WO2008126979A1

WO2008126979A1 - Pharmaceutical composition for the prevention and treatment of the ischemia-reperfusion injury in the flaps or tissues containing anthocyanin extracted from the black soybean seed coat

Info

Publication number: WO2008126979A1
Application number: PCT/KR2008/000509
Authority: WO
Inventors: Tae Hyun Choi; Ki Churl Chang; Sung Chul Shin; Jong Il Chung; Hye Jung Kim; Jun Sik Kim
Original assignee: Industry-Academic Cooperation Foundation Gyeongsang National University
Priority date: 2007-04-11
Filing date: 2008-01-28
Publication date: 2008-10-23
Also published as: KR20080092060A; KR100880876B1

Abstract

The present invention relates to pharmaceutical compositions for the prevention and treatment of the ischemia-reperfusion injury in the flaps or tissues, containing anthocyanin as an active ingredient, which is extracted from black soybeans, especially black soybean seed coats. More specifically, the anthocyanin is composed of cyanidin-3-glucoside, delphinidin-3-glucoside, and petunidin-3-glucoside.

Description

Pharmaceutical composition for the prevention and treatment of the ischemia-reperfusion injury in the flaps or tissues containing anthocyanin extracted from the black soybean seed coat

[Technical Field]

The present invention relates to pharmaceutical compositions for the prevention and treatment of the ischemia- reperfusion injury in the flaps or tissues, containing anthocyanin as an active ingredient, which is extracted from black soybeans, especially black soybean seed coats. More specifically, the anthocyanin is composed of cyanidin-3- glucoside, delphinidin-3-glucoside, and petunidin-3-glucoside .

[Background Art]

Anthocyanin is a water-soluble pigment glucoside, which exists in flowers, fruits, stems, leaves, and roots of plants.

It is usually found in vacuoles of plants and it is a natural dye representing colors such as violet, red, and blue according to the acidity of cell sap, the chemical structure of dye compounds, and binding status with other metal ions.

Anthocyanin is composed of aglycone and glycoside.

Currently the anthocyanin component is extracted from such plants as grapes, strawberries, olives, red cabbages, eggplants, roses, etc. and used as various materials. Yet, in fact, it is extracted and used almost as a co-pigment and often used with the compounds of other ingredients.

Anthocyanin, a natural vegetable dye, has recently been known to have some bioactivities . The well-known examples are anti-aging and antibacterial effect, anti-mutagenicity, cholesterol decrease, eyesight improvement, blood vessel protection, anti-ulcerative, and antioxidant effect. These bioactivities are basic for every kind of anthocyanin, but it was recently found that each anthocyanin has slightly different bioactivities.

It is known that anthocyanin is easily absorbed in intestines and it was also reported that its powerful antioxidant effect showed the protection ability for cells and tissues in animal models, in case of the ischemic infarction in the liver and heart and the acute inflammation in the lung (Takanori Tsuda et al., Archives of Biochemistry and Biophysics, 1999, 368 (2) : 361-366; Amorini A.M. et al, Free Radic. Res. 2003, 37 (4) : 453-460) . Also, in Korean Laid-Open Patent Publication No. 2007-0026284, it is disclosed as a therapeutic agent that can protect the brain cell injury resulted from the stroke due to the complexity of the mechanism of brain injury. The ischemia-reperfusion injury (IRI) occurs when the blood flow to a body part temporarily stops (ischemia) and then resumes (reperfusion) . It is presumed that the main mediators of the IRI are free radicals and neutrophils [Askar, I., Oktay, M. F., Gurlek, A., and Bac, B. Protective Effects of Some Antineoplastic Agents on Ischemia-Reperfusion Injury in Epigastric Island Skin Flaps. Microsurgery. 26: 193, 2006] . Also, polymorphonuclear neutrophils interact with vascular endothelial cells and cause the leukocyte adhesion and release of various inflammatory mediators during reperfusion [Tosa, Y., Lee, W. P. A., Kollias, N., Randolph, M. A., and May, J. W. Monoclonal Antibody to Intercellular Adhesion molecule 1 Protects Skin Flaps Against Ischemia-Reperfusion Injury: An Experimental Study in Rats. Plast. Reconstr. Surg. 101: 1586, 1998].

The IRI in the field of reconstructive surgery often occurs during the microsurgical replantation operation (Figure 3) for patients whose hands or other body parts (ears, tongue, nose, etc.) are cut, or the flap operation (Figure 4) to reconstruct tissue defects due to various reasons including traumas or cancers. Thus, there are a lot of researches underway on the mechanism of the IRI in flaps or tissues.

