KR20050080881A - New compound extrated from the seed of prunus persica and a composition of anti-inflamatory drug containing it - Google Patents

Abstract

The present invention relates to a compound M-2 and a pharmaceutical composition for treating inflammation containing the same. The M-2 is a novel compound extracted from Doin (Peach Seed), and is represented by the following Chemical Formula 1.

[Formula 1]

Inflammatory therapeutic agents containing M-2, which is a compound represented by Formula 1, have fewer side effects as a composition derived from nature.

Description

New compound extrated from the seed of Prunus persica and a composition of anti-inflamatory drug containing it}

The seeds of Prunus persica (L.) BATSCH (Rosaceae) are well known in China, Korea, and other Asian countries as the traditional folk medicine (Persicae Semen; Tounin, Taoren, China). They were frequently used as ingredients in various Chinese medicine prescriptions, especially in treating women's diseases. The components of this plant include, as well as glycerides, sterols and emulsin, cyanogenic glycosides, amygdalin and purunasin as main components. Amigdalin is also abundant in seeds of bitter almonds, apricots of the Prunus genus, and other rosaceae. Recently, glycosides extracted from these plant seeds have been reported to have anticancer activity that modulates the activity of Epstein-Bar virus initially infected with lymphocyte blast cells. However, it is not known what chemical composition of peach seeds has an anti-inflammatory effect.

On the other hand, NO, which is overproduced by inducible nitric oxide synthase (iNOS), is associated with a variety of inflammation-related diseases, and effective iNOS blockers are valuable as therapeutic agents. Macrophage acts when a living body becomes infected with a pathogen or when the immune system is hyperactive for some reason. Macrophages play an important role in defending the host against pathogens with the help of active agents of T-cells, which are pathogens and immune cells, and this activity is regulated by several intermediates. In particular, the production of nitric oxide (NO) by macrophages acts as an important mediator of killing or inhibiting growth of bacteria, fungi and parasites. Nitrogen monoxide (NO), a very unstable gas, is known as an important macrophage mediator that kills pathogens and tumor cells in the human body or regulates the immune system. [Brightbill HD, et al., Science, 285, p732-736, 1999 ].

However, when a large amount of nitrogen monoxide is produced, there are side effects that kill host cells and cause inflammation. If macrophages continue to overproduce nitric oxide even after the death of pathogens, they cause shock or severe inflammatory disease after sepsis and cause autoimmune diseases [Hobbs A.J., et al., Annu. Rev. Pharmacol. Toxicol., 39, p 191-220, 1999]. In other words, the lack of production of nitrogen monoxide is a problem in the role of the mediator of macrophages, but when produced in excess will cause inflammation or side effects. Therefore, the amount of nitrogen monoxide produced in iNOS needs to be properly adjusted.

Cyclooxygenase-2 (COX-2) is an enzyme induced and expressed by a variety of pro-inflammatory agents and cytokines. It has been found that the expression of COX-2 is abnormally increased in various cancers. It is reported that prostaglandin E2 (PGE ₂ ), produced by COX-2, is directly linked to the inflammatory response.

Current treatments for inflammation include dexamethasone and cortisone using corticosteroids. However, although they act as an inflammatory agent, they are highly toxic and can cause edema-like symptoms as a side effect. In addition, there is a case in which a problem occurs that causes severe immunosuppression due to inability to selectively act on the cause of inflammation [Check W.A and Kaliner M.A., Am. Rev. Respir. Dis., 141, p 44-51. 1990].

Since steroid drugs using corticosteroids, which are existing anti-inflammatory drugs, have severe side effects such as edema, there is an urgent need for nonsteroidal drugs with no side effects.

In recent years, interest in herbal medicines is increasing in view of in vivo safety and the like. Therefore, a lot of researches on the therapeutic agent with the active ingredient extracted from the plant, and it is known that the herbal medicine contains the active ingredient that we do not recognize. Therefore, extracting the effective pharmaceutical composition from the herbal medicine is also emerging as a new research object in modern pharmacy [Makino T., et al., Biol. Pharm. Bull., 22, p 476-480, 1999].

In view of this, the inventors have sought to develop effective inflammatory therapeutic compositions, selective nitric monoxide inhibitors and inhibitors of prostaglandin E2 with fewer side effects. In addition, in order to solve the problems of the existing anti-inflammatory drugs, the present inventors have searched for nitrogen monoxide and prostagladin E2 production inhibitors that cause inflammation in herbal ingredients, and revealed the mechanism of production inhibition to develop nonsteroidal anti-inflammatory drugs. Efforts were focused on herbal medicines.

