KR20020087570A - Paecilomyces japonica with anti-diabetic activity and its preparation method - Google Patents

Abstract

PURPOSE: A method for cultivation of Paecilomyces Japonica using a silkworm without a pupa as a host and a freeze drying method is provided. Therefore, a product having an excellent therapeutic effect on diabetic is produced with high productivity without causing the depletion of nutrients. CONSTITUTION: In the process for artificial cultivation of Paecilomyces Japonica, a 5 day old silkworm as a host is subjected to sterilization using chlorine dioxide water with a concentration of 1 to 5ppm without using heat treatment, a compound selected from the group consisting of silk amino acid, sugar and a mixture thereof is added to a medium for cultivation thereof, and then the silkworm is freeze dried and killed through an electric shock method.

Description

Cordyceps sinensis and its cultivation method of silkworm host having diabetes treatment effect {PAECILOMYCES JAPONICA WITH ANTI-DIABETIC ACTIVITY AND ITS PREPARATION METHOD}

The present invention relates to Cordyceps sinensis and its cultivation method of silkworm host having a therapeutic effect on diabetes.

Currently, the annual diabetes treatment market is 50 billion US dollars (55 trillion won), and the currently used drugs are not satisfactory in efficacy and have many side effects. There is a high expectation of effective drug development worldwide, and research in this area is active.

The name Cordyceps was originally derived from insects in winter and grasses in summer. In other words, Cordyceps fungus invades the body of an insect and dies, then uses the host's nutrients to form a fruiting body. Cordyceps sinensis belongs to the genus Cordyceps sinensis, which is widely distributed in Korea, China, and Japan. The reason why it is not known so far is that even entomologists who deal with insects were not interested in diseased insects by Cordyceps fungus, and inadvertently collected them incompletely by intentionally excluding them.

However, Cordyceps sinensis has been used to control the density of insect populations in natural ecosystems, and has been useful to humans since ancient times, and it has recently been interesting to people. In particular, it is noteworthy that it can be used as a herbal medicine or to control pests that damage crops. For example, Cordyceps sinensis, which is used as a Chinese herbal medicine in China, is collected naturally in the provinces of Xinjiang, Yuannan and Guizhou, and in the alpine region from 3000 to 4000 m above sea level from Tibet to the Himalayas. Since ancient times, Cordyceps sinensis has been treated with ginseng and antler, as one of the three most valuable herbal medicines, and has been used as an antidote for tuberculosis, jaundice and opioid poisoning as a elixir of bully. As late as in Korea, Cordyceps sinensis has been collected and tested in various clinical trials.

Today, the name Cordyceps sinensis generically refers to all fungi that are parasitic in the seeds of insects, arthropods, fungi or higher plants, and belong to the bacteriophage, incomplete, and junctional fungi. There are about 800 fungal invading insects, and most of them are known to form mushrooms. Most of them are belonging to the genus Cordyceps, and about 300 species have been reported in Korea. Up to 76 species were collected and identified. Cordyceps sinensis is a parasitic insect, and even the same insects may have different types of caterpillars that invade larvae and adults.

The importance of cordyceps fungi can be considered in terms of its potential as a medicinal aid to humans and the development of microbial agents for pest control. Herbal medicine is derived from fruiting bodies formed from mummy larvae by Cordyceps fungi, which have been used since ancient China. Its composition is composed of 10.84% moisture, 8.4% fat, 25.34% crude protein, 28.9% carbohydrate, and 4.1% ash. The fat contains 13% saturated fatty acids and 82.2% unsaturated fatty acids. Vitamin B ₁₂ contains 0.29 mg per 100 g.

Records on the efficacy of Cordyceps sinensis, Cordyceps is a nutritional tonic that protects the lungs and strengthens the kidneys, enhancing immune function. Increased immune function is not only resistant to any disease increases resistance, especially among the respiratory system of the effect is excellent.

Recently, it has been found that Cordyceps sinensis is effective as an anticancer and antidote for drug addiction with a 83% tumor suppression rate. In addition, Cordyceps sinensis has a natural healing power, which is effective in reducing the recovery time when there is a lot of physical activity due to severe exercise, and in China, athletes are taking good results.

Cordyceps sinensis used for medicinal purposes in China are Cordyceps, Larvae Cordyceps, Chrysalis Cordyceps, Mycobacterium Cordyceps, Cicada Cordyceps, White Bacillus, among them. do.

Another importance of Cordyceps sinensis is that the control of density of insect populations in natural ecosystems is carried out using leukemia. The earliest discoveries of fungal bacteria that cause disease in insects were the ancient Chinese, who regarded the mummified silkworm larva as an amulet of bully life. Because of its nature, which is related to the density section of insect populations as a natural intervention, many countries, especially developed countries, are using it to spur the development of natural biological pesticides to control pests that cause significant damage to crops.

