KR100462166B1 - A extract derived from male pupae before adults of silkworm, a preparation method thereof and foods and pharmaceutical compositions containing the same - Google Patents

Abstract

The present invention is characterized by comprising the step of selecting the male pupa by separating the male and female pupa using silkworm varieties that can be distinguished by the color of cocoon or the pattern of silkworm larvae The present invention relates to a method for preparing an extract of several chrysalis, and a lyophilized extract of several chrysalis collected by the preparation method, and a food and pharmaceutical composition having a stiffening effect, characterized by including the chrysalis extract.

Description

A extract derived from male pupae before adults of silkworm, a preparation method about and foods and pharmaceutical compositions containing the same}

The present invention relates to a pupa extract before it becomes silkworm male moth (circle), a preparation method thereof, and a food and pharmaceutical composition containing the same.

Recent economic growth places more emphasis on overall quality of life than on economic margins. Silkworms, which have contributed warmly to humanity by providing high quality fibers called silk to our humans for the past 4-5 thousand years, are now contributing greatly as a new type of natural active material production tool. In the age of treating diseases while neglecting the quality of life by synthetic medicines, there is a need for means to cure diseases while ensuring quality of life using natural active substances. In consideration of the fact that silkworm moths and pupa have been used as a suitable material, if scientific development is made, it will contribute greatly to the health of the people, the domestic sheep farming farms, and the world's sheep farming industry.

Recently, due to the high economic growth, the frequency of erectile dysfunction of organic causes is increasing due to the aging of the population and the increase of the elderly population as well as the increase of adult diseases. However, due to the tendency to pursue a better quality of life due to the improvement of economic power, the demand for treatment and improvement of this is increasing year by year.

Until now, the treatment of erectile dysfunction has been predominantly the treatment such as vasodilator injection and implant surgery, but recently, urethral vasodilator injection and oral erectile dysfunction treatment have become a new treatment.

Among them, I prefer the most effective medications. This is evident in the hot interest of the recently developed Viagra. Ideal oral medications for the treatment of erectile dysfunction should induce effective erections and their safety should be ensured even for long-term use. However, there is no effective drug that satisfies all these conditions so far.

From ancient times, silkworm moths (one-sleeping) have been found to have a strong sense of sympathy, such as bolstering Yangyang, stopping settling and bleeding, warming water, adding regularity, It is recorded that fatigue does not come even after hardening, but it is not yet clear how the jeongjeong effect is manifested. It is not.

The life history of silkworms is as follows. Silkworms are insects that completely transform, and their first generation overwinters as eggs after four periods of egg, larva, pupa and moth. Silkworm eggs hatch after a certain period of time to become ant-nue, and ant-nude to sleep after eating mulberry leaves for about 3-4 days. As such, the first state of sleep is called 1 side, and the silkworm at this time is called 1 side sleep. On the first side, sleep for about 16 hours, take off the fault, and become 2nd age silkworm. Silkworms usually sleep 4 times and are 4 sleep. Silkworms are 5 years old when they eat mulberry leaves for about 7-8 days and grow the largest. The silkworm at this time is called ripe silkworm. Ripe silkworms are made in cocoons for 2-3 days, and then into pupa, after which they are transformed into moths after 10-15 days, break through silkworm cocoons, spawn mating and finish the first generation.

As a result of focusing on the necessity of the natural substance-derived scavenger and the silkworm scavenger effect, the present inventors have found that the scavenger scavenger can be maximized from the pupa of uncoated silkworms. Thus, the present invention has been completed. Accordingly, the present invention aims to provide a fermented food and / or a pharmaceutical composition having a fermenting effect, which effectively selects unpuped pupa and contains the selected pupa extract.

Figure 1 shows a picture of a Han Hwang dog can distinguish male and female by the color of the cocoon.

Figure 2 shows a picture of Hanseongban can be distinguished male and female with or without the pattern of the cocoon.

Figure 3 shows a picture of silkworm pupa.

4 is a graph illustrating endurance measurement results by forced swimming time. Significance test P <0.5.

