Anti-inflammatory composition extracted from watermelon staminate flowers

Abstract

The present invention provides a natural product-derived substance having an anti-inflammatory effect. It was found that a composition extracted from watermelon staminate flowers has the ability to suppress production of nitric oxide by macrophages, leading to the conception of an anti-inflammatory composition extracted from watermelon staminate flowers according to the present invention. An anti-inflammatory composition extracted from watermelon staminate flowers according to the present invention was confirmed to reduce the concentration of nitric oxide in cells in a concentration-dependent manner, and is expected to be suitably used in therapeutic agents against sepsis and autoimmune diseases. In addition, the anti-inflammatory composition is considered to have few side effects because of being derived from watermelon. Further, the anti-inflammatory composition can be expected to serve as an alleviation agent for some autoimmune diseases since the anti-inflammatory composition is considered to suppress iNOS induction and TNF-α production by inhibiting the activation of NF-κB.

Description

Anti-inflammatory composition extracted from watermelon male flowers

[0001] The present invention relates to an anti-inflammatory composition extracted from watermelon male flowers. Because the anti-inflammatory composition has anti-inflammatory properties, it can be applied to pharmaceuticals and foods. It is particularly expected to be used as a therapeutic agent for sepsis.

[0002] The inflammatory response in the body is a defense mechanism for the body to defend itself against external invasion by bacteria or viruses, or against stimuli such as chemicals. This defense mechanism involves various cells, such as mast cells, neutrophils, macrophages, and lymphocytes, reacting with tissues to effectively remove foreign substances and repair tissues.

[0003] However, excessive inflammation in the body can lead to chronic inflammatory conditions known as allergies and autoimmune diseases, which can be harmful. Among these, sepsis, a systemic inflammatory response caused by bacterial or viral infections, progresses rapidly, and if not detected early and treated appropriately, can lead to multiple organ failure and death.

[0004] Sepsis is a toxic reaction to an infection. According to the Japanese Society of Intensive Care Medicine, it affects 20 to 30 million people worldwide annually and is the disease with the highest mortality rate. In particular, it is reported that in US hospitals, 750,000 people are affected annually, with over 250,000 dying. In Japan, the number of deaths in 2017 was approximately 60,000, a 2.5-fold increase from 2011.

[0005] Macrophages, immune cells, are deeply involved in this bacterial inflammation. Macrophages are activated by interferon-γ (IFN-γ), lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, and lipoteichoic acid, which is present on the surface of gram-positive bacteria, to induce the expression of inducible nitric oxide synthase (iNOS) and produce nitric oxide (NO).

[0006] When NO is overproduced, it enters the bloodstream, causing a drop in blood pressure and organ damage, leading to septic shock. The production of NO, which induces inflammation in the body, involves various signaling molecules that transmit information via receptors present on the biomembrane of macrophages.

[0007] Toll-like receptors (TLRs) on the cell surface are activated by dimerization upon recognition of ligands such as LPS, and then bind to adapter molecules such as MyD88, which trigger inflammatory signaling in the cytoplasm. As a result, transcription factors such as AP-1 (activator protein 1) and nuclear factor-kappa B (NF-κB) are activated via the mitogen-activated protein kinase (MAPK) pathway, which is involved in the induction of iNOS and inflammatory cytokine gene expression, leading to the synthesis of inflammatory cytokines such as NO and tumor necrosis factor-α (TNF-α).

[0008] IFN-γ also activates macrophages by transmitting signals from the Janus kinase / signal transducer and activator of transcription (JAK-STAT) pathway via the interferon-γ receptor (IFNGR1 and IFNGR2), a dimeric receptor present on the cell membrane.

[0009] Therefore, it is expected that inhibiting the activation of signal transduction factors involved in NO production will lead to the development of anti-inflammatory drugs for bacterial inflammation such as sepsis and autoimmune diseases.

[0010] Anti-inflammatory drugs currently being developed are mainly classified into two types: steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs. The main component of steroidal anti-inflammatory drugs is glucocorticoids, while non-steroidal anti-inflammatory drugs include aspirin and indomethacin. These are substances synthesized from natural substances. In this way, the search for natural substances with anti-inflammatory properties will lead to the development of new anti-inflammatory drugs.