Efforts to develop such natural biological pesticides have great significance in order to reduce not only pests but also environmental pollution caused by chemical pesticides. In France, biopesticides made from cordyceps are already on the market.

Silkworm Cordyceps sinensis has been recognized for its various pharmacological effects such as anti-cancer, anti-diabetic and immune-enhancing effects in herbal and folk medicine, and has been widely used in recent years.

However, these fungi currently exist in many varieties, and it has been suggested that the pharmacological effects of fungi differ depending on the type or the culture method. However, the identification of the pharmacological component and understanding of its mechanism of action have not been made at all, so there is no explanation. To this end, it is necessary to identify substances that exhibit pharmacological effects and explain their mechanism of action at the molecular biology level.

In addition, recently, advanced research groups in foreign countries have identified the active ingredients having pharmacological action from the main medicines of oriental medicine, revealing its mechanism of action, and developing a new large new drug.

One of the ways to secure international competitiveness in the development of new drugs in Korea is to discover effective pharmacological agents from drugs that have long been proven clinical effects in Korean medicine, and their mechanism of action using the latest life science research techniques. By elucidating this, the drug is either developed directly into a new drug or transformed into a more effective drug using the biochemical knowledge of the molecular target of the drug, which is expected to be enhanced, and expects enhanced pharmacological effects and fewer side effects.

Korean traditional medicine is researched using modern life science research techniques, which are modern research methods, and its efficacy is identified and proved at the molecular level to promote development as a globally recognized and proven new drug. An example of applying domestic accumulated herbal resources and knowledge to the new drug industry will be presented.

New drug development competition is driving advanced countries because of its high added value in the world, and the research is getting more advanced and competition is intensifying. In this environment, one of the ways in which new drug research in Korea can secure international competitiveness is to actively use the basis of oriental medicine, which has accumulated a lot of pharmaceutical and clinical experience in Korea.

On the other hand, in the case of Cordyceps sinensis known in the prior art was mainly based on the pupa, not silkworm, and also in the cultivation method of the drying and sterilization process through heat treatment process, it is known that a significant amount of active ingredients are lost in this process. In particular, productivity has been limited due to capacity limitations such as high pressure during heat treatment during sterilization. In the case of Cordyceps sinensis cultivated by such a prior art method, a large amount of active ingredient is inactivated in the treatment process, so that a concrete and professional review of substantially superior performance has not been made.

It is an object of the present invention to develop an excellent antidiabetic effect from cordyceps of silkworm hosts rather than cordyceps of known pupa hosts.

In addition, it is another object of the present invention to provide a method of cultivating Cordyceps sinensis so that the excellent anti-diabetic therapeutic effect can be maximally exerted.

Figure 1 shows the weight change measurement results according to an embodiment using Cordyceps sinensis of the present invention.

Figure 2 shows the blood glucose measurement results according to an embodiment using the Cordyceps Sinensis of the present invention.

Figure 3 shows the glycated hemoglobulin measurement results according to the embodiment using Cordyceps sinensis of the present invention.

Figure 4 shows the BUN measurement results according to the embodiment using the Cordyceps Sinensis of the present invention.

The present invention relates to Cordyceps sinensis host having a therapeutic effect on diabetes.

In addition, in the artificial cultivation process of Cordyceps sinensis, the artificial cultivation of Cordyceps sinensis of silkworm host, characterized in that sterilization treatment of 5 silkworms, the host, by using chlorine dioxide, to remove heat treatment in all processes. It is about a method.

In particular, the method of artificial cultivation of Cordyceps sinensis in the silkworm of the present invention is characterized in that the heat treatment is excluded in the treatment of the silkworm that is the host and finally in the treatment of Cordyceps finally obtained, for this purpose, conventional heating The method of killing silkworms by drying them by boiling, etc. may be used to kill silkworms by using electric shock, etc., and to freeze-dry them.

The sterilization process is also performed using chlorine dioxide water. Since this process can be easily used in a large-capacity container without a special device, it has been limited to productivity by treating it with a limited capacity using an expensive autoclave. Compared with the conventional prior art methods, it is possible to secure significantly superior productivity.

The concentration of the chlorine dioxide water that can be used in the present invention is not particularly limited, but is preferably about 1 to 5 ppm in consideration of the following neutralization step.

Thereafter, the method of artificial cultivation of Cordyceps sinensis of the silkworm host of the present invention is inoculated with the seedling cultured to the Cordyceps sinensis cultivation medium containing silkworms so treated, the mycelia and fruiting bodies in successive plate after forming mycelium It consists of forming.