As a result, the present inventors found that the extract of the causality has an anti-inflammatory effect, and while searching for the causative agent, the inventors found that a novel substance designated by the following Chemical Formula 1 (hereinafter referred to as M-2) has an anti-inflammatory effect. The present invention has been completed.

[Formula 1]

Accordingly, it is an object of the present invention to provide a novel substance, M-2.

In addition, it is an object of the present invention to provide a composition for treating inflammation comprising the M-2.

The present invention provides M-2 represented by the following Chemical Formula 1.

The following are the NMR results for M-2.

Compound M-2 of Chemical Formula 1 was extracted and purified according to the method and sequence described in FIG.

The dosage of M-2 depends on the patient's condition, weight, age, etc., but a person of ordinary skill in the art can easily predict the dosage by referring to M-2 activity.

The pharmaceutical composition may be prepared in various forms, namely, creams, ointments, injections, granules, and external skin preparations, depending on the mode of administration. Stabilizers, emulsifiers, buffers, salts, or small amounts of antibiotics that can inhibit further infection can be included to make a suitable drug. There are two methods of administration, such as administration by injection, oral administration of tablets, oral administration and the use of external skin preparations, depending on the condition of the patient and other circumstances.

Hereinafter, the invention will be described in more detail with reference to Examples. However, the following examples of the present invention are intended to embody the effects of M-2 according to the present invention and are not intended to limit the present invention to these examples.

Example 1 Extraction of M-2 from Doin

Doin was purchased in October 2003 from the Jeonbuk Medicinal Farming Association of Korea. Evidence specimens (no. PP777) were deposited in the herbarium at the College of Oriental Medicine, Wonkwang University (Korea). In order to obtain M-2, 600 g of phosphorus was first extracted with methanol to obtain 17.3 g of extract. This extract was separated with n-hexane, EtOAc and n-BuOH to give 1,257.5 mg of EtOAc. Finally, M-2 was extracted by performing vacuum liquid chromatography (VLC) on silica gel (0.040˜0.063 μm) with respect to EtOAc.

That is, M-2 was extracted in the order described in FIG. 1, and the analysis data of the extracted compound is as follows.

From the NMR results, it was confirmed that the compound had the structure of Formula 1.

Example 2 Measurement of the Effect of M-2 on Cell Viability of ROS 17 / 2.8 Cells

Rat osteoblastic sarcoma cell line ROS 17 / 2.8 was obtained from the American Strain Bank (American Type Culture Collection (ATCC, TIB 71, Maryland, USA)). Cells were cultured in complete RPMI 1640 medium with 10% heat-inactivated fetal bovine serum, 1% l-glutamine, 1% non-essential amino acids, 1% homeopathic / antifungal (penicillin 100 U / mL, impoterisin D 25 μg / mL, and 100 μg / mL of streptomycin), 1.5% sodium bicarbonate, and 1% minimum essential vitamins were supplemented and maintained at a concentration of 1 × 10 ⁶ cell / mL in a humid environment containing 5% carbon dioxide. To determine the effect of M-2 on the cell viability of ROS 17 / 2.8 cells, ROS 17 / 2.8 cells (2.5 × 10 ⁵ / mL) were added to the indicated concentrations of M-2 with or without the addition of CM (100). U / mL IL-1β, 200 U / mL IFN-γ, and 500 U / mL TNF-α) or separately for 24 hours. The results are shown in FIG. 2, with each bar representing the mean ± SD values of three independent experiments. * P <0.01 indicates a significant difference from the group treated in the CM.

Example 3 Determination of the Effect of M-2 on Nitrogen Production in ROS 17 / 2.8 Cells

To determine the effect of M-2 on nitrogen, the accumulated nitrogen, which is an oxide of nitrogen monoxide, was measured in the culture medium by a Greries reaction. First 100 μl of cell culture medium was mixed with 100 μl of grease reagent (1% sulfanamide, 0.1% naphthylethylenediamine dihydrochloride / 2.5% phosphoric acid), incubated at room temperature for 10 minutes, and then at 540 nm. Absorbance was measured by a plate reader. Fresh culture medium was used as a blank in all experiments. The nitrogen concentration of the samples was calculated from freshly prepared nitrogen standard curves in the culture medium. The results are shown in FIG. 3, with each bar representing the mean ± S.D. value in three independent experiments. * P <0.01 indicates a significant difference from the group treated in the CM.

