ANTI STOMACH ULCER OR STOMACH ULCER DRUG COMPOSITION

[Technical Field]

The present invention relates to a composition for preventing or treating stomach ulcers. More specifically, the present invention relates to a composition efficacious in preventing or treating stomach ulcers comprising a freeze-dried earthworm powder or an earthworm-extracted supernatant, both of which contain lumbrokinase as an active ingredient, wherein the earthworm-extracted supernatant is prepared by wet-grinding living earthworms and centrifuging the grinded earthworms, and the freeze-dried earthworm powder is obtained by filtering the supernatant, followed by freeze-drying.

[Background Art]

Dried earthworm powders or earthworm extracts have been used for antihyperlipemic, antidiabetic, blood pressure-controlling and antithrombosis drugs. Such use is based on their medical efficacies recorded in Oriental

Medicine Literatures (Shinnongbonchogyeong, etc). However, the present inventors have demonstrated that lumbrokinase contained in earthworms is efficacious in treating stomach ulcers via repeated research and tests, taking into consideration the facts that earthworms contain a great amount of lumbrokinase, which is one of the proteases, and that a great deal of protease activated receptors (PARs) which have activities for lumbrokinase are present in a human's stomach. As a result, the present invention has been finally completed.

Lumbrokinase contained in earthworms is reported to be a serine-based protease, be considerably stable against heat or pH and exhibit a specific activity for fibrin, which is one of thrombus' ingredients. Lumbrokinase administered orally must be released through the intestines into blood so that it can exert its efficacy. Research associated with this release is considered to be recently conducted.

It was reported that a great deal of protease activated receptors (PARs) present in the gastrointestines are associated with protection functions of gastrointestinal systems .

Accordingly, the present inventors ascertained efficacy of freeze-dried earthworm powders on stomach ulcer- inducing models through various methods.

[Disclosure] [Technical Problem]

The present invention has been made to solve the foregoing problems of the prior art and it is an aspect of the present invention to provide a composition for preventing and treating stomach ulcers comprising a freeze-dried earthworm powder or an earthworm-extracted supernatant, both of which contain lumbrokinase as an active ingredient, wherein the earthworm-extracted supernatant is prepared by wet-grinding living earthworms and centrifuging the grinded earthworms, and the freeze-dried earthworm powder is obtained by filtering the supernatant, followed by freeze-drying.

[Technical Solution]

In accordance with an aspect of the present invention for achieving the above aspect, there is provided a composition for preventing and treating stomach ulcers comprising a freeze-dried earthworm powder or an earthworm-extracted supernatant, both of which contain lumbrokinase as an active ingredient, wherein the earthworm-extracted supernatant is prepared by wet-grinding living earthworms and centrifuging the grinded earthworms, and the freeze-dried earthworm

powder is obtained by filtering the supernatant, followed by freeze-drying .

[Advantageous Effects]

The composition of the present invention is efficacious in preventing and treating stomach ulcers.

[Description of Drawings]

The above and other aspects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: FIGs. 1 and 2 are photographs showing the gastrointestines of a control group in Example 4 according to the present invention;

FIGs. 3 and 4 are photographs showing the gastrointestines of a white rat to which Omeprazole is administered in Example 4 according to the present invention;

FIGs. 5 and 6 are photographs showing the gastrointestines of a white rat to which Stillene is administered in Example 4 according to the present

invention; and

FIGs . 7 and 8 are photographs showing the gastrointestines of a white rat to which a freeze-dried earthworm powder is administered in Example 4 according to the present invention. [Best Mode]

The present invention will now be described with reference to the accompanying drawings in greater detail.

The anti-stomach ulcer composition according to the present invention comprises a freeze-dried earthworm powder or a supernatant extracted from earthworms (hereinafter, referred to simply as "earthworm supernatant") . Preferably, the anti-stomach ulcer composition contains lumbrokinase , which is one of the proteases, as an active ingredient.

A method for preparing the anti-stomach ulcer composition of the present invention comprises: washing raw earthworms with distilled water; wet-grinding the earthworms; centrifuging the earthworms at 2,000 ^χ g to 4,000*g for 20 to 40 min to obtain a supernatant; and preparing a supernatant-containing composition for preventing and treating stomach ulcers. Preferably, the method may further comprise filtering the

supernatant through diatomaceous silica to remove water- insoluble materials.

Preferably, the method may further comprise freeze-drying the supernatant to prepare a freeze-dried earthworm powder. The preparation of the anti-stomach ulcer composition is carried out using the freeze-dried earthworm powder.

