TW200904463A - Tumor necrosis factor-a inhibitor and prostaglandin synthase-2 inhibitor - Google Patents

Abstract

The object is to find a plant having an excellent pharmacological effect or function among plants naturally growing in Okinawa, and to extract an active ingredient contained in the plant and utilize the active ingredient in a pharmaceutical product, a functional food or the like. Disclosed are a tumor necrosis factor-a inhibitor and a prostaglandin synthase-2 inhibitor each comprising an extract of a plant Crassocephalum crepidioides S. Moore as an active ingredient.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a tumor necrosis factor-α inhibitor and a prostaglandin synthetase-2 inhibitor comprising an extract of safflower daisy as an active ingredient. [Prior Art] The islands of Okinawa belong to the maritime subtropical climate, and are distributed in the northern limit of the tropics and in the south of the temperate zone. Among the various biological resources, there are various pharmacological effects and functionalities that are foreseeable, and the unique biological resources of Okinawa have also been reported, but their research is not enough. SUMMARY OF THE INVENTION Therefore, the subject of the present invention is to use a substance having good pharmacodynamics and functionality in an organism grown in Okinawa, and extracting an active ingredient contained in the substance for use as a medicine or function. Sexual foods are the subject of the present invention. The present inventors focused on Crassocephalum crepidioides S. Moore, which is widely produced in Okinawa, and has been found to have tumor necrosis factor-α (TNF_α) after behaving for its efficacy and function. The invention is accomplished by inhibiting the action of fj and the action of anterior sputum (] adenine synthase_2 (COX_2) inhibition. J. That is, the present invention inhibits TNF-α with the extract of safflower daisy as an active ingredient. Further, the present invention is a 5- to 200904463 C Ο X - 2 inhibitor having an extract of safflower daisy as an active ingredient. [Embodiment] [Best Mode for Carrying Out the Invention] The safflower used in the present invention The extract of Chrysanthemum is obtained by extracting safflower daisy with a hydrophilic solvent such as a lower alcohol such as water, ethanol or methanol, or the like. The extract is made of safflower daisy (ground) The raw material is dried, and then subjected to fine cutting or the like, and immersed in the heated hydrophilic solvent, and the hydrophilic solvent amount is 1 to 20 L for 1 kg of the raw material. Suitable. In addition, the extraction time is 5 0 The clock should be 5 hours, and the better one is 10 minutes to 1 hour. The extract of safflower daisy (hereinafter referred to as "red flower of safflower safflower") is obtained by centrifugation and preparation. The liquid substance in which the insoluble matter is removed, or the liquid material is further freeze-dried or the like, and can be used as a dried product. The safflower extract obtained as described above has the following pharmacological activities (1) tumor necrosis factor (TNF-) α) Inhibition: In the experimental mice using the violent hepatitis, the result of beating TNF-a by administering the safflower extract to the safflower extract in advance, the amount of production was drastically reduced. This shows that the safflower extract It has the inhibitory effect of TNF_α. However, by TNF·α, after activation of neutrophils, elastic force 200904463 proteolytic enzyme inhibits vascular endothelial cells, which makes the vascular endothelium permeable, and the plasma leaks out of the blood vessels, and the blood is concentrated. Cyclical stagnation leads to microcirculatory disorders. Microcirculatory disorders cause tissue ischemia, causing insufficiency of organs. Also, after TNF-α production by macrophages, vascular endothelial cells The formation of tissue factors reduces the discovery of anticoagulant factors and reduces the activity of the protein-degrading factor. Therefore, 'microvascular embolism is formed. TNF-α inhibits mitochondorial respiration of vascular endothelial cells. When the mitochondria of nerve cells are inhibited, the excitatory amino acid is increased, causing secondary neuronal cell disorders. In addition, the permeability of the cell membrane is lost, and Ca2+ flows into the cells, resulting in