ⁴ cells, cultured in T-flask (75 cm 2) for 24 hours, fresh medium and concentrated oil powder were added to the cultured cells And cultured for 2 days. After culturing, the medium is removed, washed with phosphate buffer solution, and treated with trypsin to separate the cells. Cells are collected on a centrifuge and some are assayed for cytotoxicity by MTT assay. Some cells were treated with 5% trichloroacetic acid and then dissolved with 1N NaOH to determine the melanin content at 475 nm. The results are shown in Table 20.

[Table 20]

Claims (6)

A method for producing a powdery silkworm powder of the silkworm pupa having the constituents (fatty acid) and content of the following table consisting of the following steps 1) to 4).
1) mixing the culture medium of the complex strain of microorganisms belonging to the genus Bacillus and Aspergillus to the silkworm powder of the silkworm pupa, fermenting and extracting the mixture at pH 4.5 to 6.5, 35 to 40 ° C for 70 to 75 hours,
2) separating and purifying the silkworm pupa fermentation extract by removing hexane and activated charcoal from the silkworm pupa extract obtained in 1), adsorbing and removing impurities by adsorption, filtering and concentrating under reduced pressure to remove the organic solvent,
3) crystallizing or esterifying the silkworm purifying oil obtained in 2) and concentrating it,
4) Mixing the silkworm condensed oil obtained in 3) with a mixture of dextrose, lecithin and silk peptide to prepare an emulsion, and lyophilizing it to powder.
(table)

A health functional food for hyperlipemia improvement comprising a powdery silkworm powder obtained by the manufacturing method of claim 1 and having a constituent component (fatty acid) and content in the following table.
(table)

A health functional food having cholesterol-lowering activity, comprising a powdery silkworm powder obtained by the manufacturing method of claim 1 and having a constituent component (fatty acid) in the following table.
(table)

The composition of the present invention has the effect of enhancing collagen biosynthesis including the silkworm powder of the silkworm powder having a content of the component (fatty acid) of the following table and improving wrinkles such as collagenase inhibition, A cosmetic composition for improvement.
(table)

A cosmetic composition for skin improvement comprising a wrinkle improving and whitening effect, which comprises a silkworm powder of concentrated silkworm powder and a whitening improver of gabridine, which is prepared by the manufacturing method of claim 1 and has a content (fatty acid) in the following table.
(table)

A silkworm powder concentrate oil powder having a constituent component (fatty acid) and a content obtained by the production according to claim 1 of the following table.
(table)

Images