KR100343923B1 - Composition Comprising Xanthium strumarium L. Extract for Use in Preventing and Treating Cataract and Method for Preparation Thereof - Google Patents

Abstract

The present invention relates to a Changiza extract, which is dried and pulverized Changija, and then the fat is removed with an organic solvent, and the filtrate is extracted with water. The extract of the present invention is obtained by filtration of the water extract, coagulation of the protein to remove it, and then addition to an organic solvent to remove the material soluble in the organic solvent. Changja extract according to the present invention is effective in the prevention and treatment of cataracts.

Description

Composition Comprising Xanthium strumarium L. Extract for Use in Preventing and Treating Cataract and Method for Preparation Thereof}

The present invention relates to a material that exhibits the effect of preventing and treating cataracts using the extract of Chang-za (Sanko). More specifically, the present invention relates to a pharmaceutical composition and a method for producing Changja extracts that inhibit the development of cataracts and exhibit a therapeutic effect of cataracts.

Since vision is one of the most important parts of the human sense, cataracts, which are common in old age, have a great effect on quality of life, among other eye diseases. The incidence rate of cataracts is rapidly increasing as the age of 60, and the progression and turbidity of remarkably increases, the most common cause of blindness, the age of cataracts is increasing year by year. For example, about 1.5 million cataract surgeries are performed annually in the United States, and about 10 to 150,000 cataract surgeries are performed in Korea. The age distribution of cataracts is 4.16% between 40-49 years of age, 13.33% between 50-59 years, and 60-years. 37.5% were 69 years old and 45% were over 70 years old. In terms of cost, it is estimated that about 1 trillion won of medical expenses are consumed every year for a single disease called cataract worldwide, and about 50 billion won is spent annually in Korea.

The causes of cataracts are very complex, and systemic diseases such as diabetes mellitus and hyperparathyroidism have been reported to promote cataract progression, but cataracts in adults without systemic diseases are more difficult to determine. In this case, it is reported that many factors such as hormonal imbalance such as ultraviolet rays, heat, estrogen, and the relationship with smoking, but it is difficult to prove the effects of these factors.

During cataract formation, the theory that ultraviolet irradiation causes oxidation reactions and changes the properties of the proteins and enzyme proteins that form the structure of the lens causes lens turbidity, and the total time of exposure to ultraviolet light is cortical cataract and late development. Reports that correlate with the development of cataracts or the development of nuclear cataracts support the fact that ultraviolet light damages the lens and causes cataracts.

However, cataract is not only an old age disease in a large number of the elderly population, but also a rare disease in young people these days, it is one of the very important disease in that the frequency is gradually increasing. Cataracts are not a disease that directly affects life, but they are of increasing importance in developed countries entering the old age society because they have a great impact on quality of life.

Currently, the only method for treating cataracts is to replace the existing lens with an intraocular lens. There is no medicament for treating cataracts, and antioxidants are known to prevent cataracts, but they are not developed as drugs due to toxicity.

Accordingly, it is an object of the present invention to provide a composition that is effective in the prevention and treatment of cataracts.

Another object of the present invention is to provide a composition for preventing and treating cataract, which is substantially free of drug toxicity.

Another object of the present invention to provide a composition for the prevention and treatment of cataracts that can be administered orally.

Still another object of the present invention is to provide a composition for the prevention and treatment of cataracts which can be transdermally administered by instillation.

Another object of the present invention is to provide a method for preparing a composition that is effective in the prevention and treatment of cataracts.

1 is a graph showing the toxicity of hydrogen peroxide on human lens epithelial cells,

Figure 2 is a graph showing the cytotoxicity of Changchang extract against human lens epithelial cells,

3 is a graph showing the effect of inhibiting the toxicity of hydrogen peroxide that is a cataract-inducing substance of the extract Changsha,

4 is a graph showing the cataract inhibitory effect of the ethyl acetate layer of the extract Changchang,

5 is a graph showing the cataract inhibitory effect of the chloroform layer in the extract of Changchang,

6 is a UV search of the present inventors Changja extract,

Fig. 7 is a HPLC analysis diagram of Changza extract.

In accordance with the present invention, there is provided a Changsha extract effective for the prevention and treatment of cataracts. In addition, according to the present invention, a) adding an organic solvent to the powder of the dried Changja pulverized to dissolve and remove the fat powder; b) removing the organic solvent and extracting the remaining powder with water and filtering the extract; c) saturating the filtrate under high pressure to remove the resulting coagulation protein; d) adding an organic solvent to the filtrate to remove the material soluble in the organic solvent; And e) separating the aqueous layer to provide lyophilization.