[Mode for Invention]

The present invention will be better understood from the following examples. These examples are not to be construed as limiting the scope of the invention.

EXAMPLES Example 1 : Preparation of Freeze-dried Earthworm Powder

Of earthworms belonging to the Phylum Annelida, Subclass Oligochaeta, red worms (Lumbricus rubellus) , tiger worms (Eisenia fetida), large reddish worms (Lumbricus terrestris) , or red tiger worms (Eisenia andrei) were purchased from a farm breeding earthworms for feeds and fishing baits. 10 Kg of raw worms were thoroughly washed with distilled water to remove foreign materials and subjected to wet-grinding. The resulting worms were centrifuged at 3,000*g for 30 min to obtain a supernatant (8.5 kg). The supernatant was filtered

through diatomaceous silica to separate water-insoluble materials therefrom. The resulting residue was freeze-dried to prepare a freeze-dried earthworm powder. The weight of the freeze-dried earthworm powder obtained from the raw earthworms (10 Kg) was 1.2 Kg and a titer of a casein digestion was 870 units /g.

Example 2: Effects of Stomach Ulcer Protection on Stomach Ulcer Model Induced by Stress 6 week-old white Wistar male rats were used as subjects. Stomach ulcers were induced by a Water-Immersion Restraint Stress Model in which white rats are immersed in a bath. This model is to induce rats to have acute stomach ulcers by forcing the rats to be immersed in a stress cage containing water they hate for 6 hours. A test drug (Stillene ^® 60 mg/kg or freeze-dried earthworm powder 50 mg/kg) was administered to the rats at 30 minutes before water-immersion. The temperature of the bath was maintained at 21 ^° C to 23 ^° C. After completion of the immersion, the stomach of each specimen was enucleated and the pylorus of the stomach was then ligated. Then, 3 mL of a 2% formalin solution was injected through the cardiac orifice and the specimen was fixed for 10 min. After completion of the fixation, the greater curvature of the stomach was excised

and washed with physiological saline. The resulting greater curvature was thoroughly spread, fixed on a fixing plate and taken as a photograph. The area of injured site in the photograph was measured with a bio-imaging analyzer.

The results were shown in Table 1. An experimental group, to which 50 mg/kg of the freeze-dried earthworm powder was administered, exhibited stomach ulcer inhibition of 50.2%, as compared to a negative control group, to which no drug was administered. A positive control group, to which Stillene was administered, exhibited stomach ulcer inhibition of 18.1%.

Table 1: Effects of Freeze-dried Earthworm Powder on Stomach Ulcers Induced by Water-Immersion Restraint Stress

Stillene ^® (Positive control drug): available from

Dong-a pharmaceutical Co. , Ltd.

Stomach ulcer area is represented by "average ± standard error".

" * " represents statistical significance at 95% confidence level, in contrast with control groups.

Example 3: Effects of Stomach Ulcer Protection on Stomach Ulcer Model Induced by Drug

6 week-old white Wistar male rats were used as subjects. A stomach ulcer-inducing drug used herein was indomethacin, one of non-steroidal anti- inflammatory drugs. The indomethacin was reported to inhibit activity of cyclooxygenase that participates in prostacyclin synthesis in gastric cells, causing gastric injury. A test drug (Stillene ^* 60 mg/kg or freeze-dried earthworm powder 50 mg/kg) was administered to the Wistar rats, which had been fasted during the overnight prior to testing. After 30 minutes, 20 mg/kg of indomethacin was orally-administered to the rats to induce stomach ulcers. At 6 hours following the stomach ulcer-inducing drug administration, the rats were sacrificed. The stomach of each specimen was enucleated and the pylorus of the stomach was then ligated. 3 mL of a 2% formalin solution was injected through the cardiac orifice and the specimen was fixed for 10 min. After completion of

the fixation, the greater curvature of the stomach was excised and washed with physiological saline. The resulting greater curvature was thoroughly spread, fixed on a fixing plate and taken as a photograph. The length of injured site in the photograph was measured with a bio-imaging analyzer.

The results were shown in Table 2. An experimental group, to which 50 mg/kg of the freeze-dried earthworm powder was administered, exhibited stomach ulcer inhibition of 46.8%, as compared to a negative control group, to which no drug was administered. A positive control group, to which Stillene was administered, exhibited stomach ulcer inhibition of about 13.2%.