The present invention comprises the step of selecting the male pupa by separating the male and female pupa using silkworm varieties that can be distinguished by the color of cocoon or the pattern of silkworm larvae. It relates to a method of preparing an extract of pupa which is not mated.

The present invention also relates to a lyophilized extract of pupa pupa collected by the above production method.

The present invention relates to a pharmaceutical composition comprising a food extract having an jeongjeonggyeok effect, characterized in that it comprises a few chrysalis extract and an active ingredient in an amount effective to exhibit a jeongjeongeok effect.

First of all, in the preparation of silkworm pupae extracts to be used for erectile dysfunction and masculinity, it is necessary to select uncoated pupae. These uncoated pupa can be selected as follows.

Specifically, the method of screening the unpuped pupa of the present invention can distinguish the male and female by the color of cocoon (Hwang: ♀, white: ♂), like the silkworm breed of Hanseong Hwang dog, or the pattern of the silkworm larva like the Hansung silkworm breed ( In case of distinguishing the color of cocoon by using silkworm varieties that can distinguish male and female by ♀, civilization: ♂), it is impossible to mate male and female moths by separating white cocoons (♂) and yellow cocoons (♀) during the cocoon stage. When distinguished by the pattern of silkworm larvae, five-larva larvae were reared separately by the ♀ of the pattern (♀), and then bred by collecting them 2-4 hours before they were converted from moth (♂) to moths. Only moths that haven't been picked up. The most desirable stages of male moth and chrysalis are pupa, which is 2-4 hours before the silkworm pupa 10-15 days early sunrise (5-7 o'clock). What is not called male moths.

In addition, the pupa extract selected by the above method is most preferably an extract prepared after screening the pupa which has been almost transformed into silkworm moth, which is ultra-low temperature by treating the pupa selected by a single screening process with liquid nitrogen. It is stored in a freezer, lyophilized using a lyophilizer and made into a powder state, where it is concentrated by extracting 50% ethanol and lyophilized powder.

Specifically, the extract was treated with liquid nitrogen in selected chrysalis and frozen in a cryogenic freezer, and then powdered using a lyophilizer, followed by 50% ethanol with a volume of 20 times and repeatedly extraction with ultrasound for about 1 hour. After about five times, the filter paper was filtered and concentrated using a rotary vacuum concentrator, and then lyophilized. These extract samples were used to test the jeongjeong effect and endurance test through the following test tube and animal experiments.

The method for producing the extract of the chrysalis has its characteristics in the method of sorting the chrysalis which has not been mated, and other extraction processes can be used if it is a conventionally known method.

In the food containing the several pupa extract, it is possible to prepare a food having a firming effect of the present invention by combining the extract of the present invention with a food additive that can be added conventionally.

In addition, it will be possible to prepare a food having a stiffening effect by adding it to a conventional favorite food.

The food containing the extract of the present invention relates to a beverage containing the pupa extract of the present invention in an amount effective to exhibit a jeongjeong effect, the main ingredient is a red beet juice, DHA, and taurine in water-soluble calcium The present invention relates to a health supplement for health supplements containing conventional food additives such as oligosaccharides, liquid fructose, citric acid, β-cyclodextrin, vitamin C and vitamin D3, cinnamon extract, ginger extract, and mixed fruit flavor.

In pharmaceutical compositions comprising the pupal extract of the present invention in an amount effective to exert a sedative effect, it may comprise one or more non-toxic, pharmaceutically acceptable carriers, adjuvants or diluents, or other active ingredients.

The pharmaceutical composition of the present invention may be formulated into an oral dosage form in the form of a dry powder for use as a solution, a suspension or an emulsion in an oil or an aqueous medium, or dissolved in sterile, pyrogen-free water before use. .

The compositions of the present invention may be prepared by known methods using pharmaceutically acceptable carriers and excipients, for example tablets, troches, sugar-containing tablets, aqueous or oily suspensions, dispersible powders or particles, emulsions, soft or hard. It is formulated for oral use in the form of capsules, syrups, elixirs, which are contained in unit dosage forms and in multidose containers.