acute neuronal necrosis. The TNF-α system mediates the permeability conversion of mitochondria (PTP is an opening) through a TNF receptor. By the permeability of the inner membrane of the mitochondria, cytochrome C is released from the gap between the mitochondrial membranes, and c a s p a s e (decomposing enzyme)-3 is activated in the nucleus to induce apoptosis, and an anticancer effect can be expected. The TNF-α produced by the fat cells not only enhances the local (TNF-α concentration of adipose tissue), but also increases the concentration of TNF-〇 in the blood, causing insulin antagonism. Further, TNF-〇 inhibits infiltration into cells of glucose (muscle cells, fat cells, etc.), resulting in hyperglycemia. TNF- ct further inhibits the activity of lipoprotein lipase (LPL), causing hyperlipidemia (high-solicemia). Therefore, the TNF-α inhibitor of the present invention can be applied to vascular endothelial cell injury inhibitors, anti-micro-thrombosis agents, mitochondrial damage inhibitors, 200904463 insulin sputum resistance-rising inhibitors, hypoglycemic agents, anti-hyperlipemia Symptoms and so on. (2) Inhibition of prostaglandin synthetase (C Ο X - 2): The hepatocytes of the smoldering hepatitis mice were taken, and the C OX-2 mRNA contained therein was amplified by RT-PCR and then pre-subjected. The COX-2 mRNA was significantly reduced in the group of Safflower extracts. This proves that the safflower extract has the inhibitory effect of COX-2. COX-2 is induced at various sites by stimulation of various protoplasmic movements at the site of inflammation, resulting in major inflammation and pain-related PG. Therefore, it is expected that an ideal agent for selectively inhibiting C Ο X - 2 among the agents for anti-inflammatory action, in fact, a drug having high selectivity for COX-2, proves that it is less likely to inhibit the onset of C Ο X -1 Side effects of injury. Therefore, the COX-2 inhibitor of the present invention can be suitably used for anti-inflammatory and analgesic agents such as chronic rheumatoid arthritis, osteoarthritis, low back pain, periarthritis of the shoulder, and wrist and wrist syndrome. The TNF-α inhibitor of the present invention and a COX-2 inhibitor (hereinafter referred to as "medicine") can be produced by combining an extract of safflower daisy and a known medical carrier. The medicine can be applied to a parenteral agent such as a tablet, a capsule, a powder, a granule or the like, a liquid, a monosaccharide or the like, an injection, a drip, an external preparation, a suppository, a patch, or the like. Examples of pharmaceutical carriers used in the manufacture of the above-mentioned respective pharmaceuticals include starch, lactose, sucrose, mannitol, corn starch, crystalline cellulose-8-200904463, carboxymethylcellulose, and citric acid Agent, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl ether, ethyl cellulose, gum arabic, tragacanth, gelatin, hydroxypropyl cellulose, dextran, pectin and other binding agents, magnesium stearate, a lubricant for talc, polyethylene glycol or the like, a disintegrating agent, a disintegrating aid, a known carrier for a solid agent, a liquid component of water, ethyl alcohol, ethylene glycol, glycerin, etc., polyethylene oxide Examples of a carrier for a liquid agent such as a surfactant such as an alkyl sorbitan fatty acid ester, a taste component such as glucose or an amino acid, a dissolution aid, a coloring agent, and a preservative. Further, as for the external preparation, the suppository, and the patch, a known carrier can be used in accordance with these dosage forms. Further, the pharmaceutical of the present invention can also be used as a food additive in combination with foods and drinks. The food produced by using the food additive may also be used as a health food, and the amount of the safflower extract extracted from the medicine of the present invention varies depending on the patient, the degree of the disease, the age of the patient, etc., and must be determined according to the experiment, such as : In the case of the oral preparation of the active ingredient, one adult (60 kg body weight) should be 5 mg to 30 g per day, more preferably 20 mg to 10 g, and 1 part to several times. Further, in the case of intravascular administration, it is preferably from 2 mg to 10 g, more preferably from 10 mg to 2 g, and in this case, the same may be carried out by injection or once simultaneously with the infusion. [Examples] Hereinafter, the present invention will be described in more detail by way of Reference Examples and Examples. However, the invention is not limited thereto. 