When hyphae adhere to the appropriate humidity and temperature conditions, such conditions can be carried out as is well known in the art, by cultivating by additionally adding sugar or silk amino acid to the silkworm cultivation medium for silkworms, respectively, or in combination. The time can be shortened.

Also in the case of finally obtained Cordyceps sinensis, it is preferable to use the freeze drying method rather than the heat treatment process such as hot air drying.

In the method of cultivating the Cordyceps sinensis of the silkworm host of the present invention, the process called the silkworm and washing process neutralizes the alkalinity of the silkworms to increase the bacterial sticking rate of Cordyceps sinensis. The main feature of the above process is that the silkworms are not heated to the whole silkworms, but the nutrients of the silkworms can be inactivated when heats are applied. If you disinfect with chlorine dioxide, you can overcome the limitation of cultivation volume due to the capacity of the sterilizer to produce the desired amount according to the size of the grower. Silk amino acid and sugar mixed in the medium Enhancement of productivity by increasing the cultivation and elimination of Cordyceps sinensis by reducing the time required for cultivation by more than 2 days from the entire cultivation period. Even in the case of cultivated Cordyceps sinensis, hot air drying may cause loss of nutrients such as vitamins, so it must be lyophilized to increase diabetes treatment effect.

In addition, the present invention also relates to a composition for treating diabetes comprising such a silkworm host Cordyceps sinensis as an active ingredient.

Cordyceps sinensis developed in the present invention is characterized by having a superior diabetic therapeutic effect compared to the silkworm host with a conventional pupa.

The following examples have been described in detail the cultivation method of silkworm Cordyceps sinensis having a diabetic treatment effect to help the understanding of the present invention, which is intended to illustrate the technical spirit of the present invention, the scope of the present invention is limited to the following examples It is not.

EXAMPLE

Cultivation of Cordyceps sinensis

The kind of the bacteria: Pecilomyces japonica

Note: The silkworms of the 5th and 3rd days were dried, and the silkworms of the 5th and 3rd days were not dried, but were killed by specific techniques such as electric shock and dried by lyophilization rather than heat drying. Details are as follows.

1) Wash silkworms 5 days and 3 days alive in water,

2) After killing the washed silkworms with the electric shock of the high voltage (220 volt / 10 sec),

3) Killed silkworms were freeze-dried.

Only by doing so will most of the silkworm's original ingredients be inactivated.

Cultivation method: It was cultivated as follows.

1) Submerged in water for 48 hours and changed water at 4 hour intervals to neutralize the pH of silkworms. When the color of the silkworm was gray, sterilization was performed by immersion in chlorine dioxide (3 ppm) for 1 hour.

Dry silkworms were soaked in water, and cultured Peshiromyset japonica strains were inoculated into silkworms.

The mycelia were soaked at 95% humidity and 23-25 ° C.

2) After entering the sterile state in water, the water was poured out and exposed for 2 hours under sunlight to remove the disinfecting power.

3) In a sterilization chamber, a solution in which the silk amino acid and sugar were dissolved in a ratio of 1: 1 (1% of silkworm) was mixed.

4) The seed was inoculated.

5) The mycelia were soaked at a humidity of 95% and a temperature of 23-25 ° C.

6) transferred to the cultivation room to induce wigs.

7) Silkworms with good mycelial hung on the plate to induce fruiting bodies.

8) Fully grown Cordyceps sinensis were selected and lyophilized.

Diabetes Treatment Effectiveness Test

1. Induction of hyperglycemia from rat

end. Laboratory animals

Male SD rats of about 220 g were purchased from three test animals and used for experiments with sufficient feed and water in experimental animal breeding rooms at 20-23 ° C and 65% humidity for two weeks. .

I. Induction of hyperglycemia by streptozotoxin (STZ)

Rats fasted for 12-14 hours were intraperitoneally injected with STZ dissolved in 0.09 M citrate buffer at a concentration of 80 mg / kg to induce hyperglycemia.

On day 3 of STZ administration, blood was collected from the tail vein and blood glucose was measured using a Tinder (Sigma) reagent, and rats of 250 mg / dL or more were selected and used in the experiment.

All. Preparation and Supply of Cordyceps Sinensis Powder

A mixture of normal rat feed and Cordyceps sinensis powder and 10% starch of the feed were evenly mixed into pellets for easy feeding of experimental animals. Normal rats were treated as normal, hyperglycemic induced diabeates, and hyperglycemic rats were treated with CS-treated mice at a dose of 2000 mg / kg per horse per day. Sufficient feed was provided.