[0011] In recent years, numerous natural substances with anti-inflammatory properties have been discovered and are being used clinically. For example, ursolic acid, a type of terpenoid widely found in plants, inhibits the activation of NF-κB in macrophages stimulated with LPS, thereby suppressing the induction of iNOS and the production of TNF-α. Furthermore, curcumin, found in the plant turmeric, exhibits anti-inflammatory properties by inhibiting the activation of NF-κB in macrophages stimulated with LPS, thereby suppressing the production of TNF-α and interleukin (IL)-1β.

[0012] In particular, with regard to sepsis, quercetin, which is known to be abundant in red wine, onions, kiwi fruit, green tea, apples, berries, and Chinese cabbage (cabbage, broccoli, cauliflower, turnip, etc.), has been found to exhibit excellent antiviral effects against viral hemorrhagic sepsis.

[0013] Furthermore, Patent Document 1 discloses that dexamethasone is effective as a steroid anti-inflammatory drug for treating viral sepsis.

[0014] Special Publication No. 2023-545404

[0015] As plants grow, different substances are produced and accumulated in different parts of the plant, such as flowers, leaves, branches, stems, and roots. The present invention was discovered in the process of searching for plant-derived substances that suppress blood nitric oxide levels and can be used to treat viral inflammation, including sepsis.

[0016] In view of the above problems, the present invention was completed upon the discovery that an extract from watermelon male flowers is an anti-inflammatory composition that suppresses the ability of macrophages to produce nitric oxide and has the effect of being widely used as an anti-inflammatory pharmaceutical composition (a pharmaceutical composition that suppresses inflammation), including in the treatment of sepsis.

[0017] More specifically, the anti-inflammatory pharmaceutical composition of the present invention contains an anti-inflammatory composition extracted from watermelon male flowers as an active ingredient.

[0018] The anti-inflammatory pharmaceutical composition of the present invention has an anti-inflammatory effect because it inhibits nitric oxide production by macrophages. Therefore, it can be used to inhibit inflammatory responses caused by viruses. It is particularly expected to be applied to sepsis.

[0019] In addition, because it is derived from watermelon, it is thought to have few side effects. Furthermore, by inhibiting the activation of NF-κB, it is thought to be able to suppress the induction of iNOS and the production of TNF-α, so it is expected to be used as a mitigating agent for some autoimmune diseases.

[0020] In addition, by adjusting the concentration, a mild anti-inflammatory effect can be expected, so it can also be used in processed food compositions such as supplements.

[0021] 1 is a graph showing the ability of an anti-inflammatory composition derived from watermelon male flowers to inhibit nitric oxide production by macrophages.

[0022] The following examples are provided to explain pharmaceutical compositions and processed food compositions containing the anti-inflammatory composition of the present invention. Note that the following explanations are provided to illustrate one embodiment of the present invention and one example, and the present invention is not limited to the following explanations. The following explanations can be modified within the scope of the present invention.

[0023] The anti-inflammatory composition of the present invention is extracted from the male flowers of watermelon. Watermelon (scientific name: Citrullus lanatus) is an annual climbing plant of the Cucurbitaceae family. As is well known, its fruit is also called "suika" (watermelon), and is one of the summer fruits in Japan.

[0024] There are no particular limitations on the type of watermelon that can be used in the present invention, and varieties such as large red-fleshed varieties, seedless red-fleshed varieties, black-skinned varieties, oblong varieties, yellow-fleshed varieties, and orange-colored varieties can be suitably used.

[0025] Watermelons are dioecious, with female and male flowers blooming separately on the same vine. Female flowers have a swollen ovary beneath the petals. On the other hand, male flowers do not have an ovary, making them easy to identify. The male flowers used in this invention are those that have just bloomed, and flowers with wilted petals are not used.

[0026] The anti-inflammatory composition is obtained as follows: Harvested watermelon male flowers are immersed in ethanol for 5 hours, and the extract is subjected to liquid-liquid partitioning using n-hexane and water in a separatory funnel. This process defats the vegetable oil and removes the green plant pigment chlorophyll.

[0027] Subsequently, the aqueous layer after partitioning with n-hexane is subjected to liquid-liquid partitioning with ethyl acetate, and the ethyl acetate layer is concentrated to obtain an ethyl acetate fraction, which is the anti-inflammatory composition extracted from watermelon male flowers according to the present invention.