200904463 [Reference Example 1] (Modulation of Safflower Extract): Extracted from the farm of the Subtropical Magnetic Science Education Center of the University of the Ryukyus, the safflower daisy of 4〇~50cm in grass height, fully washed with the tap water. After the grass, 60. (The drying was carried out for 8 hours. The dried product was cut into small pieces, and each lg of the chips was immersed in a temperature of 90 ° C for 30 minutes in an amount of 10 ° C. Thereafter, the solid matter was filtered, and the filtrate was freeze-dried. A safflower extract of safflower was prepared as a powder. [Example 1] (GalN/LPS was applied to the effect of safflower extract of experimental rats).: <treatment of experimental animals> Male Sprague Dawley (SD) experimental rats were used. (body weight: 240 to 280 g) As an experimental animal, a fatty glycan compound (derived from Salmonella entenica; LPS) and D-galactosamine (G) were used to make a violent hepatitis test mouse. The experimental animals were divided into a control group (3), a GalN/LPS group (4), a GalN/LPS, and a safflower extract (cc) group (GL + Cc,) (3) obtained in Reference Example 1. In the control group and the GalN/LPS group, sterilized water and GL + C were added. “Cc (2〇mg/kg) was administered to the group, and GalN/LPS was administered into the abdominal cavity 12 hours and 1 hour respectively. Put GaIN (600 mg/kg) ' LPSiO^g/kg) into the GalN/LPS group and GL + Cc group for subcutaneous injection Intra-abdominal administration. In the control group, the same amount of physiological saline was used instead of GalN/LPS. The rats were subjected to 1 night or -10-200904463 after hunger strike, and the head was cut by G a IN / LPS for 1 hour or 12 hours. Blood was collected from the incision. In addition, in order to discuss the effect of safflower safflower alone, intraperitoneal administration was performed 12 hours and 1 hour before Cc (20 mg/kg) cutting. Saffron sinensis was administered orally. In GL + Cc·, ρ·〇_), 'cc, (20mg/kg) was administered orally in G a IN / LPS for 12 hours and one hour before oral administration. After eclipse for 1 night, by GalN /LPS is administered 12 hours later. <Modulation of cell division> The liver is removed from the slaughtered mouse's adjustment of its cell division. When the liver is removed by the serum protease, the inhibition is 5 mM. 1.5% of KB1 (ice water) of the methionine protease (Bz) was perfused. Add 4 times the weight of the isolation buffer (0.03 M trihydrochloride buffer: pH 7.0, the extracted liver sputum). 0.25M mannitol, 0.1M KC1, 0.3mM ED ΤΑ), sentenced with a Teflon glass homogenizer. The homogenate is 600xg, 10 minutes After centrifugation at 4 ° C, the supernatant was centrifuged at 5,000 x g for 10 minutes at 4 ° C for granulation. The precipitate was suspended in a separation buffer at 5 〇〇. 〇xg '1 0 minutes, centrifuge at 4 °C 'more to separate the buffer after the suspension is suspended' at 25 00xg, 2 minutes, centrifuge at 4 ° C, continue to 5 000Xg ' 8 minutes, 4 ° C The precipitate which was subjected to centrifugation was divided into granules, and the dispersion buffer of 1 _ 5 times the weight of the mitochondria was used as a cell division for the experiment. -11 - 200904463 <Measurement method> (1) Measurement of nitrogen oxide (NO) The NO of the serum was measured by N02-/N01-. The serum ι〇〇μ1 was mixed with methanol ΙΟΟμΙ, and then left to stand for 2 〇 minutes to carry out centrifugation to remove the protein. The supernatant was further centrifuged to obtain a supernatant, which was measured using a Ν analyzer (elOm). The Ν Ο analyzer measures the concentration of nitrite ions and nitrate ions in the sample by a combination of diazotization and high-speed liquid chromatography. The amount of Ν02_/Ν01_ was calculated from the calibration curve using sodium nitrite (NaN02) and sodium nitrate (NaN03) as standards. (2) Measurement of TNF-a The measurement of TNF-α in the movement of the pure pulp was carried out as follows. One and a half hours after administration by GalN/LPS, blood was collected from the incision of the cut mouse, and the serum was obtained by centrifugation at 3 000 rpm for 15 minutes. The TNF-α in this serum 50 μl