Changsha is a fruit of the Asteraceae (Xanthium strumarium L.) or large tail, which is a yearly grass that grows widely in the fields of our country. The components of the outpost include lactones xanthin, carotenoids, alkaloids and saponins, and fruit changja contains xanthostrumarin (yellow glycosides), resins, and iodine salts, and seeds contain resins and 40% oil (linoleic acid 63.4). %, Oleic acid 27%, saturated fatty acid 8.2%), and lizard is one of the plants with high iodine content.

Traditionally, outposts have been used when thyroid function is compromised due to their high iodine content. Dongbogam describes docomari as effective for diuretics, antipyretics, twitch, colds, headaches, rhinitis, sinusitis, and rheumatism. In the private sector, outposts have been used as a treatment for leprosy, as a sedative, or for stomatitis. It also used antiseptic plasters and antiseptic plasters with fruit and starch, applied to eczema, viral diseases, boils, smallpox, ulcerative skin diseases, and used as an antidote when bitten by snakes or insects. It is also reported that it is used as an analgesic agent for malignant tumors, and it is known that the concentrated liquid boiled in water for chronic rhinitis, arthritis, neuralgia.

The present inventors have experimentally confirmed that Changja extract inhibits the development of cataracts and shows excellent therapeutic effects against cataracts that have already occurred, and through the acute toxicity test, the present inventors have found that Changchang extract is a very safe drug for the human body. It was.

In the production method of the present invention, first, the Changjaja is dried and pulverized to form a powder. An organic solvent, preferably chloroform, is added thereto to dissolve and remove the fat.

Subsequently, the chloroform is removed and the remaining powder is extracted with water and filtered. The filtrate is saturated under high pressure to remove the coagulated protein, and then an organic solvent is added to the filtrate to remove soluble substances in the organic solvent.

Finally, the aqueous layer is separated and dried to obtain an extract of the present invention. At this time, as a drying method, freeze drying is preferable.

The administration of Changiza extract may be administered by subcutaneous, oral, intramuscular administration, eye drops and the like in a therapeutically effective amount alone or in a form containing a pharmacologically acceptable carrier, excipient or diluent. Formulations for oral administration suitable for the substance of the present invention are in the form of tablets, capsules, solutions, suspensions, syrups, or beverages. Agents comprising Changja extracts for oral administration may be in the form of sterile injectables, such as sterile, injectable liquids or oily suspensions. The suspension is prepared using suitable dispersing or wetting agents and suspending agents.

The sterile injectables can also be sterile injectable solutions or suspensions containing an orally acceptable diluent or solvent, such as 1,3-butanediol. Suitable diluents include, for example, water, Ringer's solution, and isotonic saline. In addition, sterile, fixed oils can be readily used as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid can likewise be used in injections.

The dosage of the mixed extract according to the present invention may vary depending on the severity of the disease, the sex, age, weight, and desired effect of the subject to be administered. Generally, in the case of oral administration to an adult, a dose of 5-50 mg, preferably 10-40 mg, per kg of body weight per day may be administered, and in the case of administration by injection orally, a person weighing 60 kg may be administered. A dose of 5-50 mg can be administered as a reference.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are provided to illustrate the present invention, and the scope of the present invention is not limited to these examples.

<Example 1>

Formulations Containing Changi Ja Extract

1. Tablet

100 mg of Changzai extract of the present invention, 120 mg of lactose for excipients and 35 mg of Avicel (microcrystalline cellulose), 15 mg of sodium starch glyconate as a disintegration aid, and 80 mg of L-HPC (Low-hydroxyprophylcell ulose) for binders. The mixture was mixed in a U-type mixer and mixed for 20 minutes. After completion of the mixing, 10 mg of the suspending agent magnesium stearate was further added and mixed for 3 minutes. Tablets containing 100 mg of extract per tablet were prepared by tableting and film coating after a quantitative test and a humidity test.

2. Syrup

An appropriate amount of sucrose was dissolved in a certain amount of water, and 80 mg of paraoxymethylbenzoate and 16 mg of paraoxypropylbenzoate were added thereto as a preservative, 1.8 g of the extract of Changjaja of the present invention was added thereto, and dissolved completely while maintaining at 60 ° C. After cooling, distilled water was added to make 150 ml to prepare a syrup.

3. Capsule

A capsule was prepared by mixing 150 mg of Changisa extract of the present invention with 200 mg of lactose as a carrier and filling the hard gelatin capsule.

4. Drink

After dissolving 200 mg of Changiza extract of the present invention in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, and high fructose as an auxiliary component were added, and an appropriate amount of sodium benzoate was added as a preservative, followed by adding water to the whole amount. 100 ml was prepared to prepare a beverage composition.

5. Injection

200 mg of Changiza extract of the present invention was dissolved in 200 ml of physiological saline containing 1% polyoxyethylene hydrogenated castro oil to prepare an injection containing the mixed extract at a concentration of 0.1%.