In oral formulations, tablets may be inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, sodium phosphate; Granulating agents such as corn, starch and alginic acid; Disintegrants; Binders such as starch, gelatin and acacia; The compound of the present invention is mixed with an excipient which can be used for the preparation of tablets, such as lubricants such as magnesium stearate, stearic acid and talc. Tablets may be used uncoated or coated to inhibit absorption and degradation of the tablets in the gastrointestinal tract. For example, you may apply time inhibiting substances, such as glyceryl monostearate and glyceryl distearate. Hard capsule is a compound of the present invention is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate, kaolin, etc., soft capsule is a solvent such as polypropylene glycol, PEGs (polyethylene glycol), ethanol, etc. that can be mixed with water And the active ingredients in oil solvents such as peanut oil, liquid paraffin and olive oil.

The water-soluble suspending agent is a mixture of excipients and active ingredients suitable for the preparation of water-soluble suspending agents. Excipients include, for example, sodium carboxymethyl cellulose, methyl cellulose, hydroxy-propyl methyl cellulose, sodium alginate, polyvinyl-pi Suspending agents such as ralidone, gum tragacanth, gum acacia and the like; Compounds condensing fatty acids such as polyoxyethylene stearate and alkylene oxides; Compounds in which alkylene oxides are condensed with long fatty acids such as heptadecaethyleneoxycetanol; Compounds which condensate partial esters derived from anhydrous hexitol anhydride and fatty acids, such as polyoxyethylene sorbitol monooleate, with ethylene oxide; Wetting agents; Or dispersing agents. Water-soluble suspensions contain preservatives, colorants, spices, sweeteners and the like.

The oily suspension is a suspension of an active ingredient in a vegetable oil such as olive oil, sesami oil, or a mineral oil such as liquid paraffin. For example, a thickening agent such as beeswax, hardened paraffin, cetyl alcohol, etc. may be used. It contains. It also contains preservatives, colorants, spices, sweeteners and the like, which can be preserved by adding antioxidants such as vitamin C.

Dispersible powders and particles have an active ingredient in a state in which a dispersing agent, a wetting agent, a suspending agent, a preservative and the like are mixed together. Suitable dispersing, wetting or suspending agents can be mentioned by way of example already mentioned above. Additional excipients are, for example, sweeteners, spices, colorants and the like.

Water-in-oil emulsions are derived from vegetable oils such as olive oil or mineral oils such as liquid paraffin, and are derived from natural phospholipids such as soy bean lecithin, and esters of anhydrous hecitol or fatty acids such as sorbitan monooleate. The active ingredient is emulsified using compounds obtained by condensation of ethylene oxide with partial esters derived from anhydrous hexitol anhydride and fatty acids such as oxyoxysorbitol monooleate.

Syrups and elixirs are a mixture of active ingredients with sweeteners such as glycerol, propylene glycol, sorbitol and sucrose.

When using the composition of the present invention, the dosage of the active ingredient may vary depending on the age, weight, general health, sex, meal, time of administration, rate of excretion, combination of drugs, degree of disease during treatment, etc. In particular, but not limited to, preferably 100 to 500 mg daily may be used daily, based on a person weighing 60 kg.

On the other hand, the amount of the compound of the present invention mixed with the carrier material to determine one dosage form depends on the mode of administration and the patient being treated.

The silkworm moth-related products seek to maximize the effects by identifying the growth effects and preparation methods, as well as elucidating the jeongjeong effect, that is, the male hormone production and erection promotion effects, the sperm productivity and the endurance test. The silkworm extract samples used in the following experiments were frozen by treating liquid nitrogen with selected chrysalis and stored in an cryogenic freezer, and then powdered using a freeze dryer. Extraction was repeated five times during the day, filtered through a filter paper, concentrated using a rotary vacuum concentrator, and then lyophilized to be powdered. In the cocoon stage, white cocoons and yellow cocoons were separated to prevent mating of male and female moths, and they were harvested before being transformed into moths to extract the maximum effect of the active substance. In addition, some of the silkworms were harvested using the varieties of silkworms with or without silkworms, and the pupa, which was separated by the above method, was gradually transformed into moths after about two weeks. You can see that. In the present invention, all of the moths are immediately collected and extracted. It can be confirmed that it is not mating and can be used as a food supplement such as health supplements because it is allowed as food. Hereinafter, silkworm extract pupa extract according to the embodiment of the present invention is obtained by the above method 'Means.