was determined by the ELIZA method of a mouse, TNF-α. kit (Biothos). -12- 1 Reverse blood • Invertase polymerase chain reaction (RT-PCR): The mice were slaughtered from the slaughtered rats to about 3 mg before the start of perfusion, and immediately frozen with liquid nitrogen and stored at -80 °C. From this liver, Kiagen's RNasey · mini kit was used for RNA extraction according to the following report. First, the 'cut liver' was placed in a 2.0 ml thin tube and added to the RLT buffer 600 μ1 attached to kit 200904463, pulverized by a syringe, and centrifuged at 1,500 x g for 3 minutes at room temperature. The supernatant was transferred to another 2 ml thin tube, and iml 70% ethanol was added thereto for pipetting, and the RNeasy·mini·column attached to the kit was placed. After centrifugation at 9000 xg for 15 seconds at room temperature, the RNA adsorbed to the column was added to 700 μl of the k i t attached washing buffer, washed, and centrifuged at 9000 g for 15 seconds at room temperature. A 500 μl kit-attached RPE buffer was further added and centrifuged at 9000 x g for 15 seconds at room temperature. The column was continuously dried by adding 5 μl of RPE buffer at 900 ° C for 2 minutes at room temperature. This was added to 30 μl of sterilized water at 900 ng, and centrifuged at room temperature for 1 minute. The eluate was diluted 100-fold, and the absorbance at 260 n and 280 nm was measured. From the RNA of 1 gg of purity and concentration, the cDNA prepared by TAKARA mRNA, Isolation, PCR, kit, (30 ° C; 1 〇 min, 45 ° C; 30 min, 5 ° C; 5 min), Using the heuristics shown below? 011. ? 011 product line and charge. Buffer (36% glycerol, 30111), butyl ketone, 0.05% bromophenol blue, 0.05% xylene, indigo) mixed at 5:1, injected into 2% agarose gel (ΙχΤΒΕ). The cells were subjected to ιοον, for 45 minutes, and stained with ethidium bromide (ethidium bromide KlOmg/ml) for 15 minutes. After decolorization for 10 minutes, the spectral band was detected under UV irradiation. As a molecular weight marker, the 100bP DNA method was used. The salt-based arrangement of the heuristics used in the PCR and the PCR conditions are shown in the following table. -13- 200904463 [Table 1] Name Heuristic (forward) (reverse) Conditional PCR product (bp) iNOS TTGGGTCTTGT TGTGCAGTCCC TAGCCTAGTC AGTGAGGAAC 94 °C-1 分59 °C-2 分72°C-3 分30 Back 264bp C0X-2 CATTCTTTGCCC GACCAGGGACCA AGCACTTCAC GACCAAAGAC 94 °C-4 minutes 94°C-15 seconds a 59 °C-30 seconds 72 °C-45 seconds a 72 °C-5 points 35 times 297bp 6APDH TCCCTCAAGAT AGATCCACAACG TGTCAGCAA GATACATT 94 °C-4 minutes 94 ° C - 15 seconds - 54 ° C - 45 seconds 72. 〇1 minute - 72 °C-7 minutes 30 times 309 bp <Measurement results> (1) - Nitric oxide (NO) Figure 1 shows the concentration of N◦ in serum. The results showed that the concentration of NO in the serum of the GaIN/LPS group was 43 0.9%, which was a significant increase in the control group, while the pre-administration of the extract of safflower extract showed a significant decrease of 2 3 7.8%, showing safflower. The effectiveness of the extract of Chrysanthemum. (2) TNF-a Figure 2 shows the concentration of TNF-α in serum after one and a half hours of GalN/LPS administration. From this figure, it was confirmed that TNF-α was not detected in the serum of the inflammatory index in the control group, and there was a significant increase in the GalN/LPS group. On the other hand, only 6% of the GalN/LPS group was used in the group in which the safflower extract was previously administered. This proves that the safflower extract can strongly inhibit the appearance of TNF- -14 - 200904463 α. (3) Reverse blood. Invertase polymerase chain reaction (rt-PCR): Fig. 3 is a diagram showing the results of RT-PCR of mRNA. An inducible nitric oxide synthase (iNOS) heuristic, a cyclooxygenase (C0X-2) heuristic, and an internally regulated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) heuristic, using RNA extracted from the liver, Perform PCR. After determining the appearance of the PCR product amplified by this PCR, iNOS, COX-2 appeared more in the GalN/LPS group than in the control group, and was previously administered to the group of safflower extracts. The appearance has decreased. On the other hand, the appearance of the internally adjusted GAPDH was a certain one, and therefore, it was confirmed that the occurrence of iNOS and COX-2 of the safflower extract was inhibited. [Example 2] Effect