<Example 2>

Toxicity test

In order to confirm the safety of the extracts of the present invention, LD ₅₀ (amount capable of killing 50% of experimental animals) was obtained by the following method. .

Thirty normal ICR mice (male, 22 ± 1 g) were divided into five groups from A to E, each six in one group, and from group A administered 5 g of Changsha extract of the present invention per kg of body weight. Equivalently, 7.5 g in group B, 10 g in group C, 12.5 g in group D, and 15 g in group E were administered orally and the Behrens-Kaber method (Reference: 高木 敬 次 郞 外:藥物實驗,南山堂, Japan , p131, 1960) by administering an extract changyija oral (po) of the present invention to measure the LD ₅₀ values. The results are shown in Table 1 below.

Lethal dose by oral administration of Changjia extract of the present invention (LD ₅₀ ) Experimental group Capacity (g / kg) Oral administration group (p.o.) * Z ** d Number of dead animals A 5 0/6 - - B 7.5 0/6 0 2.5 C 10 0/6 0 2.5 D 12.5 0/6 0 2.5 E 15 0/6 0 2.5

* Z: ½ of the number of dead animals in two consecutive doses

** d: difference between two consecutive doses

As shown in the results shown in Table 1, the LD ₅₀ value of oral administration of the Changzai extract of the present invention was 15 g / kg or more, and thus it was found to be a very safe substance with little toxicity. That is, it can be clearly seen that the extract of the present invention can be safely administered with little toxicity.

In addition, autopsies and histopathological examinations were performed on the test animals used to measure the LD ₅₀ value in the following manner. At the end of the test, all surviving animals were bled to death after ether anesthesia, and the organs were extracted and visually examined for all organ abnormalities. Autopsied whole organs were fixed in 10% neutral formalin solution for more than 10 days for pathological examination, and then dehydrated and embedded in a paraffin embedding machine (Fisher, Histomatic Tissue Processor, 166A), followed by a rotary microtome (AO Rotary Microtome). 5 μm sections were prepared and observed by hematoxylin and eosin staining.

All animals in each group were dissected, stained with H & E and histopathologically examined under the microscope. Abnormal findings due to drug administration in liver tissue, kidney, heart cardiomyocytes, gastrointestinal tract, pancreas, lung, spleen, adrenal gland, brain, testes, ovaries, bone marrow, etc. Was not observed.

Accordingly, the present invention Changchang extract was found to be a safe drug that does not cause acute toxicity to all organs, even when administered 15 g per 1 kg body weight of the highest dose, and does not cause toxicity such as any organ damage. .

<Production example>

Changiza was dried and ground to give 600 g of powder. 1 L of an organic solvent, preferably chloroform, was added thereto in sufficient amount to dissolve and remove the fat component. Subsequently, the remaining powder was removed, and the remaining powder was extracted with 8 L of water at a high temperature of 100 ° C. or higher and filtered. To this was added 8 L of water, extracted at a high temperature of 100 ° C. or higher, and filtered.

The filtrate was saturated under high pressure to remove the coagulated protein, and the filtrate was then removed twice with chloroform and twice with hexane. The aqueous layer was heated to remove the organic solvent contained in the aqueous layer, the remaining aqueous layer was concentrated, and the concentrated solution was lyophilized to obtain Changja extract 58 of the present invention.

Experimental Example 1 Inhibition Effect of Cataract Extract of Cataract

(1) Determination of the concentration of hydrogen peroxide, a cataract trigger

B3 cell line, a human epithelial cell line, is dispensed at 100,000 wells / well in 24 wells using a medium containing 20% serum, incubated for 26 hours, and then changed to a medium without serum. Hydrogen peroxide was treated with 20, 30, 40, 50, 100, 500, and 1000 μM concentrations to examine the toxicity of hydrogen peroxide against B3 cell lines, and the cells were incubated at 37 ° C. for 24 hours and then measured by MTT method. The MTT method was performed as follows.

MTT (3 (4,5-dimethylathiazol-2,5-diphenyl tetrazolium bromide) is added to about 1/10 of the total medium amount and incubated at 37 ° C. for 1 hour and 30 minutes, and then the solution is removed and a cell lysis solution such as isopropanol ( Lysing solution was added, the solution was transferred to 96 wells, and the value was measured at 570 nm with an ELISA reader, and as a result, the toxicity of hydrogen peroxide on B3 cells was as follows. At 30 μM, 40 μM, and 50 μM, there was a slight change in survival rate, and at 100 μM the survival rate was reduced by more than 50% (see FIG. 1).

Based on these results, it was decided to treat the cells with a concentration of hydrogen peroxide at a concentration of 100 μM, indicating a 50% reduction in viability.