1. Testosterone production

Testosterone production was measured by the o-phthaldehyde method. First, dissolve the silkworm extract samples in distilled water at concentrations of 100 mg / dL, 250 mg / dL, and 500 mg / dL, and take 0.1 ml each, add 0.3 ml of 33% KOH solution and 3.0 ml of 95% ethanol, and mix well. For 15 minutes, the brain cell batch is cooled after heating in a 60 ° C. water bath for 60 minutes.

5.0 ml of nucleic acid was added thereto, mixed, 3.0 ml of distilled water was added thereto, and then mixed well for 1 minute. The layers were separated, and 1.0 ml of nucleic acid layer was taken. The nucleic acid layer was concentrated to dryness with nitrogen, and 2.0 ml of o-ftaldehyde reagent was added and mixed well. After 10 minutes, 1.0 ml of concentrated sulfuric acid was added and mixed as a color developing reagent.

Within 10 to 90 minutes after sulfuric acid addition, the absorbance was measured at 550 nm using a spectrophotometer and the total testosterone content (mg / dl serum) was quantified by a standard calibration curve.

Viagra is 50mg / 70kg body weight, Kisometer extracted from seal penis is 1,500mg / 70kg body weight, silkworm pupa extract is 100mg, 250mg, 500mg / kg body weight The herbal extract mixture of Kwak + Paju + Bokbunja was also administered to the experimental animals (Sprague Dawley male rats: 250 ± 10g) for 3 weeks to weigh 250mg / kg, and then the testes were extracted and the testosterone (ng / mg protein) The amount produced was measured, and the results are shown in Table 1. Distilled water was used as a control.

As a result, the most anticipated testosterone production of Viagra was 15.76 ± 1.83ng / mg protein, which was 109.9% higher than that of the control group (100.0%), which increased about 10% of testosterone production. The production of testosterone in the group was found to be a significant increase in testosterone production of 15-33%. Among them, testosterone production of silkworm pupa extract 250mg / kg body weight administration group was 19.04 ± 1.72mg / mg protein, which was 132.8% compared to the control group (100.0%).

Testosterone production measurement division Testosterone production (ng / mg protein) % Increase Control group (distilled water) 14.34 ± 1.63 100.0 Viagra group 50mg / kg body weight 15.76 ± 1.83 109.9 * Chisometa administration group 1,500 mg / kg body weight 13.20 ± 0.94 92.1 Silkworm can pupa extract 100mg / kg body weight 17.66 ± 1.13 123.2 *** 250 mg / kg body weight 19.04 ± 1.72 132.8 *** 500mg / kg body weight 16.41 ± 1.14 114.4 ** Yin Yang 250 mg / kg body weight 17.46 ± 1.38 121.8 *** Yin Yang Kwak + Paek + Bokbunja 250 mg / kg body weight 17.16 ± 1.18 119.7 *** ※ Significance test: * P: 0.05 compared to control group; ** P: 0.01; *** P: 0.0012. Nitric Oxide in Testicles

The amount of nitric oxide produced in the testes was analyzed by some modifications of Yu et al. (1994).

NO content in testicular tissues of rats was homogenized with 0.1 g of testis tissue with 1 ml of 0.1 M phosphate buffer (pH 7.5), followed by centrifugation at 600 × g for 5 minutes, and 100 μl of 90mUnit nitrate reductase in 200 μl of supernatant. 100 μl of 0.28 mM NADPII, 100 μl of 3.5 μM FAD, and 200 μl of 0.1 M phosphate buffer (pH 7.5) are added and incubated at 25 ° C. for 1 hour. Then boil for 3 minutes to stop all reactions. Griss reagent (2% sulfanylic acid / 5% H ₃ PO ₄ : 0.2% N- (1-naphthyl) -ethylenediamine / water) was then prepared in a ratio of 1: 1 and 700 μl was added. After warming again at 60 ° C. for 10 minutes, the absorbance was measured at 546 nm to quantify the amount of nitric oxide produced according to a standard calibration curve with sodium nitrate.