on edema of red algae extract For the edema caused by administration of a red algae extract, the effect of the safflower extract was examined as follows. That is, the CrI:CD (SD)-based experimental mice as experimental animals were divided into three groups and divided into three groups. Among them, the extract of Safflower extract was administered intraperitoneally to the safflower extract dissolved in physiological saline solution at 300 mg/kg 1 hour before and 1 hour before administration of the red algae extract. In addition, in the group administered with indomethacin, an indomethacin of 10 m g / k g was orally administered 1 hour before the administration of the red algae extract. More than 2 hours before and one hour before the administration of the red algae extract in the control group, 5 mL/kg of physiological saline solution was intraperitoneally administered. -15- 200904463 The method of administering the red algae extract to each experimental mouse was carried out by putting an aqueous solution for injection of 1% red algae extract into the skin of 0·1 ml/body under the skin of the right hind foot. Further, the edema condition was measured every hour using the mouse and the hamster volume measuring device of the hind paw of the experimental mouse, before inflammation and between 1 and 5 hours after the inflammation. This result is shown in Figure 4. It is demonstrated by Figure 4 that the safflower extract has an anti-inflammatory effect. [Industrial Applicability] As shown in the above examples, the safflower extract has anti-TNF_α action, anti-COX-2 action and anti-inflammatory action. Therefore, the medicine of the present invention can be applied to vascular endothelial cell injury inhibitors, anti-micro-thrombosis agents, mitochondrial damage inhibitors, insulin sputum resistance-suppressing inhibitors, hypoglycemic agents, anti-hyperlipidemia, chronic rheumatoid Anti-inflammatory and analgesic agents for arthritis, anti-inflammatory and analgesic agents for osteoarthritis, anti-inflammatory and analgesic agents for low back pain, anti-inflammatory and analgesic agents for inflammation around the shoulder joints, and anti-inflammatory and analgesic agents for shoulder and neck wrist syndrome. Further, the medicine containing the safflower extract of the present invention can be used as a food additive to be added to various foods to obtain a health food having the above-mentioned effects. BRIEF DESCRIPTION OF THE DRAWINGS [Fig. 1] A graph showing the concentration of N 0 in the serum of the GaIN/LPS group of Example 1, the safflower extract and the control group. Fig. 2 is a diagram showing the concentration of TNF-α in the serum of the GalN/LPS group of Example 1, the safflower extract group and the control group. -16-200904463 [Fig. 3] A graph showing the results of RT-PCR of the mRNA of Example 1. Fig. 4 is a view showing the effect of the safflower extract on the edema of the red algae extract.

Claims (1)

200904463 X. Patent application scope 1 · A tumor necrosis factor-α inhibitor characterized by the extract of safflower 褛 褛 昭 (Showa grass) as an active ingredient. 2 · The tumor necrosis factor-α inhibitor as claimed in claim 1 is a vascular endothelial cell injury inhibitor, an anti-micro-thrombosis agent, a mitochondrial damage inhibitor, an insulin antagonistic increase inhibitor, a hypoglycemic agent Or anti-hyperlipidemic agents. 3. A prostaglandin synthetase-2 inhibitor characterized by the extract of safflower and chrysanthemum as an active ingredient. 4. For example, the prostaglandin synthetase-2 inhibitor of the third paragraph of the patent application is an anti-inflammatory, analgesic agent for chronic rheumatoid arthritis, an anti-inflammatory and analgesic agent for osteoarthritis, and an anti-inflammatory for low back pain. Analgesic, anti-inflammatory, analgesic or shoulder and neck wrist syndrome with anti-inflammatory and analgesic agents. 5. The medicine according to any one of claims 1 to 4, wherein the safflower daisy extract is an extract of safflower daisy whole grass. 6. The medicine according to any one of claims 1 to 5, wherein the safflower extract is a hot water extract of 80 ° C or higher. 7. The medicine according to any one of the first to sixth aspects of the invention, which is a food additive. 8. A kind of food ’ is characterized by containing the medicine of the seventh item of the patent application. -18 -