Table 2: Effects of Freeze-dried Earthworm Powder on Stomach Ulcers Induced by Drug

Stillene ^® (Positive control drug) : available from Dong-a pharmaceutical Co. , Ltd.

Stomach ulcer area is represented by "average ± standard error". " * " represents statistical significance at 95% confidence level, in contrast with control groups.

Example 4: Stomach Ulcer Treatment Effects of Freeze-dried Earthworm Powder on Stomach Ulcer Model 6 week-old white Wistar male rats were used as subjects. Stomach ulcers were induced by a Water-Immersion Restraint Stress Model in the same manner as Example 2. Stomach ulcer-induction (water-immersion) was performed for three days, once per day, that is, three times in total. In the first day, after the rats were subjected to water- immersion for 3 hours, they were isolated from a bath and transferred into a breeding cage. At 3 hours following the water-immersion, a test drug (Omeprazole 10 mg/kg, Stillene ^® 60 mg/kg or freeze-dried earthworm powder 50 mg/kg) was orally-administered to the rats. In the second day, the water-immersion was carried out for 2 hours and the test drugs were administered to the rats in the same dosage as in the first day at one hour prior to water-immersion and at three hours after the completion of water-immersion. In the

third day, water-immersion and drug administration were carried out in the same manner as in the second day. At 3 hours following the drug administration, the rats were sacrificed and the stomach thereof was enucleated. The pylorus of the stomach was then ligated. Then, 3 mL of a 2% formalin solution was injected through the cardiac orifice and the specimen was fixed for 10 min. After completion of the fixation, the greater curvature of the stomach was excised and washed with physiological saline. The resulting greater curvature was thoroughly spread, fixed on a fixing plate and taken as a photograph. The area of injured site in the photograph was measured with a bio-imaging analyzer.

The results were shown in Table 3 and FIGs. 1 to 8. An experimental group, to which 50 mg/kg of the freeze-dried earthworm powder was administered, exhibited stomach ulcer inhibition of 72.2%, as compared to a negative control group, to which no drug was administered. One positive control group, to which Omeprazole was administered, exhibited stomach ulcer inhibition of 71.6%. Another positive control group, to which Stillene was administered, exhibited stomach ulcer inhibition of about 18%. These results indicate that the freeze-dried earthworm powder exhibits excellent inhibitory activity for stomach ulcers induced by water- immersion restraint stress, which is higher than that of

Stillene, and furthermore, comparable to that of Omeprazole.

Table 3: Stomach Ulcer Treatment Effects of Freeze-dried Earthworm Powder on Stomach Ulcers Induced by Water- Immersion Restraint Stress

Stillene ^® (Positive control drug) : available from Dong-a pharmaceutical Co., Ltd.

Omeprazole (Positive control drug): used in only Example 4, where a stomach ulcer treatment effect is tested, as ingredient of stomach ulcer drug available from Sigma Co., Ltd.

Stomach ulcer area is represented by "average ± standard error".

^w * ^Ii represents statistical significance at 95% confidence level, in contrast with control groups.

Example 5 : Tests with Earthworm-extracted Supernatant The tests were performed in the same manner as in Examples 2 to 4 , except that 8.5 kg of the earthworm- extracted supernatant prepared in Example 1 was used instead of the freeze-dried earthworm powder. Since the earthworm- extracted supernatant has a weight, about 7-fold higher than that of the freeze-dried earthworm powder, it was used in an amount corresponding to 7-fold of the weight of the freeze- dried earthworm powder used in Examples 2 to 4.

The results obtained from this Example were substantially the same as in Examples 2 to 4.

[Industrial Applicability]

As apparent from the foregoing, the anti-stomach ulcer composition of the present invention is efficacious in preventing and treating stomach ulcers, thus being industrially applicable.

^w * " represents statistical significance at 95% confidence level, in contrast with control groups.

Example 5 : Tests with Earthworm-extracted Supernatant The tests were performed in the same manner as in Examples 2 to 4 , except that 8.5 kg of the earthworm- extracted supernatant prepared in Example 1 was used instead of the freeze-dried earthworm powder. Since the earthworm- extracted supernatant has a weight, about 7-fold higher than that of the freeze-dried earthworm powder, it was used in an amount corresponding to 7-fold of the weight of the freeze- dried earthworm powder used in Examples 2 to 4.

The results obtained from this Example were substantially the same as in Examples 2 to 4.

[Industrial Applicability]

As apparent from the foregoing, the anti-stomach ulcer composition of the present invention is efficacious in preventing and treating stomach ulcers, thus being industrially applicable.

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