Also, lots of agents have been used for the experiment of skin flap models to decrease the IRI. The examples include Trimetazidine (antioxidants) , anticancer agents, FK506 (immunosuppressants), Abciximab (platelet glycoprotein Ilb/IIIa receptor antagonist) , and monoclonal antibodies against ICAM-I. However, when these drugs are used for the IRI of skin flaps, side effects may occur and they are not economical since they are very pricy. Accordingly, those drugs are now used only for research. Therefore, there is an increasing need for the development of agents that are easy to find, inexpensive, and relatively safe.

On the other hand, there have been many studies on black soybeans since the Food and Drug Administration approved that the soy protein helps prevent the coronary heart disease in 1998. It is known that black soybeans contain essential fatty acids, lecithin, fiber, saponin, etc., so that they have the effect to prevent and treat adult diseases such as hypertension, diabetes, arteriosclerosis, and obesity. The vegetable fiber contained in black soybeans is known to have the effect to prevent not only constipation but also colon cancer. Donguibogam, the Korean medical book written in the 17^th century, describes black soybeans as "a drug that protects the five viscera, keeps intestines warm by helping twelve meridians, gets rid of boils, and treats radiating pain". Also, anthocyanin, a pigment contained in black soybeans, has the anti-age effect in case of a constant use as antioxidants. Isoflavone, the soy protein, contains saponin and phytosterol as well as genistin that have powerful anticancer activities, and helps calcium absorption into bones. Particularly, the seed coats of black soybeans contain an anticancer material called glycitein.

Anthocyanin is a water-soluble pigment glucoside and largely divided into six kinds of anthocyanidins depending on the number of the hydroxyl groups and the methylation of specific hydroxyl groups. It is also divided into hundreds of kinds depending on the sort of sugar, which is bonded with anthocyanidins, and the sort and bonding location of the acyl group. Anthocyanin is currently getting a lot of attentions due to its various health promoting advantages such as potential antioxidant and anti-inflammatory effect and inhibition of chronic diseases.

[Disclosure of Invention] [Technical Subject]

While the present inventors were conducting the studies on the protection and treatment of the IRI in the flaps and tissues, remarkably it was found that anthocyanin extracted from black soybeans, especially from their seed coats was effective in protecting and treating the IRI in the flaps, and so the present invention was completed.

Therefore, the object of the present invention is to provide pharmaceutical compositions that are usable for the prevention and treatment of the ischemia-reperfusion injury in the flaps or tissues, containing anthocyanin extracted from black soybean seed coats.

[Technical Solution]

The present invention relates to pharmaceutical compositions for the prevention and treatment of the ischemia- reperfusion injury in the flaps or tissues, containing anthocyanin as an active ingredient, which is extracted from black soybeans, especially black soybean seed coats. More specifically, the anthocyanin is composed of cyanidin-3- glucoside, delphinidin-3-glucoside, and petunidin-3-glucoside.

The purified anthocyanin of the present invention is composed of 65~80 weight% of cyanidin-3-glucoside, 15~25 weight% of delphinidin-3-glucoside, and 5~10 weight% of petunidin-3-glucoside .

The pharmaceutical compositions containing the pharmaceutically effective amount of purified anthocyanin, as an active ingredient of the present invention, which is for the prevention and treatment of the IRI in the flaps or tissues, can be formulated into various forms along with pharmaceutically permitted media. The formulation can be prepared according to the methods notified in the field.

The pharmaceutical compositions for the prevention and treatment of the IRI in the flaps or tissues can be formulated into various oral or parenteral medication types. For oral medications, the examples are tablets, pills, hard and soft capsules, liquids, suspending agents, emulsifiers, syrups, granules, etc. These formulations may contain, other than active ingredients, diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and/or glycine) and lubricants (e.g. silica, talc, stearic acid and its magnesium or calcium salt, and/or polyethylene glycol) . Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidine. In some cases, they can contain tablet disintegrants such starch, agar, alginic acid, or its sodium salt, or boiling mixture and/or absorbents, colorants, flavor enhancers, and sweetening agents. Those formulations can be prepared by the usual methods such as mixing, granulating, or coating. Also, the typical formulation for parenteral administration is injections using water, Ringer's solution, isotonic physiological saline solution, or suspending solution. Sterile fixed oils can be used as a solvent or suspended medium. Any bland fixed oils including mono-, di-glyceride can be used for this purpose and fatty acids such as oleic acid may be used for injectable preparations.

Moreover, aqueous or oily ointments, antioxidants, preservatives, bulking agents etc. can be employed to make external preparations such as ointment, cream, gel, and lotion. They can be also formulated into transdermal patches composed of protective foils (release liners) and matrix that contain supporting layers or film materials, active ingredients, media, and special adhesives to adhere to skins. The dosage of anthocyanin of the present invention depends on the patients' age, gender, weight and severity of illness, but for the present invention, it is administrated 0.1 mg ~ 1 g per 1 kg of patients' weight, once to four times a day.