[0028] As shown in the examples below, this anti-inflammatory composition can suppress the NO production ability of macrophages with viral disease when they are stimulated with LPS and IFN-γ and enter a state of excessive NO production. Therefore, it is expected that the anti-inflammatory composition will inhibit the activation of NF-κB, suppress the production of TNF-α and interleukin (IL)-1β, and exhibit anti-inflammatory effects. In other words, it is possible to obtain an anti-inflammatory pharmaceutical composition (hereinafter simply referred to as "anti-inflammatory pharmaceutical composition") containing the anti-inflammatory composition extracted from watermelon male flowers as an active ingredient. Of course, the anti-inflammatory composition extracted from watermelon male flowers can also directly constitute a composition that inhibits nitric oxide production in macrophages.

[0029] The anti-inflammatory pharmaceutical composition of the present invention may be converted into a salt by mixing with a pharmaceutically acceptable acid in a solvent such as water, methanol, ethanol, acetone, etc. Examples of pharmaceutically acceptable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfate, phosphoric acid, and nitric acid, and organic acids such as acetic acid, propionic acid, oxalic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, maleic acid, fumaric acid, methanesulfonic acid, p-toluenesulfonic acid, and ascorbic acid.

[0030] The anti-inflammatory pharmaceutical composition can also be administered orally or parenterally (eg, by intravenous, subcutaneous, or intramuscular injection, topically, rectally, transdermally, or intranasally).

[0031] Pharmaceutical compositions for oral administration can be prepared by adding pharmaceutically acceptable and commonly used excipients, binders, lubricants, disintegrants, surfactants, flow enhancers, etc. Examples of excipients that can be used include lactose, fructose, glucose, corn starch, sorbitol, and crystalline cellulose.

[0032] As the binder, methyl cellulose, ethyl cellulose, gum arabic, gelatin, hydroxypropyl cellulose, polyvinylpyrrolidone, etc. can be used. As the lubricant, talc, magnesium stearate, polyethylene glycol, hardened vegetable oil, etc. can be suitably used.

[0033] Disintegrants that can be used include starch, sodium alginate, gelatin, calcium carbonate, calcium citrate, dextrin, magnesium carbonate, synthetic magnesium silicate, etc. Surfactants that can be used include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80, etc.

[0034] Fluidity promoters that can be used include light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate, etc. Other additives that can be used include syrup, petrolatum, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium nitrite, sodium phosphate, etc.

[0035] In addition, the anti-inflammatory pharmaceutical composition according to the present invention may be prepared in various dosage forms depending on the mode of administration, such as oral preparations such as capsules, tablets, granules, powders, pills, and fine granules, and parenteral preparations such as injections, rectal preparations, oily suppositories, and aqueous suppositories. In addition to the anti-inflammatory composition extracted from watermelon male flowers, the anti-inflammatory pharmaceutical composition according to the present invention may contain other pharmacologically active ingredients.

[0036] The anti-inflammatory pharmaceutical composition of the present invention can be used as a therapeutic agent for known conditions that are improved by NO suppression, such as autoimmune diseases accompanied by inflammation, such as sepsis.

[0037] The anti-inflammatory composition extracted from the watermelon male flowers of the present invention can also be provided as a processed food composition. The processed food composition of the present invention may also be referred to as an anti-inflammatory processed food composition. Processed food compositions include not only general processed foods, including luxury foods and health foods such as candy, gum, jelly, biscuits, cookies, rice crackers, bread, noodles, fish and meat paste products, tea, soft drinks, coffee drinks, dairy drinks, whey drinks, lactic acid bacteria drinks, yogurt, ice cream, and pudding, but also foods with health claims, such as foods for specified health uses and foods with nutrient functions specified in the Ministry of Health, Labor, and Welfare's Food with Health Claims System, as well as nutritional supplements, feed, food additives, and the like.

[0038] The anti-inflammatory composition of the present invention can be used with varying ratios of its active ingredients depending on the situation in which it is used. In particular, when used as a pharmaceutical composition (including a therapeutic agent) for the treatment of obvious inflammatory symptoms, the concentration can be increased to an amount that reduces blood nitric oxide levels.

[0039] Additionally, when the anti-inflammatory composition is included in a processed food composition such as a supplement, it is advisable to use the anti-inflammatory composition in a low ingredient ratio.

[0040] <Production of anti-inflammatory composition extracted from watermelon male flowers> The ethanol extract of watermelon male flowers was subjected to liquid-liquid partitioning using n-hexane and water in a separatory funnel. In this process, the vegetable oil was defatted and the green plant pigment chlorophyll was removed. Subsequently, the aqueous layer after partitioning with n-hexane was subjected to liquid-liquid partitioning using ethyl acetate, and this ethyl acetate layer was concentrated using a rotary evaporator to obtain 30 g of an ethyl acetate fraction. In this example, this is tentatively referred to as the "male flower extract."