Example 4 PGE of ROS 17 / 2.8 Cells ₂ Measure the influence of M-2 on production

To determine the effect of M-2 on PGE ₂ production of ROS 17 / 2.8 cells, cells (1 × 10 ⁶ cells / mL) were pre-incubated with M-2 for 2 hours and CM (100 U / further incubated for 6-18 hours in a 24-hole plate incubator with mL IL-1β, 200 U / mL IFN-γ, and 500 U / mL TNF-α). Supernatants were removed at defined times and PGE ₂ concentrations were quantified using an immunoassay kit according to the preparation sequence (R & D System, Minneapolis, MN, USA). The results are shown in FIG. 4, with each bar representing the mean ± SD values in three independent experiments. * P <0.01 indicates a significant difference from the group treated in the CM.

Example 5 Measurement of the Effect of M-2 on iNOS and COX-2 in ROS 17 / 2.8 Cells

To determine the effect of M-2 on iNOS and COX-2 of ROS 17 / 2.8 cells, cellular proteins were extracted from control and M-2 treated ROS 17 / 2.8 cells. Washed cell precipitates in cold lysis buffer [10 mM Tris-base, 5 mM EDTA, 50 mM NaCl, 1% triton X-100, 5 mM phenylmethylsulfon fluoride, 2 mM sodium orthovanadate, 10 [Mu] g / mg leupeptin, 25 [mu] g / mL aprotinin] was resuspended and incubated at 4 [deg.] C. for 30 minutes. Nuclei and cell debris were removed using microcentrifugation, and the supernatant was quenched. Treated and untreated cell proteins 30 μg, respectively, were subjected to electroprotein detection with a nitrocellulose membrane and separated by 10% sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis. Primary antibodies (primary antibodies of rabbit anti-iNOS, rabbit anti-COX-1, and rabbit anti-COX-2) were diluted appropriately and incubated for 4 hours, followed by blocking (5%) at 4 ° C for immunoprotein detection. Skim milk) overnight. The blot was washed twice with PBS and 1 at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG isolated from wasabi diluted 1: 5000. Incubated for hours. The blot was again washed three times with tween20 / tris-buffered saline (TTBS), developed for 1 hour with 10 mL of a 1: 1 mixed solution of the ECL detection system, followed by rapid drying and 2 The film was exposed to ˜20 minutes. Protein concentration was analyzed according to the manufacturer's instructions using the Bio-Rad protein assay reagent. Each bar represents the mean ± S.D. value in three independent experiments.

As stated above, when using M-2 or a composition containing the same extracted from the intestine according to the present invention as an inflammatory treatment, it is possible to provide a natural inflammatory treatment with fewer side effects.

Figure 1 shows the process of extracting a novel compound from the phosphorus.

FIG. 2 shows ROS 17 / 2.8 cells (2.5 × 10 ⁵ / mL) with or without CM-2 at the indicated concentrations (100 U / mL IL-1β, 200 U / mL IFN-γ, and Cell viability was measured after incubation with 500 U / mL TNF-α for 24 hours.

Figure 3 shows ROS 17 / 2.8 cells (2.5 × 10 ⁵ / mL) with or without M-2 (1-40 μg / mL) (100 U / mL IL-1β, 200 U / mL). After incubation with IFN-γ and 500 U / mL TNF-α for 24 hours or 18 hours, nitrogen concentration and iNOS expression levels were measured.

4 shows ROS 17 / 2.8 cells (1 × 10 ⁶ / mL) with or without M-2 (1-40 μg / mL) (100 U / mL IL-1β, 200 U / mL) After incubation with IFN- [gamma] and 500 U / mL TNF- [alpha] for 24 hours or 18 hours, the expression level of intracellular prostaglandin E2 [Prostaglandin E2 (PGE ₂ )] was measured.

FIG. 5 shows 17 / 2.8 cells (1 × 10 ⁶ / mL) with or without CM-2 at the indicated concentrations (100 U / mL IL-1β, 200 U / mL IFN-γ, and 500 U / mL TNF-α) and incubated for 18 hours, the COX-2 activity and expression was measured.

Claims (6)

The novel compound (M-2) represented by the following formula (1). [Formula 1] Inflammation treatment composition comprising M-2 represented by the formula (1). The composition of claim 2, wherein the M-2 is extracted from a phosphorus. The composition according to claim 2, wherein the composition is used in the form of a cream, an ointment, an injection, a granule, or an external dermal solution. The composition according to any one of claims 2 to 4, wherein the M-2 inhibits the production of nitrogen monoxide. The composition according to any one of claims 2 to 4, wherein the M-2 inhibits the expression of prostaglandin E2 (PGE ₂ ).