[Brief Description of Drawings]

Figure 1 shows the picture of the section measurement of the survived flaps after the IRI in skin flaps of rats.

Figure 2 shows the picture of flaps from the control group, the experimental group A and the experimental group B after seven days of the IRI in Experimental Example 1.

Figure 3 shows the picture of the microsurgical replantation surgery for a cut finger and another one after surgery.

Figure 4 shows the picture of the free flap operation to reconstruct the necrotic skin and tissue in the foot and another one after surgery.

[Best Mode for Carrying Out the Invention]

Hereinafter, the present invention is described in more detail based on the following examples. But, these examples are not intended to limit the scope of the present invention.

[Example 1] Extraction and Purification of Anthocyanin

Using 200 g of black soybean seed coats (Glycine max (L) Merr.) cropped in the farmland of Gyeongsang National University at Jinju, it was extracted by methanol at 4 ^°C for 24 hours, which was repeated three times. The extraction was concentrated under reduced pressure and diluted until its volume reached 200ml. The solution containing anthocyanin was concentrated to be 100 ml and purified using Amberlite XAD-7 column and Spephadex LH-20. The purified anthocyanin was then composed of 72% of cyanidin-3-glucoside, 20% of cyanidin-3- glucoside, and 6% of petunidin-3-glucoside.

[Experimental Example 1] The Protective Effect on the IRI in Epigastric Flaps after Anthocyanin Treatment To find out the protective effect of anthocyanin on the IRI, sodium pentobarbital of 45mg/kg was injected into the coeliac of thirty rats (Spaugue-Dawley rat, 25Og) for anesthesia. The rats were divided into three groups and given anthocyanin in Example 1 or water twelve hours before the IRI. The control group: water (0.25ml/250g) was given

The experimental group A: anthocyanin (50mg/kg) was given The experimental group B: anthocyanin (100mg/kg) was given

The chest and abdominal hair of the rats were eliminated from the three groups (ten rats for each) and the flaps of 3x5 cm were designed on the right side. Except for inferior epigastric vessels underneath, the flaps were completely aroused. Then femoral vessels were well skeletonized at proximal and distal part of inferior epigastric vessels. The femoral vessel in the distal part was ligated with 6-0 nylon and that in the proximal part was temporarily ligated with microvascular Acland 2V clamp. After flaps were replaced into the original place, they were fixed with 3-0 nylon. After intramusculer injection of antibiotics, flaps were covered with transparent vinyl to keep rats from hurting their own flaps. After ten hours, rats were anesthetized again and the clamps were eliminated. Then the flaps were fully stitched where they were. After seven days of operation, the rats were killed to measure the survived part of the flaps and the section of necrosis. The section of the survived flaps was shown with percentage, considering the flaps before surgery as 100. The result is shown in Figure 1. Figure 1 shows that the experimental group A and B, which were given anthocyanin twelve hours before the operation, had increased section of survived flaps up to 74.5% and 83%, compared to the control group (62%) . Thus, it was found that the more concentration of anthocyanin, the more sections of survived flaps.

[industrial Applicability]

The anthocyanin extracted from black soybeans, especially black soybean seed coats, which is composed of 65~80 weight% of cyanidin-3-glucoside, 15~25 weight% of delphinidin-3- glucoside, and 5~10 weight% of petunidin-3-glucoside, has the antioxidant and anti-inflammatory property and shows great effect on improving the survival of flaps or tissues from the ischemia-reperfusion injury, so it can be used as an excellent agent to prevent and treat the ischemia-reperfusion injury.

Claims

[CLAIMS]

[Claim l]

Pharmaceutical compositions for the prevention and treatment of the ischemia-reperfusion injury in the flaps or tissues, containing anthocyanin as an active ingredient, which is extracted from black soybeans.

[Claim 2]

The pharmaceutical compositions for the prevention and treatment of the ischemia-reperfusion injury in the flaps or tissues of claim 1, wherein said anthocyanin is extracted from black soybean seed coats.

[Claim 3] The pharmaceutical compositions for the prevention and treatment of the ischemia-reperfusion injury in the flaps or tissues of claim 2, wherein said anthocyanin is composed of cyanidin-3-glucoside, delphinidin-3-glucoside, and petunidin- 3-glucoside .

[Claim 4]

The pharmaceutical compositions for the prevention and treatment of the ischemia-reperfusion injury in the flaps or tissues of claim 3, wherein said anthocyanin is composed of 65-80 weight% of cyanidin-3-glucoside, 15-25 weight% of delphinidin-3-glucoside, and 5-10 weight% of petunidin-3- glucoside .