[0041] <Cell Preparation> Mouse macrophage RAW264.7 cells were obtained from RIKEN (Tsukuba, Ibaraki Prefecture). RAW264.7 is a macrophage-like cell line established from a tumor in a male mouse induced with Abelson murine leukemia virus. RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GE Healthcare UK Ltd., UK) containing 10% fetal bovine serum, 50 U / mL penicillin, and 50 μg / mL streptomycin at 37°C under 95% air and 5% CO. ２ It was cultivated under the environment.

[0042] Mouse macrophage-like cell line RAW264.7 cells (2 × 10 ５ 500 μL of the culture medium was seeded into a 24-well plate and incubated for 24 hours (37°C, 95% air-5% CO ２ Then, male flower extract was added to the plants to a final concentration of 10, 25, 50, 100, 200, 300, 400, or 500 μg / mL, and the plants were pretreated for 2 hours.

[0043] After pretreatment, LPS (Sigma-Aldrich, MO, USA) at a final concentration of 200 ng / mL and IFN-γ (Merck Millipore, MA, USA) at a final concentration of 5 ng / mL were simultaneously added, and stimulation was carried out for 24 hours. After stimulation, nitrite (NO), an oxidation product of NO produced by RAW264.7 cells, was detected. ２ － ) was developed with Griess reagent (Promega, WI, USA), and the absorbance at 540 nm was measured using a microplate reader (Varioskan LUX, Thermo Fisher Scientific, MA, USA) to indirectly measure the amount of NO production.

[0044] The control was RAW264.7 cells to which no male flower extract, LPS, or IFN-γ had been added. Quercetin and dexamethasone were used as a comparative example. Quercetin and dexamethasone were added to RAW264.7 cells at the same time as the male flower extract.

[0045] The results are shown in Figure 1. Referring to Figure 1, the horizontal axis indicates the type of sample, and the vertical axis indicates the nitrite concentration ratio calculated from absorbance. Compared to the control, the sample to which only LPS and INF-γ were added showed a nitrite concentration ratio of 7 or more. On the other hand, quercetin and dexamethasone, which are believed to be effective against sepsis, showed a clear inhibitory effect on nitrite concentration at 20 μM and 200 μM, respectively (right end of graph). Note that quercetin and dexamethasone correspond to 6 μg / ml and 78 μg / ml, respectively.

[0046] When male flower extract was added to RAW264.7 cells stimulated with both LPS and IFN-γ, nitrite levels were suppressed concentration-dependently, even though the dose was one order of magnitude higher than that of quercetin and dexamethasone. This indicates that the anti-inflammatory composition extracted from watermelon male flowers according to the present invention has the same inhibitory effect on nitrite levels as quercetin and dexamethasone, which are known to be effective against sepsis, and also has the effect of suppressing blood nitric oxide levels, suggesting that it is also effective against sepsis. In other words, it can be said to be an anti-inflammatory composition (a composition that can suppress inflammation).

[0047] The anti-inflammatory composition extracted from the watermelon male flowers of the present invention is expected to have the effect of suppressing blood nitric oxide levels and to have an inhibitory effect on viral autoimmune diseases such as sepsis, and is expected to be used in various aspects such as pharmaceutical compositions and processed food compositions.

Claims

1. An anti-inflammatory pharmaceutical composition containing as an active ingredient an anti-inflammatory composition extracted from watermelon male flowers.

2. A drug for treating sepsis, the active ingredient of which is an anti-inflammatory composition extracted from watermelon male flowers.

3. A composition for inhibiting nitric oxide production in macrophages, the active ingredient of which is an anti-inflammatory composition extracted from watermelon male flowers.

4. A processed food composition comprising an anti-inflammatory composition extracted from watermelon male flowers.

5. A method for producing an anti-inflammatory composition extracted from watermelon male flowers, comprising the steps of: performing liquid-liquid partitioning of an ethanol extract of watermelon male flowers with n-hexane and water in a separatory funnel; and performing liquid-liquid partitioning of the aqueous layer obtained after partitioning with n-hexane using ethyl acetate, and then concentrating the ethyl acetate layer using a rotary evaporator to obtain an ethyl acetate fraction.

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