Nitric oxide (NO) is known to play a role in blood pressure regulation, neurotransmission, blood coagulation, immune function, and is synthesized by nitric oxide synthase (NOS) from L-arginine of various tissues and cells. NO is also known as an erectile facilitation factor due to vasodilation. In order to evaluate the amount of nitric oxide (NO) found to be a sex expression promoting factor directly related to the production of cGMP, a direct erectile component, testosterone in the testes In the same way as the production evaluation, silkworm pupa extract, Viagra, Kisometer, Yin Yang and Yak Kak + Pokpo + Bokbun herbal extracts were extracted in experimental animals (Sprague Dawley male rats: 250 ± 10g) for 3 weeks. After administration, the testicles were extracted and the amount of nitric oxide (nmol / mg protein) in the testicles was measured, and the results are shown in Table 2.

As shown in Table 2, the production of nitric oxide was lower than Viagra or chisometa at all, while the nitric oxide production of the herbal mixture extracts such as yin and yang were 23.98 ± 0.65 and 24.06 ± 0.22 nmol, respectively. As / mg protein, 118.7% and 119.0% of the control group (100.0%) were confirmed to be increased by about 20%. At this time, distilled water was used as a control. However, the nitric oxide production of silkworm pupa 250mg and 500mg / kg body weight group was 23.56 ± 1.35 and 23.42 ± 0.93 nmol / mg protein, respectively, 116.6% and 115.9%, respectively, about 16-17% of the control group (100.0%). It was confirmed that the production of significant nitric oxide was increased.

Measurement of Nitric Oxide Production in Testicles division Nitric oxide (NO) production amount (nmol / mg protein) % Increase Control group (distilled water) 20.21 ± 1.12 100.0 Viagra group 50mg / kg body weight 21.28 ± 2.01 105.3 Chisometa administration group 1,500 mg / kg body weight 21.33 ± 0.42 105.5 Silkworm can pupa extract 100mg / kg body weight 21.62 ± 2.23 106.9 250 mg / kg body weight 23.56 ± 1.35 116.6 ** 500mg / kg body weight 23.42 ± 0.93 115.9 ** Yin Yang 250 mg / kg body weight 23.98 ± 0.65 118.7 *** Yin Yang Kwak + Paek + Bokbunja 250 mg / kg body weight 24.06 ± 0.22 119.0 *** ※ Significance test: * P: 0.05 compared to control group; ** P: 0.01; *** P: 0.0013. Sperm count in epididymis was measured in the epididymis according to the method of Morrissey et al. (1988). The left epididymis of the rat was extracted and stored at −70 ° C. until measured. After dissolution at room temperature, homogenization was performed by adding HBSS solution (Hanks' Balanced Salt Solution) 10 times the weight of epididymis. After diluting the homogenate 40 times with HBSS solution again, a certain amount was taken and analyzed by quantum sperm count (× 10 ⁶ / g epididymis) using an inverted microscope using a hemocytometer. Viagra, Kisumometer extracted from seal penis, silkworm pupa extract, herbal extract mixture of Yin-Yang and Yin-Kwak + Paju + Bokbunja were extracted in the same way (Sprague Dawley male rats: 250 ± 10g ) Was administered for 3 weeks, and sperm count (× 10 ⁶ / g epididymis) was measured in the epididymis, and the results are shown in Table 3.

As shown in Table 3, the silkworm pupa extract administration group was found to have a superior increase in sperm count compared to Viagra or other herbal mixtures. The sperm count of silkworm pupa extract 250mg / kg body weight group was 75.47 ± 0.09 × 10 ⁶ / g epididymis, which was 141.4% compared to the control group (100.0%), which showed a 41% increase in sperm count, and 100mg and 500 mg / The kg body weight group also showed a significant increase in sperm count of 135.6% and 124.5%, respectively, compared to the control group. Distilled water was used as a control.