(2) Investigation of Cytotoxicity of Changi Ja Extract

B3 cells (human lens epithelial cells) were cultured for 26 hours after dispensing 100,000 cells per well into 24 wells using a medium containing 20% serum to investigate the toxicity of the extract. After 26 hours, the medium was changed to a medium containing 1% serum, followed by incubation for 20 hours, and then Changchang extract was treated at various concentrations without serum. The concentration of non-toxic to Changiza extract was determined, and the concentration was subdivided to examine the viability of cells by the MTT method as described above (see FIG. 2).

(3) Inhibition Effect of Cataract Extract of Cataract

The following experiments were conducted to determine the effect of Changjaza extracts on inhibiting the toxicity of hydrogen peroxide, a cataract trigger.

The B3 cell line was dispensed at 100,000 wells / well in 24 wells using a medium containing 20% serum, incubated for 26 hours, and then changed to a medium containing 1% serum, and then incubated at 37 ° C. for 20 hours. After 20 hours, change the medium to serum-free medium, and examine the cytotoxicity of the extract, and then incubate the extract for 3 hours and 30 minutes at a concentration within the range that does not affect the cells. After treatment for 24hr and then the effect of cataract inhibition of each material was measured by MTT method. As a positive control, a catalyst having a hydrogen peroxide decomposition effect was used. As a result, Changja extract showed an effect of blocking the effect of hydrogen peroxide 12% when administered 500 ng (see Fig. 3). Therefore, Changzai extract of the present invention is recognized as an effective drug capable of preventing and treating cataracts.

(4) Cataract Inhibitory Effects of Fractions According to Solvent Extraction of Changi Ja Extract

Changjaza extract was divided into chloroform, ethyl acetate, butanol and water layers to measure the effect of cataract inhibition using the same method as in (3) above. As a result, the ethyl acetate layer in the extract showed 21% cataract suppression at 100 ng / ml (see Figure 4) and the chloroform layer in the extract showed 17% inhibition at 100 ng / ml. See FIG. 5). The butanol layer and the water layer were found to be ineffective.

Experimental Example 2 Characteristics of Extracts of the Invention According to UV Absorbance

Changja extract of the present invention was investigated using an ultraviolet spectrophotometer (Spectrophotometer; Pharmacia, Ultrospec 2000) and HPLC (High Performance Liquid Chromatography; HP1090). FIG. 5 shows the results of UV absorbance search of the extract of Changchang (1 mg / ml), where the absorbance was increased at 287 and 318 nm (FIG. 6). This indicates that the extract of Changchang has an effect on absorption of ultraviolet rays.

Experimental Example 3 Properties of Chang-Ja Extract of the Present Invention According to HPLC Analysis

Changzai extract of the present invention was dissolved in water at a concentration of 1 mg / ml for detection by HPLC. 10 μL of the dissolved extract solution was taken and analyzed using HPLC (HP1090).

(Analysis condition)

Column: C18 ODS; 4.6 mm i.d. x 100 mm L

Mobile phase: 1% acetic acid and methanol gradient system

Flow rate: 1 ml / min

Detection: measured at 254 nm with photodioid array detector

In repeated experiments, characteristic peaks were observed in the extract. Fig. 7 shows that the Changzai extract as the HPLC profile of the extract was contained 0.14% (RT = 10.932) and 0.25% (RT = 18.739) of 3,4-dihydroxybenzoic acid under the above analysis conditions. . In addition, characteristic peaks were observed at 4.408, 16.904, 17.432, 19.610, and 27.603.

The present invention is a herbal extract for the prevention and treatment of cataracts using Changza extract, less side effects, long-term administration and administration is possible. In addition, the present invention does not lead to dependencies and resistance.

The present invention is to prevent or treat cataracts, for example, it can prevent and treat diabetic cataracts, senile cataracts, cataracts caused by ultraviolet light.

Claims (6)

A cataract prophylactic and therapeutic agent comprising a Changiza extract. A cataract prevention and treatment agent formulated in a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient or diluent to Changiza extract. The method of claim 2, Wherein said pharmaceutical unit dosage form is selected from the group consisting of tablets, capsules, solutions, suspensions, syrups, beverages, and injections. The method of claim 2 or 3, A cataract prophylactic and therapeutic agent formulated to administer Changiza extract in an amount of 5 to 50 mg per kg of adult body weight per day. a) adding an organic solvent to the dried changja powder, dissolving and removing the fat powder; b) removing the organic solvent and extracting the remaining powder with water and filtering the extract; c) saturating the filtrate under high pressure to remove the resulting coagulation protein; d) adding an organic solvent to the filtrate to remove the material soluble in the organic solvent; And e) separating and drying the aqueous layer Changja extract manufacturing method comprising a. The method of claim 5, The organic solvent is chloroform or hexane.