2. Biochemical Tests in Blood and Plasma

end. Blood separation

Rats were anesthetized with ether, collected in heparinized test tubes for blood glucose separation, centrifuged at 3000 rpm for 10 minutes, and stored at -40 ° C. Whole blood was collected separately for storage of glycated hemoglobin and stored at 4 ° C. Used.

I. Blood glucose measurement

3 mL of a binder reagent (Sigma, 315-100) was added to 10 μl of plasma, and the mixture was left at room temperature for 15 minutes, and the gloss was measured at 505 nm. A calibration curve was drawn with the standard solution to calculate the blood glucose content.

All. Glycation Hemoglobin Measurement

50 [mu] l of blood was added to 200 [mu] l of the reagent, carefully mixed, and left at room temperature for 10 minutes. Take 50 μl of this solution and drop it into a disc in a disposable column filled with cation-exchange resin. Then, stabilize it by flowing 200 μl of HbA1 Wash Buffer, and then evaporate 4 mL of HbA1 Wash Buffer. The elution fraction (E) was obtained by diluting the fractions collected by developing the mL 4 times. Each fraction was measured for absorbance at a spectrophotometer of 415 nm to calculate the glycated hemoglobin amount by the following equation.

la. BUN measurement

20 μl of serum is added to 2 mL of urease enzyme buffer and reacted at 37 ° C. for 15 minutes to decompose urea with ammonia and carbonic acid. The ammonia is colored with 2 mL of color solution and colorimetrically determined at 570 nm.

3. Measurement of weight and height of experimental animals

Cordyceps sinensis is administered daily to weigh the rats, and sacrifice the rats on the tenth day to separate the kidneys, wash the saline solution, weigh the kidneys, and display the weight comparison of the kidneys.

4. Experimental Results

end. Weight change

The normal control group showed continuous weight gain from day 1 to day 10 and showed little weight gain in diabetic induction countries. In the Cordyceps sinensis group, the weight gain was slightly higher than that of the diabetes group, but there was no significant difference. The measurement result is as showing in FIG.

I. Height and weight

In the diabetic induction group, the renal function was lowered (30%) than the normal control group on day 10 due to the deterioration of renal function. However, it was significantly reduced to 20% by the administration of Cordyceps sinensis. However, normal control group, diabetes-induced group, Cordyceps sinensis group did not show any difference in the weight of the kidney.

All. Blood glucose concentration

Compared with the normal control group, the diabetic induction group had a 2.28-fold increase in blood glucose concentration and 1.34 times the normal control group in the Cordyceps sinensis group.

Considering the oral administration of Cordyceps sinensis, the markedly reduced effect of glucose concentration in blood suggests the possibility of suppressing glucose absorption itself.

la. Blood glycated hemoglobin level

When the amount of glucose in the blood increases due to diabetes, glucose is bound to hemoglobin in the blood, thereby increasing the amount of glycated hemoglobin. In this experiment, the diabetic induction group was found to have a 45% increase in glycated hemoglobin compared to the normal control group and decreased to about 30% by administration of Cordyceps sinensis.

hemp. Effects on Kidney Function Decreased by Diabetes

In diabetic induction group, the renal function is decreased and blood excretion of blood urea nitrogen (BUN) is not smooth, which increases blood BUN level. In this experiment, the diabetic induction group showed an increase in BUN value of about 30% compared to the normal control group, and it could be seen that it was completely restored to normal level by the administration of Cordyceps sinensis.

As described above, silkworm host Cordyceps sinensis according to the present invention has a very high activity of excellent diabetes treatment effect, and at the same time very low side effects such as kidney disease, it can be seen that it has a very excellent performance in terms of diabetes treatment effect, By eliminating heat treatment in the process and performing a sterilization treatment process using chlorine dioxide, Cordyceps sinensis having excellent activity can be grown with high productivity, and thus, industrial applicability is very high. In addition, one of ordinary skill in the art to which the present invention pertains, it will be appreciated that various forms of imitation and modification are possible within the scope of the technical idea of the present invention based on the above specification.

Claims (6)

Cordyceps sinensis with diabetes treatment effect. In the artificial cultivation process of Cordyceps sinensis, the method of artificial cultivation of Cordyceps sinensis, which is characterized in that sterilization of the silkworm of the five-year-old silkworm, which is the host, using chlorine dioxide. The method of claim 2 wherein said silkworm is lyophilized. The method of claim 2 or 3, wherein the silkworm is killed by an electric shock method. The method of claim 2 wherein said chlorine dioxide water has a concentration of 1 to 5 ppm. The method of claim 2, wherein the silkworm cordyceps cultivation medium comprising silkworms is further mixed with one selected from the group consisting of silk amino acids, sugars, and mixtures thereof.