Sperm Count in the Epididymis division Sperm number (× 10 ⁶ / g epididymis) % Increase Control group (distilled water) 53.38 ± 2.18 100.0 Viagra group 50mg / kg body weight 59.72 ± 0.80 111.9 Chisometa administration group 1,500 mg / kg body weight 51.51 ± 1.68 96.5 Silkworm can pupa extract 100mg / kg body weight 70.22 ± 2.37 135.6 *** 250 mg / kg body weight 75.47 ± 0.09 141.4 *** 500mg / kg body weight 66.47 ± 4.71 124.5 *** Yin Yang 250 mg / kg body weight 70.95 ± 2.05 132.9 *** Yin Yang Kwak + Paek + Bokbunja 250 mg / kg body weight 66.73 ± 1.11 125.0 *** ※ Significance test: * P: 0.05 compared to control group; ** P: 0.01; *** P: 0.0014. Endurance measurement

The endurance measurement was performed according to the method of Avakyan et al. (Byullen Experimental noi medisiny, 84: 375). A male ICR mouse is used as an experimental animal, and the tail of the animal is fixed upside down from the surface to a height of 10 to 12 cm on a 100 × 50 × 50 cm water bath so that the head and upper limbs of the mouse are submerged so as to induce movement. Measure the amount of time your head is submerged with maximum fatigue. At this time, the temperature of the water bath was maintained at 14 to 16 ℃, the sample was continuously administered for 4 days and the experiment was conducted only 24 hours after the final sample administration.

The silkworm powder used in this endurance experiment was a lyophilized powder of 5-day-old larvae of silkworms.

We examined the effects of endurance on masculinity or strength rather than directly related to erectile dysfunction. The animals were measured for the maximum fatigue and the time the head was submerged. As a result, as shown in Table 5, Viagra has a lower endurance than the control group, and the male moth extract of the present invention has increased the endurance more than the silkworm powder, which is about 60% more effective than the control group.

Tocopherol has also been shown to be somewhat effective in improving endurance.

Endurance measurement by forced swimming time division Control Pupa extract Silkworm powder Viagra Tocopherol Swimming time (minutes) 14.42 ± 3.71 15.33 ± 8.71 20.37 ± 8.71 12.16 ± 8.71 17.21 ± 8.71 % Increase 100 106 141 84 119

As described and demonstrated in detail above, the present invention provides a method for effectively extracting erectile dysfunction and masculinity from silkworm pupa.

The silkworm pupae of the present invention uses silkworm varieties that can be distinguished by the color of cocoons or silkworm larvae, so that the silkworm pupae become moths from the beginning, so only male moths can be collected effectively. I could not solve the problem. In addition, since only a few moths can not determine whether or not mating, using a few chrysalis immediately before the moth can not only obtain a certain effect of the male moths, but also has the advantage of ensuring that the number of uncoated chrysalis.

And male moths are not allowed as food, but silkworm pupae has the advantage that it can be used as a food because it can be used as a food regardless of gender. Male moths can be used as pharmaceutical materials by purely separating substances directly related to erectile dysfunction or male history.

Claims (6)

Selecting male pupa by separating male and female pupa using silkworm varieties capable of distinguishing male and female silkworms by the color of cocoon or the presence or absence of a pattern of silkworm larvae; Freeze-drying the selected chrysalis and grinding to make powder; And extracting the chrysalis powder with alcohol. Water pupa extract prepared by the method of claim 1. Gangjeong (强 精) food composition, characterized in that it comprises the extract of the pupa of claim 2. Pharmaceutical composition, characterized in that the active ingredient contains the pupa extract of claim 2 in an amount effective to exhibit a jeongjeong effect. A food composition for gangjeong, characterized in that it contains silkworm pupa or extract of pupa. Pharmaceutical composition, characterized in that it comprises silkworm pupa or pupa extract as an active ingredient in an amount effective to exhibit a scavenger effect.